Tom's Thesis Submitted Copy

Design, Synthesis and Application of Enzyme Responsive Hydrogel Particles using Peptide Actuators A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Engineering and Physical Sciences

2009 Tom O. McDonald School of Materials

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Contents DECLARATION............................................................................................................14 ABSTRACT.................................................................................................................15 ACKNOWLEDGEMENTS.................................................................................................16 1

2

INTRODUCTION...........................................................................................18 1.1.

MOTIVATION OF THE PROJECT...........................................................................19

1.2.

LAYOUT OF THE THESIS...................................................................................19

LITERATURE REVIEW...............................................................................22 2.1.

INTRODUCTION...............................................................................................22

2.2.

STIMULI RESPONSIVE MATERIALS.......................................................................22

2.2.1. Chemical hydrogels...............................................................................22 2.3.

BIORESPONSIVE HYDROGELS............................................................................23

2.3.1. Introduction...........................................................................................23 2.3.2. Actuation based on changes in crosslinking density.............................24 2.3.2.1.

Systems incorporating peptide crosslinkers...................................24

2.3.2.2.

Non-covalent crosslinking interactions.........................................29

2.3.3. Actuation based on electrostatic interactions.......................................34 2.3.3.1.

Electrostatic interactions between charges on the polymer

network..........................................................................................................35 2.3.3.2.

Electrostatic interactions between charges present in pendant

actuators .......................................................................................................39 2.3.4. Actuation based on conformational changes........................................42 2.3.5. Summary................................................................................................45 2.4.

PEGA.........................................................................................................46

2.4.1. The history of PEGA..............................................................................46 2.4.2. Enzyme catalysed synthesis on PEGA...................................................53 2.4.3. Summary................................................................................................56 2.5.

MICROFLUIDIC POLYMERISATION OF PARTICLES....................................................57

2.5.1. Summary................................................................................................67 2.6.

AIMS OF THESIS..............................................................................................69

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3

PEGA POLYMERISATION, CHARACTERISATION AND ENZYME

RESPONSIVE SWELLING THROUGH FUNCTIONALISATION WITH PEPTIDE ACTUATORS........................................................................................70 3.1.

ABSTRACT.....................................................................................................70

3.2.

INTRODUCTION...............................................................................................70

3.3.

EXPERIMENTAL..............................................................................................74

3.3.1. Materials...............................................................................................74 3.3.2. Inverse suspension polymerisation........................................................74 3.3.3. Charged PEGA polymerisation.............................................................74 3.3.4. Microscopy and particle size analysis...................................................75 3.3.5. Solid phase peptide synthesis................................................................76 3.3.5.1.

Solid phase synthesis of dipeptides and enzyme treatment...........77

3.3.5.2.

Solid phase peptide synthesis of peptide actuators........................78

3.3.6. HPLC.....................................................................................................78 3.3.7. Two-photon microscopy........................................................................78 3.3.8. Determining particle swelling...............................................................79 3.3.9. Assessing accessibility...........................................................................79 3.3.10.

Entrapping payload...........................................................................79

3.3.11.

Fluorimetry........................................................................................79

3.3.12.

Microscopy and determination of swelling.......................................80

3.4.

RESULTS AND DISCUSSION...............................................................................81

3.4.1. Production of µPEGA800........................................................................81 3.4.1.1.

Particle morphology and size characterisation of µPEGA............82

3.4.2. Production of µPEGA+ and µPEGA-.....................................................83 3.4.3. Further characterisation of µPEGA......................................................88 3.4.3.1.

Amine characterisation by two-photon microscopy and enzyme

compatibility..................................................................................................88 3.4.3.2.

Comparison of enzymatic hydrolysis within µPEGA800 and

macroparticles................................................................................................90 3.4.4. Functionalisation with peptide actuators..............................................91

3.5.

3.4.4.1.

Enzyme responsive increase in accessibility.................................92

3.4.4.2.

Demonstration of the encapsulation of a payload..........................95

3.4.4.3.

Enzyme specific release.................................................................96

3.4.4.4.

Enzyme responsive increase in swelling; effect of ionic strength. 97

CONCLUSIONS................................................................................................98 3

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

4

DESIGNING NEW PEPTIDE ACTUATORS FOR IMPROVED

ENZYME RESPONSIVE BEHAVIOUR..............................................................99 4.1.

ABSTRACT.....................................................................................................99

4.2.

INTRODUCTION...............................................................................................99

4.2.1. Branched peptide actuators...................................................................99 4.3.

MATERIALS AND METHODS.............................................................................102

4.3.1. Materials.............................................................................................102 4.3.2. Inverse suspension polymerisation and polymer characterisation.....102 4.3.3. Solid phase peptide synthesis..............................................................103 4.3.4. Microscopy and determination of swelling.........................................103 4.3.5. Two-photon microscopy......................................................................103 4.3.6. Entrapping payload.............................................................................104 4.3.7. Release measurements.........................................................................104 4.3.8. HPLC and LCMS.................................................................................104 4.4.

RESULTS AND DISCUSSION..............................................................................106

4.4.1. Microparticle characterisation and peptide functionalisation............106 4.4.2. Actuator design and responsiveness of peptide functionalised particles. . .............................................................................................................107 4.4.3. Characterisation of enzyme action on peptide actuators....................111 4.4.4. Enzyme triggered release....................................................................113 4.5. 5

CONCLUSIONS..............................................................................................115

MICROFLUIDIC PREPARATION OF LOW POLYDISPERSITY PEGA

PARTICLES...........................................................................................................116 5.1.

ABSTRACT...................................................................................................116

5.2.

INTRODUCTION.............................................................................................116

5.3.

MATERIALS AND METHODS.............................................................................119

5.3.1. Microfluidic system.............................................................................119 5.3.2. Flow focussing setup...........................................................................119 5.3.3. Monomer and continuous phase preparation.....................................120 5.3.4. Particle size measurement...................................................................120 5.4.

RESULTS AND DISCUSSION..............................................................................121

5.4.1. Microfluidic system.............................................................................121 5.4.1.1.

Variation of particle size with flow rate ratios and surfactant

concentration................................................................................................121

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

5.4.1.2.

Effect of increasing total flow rate on particle size.....................126

5.4.1.3.

Effect of flow ratios and surfactant concentration on polydispersity .....................................................................................................127

5.4.1.4.

Variation of particle production rates with the conditions assessed. . .....................................................................................................127

5.4.2. Using a simplified microfluidic flow-focussing device to produce polymer particles.............................................................................................129 5.4.2.1.

Orientation of device...................................................................129

5.4.2.2.

Optimising conditions with the FF device...................................131

5.4.2.3.

Effect of changing oil phase........................................................133

5.4.3. Optimum conditions for particle production.......................................134 5.4.3.1.

Particle size distribution...............................................................134

5.4.4. Morphology of particles......................................................................135 5.5.

CONCLUSIONS..............................................................................................136

6

CONCLUSIONS............................................................................................137

7

FUTURE WORK...........................................................................................139

8

APPENDICES................................................................................................141 8.1.

BACKGROUND..............................................................................................141

8.1.1. Polymers..............................................................................................141 8.1.1.1.

Peptides and proteins...................................................................141

8.1.2. Two-photon microscopy......................................................................143 8.1.3. Fluorescence resonance energy transfer.............................................144 8.2.

SUPPLEMENTARY DATA..................................................................................145

8.2.1. HPLC solvent gradient........................................................................145 8.2.2. Synthesis of activated disulfide-methacrylamide monomer................145 9

REFERENCES...............................................................................................147

References

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

List of figures Figure 1–1. The inverse suspension polymerisation of µPEGAs..............................20 Figure 1–2. Enzyme responsive µPEGA...................................................................20 Figure 1–3. Enzyme responsive µPEGA through the incorporation of branched peptide actuators........................................................................................................21 Figure 1–4. Production of PEGA particles by microfluidic polymerisation.............21 Figure 2–1. Schematic representation of the different categories of bioresponsive hydrogels....................................................................................................................24 Figure 2–2. Cell responsive synthetic hydrogels.......................................................26 Figure 2–3. Antigen responsive hydrogel..................................................................31 Figure 2–4. Displacement-Induced Switching Rates of Bioresponsive Hydrogel Microlenses................................................................................................................32 Figure 2–5. Novel synthesis of HPMA copolymers containing peptide grafts and their self assembly into hybrid hydrogels..................................................................34 Figure 2–6. The enzymes and the reactions they catalyse used in glucose responsive hydrogels....................................................................................................................36 Figure 2–7. Characterization of glucose-sensitive insulin release systems in simulated in vivo conditions......................................................................................37 Figure 2–8. Enzyme-responsive hydrogel particles for the controlled release of proteins: Designing peptide actuators to match payload...........................................41 Figure 2–9. Peptide actuator designed for the release of negatively charged protein molecules...................................................................................................................42 Figure 2–10. Ligand responsive hydrogel that relies on conformational changes... .44 Figure 2–11. Chemical structure of PEGA................................................................46 Figure 2–12. Inhibition of cruzipain visualized in a fluorescence quenched solidphase inhibitor library assay. D-amino acid inhibitors for cruzipain, cathepsin B and cathepsin L.................................................................................................................48 Figure 2–13. Using two photon microscopy to quantify enzymatic reaction rates on polymer beads............................................................................................................50 Figure 2–14. A micropatterned hydrogel platform for chemical Synthesis and biological analysis......................................................................................................52 Figure 2–15. Real-time imaging of protease action on substrates covalently immobilised to polymer supports..............................................................................53

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 2–16. Biomimetic synthesis and optimization of cyclic peptide antibiotics...... ...................................................................................................................................55 Figure 2–17. Formation of dispersions using "flow focusing" in microchannels......58 Figure 2–18. An axisymmetric flow-focusing microfluidic device (AFFD).............60 Figure 2–19. Interfacial Polymerization within a Simplified Microfluidic Device: Capturing Capsules....................................................................................................61 Figure 2–20. Polymer particles with various shapes and morphologies produced in continuous microfluidic reactors...............................................................................63 Figure 2–21. Continuous microfluidic reactors for polymer particles.......................65 Figure 2–22. A micro-reactor for preparing uniform molecularly imprinted polymer beads..........................................................................................................................66 Figure 2–23. A predictive approach of the influence of the operating parameters on the size of polymer particles synthesized in a simplified microfluidic system.........67 Figure 3–1. Schematic of the enzyme responsive swelling of PEGA particles functionalised with linear peptide actuators. ...........................................................73 Figure 3–2. Monomers used in the synthesis of PEGA and its charged variants......75 Figure 3–3. Solid phase peptide synthesis scheme....................................................76 Figure 3–4. Chemical reactions involved in peptide synthesis..................................77 Figure

3–5.

Polymerisation

of

PEGA

particles

by

inverse

suspension

polymerisation...........................................................................................................82 Figure 3–6. Optical micrograph of microparticles is water.......................................83 Figure 3–7. Chemical structure of PEGA and its charged variants.72........................84 Figure 3–8. PEGA+ microparticles............................................................................85 Figure 3–9. Optical micrographs PEGA-,..................................................................86 Figure 3–10. The uptake of water by the three different types of dry PEGA microparticles and the effect of ionic strength on the swelling of the three different types of PEGA microparticles...................................................................................90 Figure 3–12. Comparison of time dependence of enzyme reactions on µPEGA and commercially available macroparticles.....................................................................91 Figure 3–13. A schematic of enzyme responsive microparticles illustrating both successful and unsuccessful

cleavage of the ECP resulting in a change of

accessibility to a 40 kDa FITC labelled dextran........................................................95 Figure 3–14. Confocal microscopy images of representative microparticles in an aqueous solution of 40 kDa fluorescently labelled dextran.......................................95

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 3–15. The pH responsive loading of the 40 kDa FITC labelled dextran (1 mg/ml) into the PEGA particles, gain of images varied............................................97 Figure 4–1. Schematic of enzyme responsive branched peptide actuator...............101 Figure 4–2. HPLC quantification of Fmoc removed after each coupling step for linear and branched peptide actuators......................................................................106 Figure 4–3. pH responsive swelling behaviour of peptide actuators on PEGA microparticles...........................................................................................................107 Figure 4–4. Enzyme responsive swelling behaviour of peptide actuator functionalised µPEGA at pH 7................................................................................109 Figure 4–5. HPLC and MS analysis of enzyme hydrolysis of branched peptide actuators...................................................................................................................110 Figure 4–6. Effect of ionic strength on the maximal enzyme responsive swelling of branched peptide actuator functionalised µPEGA at pH 7......................................111 Figure 4–7. Comparison of thermolysin action on both linear and branched peptide actuators on µPEGA................................................................................................112 Figure 4–8. µPEGA particles functionalised with the branched peptide actuator at pH 7..........................................................................................................................114 Figure 5–1. Schematic of microfluidic setup for the synthesis of controlled-size polymer particles......................................................................................................118 Figure 5–2. Effect of Qc/Qd on mean particle diameter with no surfactant in the continuous phase,.....................................................................................................122 Figure 5–3. Effect of surfactant concentration on particle size...............................124 Figure 5–4. Effect of total flow rate (at constant Qc/Qd) on mean particle diameter. .................................................................................................................................126 Figure 5–5. Effect of Qc/Qd and surfactant concentration on polydispersity...........128 Figure 5–6. Effect of Qc/Qd and surfactant concentration on particle production rate. .................................................................................................................................128 Figure 5–7. Schematic representation of the ‘flow-focussing’ device....................129 Figure 5–8. Effect device orientation on droplet and resulting particle formation..131 Figure 5–9. Development of flow-focussing setup..................................................132 Figure 5–10. Optical micrographs of PEGA particles produced using FF device with silicone oil at a variety of conditions......................................................................133 Figure 5–11. PEGA Particles produced under the optimum conditions..................135 Figure 5–12. The typically slightly asymmetric shape of particles produced by microfluidics each image is of particles produced under different conditions........136 8

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 8–1. The structures of the twenty DNA encoded amino acids. The single letter abbreviation is indicated with the brackets.....................................................143 Figure 8–2. Jablonski energy diagram showing a comparison of the excitation of a fluorophore with a single photon (confocal microscopy) and two photons (twophoton microscopy).................................................................................................144 Figure 8–3. HPLC solvent gradient used in analytical runs....................................145 Figure 8–4. Synthesis of PDTEMA.130....................................................................146 Figure 8–5. Characterisation of PDTEMA by NMR...............................................146 Figure 8–5. Characterisation of PDTEMA by NMR. 1H NMR (CDCl3, 200 MHz): δ (ppm) 7.0-8.6 (m, 5H, Ar-H and -NH), 5.809 (s, 1H, one of d, CH2), 5.361 (s, 1H, one of dCH2), 3.603 (m, 2H, -CH2-NR), 2.960 (t, 2H, -S-CH2-), 2.002 (d, 3H, -CH3, the split of this peak is due to the tautomerization of the adjacent double bond).

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

List of tables Table 2–1. Selected studies of bioresponsive hydrogels based on peptide crosslinkers................................................................................................................25 Table 2–2. Selected studies of bioresponsive hydrogels based on non-covalent crosslinking interactions............................................................................................30 Table 2–3. Selected studies of bioresponsive hydrogels based on electrostatic interactions.................................................................................................................35 Table 3–2. Specificity of the enzymes used towards amino acids (AA) in the substrate and their molecular weight.........................................................................93 Table 3–1. Values for HPLC relative enzyme cleavage for each ECP......................94 Table 3–2. Values for HPLC relative enzyme cleavage for each ECP.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

List of abbreviations α-CD

α-cyclodextrin

ABP

Aminobenzophenone

AFFD

Axisymmetric flow-focusing microfluidic device

APS

Ammonium persulfate

APTMS

3-aminopropyltrimethoxysilane

BDDA

1,4-butanediol diacrylate

CaM

Calmodulin

CDDP

Cisplatin

CPGs

Controlled pore glasses

CV

Coefficient of variation

DCM

Dichloromethane

DEAEMA

Diethylaminoethyl methacrylate

DIC

Differential interference contrast

DIPEA

N,N-Diisopropylethylamine

DMAEMA

N,N-dimethylaminoethyl methacrylate

DMSO

Dimethyl sulfoxide

DOPA

L-3,4-dihydroxylphenylalanine

DTT

Dithiothreitol

ECM

Extracellular matrix

ECP

Enzyme cleavable peptide

EG

Ethylene glycol

EGDMA

Ethyleneglycol dimethacrylate

ESEM

Environmental scanning electron microscopy

FF

Flow-focussing

FITC

Fluorescein isothiocyanate

Fmoc

9-fluorenylmethoxycarbonyl

FRET

Fluorescence quenched peptide substrate

GOx

Glucose oxidase

HBTU

O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate

HOBt

Hydroxybenzotriazole

HPLC

High-performance liquid chromatography

HPMC

Hydroxypropyl methyl cellulose ether

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

HPMA

N-(2-hydroxypropyl)methacrylamide methacryoyl

ID

Internal diameter

IVD

Intervertebral discs

LbL

Layer-by-layer assembly

LCST

Lower-critical solution temperature

µPEGA

Microparticular PEGA

MA

Methacrylate

MAA

Methacrylic acid

MALDI

Matrix-assisted laser desorption/ionization

MBAA

N,N’-methylenebisacrylamide

MFFD

Microfluidic flow-focusing device

MMA

Methyl methacrylate

MMP

Matrix metalloproteinase

MS

Mass spectrometry

NSA

N-succinimidylacrylate

PAAc

Poly(acrylic acid)

PCP

Phosphotpanteheine

PDMA

Poly(dimethylsiloxane)

PDTEMA

N-[2-(2-pyridyldithio)]ethyl methacrylamide

PEG

Poly(ethylene glycol)

PEGA

Poly(ethylene glycol)-co-acrylamide

PEGMA

Poly(ethylene glycol) monomethacylate

PETA-3

Pentaerythritol triacryalte

PGA

Penicillin G amidase

PNIPAAm

Poly(N-isopropylacrylamide)

poly(HEMA)

poly(2-hydroxyethyl methacrylate)

PLE

Porcine liver esterase

PU

Polyurethane

PVDF

polyvinylidene fluoride

Qc

Flow rate of the continuous phase

Qd

Flow rate of the dispersed phase

SEM

Scanning electron micrscopy

Semi-IPN

Semi-interpenetrating network

Span 20

Sorbitan monolaurate

SPPS

Solid-phase peptide synthesis 12

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

TEGDMA

Tetraethylene glycol dimethacrylate

TEMED

N,N,N′,N′- tetramethylethylenediamine

TFA

Trifluoroacetic acid

TFP

Trifluoperazine ligand

TG

Transglutaminase

THF

Tetrahydrofuran

TPGDA

Tripropyleneglycol diacrylate

TPM

Two-photon microscopy

tTG

Tissue transglutaminase

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Declaration No portion of this work has been submitted in support of an application for another degree or qualification of this or any other university or institute of learning.

Copyright (i) The author of this thesis (including any appendices and/or schedules to this thesis) owns any copyright in it (the “Copyright”) and s/he has given The University of Manchester the right to use such Copyright for any administrative, promotional, educational and/or teaching purposes. (ii) Copies of this thesis, either in full or in extracts, may be made only in accordance with the regulations of the John Rylands University Library of Manchester. Details of these regulations may be obtained from the Librarian. This page must form part of any such copies made. (iii) The ownership of any patents, designs, trade marks and any and all other intellectual property rights except for the Copyright (the “Intellectual Property Rights”) and any reproductions of copyright works, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property Rights and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property Rights and/or Reproductions. (iv) Further information on the conditions under which disclosure, publication and exploitation of this thesis, the Copyright and any Intellectual Property Rights and/or Reproductions described in it may take place is available from the Head of the School of Materials.

14

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Abstract Stimuli responsive materials are well documented and function by translating a molecular event into a macroscopic transition. Polymer hydrogels are a particular type of material that offers excellent potential for applications within the field of biomaterials. In this thesis an enzyme responsive polymer hydrogel has been demonstrated that functions through the incorporation of designed peptide actuators. The experimental findings within this thesis are separated into three separate chapters. The first experimental chapter describes the synthesis of poly(ethylene glycol)-coacrylamide microparticles (μPEGA) by inverse suspension polymerisation. Particles with neutral, positive or negative charge were prepared and characterised. Neutral µPEGA were selected were chosen as the preferred polymer particles. These particles were then utilised as the basis of the enzyme responsive system. Here linear peptide actuators were incorporated into the particles. These particles demonstrated an enzyme specific increase in accessibility. Additionally, functionalised particles displayed pH responsive behaviour allowing for the physical entrapment of a payload. The release of this payload was only possible when the functionalised particles were exposed to the target enzyme. However, at physiological ionic strength no response of the particles was observed due to electrostatic screening. In order to overcome electrostatic screening, branched peptide actuators were developed and incorporated into μPEGA. These peptide actuators provided enhanced charge density and the functionalised particles were able to respond through an increase in swelling to the target enzyme at physiological ionic strength. Using this system it was possible to selectively release a macromolecule at physiological ionic strength in response to the target enzyme. The large size distribution of μPEGA was addressed in the final experimental chapter. Here, PEGA particles were prepared by a simplified microfluidic setup assembled using a needle and tubing. The size of the particles produced was determined by the surfactant concentration and the relative flow rates of the dispersed and continuous phases.

15

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Acknowledgements The last three years of my PhD have been an invaluable time in my life, I feel I have greatly improved a variety of my skills and abilities as well as maturing as a person. I have to firstly thank Rein (now Professor Ulijn!) for all his time, comments, feedback and assistance he has given me throughout my PhD. The opportunities I have had and a great number of my achievements have only been possible due to him. I was also very fortunate to have Brian Saunders as my second supervisor, he has always been available to help me as well as offering me support whenever it was needed. His constructive criticism regarding my thesis as been invaluable, thank you Brian! There were, of course, numerous other individuals who have been very influential in the completion of this PhD. These people have taught me new skills along with refining other aspects on my research for which I am very grateful: Paul Thornton, my mentor at the start of my PhD, without whom I would have been lost. Rob Mart for his continuous technical advice (no matter how many times I asked...). Last but not least, Andrew Hirst for his exceptionally useful advice with my writing, along with his thorough proof reading of this thesis. All my friends I have made during my studies have made the last three years very enjoyable and memorable: Richard (our trips to Fab Café), Simon (gym visits and trading sporting notes), Grace (discussing world politics), Rumana (the cat ‘jokes’), Alison (all the conference trips), Claire (teaching me so many ‘useful’ French phrases), Kapil, Riaz (no you can’t take the eighth decimal place into account!) Louise, Andy T, Bobby T (stay off the T120s...) Nurguse, Andrew, Apurba and Kate it wouldn’t have been the same without you. Two technicians have been particularly helpful with my experimental work: Robert Fernandez for his continuous help with confocal and TPM. Also, Andy Wallwork for his time and assistance in my attempts to use stereolithography to produce microfluidic devices.

16

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

The support I have received from my family (including the regular questioning asking when I would get a job!) has been very important. I am very grateful for the encouragement that my parents gave me throughout my time at university. Finally I have to say many thanks to my girlfriend Jenny Hayes, she has put up with my last seven years at university! She has also given me enormous moral and financial support. Hopefully I can repay this in kind during her upcoming teacher training.

17

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1 Introduction Biomaterials science is an interdisciplinary field that encompasses medicine, biology,

chemistry

and

materials

science.

BiomaterialsPEVuZE5vdGU+PENpdGU+PEF1dGhvcj5SYXRuZXI8L0F1dGhvcj4 8WWVhcj4yMDA0PC9ZZWFyPjxS ZWNOdW0+MzY5PC9SZWNOdW0+PHJlY29yZD48cmVjLW51bWJlcj4zNjk8L3 JlYy1udW1iZXI+PGZv cmVpZ24ta2V5cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJmYTl6ZHpzdmtzeDJwcm VyZTk4eGQ1MnFk Zndmd3I5MHA1czIiPjM2OTwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYtdHlwZS BuYW1lPSJKb3Vy bmFsIEFydGljbGUiPjE3PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxhdXRob3J zPjxhdXRob3I+ UmF0bmVyLCBCLiBELjwvYXV0aG9yPjxhdXRob3I+QnJ5YW50LCBTLiBKLj wvYXV0aG9yPjwvYXV0 aG9ycz48L2NvbnRyaWJ1dG9ycz48YXV0aC1hZGRyZXNzPlVuaXYgV2FzaGluZ 3RvbiwgRGVwdCBC aW9lbmduLCBTZWF0dGxlLCBXQSA5ODE5NSBVU0EuIFVuaXYgV2FzaGluZ 3RvbiwgRGVwdCBDaGVt IEVuZ24sIFNlYXR0bGUsIFdBIDk4MTk1IFVTQS4mI3hEO1JhdG5lciwgQkQsIF VuaXYgV2FzaGlu Z3RvbiwgRGVwdCBCaW9lbmduLCBTZWF0dGxlLCBXQSA5ODE5NSBVU0Eu JiN4RDtyYXRuZXJAdXdl Yi5lbmdyLndhc2hpbmd0b24uZWR1PC9hdXRoLWFkZHJlc3M+PHRpdGxlcz48d Gl0bGU+QmlvbWF0 ZXJpYWxzOiBXaGVyZSB3ZSBoYXZlIGJlZW4gYW5kIHdoZXJlIHdlIGFyZSBn b2luZzwvdGl0bGU+ PHNlY29uZGFyeS10aXRsZT5Bbm51YWwgUmV2aWV3IG9mIEJpb21lZGljYW wgRW5naW5lZXJpbmc8 L3NlY29uZGFyeS10aXRsZT48YWx0LXRpdGxlPkFubnUuIFJldi4gQmlvbWVkLi BFbmcuPC9hbHQt dGl0bGU+PC90aXRsZXM+PHBlcmlvZGljYWw+PGZ1bGwtdGl0bGU+QW5ud 18

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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effectively

and

improve

the

quality

of

life

for

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

VzPjxwZXJpb2Rp Y2FsPjxmdWxsLXRpdGxlPkJpb21hdGVyaWFscyBzY2llbmNlOiBBbiBpbnRyb2 R1Y3Rpb24gdG8g bWF0ZXJpYWxzIGluIG1lZGljaW5lPC9mdWxsLXRpdGxlPjwvcGVyaW9kaWNh bD48cGFnZXM+MS0x MDwvcGFnZXM+PHNlY3Rpb24+MTwvc2VjdGlvbj48ZGF0ZXM+PHllYXI+MT k5NjwveWVhcj48L2Rh dGVzPjxwdWItbG9jYXRpb24+U2FuIERpZWdvLCBDYWxpZm9ybmlhPC9wdW ItbG9jYXRpb24+PHB1 Ymxpc2hlcj5BY2FkZW1pYyBQcmVzczwvcHVibGlzaGVyPjx1cmxzPjwvdXJscz4 8L3JlY29yZD48 L0NpdGU+PENpdGU+PEF1dGhvcj5MYW5nZXI8L0F1dGhvcj48WWVhcj4yMD A0PC9ZZWFyPjxSZWNO dW0+MzE5PC9SZWNOdW0+PHJlY29yZD48cmVjLW51bWJlcj4zMTk8L3JlYy1 udW1iZXI+PGZvcmVp Z24ta2V5cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJmYTl6ZHpzdmtzeDJwcmVyZTk 4eGQ1MnFkZndm d3I5MHA1czIiPjMxOTwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYtdHlwZSBuY W1lPSJKb3VybmFs IEFydGljbGUiPjE3PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxhdXRob3JzPjxh dXRob3I+TGFu Z2VyLCBSLjwvYXV0aG9yPjxhdXRob3I+VGlycmVsbCwgRC4gQS48L2F1dGhv cj48L2F1dGhvcnM+ PC9jb250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5NSVQsIERlcHQgQ2hlbSBFb mduLCBDYW1icmlk Z2UsIE1BIDAyMTM5IFVTQS4gQ0FMVEVDSCwgRGl2IENoZW0gJmFtcDsgQ 2hlbSBFbmduLCBQYXNh ZGVuYSwgQ0EgOTExMjUgVVNBLiYjeEQ7TGFuZ2VyLCBSLCBNSVQsIERlc HQgQ2hlbSBFbmduLCBC bGRnIEUyNS0zNDIsIENhbWJyaWRnZSwgTUEgMDIxMzkgVVNBLiYjeEQ7cm xhbmdlckBtaXQuZWR1 PC9hdXRoLWFkZHJlc3M+PHRpdGxlcz48dGl0bGU+RGVzaWduaW5nIG1hdGV yaWFscyBmb3IgYmlv bG9neSBhbmQgbWVkaWNpbmU8L3RpdGxlPjxzZWNvbmRhcnktdGl0bGU+Tm F0dXJlPC9zZWNvbmRh 26

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cnktdGl0bGU+PGFsdC10aXRsZT5OYXR1cmU8L2FsdC10aXRsZT48L3RpdGxlc z48cGVyaW9kaWNh bD48ZnVsbC10aXRsZT5OYXR1cmU8L2Z1bGwtdGl0bGU+PGFiYnItMT5OYX R1cmU8L2FiYnItMT48 L3BlcmlvZGljYWw+PGFsdC1wZXJpb2RpY2FsPjxmdWxsLXRpdGxlPk5hdHVy ZTwvZnVsbC10aXRs ZT48YWJici0xPk5hdHVyZTwvYWJici0xPjwvYWx0LXBlcmlvZGljYWw+PHBh Z2VzPjQ4Ny00OTI8 L3BhZ2VzPjx2b2x1bWU+NDI4PC92b2x1bWU+PG51bWJlcj42OTgyPC9udW1iZ XI+PGtleXdvcmRz PjxrZXl3b3JkPlNIQVBFLU1FTU9SWSBQT0xZTUVSUzwva2V5d29yZD48a2V5 d29yZD5EUlVHLURF TElWRVJZPC9rZXl3b3JkPjxrZXl3b3JkPkdFTkUgREVMSVZFUlk8L2tleXdvcm Q+PGtleXdvcmQ+ UE9MWShMQUNUSUM8L2tleXdvcmQ+PGtleXdvcmQ+QUNJRC1DTy1MWVN JTkUpPC9rZXl3b3JkPjxr ZXl3b3JkPlNZU1RFTTwva2V5d29yZD48a2V5d29yZD5USVNTVUU8L2tleXdvc mQ+PGtleXdvcmQ+ Q0hJUDwva2V5d29yZD48a2V5d29yZD5IWURST0dFTFM8L2tleXdvcmQ+PGtle XdvcmQ+UFJPVEVJ TlM8L2tleXdvcmQ+PGtleXdvcmQ+QVJSQVlTPC9rZXl3b3JkPjwva2V5d29yZH M+PGRhdGVzPjx5 ZWFyPjIwMDQ8L3llYXI+PHB1Yi1kYXRlcz48ZGF0ZT5BcHI8L2RhdGU+PC9 wdWItZGF0ZXM+PC9k YXRlcz48aXNibj4wMDI4LTA4MzY8L2lzYm4+PGFjY2Vzc2lvbi1udW0+SVNJO jAwMDIyMDU0MDEw MDAzMTwvYWNjZXNzaW9uLW51bT48d29yay10eXBlPlJldmlldzwvd29yay10e XBlPjx1cmxzPjxy ZWxhdGVkLXVybHM+PHVybD4mbHQ7R28gdG8gSVNJJmd0OzovLzAwMDIy MDU0MDEwMDAzMTwvdXJs PjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxlY3Ryb25pYy1yZXNvdXJjZS1ud W0+MTAuMTAzOC9u YXR1cmUwMjM4ODwvZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+PGxhbmd1Y WdlPkVuZ2xpc2g8L2xh bmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+ 7,8 In the majority of 27

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cases current biomaterials are inert i.e. they do not interact with their surroundings. This characteristic is somewhat limiting when the material is used to replace a function in the dynamic and changing environment of biological systems. For this reason, there has been great interest in designed biomaterials that can respond (e.g. swelling,9 payload release10 and dissolution11) to an environmental stimulus. A particularly interesting choice of stimulus would be enzymes, nature’s catalysts, because of their vital importance to living systems. Enzymes drive and control almost every process that occurs in the human body from respiration to growth.12 Of special interest is the role enzymes have within disease processes, these include: the

metastasis

of

tumours,13

chronic

wounds14

and

arthritis.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5WaXNzZTwvQXV0aG9yPjxZ ZWFyPjIwMDM8L1llYXI+PFJl Y051bT4zMTc8L1JlY051bT48cmVjb3JkPjxyZWMtbnVtYmVyPjMxNzwvcmVjL W51bWJlcj48Zm9y ZWlnbi1rZXlzPjxrZXkgYXBwPSJFTiIgZGItaWQ9ImZhOXpkenN2a3N4MnByZ XJlOTh4ZDUycWRm d2Z3cjkwcDVzMiI+MzE3PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG 5hbWU9IkpvdXJu YWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcn M+PGF1dGhvcj5W aXNzZSwgUi48L2F1dGhvcj48YXV0aG9yPk5hZ2FzZSwgSC48L2F1dGhvcj48L2 F1dGhvcnM+PC9j b250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5Vbml2IExvbmRvbiBJbXBlcmlhbC BDb2xsIFNjaSBU ZWNobm9sICZhbXA7IE1lZCwgRmFjIE1lZCwgRGl2IFJoZXVtYXRvbCwgS2Vu bmVkeSBJbnN0LCBM b25kb24gVzYgOExILCBFbmdsYW5kLiYjeEQ7TmFnYXNlLCBILCBVbml2IExv bmRvbiBJbXBlcmlh bCBDb2xsIFNjaSBUZWNobm9sICZhbXA7IE1lZCwgRmFjIE1lZCwgRGl2IFJoZ XVtYXRvbCwgS2Vu bmVkeSBJbnN0LCAxIEFzcGVubGVhIFJkLCBMb25kb24gVzYgOExILCBFbmds YW5kLjwvYXV0aC1h ZGRyZXNzPjx0aXRsZXM+PHRpdGxlPk1hdHJpeCBtZXRhbGxvcHJvdGVpbmFz ZXMgYW5kIHRpc3N1 ZSBpbmhpYml0b3JzIG9mIG1ldGFsbG9wcm90ZWluYXNlcyAtIFN0cnVjdHVyZ 28

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

dGU+TWF5PC9kYXRlPjwvcHViLWRhdGVzPjwvZGF0ZXM+PGlzYm4+MDAw OS03MzMwPC9pc2JuPjxh Y2Nlc3Npb24tbnVtPklTSTowMDAxODI1ODAwMDAwMDQ8L2FjY2Vzc2lvbi1 udW0+PHdvcmstdHlw ZT5SZXZpZXc8L3dvcmstdHlwZT48dXJscz48cmVsYXRlZC11cmxzPjx1cmw+Jm x0O0dvIHRvIElT SSZndDs6Ly8wMDAxODI1ODAwMDAwMDQ8L3VybD48L3JlbGF0ZWQtdXJs cz48L3VybHM+PGVsZWN0 cm9uaWMtcmVzb3VyY2UtbnVtPjEwLjExNjEvMDEucmVzLjAwMDAwNzAxM TIuODA3MTEuM2Q8L2Vs ZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjxsYW5ndWFnZT5FbmdsaXNoPC9sYW 5ndWFnZT48L3JlY29y

ZD48L0NpdGU+PC9FbmROb3RlPgB=

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZXMgYW5kIHRpc3N1 ZSBpbmhpYml0b3JzIG9mIG1ldGFsbG9wcm90ZWluYXNlcyAtIFN0cnVjdHVyZ SwgZnVuY3Rpb24s IGFuZCBiaW9jaGVtaXN0cnk8L3RpdGxlPjxzZWNvbmRhcnktdGl0bGU+Q2lyY3 VsYXRpb24gUmVz ZWFyY2g8L3NlY29uZGFyeS10aXRsZT48YWx0LXRpdGxlPkNpcmMuUmVzLj wvYWx0LXRpdGxlPjwv dGl0bGVzPjxwZXJpb2RpY2FsPjxmdWxsLXRpdGxlPkNpcmN1bGF0aW9uIFJlc2 VhcmNoPC9mdWxs LXRpdGxlPjxhYmJyLTE+Q2lyYy5SZXMuPC9hYmJyLTE+PC9wZXJpb2RpY2F sPjxhbHQtcGVyaW9k aWNhbD48ZnVsbC10aXRsZT5DaXJjdWxhdGlvbiBSZXNlYXJjaDwvZnVsbC10a XRsZT48YWJici0x PkNpcmMuUmVzLjwvYWJici0xPjwvYWx0LXBlcmlvZGljYWw+PHBhZ2VzPjg yNy04Mzk8L3BhZ2Vz Pjx2b2x1bWU+OTI8L3ZvbHVtZT48bnVtYmVyPjg8L251bWJlcj48a2V5d29yZH M+PGtleXdvcmQ+ ZXh0cmFjZWxsdWxhciBtYXRyaXg8L2tleXdvcmQ+PGtleXdvcmQ+cHJvdGVhc2 U8L2tleXdvcmQ+ PGtleXdvcmQ+cHJvdGVhc2UgaW5oaWJpdG9yczwva2V5d29yZD48a2V5d29yZ D5TT1JTQllTLUZV TkRVUy1EWVNUUk9QSFk8L2tleXdvcmQ+PGtleXdvcmQ+RVJZVEhST0lELV BPVEVOVElBVElORyBB Q1RJVklUWTwva2V5d29yZD48a2V5d29yZD5HUk9XVEgtUFJPTU9USU5HIEF DVElWSVRZPC9rZXl3 b3JkPjxrZXl3b3JkPk1BTU1BUlkgRVBJVEhFTElBTC1DRUxMUzwva2V5d29yZ D48a2V5d29yZD5G SUJST05FQ1RJTi1MSUtFPC9rZXl3b3JkPjxrZXl3b3JkPkRPTUFJTjwva2V5d29y ZD48a2V5d29y ZD5FWFRSQUNFTExVTEFSLU1BVFJJWDwva2V5d29yZD48a2V5d29yZD5HR UxBVElOQVNFLUE8L2tl eXdvcmQ+PGtleXdvcmQ+UFJPR0VMQVRJTkFTRS1BPC9rZXl3b3JkPjxrZXl3b 3JkPkNBVEFMWVRJ Qzwva2V5d29yZD48a2V5d29yZD5ET01BSU48L2tleXdvcmQ+PGtleXdvcmQ+SS BDT0xMQUdFTjwv 31

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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b3RlPgB=

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Pjxpc2JuPjEzNjct NTkzMTwvaXNibj48YWNjZXNzaW9uLW51bT5JU0k6MDAwMTc5NjM4NDA wMDIyPC9hY2Nlc3Npb24t bnVtPjx3b3JrLXR5cGU+UmV2aWV3PC93b3JrLXR5cGU+PHVybHM+PHJlbGF 0ZWQtdXJscz48dXJs PiZsdDtHbyB0byBJU0kmZ3Q7Oi8vMDAwMTc5NjM4NDAwMDIyPC91cmw+P C9yZWxhdGVkLXVybHM+ PC91cmxzPjxsYW5ndWFnZT5FbmdsaXNoPC9sYW5ndWFnZT48L3JlY29yZD48 L0NpdGU+PC9FbmRO b3RlPgB= 17-19 The former, chemical hydrogels are an area of focus in this thesis. These crosslinked (either physically of chemically) hydrophilic polymers give polymer networks that can contain between ten to many thousand times their dry mass in water.16 This highly hydrated nature of hydrogels presents similar characteristics to biological tissue. Additionally, the properties of these polymer networks can be altered by changing the polymer’s interactions with neighbouring chains or the water molecules surrounding the polymer chains. This thesis consists of two main areas of research, (i) the development of a hydrogel system amenable to chemical modification thereby incorporating enzyme responsiveness and (ii), the design and development of enzyme responsive functionalities.

1.1. Motivation of the project The motivation of this research was to develop enzyme responsive hydrogel particles based on peptide actuators. This proof-of-concept research would demonstrate the potential application of peptide actuators in the enzyme specific release of a payload. In the future systems such as these might serve a role in the triggered delivery of drugs.

1.2. Layout of the thesis Within this thesis the experimental chapters are presented separately, each with its own introduction, results, discussion, and conclusion sections. By separating the chapters in this manner it is intended to give the reader a clear view to each section of research.

42

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

The first experimental chapter, Chapter PEGA polymerisation, characterisation and enzyme responsive swelling through functionalisation with peptide actuators2 investigates the synthesis and characterisation of poly(ethylene glycol)-coacrylamide (PEGA) microparticles (µPEGA) by way of inverse suspension polymerisation (Figure 1–1). The resulting microparticles were the functionalisation with peptide actuators. Finally, the responsive behaviour of these particles was then determined and exploited for enzyme specific release of a payload Figure 1–2.

Figure 1–1. The inverse suspension polymerisation of µPEGAs.

Figure 1–2. Enzyme responsive µPEGA. Scheme of enzyme responsiveness of linear peptide actuators.

43

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Chapter Designing new peptide actuators for improved enzyme responsive behaviour goes on to tackle the limitations of the linear peptide actuators with the design and development of branched peptide actuators. µPEGA functionalised with these peptide actuators demonstrate enzyme specific swelling at physiological ionic strength (Figure 1–3).

Figure 1–3. Enzyme responsive µPEGA through the incorporation of branched peptide actuators. Schematic of enzyme responsive branched peptide actuator.

The final experimental chapter explores the application of microfluidic devices in the production of PEGA particles, with the aim of addressing the high polydispersity of the particles when made by inverse suspension polymerisation (Figure 1–4).

44

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald Figure 1–4. Production of PEGA particles by microfluidic polymerisation. Schematic representation of microfluidic setup.

45

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2 Literature review 2.1. Introduction This chapter aims to give the reader a good understanding of the literature predominately from the last ten years in three separate fields: (i) Bioresponsive hydrogels. (ii) The previous research undertaken on the polymer poly(ethylene glycol)-co-acrylamide (PEGA), on which most of the experimental work is based. (iii) The use of microfluidic devices to produce polymer particles.

2.2. Stimuli responsive materials Stimuli

responsive

materials

are

very

well

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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20-25

these materials function by translating a molecular event into a macroscopic transition. Polymer (chemical) hydrogels are a particular type of responsive material that offers excellent potential for applications (e.g. sensing,26 drug delivery27 or tissue engineeringPEVuZE5vdGU+PENpdGU+PEF1dGhvcj5FaHJiYXI8L0F1dGhvcj48 WWVhcj4yMDA3PC9ZZWFyPjxS ZWNOdW0+MjkxPC9SZWNOdW0+PHJlY29yZD48cmVjLW51bWJlcj4yOTE8L 3JlYy1udW1iZXI+PGZv cmVpZ24ta2V5cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJmYTl6ZHpzdmtzeDJwcm VyZTk4eGQ1MnFk Zndmd3I5MHA1czIiPjI5MTwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYtdHlwZSB uYW1lPSJKb3Vy bmFsIEFydGljbGUiPjE3PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxhdXRob3J zPjxhdXRob3I+ RWhyYmFyLCBNLjwvYXV0aG9yPjxhdXRob3I+Uml6emksIFMuIEMuPC9hdXR ob3I+PGF1dGhvcj5I bHVzaGNodWssIFIuPC9hdXRob3I+PGF1dGhvcj5Eam9ub3YsIFYuPC9hdXRob3I +PGF1dGhvcj5a aXNjaCwgQS4gSC48L2F1dGhvcj48YXV0aG9yPkh1YmJlbGwsIEouIEEuPC9hdX Rob3I+PGF1dGhv cj5XZWJlciwgRi4gRS48L2F1dGhvcj48YXV0aG9yPkx1dG9sZiwgTS4gUC48L2F 1dGhvcj48L2F1 dGhvcnM+PC9jb250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5FY29sZSBQb2x5d GVjaCBGZWQgTGF1 73

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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dGU+

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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85

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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23,29 2.2.1. Chemical hydrogels Chemical hydrogels are hydrophilic polymers that are highly crosslinked resulting in insoluble 3-D networks. The first synthetic hydrogels were developed in 1960 by Wichterle and Lim30 when they produced poly(2-hydroxyethyl methacrylate) poly(HEMA). They showed that these new hydrophilic networks overcame many problems associated with polymers that were previously being used in medical applications. The generation of materials available at that time had poor biocompatibility and upon implantation caused severe local inflammation. These chemical hydrogels offered completely new properties due to their high water content; which was similar to surrounding tissues, gave mechanical properties comparable to biological materials and allowed the diffusion of metabolites through the material. At the time this was published Wichterle and Lim suggested that these polymers could be used to manufacture contact lenses and arteries.30 Subsequently there has been great interest in exploiting hydrogels within the field of biomedical applications. Currently the main application of these biomaterials in medicine is as contact lenses. Indeed, contact lenses are now used by approximately 125 million people worldwide to augment eye sight.31 Stimuli responsive hydrogels have been shown to respond to numerous stimuli including pH,32 temperature,33 electric fields,34 ionic strength35 and small molecules.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5UcmFpdGVsPC9BdXRob3I+ PFllYXI+MjAwMDwvWWVhcj48 UmVjTnVtPjI3MzwvUmVjTnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+MjczPC9y ZWMtbnVtYmVyPjxm b3JlaWduLWtleXM+PGtleSBhcHA9IkVOIiBkYi1pZD0iZmE5emR6c3Zrc3gycHJl cmU5OHhkNTJx ZGZ3ZndyOTBwNXMyIj4yNzM8L2tleT48L2ZvcmVpZ24ta2V5cz48cmVmLXR5 cGUgbmFtZT0iSm91 cm5hbCBBcnRpY2xlIj4xNzwvcmVmLXR5cGU+PGNvbnRyaWJ1dG9ycz48YXV 0aG9ycz48YXV0aG9y PlRyYWl0ZWwsIFQuPC9hdXRob3I+PGF1dGhvcj5Db2hlbiwgWS48L2F1dGhvcj 48YXV0aG9yPktv

86

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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of

excellent

review

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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2.3. Bioresponsive Hydrogels1 Bioresponsive hydrogels allow for the potential treatment of diseases by responding directly to a biological marker for the disease. In the future these systems may provide more effective medical treatment through highly targeted specific responses. 2.3.1. Introduction There is a huge range of biological stimuli, these generally fall into two main categories: small molar mass substances (such as glucose or calcium ions) or macromolecules (including enzymes and proteins). Through the incorporation of a moiety capable of recognising a specific biological stimulus and utilising this recognition event to produce a response it is possible to make a material 1 Short parts of this section have been published by the author in: T.O. McDonald, R.J. Williams, A.G. Patrick, B.G. Cousins and R.V. Ulijn, Bio-responsive chemically cross-linked and physical hydrogels. Biomedical applications of electroactive polymer actuators. Wiley. 2009.

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‘bioresponsive’. In this thesis the term ‘bioresponsive’ is defined as stimuli responsive materials that change properties in response to a biological molecular recognition event.40 These molecular interactions are then translated through molecular actuation into macroscopic changes in the properties of the material. This section will focus on different methods used to actuate macroscopic responses of chemical hydrogels to a biological stimulus. The preparation of these devices will be covered in detail along with how these systems have been used in the development of biosensor, drug delivery and tissue scaffold systems. The methods of actuation within bioresponsive materials can generally be divided into three categories (Figure 2–1), changes in: (i) cross-linking density, (ii) electrostatic interactions and (iii) molecular conformation.

Figure 2–1. Schematic representation of the different categories of bioresponsive hydrogels.

2.3.2. Actuation based on changes in crosslinking density Systems based on actuation through changes in cross-linking density can be separated into two main categories: covalent crosslinking of polymers by peptides which are modified by enzymes, or, non-covalent interactions between crosslinking

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moieties in which these interactions can be disrupted by competition between free moieties and those responsible for crosslinking. 2.3.2.1. Systems incorporating peptide crosslinkers The incorporation of peptide crosslinkers into the hydrogel offers the potential for enzymes that hydrolyse peptide bonds (proteases) to specifically degrade the polymer network. There are a number of different approaches by which these peptides have been integrated into the various polymers to prepare responsive systems (Table 2–1).

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Table 2–1. Selected studies of bioresponsive hydrogels based on peptide crosslinkers.

# 1

Polymer PEG

2 Poly(NIPAMco-AAC)

Stimulus MMP-1

Biorecognition Enzyme hydrolysis of

Response Gel

Ref 41

MMP-13

peptide crosslinks Enzyme hydrolysis of

dissolution Gel

PE

peptide crosslinks

dissolution

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

tc Ds gR W 5n bi wg Q m Vy a2 Vs ZX ksI EN BI Dk 0N zI wI FV TQ S4 mI 3h EO 0hl Y W x5 LC BL RS wg V 123

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W 5p di BD Y W xp Zi BC ZX JrZ W xle Sw gR G V wd CB Ca W 9lb md uL CA zN zA gS E1 N Qi Ax Nz Y wL 124

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

CB CZ XJ rZ W xle Sw g Q0 Eg OT Q3 Mj Ag V V NB Li Yj eE Q7 a2 Vo Z W Fse UB zb 2N yY XR lcy 5iZ XJ rZ 125

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W xle S5l ZH U8 L2 F1 dG gt Y W Rk cm Vz cz4 8d Gl 0b G Vz Pjx 0a XR sZ T5 Te W 50 aG Vz aX Mg Y W 5kI 126

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

G No Y XJ hY 3R lc ml 6Y XR pb 24 gb 2Y ga W 5q Z W N0 Y WJ sZ SB wb 2x 5K E4t aX Nv cH Jvc Hls Y W 127

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Ny eW xh b Wl kZ S1j by 1h Y3 J5b Glj IG Fja W Qp IG h5 ZH Jv Z2 Vs cy B3 aX Ro IH By b3 Rl b2 x5 dG ljY W 128

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

xse SB kZ W dy Y W Rh Y mx lIG Ny b3 Nz L W xp bm tzP C9 0a XR sZ T4 8 c2 Vj b2 5k Y XJ 5L XR pd Gx 129

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

lPk Jpb 21 hY 3Jv b W 9s Z W N1 bG Vz PC 9z Z W Nv bm Rh cn ktd Gl 0b G U+ PG Fs dC 10 aX Rs ZT 5C aW 130

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

9t Y W Ny b2 1v bG Vj d W xlc zw vY W x0 LX Rp dG xlP jw vd Gl 0b G Vz Pjx wZ XJ pb 2R pY 2F sPj xm d 131

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W xs LX Rp dG xlP kJp b2 1h Y3 Jvb W 9s Z W N1 bG Vz PC 9m d W xs LX Rp dG xlP jxh Y mJ yL TE +Q ml vb 132

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W Fjc m9 t b2 xl Y3 Vs ZX M8 L2 Fi Yn It M T4 8L 3B lc ml vZ Glj Y W w+ PG Fs dC 1w ZX Jpb 2R pY 2F sPj 133

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

xm d W xs LX Rp dG xl Pk Jpb 21 hY 3Jv b W 9s Z W N1 bG Vz PC 9m d W xs LX Rp dG xlP jxh Y mJ yL TE 134

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

+Q ml vb W Fjc m9 tb2 xl Y3 Vs ZX M8 L2 Fi Yn It M T4 8L 2F sd C1 wZ XJ pb 2R pY 2F sPj xw Y W dlc z4 xM 135

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

jE0 LT Ey Mj M8 L3 Bh Z2 Vz Pjx 2b 2x 1b W U+ N D wv dm 9sd W 1lP jxu d W 1iZ XI +N Tw vb nV tY m Vy Pjx 136

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

rZ Xl 3b 3Jk cz4 8a 2V 5d 29 yZ D5 DR Ux M LU FE SE VT SU 9O IF BF UF RJ RE VT PC 9r ZX l3b 3Jk Pjx rZ Xl 3b 137

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3Jk Pl BP TF ko Q Ux ER Uh ZR EU gR 1V M V VJ PT kF UR Sk g SFl EU k9 HR Ux TP C9 rZ Xl 3b 3Jk Pjx rZ Xl 138

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3b 3Jk Pkl TT 1B ST 1B ZT EF D Ull M Q U1 JR EU gQ 09 QT 0x ZT U VS IE dF TF M8 L2t le Xd vc m Q+ PG tle 139

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Xd vc m Q+ UE hB U0 Ut VF JB Tl NJ VE lP Ti BU RU 1Q RV JB VF VS RT wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 140

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

5T T0 ZU LV RJ U1 N VR SB BV Ud NR U5 U Q VR JT 04 8L 2tl eX dv cm Q+ PG tle Xd vc m Q+ Ti1 JU 09 Q Uk 141

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

9Q W Ux BQ 1J ZT EF NS UR FP C9 rZ Xl 3b 3Jk Pjx rZ Xl 3b 3Jk Pl BP TF lN RV JJ Qz wv a2 V5 d2 9y ZD 48 a2 142

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

V5 d2 9y ZD 5C SU 9N Q VR FU kl BT F M8 L2t le Xd vc m Q+ PG tle Xd vc m Q+ TU FU Uk lYI E1 FV EF M TE 143

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

9Q Uk 9U RU lO Q V NF Uz wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5F W FR SQ U NF TE xV TE FS LU 1B VF JJ 144

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W D wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5T V0 V M TE lO Rz wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5C RU 145

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

hB Vk lP Uj wv a2 V5 d2 9y ZD 48 L2t le Xd vc m Rz Pjx kY XR lcz 48 eW Vh cj4 yM D Az PC 95 Z W Fy Pjx wd 146

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

WI tZ GF 0Z X M +P GR hd G U+ U2 V wL U9 j dD wv ZG F0 ZT 48 L3 B1 Yi 1k Y XR lcz 48 L2 Rh dG Vz Pjx 147

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

pc 2Ju Pj E1 Mj Ut Nz c5 Nz wv aX Ni bj4 8Y W Nj ZX Nz aW 9u L W 51 bT 5J U0 k6 M D A w M Tg 1M zg 148

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2O TA w M DE 1P C9 hY 2N lc3 Np b2 4tb nV tPj x3 b3J rL XR 5c G U+ Q XJ 0 aW Ns ZT wv d2 9y ay 10 eX Bl 149

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Pjx 1c mx zPj xy Z W xh dG Vk LX Vy bH M +P H Vy bD 4m bH Q7 R2 8g dG 8g SV NJ Jm d0 Oz ov Lz A w M 150

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

DE 4N T M4 Nj kw M D Ax NT wv dX JsP jw vc m Vs Y XR lZ C1 1c mx zPj wv dX Jsc z4 8Z W xl Y3 Ry b2 5p 151

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Yy 1y ZX Nv dX JjZ S1 ud W 0+ M TA uM TA yM S9i bT Az N D A0 Nj c8 L2 Vs Z W N0 cm 9u aW Mt cm Vz b3 152

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Vy Y2 Ut bn Vt Pjx sY W 5n d W Fn ZT 5F bm dsa X No PC 9s Y W 5n d W Fn ZT 48 L3 JlY 29 yZ D4 8L 0N 153

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

pd G U+ PC 9F bm RO b3 Rl Pn == PE Vu ZE 5v dG U+ PE Np dG U+ PE F1 dG hv cj5 La W 08 L0 F1 dG hv cj4 8 154

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W W Vh cj4 yM D Az PC 9Z Z W Fy Pjx SZ W N O d W 0+ Mj g3 PC 9S Z W N Od W 0+ PH JlY 29 yZ D4 155

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

8c m Vj L W 51 b WJ lcj 4y O Dc 8L 3Jl Yy 1u d W 1iZ XI +P GZ vc m Vp Z2 4ta 2V 5cz 48 a2 V5 IG Fw cD 156

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

0i RU 4iI GR iL Wl kP SJ m YT l6Z Hp zd mt ze DJ wc m Vy ZT k4 eG Q1 Mn Fk Zn dm d3I 5M H A1 czI iPj I4 Nz 157

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

wv a2 V5 Pj wv Zm 9y Z Wl nbi 1r ZX lzP jxy Z W Yt dH lw ZS Bu Y W 1lP SJ Kb 3V yb mF s IE Fy dG ljb G 158

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Ui Pj E3 PC 9y Z W Yt dH lw ZT 48 Y2 9u dH Jp Yn V0 b3J zPj xh dX Ro b3J zPj xh dX Ro b3I +S 2lt LC BT Lj wv 159

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Y X V0 aG 9y Pjx hd XR ob 3I+ SG Vh bH ksI Es uI EU uP C9 hd XR ob 3I+ PC 9h dX Ro b3J zPj wv Y2 9u dH Jp Yn 160

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

V0 b3J zPj xh dX Ro L W Fk ZH Jlc 3M +V W 5p di BD Y W xp Zi BC ZX JrZ W xle Sw gR G V wd CB Ca W 9lb 161

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

md u LC BC ZX JrZ W xle Sw gQ 0E gO TQ 3M jA gV V NB Li BV bm l2I EN hb Gl mI EJl cm tlb G V5 LC BE ZX B0 162

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

IE 1h dC BT Y2 kgJ mF tc Ds gR W 5n bi wg Q m Vy a2 Vs ZX ksI EN BI Dk 0N zI wI FV TQ S4 mI 3h EO 0hl Y 163

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W x5 LC BL RS wg V W 5p di BD Y W xp Zi BC ZX JrZ W xle Sw gR G V wd CB Ca W 9lb md uL CA zN zA gS 164

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

E1 N Qi Ax Nz Y wL CB CZ XJ rZ W xle Sw g Q0 Eg OT Q3 Mj Ag V V NB Li Yj eE Q7 a2 Vo Z W Fse UB zb 165

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2N yY XR lcy 5iZ XJ rZ W xle S5l ZH U8 L2 F1 dG gt Y W Rk cm Vz cz4 8d Gl 0b G Vz Pjx 0a XR sZ T5 Te W 50 166

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

aG Vz aX Mg Y W 5kI G No Y XJ hY 3R lc ml 6Y XR pb 24 gb 2Y ga W 5q Z W N0 Y WJ sZ SB wb 2x 5K E4t 167

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

aX Nv cH Jvc Hls Y W Ny eW xh b Wl kZ S1j by 1h Y3 J5b Glj IG Fja W Qp IG h5 ZH Jv Z2 Vs cy B3 aX Ro IH By 168

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

b3 Rl b2 x5 dG ljY W xse SB kZ W dy Y W Rh Y mx lIG Ny b3 Nz L W xp bm tzP C9 0a XR sZ T4 8 c2 Vj b2 169

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

5k Y XJ 5L XR pd Gx lPk Jpb 21 hY 3Jv b W 9s Z W N1 bG Vz PC 9z Z W Nv bm Rh cn ktd Gl 0b G U+ PG Fs 170

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

dC 10 aX Rs ZT 5C aW 9t Y W Ny b2 1v bG Vj d W xlc zw vY W x0 LX Rp dG xlP jw vd Gl 0b G Vz Pjx wZ XJ 171

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

pb 2R pY 2F sPj xm d W xs LX Rp dG xlP kJp b2 1h Y3 Jvb W 9s Z W N1 bG Vz PC 9m d W xs LX Rp dG xlP jxh 172

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Y mJ yL TE +Q ml vb W Fjc m9 t b2 xl Y3 Vs ZX M8 L2 Fi Yn It M T4 8L 3B lc ml vZ Glj Y W w+ PG Fs dC 173

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1w ZX Jpb 2R pY 2F sPj xm d W xs LX Rp dG xl Pk Jpb 21 hY 3Jv b W 9s Z W N1 bG Vz PC 9m d W xs LX Rp 174

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

dG xlP jxh Y mJ yL TE +Q ml vb W Fjc m9 tb2 xl Y3 Vs ZX M8 L2 Fi Yn It M T4 8L 2F sd C1 wZ XJ pb 2R pY 2F 175

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

sPj xw Y W dlc z4 xM jE0 LT Ey Mj M8 L3 Bh Z2 Vz Pjx 2b 2x 1b W U+ N D wv dm 9sd W 1lP jxu d W 1iZ XI +N 176

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Tw vb nV tY m Vy Pjx rZ Xl 3b 3Jk cz4 8a 2V 5d 29 yZ D5 DR Ux M LU FE SE VT SU 9O IF BF UF RJ RE VT PC 9r 177

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZX l3b 3Jk Pjx rZ Xl 3b 3Jk Pl BP TF ko Q Ux ER Uh ZR EU gR 1V M V VJ PT kF UR Sk g SFl EU k9 HR Ux TP C9 178

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

rZ Xl 3b 3Jk Pjx rZ Xl 3b 3Jk Pkl TT 1B ST 1B ZT EF D Ull M Q U1 JR EU gQ 09 QT 0x ZT U VS IE dF TF M8 L2t 179

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

le Xd vc m Q+ PG tle Xd vc m Q+ UE hB U0 Ut VF JB Tl NJ VE lP Ti BU RU 1Q RV JB VF VS RT wv a2 V5 d2 9y 180

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZD 48 a2 V5 d2 9y ZD 5T T0 ZU LV RJ U1 N VR SB BV Ud NR U5 U Q VR JT 04 8L 2tl eX dv cm Q+ PG tle Xd vc 181

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

m Q+ Ti1 JU 09 Q Uk 9Q W Ux BQ 1J ZT EF NS UR FP C9 rZ Xl 3b 3Jk Pjx rZ Xl 3b 3Jk Pl BP TF lN RV JJ Qz wv 182

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5C SU 9N Q VR FU kl BT F M8 L2t le Xd vc m Q+ PG tle Xd vc m Q+ TU FU 183

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Uk lYI E1 FV EF M TE 9Q Uk 9U RU lO Q V NF Uz wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5F W FR SQ U NF TE 184

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

xV TE FS LU 1B VF JJ W D wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5T V0 V M TE lO Rz wv a2 V5 d2 9y ZD 48 185

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

a2 V5 d2 9y ZD 5C RU hB Vk lP Uj wv a2 V5 d2 9y ZD 48 L2t le Xd vc m Rz Pjx kY XR lcz 48 eW Vh cj4 yM D Az 186

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PC 95 Z W Fy Pjx wd WI tZ GF 0Z X M +P GR hd G U+ U2 V wL U9 j dD wv ZG F0 ZT 48 L3 B1 Yi 1k Y XR 187

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

lcz 48 L2 Rh dG Vz Pjx pc 2Ju Pj E1 Mj Ut Nz c5 Nz wv aX Ni bj4 8Y W Nj ZX Nz aW 9u L W 51 bT 5J U0 k6 M 188

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

D A w M Tg 1M zg 2O TA w M DE 1P C9 hY 2N lc3 Np b2 4tb nV tPj x3 b3J rL XR 5c G U+ Q XJ 0 aW Ns ZT 189

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

wv d2 9y ay 10 eX Bl Pjx 1c mx zPj xy Z W xh dG Vk LX Vy bH M +P H Vy bD 4m bH Q7 R2 8g dG 8g SV NJ Jm 190

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

d0 Oz ov Lz A w M DE 4N T M4 Nj kw M D Ax NT wv dX JsP jw vc m Vs Y XR lZ C1 1c mx zPj wv dX Jsc z4 191

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

8Z W xl Y3 Ry b2 5p Yy 1y ZX Nv dX JjZ S1 ud W 0+ M TA uM TA yM S9i bT Az N D A0 Nj c8 L2 Vs Z W N0 192

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cm 9u aW Mt cm Vz b3 Vy Y2 Ut bn Vt Pjx sY W 5n d W Fn ZT 5F bm dsa X No PC 9s Y W 5n d W Fn ZT 48 193

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

L3 JlY 29 yZ D4 8L 0N pd G U+ PC 9F bm RO b3 Rl Pn == 3

PEG

4 Polyacrylamide 5

PEG

TG

42 43

Enzymatic

Hydrogel

crosslinking of

formation

Chymotrypsin

peptides Enzyme hydrolysis of

Hydrogel

11

Factor XIIIa

peptide crosslinks Enzymatic

dissolution Hydrogel

PE

& MMP

crosslinking of

formation or

Vu

peptides & Enzyme

hydrogel

ZE

hydrolysis of peptide

dissolution

5v

crosslinks

dG U+ PE Np dG U+ PE F1

194

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

dG hv cj5 Fa HJi Y XI 8L 0F 1d Gh vcj 48 W W Vh cj4 yM D A3 PC 9Z Z W Fy Pjx S Z W N Od W 0+ Mj kw 195

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PC 9S Z W N Od W 0+ PH JlY 29 yZ D4 8c m Vj L W 51 b WJ lcj 4y OT A8 L3 JlY y1 ud W 1iZ XI +P GZ v 196

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cm Vp Z2 4ta 2V 5cz 48 a2 V5 IG Fw cD 0i RU 4iI GR iL Wl kP SJ m YT l6Z Hp zd mt ze DJ wc m Vy ZT k4 eG Q1 197

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Mn Fk Zn dm d3I 5M H A1 czI iPj I5 M D wv a2 V5 Pj wv Zm 9y Z Wl nbi 1r ZX lzP jxy Z W Yt dH lw ZS Bu Y 198

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W 1lP SJ Kb 3V y bm FsI EF yd Glj bG Ui Pj E3 PC 9y Z W Yt dH lw ZT 48 Y2 9u dH Jp Yn V0 b3J zPj xh dX Ro 199

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

b3J zPj xh dX Ro b3I + R W hy Y mF yL CB NL jw vY X V0 aG 9y Pjx hd XR ob 3I+ U ml 6e mk sIF Mu IE Mu PC 200

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

9h dX Ro b3I +P GF 1d Gh vcj 5T Y2 hv Z W 5t Y Wt lcn Ms IFI uI Ec uP C9 hd XR ob 3I+ PG F1 dG hv cj5 TY W 201

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

4g T Wl nd W Vs LC BC Lj wv Y X V0 aG 9y Pjx hd XR ob 3I+ SH Vi Y m Vs bC wg Si4 gQ S4 8L 2F 1d Gh vcj 202

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

48 Y X V0 aG 9y Pld lY m Vy LC BG Li BF Lj wv Y X V0 aG 9y Pjx hd XR ob 3I+ TH V0 b2 xm LC BN Li BQ Lj 203

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

wv Y X V0 aG 9y Pj wv Y X V0 aG 9y cz4 8L 2N vb nR ya WJ 1d G9 y cz4 8Y X V0 aC 1h ZG Ry ZX Nz Pk Vj 204

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

b2 xlI FB vb Hl 0Z W No IE Zl ZC B M Y X Vz Y W 5u ZS wg S W 5z dC BC aW 9lb md u LC BT dG 4g M 205

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

TU sIE xh dX Nh bm 5lL CB Td 2l0 em Vy bG Fu ZC 4g R W Nv bG Ug U G9 se XR lY 2g gR m Vk IE xh dX Nh bm 206

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

5lL CB M Y WI gU 3R lbS BD Z W xsI EJ pb 2V uZ 24s IE xh dX Nh bm 5lL CB Td 2l0 em Vy bG Fu ZC 4g V W 5p 207

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

di Ba dX Jp Y2 gg SG 9zc Cw gQ ml vZ W 5n bi BT Z W N0 LC BD SC 04 M Dk xIF p1 cm lja Cw gU 3d pd Hp lc 208

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

mx hb m Qu IF Vu aX Yg W nV ya W No IE hv c3 As IE Rlc H Qg Q3 Jhb ml vI E1 he Gls bG 9m Y W Np Y W 209

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

wg U3 Vy Zy wg Q0 gt O D A5 M SB a dX Jp Y2 gsI FN 3a XR 6Z XJ sY W 5k Li Yj eE Q7 TH V0 b2 xm LC BN 210

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

UC wg R W Nv bG Ug U G9 se XR lY 2g gR m Vk IE xh dX Nh bm 5lL CB Jbn N0 IEJ pb 2V uZ 24s IF N0 bi Ax NS 211

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

wg Q mx kZ yB BS SA zM T M4 LC B M Y X Vz Y W 5u ZS wg U3 dp dH plc mx hb m Qu JiN 4R Dtt Y XR 0a 212

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Gl hc y5s dX Rv bG ZA ZX B mb C5 ja D wv Y X V0 aC 1h ZG Ry ZX Nz Pjx 0a XR s ZX M +P HR pd Gx lPk Jpb 213

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

21 vb G Vj d W xh ciB oe W Ry b2 dlb H Mg Zm 9y b W Vk IG Fu ZC Bk Z W dy Y W Rl ZC B2 aW Eg c2l 214

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

0Z S1 zc G Vj aW Zp Yy Bl bn p5 b W F0 aW Mg cm Vh Y3 Rp b2 5z PC 90 aX Rs ZT 48 c2 Vj b2 5k Y XJ 5L 215

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

XR pd Gx l Pk Jpb 21 hY 3Jv b W 9s Z W N1 bG Vz PC 9z Z W Nv bm Rh cn ktd Gl 0b G U+ PG Fs dC 10 aX 216

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Rs ZT 5C aW 9t Y W Ny b2 1v bG Vj d W xlc zw vY W x0 LX Rp dG xlP jw vd Gl 0b G Vz Pjx wZ XJ pb 2R pY 217

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2F sPj xm d W xs LX Rp dG xlP kJp b2 1h Y3 Jvb W 9s Z W N1 bG Vz PC 9m d W xs LX Rp dG xlP jxh Y mJ yL 218

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

TE +Q ml vb W Fjc m9 tb2 xl Y3 Vs ZX M8 L2 Fi Yn It M T4 8L 3B lc ml vZ Glj Y W w+ PG Fs dC 1w ZX Jpb 2R 219

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

pY 2F sPj xm d W xs LX Rp dG xlP kJp b2 1h Y3 Jvb W 9s Z W N1 bG Vz PC 9m d W xs LX Rp dG xlP jxh Y mJ 220

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

yL TE +Q ml vb W Fjc m9 tb2 xl Y3 Vs ZX M8 L2 Fi Yn It M T4 8L 2F sd C1 w ZX Jpb 2R pY 2F sPj xw Y W dlc 221

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

z4z M D A wL T M w M Dc 8L 3B hZ 2V zPj x2 b2 x1 b W U+ O D wv dm 9sd W 1lP jxu d W 1i ZX I+ M 222

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

TA 8L 25 1b WJ lcj 48 a2 V5 d2 9y ZH M +P Gtl eX dv cm Q+ Q1 JP U1 Mt TE lO S0l OR zw va 2V 5d 29 yZ D4 8 223

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

a2 V5 d2 9y ZD 5D RU xM LU 1J R1 JB VE lP Tj wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5H TF lD T0 wp IE hZ RF 224

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

JP R0 V M Uz wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5G Q U N UT 1It W ElJ ST wv a2 V5 d2 9y ZD 48 a2 V5 225

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

d2 9y ZD 5Q RV BU SU RF PC 9r ZX l3b 3Jk Pjx rZ Xl 3b 3Jk Pk 1B VF JJ W D wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y 226

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZD 5G SU JS SU 48 L2t le Xd v cm Q+ PG tle Xd vc m Q+ Qk lP TU FU RV JJ Q Ux TP C9 rZ Xl 3b 3Jk Pjx rZ Xl 227

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3b 3Jk Pk RF R1 JB RE FU SU 9O PC 9r ZX l3b 3Jk Pjx rZ Xl 3b 3Jk Pk 1P TE V D V Ux FU zw va 2V 5d 29 yZ D4 228

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

8L 2tl eX dv cm Rz Pjx kY XR lcz 48 eW Vh cj4 yM D A3 PC 95 Z W Fy Pjx wd WI tZ GF 0Z X M +P GR hd G U+ 229

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

T2 N0 PC 9k Y XR lPj wv cH Vi L W Rh dG Vz Pj wv ZG F0 ZX M +P Gl zY m4 + M TU yN S0 3N zk 3P C9 pc 230

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2Ju Pjx hY 2N lc3 Np b2 4tb nV tPk lT ST ow M D Ay NT A w M Dk 5M D A w M D U8 L2 Fj Y2 Vz c2l vbi 1u 231

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

d W 0+ PH dv cm std Hl wZ T5 Bc nR pY 2xl PC 93 b3J rL XR 5c G U+ PH Vy bH M +P HJl bG F0 Z W Qt dX Jsc 232

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

z4 8d XJ sPi Zs dD tH by B0 by BJ U0 km Z3 Q7 Oi 8v M D A w Mj U w M D A5 OT A w M D A1 PC 91 233

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cm w+ PC 9y Z W xh dG Vk LX Vy bH M +P C9 1c mx zPj xlb G Vj dH Jvb mlj LX Jlc 29 1c m Nl L W 51 bT 4x 234

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

M C4 xM DI xL 2Jt M Dc w Mj I4 Zj wv Z W xl Y3 Ry b2 5p Yy 1y ZX Nv dX JjZ S1 ud W 0+ PG xh bm d1 Y 235

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W dlP kV uZ 2x pc 2g 8L 2x hb md 1 Y W dlP jw vc m Vj b3J kPj wv Q2 l0Z T4 8L 0V uZ E5 vd G U+ A G= = 236

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PE Vu ZE 5v dG U+ PE Np dG U+ PE F1 dG hv cj5 Fa HJi Y XI 8L 0F 1d Gh vcj 48 W W Vh cj4 yM D A3 PC 9Z Z 237

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W Fy Pjx S Z W N Od W 0+ Mj kw PC 9S Z W N Od W 0+ PH JlY 29 yZ D4 8c m Vj L W 51 b WJ lcj 4y 238

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

OT A8 L3 JlY y1 ud W 1iZ XI +P GZ v cm Vp Z2 4ta 2V 5cz 48 a2 V5 IG Fw cD 0i RU 4iI GR iL Wl kP SJ m YT l6Z 239

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Hp zd mt ze DJ wc m Vy ZT k4 eG Q1 Mn Fk Zn dm d3I 5M H A1 czI iPj I5 M D wv a2 V5 Pj wv Zm 9y Z Wl nbi 240

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1r ZX lzP jxy Z W Yt dH lw ZS Bu Y W 1lP SJ Kb 3V y bm FsI EF yd Glj bG Ui Pj E3 PC 9y Z W Yt dH lw ZT 241

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

48 Y2 9u dH Jp Yn V0 b3J zPj xh dX Ro b3J zPj xh dX Ro b3I + R W hy Y mF yL CB NL jw vY X V0 aG 9y Pjx hd 242

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

XR ob 3I+ U ml 6e mk sIF Mu IE Mu PC 9h dX Ro b3I +P GF 1d Gh vcj 5T Y2 hv Z W 5t Y Wt lcn Ms IFI uI Ec uP 243

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

C9 hd XR ob 3I+ PG F1 dG hv cj5 TY W 4g T Wl nd W Vs LC BC Lj wv Y X V0 aG 9y Pjx hd XR ob 3I+ SH Vi Y 244

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

m Vs bC wg Si4 gQ S4 8L 2F 1d Gh vcj 48 Y X V0 aG 9y Pld lY m Vy LC BG Li BF Lj wv Y X V0 aG 9y Pjx hd 245

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

XR ob 3I+ TH V0 b2 xm LC BN Li BQ Lj wv Y X V0 aG 9y Pj wv Y X V0 aG 9y cz4 8L 2N vb nR ya WJ 1d G9 y 246

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cz4 8Y X V0 aC 1h ZG Ry ZX Nz Pk Vj b2 xlI FB vb Hl 0Z W No IE Zl ZC B M Y X Vz Y W 5u ZS wg S W 247

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

5z dC BC aW 9lb md u LC BT dG 4g M TU sIE xh dX Nh bm 5lL CB Td 2l0 em Vy bG Fu ZC 4g R W Nv bG Ug U G9 248

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

se XR lY 2g gR m Vk IE xh dX Nh bm 5lL CB M Y WI gU 3R lbS BD Z W xsI EJ pb 2V uZ 24s IE xh dX Nh bm 5lL 249

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

CB Td 2l0 em Vy bG Fu ZC 4g V W 5p di Ba dX Jp Y2 gg SG 9zc Cw gQ ml vZ W 5n bi BT Z W N0 LC BD SC 04 250

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

M Dk xIF p1 cm lja Cw gU 3d pd Hp lc mx hb m Qu IF Vu aX Yg W nV ya W No IE hv c3 As IE Rlc H Qg Q3 Jhb 251

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ml vI E1 he Gls bG 9m Y W Np Y W wg U3 Vy Zy wg Q0 gt O D A5 M SB a dX Jp Y2 gsI FN 3a XR 6Z XJ sY 252

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W 5k Li Yj eE Q7 TH V0 b2 xm LC BN UC wg R W Nv bG Ug U G9 se XR lY 2g gR m Vk IE xh dX Nh bm 5lL CB 253

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Jbn N0 IEJ pb 2V uZ 24s IF N0 bi Ax NS wg Q mx kZ yB BS SA zM T M4 LC B M Y X Vz Y W 5u ZS wg U3 dp 254

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

dH plc mx hb m Qu JiN 4R Dtt Y XR 0a Gl hc y5s dX Rv bG ZA ZX B mb C5 ja D wv Y X V0 aC 1h ZG Ry ZX Nz 255

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Pjx 0a XR s ZX M +P HR pd Gx lPk Jpb 21 vb G Vj d W xh ciB oe W Ry b2 dlb H Mg Zm 9y b W Vk IG Fu ZC 256

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Bk Z W dy Y W Rl ZC B2 aW Eg c2l 0Z S1 zc G Vj aW Zp Yy Bl bn p5 b W F0 aW Mg cm Vh Y3 Rp b2 5z PC 257

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

90 aX Rs ZT 48 c2 Vj b2 5k Y XJ 5L XR pd Gx l Pk Jpb 21 hY 3Jv b W 9s Z W N1 bG Vz PC 9z Z W Nv bm 258

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Rh cn ktd Gl 0b G U+ PG Fs dC 10 aX Rs ZT 5C aW 9t Y W Ny b2 1v bG Vj d W xlc zw vY W x0 LX Rp dG xlP 259

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

jw vd Gl 0b G Vz Pjx wZ XJ pb 2R pY 2F sPj xm d W xs LX Rp dG xlP kJp b2 1h Y3 Jvb W 9s Z W N1 bG Vz PC 260

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

9m d W xs LX Rp dG xlP jxh Y mJ yL TE +Q ml vb W Fjc m9 tb2 xl Y3 Vs ZX M8 L2 Fi Yn It M T4 8L 3B lc ml 261

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

vZ Glj Y W w+ PG Fs dC 1w ZX Jpb 2R pY 2F sPj xm d W xs LX Rp dG xlP kJp b2 1h Y3 Jvb W 9s Z W N1 bG Vz 262

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PC 9m d W xs LX Rp dG xlP jxh Y mJ yL TE +Q ml vb W Fjc m9 tb2 xl Y3 Vs ZX M8 L2 Fi Yn It M T4 8L 2F sd 263

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

C1 w ZX Jpb 2R pY 2F sPj xw Y W dlc z4z M D A wL T M w M Dc 8L 3B hZ 2V zPj x2 b2 x1 b W U+ O D 264

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

wv dm 9sd W 1lP jxu d W 1i ZX I+ M TA 8L 25 1b WJ lcj 48 a2 V5 d2 9y ZH M +P Gtl eX dv cm Q+ Q1 JP U1 Mt 265

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

TE lO S0l OR zw va 2V 5d 29 yZ D4 8 a2 V5 d2 9y ZD 5D RU xM LU 1J R1 JB VE lP Tj wv a2 V5 d2 9y ZD 48 a2 266

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

V5 d2 9y ZD 5H TF lD T0 wp IE hZ RF JP R0 V M Uz wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5G Q U N UT 1It 267

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W ElJ ST wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5Q RV BU SU RF PC 9r ZX l3b 3Jk Pjx rZ Xl 3b 3Jk Pk 1B VF JJ W 268

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

D wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5G SU JS SU 48 L2t le Xd v cm Q+ PG tle Xd vc m Q+ Qk lP TU FU RV 269

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

JJ Q Ux TP C9 rZ Xl 3b 3Jk Pjx rZ Xl 3b 3Jk Pk RF R1 JB RE FU SU 9O PC 9r ZX l3b 3Jk Pjx rZ Xl 3b 3Jk Pk 1P TE 270

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

V D V Ux FU zw va 2V 5d 29 yZ D4 8L 2tl eX dv cm Rz Pjx kY XR lcz 48 eW Vh cj4 yM D A3 PC 95 Z W Fy Pjx 271

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

wd WI tZ GF 0Z X M +P GR hd G U+ T2 N0 PC 9k Y XR lPj wv cH Vi L W Rh dG Vz Pj wv ZG F0 ZX M +P Gl 272

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

zY m4 + M TU yN S0 3N zk 3P C9 pc 2Ju Pjx hY 2N lc3 Np b2 4tb nV tPk lT ST ow M D Ay NT A w M Dk 5M D 273

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

A w M D U8 L2 Fj Y2 Vz c2l vbi 1u d W 0+ PH dv cm std Hl wZ T5 Bc nR pY 2xl PC 93 b3J rL XR 5c G U+ PH 274

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Vy bH M +P HJl bG F0 Z W Qt dX Jsc z4 8d XJ sPi Zs dD tH by B0 by BJ U0 km Z3 Q7 Oi 8v M D A w Mj U 275

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

w M D A5 OT A w M D A1 PC 91 cm w+ PC 9y Z W xh dG Vk LX Vy bH M +P C9 1c mx zPj xlb G Vj dH Jvb 276

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

mlj LX Jlc 29 1c m Nl L W 51 bT 4x M C4 xM DI xL 2Jt M Dc w Mj I4 Zj wv Z W xl Y3 Ry b2 5p Yy 1y ZX 277

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Nv dX JjZ S1 ud W 0+ PG xh bm d1 Y W dlP kV uZ 2x pc 2g 8L 2x hb md 1 Y W dlP jw vc m Vj b3J kPj wv Q2 278

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

l0Z T4 8L 0V uZ E5 vd G U+ A G= = 6

Poly(HPMA)

Calcium ions

Enzymatic degradation

Release of

44 PE

of polymer

payload

Vu ZE 5v dG U+ PE Np dG U+ PE F1 dG hv cj5 Hb 2x kY mF yd D wv

279

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Q X V0 aG 9y Pjx ZZ W Fy PjI w M DI 8L 1ll Y XI + PF JlY 05 1b T4 yO Tc 8L 1Jl Y0 51 bT 48 cm Vj b3J kPj 280

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

xy Z W Mt bn Vt Y m Vy PjI 5N zw vc m Vj L W 51 b WJ lcj 48 Zm 9y Z Wl nbi 1r ZX lzP jxr ZX kg Y XB 281

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

wP SJ FT iIg ZG Ita W Q9 Im Zh O Xp ke nN 2a 3N 4M nB yZ XJl OT h4 ZD Uy cW R md 2Z 3cj kw cD Vz Mi I+ Mj 282

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

k3 PC 9r ZX k+ PC 9m b3J la W du L Wt le X M +P HJl Zi1 0e XB lIG 5h b W U9 Ikp v dX Ju Y W wg Q XJ 283

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

0a W Ns ZS I+ M Tc 8L 3Jl Zi1 0e XB lPj xjb 25 0c mli dX Rv cn M +P GF 1d Gh vc nM +P GF 1d Gh v cj5 Hb 2x 284

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

kY mF yd Cw gU i48 L2 F1 dG hv cj4 8Y X V0 aG 9y Pl Ry Y Wl 0Z W ws IF Qu PC 9h dX Ro b3I +P GF 1d Gh v 285

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cj5 M Y XB pZ G9 0L CB TL iB BL jw vY X V0 aG 9y Pjx hd XR ob 3I+ S2 9z dC wg Si4 8L 2F 1d Gh vcj 48 L2 F1 286

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

dG hv cn M +P C9 jb2 50 cm lid XR vc nM +P GF 1d Gg tY W Rk cm Vz cz5 CZ W 4g R3 Vy aW 9uI FV ua X Yg Tm 287

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Vn ZX Ys IE Rl cH Qg Q2 hlb SB Fb md uL CB JT C0 4N DE w NS BC Z W Vy IF No ZX Zh LC BJ c3J hZ W wu JiN 288

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

4R Dt Lb 3N 0L CB KL CB C Z W 4g R3 Vy aW 9uI FV ua X Yg Tm Vn ZX Ys IE Rlc H Qg Q2 hlb SB Fb md uL CB 289

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

QT 0Ig Nj Uz LC BJ TC 04 N DE w NS BC Z W Vy IF No ZX Zh LC BJ c3J hZ W wu PC 9h dX Ro L W Fk ZH Jlc 290

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3M +P HR pd Gx lcz 48 dG l0b G U+ R W 56 eW 1h dG lj Y W xse SB jb2 50 cm 9sb G Vk IH Jlc 3B vb nN pd m 291

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Ug ZH J1 Zy Bk Z W xp dm Vy eS Bz eX N0 Z W 1z PC 90 aX Rs ZT 48 c2 Vj b2 5k Y XJ 5L XR pd Gx lPl Bv 292

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

bH ltZ XJ zI GZ vci BB ZH Zh bm Nl ZC BU Z W No bm 9sb 2d pZ X M8 L3 Nl Y2 9u ZG Fy eS 10 aX Rs ZT 48 Y 293

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W x0 LX Rp dG xlP lB vb Hlt Li BB ZH Yu IF Rl Y2 hu b2 wu PC 9h bH Qt dG l0b G U+ PC 90 aX Rs ZX M +P HB 294

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

lc ml vZ Glj Y W w+ PG Z1 bG wt dG l0b G U+ U G9 se W 1lc nM gZ m9 yI EF kd mF uY 2V kIF Rl Y2 hu b2 xv 295

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Z2l lcz wv Zn Vs bC 10 aX Rs ZT 48 Y WJ ici 0x Pl Bv bH ltL iB BZ H Yu IF Rl Y2 hu b2 wu PC 9h Y mJ yL TE 296

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

+ PC 9w ZX Jpb 2R pY 2F sPj xh bH Qt cG Vy aW 9k aW Nh bD 48 Zn Vs bC 10 aX Rs ZT 5Q b2 x5 b W Vy cy B 297

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

mb 3Ig Q W R2 Y W 5jZ W Qg V G Vj aG 5v bG 9n aW Vz PC 9m d W xs LX Rp dG xlP jxh Y mJ yL TE +U G9 298

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

se W 0uI EF kdi 4g V G Vj aG 5v bC 48 L2 Fi Yn It M T4 8L 2F sd C1 wZ XJ pb 2R pY 2F sPj xw Y W dlc z4 299

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

xM D A2 LT Ew M Tg 8L 3B hZ 2V zPj x2 b2 x1 b W U+ M T M8 L3 Zv bH Vt ZT 48 bn Vt Y m Vy Pj Ew LT 300

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Ey PC 9u d W 1iZ XI +P Gtl eX dv cm Rz Pjx rZ Xl 3b 3Jk Pm Vu enl tY XR pY 2F sb Hk gY 29 ud HJ vb Gx lZ D 301

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5jb 25 0c m9 sb G Vk IG Ry d Wc g ZG Vs aX Zlc nk 8L 2tl eX dv cm Q+ 302

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PG tle Xd vc m Q+ Y2 Fs Y2 l1b S1 yZ X N wb 25 za XZ lP C9 rZ Xl 3b 3Jk Pjx r ZX l3b 3Jk Pm dsd W Nv c2 Ut 303

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cm Vz cG 9u c2l 2Z Tw va 2V 5d 29 yZ D4 8a 2V 5d 29 yZ D5 pb nN 1b Gl uI HJl bG Vh c2 U8 L2t le Xd vc m Q+ 304

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PG tle Xd vc m Q+ R0 xV Q0 9T RS 1T RU 5T SV RJ Vk Ug TU V N QlJ BT kV TP C9 rZ Xl 3b 3Jk Pjx rZ Xl 3b 3Jk 305

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Pk NB VE lP Tkl DI EN PU E9 M W U1 FU iBI W UR ST 0d FT F M8 L2t le Xd vc m Q+ PG tle Xd vc m Q+ SU 5T 306

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

V Ux JTj wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5S RU xF Q V NF PC 9r ZX l3b 3Jk Pjx rZ Xl 3b 3Jk Pk 9Y SU RB 307

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

U0 U8 L2t le Xd vc m Q+ PG tle Xd vc m Q+ V Ux U Uk FT T1 V OR D wv a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 308

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

5U Uk FO U1 BP Ul Q8 L2t le Xd vc m Q+ PG tle Xd vc m Q+ Q0 FU Q Ux BU 0U 8L 2tl eX dv cm Q+ PG tle Xd vc 309

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

m Q+ Q U1 ZT EF TR Tw v a2 V5 d2 9y ZD 48 a2 V5 d2 9y ZD 5D Q Ux DS V V NP C9 rZ Xl 3b 3Jk Pjx rZ Xl 310

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3b 3Jk Pk RF Vk lD RV M8 L2t le Xd v cm Q+ PC 9r ZX l3b 3Jk cz4 8Z GF 0Z X M +P Hll Y XI + Mj A w Mj wv 311

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

eW Vh cj4 8c H Vi L W Rh dG Vz Pjx kY XR lPk 9j dC 1E Z W M8 L2 Rh dG U+ PC 9w d WI tZ GF 0Z X M +P 312

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

C9 kY XR lcz 48 aX Ni bj4 xM D Qy LT cx N Dc 8L 2lz Y m4 +P GF j Y2 Vz c2l vbi 1u d W 0+ SV NJ Oj A w 313

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

M DE 4M D Y3 OT A w M D Az OT wv Y W Nj ZX Nz aW 9u L W 51 bT 48 d2 9y ay 10 eX Bl Pk Fy dG ljb 314

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

G U8 L3 dv cm std Hl wZ T4 8d XJ scz 48 cm Vs Y XR lZ C1 1c mx zPj x1 cm w+ Jm x0 O0 dvI HR vI El T SS Zn 315

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

dD s6 Ly 8w M D Ax O D A2 Nz kw M D A w Mz k8 L3 Vy bD 48 L3 Jlb GF 0Z W Qt dX Jsc z4 8L 3V yb H 316

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

M +P G Vs Z W N0 cm 9u aW Mt cm Vz b3 Vy Y2 Ut bn Vt Pj Ew Lj Ew M DI vc GF 0Lj I3 NT wv Z W xl Y3 317

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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2.3.2.1.1 Enzyme catalysed changes in peptide crosslinker density The innovative development of systems based on enzyme catalysed changes in peptide crosslinker density (Table 2–1, entries 1-5) was by Hubbell and coworkers.41Figure

2–2

In the designing of a cell-responsive hydrogel, in which the

hydrated environment was remodelled by cell-secreted enzymes. The main structural component of the hydrogel was polyethylene glycol (PEG), offering a hydrophilic polymer network that resists protein adsorption. Other biological functionalities were introduced in the form of peptides (Figure 2–2 A). Four-armed PEG molecules with vinyl sulfone end groups were first reacted with a low stoichiometric ratio of mono-cysteine peptide based on RGD (the tripeptide determined to be the cellattachment domain in fibronectin).46 This partially derivatised PEG was then crosslinked by reacting with a bis-cysteine peptide (Ac-GCRD-GPQG↓IWGQDRCG-NH2) (arrow indicates enzyme cleavage site). The flanking of the cysteine residues with the RD sequence provides water solubility and an optimal pKa for the formation of cysteine thiolate. This system formed elastic hydrogels under

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physiological conditions. Dynamic rheometry (in which the material is subjected to an oscillating strain)47Figure 2–2 was used to observe gel formation showing that at higher pH the gel point occurred more rapidly (Figure 2–2B).

Figure 2–2. Cell responsive synthetic hydrogels. A:(1) A Michael type addition reaction between vinyl-sulfone functionalised multiarm PEGs and mono-cysteine adhesion peptides (2) and bis-cysteine MMP substrate peptides was used to form gels from aqueous solutions in the presence of cells. These hydrogel networks were designed to respond to local protease activity at the cell surface (3). B: Dynamic rheometry of a typical gelation reaction, showing the elastic modulus (G’) and loss modulus (G’’) and the gel point’s sensitivity to pH. C: The swelling and degradation of the gel network in responsive to incubation with MMP-1. 41

Addition of matrix metalloproteinase (MMP)-1 solution to the gel induced a volume increase until network dissolution was attained as a result of enzyme catalysed peptide hydrolysis. Conversely, a hydrogel formed using a peptide crosslinker with a sequence that the protease does not have specificity for did not show any change in volume upon MMP-1 treatment (Figure 2–2 C). MMP sensitive hydrogels allowed cells to migrate with the hydrogel and in vivo studies showed that extensive vascularisation only occurred with the MMP sensitive peptide crosslinker.41

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Furthermore, Healy and co-workers demonstrated a similar system incorporating protease

cleavable

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

7R28gdG8gSVNJJmd0 OzovLzAwMDE4NTM4NjkwMDAxNTwvdXJsPjwvcmVsYXRlZC11cmxzPjwvd XJscz48ZWxlY3Ryb25p Yy1yZXNvdXJjZS1udW0+MTAuMTAyMS9ibTAzNDA0Njc8L2VsZWN0cm9ua WMtcmVzb3VyY2UtbnVt PjxsYW5ndWFnZT5FbmdsaXNoPC9sYW5ndWFnZT48L3JlY29yZD48L0NpdGU +PC9FbmROb3RlPn== 42 This system was based NIPAAm and acrylic acid (AAc) monomers. The peptide crosslinker (QPQG-LAK-NH2) was synthesised through the acrylation of the amine groups of the lysine residues and the (N-terminal amine) of glutamine with acryloyl chloride. The resulting hydrogels (by free-radical polymerisation) were injected through a 2 mm aperture without demonstrating appreciable macroscopic fracture. Enzyme degradation of the gels occurred when exposed to MMP-13 with degradation time dependant on cross-linking density. In this way, degradation times could be modulated, ranging from approximately 100 hours for the lowest crosslinking density up to 300 hours with the highest degree of crosslinking.42 In another study a system that responded to enzymes by hydrogel formation rather than degradation was shown by Messersmith and co-workers.43 The protein crosslinking enzyme transglutaminase (TG) was used to catalyse reactions between short peptides conjugated to PEG. This enzyme catalysed an acyl-transfer reaction between the γ-carboxamide group of protein-bound glutaminyl residues and the εamino group of Lys residues, resulting in the formation of ε-(γ-glutamyl)lysine isopeptide sidechain bridges. Through the rational design of peptide substrates for TG rapid formation of a hydrogel was observed when multifunctional PEG molecules with conjugated peptides were exposed to the enzyme. The incorporation of the adhesive amino acid L-3,4-dihydroxylphenylalanine (DOPA) was also demonstrated for the first time with the best substrates for TG crosslinking found to be DOPA-FKG-NH2 and DOPA-GQQQLG-NH2.43 In 2004 Moore and co-workers published an enzyme responsive hydrogel utilising a novel conjugation technique.11 Earlier methods based on reacting peptide amine groups with acryloyl chloride or the Michael addition of cysteines to vinyl sulfones described by Hubbell had clear limitations. The acrylol chloride reactions lacked selectivity while Michael additions required a basic residue to be near to the cysteine 365

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

(in order to lower the pKa of the thiol giving increased rates of reaction). In the work presented by Moore and co-workers a scheme that allowed for the selective coupling of acrylamides to peptide sequences was demonstrated. The disulphide exchange between an active thiol containing monomer and thiol on a cysteine side chain occurred under acidic conditions and was selective for only thiols. The acidic environment firstly protonated the pyridyl group providing a good leaving group and secondly protonated the lysine residues preventing them from performing Michael addition to the methacrylamide functionality. The activated disulphide monomers were conjugated to the peptide and these peptide crosslinkers was then copolymerised with acrylamide by way of UV initiators. With this methodology poly(acrylamide) hydrogels crosslinked with peptides (CYKC) were prepared that would dissolve when subjected to chymotrypsin solution. Chymotrypsin solutions were flowed through microchannels containing hydrogel disks while the sizes of these disks were monitored. The control sample (containing a peptide for which chymotrypsin does not have specificity, CSKC) remained unaffected by the enzyme solution while the test sample containing the enzyme cleavable peptide shrank until complete dissolution occurred at 20 minutes. 11 More recently, Lutolf and co-workers have further developed the concept of cell responsive hydrogels with the aim of further mimicking the dynamic remodelling of the

ECM

that

occurs

in

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PjxhdXRob3I+SHViYmVsbCwgSi4gQS48L2F1dGhvcj48YXV0aG9yPldlYmVyLCBG LiBFLjwvYXV0 aG9yPjxhdXRob3I+THV0b2xmLCBNLiBQLjwvYXV0aG9yPjwvYXV0aG9ycz48L2N vbnRyaWJ1dG9y cz48YXV0aC1hZGRyZXNzPkVjb2xlIFBvbHl0ZWNoIEZlZCBMYXVzYW5uZSwgSW 5zdCBCaW9lbmdu LCBTdG4gMTUsIExhdXNhbm5lLCBTd2l0emVybGFuZC4gRWNvbGUgUG9seXRl Y2ggRmVkIExhdXNh bm5lLCBMYWIgU3RlbSBDZWxsIEJpb2VuZ24sIExhdXNhbm5lLCBTd2l0emVybGF uZC4gVW5pdiBa dXJpY2ggSG9zcCwgQmlvZW5nbiBTZWN0LCBDSC04MDkxIFp1cmljaCwgU3dpd HplcmxhbmQuIFVu aXYgWnVyaWNoIEhvc3AsIERlcHQgQ3JhbmlvIE1heGlsbG9mYWNpYWwgU3VyZy wgQ0gtODA5MSBa dXJpY2gsIFN3aXR6ZXJsYW5kLiYjeEQ7THV0b2xmLCBNUCwgRWNvbGUgUG9s eXRlY2ggRmVkIExh dXNhbm5lLCBJbnN0IEJpb2VuZ24sIFN0biAxNSwgQmxkZyBBSSAzMTM4LCBMY XVzYW5uZSwgU3dp dHplcmxhbmQuJiN4RDttYXR0aGlhcy5sdXRvbGZAZXBmbC5jaDwvYXV0aC1hZG RyZXNzPjx0aXRs ZXM+PHRpdGxlPkJpb21vbGVjdWxhciBoeWRyb2dlbHMgZm9ybWVkIGFuZCBkZ WdyYWRlZCB2aWEg c2l0ZS1zcGVjaWZpYyBlbnp5bWF0aWMgcmVhY3Rpb25zPC90aXRsZT48c2Vjb25k YXJ5LXRpdGxl PkJpb21hY3JvbW9sZWN1bGVzPC9zZWNvbmRhcnktdGl0bGU+PGFsdC10aXRsZ T5CaW9tYWNyb21v bGVjdWxlczwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2RpY2FsPjxmdWxsLXRp dGxlPkJpb21h Y3JvbW9sZWN1bGVzPC9mdWxsLXRpdGxlPjxhYmJyLTE+QmlvbWFjcm9tb2xlY3 VsZXM8L2FiYnIt MT48L3BlcmlvZGljYWw+PGFsdC1wZXJpb2RpY2FsPjxmdWxsLXRpdGxlPkJpb21 hY3JvbW9sZWN1 bGVzPC9mdWxsLXRpdGxlPjxhYmJyLTE+QmlvbWFjcm9tb2xlY3VsZXM8L2FiYnIt MT48L2FsdC1w ZXJpb2RpY2FsPjxwYWdlcz4zMDAwLTMwMDc8L3BhZ2VzPjx2b2x1bWU+ODwvd 367

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

m9sdW1lPjxudW1i ZXI+MTA8L251bWJlcj48a2V5d29yZHM+PGtleXdvcmQ+Q1JPU1MtTElOS0lORz wva2V5d29yZD48 a2V5d29yZD5DRUxMLU1JR1JBVElPTjwva2V5d29yZD48a2V5d29yZD5HTFlDT0 wpIEhZRFJPR0VM Uzwva2V5d29yZD48a2V5d29yZD5GQUNUT1ItWElJSTwva2V5d29yZD48a2V5d29 yZD5QRVBUSURF PC9rZXl3b3JkPjxrZXl3b3JkPk1BVFJJWDwva2V5d29yZD48a2V5d29yZD5GSUJS SU48L2tleXdv cmQ+PGtleXdvcmQ+QklPTUFURVJJQUxTPC9rZXl3b3JkPjxrZXl3b3JkPkRFR1J BREFUSU9OPC9r ZXl3b3JkPjxrZXl3b3JkPk1PTEVDVUxFUzwva2V5d29yZD48L2tleXdvcmRzPjxkYX Rlcz48eWVh cj4yMDA3PC95ZWFyPjxwdWItZGF0ZXM+PGRhdGU+T2N0PC9kYXRlPjwvcHVi LWRhdGVzPjwvZGF0 ZXM+PGlzYm4+MTUyNS03Nzk3PC9pc2JuPjxhY2Nlc3Npb24tbnVtPklTSTowMDA yNTAwMDk5MDAw MDU8L2FjY2Vzc2lvbi1udW0+PHdvcmstdHlwZT5BcnRpY2xlPC93b3JrLXR5cGU +PHVybHM+PHJl bGF0ZWQtdXJscz48dXJsPiZsdDtHbyB0byBJU0kmZ3Q7Oi8vMDAwMjUwMDA5O TAwMDA1PC91cmw+ PC9yZWxhdGVkLXVybHM+PC91cmxzPjxlbGVjdHJvbmljLXJlc291cmNlLW51bT4 xMC4xMDIxL2Jt MDcwMjI4ZjwvZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+PGxhbmd1YWdlPkVuZ2x pc2g8L2xhbmd1

YWdlPjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+AG==

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

RWhyYmFyLCBNLjwvYXV0aG9yPjxhdXRob3I+Uml6emksIFMuIEMuPC9hdXRob 3I+PGF1dGhvcj5T Y2hvZW5tYWtlcnMsIFIuIEcuPC9hdXRob3I+PGF1dGhvcj5TYW4gTWlndWVsLCB CLjwvYXV0aG9y PjxhdXRob3I+SHViYmVsbCwgSi4gQS48L2F1dGhvcj48YXV0aG9yPldlYmVyLCBG LiBFLjwvYXV0 aG9yPjxhdXRob3I+THV0b2xmLCBNLiBQLjwvYXV0aG9yPjwvYXV0aG9ycz48L2N vbnRyaWJ1dG9y cz48YXV0aC1hZGRyZXNzPkVjb2xlIFBvbHl0ZWNoIEZlZCBMYXVzYW5uZSwgSW 5zdCBCaW9lbmdu LCBTdG4gMTUsIExhdXNhbm5lLCBTd2l0emVybGFuZC4gRWNvbGUgUG9seXRl Y2ggRmVkIExhdXNh bm5lLCBMYWIgU3RlbSBDZWxsIEJpb2VuZ24sIExhdXNhbm5lLCBTd2l0emVybGF uZC4gVW5pdiBa dXJpY2ggSG9zcCwgQmlvZW5nbiBTZWN0LCBDSC04MDkxIFp1cmljaCwgU3dpd HplcmxhbmQuIFVu aXYgWnVyaWNoIEhvc3AsIERlcHQgQ3JhbmlvIE1heGlsbG9mYWNpYWwgU3VyZy wgQ0gtODA5MSBa dXJpY2gsIFN3aXR6ZXJsYW5kLiYjeEQ7THV0b2xmLCBNUCwgRWNvbGUgUG9s eXRlY2ggRmVkIExh dXNhbm5lLCBJbnN0IEJpb2VuZ24sIFN0biAxNSwgQmxkZyBBSSAzMTM4LCBMY XVzYW5uZSwgU3dp dHplcmxhbmQuJiN4RDttYXR0aGlhcy5sdXRvbGZAZXBmbC5jaDwvYXV0aC1hZG RyZXNzPjx0aXRs ZXM+PHRpdGxlPkJpb21vbGVjdWxhciBoeWRyb2dlbHMgZm9ybWVkIGFuZCBkZ WdyYWRlZCB2aWEg c2l0ZS1zcGVjaWZpYyBlbnp5bWF0aWMgcmVhY3Rpb25zPC90aXRsZT48c2Vjb25k YXJ5LXRpdGxl PkJpb21hY3JvbW9sZWN1bGVzPC9zZWNvbmRhcnktdGl0bGU+PGFsdC10aXRsZ T5CaW9tYWNyb21v bGVjdWxlczwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2RpY2FsPjxmdWxsLXRp dGxlPkJpb21h Y3JvbW9sZWN1bGVzPC9mdWxsLXRpdGxlPjxhYmJyLTE+QmlvbWFjcm9tb2xlY3 VsZXM8L2FiYnIt MT48L3BlcmlvZGljYWw+PGFsdC1wZXJpb2RpY2FsPjxmdWxsLXRpdGxlPkJpb21 369

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

hY3JvbW9sZWN1 bGVzPC9mdWxsLXRpdGxlPjxhYmJyLTE+QmlvbWFjcm9tb2xlY3VsZXM8L2FiYnIt MT48L2FsdC1w ZXJpb2RpY2FsPjxwYWdlcz4zMDAwLTMwMDc8L3BhZ2VzPjx2b2x1bWU+ODwvd m9sdW1lPjxudW1i ZXI+MTA8L251bWJlcj48a2V5d29yZHM+PGtleXdvcmQ+Q1JPU1MtTElOS0lORz wva2V5d29yZD48 a2V5d29yZD5DRUxMLU1JR1JBVElPTjwva2V5d29yZD48a2V5d29yZD5HTFlDT0 wpIEhZRFJPR0VM Uzwva2V5d29yZD48a2V5d29yZD5GQUNUT1ItWElJSTwva2V5d29yZD48a2V5d29 yZD5QRVBUSURF PC9rZXl3b3JkPjxrZXl3b3JkPk1BVFJJWDwva2V5d29yZD48a2V5d29yZD5GSUJS SU48L2tleXdv cmQ+PGtleXdvcmQ+QklPTUFURVJJQUxTPC9rZXl3b3JkPjxrZXl3b3JkPkRFR1J BREFUSU9OPC9r ZXl3b3JkPjxrZXl3b3JkPk1PTEVDVUxFUzwva2V5d29yZD48L2tleXdvcmRzPjxkYX Rlcz48eWVh cj4yMDA3PC95ZWFyPjxwdWItZGF0ZXM+PGRhdGU+T2N0PC9kYXRlPjwvcHVi LWRhdGVzPjwvZGF0 ZXM+PGlzYm4+MTUyNS03Nzk3PC9pc2JuPjxhY2Nlc3Npb24tbnVtPklTSTowMDA yNTAwMDk5MDAw MDU8L2FjY2Vzc2lvbi1udW0+PHdvcmstdHlwZT5BcnRpY2xlPC93b3JrLXR5cGU +PHVybHM+PHJl bGF0ZWQtdXJscz48dXJsPiZsdDtHbyB0byBJU0kmZ3Q7Oi8vMDAwMjUwMDA5O TAwMDA1PC91cmw+ PC9yZWxhdGVkLXVybHM+PC91cmxzPjxlbGVjdHJvbmljLXJlc291cmNlLW51bT4 xMC4xMDIxL2Jt MDcwMjI4ZjwvZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+PGxhbmd1YWdlPkVuZ2x pc2g8L2xhbmd1 YWdlPjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+AG== 44 This includes the formation of new bonds as well as degradation. Here, much like Messersmith and co-workers, they made use of a type of transglutaminase; the crosslinking enzyme factor XIIIa. This enzyme plays a key role in fibrin clot formation upon tissue damage by catalysing acyl-transfer between the carboxamide group of a protein-bound glutamine (Q) residue and the ε amino group in a lysine (K) residue. Multiarm PEG molecules were functionalised with either Gln in the 370

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

form of its XIIIa acceptor substrate (NQEQVSPL) or an MMP cleavable peptide terminated at the C-terminus with Lys. A mixture of these precursors formed a hydrogel in the presence of factor XIIIa with a gel point time of about 750 seconds as determined by real-time shear rheometric measurements. The bioactive binding ligand RGD could also be incorporated in the network by having the XIIIa acceptor substrate at the C-terminus of RGD. Upon treatment of these hydrogels with an MMP solution rapid dissolution was observed (within 10 minutes) dependant on the peptide

crosslinker

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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of

the

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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respectively

were

not

coupled

to

the

cartilage.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Kb25lczwvQXV0aG9yPjxZZ WFyPjIwMDc8L1llYXI+PFJl Y051bT4zMDk8L1JlY051bT48cmVjb3JkPjxyZWMtbnVtYmVyPjMwOTwvcmVj LW51bWJlcj48Zm9y ZWlnbi1rZXlzPjxrZXkgYXBwPSJFTiIgZGItaWQ9ImZhOXpkenN2a3N4MnByZ XJlOTh4ZDUycWRm d2Z3cjkwcDVzMiI+MzA5PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG 5hbWU9IkpvdXJu 380

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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2.3.2.1.2 Enzyme hydrolysis of hydrogel network A different approach to using enzymes in a responsive system based on hydrogel degradation has been described by Kost and co-workers.49 In that work a calcium responsive system was prepared through the incorporation of a non-active enzyme into a polymer matrix consisting of the enzyme’s substrate. The enzyme used was the starch hydrolysing, α-amylase, while the polymer matrix was a mixture of starch and hydroxypropyl methyl cellulose ether (HPMC). α-amylase’s are known to contain at least one atom of calcium firmly and specifically bound to the enzyme molecule (protein) that is essential for its activity. Therefore, firstly de-activated αamylase was prepared by chelating the calcium ions without denaturating the enzyme. Enzyme containing tablets were then prepared by wet granulation in which all ingredients were ground, mixed then dried before being pressed into tablets. Upon incubation of the tablets a concentration dependant release of the model drug myoglobin

was

obtained

with

a

constant

rate

of

release

over

30

hours.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Hb2xkYmFydDwvQXV0aG9yPj xZZWFyPjIwMDI8L1llYXI+ PFJlY051bT4yOTc8L1JlY051bT48cmVjb3JkPjxyZWMtbnVtYmVyPjI5NzwvcmV jLW51bWJlcj48 Zm9yZWlnbi1rZXlzPjxrZXkgYXBwPSJFTiIgZGItaWQ9ImZhOXpkenN2a3N4Mn ByZXJlOTh4ZDUy cWRmd2Z3cjkwcDVzMiI+Mjk3PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10e XBlIG5hbWU9Ikpv dXJuYWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dG hvcnM+PGF1dGhv cj5Hb2xkYmFydCwgUi48L2F1dGhvcj48YXV0aG9yPlRyYWl0ZWwsIFQuPC9hd XRob3I+PGF1dGhv cj5MYXBpZG90LCBTLiBBLjwvYXV0aG9yPjxhdXRob3I+S29zdCwgSi48L2F1d Ghvcj48L2F1dGhv

385

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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389

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

competition between the free moieties and those crosslinking the polymer (Table 2– 2). Table 2–2. Selected studies of bioresponsive hydrogels based on non-covalent crosslinking interactions

# Polymer 1 Polyacrylamide

Stimulus Antigen

Biorecognition Antigen-antibody

Response Reversible

Ref 27

2 Poly(NIPAM-

Antigen

bonding Biotin-Antibiotin

swelling Reversible

26

Coiled-coil

binding Self-assembly through

swelling Hydrogel

PEV

forming

coiled-coil formation

formation

uZE

3

co-AAC) Poly(HPMA)

peptides

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448

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

An excellent early example of this type of system is the antigen responsive hydrogel described by Uragami and co-workers.27 In this study a semi-interpenetrating network (semi-IPN) hydrogel containing both grafted antigens and their corresponding specific antibody was prepared. Vinyl functionality was introduced to both of the biological moieties by reacting them with N-succinimidylacrylate (NSA) (which reacts with the primary amines). The modified goat-anti-rabbit antibody (GAR) IgG was then copolymerised with acrylamide to give a polymer solution. Vinyl rabbit IgG (antigen) was mixed with the GAR IgG polymer solution, acrylamide, a crosslinker N,N’-methylenebisacrylamide (MBAA) and polymerised with free-radical redox initiators (Figure 2–3 A). Antigen-antibody binding then caused the formation of further cross-links within the polymer reducing the swelling (Figure 2–3 B). The presence of free antigens in the solution around the hydrogel created competition between the grafted antigens leading to a decrease in crosslinking density, thus an increase in swelling of the hydrogel (Figure 2–3 C). This increase in swelling was reversible. By removing the hydrogel from the antigen solution and washing, the polymer would return to approximately its original volume. This response also corresponds to an increase in permeability of the polymer and by using the polymer as a membrane, selective permeation of a protein was shown in response to the specific antibody (Figure 2–3 D). More recently a similar design was employed to produce an antigen-responsive membrane that has gating properties (allow for the opening of closing of pores) for selective diffusion in response to the presence of a free antigen.51

449

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald Figure 2–3. Antigen responsive hydrogel. A: Synthesis of the antigen-antibody semi-IPN hydrogel. B: Diagram of a suggested mechanism for the swelling of an antigen-antibody semiIPN hydrogel in response to a free antigen. C: Antigen recognition by antigen-antibody semiIPN hydrogel. D: Reversible swelling changes and antigen-responsive permeation profiles. 27

Lyon and co-workers have demonstrated an antigen responsive hydrogel, 26 this system consists of a co-polymer of pNIPAAm and acrylic acid with N,N’methylenebisacrylamide as the crosslinker prepared by free radical initiated precipitation polymerisation. In this process the monomer(s) and initiator are soluble in the solvent and initiation takes place in solution. The polymer chains then grow until they reach a critical chain length where they exceed their solubility and precipitate from solution.52Figure 2–4Figure 2–4 Lyon and co-workers covalently incorporated a biotin moiety into the microgels by coupling biotin hydrazide to the carboxyl group of the acrylic acid using a water-soluble carbodiimide (1-ethyl-3-(3dimethylaminopropyl)carbodiimide). Aminobenzophenone (ABP) was then also coupled to the polymer using N,N’-dicyclohexylcarbodiimide in dimethyl sulfoxide (DMSO).

Glass

coverslips

functionalised

with

the

cationic

silane

3-

aminopropyltrimethoxysilane (APTMS) were then placed in an aqueous solution of biotin/ABP functionalised polymer particles which strongly attached to the glass surface via Coulombic interactions. These ‘microlens’ covered surfaces were then exposed to a solution of anti-biotin (antibody), leading to antigen-antibody binding which was followed by photoligation of the antibody to the ABP. This treatment effectively led to a crosslinking of the surface of the microlenses by antigenantibody binding. Exposure of these microlenses to biocytin (antigen) led to competition in binding with anti-biotin resulting in a decrease in the crosslinking density of the surface of the microlenses (Figure 2–4 A). This resulted in an increase in swelling of the polymer and a corresponding decrease in the microlens focussing power. This was determined using brightfield transmission and differential interference contrast (DIC) optical microscopy (Figure 2–4 B). The response rate of the hydrogel microlenses were found to be strongly coupled to analyte concentration.26

450

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 2–4. Displacement-Induced Switching Rates of Bioresponsive Hydrogel Microlenses. A: The microgels are functionalized with biotin and ABP (top left), are cross-linked by anti-biotin (top centre) and exposed to a solution of biocytin (top right). B: Response of hydrogel microlenses to incubation with anti-biotin antibodies. Microscopic images of hydrogel microlenses (left element in each image) and biotin/ABP functionalized hydrogel microlenses (right element in each image) are shown, (a) before and (b) after incubation with anti-biotin. The scale bar is 2 µm.26

Kopeček and co-workers describe a hybrid hydrogel (a hydrogel system consisting of at least two distinct classes of molecules connected either covalently or noncovalently) in which the crosslinking was achieved through the self-assembly of coiled-coil forming peptides (Figure 2–5 A). These peptides were grafted onto a polymer backbone of N-(2-hydroxypropyl)methacrylamide (HPMA). Coiled-coils are a native protein conformation that consists of two or more α-helices wound together to form a super-helix.53 The attraction of incorporating these types of peptides into a polymer is that the association and disassociation of these coiledcoils is determined by the primary structure of the amino acid sequence. In this system polymerisable functionality was imparted into the peptides by capping the Ntermini of the peptide with N-methacyloylglycyl-glycyltryptophan. This offered the same functionality as HPMA ensuring the compatibility of the comonomers in free radical copolymerisation. Examination of the self-assembly of these copolymers into hydrogels was achieved using microrheology and dynamic light scattering. Dynamic light scattering evaluated the size of nanoparticles formed as association of coiledcoil forming peptides led to gelation. Analysis of the hydrodynamic radius, Rh 451

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

showed that particle size was independent of graft copolymer concentration at low concentrations but increased dramatically as the concentration was increased above 5.52 mg/ml (Figure 2–5 B). These results indicated that the self-assembly process was strongly governed by polymer concentration. Temperature also had an effect on the self-assembly process, as temperature was increased (Figure 2–5 C) there is a gradual increase in the Rh. The increased thermal energy of the system led to an increase in the probably of a collision of the peptide grafts making the self-assembly process

more

effective

at

higher

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 2–5. Novel synthesis of HPMA copolymers containing peptide grafts and their self assembly into hybrid hydrogels. A: the self-assembly of copolymers into hydrogels. B: Concentration dependence of the hydrodynamic radii Rh of polymer clusters. C: Temperature dependence

of

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2.3.3. Actuation based on electrostatic interactions A second mechanism to actuate a change in swelling is through electrostatic repulsion/attraction within the polymeric network. These charges are typically induced on the polymer chain but it has also been demonstrated that grafting specific actuators on to the polymer is possible ().

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Table 2–3. Selected studies of bioresponsive hydrogels based on electrostatic interactions

# 1

Polymer Poly(HEMA-co-

Stimulus Glucose

Biorecognition Conversion of glucose

Response Increased

Ref 36

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

dsd WN vc2 Utc2 Vuc 2l0a XZp dHk 8L2t leXd vcm Q+P Gtle Xdv cmQ +ZX F1a Wxp YnJ pdW 0g c3dl bGx pbm c8L 2tle Xdv cmQ +PG tleX dvc mQ +U1 dFT ExJ 476

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Tkc gUF JPU EVS VEl FUz wva 2V5 d29 yZD 48 a2V 5d2 9yZ D5J TlN VTE lOIF JFT EV BU0 U8L 2tle Xdv cmQ +PG tleX dvc mQ +RF JVR y1E RUx JVk VS 477

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

WT wv a2V 5d2 9yZ D48 a2V 5d2 9yZ D5N RU1 CUk FOR VM 8L2t leXd vcm Q+P Gtle Xdv cmQ +VF JBTl NQ T1J UPC 9r ZXl 3b3J kPjx rZXl 3b3J kPkJ FSE FW 478

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

SU9 SPC 9rZ Xl3 b3Jk Pjxr ZXl 3b3J kPl NZ U1R FTV M8 L2tl eXd v cmQ +PG tleX dvc mQ +R0 VM Uzw va2 V5d 29y ZD4 8L2t leXd vcm RzPj xkY XRl cz48 479

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

eW Vhcj 4yM DA wPC 95 ZW FyPj xwd WIt ZGF 0ZX M+ PGR hdG U+T WF 5PC 9kY XRl Pjw vcH ViL WR hdG VzP jwv ZGF 0ZX M+ PGl zYm 4+ MD Az 480

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Mi0 zOD YxP C9p c2Ju Pjxh Y2N lc3N pb2 4tbn VtP klTS Tow MD Aw OD U1N jUy MD Aw MD k8L 2Fj Y2V z c2lv bi1u dW0 +PH dvc mstd Hlw ZT5 Bcn RpY 481

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2xlP C93 b3Jr LX R5c GU +PH Vyb HM +PH JlbG F0Z WQt dXJ s cz48 dXJ sPiZ sdDt Hby B0b yBJ U0k mZ3 Q7O i8v MD Aw MD g1N TY1 MjA wM DA5 PC9 482

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1cm w+P C9y ZW xhd GVk LX Vyb HM +PC 91c mxz Pjxs YW 5nd WF nZT 5Fb mds aXN oPC 9sY W5n dW FnZ T48 L3Jl Y29 yZD 48L 0Np dGU + PEN pdG 483

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

U+P EF1 dGh vcj5 Qb2 R1Y Ww 8L0 F1d Ghv cj48 WW Vhcj 4yM DA wPC 9ZZ WF yPjx SZ WN Od W0 +Mz Ay PC9 SZ WN Od W0 +PH JlY2 9yZ D48 cmV 484

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

jLW 51b WJl cj4z MDI 8L3 JlYy 1ud W1i ZXI +PG Zvc mV pZ2 4ta2 V5 cz48 a2V 5IG Fwc D0i RU4 iIG RiL Wlk PSJ mY Tl6 ZHp zdm tzeD Jwc mV yZT k4e 485

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

GQ1 MnF kZn dmd 3I5 MH A1 czIi PjM wMj wva 2V5 Pjw vZm 9yZ Wln bi1r ZXl zPjx yZ WYt dHl wZS BuY W1l PSJ Kb3 Vyb mFs IEF ydG lj bGU iPjE 3PC 486

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

9yZ WYt dHl wZT 48Y 29u dHJ pYn V0b 3JzP jxhd XRo b3Jz Pjxh dXR ob3I +U G9k dW FsL CBL Ljw vYX V0a G9y Pjxh dXR ob3I +RG 95b GUs IEY uIEo uPC 9hd 487

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

XRo b3I+ PGF 1dG hvcj 5QZ XB wY XM sIE4 u IEE uPC 9hd XRo b3I+ PC9 hdX Rob 3JzP jwv Y29 udH JpY nV0 b3Jz Pjxh dXR oL WF kZH Jlc3 M+ UH VyZ 488

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

HVl IFV uaX YsI FNj aCB DaG VtIE VuZ 24sI EJp b21 hdC Am YW 1wO yBE cnV nIE Rlb Gl2 ZXJ 5IEx hYn MsI Fcg TGF mY Xlld HRl LCB JTi A0N zkw NyB 489

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

VU0 EuJi N4R DtQ ZX Bw YX MsI E5B LCB QdX Jkd WU gV W5p diw gU2 No IEN oZ W0g RW 5nbi wgQ mlv bW F0I CZh bXA 7IE Ryd Wcg RG Vsa XZl 490

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cnkg TGF icyw gVy BM YW Zhe WV 0dG Us IElO IDQ 3OT A3I FVT QS4 8L2 F1d Ggt YW Rkc mVz cz48 dGl 0bG VzP jx0a XRs ZT5 Ee W5h bWl jIGJ laG F2 491

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

aW9 yIG 9mI Gds dW Nvc 2Ug b3h pZG FzZ S1jb 250 YWl uaW 5nI G1p Y3J vcG Fyd Gljb GVz IG9 mIH Bvb Hko ZX Roe Wxl bm UgZ 2x5 Y29 sKS 1nc mF 492

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

md GVk IGN hdG lvb mljI Gh5 ZHJ vZ2 Vsc yBp biBh biBl bnZ pcm 9u bW Vud CBv ZiBj aGF uZ2l uZy BwS Dwv dGl 0bG U+P HNl Y29 uZG Fye S10 aXR sZT 493

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

5Ca W9t YX Rlc mlh bH M8 L3N lY2 9uZ GFy eS1 0aX RsZ T48 YW x0L XRp dGx lPkJ pb2 1hd GVy aWF scz wvY Wx0 LX Rpd Gxl Pjw vdG l0 bGV zPjx 494

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

wZ XJp b2R pY2 FsPj xmd Wxs LX Rpd Gxl PkJp b21 hdG Vya WFs czw vZn Vsb C10 aXR sZT 48Y WJi ci0x PkJp b21 hdG Vya WFs czw vY WJi ci0x Pjw vcG 495

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Vya W9k aW Nhb D48 YW x0L XBl cml vZG ljY Ww +PG Z1 bGw tdGl 0bG U+ Qml vbW F0Z XJp YW xzP C9m dWx sLX Rpd Gxl Pjxh YmJ yLT E+Q mlv bW 496

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

F0Z XJp YW xzP C9h YmJ yLT E+P C9h bHQ tcG Vya W9k aW Nhb D48 cGF nZX M+ MT QzO S0x ND Uw PC9 wY Wdl cz48 dm9 sdW 1lPjI x PC9 2b2 x1b 497

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

WU +PG 51b WJl cj4x ND wvb nVt Ym VyP jxrZ Xl3 b3Jk cz48 a2V 5d2 9yZ D5j YX Rpb 25p YyB o eW Ryb 2dlb HM 8L2t leXd vcm Q+P Gtle Xdv cmQ +b 498

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Wljc m9 wY XJ0 aW NsZ XM 8L2t leXd vcm Q+P Gtle Xdv cmQ +cH Vsc 2F0 aWx lIH N3Z Wxs aW5 nPC 9rZ Xl3 b3Jk Pjxr ZXl 3b3J kPnJ lbG F4Y XRp b24 8L2t 499

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

leXd v cmQ +PG tleX dvc mQ +RE VM SVZ FUl kgU 1lT VE VN Uzw va2 V5d 29y ZD4 8a2 V5d 29y ZD5 NS UN ST1 NQ SEV S RV M8 L2tl eXd vcm 500

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Q+P Gtle Xdv cmQ +Uk VM RUF TRT wva 2V5 d29 yZD 48a2 V5d 29y ZD5 OQ U5P UEF SVE lD TEV TPC 9rZ Xl3 b3Jk Pjxr ZXl 3b3J kPk 1JQ 1JP Q0F QU1 VM 501

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

RV M8 L2tl eXd vcm Q+P C9r ZXl 3b3J kcz4 8 ZGF 0ZX M+ PHll YXI +Mj Aw MD wve WV hcj4 8cH ViL WR hdG VzP jxk YX RlP kp1 bDw vZG F0Z T48 502

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

L3B 1Yi 1k YX Rlcz 48L 2Rh dGV zPjx pc2J uPj AxN DIt OT Yx Mjw vaX Nibj 48Y WNj ZX Nza W9u LW 51b T5J U0k 6M DA w MD g3M zk5 OT Aw 503

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

MD A0P C9h Y2N lc3N pb2 4tbn VtPj x3b 3JrL XR5 cGU +Q XJ0 aW NsZ Twv d29 yay1 0eX Bl Pjx1 cmx zPjx yZ Wxh dGV kLX Vyb HM +PH Vyb D4 mb HQ7 504

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

R28 gdG 8gS VNJ Jmd 0Oz ovL zAw MD A4N zM5 OTk w MD Aw ND wvd XJs Pjw vcm VsY XRl ZC1 1cm xzPj wvd XJsc z48b GFu Z3V hZ2 U+R W5n bGlz aDw 505

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

vbG FuZ 3Vh Z2U +PC 9yZ WN vcm Q+P C9D aXR lPjw vR W5k Tm9 0ZT 4A PEV uZE 5vd GU +PE Npd GU +PE F1d Ghv cj5Q b2R 1Y Ww 8L0 F1d Ghv cj48 506

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

WW Vhcj 4yM DA wPC 9ZZ WF yPjx S ZW NOd W0 +Mj gwP C9S ZW NOd W0 +PH JlY2 9yZ D48 cmV jLW 51b WJl cj4y OD A8L 3JlY y1u dW1 iZXI +PG Zv 507

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cmV pZ2 4ta2 V5c z48a 2V5 IGF wcD 0iR U4iI GRi LWl kPS JmY Tl6 ZHp zdm tzeD Jwc mV yZT k4e GQ1 MnF k Znd md3 I5M HA1 czIi PjI4 MD wva 2V5 Pjw 508

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

vZm 9yZ Wln bi1r ZXl zPjx yZ WYt dHl wZS BuY W1l PSJ Kb3 Vy bmF sIEF ydG ljbG UiPj E3P C9y ZW Ytd Hlw ZT4 8Y2 9ud HJp YnV 0b3J zPjx hdX Rob 3JzP 509

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

jxhd XRo b3I+ UG9 kdW FsL CBL Ljw vYX V0a G9y Pjxh dXR ob3I +RG 95b GUs IEY uIEo uPC 9hd XRo b3I+ PGF 1dG hvcj 5Q ZX Bw YX MsI E4uI EEu PC9 hdX 510

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Rob 3I+P C9h dXR ob3J zPj wvY 29u dHJ pYn V0b 3JzP jxhd XRo LW FkZ HJl c3M +U HVy ZH VlIF Vua XYs IFNj aCB DaG VtIE VuZ 24sI EJp b21 hdC Am YW 511

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1wO yBE cnV nIE Rlb Gl2 ZXJ 5 IExh Yn MsI Fcg TGF mY Xlld HRl LCB JTi A0N zkw NyB VU0 EuJi N4R DtQ ZX Bw YX MsI E5B LCB QdX Jkd WU g 512

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

VW 5pdi wgU 2No IEN oZ W0g RW 5nbi wgQ mlv bW F0I CZh bXA 7IE Ryd Wcg RG Vsa XZl cnkg TGF icyw gVy BM YW Zhe WV 0dG UsI ElOI DQ3 OT A3I 513

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

FVT QS4 8L2 F1d Ggt YW Rkc mVz cz48 dGl 0bG VzP jx0a XRs ZT5 Qcm Vw YXJ hdG lvbi Bhb mQ gZH luY W1p YyB yZX Nwb 25z ZSB vZi BjY XRp b25 pYy 514

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Bjb3 Bvb HltZ XIg aHlk cm9 n ZW xzI GNv bnR haW 5pb mcg Z2x 1Y2 9zZ SBv eGlk YX NlP C90 aXR sZT 48c2 Vjb 25k YXJ 5LX Rpd Gxl PlB v bHlt ZXI 515

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

8L3 NlY 29u ZGF yeS 10a XRs ZT4 8Y Wx0 LX Rpd Gxl PlB vbH ltZX I8L2 Fsd C10 aXR sZT 48L 3Rp dGx lcz4 8cG Vya W9k aW Nhb D48 ZnV sbC 10a XRs 516

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZT5 Qb2 x5b WV yPC 9md Wxs LX Rpd Gxl Pjxh YmJ yLT E+ UG9 seW 1lcj wvY WJi ci0x Pjw vcG Vya W9k aW Nhb D48 YW x0L XBl cml vZG ljY Ww +PG 517

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Z1b Gwt dGl 0bG U+ UG9 seW 1lcj wvZ nVs bC1 0aX RsZ T48 YW Jici0 xPl Bvb HltZ XI8 L2Fi YnIt MT 48L 2Fsd C1w ZXJ pb2 Rp Y2F sPjx wY Wdl cz4z OTc 518

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1LT M5 OD M8 L3B hZ2 VzP jx2b 2x1 bW U+ ND E8L 3Zv bHV tZT 48b nVt Ym VyP jEx PC9 udW 1iZ XI+ PGtl eXd vcm RzPj xrZ Xl3 b3Jk Pm Nhd Glv 519

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

bmlj IGh 5ZH JvZ 2Vs PC9 rZXl 3b3J kPjx r ZXl 3b3J kPm dsd WN vc2 Utc2 Vuc 2l0a XZp dHk 8L2t leXd vcm Q+P Gtle Xdv cmQ +ZX F1a Wxp YnJ pdW 0g c3dl 520

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

bGx pbm c8L 2tle Xdv cmQ +PG tleX dvc mQ +U1 dFT ExJ Tkc gUF JPU EVS VEl FUz wva 2V5 d29 yZD 48 a2V 5d2 9yZ D5J TlN VTE lOIF JFT EV BU0 U8L 521

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2tle Xdv cmQ +PG tleX dvc mQ +RF JVR y1E RUx JVk VS WT wv a2V 5d2 9yZ D48 a2V 5d2 9yZ D5N RU1 CUk FOR VM 8L2t leXd vcm Q+P Gtle Xdv cmQ +VF 522

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

JBTl NQ T1J UPC 9r ZXl 3b3J kPjx rZXl 3b3J kPkJ FSE FW SU9 SPC 9rZ Xl3 b3Jk Pjxr ZXl 3b3J kPl NZ U1R FTV M8 L2tl eXd v cmQ +PG tleX dvc mQ +R0 523

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

VM Uzw va2 V5d 29y ZD4 8L2t leXd vcm RzPj xkY XRl cz48 eW Vhcj 4yM DA wPC 95 ZW FyPj xwd WIt ZGF 0ZX M+ PGR hdG U+T WF 5PC 9kY XRl Pjw vcH 524

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ViL WR hdG VzP jwv ZGF 0ZX M+ PGl zYm 4+ MD Az Mi0 zOD YxP C9p c2Ju Pjxh Y2N lc3N pb2 4tbn VtP klTS Tow MD Aw OD U1N jUy MD Aw MD k8L 525

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2Fj Y2V z c2lv bi1u dW0 +PH dvc mstd Hlw ZT5 Bcn RpY 2xlP C93 b3Jr LX R5c GU +PH Vyb HM +PH JlbG F0Z WQt dXJ s cz48 dXJ sPiZ sdDt Hby B0b yBJ 526

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

U0k mZ3 Q7O i8v MD Aw MD g1N TY1 MjA wM DA5 PC9 1cm w+P C9y ZW xhd GVk LX Vyb HM +PC 91c mxz Pjxs YW 5nd WF nZT 5Fb mds aXN oPC 9sY 527

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W5n dW FnZ T48 L3Jl Y29 yZD 48L 0Np dGU + PEN pdG U+P EF1 dGh vcj5 Qb2 R1Y Ww 8L0 F1d Ghv cj48 WW Vhcj 4yM DA wPC 9ZZ WF yPjx SZ WN Od 528

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W0 +Mz Ay PC9 SZ WN Od W0 +PH JlY2 9yZ D48 cmV jLW 51b WJl cj4z MDI 8L3 JlYy 1ud W1i ZXI +PG Zvc mV pZ2 4ta2 V5 cz48 a2V 5IG Fwc D0i RU4 529

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

iIG RiL Wlk PSJ mY Tl6 ZHp zdm tzeD Jwc mV yZT k4e GQ1 MnF kZn dmd 3I5 MH A1 czIi PjM wMj wva 2V5 Pjw vZm 9yZ Wln bi1r ZXl zPjx yZ WYt dHl 530

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

wZS BuY W1l PSJ Kb3 Vyb mFs IEF ydG lj bGU iPjE 3PC 9yZ WYt dHl wZT 48Y 29u dHJ pYn V0b 3JzP jxhd XRo b3Jz Pjxh dXR ob3I +U G9k dW FsL CBL Ljw 531

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

vYX V0a G9y Pjxh dXR ob3I +RG 95b GUs IEY uIEo uPC 9hd XRo b3I+ PGF 1dG hvcj 5QZ XB wY XM sIE4 u IEE uPC 9hd XRo b3I+ PC9 hdX Rob 3JzP jwv Y29 532

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

udH JpY nV0 b3Jz Pjxh dXR oL WF kZH Jlc3 M+ UH VyZ HVl IFV uaX YsI FNj aCB DaG VtIE VuZ 24sI EJp b21 hdC Am YW 1wO yBE cnV nIE Rlb Gl2 ZXJ 533

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

5IEx hYn MsI Fcg TGF mY Xlld HRl LCB JTi A0N zkw NyB VU0 EuJi N4R DtQ ZX Bw YX MsI E5B LCB QdX Jkd WU gV W5p diw gU2 No IEN oZ W0g RW 534

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

5nbi wgQ mlv bW F0I CZh bXA 7IE Ryd Wcg RG Vsa XZl cnkg TGF icyw gVy BM YW Zhe WV 0dG Us IElO IDQ 3OT A3I FVT QS4 8L2 F1d Ggt YW Rkc mVz 535

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cz48 dGl 0bG VzP jx0a XRs ZT5 Ee W5h bWl jIGJ laG F2 aW9 yIG 9mI Gds dW Nvc 2Ug b3h pZG FzZ S1jb 250 YWl uaW 5nI G1p Y3J vcG Fyd Gljb GVz IG9 536

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

mIH Bvb Hko ZX Roe Wxl bm UgZ 2x5 Y29 sKS 1nc mF md GVk IGN hdG lvb mljI Gh5 ZHJ vZ2 Vsc yBp biBh biBl bnZ pcm 9u bW Vud CBv ZiBj aGF uZ2l 537

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

uZy BwS Dwv dGl 0bG U+P HNl Y29 uZG Fye S10 aXR sZT 5Ca W9t YX Rlc mlh bH M8 L3N lY2 9uZ GFy eS1 0aX RsZ T48 YW x0L XRp dGx lPkJ pb2 1hd 538

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

GVy aWF scz wvY Wx0 LX Rpd Gxl Pjw vdG l0 bGV zPjx wZ XJp b2R pY2 FsPj xmd Wxs LX Rpd Gxl PkJp b21 hdG Vya WFs czw vZn Vsb C10 aXR sZT 48Y 539

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

WJi ci0x PkJp b21 hdG Vya WFs czw vY WJi ci0x Pjw vcG Vya W9k aW Nhb D48 YW x0L XBl cml vZG ljY Ww +PG Z1 bGw tdGl 0bG U+ Qml vbW F0Z XJp 540

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

YW xzP C9m dWx sLX Rpd Gxl Pjxh YmJ yLT E+Q mlv bW F0Z XJp YW xzP C9h YmJ yLT E+P C9h bHQ tcG Vya W9k aW Nhb D48 cGF nZX M+ MT QzO S0x 541

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ND Uw PC9 wY Wdl cz48 dm9 sdW 1lPjI x PC9 2b2 x1b WU +PG 51b WJl cj4x ND wvb nVt Ym VyP jxrZ Xl3 b3Jk cz48 a2V 5d2 9yZ D5j YX Rpb 25p YyB 542

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

o eW Ryb 2dlb HM 8L2t leXd vcm Q+P Gtle Xdv cmQ +b Wljc m9 wY XJ0 aW NsZ XM 8L2t leXd vcm Q+P Gtle Xdv cmQ +cH Vsc 2F0 aWx lIH N3Z Wxs aW5 543

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

nPC 9rZ Xl3 b3Jk Pjxr ZXl 3b3J kPnJ lbG F4Y XRp b24 8L2t leXd v cmQ +PG tleX dvc mQ +RE VM SVZ FUl kgU 1lT VE VN Uzw va2 V5d 29y ZD4 8a2 V5d 544

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

29y ZD5 NS UN ST1 NQ SEV S RV M8 L2tl eXd vcm Q+P Gtle Xdv cmQ +Uk VM RUF TRT wva 2V5 d29 yZD 48a2 V5d 29y ZD5 OQ U5P UEF SVE lD TEV 545

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

TPC 9rZ Xl3 b3Jk Pjxr ZXl 3b3J kPk 1JQ 1JP Q0F QU1 VM RV M8 L2tl eXd vcm Q+P C9r ZXl 3b3J kcz4 8 ZGF 0ZX M+ PHll YXI +Mj Aw MD wve WV hcj4 546

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

8cH ViL WR hdG VzP jxk YX RlP kp1 bDw vZG F0Z T48 L3B 1Yi 1k YX Rlcz 48L 2Rh dGV zPjx pc2J uPj AxN DIt OT Yx Mjw vaX Nibj 48Y WNj ZX Nza 547

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W9u LW 51b T5J U0k 6M DA w MD g3M zk5 OT Aw MD A0P C9h Y2N lc3N pb2 4tbn VtPj x3b 3JrL XR5 cGU +Q XJ0 aW NsZ Twv d29 yay1 0eX Bl Pjx1 548

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cmx zPjx yZ Wxh dGV kLX Vyb HM +PH Vyb D4 mb HQ7 R28 gdG 8gS VNJ Jmd 0Oz ovL zAw MD A4N zM5 OTk w MD Aw ND wvd XJs Pjw vcm VsY XRl 549

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZC1 1cm xzPj wvd XJsc z48b GFu Z3V hZ2 U+R W5n bGlz aDw vbG FuZ 3Vh Z2U +PC 9yZ WN vcm Q+P C9D aXR lPjw vR W5k Tm9 0ZT 4A 54,5 3 Poly(AAC) grafted onto porous

Glucose

Conversion of glucose

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permeabilit

membrane of PVDF

y of

550

5 56

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

4

PEGA

Protease

Enzymatic hydrolysis

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of peptide actuators

swelling

uZE 5vd GU +PE Npd GU +PE F1d Ghv cj5U aG9 ybn Rvbj wvQ XV0 aG9 yPjx ZZ WF yPjI wM Dc8 L1ll YXI + PFJl Y05 1bT 4xN jwv Um VjT nVt

551

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Pjxy ZW Nvc mQ +PH JlYy 1ud W1i ZXI +M TY8 L3Jl Yy1 udW 1iZ XI+ PGZ v cmV pZ2 4ta2 V5c z48a 2V5 IGF wcD 0iR U4iI GRi LWl kPS JmY Tl6 ZHp zdm 552

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

tzeD Jwc mV yZT k4e GQ1 MnF k Znd md3 I5M HA1 czIi PjE2 PC9 rZX k+P C9m b3Jl aWd uL Wtle XM +PH JlZi 10e XBl IG5 hbW U9I kpv dXJ u YW wgQ 553

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

XJ0 aW NsZ SI+ MTc 8L3 JlZi 10e XBl Pjxj b25 0cm lidX Rvc nM+ PGF 1dG hvcn M+ PGF 1dG hvcj 5U aG9 ybn Rvbi wgU C4g RC4 8L2 F1d Ghv cj48 YX V0a 554

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

G9y Pk1 hcn QsI FIuI Eou PC9 hdX Rob 3I+P GF1 dGh v cj5V bGl qbi wgU i4g Vi4 8L2 F1d Ghv cj48 L2F 1dG hvcn M+ PC9 jb25 0cm lidX Rvc nM+ PGF 1dG 555

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

gtY WR k cmV zcz5 TY2 ggT WF 0LC BN YW 5jaG Vzd GVy IE0x IDdI Uyw gTG FuY 3Ms IEV uZ2 xhb mQ uIE1 JQi wgT WF u Y2h lc3R lciB NM SA3 SF 556

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

MsI Exh bm NzL CBF bmd sY W5k LiYj eEQ 7V Wxp am4 sIFJ WL CBT Y2g gT WF 0LC BH cm9 zdm Vub 3Ig U3Q sIE1 hbm NoZ XN0 ZXI gTT EgN 0hT LCB 557

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

MY W5j cyw gR W5n bGF uZC 4mI 3hE O3Jl aW4 u dWx pam 5Ab WF uY2 hlc3 Rlci 5hY y51a zwv YX V0a C1h ZG RyZ XNz Pjx0 aXR sZX M+ PHR pdG xlPk 558

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Vue nlt ZS1 yZX Nwb 25za XZlI HBv bHlt ZXI gaHl kcm 9nZ Ww gcG Fyd Gljb GVz IGZ vciB jb25 0cm 9sb GVk IHJl bGV hc2 U8L 3Rp dGx lPjx zZ WN vbm Rhc 559

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

nktd Gl0 bGU +Q WR 2Y W5j ZW QgT WF 0ZX JpY Wxz PC9 zZ WN vbm Rh cnkt dGl 0bG U+P GFs dC1 0aX RsZ T5B ZH YuI E1h dGV yLj wvY Wx0 LX 560

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Rpd Gxl Pjw vdG l0b GVz Pjx wZ XJp b2R pY2 FsPj xmd Wxs LX Rpd Gxl PkF kdm FuY 2Vk IE1h dGV yaW Fscz wvZ nVs bC1 0aX RsZ T48 YW Jici0 x PkF 561

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

kdi4 gT WF 0ZX IuP C9h YmJ yLT E+P C9w ZXJ pb2 RpY 2Fs Pjxh bHQ tcG Vya W9k aW Nhb D48 ZnV sbC 10 aXR sZT 5BZ HZh bm NlZ CB NY XRl cml 562

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

hbH M8 L2Z 1bG wtd Gl0 bGU +PG FiY nIt MT 5BZ HYu IE1h dGV yLj wv YW Jici0 xPj wvY Wx0 LX Blc mlv ZGlj YW w+P HBh Z2V zPjE yNT ItKz wvc GFn 563

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZX M+ PHZ vbH VtZ T4x OT wv dm9 sdW 1lPj xud W1i ZXI +OT wvb nVt Ym VyP jxrZ Xl3 b3Jk cz48 a2V 5d2 9yZ D4y LV BIT 1RP TiB NS UN S T1N 564

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

DT1 BZP C9r ZXl 3b3J kPjx rZXl 3b3J kPl BFU FRJ RE UtU 1lO VEh FU0 lTP C9r ZXl 3b3J kPjx rZXl 3 b3Jk PlN PTE lEIF NV UFB PUl RTP C9r ZXl 3b3J kPjx 565

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

rZXl 3b3J kPk RSV Uct RE VM SVZ FUl k8L 2tle Xdv cmQ +PG tleX dvc mQ +UF JPV EV BU0 U8L 2tle Xdv cmQ +PG tleX dvc mQ +Qk lPT UF UR VJJ QUx 566

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

TPC 9rZ Xl3 b3Jk Pjxr ZXl 3b3J kPl BST 1RF SU5 BU0 VTP C9r ZXl 3b3J kPjx rZXl 3b3J kPkl OSE lCS VRP UlM 8L2t l eXd vcm Q+P Gtle Xdv cmQ +Qk lPT E9H 567

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

WT wva 2V5 d29 yZD 48a2 V5d 29y ZD5 MS UJS QVJ JRV M8 L2tl eXd v cmQ +PC 9rZ Xl3 b3Jk cz48 ZGF 0ZX M+ PHll YXI +Mj Aw Nzw veW Vhcj 48c HVi 568

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

LW Rhd GVz Pjxk YX RlP k1h eTw vZG F0Z T48 L3B 1Yi 1kY XRl cz48 L2R hdG VzP jxpc 2JuP jA5 Mz UtO TY0 OD wva XNi bj48 YW NjZ XNz aW9 uL W51 569

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

bT5 JU0 k6M DA wMj Q2N jU4 MT Aw MD E2P C9h Y2N lc3N pb2 4tbn VtPj x3b 3JrL XR5 cGU +Q XJ0 aW NsZ Twv d29 yay1 0eX BlPj x1c mxz Pjxy ZW xhd 570

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

GVk LX Vyb HM +PH Vyb D4 mb HQ7 R28 gdG 8gS VNJ Jmd 0 Ozo vLz Aw MDI 0Nj Y1O DE wM DAx Njw vdX JsPj wvc mVs YX RlZ C11 cmx zPj wvd 571

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

XJsc z48 ZW xlY 3Ry b25 p Yy1 yZX Nvd XJj ZS1 udW 0+M TAu MT Aw Mi9 hZG 1hLj IwM DY wM Tc4 ND wvZ Wxl Y3R yb2 5pY y1y ZX Nvd XJj ZS1 572

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

udW 0+P Gxh bmd 1Y Wdl PkV uZ2 xpc2 g8L 2xh bmd 1Y Wdl Pjw vcm Vjb 3JkP jwv Q2l 0ZT 48Q 2l0Z T48 QX V0a G9y PlR ob3J udG 9uP C9B dXR ob3I +PFl 573

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

lYX I+M jAw OD wv WW Vhcj 48U mVj TnV tPjI3 NT wvU mVj TnV tPjx yZ WN vcm Q+P HJl Yy1 udW 1iZ XI+ Mjc 1PC 9yZ WM tbn VtY mV yPjx mb3 Jla 574

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Wdu LWt leX M+ PGtl eSB hcH A9I kVO IiBk Yi1 pZD 0iZ mE5 emR 6c3 Zrc3 gyc HJlc mU 5OH hkN TJx ZGZ 3Zn dyO TB wN XM yIj4 y NzU 8L2t leT4 8L2 575

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Zvc mV pZ2 4ta2 V5c z48c mV mL XR5 cGU gbm FtZ T0iS m91 cm5 hbC BBc nRp Y2x lIj4x Nzw vcm Vm LX R5c GU +PG Nvb nRy aWJ 1dG 9ycz 48Y XV0 aG9 576

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ycz4 8YX V0a G9y PlR ob3J udG 9uL CB QLi BE Ljw vYX V0a G9y Pjxh dXR ob3I +T WF ydC wgU i4gS i48L 2F1 dGh vcj4 8YX V0a G9y Pldl YmI sIF MuI Eou 577

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PC9 hdX Rob 3I+P GF1 dGh vcj5 VbG lqbi wgU i4g Vi4 8L2 F1d Ghv cj48 L2F 1dG hvcn M+ PC9 jb25 0cm li dXR vcn M+ PGF 1dG gtY WR kcm Vzc z5V bml 578

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2IE1 hbm NoZ XN0 ZXI sIF Nja CB NY XQs IE1h bm NoZ XN0 ZXI g TTE gN0 RO LCB MY W5j cyw gR W5n bGF uZC 4gV W5p diB NY W5j aGV zdG VyL 579

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

CB NS UIsI E1h bm NoZ XN0 ZXI g TTE gN0 RO LCB MY W5j cyw gR W5n bGF uZC 4mI 3hE O1R ob3J udG 9uL CB QR Cwg VW 5pdi BN YW 5jaG Vzd 580

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

GVy LCB TY2 ggT WF 0LC BN YW 5jaG Vzd GVy IE0x IDd ETi wgT GFu Y3 MsI EVu Z2x hbm QuJi N4R Dty ZWl uLn Vs aWp uQG 1hb mN oZX N0Z XIu YW 581

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Mud Ws8 L2F 1dG gtY WR kcm Vzc z48d Gl0 bGV zPjx 0aX RsZ T5F bnp 5bW Ut cmV zcG 9uc2 l2ZS Boe WR yb2 dlbC Bw YXJ 0aW NsZ XM gZm 9yI HRo ZSB 582

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

jb25 0cm 9sb GVk IHJl bGV hc2 Ug b2Y gcH Jvd GVp bnM 6IG Rlc2 lnb mlu ZyB wZ XB0 aW RlI GFj dHV hdG 9ycy B0b yBt YX Rja CB wY Xlsb 2Fk PC9 583

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

0aX RsZ T48 c2Vj b25 kYX J5L XRp dGx lPlN vZn QgT WF 0dG VyP C9z ZW Nvb mRh cnkt dGl 0bG U+P GFs dC1 0aX RsZ T5T b2Z 0IE1 hdH Rlcj wvY Wx0 LX 584

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Rpd Gxl Pjw vdG l0b GVz Pjx wZ XJp b2R pY2 FsPj xmd Wxs LX Rpd Gxl PlN vZn QgT WF 0dG VyP C9m dWx sLX Rpd Gxl Pjxh YmJ yLT E+U 29m dCB NY 585

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

XR0 ZXI 8L2 FiY nIt MT 48L 3Blc mlv ZGlj YW w+P GFs dC1 wZ XJp b2R pY2 FsPj xmd Wxs LX Rpd Gxl PlN vZn QgT WF 0dG VyP C9m dWx sLX Rpd Gxl 586

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Pjxh YmJ yLT E+U 29m dCB NY XR0 ZXI 8L2 FiY nIt MT 48L 2Fsd C1w ZXJ pb2 RpY 2Fs Pjx w YW dlcz 44M jEtO DI3 PC9 wY Wdl cz48 dm9 sdW 1lPj Q8L 587

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3Zv bHV tZT 48b nVt Ym VyP jQ8 L25 1bW Jlcj4 8 a2V 5d2 9yZ HM +PG tleX dvc mQ +UE 9M WU 1FU iBU SEV SQ VBF VV RJQ 1M8 L2tl eXd vcm Q+P 588

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Gtle Xdv cmQ + U01 BUl QgQ klPT UF UR VJJ QUx TPC 9rZ Xl3 b3Jk Pjxr ZXl 3b3J kPk RSV Uct RE VM SVZ FUl k8L 2tle Xdv cmQ +PG tleX dvc mQ +TU 589

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

lDU k9T Q09 QW Twv a2V 5d2 9yZ D48 a2V 5d2 9yZ D5D T1B PTF lNR VI8 L2tl eXd v cmQ +PG tleX dvc mQ +TE lCU kFS SU VTP C9r ZXl 3b3J kPjx rZXl 590

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3b3J kPl NV UFB PUl RTP C9r ZXl 3b3J k Pjxr ZXl 3b3J kPl BST 0RS VUd TPC 9rZ Xl3 b3Jk Pjxr ZXl 3b3J kPk NB UlJJ RVJ TPC 9rZ Xl3 b3Jk Pjxr ZXl 3b3J 591

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

kPl ND SU VO Q0U 8L2t leXd vcm Q+P C9r ZXl 3b3J kcz4 8ZG F0Z XM +PH llY XI+ MjA wO Dwv eW Vh cj48 L2R hdG VzP jxpc 2JuP jE3 ND QtN jgz WD 592

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

wva XNi bj48 YW NjZ XNz aW9 uL W51 bT5 JU0 k6M DA wMj U0 Mzg 2OD Aw MDI zPC 9hY 2Nlc 3Np b24t bnV tPjx 3b3J rLX R5c GU +Q XJ0 aW NsZ Twv 593

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

d29 yay1 0eX BlPj x1 cmx zPjx yZ Wxh dGV kLX Vyb HM +PH Vyb D4 mb HQ7 R28 gdG 8gS VNJ Jmd 0Oz ovL zAw MDI 1ND M4 Njg wM DAy Mz wvd XJs 594

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Pjw vcm VsY XRl ZC1 1cm xzPj wvd XJsc z48 ZW xlY 3Ry b25 pYy 1yZ XNv dXJj ZS1 udW 0+M TAu MT AzO S9i NzE 0Nz Uw Yzw vZ Wxl Y3R yb2 5pY y1y 595

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZX Nvd XJj ZS1 udW 0+P Gxh bmd 1Y Wdl PkV uZ2 xpc2 g8 L2x hbm d1Y Wdl Pjw vcm Vjb 3JkP jwv Q2l 0ZT 48L 0Vu ZE5 vdG U+ PEV uZE 5vd GU +PE 596

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Npd GU +PE F1d Ghv cj5U aG9 ybn Rvbj wvQ XV0 aG9 yPjx ZZ WF yPjI wM Dc8 L1ll YXI + PFJl Y05 1bT 4xN jwv Um VjT nVt Pjxy ZW Nvc mQ +PH JlYy 597

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1ud W1i ZXI +M TY8 L3Jl Yy1 udW 1iZ XI+ PGZ v cmV pZ2 4ta2 V5c z48a 2V5 IGF wcD 0iR U4iI GRi LWl kPS JmY Tl6 ZHp zdm tzeD Jwc mV yZT k4e GQ1 598

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

MnF k Znd md3 I5M HA1 czIi PjE2 PC9 rZX k+P C9m b3Jl aWd uL Wtle XM +PH JlZi 10e XBl IG5 hbW U9I kpv dXJ u YW wgQ XJ0 aW NsZ SI+ MTc 8L3 599

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

JlZi 10e XBl Pjxj b25 0cm lidX Rvc nM+ PGF 1dG hvcn M+ PGF 1dG hvcj 5U aG9 ybn Rvbi wgU C4g RC4 8L2 F1d Ghv cj48 YX V0a G9y Pk1 hcn QsI FIuI Eou 600

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PC9 hdX Rob 3I+P GF1 dGh v cj5V bGl qbi wgU i4g Vi4 8L2 F1d Ghv cj48 L2F 1dG hvcn M+ PC9 jb25 0cm lidX Rvc nM+ PGF 1dG gtY WR k cmV zcz5 TY2 601

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ggT WF 0LC BN YW 5jaG Vzd GVy IE0x IDdI Uyw gTG FuY 3Ms IEV uZ2 xhb mQ uIE1 JQi wgT WF u Y2h lc3R lciB NM SA3 SF MsI Exh bm NzL CBF bmd 602

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

sY W5k LiYj eEQ 7V Wxp am4 sIFJ WL CBT Y2g gT WF 0LC BH cm9 zdm Vub 3Ig U3Q sIE1 hbm NoZ XN0 ZXI gTT EgN 0hT LCB MY W5j cyw gR W5n bGF 603

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

uZC 4mI 3hE O3Jl aW4 u dWx pam 5Ab WF uY2 hlc3 Rlci 5hY y51a zwv YX V0a C1h ZG RyZ XNz Pjx0 aXR sZX M+ PHR pdG xlPk Vue nlt ZS1 yZX Nwb 25za 604

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

XZlI HBv bHlt ZXI gaHl kcm 9nZ Ww gcG Fyd Gljb GVz IGZ vciB jb25 0cm 9sb GVk IHJl bGV hc2 U8L 3Rp dGx lPjx zZ WN vbm Rhc nktd Gl0 bGU +Q WR 2Y 605

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W5j ZW QgT WF 0ZX JpY Wxz PC9 zZ WN vbm Rh cnkt dGl 0bG U+P GFs dC1 0aX RsZ T5B ZH YuI E1h dGV yLj wvY Wx0 LX Rpd Gxl Pjw vdG l0b GVz 606

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Pjx wZ XJp b2R pY2 FsPj xmd Wxs LX Rpd Gxl PkF kdm FuY 2Vk IE1h dGV yaW Fscz wvZ nVs bC1 0aX RsZ T48 YW Jici0 x PkF kdi4 gT WF 0ZX IuP C9h 607

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

YmJ yLT E+P C9w ZXJ pb2 RpY 2Fs Pjxh bHQ tcG Vya W9k aW Nhb D48 ZnV sbC 10 aXR sZT 5BZ HZh bm NlZ CB NY XRl cml hbH M8 L2Z 1bG wtd Gl0 608

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

bGU +PG FiY nIt MT 5BZ HYu IE1h dGV yLj wv YW Jici0 xPj wvY Wx0 LX Blc mlv ZGlj YW w+P HBh Z2V zPjE yNT ItKz wvc GFn ZX M+ PHZ vbH VtZ T4x 609

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

OT wv dm9 sdW 1lPj xud W1i ZXI +OT wvb nVt Ym VyP jxrZ Xl3 b3Jk cz48 a2V 5d2 9yZ D4y LV BIT 1RP TiB NS UN S T1N DT1 BZP C9r ZXl 3b3J kPjx 610

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

rZXl 3b3J kPl BFU FRJ RE UtU 1lO VEh FU0 lTP C9r ZXl 3b3J kPjx rZXl 3 b3Jk PlN PTE lEIF NV UFB PUl RTP C9r ZXl 3b3J kPjx rZXl 3b3J kPk RSV Uct RE 611

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

VM SVZ FUl k8L 2tle Xdv cmQ +PG tleX dvc mQ +UF JPV EV BU0 U8L 2tle Xdv cmQ +PG tleX dvc mQ +Qk lPT UF UR VJJ QUx TPC 9rZ Xl3 b3Jk Pjxr ZXl 612

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3b3J kPl BST 1RF SU5 BU0 VTP C9r ZXl 3b3J kPjx rZXl 3b3J kPkl OSE lCS VRP UlM 8L2t l eXd vcm Q+P Gtle Xdv cmQ +Qk lPT E9H WT wva 2V5 d29 yZD 48a2 613

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

V5d 29y ZD5 MS UJS QVJ JRV M8 L2tl eXd v cmQ +PC 9rZ Xl3 b3Jk cz48 ZGF 0ZX M+ PHll YXI +Mj Aw Nzw veW Vhcj 48c HVi LW Rhd GVz Pjxk YX RlP 614

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

k1h eTw vZG F0Z T48 L3B 1Yi 1kY XRl cz48 L2R hdG VzP jxpc 2JuP jA5 Mz UtO TY0 OD wva XNi bj48 YW NjZ XNz aW9 uL W51 bT5 JU0 k6M DA wMj Q2N 615

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

jU4 MT Aw MD E2P C9h Y2N lc3N pb2 4tbn VtPj x3b 3JrL XR5 cGU +Q XJ0 aW NsZ Twv d29 yay1 0eX BlPj x1c mxz Pjxy ZW xhd GVk LX Vyb HM +PH Vyb 616

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

D4 mb HQ7 R28 gdG 8gS VNJ Jmd 0 Ozo vLz Aw MDI 0Nj Y1O DE wM DAx Njw vdX JsPj wvc mVs YX RlZ C11 cmx zPj wvd XJsc z48 ZW xlY 3Ry b25 617

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

p Yy1 yZX Nvd XJj ZS1 udW 0+M TAu MT Aw Mi9 hZG 1hLj IwM DY wM Tc4 ND wvZ Wxl Y3R yb2 5pY y1y ZX Nvd XJj ZS1 udW 0+P Gxh bmd 1Y Wdl 618

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PkV uZ2 xpc2 g8L 2xh bmd 1Y Wdl Pjw vcm Vjb 3JkP jwv Q2l 0ZT 48Q 2l0Z T48 QX V0a G9y PlR ob3J udG 9uP C9B dXR ob3I +PFl lYX I+M jAw OD wv WW 619

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Vhcj 48U mVj TnV tPjI3 NT wvU mVj TnV tPjx yZ WN vcm Q+P HJl Yy1 udW 1iZ XI+ Mjc 1PC 9yZ WM tbn VtY mV yPjx mb3 Jla Wdu LWt leX M+ PGtl eSB 620

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

hcH A9I kVO IiBk Yi1 pZD 0iZ mE5 emR 6c3 Zrc3 gyc HJlc mU 5OH hkN TJx ZGZ 3Zn dyO TB wN XM yIj4 y NzU 8L2t leT4 8L2 Zvc mV pZ2 4ta2 V5c z48c 621

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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643

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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653

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

FjY2Vzc2lvbi1udW0+ SVNJOjAwMDIyMTg5NzAwMDAwNDwvYWNjZXNzaW9uLW51bT48d29yay1 0eXBlPkFydGljbGU8L3dv cmstdHlwZT48dXJscz48cmVsYXRlZC11cmxzPjx1cmw+Jmx0O0dvIHRvIElTSSZ ndDs6Ly8wMDAy MjE4OTcwMDAwMDQ8L3VybD48L3JlbGF0ZWQtdXJscz48L3VybHM+PGVsZ WN0cm9uaWMtcmVzb3Vy Y2UtbnVtPjEwLjEwMTYvai5qY29ucmVsLjIwMDQuMDIuMDI2PC9lbGVjdHJv bmljLXJlc291cmNl LW51bT48bGFuZ3VhZ2U+RW5nbGlzaDwvbGFuZ3VhZ2U+PC9yZWNvcmQ+P C9DaXRlPjwvRW5kTm90 ZT5= 36,54-56,59-61 Generally glucose oxidase (GOx) is immobilised in the hydrogel, when glucose is present it is converted to gluconic acid, which lowers the pH within the microenvironment of the hydrogel. There are two different macroscopic designs in which this decrease in pH is used to actuate a change in swelling: matrix type systems where the enzyme and insulin are contained within a bulk polymer, or membrane type systems where the drug is contained in a reservoir within a membrane. Kost and co-workers produced an example of the matrix system where the insulin and enzyme were contained uniformly throughout a hydrogel. The hydrogel was made from 2-hydroxyethyl methacrylate (HEMA), N,Ndimethylaminoethyl

methacrylate

(DMAEMA)

with

tetraethylene

glycol

dimethacrylate (TEGDMA) as a crosslinking agent. The presence of amines and the low crosslinker concentration (between 0-0.95 %) meant that at low pH the amines become ionised leading to an increase in swelling (schematic shown in Figure 2–7).

Figure 2–6. The enzymes and the reactions they catalyse used in glucose responsive hydrogels.

GOx, catalase and insulin were incorporated into the hydrogel during the polymerisation step (free-radical initiation at room temperature). Figure 2–6 shows the reactions driven by these enzymes, GOx catalyses the reaction of glucose to gluconic acid forming hydrogen peroxide. A build up of hydrogen peroxide leads to inhibition of the enzyme, and because oxygen is needed to form gluconic acid a shortage of oxygen leads to slower swelling rates. For this reason catalase was also 666

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

incorporated into this system which serves to convert hydrogen peroxide to water and oxygen. The effect of crosslinker concentration on the responsive swelling behaviour was investigated and it was found that polymers prepared without crosslinker led to the greatest increase in swelling. These polymers did not dissolve in water even over prolonged periods, Kost and co-workers suggest that this stability may be due to entanglements and non-covalent interactions between the polymer chains. Due to the dynamic nature of conditions in vivo non-steady state experiments investigating swelling changes in response to glucose were examined. The glucose concentration was switched between a hyperglycaemic blood glucose concentration and a normal blood glucose concentration, in these experiments they found that deswelling occurred more rapidly than a further increase in swelling. Analysis of glucose triggered release of insulin in matrices with varying crosslinker concentration also showed that the shortest response time and the greatest amount of insulin was released in matrices without crosslinking agent. The device was implanted in rats and these in vivo experiments indicated that some of the entrapped insulin retained its active form and was effective in reducing blood glucose levels. Additionally, over the 2-3 weeks the matrices were implanted no fibrotic encapsulation was observed demonstrating the biocompatibility of the devices. 36

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 2–7. Characterization of glucose-sensitive insulin release systems in simulated in vivo conditions. Schematic presentation of a matrix system based on poly(HEMA-co-DMAEMA). (top) unswollen matrix at time t=0, (bottom) swollen matrix.

Peppas and co-workers have previously developed a similar system consisting a copolymer of diethylaminoethyl methacrylate (DEAEMA) and poly(ethylene glycol) monomethacylate (PEGMA) using tetra(ethylene glycol) dimethacryalte (TEGMA) as the crosslinker. GOx and catalase were given vinyl functionality by reacting with acryloyl chloride and mixed with the monomer solution prior to UV initiated polymerisation to give hydrogel films. With this system they demonstrated pulsatile pH-responsive swelling, but did not show glucose-responsive behaviour.54 Within their following publication microparticles with the same chemistry were produced by inverse suspension polymerisation with redox-initiators. These microparticles demonstrated rapid swelling/deswelling dynamics in response to changes in pH and it was determined that faster responses could be obtained from smaller particles.55

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

A membrane type system for insulin release was developed by Liang and coworkers. Here, poly(acrylic acid) (PAAc) was grafted to a porous membrane of polyvinylidene fluoride (PVDF), GOx was covalently bound to the PAAc by firstly activating the carboxyl groups with a water soluble carbodiimide then immersing the membrane in a aqueous solution of GOx. The PAAc with GOx covalently immobilised effectively ‘gate’ the pores of the PVDF. At neutral pH and when there is no glucose in the surrounding environment the pores within the membrane are ‘closed’. When the carboxyl groups present in the PAAc chains are dissociated and negatively charged, these charges along the polymer chains electrostatically repel one another forcing the chain to lengthen and extend. When glucose was present it was oxidised into gluconic acid by the immobilised GOx, this led to a reduction of pH in the microenvironment of the pores protonating the carboxylate groups on the grafted PAAc chains. The gates then ‘opened’ because the PAAc chains were collapsed on the removal of the electrostatic repulsion allowing the insulin to diffuse out of the membrane. The grafting density of PAAc was varied to find the ideal value for insulin release, at low values it was found that the PAAc chains were too short/sparse to effectively close the pores. At higher densities the PAAc became too long/dense and it was no longer possible for a conformation change to occur. Using a grafting yield of 1.55% the insulin permeation coefficient after glucose addition was 9.37 times greater than without glucose.56 Recently there have been fewer publications in which GOx has been utilised to actuate insulin release (50 % decrease from 2003), it appears that this method has reached it limits and recent approaches by researchers are based on effective oral delivery of insulin rather than the development of glucose responsive polymers. An obvious limitation within GOx hydrogel systems is that the treatment of diabetes is a continuous long term process; any implanted devices must be able to contain large quantities of insulin for release over this time frame in addition to maintaining their dynamic response.

2.3.3.2. Electrostatic interactions between charges present in pendant actuators The design of actuators in which the molecular actuation involves a change in the net charge offers the potential to develop more intricate responsive systems. Current approaches utilise enzyme cleavable peptides as the sensing element. Thornton et al

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

described the development of a functionalised hydrogel that alters its accessibility in response

to

an

enzyme

with

a

selected

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZCBNYXRlcmlhbHM8L3NlY29uZGFyeS10aXRsZT48YWx0LXRpdGxlPkFkdi4g TWF0ZXIuPC9hbHQt dGl0bGU+PC90aXRsZXM+PHBlcmlvZGljYWw+PGZ1bGwtdGl0bGU+QWR2Y W5jZWQgTWF0ZXJpYWxz PC9mdWxsLXRpdGxlPjxhYmJyLTE+QWR2LiBNYXRlci48L2FiYnItMT48L3Blc mlvZGljYWw+PGFs dC1wZXJpb2RpY2FsPjxmdWxsLXRpdGxlPkFkdmFuY2VkIE1hdGVyaWFsczwv ZnVsbC10aXRsZT48 YWJici0xPkFkdi4gTWF0ZXIuPC9hYmJyLTE+PC9hbHQtcGVyaW9kaWNhbD48 cGFnZXM+MTI1Mi0r PC9wYWdlcz48dm9sdW1lPjE5PC92b2x1bWU+PG51bWJlcj45PC9udW1iZXI+P GtleXdvcmRzPjxr ZXl3b3JkPjItUEhPVE9OIE1JQ1JPU0NPUFk8L2tleXdvcmQ+PGtleXdvcmQ+UE VQVElERS1TWU5U SEVTSVM8L2tleXdvcmQ+PGtleXdvcmQ+U09MSUQgU1VQUE9SVFM8L2tleX dvcmQ+PGtleXdvcmQ+ RFJVRy1ERUxJVkVSWTwva2V5d29yZD48a2V5d29yZD5QUk9URUFTRTwva2 V5d29yZD48a2V5d29y ZD5CSU9NQVRFUklBTFM8L2tleXdvcmQ+PGtleXdvcmQ+UFJPVEVJTkFTRV M8L2tleXdvcmQ+PGtl eXdvcmQ+SU5ISUJJVE9SUzwva2V5d29yZD48a2V5d29yZD5CSU9MT0dZPC9r ZXl3b3JkPjxrZXl3 b3JkPkxJQlJBUklFUzwva2V5d29yZD48L2tleXdvcmRzPjxkYXRlcz48eWVhcj4y MDA3PC95ZWFy PjxwdWItZGF0ZXM+PGRhdGU+TWF5PC9kYXRlPjwvcHViLWRhdGVzPjwvZ GF0ZXM+PGlzYm4+MDkz NS05NjQ4PC9pc2JuPjxhY2Nlc3Npb24tbnVtPklTSTowMDAyNDY2NTgxMDAw MTY8L2FjY2Vzc2lv bi1udW0+PHdvcmstdHlwZT5BcnRpY2xlPC93b3JrLXR5cGU+PHVybHM+PHJlb GF0ZWQtdXJscz48 dXJsPiZsdDtHbyB0byBJU0kmZ3Q7Oi8vMDAwMjQ2NjU4MTAwMDE2PC91cm w+PC9yZWxhdGVkLXVy bHM+PC91cmxzPjxlbGVjdHJvbmljLXJlc291cmNlLW51bT4xMC4xMDAyL2Fkb WEuMjAwNjAxNzg0 PC9lbGVjdHJvbmljLXJlc291cmNlLW51bT48bGFuZ3VhZ2U+RW5nbGlzaDwvb 676

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

MT5Tb2Z0IE1hdHRlcjwvYWJici0xPjwvcGVyaW9kaWNhbD48YWx0LXBlcmlv ZGljYWw+PGZ1bGwt dGl0bGU+U29mdCBNYXR0ZXI8L2Z1bGwtdGl0bGU+PGFiYnItMT5Tb2Z0IE1h dHRlcjwvYWJici0x PjwvYWx0LXBlcmlvZGljYWw+PHBhZ2VzPjgyMS04Mjc8L3BhZ2VzPjx2b2x1b WU+NDwvdm9sdW1l PjxudW1iZXI+NDwvbnVtYmVyPjxrZXl3b3Jkcz48a2V5d29yZD5QT0xZTUVSIF RIRVJBUEVVVElD Uzwva2V5d29yZD48a2V5d29yZD5TTUFSVCBCSU9NQVRFUklBTFM8L2tleXd vcmQ+PGtleXdvcmQ+ RFJVRy1ERUxJVkVSWTwva2V5d29yZD48a2V5d29yZD5NSUNST1NDT1BZP C9rZXl3b3JkPjxrZXl3 b3JkPkNPUE9MWU1FUjwva2V5d29yZD48a2V5d29yZD5MSUJSQVJJRVM8L2t leXdvcmQ+PGtleXdv cmQ+U1VQUE9SVFM8L2tleXdvcmQ+PGtleXdvcmQ+UFJPRFJVR1M8L2tleXd vcmQ+PGtleXdvcmQ+ Q0FSUklFUlM8L2tleXdvcmQ+PGtleXdvcmQ+U0NJRU5DRTwva2V5d29yZD48 L2tleXdvcmRzPjxk YXRlcz48eWVhcj4yMDA4PC95ZWFyPjwvZGF0ZXM+PGlzYm4+MTc0NC02O DNYPC9pc2JuPjxhY2Nl c3Npb24tbnVtPklTSTowMDAyNTQzODY4MDAwMjM8L2FjY2Vzc2lvbi1udW0 +PHdvcmstdHlwZT5B cnRpY2xlPC93b3JrLXR5cGU+PHVybHM+PHJlbGF0ZWQtdXJscz48dXJsPiZsdD tHbyB0byBJU0km Z3Q7Oi8vMDAwMjU0Mzg2ODAwMDIzPC91cmw+PC9yZWxhdGVkLXVybHM +PC91cmxzPjxlbGVjdHJv bmljLXJlc291cmNlLW51bT4xMC4xMDM5L2I3MTQ3NTBjPC9lbGVjdHJvbmljL XJlc291cmNlLW51 bT48bGFuZ3VhZ2U+RW5nbGlzaDwvbGFuZ3VhZ2U+PC9yZWNvcmQ+PC9Da XRlPjwvRW5kTm90ZT5= 57,58,62 This was achieved through modification of poly(ethylene glycol)-co-acrylamide (PEGA) particles by solid phase peptide synthesis with peptide actuators. Earlier work highlighted tri-peptides consisting of an enzyme cleavable (di)peptide (ECP) and the charged amino acid arginine. The presence of these charged groups within the polymer led to electrostatically induced swelling. Upon cleavage of the ECP by an enzyme with matching specificity these 678

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cationic groups were removed resulting in a decrease in swelling. The highly hydrated and crosslinked structure of PEGA makes the interior of the polymer network accessible to macromolecules. The maximum size (molecular weight) of macromolecule that can diffuse into PEGA is well defined and termed the molecular cut-off weight. Above this molecular weight molecules are unable to diffuse into the interior

of

the

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

a2V5d29yZD5MSU5LRVI8L2tleXdvcmQ+PGtleXdvcmQ+U1BFQ0lGSUNJVFk8 L2tleXdvcmQ+PGtl eXdvcmQ+Q0hFTUlTVFJZPC9rZXl3b3JkPjxrZXl3b3JkPkJFQURTPC9rZXl3b3Jk PjxrZXl3b3Jk PkdFTkVSQVRJT048L2tleXdvcmQ+PGtleXdvcmQ+UkVTSU48L2tleXdvcmQ+P C9rZXl3b3Jkcz48 ZGF0ZXM+PHllYXI+MjAwMjwveWVhcj48cHViLWRhdGVzPjxkYXRlPkF1Zz wvZGF0ZT48L3B1Yi1k YXRlcz48L2RhdGVzPjxpc2JuPjA5NDctNjUzOTwvaXNibj48YWNjZXNzaW9uL W51bT5JU0k6MDAw MTc3NjUyODAwMDIxPC9hY2Nlc3Npb24tbnVtPjx3b3JrLXR5cGU+QXJ0aWNs ZTwvd29yay10eXBl Pjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD4mbHQ7R28gdG8gSVNJJmd0Oz ovLzAwMDE3NzY1Mjgw MDAyMTwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48bGFuZ3VhZ2U+RW5 nbGlzaDwvbGFuZ3Vh Z2U+PC9yZWNvcmQ+PC9DaXRlPjwvRW5kTm90ZT5= 63 An increase in swelling caused a corresponding increase in this cut-off weight. Through the use of different molecular weight Fluorescein isothiocyanate (FITC) labelled dextrans it was possible to monitor the changes in polymer accessibility using two-photon microscopy (this technique is described in section Two-photon microscopy). If a dextran with a molecular weight greater than the cut-off for the polymer could diffuse into the interior of the particles it indicated there was an increase in accessibility (due to charge induced swelling). It was found that enzymes with the correct specificity for the ECP (thus removing the charged groups) led to a decrease in accessibility and diameter of the particles.62 Thornton et al went on to develop this system of hydrogel particles functionalised with peptides for the controlled release of entrapped payloads. Here, the peptide actuator consisted of two opposing charged amino acids separated by an uncharged ECP, corresponding to the sequence Fmoc-D-(ECP)-R-PEGA (Figure 2–8 A). This zwitterionic peptide had a net neutral charge but upon enzyme cleavage of the ECP only the cationic amino acid arginyl and half the ECP remained covalently attached to the hydrogel. With pKa of the amino group being 7.5, at pH values below this the magnitude of response was greater due to the ionisation of amine group. This enzyme responsive increase in swelling (a maximum increase in volume of 100 %) corresponded to increase in accessibility which was used to release an entrapped payload (Figure 2–8 B). 683

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Approximately 50 % of the payload being released in 30 minutes in response to an enzyme with the correct specificity.57 This type of release system has a number of advantages over other release methods: the payload molecule does not need to be covalently modified, enzymatic cleavage of the ECP results in a tunable number of payload molecules being released and the payload loading method is relatively mild (a pH switch). In a recent publication58 Thornton et al modified the system to release proteins with different charges at physiological pH (avidin pI= 10.0 and albumin pI= 4.7). This was achieved by tailoring the design of the peptide actuator to give a net charge after ECP cleavage that was matched to the charge on the protein. It was possible to release the positivity charged avidin with the conventional peptide actuator (Fmoc-D-(ECP)-R-PEGA) due to the electrostatic repulsion between the actuators and the protein as observed by two-photon microscopy (Figure 2–9). In order to release albumin a new actuator was designed (Fmoc-R-R(ECP)-D-D-PEGA), again, this actuator had a net overall neutral charge but upon enzyme specific hydrolysis of the ECP a net negative charge remained coupled to the polymer (two negative carboxyl groups on the aspartic acid one positive amine group). This allowed for release of the payload (albumin) due to electrostatic repulsion. Analysis of the proteins released from the particles showed that although some proteolysis did occur it was not a major concern. Another limitation of this approach was the release of Fmoc-peptide fragments upon enzymatic hydrolysis.

684

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald Figure 2–8. Enzyme-responsive hydrogel particles for the controlled release of proteins: Designing peptide actuators to match payload. A: The peptide designed for the release of positively charged proteins was comprised of Fmoc–D–AA–R, where the amide bond between the two alanine residues is particularly liable to cleavage by our target enzyme. B: Generation of positive charges by enzymatic cleavage of the bond between alanine residues allows protein molecules to diffuse through the polymer pores for payload release.58

However, the main limitation of this concept was its inability to respond at physiological ionic strength. When, counter-ions present in the solution screened the electrostatic interactions between the peptide actuators, this led to an insignificant release rate.58

Figure 2–9. Peptide actuator designed for the release of negatively charged protein molecules. A: Two N-terminal arginine units are separated from two aspartic acid groups by two alanine residues. A net negative charge remains on the particle following enzymatic hydrolysis. B: Exclusion of albumin from the negatively charged swollen particle occurs following hydrolysis of the bond between alanine residues.58

2.3.4. Actuation based on conformational changes The third mechanism of actuation is based on changes in conformation of natural proteins. These systems incorporate a natural protein into the hydrogel that undergoes a conformation change and thus alters the characteristics of the material.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

The use of proteins as actuators is a new development in bioresponsive hydrogels.64 An example of this system was developed by Mrksich and co-workers 65. In that study the functional nature of the hydrogel was conferred to the system through the introduction of the protein calmodulin (CaM). CaM is a 16.5-kDa protein with two distinct conformational states. In the presence of calcium ions, CaM has an extended, dumbbell-shaped conformation where the distance between the ends of the protein is approximately 50 Å (extended CaM). This calcium-bound extended CaM undergoes a transition from an extended dumbbell to a collapsed conformation (collapsed CaM) upon the binding of ligands (with a distance between the protein ends of approximately 15 Å). An engineered version of the CaM protein was prepared in which the tyrosine residues at the ends of protein are replaced with cysteine residues. This modification meant it was possible to selectively react the acrylate end groups on the four-armed PEG molecules to the protein through a Michael-type addition. This formed a water soluble conjugate. The success of this reaction was shown by MALDI-TOF MS. A hydrogel was then formed by mixing the conjugate with dithiothreitol (DTT) at room temperature crosslinking the remaining acrylate groups to give a solid hydrogel. The hydrogel showed a macroscopic decrease in volume when exposed to the trifluoperazine ligand (TFP). TFP binds specifically to CaM, causing the CaM to undergo a conformation change from extended to collapsed (Figure 2–10 A & B). This decrease in volume could be reversed by chelating the calcium ions thus removing the calcium bound ligand. Numerous cycles between and extended and collapsed material were possible demonstrating the reversible nature of the hydrogel (Figure 2–10 C). More recently this concept has been developed to incorporate a photochemical assembly allowing spatial control of the location of the dynamic proteins.66

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 2–10. Ligand responsive hydrogel that relies on conformational changes. A: The two conformational states of CaM, an extended conformation in the presence of calcium ions (left), and a collapsed conformation upon binding to a ligand (right). B: A hydrogel with CaM in a ligand-free state (left) and the same gel with CaM in a ligand-bound state (right) (scale bars: 1 mm). C: Hydrogels were exposed to TFP ligand, and the volume was measured at various intervals for 2 h. The gel was then washed repeatedly and incubated in a calcium-containing buffer to restore the extended CaM conformation. 65

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2.3.5. Summary Bioresponsive hydrogels are a relatively new area of research but there have already been a range of different successful approaches to the design of these materials. These systems present methods of: detecting biological compounds, controlling cell modelling of scaffolds and the targeted/controlled release of active agents for disease specific treatment. However, there are two main obstacles that need to be overcome for many of these systems to provide actual medical usage: Few of these systems offer true reversible responses, rather than one thermodynamically favoured direction. Secondly, the high degrees of complexity within some of these materials makes acquiring approval to use within the body difficult as it is essential to understand what happens to all compounds once within the body.40 That said, the developments within this field help outline the design rules for future researchers to continue to progress and refine the concepts. In the future, research built upon this groundwork should help to provide more effective treatments within the medical field.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2.4. PEGA 2.4.1. The history of PEGA In 1992, Meldal developed PEGA, a copolymer of poly(ethylene glycol) (PEG) and acrylamide (Figure 2–11).67 This material was a highly polar solid support for solid phase peptide synthesis particular continuous flow peptide synthesis. PEGA offered a number advantageous properties: It was transparent with no absorbance in the aromatic region allowing for easy spectrophotometric monitoring of the reaction process. It had a highly branched polymer network with swelling in both organic and polar solvents. Finally, the resin was highly polar assisting peptide solvation.67 This section covers developments within the field of PEGA detailing methods and techniques from the literature that have arisen since the first publication of the material.

Figure 2–11. Chemical structure of PEGA.

PEGA provided an important new property to the field of solid phase peptide synthesis (SPPS)68 in its compatibility with both organic and aqueous solvents. This property allowed peptides to be incorporated onto the polymer which could then be placed in an aqueous environment. The open structure of the polymer allowed

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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cmQ+PC9DaXRlPjwvRW5kTm90ZT5=

69,70

This

development inspired a number of publications in which techniques used in SPPS such as split and mix synthesis were exploited to prepare libraries of peptides. These peptide libraries were then exposed to selected enzymes and their effect determined. An early example of this approach was demonstrated by Meldal and co-workers. Here, they prepared a ‘one bead, two compounds’ (Figure 2–12) library to screen for inhibitors for proteolytic enzymes that are essential for parasite development. Two different peptides were incorporated into individual particles (referred to as beads 697

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

within SPPS literature) by firstly temporarily protecting a fraction of the amine groups in the PEGA particles with hydroxymethylbenzoic acid, while using the remaining amines to synthesise the fluorescence quenched peptide substrate (FRET). These substrates allow for the detection of enzyme action; upon hydrolysis of the substrate the fluorescence quencher is cleaved resulting in a fluorescent molecule. The hydroxyl function on the PEGA particles was then esterificated and a second peptide (to screen for inhibition) prepared by split synthesis from the ester bond to the hydroxymethyl benzamide. These particles were then exposed to the enzyme solution (cruzipain) and hydrolysis of the fluorescence quenched peptide substrate resulted in highly fluorescent particles. The fluorescence was only seen on the surface of the particles indicating that the 57 kDa enzyme was not able to diffuse inside into the polymer network. The darkest particles were manually collected and the peptides sequenced by Edman degradation-gas phase sequencing. This method yielded

a

first

generation

of

effective

cruzipain

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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699

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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702

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 2–12. Inhibition of cruzipain visualized in a fluorescence quenched solid-phase inhibitor library assay. D-amino acid inhibitors for cruzipain, cathepsin B and cathepsin L. The strategy used

in

the

synthesis

of

the

‘one

bead,

two

compounds’

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald ENhcmxz YmVyZyBWZWogMTAsIERLLTI1MDAgQ29wZW5oYWdlbiwgRGVubWFyay48L2F1dGgt YWRkcmVzcz48 dGl0bGVzPjx0aXRsZT5JbmhpYml0aW9uIG9mIGNydXppcGFpbiB2aXN1YWxpemVkIGluI GEgZmx1 b3Jlc2NlbmNlIHF1ZW5jaGVkIHNvbGlkLXBoYXNlIGluaGliaXRvciBsaWJyYXJ5IGFzc2F5 LiBE LWFtaW5vIGFjaWQgaW5oaWJpdG9ycyBmb3IgY3J1emlwYWluLCBjYXRoZXBzaW4gQiB hbmQgY2F0 aGVwc2luIEw8L3RpdGxlPjxzZWNvbmRhcnktdGl0bGU+Sm91cm5hbCBvZiBQZXB0aWRlI FNjaWVu Y2U8L3NlY29uZGFyeS10aXRsZT48YWx0LXRpdGxlPkouIFBlcHQuIFNjaS48L2FsdC10aX RsZT48 L3RpdGxlcz48cGVyaW9kaWNhbD48ZnVsbC10aXRsZT5Kb3VybmFsIG9mIFBlcHRpZGUg U2NpZW5j ZTwvZnVsbC10aXRsZT48YWJici0xPkouIFBlcHQuIFNjaS48L2FiYnItMT48L3BlcmlvZGljY Ww+ PGFsdC1wZXJpb2RpY2FsPjxmdWxsLXRpdGxlPkpvdXJuYWwgb2YgUGVwdGlkZSBTY2ll bmNlPC9m dWxsLXRpdGxlPjxhYmJyLTE+Si4gUGVwdC4gU2NpLjwvYWJici0xPjwvYWx0LXBlcmlvZ GljYWw+ PHBhZ2VzPjgzLTkxPC9wYWdlcz48dm9sdW1lPjQ8L3ZvbHVtZT48bnVtYmVyPjI8L251bW Jlcj48 a2V5d29yZHM+PGtleXdvcmQ+Zmx1b3Jlc2NlbmNlIHF1ZW5jaGVkIGFzc2F5PC9rZXl3b3J kPjxr ZXl3b3JkPmluaGliaXRvciBsaWJyYXJ5PC9rZXl3b3JkPjxrZXl3b3JkPlRyeXBhbm9zb21hIG Ny dXppPC9rZXl3b3JkPjxrZXl3b3JkPmNhdGhlcHNpbiBCIGFuZCBMIGluaGliaXRvcnM8L2tl eXdv cmQ+PGtleXdvcmQ+cGFyYXNpdGljIHByb3RlYXNlIGluaGliaXRpb248L2tleXdvcmQ+PGtle Xdv cmQ+VFJZUEFOT1NPTUEtQ1JVWkk8L2tleXdvcmQ+PGtleXdvcmQ+Q1lTVEVJTkUgUFJ PVEVJTkFT RTwva2V5d29yZD48a2V5d29yZD5FTlpZTUFUSUMtU1lOVEhFU0lTPC9rZXl3b3JkPjxrZXl 3b3Jk PkNSWVNUQUwtU1RSVUNUVVJFPC9rZXl3b3JkPjxrZXl3b3JkPlBFUFRJREUtU1lOVEh FU0lTPC9r ZXl3b3JkPjxrZXl3b3JkPk5PVk8gREVTSUdOPC9rZXl3b3JkPjxrZXl3b3JkPk5PTlBFUFRJ REU8 L2tleXdvcmQ+PGtleXdvcmQ+UEVHQTwva2V5d29yZD48a2V5d29yZD5JREVOVElGSUNB VElPTjwv a2V5d29yZD48a2V5d29yZD5TUEVDSUZJQ0lUWTwva2V5d29yZD48L2tleXdvcmRzPjxkYX

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald Rlcz48 eWVhcj4xOTk4PC95ZWFyPjxwdWItZGF0ZXM+PGRhdGU+QXByPC9kYXRlPjwvcHViL WRhdGVzPjwv ZGF0ZXM+PGlzYm4+MTA3NS0yNjE3PC9pc2JuPjxhY2Nlc3Npb24tbnVtPklTSTowMDAwN zMxMDg5 MDAwMDE8L2FjY2Vzc2lvbi1udW0+PHdvcmstdHlwZT5BcnRpY2xlPC93b3JrLXR5cGU+P HVybHM+ PHJlbGF0ZWQtdXJscz48dXJsPiZsdDtHbyB0byBJU0kmZ3Q7Oi8vMDAwMDczMTA4OTA wMDAxPC91 cmw+PC9yZWxhdGVkLXVybHM+PC91cmxzPjxsYW5ndWFnZT5FbmdsaXNoPC9sYW5nd WFnZT48L3Jl

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705

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald LiBE LWFtaW5vIGFjaWQgaW5oaWJpdG9ycyBmb3IgY3J1emlwYWluLCBjYXRoZXBzaW4gQiB hbmQgY2F0 aGVwc2luIEw8L3RpdGxlPjxzZWNvbmRhcnktdGl0bGU+Sm91cm5hbCBvZiBQZXB0aWRlI FNjaWVu Y2U8L3NlY29uZGFyeS10aXRsZT48YWx0LXRpdGxlPkouIFBlcHQuIFNjaS48L2FsdC10aX RsZT48 L3RpdGxlcz48cGVyaW9kaWNhbD48ZnVsbC10aXRsZT5Kb3VybmFsIG9mIFBlcHRpZGUg U2NpZW5j ZTwvZnVsbC10aXRsZT48YWJici0xPkouIFBlcHQuIFNjaS48L2FiYnItMT48L3BlcmlvZGljY Ww+ PGFsdC1wZXJpb2RpY2FsPjxmdWxsLXRpdGxlPkpvdXJuYWwgb2YgUGVwdGlkZSBTY2ll bmNlPC9m dWxsLXRpdGxlPjxhYmJyLTE+Si4gUGVwdC4gU2NpLjwvYWJici0xPjwvYWx0LXBlcmlvZ GljYWw+ PHBhZ2VzPjgzLTkxPC9wYWdlcz48dm9sdW1lPjQ8L3ZvbHVtZT48bnVtYmVyPjI8L251bW Jlcj48 a2V5d29yZHM+PGtleXdvcmQ+Zmx1b3Jlc2NlbmNlIHF1ZW5jaGVkIGFzc2F5PC9rZXl3b3J kPjxr ZXl3b3JkPmluaGliaXRvciBsaWJyYXJ5PC9rZXl3b3JkPjxrZXl3b3JkPlRyeXBhbm9zb21hIG Ny dXppPC9rZXl3b3JkPjxrZXl3b3JkPmNhdGhlcHNpbiBCIGFuZCBMIGluaGliaXRvcnM8L2tl eXdv cmQ+PGtleXdvcmQ+cGFyYXNpdGljIHByb3RlYXNlIGluaGliaXRpb248L2tleXdvcmQ+PGtle Xdv cmQ+VFJZUEFOT1NPTUEtQ1JVWkk8L2tleXdvcmQ+PGtleXdvcmQ+Q1lTVEVJTkUgUFJ PVEVJTkFT RTwva2V5d29yZD48a2V5d29yZD5FTlpZTUFUSUMtU1lOVEhFU0lTPC9rZXl3b3JkPjxrZXl 3b3Jk PkNSWVNUQUwtU1RSVUNUVVJFPC9rZXl3b3JkPjxrZXl3b3JkPlBFUFRJREUtU1lOVEh FU0lTPC9r ZXl3b3JkPjxrZXl3b3JkPk5PVk8gREVTSUdOPC9rZXl3b3JkPjxrZXl3b3JkPk5PTlBFUFRJ REU8 L2tleXdvcmQ+PGtleXdvcmQ+UEVHQTwva2V5d29yZD48a2V5d29yZD5JREVOVElGSUNB VElPTjwv a2V5d29yZD48a2V5d29yZD5TUEVDSUZJQ0lUWTwva2V5d29yZD48L2tleXdvcmRzPjxkYX Rlcz48 eWVhcj4xOTk4PC95ZWFyPjxwdWItZGF0ZXM+PGRhdGU+QXByPC9kYXRlPjwvcHViL WRhdGVzPjwv ZGF0ZXM+PGlzYm4+MTA3NS0yNjE3PC9pc2JuPjxhY2Nlc3Npb24tbnVtPklTSTowMDAwN zMxMDg5 MDAwMDE8L2FjY2Vzc2lvbi1udW0+PHdvcmstdHlwZT5BcnRpY2xlPC93b3JrLXR5cGU+P

706

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald HVybHM+ PHJlbGF0ZWQtdXJscz48dXJsPiZsdDtHbyB0byBJU0kmZ3Q7Oi8vMDAwMDczMTA4OTA wMDAxPC91 cmw+PC9yZWxhdGVkLXVybHM+PC91cmxzPjxsYW5ndWFnZT5FbmdsaXNoPC9sYW5nd WFnZT48L3Jl Y29yZD48L0NpdGU+PC9FbmROb3RlPgB= 70

The mesh size of the polymer matrix in PEGA is controlled by the length (molecular weight) of the PEG chains cross-linking the polymer. Most earlier work on had been carried out on PEGA1900 (subscript refers to the molecular weight of the PEG in g/mol), it was found that enzymes up to 50 kDa could diffuse into the polymer network.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5NZWxkYWw8L0F1dGhvcj48 WWVhcj4xOTk0PC9ZZWFyPjxS ZWNOdW0+MjwvUmVjTnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+MjwvcmVjL W51bWJlcj48Zm9yZWln bi1rZXlzPjxrZXkgYXBwPSJFTiIgZGItaWQ9IjIwcDByMmRwOWY5dGQyZTJy YTl2cjJwbnMwenh2 d3BzMHg1ZSI+Mjwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYtdHlwZSBuYW1lP SJKb3VybmFsIEFy dGljbGUiPjE3PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxhdXRob3JzPjxhdXR ob3I+TWVsZGFs LCBNLjwvYXV0aG9yPjxhdXRob3I+QXV6YW5uZWF1LCBGLiBJLjwvYXV0a G9yPjxhdXRob3I+SGlu ZHNnYXVsLCBPLjwvYXV0aG9yPjxhdXRob3I+UGFsY2ljLCBNLiBNLjwvYX V0aG9yPjwvYXV0aG9y cz48L2NvbnRyaWJ1dG9ycz48YXV0aC1hZGRyZXNzPlVOSVYgQUxCRVJUQS xERVBUIENIRU0sRURN T05UT04sQUIsQ0FOQURBLiYjeEQ7TUVMREFMLCBNLCBDQVJMU0JFUkcg TEFCLERFUFQgQ0hFTSxH QU1MRSBDQVJMU0JFUkcgVkVKIDEwLERLLTI1MDAgVkFMQlksREVOTU FSSy48L2F1dGgtYWRkcmVz cz48dGl0bGVzPjx0aXRsZT5BIFBFR0EgUkVTSU4gRk9SIFVTRSBJTiBUSEUg U09MSUQtUEhBU0Ug Q0hFTUlDQUwtRU5aWU1BVElDIFNZTlRIRVNJUyBPRiBHTFlDT1BFUFRJR EVTPC90aXRsZT48c2Vj b25kYXJ5LXRpdGxlPkpvdXJuYWwgb2YgdGhlIENoZW1pY2FsIFNvY2lldHktQ 2hlbWljYWwgQ29t 707

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

48L3JlbGF0ZWQtdXJs cz48L3VybHM+PGxhbmd1YWdlPkVuZ2xpc2g8L2xhbmd1YWdlPjwvcmVjb3JkP jwvQ2l0ZT48L0Vu ZE5vdGU+AG== 69,71 Meldal and co-workers then went on to address this limitation of the earlier PEGA resin by preparing PEGA cross-linked with PEG with molecular weights of 4000, 6000, 8000. Libraries of fluorescence quenched peptide substrates (FRET) (described in more detail in section Fluorescence resonance energy transfer) were then prepared on these resins and incubated with the enzyme, MMP-9 which has active forms of 67-83 kDa. Particles that appeared bright were manually isolated and sequenced to identify substrates for MMP-9, effectively identifying MMP-9 substrates.72 This method of fluorescencequenched peptide substrates was later used to screen for substrates the cysteine protease, Papain. Here, the limited loading of the PEGA4000 was doubled through the incorporation of a K-K dipeptide as the first functionalisation step on the resin.73 Interest in utilising PEGA in the preparation of peptide libraries for screening with enzymes soon increased. This led to a drive to further understand the polymer’s structure/property relationship with enzyme catalysed reactions. Bradley and coworkersPEVuZE5vdGU+PENpdGU+PEF1dGhvcj5LcmVzczwvQXV0aG9yPjxZZ WFyPjIwMDI8L1llYXI+PFJl Y051bT4xNTc8L1JlY051bT48cmVjb3JkPjxyZWMtbnVtYmVyPjE1NzwvcmVjL W51bWJlcj48Zm9y ZWlnbi1rZXlzPjxrZXkgYXBwPSJFTiIgZGItaWQ9ImZhOXpkenN2a3N4MnByZ XJlOTh4ZDUycWRm d2Z3cjkwcDVzMiI+MTU3PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG 5hbWU9IkpvdXJu YWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcn M+PGF1dGhvcj5L cmVzcywgSi48L2F1dGhvcj48YXV0aG9yPlphbmFsZXR0aSwgUi48L2F1dGhvcj4 8YXV0aG9yPkFt b3VyLCBBLjwvYXV0aG9yPjxhdXRob3I+TGFkbG93LCBNLjwvYXV0aG9yPjxh dXRob3I+RnJleSwg Si4gRy48L2F1dGhvcj48YXV0aG9yPkJyYWRsZXksIE0uPC9hdXRob3I+PC9hdX Rob3JzPjwvY29u dHJpYnV0b3JzPjxhdXRoLWFkZHJlc3M+VW5pdiBTb3V0aGFtcHRvbiwgRGVw dCBDaGVtLCBTb3V0 aGFtcHRvbiBTTzE3IDFCSiwgSGFudHMsIEVuZ2xhbmQuIEdsYXhvU21pdGhLb 713

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

GluZSwgTWVkIFJl cyBDdHIsIFN0ZXZlbmFnZSBTRzEgMk5ZLCBIZXJ0cywgRW5nbGFuZC4gVW 5pdiBDYW1icmlkZ2Us IERlcHQgQ2hlbSwgR2xheG9TbWl0aEtsaW5lLCBDaGVtIFRlY2hub2wsIENhbW JyaWRnZSwgRW5n bGFuZC4mI3hEO0JyYWRsZXksIE0sIFVuaXYgU291dGhhbXB0b24sIERlcHQgQ 2hlbSwgU291dGhh bXB0b24gU08xNyAxQkosIEhhbnRzLCBFbmdsYW5kLjwvYXV0aC1hZGRyZXN zPjx0aXRsZXM+PHRp dGxlPkVuenltZSBhY2Nlc3NpYmlsaXR5IGFuZCBzb2xpZCBzdXBwb3J0czogV2h pY2ggbW9sZWN1 bGFyIHdlaWdodCBlbnp5bWVzIGNhbiBiZSB1c2VkIG9uIHNvbGlkIHN1cHBvcn RzPyBhbiBpbnZl c3RpZ2F0aW9uIHVzaW5nIGNvbmZvY2FsIFJhbWFuIG1pY3Jvc2NvcHk8L3Rpd GxlPjxzZWNvbmRh cnktdGl0bGU+Q2hlbWlzdHJ5LWEgRXVyb3BlYW4gSm91cm5hbDwvc2Vjb25k YXJ5LXRpdGxlPjxh bHQtdGl0bGU+Q2hlbS4tRXVyLiBKLjwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZ XJpb2RpY2FsPjxm dWxsLXRpdGxlPkNoZW1pc3RyeS1hIEV1cm9wZWFuIEpvdXJuYWw8L2Z1bG wtdGl0bGU+PGFiYnIt MT5DaGVtLi1FdXIuIEouPC9hYmJyLTE+PC9wZXJpb2RpY2FsPjxhbHQtcGVya W9kaWNhbD48ZnVs bC10aXRsZT5DaGVtaXN0cnktYSBFdXJvcGVhbiBKb3VybmFsPC9mdWxsLXR pdGxlPjxhYmJyLTE+ Q2hlbS4tRXVyLiBKLjwvYWJici0xPjwvYWx0LXBlcmlvZGljYWw+PHBhZ2VzP jM3NjktMzc3Mjwv cGFnZXM+PHZvbHVtZT44PC92b2x1bWU+PG51bWJlcj4xNjwvbnVtYmVyPjxr ZXl3b3Jkcz48a2V5 d29yZD5jb21iaW5hdG9yaWFsIGNoZW1pc3RyeTwva2V5d29yZD48a2V5d29yZ D5lbnp5bWVzPC9r ZXl3b3JkPjxrZXl3b3JkPlJhbWFuIHNwZWN0cm9zY29weTwva2V5d29yZD48a2 V5d29yZD5zb2xp ZCBzdXBwb3J0czwva2V5d29yZD48a2V5d29yZD5DT01CSU5BVE9SSUFMIExJ QlJBUklFUzwva2V5 714

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

d29yZD48a2V5d29yZD5QT0xZTUVSSUMgU1VQUE9SVDwva2V5d29yZD48a2 V5d29yZD5PUkdBTklD LVNZTlRIRVNJUzwva2V5d29yZD48a2V5d29yZD5QRVBUSURFLVNZTlRIRV NJUzwva2V5d29yZD48 a2V5d29yZD5MSU5LRVI8L2tleXdvcmQ+PGtleXdvcmQ+U1BFQ0lGSUNJVFk8 L2tleXdvcmQ+PGtl eXdvcmQ+Q0hFTUlTVFJZPC9rZXl3b3JkPjxrZXl3b3JkPkJFQURTPC9rZXl3b3Jk PjxrZXl3b3Jk PkdFTkVSQVRJT048L2tleXdvcmQ+PGtleXdvcmQ+UkVTSU48L2tleXdvcmQ+P C9rZXl3b3Jkcz48 ZGF0ZXM+PHllYXI+MjAwMjwveWVhcj48cHViLWRhdGVzPjxkYXRlPkF1Zz wvZGF0ZT48L3B1Yi1k YXRlcz48L2RhdGVzPjxpc2JuPjA5NDctNjUzOTwvaXNibj48YWNjZXNzaW9uL W51bT5JU0k6MDAw MTc3NjUyODAwMDIxPC9hY2Nlc3Npb24tbnVtPjx3b3JrLXR5cGU+QXJ0aWNs ZTwvd29yay10eXBl Pjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD4mbHQ7R28gdG8gSVNJJmd0Oz ovLzAwMDE3NzY1Mjgw MDAyMTwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48bGFuZ3VhZ2U+RW5 nbGlzaDwvbGFuZ3Vh Z2U+PC9yZWNvcmQ+PC9DaXRlPjwvRW5kTm90ZT5= PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5LcmVzczwvQXV0aG9yPjxZZWFyPjI wMDI8L1llYXI+PFJl Y051bT4xNTc8L1JlY051bT48cmVjb3JkPjxyZWMtbnVtYmVyPjE1NzwvcmVjL W51bWJlcj48Zm9y ZWlnbi1rZXlzPjxrZXkgYXBwPSJFTiIgZGItaWQ9ImZhOXpkenN2a3N4MnByZ XJlOTh4ZDUycWRm d2Z3cjkwcDVzMiI+MTU3PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG 5hbWU9IkpvdXJu YWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcn M+PGF1dGhvcj5L cmVzcywgSi48L2F1dGhvcj48YXV0aG9yPlphbmFsZXR0aSwgUi48L2F1dGhvcj4 8YXV0aG9yPkFt b3VyLCBBLjwvYXV0aG9yPjxhdXRob3I+TGFkbG93LCBNLjwvYXV0aG9yPjxh dXRob3I+RnJleSwg Si4gRy48L2F1dGhvcj48YXV0aG9yPkJyYWRsZXksIE0uPC9hdXRob3I+PC9hdX 715

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Rob3JzPjwvY29u dHJpYnV0b3JzPjxhdXRoLWFkZHJlc3M+VW5pdiBTb3V0aGFtcHRvbiwgRGVw dCBDaGVtLCBTb3V0 aGFtcHRvbiBTTzE3IDFCSiwgSGFudHMsIEVuZ2xhbmQuIEdsYXhvU21pdGhLb GluZSwgTWVkIFJl cyBDdHIsIFN0ZXZlbmFnZSBTRzEgMk5ZLCBIZXJ0cywgRW5nbGFuZC4gVW 5pdiBDYW1icmlkZ2Us IERlcHQgQ2hlbSwgR2xheG9TbWl0aEtsaW5lLCBDaGVtIFRlY2hub2wsIENhbW JyaWRnZSwgRW5n bGFuZC4mI3hEO0JyYWRsZXksIE0sIFVuaXYgU291dGhhbXB0b24sIERlcHQgQ 2hlbSwgU291dGhh bXB0b24gU08xNyAxQkosIEhhbnRzLCBFbmdsYW5kLjwvYXV0aC1hZGRyZXN zPjx0aXRsZXM+PHRp dGxlPkVuenltZSBhY2Nlc3NpYmlsaXR5IGFuZCBzb2xpZCBzdXBwb3J0czogV2h pY2ggbW9sZWN1 bGFyIHdlaWdodCBlbnp5bWVzIGNhbiBiZSB1c2VkIG9uIHNvbGlkIHN1cHBvcn RzPyBhbiBpbnZl c3RpZ2F0aW9uIHVzaW5nIGNvbmZvY2FsIFJhbWFuIG1pY3Jvc2NvcHk8L3Rpd GxlPjxzZWNvbmRh cnktdGl0bGU+Q2hlbWlzdHJ5LWEgRXVyb3BlYW4gSm91cm5hbDwvc2Vjb25k YXJ5LXRpdGxlPjxh bHQtdGl0bGU+Q2hlbS4tRXVyLiBKLjwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZ XJpb2RpY2FsPjxm dWxsLXRpdGxlPkNoZW1pc3RyeS1hIEV1cm9wZWFuIEpvdXJuYWw8L2Z1bG wtdGl0bGU+PGFiYnIt MT5DaGVtLi1FdXIuIEouPC9hYmJyLTE+PC9wZXJpb2RpY2FsPjxhbHQtcGVya W9kaWNhbD48ZnVs bC10aXRsZT5DaGVtaXN0cnktYSBFdXJvcGVhbiBKb3VybmFsPC9mdWxsLXR pdGxlPjxhYmJyLTE+ Q2hlbS4tRXVyLiBKLjwvYWJici0xPjwvYWx0LXBlcmlvZGljYWw+PHBhZ2VzP jM3NjktMzc3Mjwv cGFnZXM+PHZvbHVtZT44PC92b2x1bWU+PG51bWJlcj4xNjwvbnVtYmVyPjxr ZXl3b3Jkcz48a2V5 d29yZD5jb21iaW5hdG9yaWFsIGNoZW1pc3RyeTwva2V5d29yZD48a2V5d29yZ D5lbnp5bWVzPC9r 716

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZXl3b3JkPjxrZXl3b3JkPlJhbWFuIHNwZWN0cm9zY29weTwva2V5d29yZD48a2 V5d29yZD5zb2xp ZCBzdXBwb3J0czwva2V5d29yZD48a2V5d29yZD5DT01CSU5BVE9SSUFMIExJ QlJBUklFUzwva2V5 d29yZD48a2V5d29yZD5QT0xZTUVSSUMgU1VQUE9SVDwva2V5d29yZD48a2 V5d29yZD5PUkdBTklD LVNZTlRIRVNJUzwva2V5d29yZD48a2V5d29yZD5QRVBUSURFLVNZTlRIRV NJUzwva2V5d29yZD48 a2V5d29yZD5MSU5LRVI8L2tleXdvcmQ+PGtleXdvcmQ+U1BFQ0lGSUNJVFk8 L2tleXdvcmQ+PGtl eXdvcmQ+Q0hFTUlTVFJZPC9rZXl3b3JkPjxrZXl3b3JkPkJFQURTPC9rZXl3b3Jk PjxrZXl3b3Jk PkdFTkVSQVRJT048L2tleXdvcmQ+PGtleXdvcmQ+UkVTSU48L2tleXdvcmQ+P C9rZXl3b3Jkcz48 ZGF0ZXM+PHllYXI+MjAwMjwveWVhcj48cHViLWRhdGVzPjxkYXRlPkF1Zz wvZGF0ZT48L3B1Yi1k YXRlcz48L2RhdGVzPjxpc2JuPjA5NDctNjUzOTwvaXNibj48YWNjZXNzaW9uL W51bT5JU0k6MDAw MTc3NjUyODAwMDIxPC9hY2Nlc3Npb24tbnVtPjx3b3JrLXR5cGU+QXJ0aWNs ZTwvd29yay10eXBl Pjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD4mbHQ7R28gdG8gSVNJJmd0Oz ovLzAwMDE3NzY1Mjgw MDAyMTwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48bGFuZ3VhZ2U+RW5 nbGlzaDwvbGFuZ3Vh Z2U+PC9yZWNvcmQ+PC9DaXRlPjwvRW5kTm90ZT5= 63 presented a paper in which confocal Raman microscopy was used to investigate enzyme accessibility. Three different supports for SPPS; TentaGel, PEGA1900 and controlled pore glasses (CPGs) were functionalised with a peptide substrate terminated with 4-cyanobenzamide and incubated with five different enzymes with a range of molecular weights. The library of particles were then investigated by confocal Raman microscopy to ascertain if the peptide sequence was hydrolysed by monitoring the stretching frequency of the cyano group. TentaGel was not accessible to any of the enzymes, indeed, there was no detectable effect on the peptide with even the smallest enzyme (MMP-12, 22 kDa). PEGA1900 displayed peptide cleavage with all enzymes below 35 kDa but not enzymes above 42.5 kDa. Finally, beaded CPG showed complete cleavage of the peptide for all enzymes tested. This was 717

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

because the pores in the CPG used in the study were 100 nm in diameter which was much larger than the hydrodynamic radius of the enzymes. This study provided a well defined accessibility for PEGA to enzymes from which the research community to work.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5LcmVzczwvQXV0aG9yPjxZZWF yPjIwMDI8L1llYXI+PFJl Y051bT41PC9SZWNOdW0+PHJlY29yZD48cmVjLW51bWJlcj41PC9yZWMtbnV tYmVyPjxmb3JlaWdu LWtleXM+PGtleSBhcHA9IkVOIiBkYi1pZD0iMjBwMHIyZHA5Zjl0ZDJlMnJhO XZyMnBuczB6eHZ3 cHMweDVlIj41PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG5hbWU9Ik pvdXJuYWwgQXJ0 aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcnM+PGF1dGh vcj5LcmVzcywg Si48L2F1dGhvcj48YXV0aG9yPlphbmFsZXR0aSwgUi48L2F1dGhvcj48YXV0aG9 yPkFtb3VyLCBB LjwvYXV0aG9yPjxhdXRob3I+TGFkbG93LCBNLjwvYXV0aG9yPjxhdXRob3I+ RnJleSwgSi4gRy48 L2F1dGhvcj48YXV0aG9yPkJyYWRsZXksIE0uPC9hdXRob3I+PC9hdXRob3JzPj wvY29udHJpYnV0 b3JzPjxhdXRoLWFkZHJlc3M+VW5pdiBTb3V0aGFtcHRvbiwgRGVwdCBDaGVt LCBTb3V0aGFtcHRv biBTTzE3IDFCSiwgSGFudHMsIEVuZ2xhbmQuIEdsYXhvU21pdGhLbGluZSwgT WVkIFJlcyBDdHIs IFN0ZXZlbmFnZSBTRzEgMk5ZLCBIZXJ0cywgRW5nbGFuZC4gVW5pdiBDY W1icmlkZ2UsIERlcHQg Q2hlbSwgR2xheG9TbWl0aEtsaW5lLCBDaGVtIFRlY2hub2wsIENhbWJyaWRnZ SwgRW5nbGFuZC4m I3hEO0JyYWRsZXksIE0sIFVuaXYgU291dGhhbXB0b24sIERlcHQgQ2hlbSwgU2 91dGhhbXB0b24g U08xNyAxQkosIEhhbnRzLCBFbmdsYW5kLjwvYXV0aC1hZGRyZXNzPjx0aXR sZXM+PHRpdGxlPkVu enltZSBhY2Nlc3NpYmlsaXR5IGFuZCBzb2xpZCBzdXBwb3J0czogV2hpY2ggbW 9sZWN1bGFyIHdl aWdodCBlbnp5bWVzIGNhbiBiZSB1c2VkIG9uIHNvbGlkIHN1cHBvcnRzPyBhbi 718

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

BpbnZlc3RpZ2F0 aW9uIHVzaW5nIGNvbmZvY2FsIFJhbWFuIG1pY3Jvc2NvcHk8L3RpdGxlPjxzZ WNvbmRhcnktdGl0 bGU+Q2hlbWlzdHJ5LWEgRXVyb3BlYW4gSm91cm5hbDwvc2Vjb25kYXJ5LXR pdGxlPjxhbHQtdGl0 bGU+Q2hlbS4tRXVyLiBKLjwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2Rp Y2FsPjxmdWxsLXRp dGxlPkNoZW1pc3RyeS1hIEV1cm9wZWFuIEpvdXJuYWw8L2Z1bGwtdGl0bGU +PGFiYnItMT5DaGVt Li1FdXIuIEouPC9hYmJyLTE+PC9wZXJpb2RpY2FsPjxhbHQtcGVyaW9kaWNhb D48ZnVsbC10aXRs ZT5DaGVtaXN0cnktYSBFdXJvcGVhbiBKb3VybmFsPC9mdWxsLXRpdGxlPjxh YmJyLTE+Q2hlbS4t RXVyLiBKLjwvYWJici0xPjwvYWx0LXBlcmlvZGljYWw+PHBhZ2VzPjM3Njkt Mzc3MjwvcGFnZXM+ PHZvbHVtZT44PC92b2x1bWU+PG51bWJlcj4xNjwvbnVtYmVyPjxrZXl3b3Jkcz4 8a2V5d29yZD5j b21iaW5hdG9yaWFsIGNoZW1pc3RyeTwva2V5d29yZD48a2V5d29yZD5lbnp5b WVzPC9rZXl3b3Jk PjxrZXl3b3JkPlJhbWFuIHNwZWN0cm9zY29weTwva2V5d29yZD48a2V5d29yZ D5zb2xpZCBzdXBw b3J0czwva2V5d29yZD48a2V5d29yZD5DT01CSU5BVE9SSUFMIExJQlJBUklFU zwva2V5d29yZD48 a2V5d29yZD5QT0xZTUVSSUMgU1VQUE9SVDwva2V5d29yZD48a2V5d29yZD 5PUkdBTklDLVNZTlRI RVNJUzwva2V5d29yZD48a2V5d29yZD5QRVBUSURFLVNZTlRIRVNJUzwva2 V5d29yZD48a2V5d29y ZD5MSU5LRVI8L2tleXdvcmQ+PGtleXdvcmQ+U1BFQ0lGSUNJVFk8L2tleXdvc mQ+PGtleXdvcmQ+ Q0hFTUlTVFJZPC9rZXl3b3JkPjxrZXl3b3JkPkJFQURTPC9rZXl3b3JkPjxrZXl3b 3JkPkdFTkVS QVRJT048L2tleXdvcmQ+PGtleXdvcmQ+UkVTSU48L2tleXdvcmQ+PC9rZXl3b3 Jkcz48ZGF0ZXM+ PHllYXI+MjAwMjwveWVhcj48cHViLWRhdGVzPjxkYXRlPkF1ZzwvZGF0ZT48 L3B1Yi1kYXRlcz48 719

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

sZXM+PHRpdGxlPkVu enltZSBhY2Nlc3NpYmlsaXR5IGFuZCBzb2xpZCBzdXBwb3J0czogV2hpY2ggbW 9sZWN1bGFyIHdl aWdodCBlbnp5bWVzIGNhbiBiZSB1c2VkIG9uIHNvbGlkIHN1cHBvcnRzPyBhbi BpbnZlc3RpZ2F0 aW9uIHVzaW5nIGNvbmZvY2FsIFJhbWFuIG1pY3Jvc2NvcHk8L3RpdGxlPjxzZ WNvbmRhcnktdGl0 bGU+Q2hlbWlzdHJ5LWEgRXVyb3BlYW4gSm91cm5hbDwvc2Vjb25kYXJ5LXR pdGxlPjxhbHQtdGl0 bGU+Q2hlbS4tRXVyLiBKLjwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2Rp Y2FsPjxmdWxsLXRp dGxlPkNoZW1pc3RyeS1hIEV1cm9wZWFuIEpvdXJuYWw8L2Z1bGwtdGl0bGU +PGFiYnItMT5DaGVt Li1FdXIuIEouPC9hYmJyLTE+PC9wZXJpb2RpY2FsPjxhbHQtcGVyaW9kaWNhb D48ZnVsbC10aXRs ZT5DaGVtaXN0cnktYSBFdXJvcGVhbiBKb3VybmFsPC9mdWxsLXRpdGxlPjxh YmJyLTE+Q2hlbS4t RXVyLiBKLjwvYWJici0xPjwvYWx0LXBlcmlvZGljYWw+PHBhZ2VzPjM3Njkt Mzc3MjwvcGFnZXM+ PHZvbHVtZT44PC92b2x1bWU+PG51bWJlcj4xNjwvbnVtYmVyPjxrZXl3b3Jkcz4 8a2V5d29yZD5j b21iaW5hdG9yaWFsIGNoZW1pc3RyeTwva2V5d29yZD48a2V5d29yZD5lbnp5b WVzPC9rZXl3b3Jk PjxrZXl3b3JkPlJhbWFuIHNwZWN0cm9zY29weTwva2V5d29yZD48a2V5d29yZ D5zb2xpZCBzdXBw b3J0czwva2V5d29yZD48a2V5d29yZD5DT01CSU5BVE9SSUFMIExJQlJBUklFU zwva2V5d29yZD48 a2V5d29yZD5QT0xZTUVSSUMgU1VQUE9SVDwva2V5d29yZD48a2V5d29yZD 5PUkdBTklDLVNZTlRI RVNJUzwva2V5d29yZD48a2V5d29yZD5QRVBUSURFLVNZTlRIRVNJUzwva2 V5d29yZD48a2V5d29y ZD5MSU5LRVI8L2tleXdvcmQ+PGtleXdvcmQ+U1BFQ0lGSUNJVFk8L2tleXdvc mQ+PGtleXdvcmQ+ Q0hFTUlTVFJZPC9rZXl3b3JkPjxrZXl3b3JkPkJFQURTPC9rZXl3b3JkPjxrZXl3b 3JkPkdFTkVS 721

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

QVRJT048L2tleXdvcmQ+PGtleXdvcmQ+UkVTSU48L2tleXdvcmQ+PC9rZXl3b3 Jkcz48ZGF0ZXM+ PHllYXI+MjAwMjwveWVhcj48cHViLWRhdGVzPjxkYXRlPkF1ZzwvZGF0ZT48 L3B1Yi1kYXRlcz48 L2RhdGVzPjxpc2JuPjA5NDctNjUzOTwvaXNibj48YWNjZXNzaW9uLW51bT5J U0k6MDAwMTc3NjUy ODAwMDIxPC9hY2Nlc3Npb24tbnVtPjx3b3JrLXR5cGU+QXJ0aWNsZTwvd29ya y10eXBlPjx1cmxz PjxyZWxhdGVkLXVybHM+PHVybD4mbHQ7R28gdG8gSVNJJmd0OzovLzAwM DE3NzY1MjgwMDAyMTwv dXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48bGFuZ3VhZ2U+RW5nbGlzaDwvb GFuZ3VhZ2U+PC9y ZWNvcmQ+PC9DaXRlPjwvRW5kTm90ZT5= 63 An alternative approach to that used by Meldal and co-workers to increase the accessibility of PEGA was described by the groups of Flitsch and Gardossi in a series

of

papers.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5CYXNzbzwvQXV0aG9yPjxZZ WFyPjIwMDM8L1llYXI+PFJl Y051bT4yNTQ8L1JlY051bT48cmVjb3JkPjxyZWMtbnVtYmVyPjI1NDwvcmVjL W51bWJlcj48Zm9y ZWlnbi1rZXlzPjxrZXkgYXBwPSJFTiIgZGItaWQ9ImZhOXpkenN2a3N4MnByZ XJlOTh4ZDUycWRm d2Z3cjkwcDVzMiI+MjU0PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG 5hbWU9IkpvdXJu YWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcn M+PGF1dGhvcj5C YXNzbywgQS48L2F1dGhvcj48YXV0aG9yPkRlIE1hcnRpbiwgTC48L2F1dGhvcj4 8YXV0aG9yPkdh cmRvc3NpLCBMLjwvYXV0aG9yPjxhdXRob3I+TWFyZ2V0dHMsIEcuPC9hdXR ob3I+PGF1dGhvcj5C cmF6ZW5kYWxlLCBJLjwvYXV0aG9yPjxhdXRob3I+Qm9zbWEsIEEuIFkuPC9h dXRob3I+PGF1dGhv cj5VbGlqbiwgUi4gVi48L2F1dGhvcj48YXV0aG9yPkZsaXRzY2gsIFMuIEwuPC9h dXRob3I+PC9h dXRob3JzPjwvY29udHJpYnV0b3JzPjxhdXRoLWFkZHJlc3M+VW5pdiBUcmllc3 722

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

acrylamide in the polymerisation mixture with a permanently charged acrylamide based monomer. These PEGA+ and PEGA- particles demonstrated greater swelling than the neutral PEGA1900, although as the ionic strength of aqueous solution was increased the swelling of the charged PEGAs reduced to the same value as that of PEGA1900. The swelling behaviour of the charged polymers was due to the electrostatic repulsion between adjacent polymer chains, this resulted in a increase in pore size as determined by an increase in enzyme accessibility. The substrate for the enzyme used (penicillin G amidase, PGA, 88 kDa) was N-phenylacetylated L-Phe, and was only cleaved to low conversions (10 %) when coupled onto PEGA1900 and PEGA- however, for PEGA+ much greater conversions of 50 % were observed.74 In their following paper these electrostatic effects were further investigated with hydrolytic yields as high as 80 % being obtained by increasing the amount of positive charges in the polymer network. These electrostatically attracted the negatively charged enzyme (PGA, pI = 5.2-5.4) thus favouring the accessibility of the

bulky

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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dGU+AG==

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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743

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

FyPjIwMDU8L3ll YXI+PHB1Yi1kYXRlcz48ZGF0ZT5KYW48L2RhdGU+PC9wdWItZGF0ZXM+P C9kYXRlcz48aXNibj4w MDQwLTQwMjA8L2lzYm4+PGFjY2Vzc2lvbi1udW0+SVNJOjAwMDIyNjMzMj QwMDAyMzwvYWNjZXNz aW9uLW51bT48d29yay10eXBlPkFydGljbGU8L3dvcmstdHlwZT48dXJscz48cmV sYXRlZC11cmxz Pjx1cmw+Jmx0O0dvIHRvIElTSSZndDs6Ly8wMDAyMjYzMzI0MDAwMjM8L3 VybD48L3JlbGF0ZWQt dXJscz48L3VybHM+PGVsZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjEwLjEwMT Yvai50ZXQuMjAwNC4x MS4wMTU8L2VsZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjxsYW5ndWFnZT5Fb mdsaXNoPC9sYW5ndWFn ZT48L3JlY29yZD48L0NpdGU+PC9FbmROb3RlPgB= 77 In a final publication on this topic, they made use of the improved yields of PGA catalysed reaction on PEGA+

to

demonstrate

a

hydrazide

enzyme

cleavable

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Overall, the introduction of permanent charges to the polymer network offers an interesting method for the adjustment of the polymers properties and interaction with surrounding proteins.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Another analytical technique was introduced into the area of enzyme catalysed reactions on PEGA in 2003 by Flitsch and co-workers in the form of two photon microscopy (TPM) (described in section Two-photon microscopy).78 This technique allows the production of images detailing the spatial resolution of fluorophores within polymer particles. PEGA1900 was functionalised with Fmoc-F-F and exposed to the protease thermolysin for different lengths of time. Where hydrolysis had occurred free amine groups were present, these were chemically acylated with dansyl chloride (Figure 2–13 A). These areas then showed as fluorescent, revealing where enzyme hydrolysis has occurred. Thermolysin treated particles initially displayed a bright ring on the outside of particle, which then expanded into the particle until after approximately 45 minutes when the whole particle was fluorescent (Figure 2–13 B). This indicated that the enzymatic action was limited by diffusion of the enzyme into in the polymer particle. Indeed, it was determined that for the enzyme to diffuse the distance from the outside of the particle to the centre (100 µm) in an aqueous environment would take approximately one minute.79 The use of TPM presented an effective method to determine spatial and temporal resolution of enzyme hydrolysis within polymer particles.

Figure 2–13. Using two photon microscopy to quantify enzymatic reaction rates on polymer beads. A: Thermolysin catalysed hydrolysis of solid supported Fmoc–Phe–Phe. B: Thermolysin catalysed hydrolysis of PEGA1900 bound dipeptide 1 as examined by TPM. From left to right the images represent 5, 10, 20, 45, 60, 90, 120 and 240 min.

In 2003 a new development in the use of combinatorial libraries on PEGA was developed. With the establishment of large libraries of PEGA-bound peptide for screening, a faster and effective method was needed to separate particles with ‘hits’ from particles that were ‘non-hits’. Previously, this process had been carried out manually on PEGA. Meldal and co-workers prepared a ‘one bead, two compound’ combinatorial library; one compound consisted of a fluorescence quenched peptide substrate (FRET) while the second compound was a randomly synthesised peptide (by split and mix synthesis) to screen for inhibition of their test enzymes,

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

metalloproteinases (MMPs). These two compounds then competed for binding with the same enzyme and if the FRET substrate was not cleaved this indicated inhibition of the enzyme by the library compound. Once incubated with the enzyme the particles were analysed with an instrument developed originally developed for high throughput screening and sorting of different transgenic-fluorescent tagged organisms. It was further adapted for the purpose of sorting labelled particles. This device allowed ‘hits’, dark particles, to be separated from ‘non-hits’ in a very fast and reliable manner. Using this approach ten dark particles were selected, sequenced and resynthesised to provide very potent inhibitor activity towards a number of MMPs.80 In 2006 Ulijn and co-workers described a different morphology of PEGA in the form a micropatterned PEGA surface.81 Within this work three different techniques were utilised to prepare a micropatterned surface: photolithography, capillary force lithography (using a patterned stamp) and spotting with a manual microarray (Figure 2–14). These patterned surfaces maintained the same chemistry as PEGA particles allowing for functionalisation with peptides by SPPS. Using this methodology PEGA surfaces were prepared that could direct cell adhesion (using the peptide RGD) or screen for protease specificity with FRET peptide substrates.81

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald Figure 2–14. A micropatterned hydrogel platform for chemical Synthesis and biological analysis. Optical micrographs of fibroblasts cultured in serum-containing media for 48 h on (functionalized) PEGA-patterned surfaces. (a) Unmodified PEGA surface; fibroblast cells strictly adhere and spread only on the glass surroundings, and not to the PEGA surface. (b) RGE-modified control surface that resists cell adhesion. (c) RGD-modified PEGA surface that promotes cell adhesion.81

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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82

Within this work they made use of TPM to demonstrate real-time spatially resolved measurement of enzyme activity on polymer particles. Aminocoumarin-carboxylic acid (a fluorescent derivative used extensively in biological assays) was coupled onto PEGA1900 via a hexa-glycine linker. A peptide or amino acid was then coupled to the aminocumarin resulting in quenching of its fluorescence (Figure 2–15 A). When Bz-R-OH was coupled onto the aminocumarin and these particles were treated with trypsin fluorescence appeared as an annular ring around the outside of the particle which then gradually progressed inwards. This indicated that enzyme hydrolysis was initially confined to the outside of the particles gradually diffusing towards the centre of the particle (Figure 2–15 B). This was in agreement with the non-real-time studies using TPM that were previously carried out by Flitsch and coworkers.79 Indeed, it took over three hours for the fluorescence values of the centre of the particle to match those of the outside, this delay was likely a result of the electrostatic repulsion between the positively charged trypsin (pI = 8.69) and the positively charged arginine covalently attached to the aminocumarin. When a different enzyme (Subtilisin Carlsberg) was incubated with the functionalised particles (Bz-R-OH was replaced with Z-G-G-L-OH) different behaviour was observed; there were no defined rings noted and increase in fluorescence upon enzyme action was relatively homogeneous across the particles (Figure 2–15 C). This was presumably due to the smaller size of the Subtilisin Carlsberg (although this is not detailed within the paper) resulting in fast enzyme diffusion that was not rate limiting. In summary, TPM provides a useful tool for the real-time analyse of enzyme hydrolysis although it not shown whether the aminocumarin itself effects enzyme

rate

or

hydrolysis

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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765

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

T4xMC4xMDAyL2Fk c2MuMjAwNzAwMDQ0PC9lbGVjdHJvbmljLXJlc291cmNlLW51bT48bGFuZ3Vh Z2U+RW5nbGlzaDwv bGFuZ3VhZ2U+PC9yZWNvcmQ+PC9DaXRlPjwvRW5kTm90ZT4A 82

Figure 2–15. Real-time imaging of protease action on substrates covalently immobilised to polymer supports. A: Schematic of coupling and enzymatic reaction processes on PEGA1900 particles. B & C: Two photon cross-section images of PEGA1900 particles treated with B; trypsin and C; Subtilisin Carlsberg respectively.

2.4.2. Enzyme catalysed synthesis on PEGA A different area of research on PEGA was the synthesis of peptides on solid support by enzymatic means, the first example of this was published by Flitsch and coworkers.83 In this research, PEGA particles functionalised with phenylalanine were prepared using solid phase methodology, enzymatically catalysed amide bond formation then occurred upon treatment of these particles with an excess of amino acids (with their amines protected with Fmoc) in the presence of thermolysin. High yields were found for more hydrophobic amino acids while more polar residues led to lower yields. The authors suggested that amide formation was driven by three main factors: the large excess of substrates, the removal of ionisation (amines) within the solid support and the improved solvation of the hydrophobic (Fmoc protected) acyl donors within the PEGA. This publication provided a method for enzymatic synthesis of peptides allowing for high enantioselectivity and without the need of side chain protection that is required in conventional chemical synthesis.83 766

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Within a following publication Flitsch et al84 went on to develop this method to obtain L,L and L,D diastereoisomers of dipeptides and L-amino acids in good yields starting from enantiomeric mixtures of amino acids using the enantioselectivity of the enzyme (thermolysin) catalysed reactions.84 Recently, Flitsch et al85 investigated the main factors controlling the enzymatic synthesis of peptides. From the three main factors described in their first publication it was determined that reduction in the unfavourable hydrophobic hydration of the Fmoc group within the solid support compared with the free amino acid in solution was the most important driving force in the enzyme catalysed synthesis.85 A separate publication was also produced documenting enzymatic synthesis on PEGA by another group of researchers. Burkart and co-workers published describing a biomimetic approach to the synthesis of a cyclic peptide antibiotic,86Figure 2–16 a scheme is shown in Figure 2–16 A. A peptidic linkage was first synthesised on the resin by traditional chemical means, this was structurally homologous to the tether found naturally (phosphotpanteheine, PCP). A linear decapeptide, the substrate for the enzymatic cyclisation was constructed from the linker (via an ester bond) via conventional SPPS. Incubation of these functionalised PEGA particles with TycC TE (the isolated C-terminal thioesterase domain excised from the larger synthetase protein found naturally) resulted in the release of the cyclised product (tyrocidine A, a cationic peptide antimicrobial). By employing SPPS to produce a library of decapeptides in which the fourth amino acid (Dphenylalanine) was substituted for either natural or non-natural amino acids an insight of the enzymology of TycC TE was obtained (Figure 2–16 B). Furthermore, the effective substrates for the enzymatic cyclisation were investigated as artificial analogies of the cyclic peptide antibiotic.86 This paper demonstrated the enzymatic synthesis of cyclic peptides on solid supports (PEGA) and how this methodology may be exploited to investigate the specificity of the enzyme. This allowed for the production chemical analogies of the peptide offering the possibility of finding peptides with greater therapeutic effect.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 2–16. Biomimetic synthesis and optimization of cyclic peptide antibiotics. A: Natural versus biomimetic macrocycle synthesis. The enzymatic assembly line involved in biosynthesis of the cyclic cationic antimicrobial peptide, tyrocidine A. A carrier protein (PCP) in each module is loaded with a phosphopantetheine prosthetic group (red). The individual modules contain domains that load amino-acid building blocks onto the thioester tether and condense via successive peptide bond giving the terminal PCP domain loaded with a linear decapeptide. The terminal thioesterase domain (TE) catalyses head-to-tail cyclisation. In the biomimetic synthetic strategy, a linker (red) that mimics phosphopantetheine was chemically synthesized onto a solid-phase resin. Solid-phase peptide synthesis is used to construct a tethered linear peptide, which can then serve as a substrate for cyclisation by the TE domain excised from the synthetase proteins. B: Addition of the TE catalyses formation of the cyclisation product or the hydrolysis product.

2.4.3. Summary PEGA, a material initially developed as a new resin for use in continuous SPPS, offers a number of unique properties that make it highly suitable for the preparation of peptides. These peptides covalently attached to solid supports can then be exposed to enzymes in aqueous solutions. This concept has been used to screen for protease inhibitors and determine protease specificity. Effective methods have been shown that have allowed enzyme activity within the hydrated polymer to be better understood, as well as providing useful tools for future researchers. Additionally, PEGA polymers have been created that contain permanent charges that give increased yields for reactions catalysed by enzymes with the opposing charge.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Protease responsive PEGA particles have been demonstrate through the incorporation of charged peptide actuators (described in detail in section Electrostatic interactions between charges present in pendant actuators) Finally, PEGA has been used to demonstrate enzymatic synthesis of peptides on solid supports and employed to use a biomimetic approach for the partially enzymatic synthesis of cyclic peptides allowing for the screening of effective substrates and production of analogies.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2.5. Microfluidic polymerisation of particles Polymerisation processes in which a monomer solution is dispersed within another immiscible solvent may overcome many problems that occur with other polymerisation techniques such as autoacceleration and heat transfer limitations.87 There

are

numerous

techniques

(such

as

suspension

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PjxrZXl3b3JkPkVNVUxTSUZJQ0FUSU9OIFRFQ0hOSVFVRTwva2V5d29yZD4 8a2V5d29yZD5NQUNS T1BPUk9VUyBDT1BPTFlNRVJTPC9rZXl3b3JkPjwva2V5d29yZHM+PGRhdGV zPjx5ZWFyPjIwMDA8 L3llYXI+PHB1Yi1kYXRlcz48ZGF0ZT5KYW48L2RhdGU+PC9wdWItZGF0ZX M+PC9kYXRlcz48aXNi bj4wOTI3LTc3NTc8L2lzYm4+PGFjY2Vzc2lvbi1udW0+SVNJOjAwMDA4NDc3 NDIwMDAwNDwvYWNj ZXNzaW9uLW51bT48d29yay10eXBlPlJldmlldzwvd29yay10eXBlPjx1cmxzPjxyZ WxhdGVkLXVy bHM+PHVybD4mbHQ7R28gdG8gSVNJJmd0OzovLzAwMDA4NDc3NDIwMDA wNDwvdXJsPjwvcmVsYXRl ZC11cmxzPjwvdXJscz48bGFuZ3VhZ2U+RW5nbGlzaDwvbGFuZ3VhZ2U+PC9y ZWNvcmQ+PC9DaXRl PjwvRW5kTm90ZT5= 88 and emulsion polymerisation89) used to form discrete polymer particles through the formation of a monomer emulsion however, these are “top-down” approaches where the mixing of the two liquids occurs as a bulk process. This leads to little control over the formation of individual droplet dimensions and therefore typically results in particles with a relatively broad size distribution. Ideally a “bottom-up” approach to emulsification in which the formation of each individual droplet is controlled and defined would be ideal. One strategy to achieve these objectives is based on microfluidic devices. A early example of the controlled formation of liquid dispersions using a microfluidic flow-focusing device (MFFD) was shown by Stone and co-workers.90Figure 2– 17Figure 2–17 Here, a pressure gradient along the long axis of the device forced the two immiscible liquids through the orifice of the MFFD. The continuous phase (supplied by the two outside channels) surrounded the dispersed phase (flowing through the central channel) (Figure 2–17 A) causing the inner flow to become unstable. This flow then broke in the orifice to give discrete droplets which then entered the outlet channel (Figure 2–17 B & C). The planar microchannel design was made by soft lithography in poly(dimethylsiloxane) (PDMS) and the smallest droplets produced were much smaller than the orifice radius. Ultimately the flow ratios of the water and oil through the device determined the size of droplets sizes formed.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 2–17. Formation of dispersions using "flow focusing" in microchannels. A: Flowfocusing geometry implemented in a microfluidic device. An orifice is placed downstream of three coaxial inlet streams. Water flows in the central channel and oil flows in the two outer channels. B & C: Droplets are formed at the orifice and move into the outlet channel 90

In 2005 the idea of combining microfluidic droplet formation with a polymerisation process was utilised to produce polymer particles.91 By dissolving the monomer in the dispersed phase uniform monomer droplets were produced, a method of initiation was then included to give polymer particles. These systems typically use UV radiation to initiate polymerisation although interfacial

polymerisation and

thermally initiation have also been used.92 Whitesides and co-workers produced a MFFD in either PDMS or polyurethane, this allowed both water-in-oil and oil-in-water dispersion to be produced.91 They found that it was possible to also produce uniform non-spherical particles by restricting the dimensions of the outlet channel. If one of the dimensions is less than that of the diameter of a regular droplet then disks were formed, by restricting both the height and the width of the channel polymer ellipsoids or rods were produced. A number of different monomer were used and with a single MFFD device they produced up to

775

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

250 polymer particles per second with a polydispersity (defined as the standard deviation in the particle diameter divided by the mean particle diameter) of 1.5 %.91 At the same time Whitesides and co-workers also published a variation of the flowfocusing microfluidic device in the form of an axisymmetric flow-focusing microfluidic device (AFFD) (Figure 2–18 A).93 This device was fabricated from PDMS but rather than using photolithography to create microchannels, glass microfibres and polyethylene tubing were used to template the design (Figure 2–18 B). The main advantage of an axisymmetric design over the conventional setup was that AFFD confines droplets to the central axis of the channel, protecting droplets from shear or damage resulting from adhesion or wetting of the walls of the outlet channel. Polymer membranes of Nylon-6,6 enclosing aqueous solutions of either ions or superparamagnetic particles were produced by interfacial polymerisation. The polydispersity or coefficient of variation (CV) of these microcapsules was approximately 5 %. The orientation of the device was found to be very important due to the relative densities of the two liquids, the dispersed phase (water) had a higher density than the continuous phase (hexadecane). This would lead to the droplets settling against the floor of the channel where they formed a high volume fraction emulsion, this altered the flow of the continuous phase providing a higher resistance to flow in the outlet channel, increasing the pressure in the orifice region. Through the vertical orientation of the device these problems were avoided (as the flow and gravity were in the same orientation) (Figure 2–18 C). The interfacial polymerisation could be quenched by flowing the nylon coated droplets into a beaker of dodecan-1-ol in hexadecane (Figure 2–18 D).93

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 2–18. An axisymmetric flow-focusing microfluidic device (AFFD) A: An axisymmetrical flow focussing channel, composed of a cylindrical tube with a narrow cross-section halfway along its length. The narrow region serves as the orifice where fluid is focused and breaks into aqueous droplets. B: A scheme depicting the fabrication process of an AFFD. C: a) The diameter of droplets at various flow rates of continuous phase. (b & c) images showing droplets created in the AFFD oriented (b) horizontally and (c) vertically. D: a) A collection of nylon-6,6coated aqueous droplets. b) A coated-droplet containing 50 nm diameter magnetic particles in an applied magnetic field.93

McQuade and co-workers described a simplified microfluidic device that produced capsules by interfacial polymerisation without the need for any form of microfabrication.92 This setup required only needles and tubing (Figure 2–19 A); the continuous phase (30 % w/v aqueous solution of glycerol) flowed through the tubing and the dispersed phase (3:1 cyclohexane/chloroform with 2 % Tween 80 (surfactant)) was introduced via a 30 gauge needle inserted through the wall of the tubing into the middle of the channel (Figure 2–19 B). Analogous to Whiteside and co-workers’ axisymmetric device, droplets produced with the needle and tubing were entirely surrounded by continuous phase (coaxial flow) with the additional advantage that any blocking of the device was very simple to overcome by replacing the tubing and/or needle. Oil filled polyamide capsules were produced by introducing polyethyeneimine (PEI) to the continuous phase and sebacoyl and trimesoyl chloride to the dispersed phase. By varying the continuous flow rate

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

capsules between 313 - 865 µm in diameter with a coefficient of variation (CV) of 3.3 - 8.6 % were obtained (Figure 2–19 C).92

Figure 2–19. Interfacial Polymerization within a Simplified Microfluidic Device: Capturing Capsules. A: Photograph of fluidic device including needle and dye-filled organic droplets dispersed in the continuous aqueous phase. B: Schematic of fluidic device. C: Light microscope images of capsules in water formed with constant organic flow rate (0.141 mL min-1) and increasing aqueous flow rate (clockwise). 92

Kumacheva and co-workers published two papers on the use of microfluidic reactors for

the

production

of

polymer

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

kt1bWFjaGV2YSwg RS48L2F1dGhvcj48L2F1dGhvcnM+PC9jb250cmlidXRvcnM+PGF1dGgtYWRkc mVzcz5Vbml2IFRv cm9udG8sIERlcHQgQ2hlbSwgVG9yb250bywgT04gTTVTIDNINiwgQ2FuYWRh LiYjeEQ7S3VtYWNo ZXZhLCBFLCBVbml2IFRvcm9udG8sIERlcHQgQ2hlbSwgODAgU3QgR2Vvcmd lIFN0LCBUb3JvbnRv LCBPTiBNNVMgM0g2LCBDYW5hZGEuJiN4RDtla3VtYWNoZUBjaGVtLnV0b 3JvbnRvLmNhPC9hdXRo LWFkZHJlc3M+PHRpdGxlcz48dGl0bGU+UG9seW1lciBwYXJ0aWNsZXMgd2l0 aCB2YXJpb3VzIHNo YXBlcyBhbmQgbW9ycGhvbG9naWVzIHByb2R1Y2VkIGluIGNvbnRpbnVvdXM gbWljcm9mbHVpZGlj IHJlYWN0b3JzPC90aXRsZT48c2Vjb25kYXJ5LXRpdGxlPkpvdXJuYWwgb2Ygd GhlIEFtZXJpY2Fu IENoZW1pY2FsIFNvY2lldHk8L3NlY29uZGFyeS10aXRsZT48YWx0LXRpdGxlP kouIEFtLiBDaGVt LiBTb2MuPC9hbHQtdGl0bGU+PC90aXRsZXM+PHBlcmlvZGljYWw+PGZ1bG wtdGl0bGU+Sm91cm5h bCBvZiB0aGUgQW1lcmljYW4gQ2hlbWljYWwgU29jaWV0eTwvZnVsbC10aXR sZT48YWJici0xPkou IEFtLiBDaGVtLiBTb2MuPC9hYmJyLTE+PC9wZXJpb2RpY2FsPjxhbHQtcGVya W9kaWNhbD48ZnVs bC10aXRsZT5Kb3VybmFsIG9mIHRoZSBBbWVyaWNhbiBDaGVtaWNhbCBTb2 NpZXR5PC9mdWxsLXRp dGxlPjxhYmJyLTE+Si4gQW0uIENoZW0uIFNvYy48L2FiYnItMT48L2FsdC1wZ XJpb2RpY2FsPjxw YWdlcz44MDU4LTgwNjM8L3BhZ2VzPjx2b2x1bWU+MTI3PC92b2x1bWU+PG 51bWJlcj4yMjwvbnVt YmVyPjxrZXl3b3Jkcz48a2V5d29yZD5NSUNST0NIQU5ORUwgRU1VTFNJRkl DQVRJT048L2tleXdv cmQ+PGtleXdvcmQ+UE9MWUVMRUNUUk9MWVRFIE1JQ1JPQ0FQU1VMR VM8L2tleXdvcmQ+PGtleXdv cmQ+QVFVRU9VUzwva2V5d29yZD48a2V5d29yZD5ESVNQRVJTSU9OUzwva 2V5d29yZD48a2V5d29y 783

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZD5TSEVMTCBNSUNST0NBUFNVTEVTPC9rZXl3b3JkPjxrZXl3b3JkPlBIQVN FLVNFUEFSQVRJT048 L2tleXdvcmQ+PGtleXdvcmQ+QlVJTERJTkctQkxPQ0tTPC9rZXl3b3JkPjxrZXl3b 3JkPkxBVEVY LVBBUlRJQ0xFUzwva2V5d29yZD48a2V5d29yZD5MSVFVSUQgSkVUPC9rZXl 3b3JkPjxrZXl3b3Jk PkZMT1c8L2tleXdvcmQ+PGtleXdvcmQ+TUlDUk9FTkNBUFNVTEFUSU9OPC 9rZXl3b3JkPjwva2V5 d29yZHM+PGRhdGVzPjx5ZWFyPjIwMDU8L3llYXI+PHB1Yi1kYXRlcz48ZGF0 ZT5KdW48L2RhdGU+ PC9wdWItZGF0ZXM+PC9kYXRlcz48aXNibj4wMDAyLTc4NjM8L2lzYm4+PGF jY2Vzc2lvbi1udW0+ SVNJOjAwMDIyOTYxOTUwMDA0MDwvYWNjZXNzaW9uLW51bT48d29yay1 0eXBlPkFydGljbGU8L3dv cmstdHlwZT48dXJscz48cmVsYXRlZC11cmxzPjx1cmw+Jmx0O0dvIHRvIElTSSZ ndDs6Ly8wMDAy Mjk2MTk1MDAwNDA8L3VybD48L3JlbGF0ZWQtdXJscz48L3VybHM+PGVsZ WN0cm9uaWMtcmVzb3Vy Y2UtbnVtPjEwLjEwMjEvamEwNDI0OTR3PC9lbGVjdHJvbmljLXJlc291cmNlL W51bT48bGFuZ3Vh Z2U+RW5nbGlzaDwvbGFuZ3VhZ2U+PC9yZWNvcmQ+PC9DaXRlPjxDaXRlPjx BdXRob3I+U2VvPC9B dXRob3I+PFllYXI+MjAwNTwvWWVhcj48UmVjTnVtPjIyNDwvUmVjTnVtPjxy ZWNvcmQ+PHJlYy1u dW1iZXI+MjI0PC9yZWMtbnVtYmVyPjxmb3JlaWduLWtleXM+PGtleSBhcHA9I kVOIiBkYi1pZD0i ZmE5emR6c3Zrc3gycHJlcmU5OHhkNTJxZGZ3ZndyOTBwNXMyIj4yMjQ8L2tle T48L2ZvcmVpZ24t a2V5cz48cmVmLXR5cGUgbmFtZT0iSm91cm5hbCBBcnRpY2xlIj4xNzwvcmVm LXR5cGU+PGNvbnRy aWJ1dG9ycz48YXV0aG9ycz48YXV0aG9yPlNlbywgTS48L2F1dGhvcj48YXV0a G9yPk5pZSwgWi4g SC48L2F1dGhvcj48YXV0aG9yPlh1LCBTLiBRLjwvYXV0aG9yPjxhdXRob3I+T W9rLCBNLjwvYXV0 aG9yPjxhdXRob3I+TGV3aXMsIFAuIEMuPC9hdXRob3I+PGF1dGhvcj5HcmFoY 784

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

W0sIFIuPC9hdXRo b3I+PGF1dGhvcj5LdW1hY2hldmEsIEUuPC9hdXRob3I+PC9hdXRob3JzPjwvY29 udHJpYnV0b3Jz PjxhdXRoLWFkZHJlc3M+VW5pdiBUb3JvbnRvLCBEZXB0IENoZW0sIFRvcm9 udG8sIE9OIE01UyAz SDYsIENhbmFkYS4mI3hEO0t1bWFjaGV2YSwgRSwgVW5pdiBUb3JvbnRvLCB EZXB0IENoZW0sIDgw IFN0IEdlb3JnZSBTdCwgVG9yb250bywgT04gTTVTIDNINiwgQ2FuYWRhLiYje EQ7ZWt1bWFjaGVA YWxjaGVteS5jaGVtLnV0b3JvbnRvLmNhPC9hdXRoLWFkZHJlc3M+PHRpdGxl cz48dGl0bGU+Q29u dGludW91cyBtaWNyb2ZsdWlkaWMgcmVhY3RvcnMgZm9yIHBvbHltZXIgcGFy dGljbGVzPC90aXRs ZT48c2Vjb25kYXJ5LXRpdGxlPkxhbmdtdWlyPC9zZWNvbmRhcnktdGl0bGU+P GFsdC10aXRsZT5M YW5nbXVpcjwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2RpY2FsPjxmdWxs LXRpdGxlPkxhbmdt dWlyPC9mdWxsLXRpdGxlPjxhYmJyLTE+TGFuZ211aXI8L2FiYnItMT48L3Blc mlvZGljYWw+PGFs dC1wZXJpb2RpY2FsPjxmdWxsLXRpdGxlPkxhbmdtdWlyPC9mdWxsLXRpdGxl PjxhYmJyLTE+TGFu Z211aXI8L2FiYnItMT48L2FsdC1wZXJpb2RpY2FsPjxwYWdlcz4xMTYxNC0xM TYyMjwvcGFnZXM+ PHZvbHVtZT4yMTwvdm9sdW1lPjxudW1iZXI+MjU8L251bWJlcj48a2V5d29yZ HM+PGtleXdvcmQ+ RU1QTE9ZSU5HIE1JQ1JPQ0hBTk5FTCBFTVVMU0lGSUNBVElPTjwva2V5d2 9yZD48a2V5d29yZD5E Uk9QTEVUIEZPUk1BVElPTjwva2V5d29yZD48a2V5d29yZD5QT0xZR09OQUw 8L2tleXdvcmQ+PGtl eXdvcmQ+Q0FQSUxMQVJJRVM8L2tleXdvcmQ+PGtleXdvcmQ+U0laRSBESV NUUklCVVRJT05TPC9r ZXl3b3JkPjxrZXl3b3JkPkxPTkcgQlVCQkxFUzwva2V5d29yZD48a2V5d29yZD5 GTE9XPC9rZXl3 b3JkPjxrZXl3b3JkPk1JQ1JPU1BIRVJFUzwva2V5d29yZD48a2V5d29yZD5HRU5 FUkFUSU9OPC9r 785

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZXl3b3JkPjxrZXl3b3JkPkRFVklDRTwva2V5d29yZD48a2V5d29yZD5DSElQPC9 rZXl3b3JkPjwv a2V5d29yZHM+PGRhdGVzPjx5ZWFyPjIwMDU8L3llYXI+PHB1Yi1kYXRlcz48 ZGF0ZT5EZWM8L2Rh dGU+PC9wdWItZGF0ZXM+PC9kYXRlcz48aXNibj4wNzQzLTc0NjM8L2lzYm4 +PGFjY2Vzc2lvbi1u dW0+SVNJOjAwMDIzMzczMDIwMDAxNTwvYWNjZXNzaW9uLW51bT48d29 yay10eXBlPkFydGljbGU8 L3dvcmstdHlwZT48dXJscz48cmVsYXRlZC11cmxzPjx1cmw+Jmx0O0dvIHRvIEl TSSZndDs6Ly8w MDAyMzM3MzAyMDAwMTU8L3VybD48L3JlbGF0ZWQtdXJscz48L3VybHM+ PGVsZWN0cm9uaWMtcmVz b3VyY2UtbnVtPjEwLjEwMjEvbGEwNTA1MTllPC9lbGVjdHJvbmljLXJlc291cm NlLW51bT48bGFu Z3VhZ2U+RW5nbGlzaDwvbGFuZ3VhZ2U+PC9yZWNvcmQ+PC9DaXRlPjwvR W5kTm90ZT5= 94,95Figure 2–20Figure 2–20 The first paper demonstrated a novel approach to the continuous production of core-shell droplets and polymer capsules. In this work, the microfluidic reactor was fabricated in polyurethane by softlithography to give a MFFD with five separate channels approaching the orifice. This design allowed three immiscible liquids to be supplied to the flow-focussing region; the outer channels contained an aqueous solution containing surfactant (sodium dodecyl sulphate) while the middle channels contained the oil phase (silicone oil) and the monomer phase (tripropyleneglycol diacrylate (TPGDA) or ethyleneglycol dimethacrylate (EGDMA)) flowed through the centre channel. Photoinitiator was contained in the monomer phase and SPAN 80 in the oil phase. Upon a pressure gradient along the long axis of the axis of the MFFD the three liquids were forced through the orifice and the continuous water phase surrounded the monomer-oil thread which adopted a circular cross-section (Figure 2–20 A). To minimise interfacial tension this coaxial jet broke into segments of with a spherical shape. These monomer droplets were then photopolymerised in the wavy channel (this shape maximises the curing time with minimum size) of the microfluidic reactor. By controlling the flow rates of the three phases the diameter of the cores, the size of the core-shell droplets and the thickness of the shells were controlled. Generally if the ratio of flow rates of outer to inner phases was increased the size of droplets was reduced due to the increased shear stress imposed on the undulated jet 786

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of the dispersant liquid. If the flow rate of the aqueous phase was increased smaller droplets with thinner cores were formed (with thicker shells). While at higher oil phase flow rates the diameter of the cores increased and the shell thickness decreased. It was also demonstrated that it was possible to control the number of cores within the droplets. This was achieved by varying the value of interfacial capillary wavelength and shifting the length and phases of capillary waves (undulations). The oil cores could be removed after the photopolymerisation of the monomer by using acetone to obtain particles with different shapes (Figure 2–20 B). Overall productivity of the microfluidic reactor was 200 to 1000 s -1 with particles polydispersity not exceeding 2.5%.94

Figure 2–20. Polymer particles with various shapes and morphologies produced in continuous microfluidic reactors. A: (a) Schematic of production of droplets in MFFD by laminar co-flow of silicone oil (A), monomer (B), and aqueous (C) phases. (b) Schematic of the wavy channel used for photopolymerization of monomer in core-shell droplets. (c) Optical microscopy image of core-shell droplets. The scale bar is 200 µm. (d) Photograph of a PU microfluidic system. The arrow is pointing to the orifice. B: (a-e) Scanning electron microscopy images of polymer

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald microparticles obtained by polymerizing monomer in droplets, after removing a silicon oil core. (Inset) Cross section of the core-shell particle. (f) Cross section of a polymer particle with three cores obtained by polymerizing core-shell droplets with three cores. Scale bar is 40 µm.94

A second paper by the Kumacheva group95Figure 2–21Figure 2–21Figure 2– 21Figure 2–21 that was published at the same time was a comprehensive paper detailing the use of MFFD to produce polymer particles (Figure 2–21 A). As in the Whitesides and co-workers MFFD publication, these devices were produced using soft lithography in either poly(dimethylsiloxane) (PDMS) or polyurethane (PU). Here, four different multifunctional acrylate monomers were emulsified. Three major regimes in the formation of monomer droplets were found; at low flow rates for the aqueous continuous phase (Qc) and low flow ratios of continuous to dispersed phases (Qc/Qd) the device operated in a dripping regime with the monomeric thread breaking into monomer droplets behind the orifice with droplet size significantly larger than the orifice size. The second flow regime observed was at moderate values of Qc and Qc/Qd in which the monomer droplets were generated by the breaking up of the monomer thread in or behind the orifice, upon releasing a droplet the monomeric thread retracted break upstream. The final flow regime was jetting mode and this was observed at high flow rates and high Qc/Qd, the monomeric thread remained behind the orifice breaking into droplets (Figure 2–21 B). Under certain conditions the formation of smaller satellite droplets were observed along with the main droplet population. The size of the droplets produced was shown to be governed by the properties of the monomer liquid (viscosity and interfacial tension) and the flow rates of the continuous and dispersed phases. Therefore, the conditions with which monomer droplets with a very narrow size distribution varied for each monomer and by varying these conditions along with the design of the MFFD particularly the size of the orifice droplets (Figure 2–21 C) as small as 18 µm were formed. By adding photoinitiator to the monomer liquid and exposing the monomer droplets to UV light after the orifice polymer particles were formed, additionally, it was demonstrated that by changing the dimensions of the channel in which polymerisation took place different shape particles could be obtained. These included discs of various morphologies if the height of the channel was less that the initial diameter of the monomer droplet or rods if both the height and width of the channel were less than the diameter (Figure 2–21 D). This paper provided an indepth study of the use of MFFDs to produce monodisperse polymer particles.95

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Figure 2–21. Continuous microfluidic reactors for polymer particles. A:(a) Schematic of droplet formation

in

the

microfluidic

flow-focusing

device.

(b)

Wavy

channel

for

the

photopolymerization of monomer droplets. B: Breakup of the monomer thread in 2 wt % aqueous SDS solution. (a) Regime 1, (b) Regime 2, (c) Regime 3. C: SEM image of polymer particles. Scale bar is 100 µm. D: Schematic (a-c) and optical microscopy (a’-c’) images of polymer particles with different shapes: microspheres (a, a’), disks (b, b’), (c, c’) and rods of obtained via photopolymerization of droplets. Scale bar is 50 µm.95

An alternative microfluidic reactor has been described by Goddard et al,96 this system utilises a 35 µm diameter hole through which the dispersed phase was introduced into spiral channel where photopolymerisation took place (Figure 2–22 A & B). This device was fashioned from a polycarbonate sheet using a precision milling machine. Using this device it was possible to produce poly(methacrylate) (pMA) particles with a low CV (typically around 2%) in a range of diameters between 10-115 µm (Figure 2–22 C). Additionally, it was possible to produce molecularly imprinted particles through the incorporation of propranolol into the monomer solution prior to polymerisation, these particles showed the same uptake of propranolol as particles produced by conventional suspension polymerisations.96

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Figure 2–22. A micro-reactor for preparing uniform molecularly imprinted polymer beads A: Schematic of the spiral micro-flow-reactor showing the overall layout and the sample and oil inlet points. B: Detail of the tapered region of the reactor where the monomer is introduced into the flowing continuous phase. Monomer is extruded into the flowing oil, where it breaks off into droplets. These are swept into the spiral polymerisation reactor where they are polymerised by UV light. C: SEM of typical particles produced in oil using the micro-reactor.96

A different route to prepare near monodisperse particles was demonstrated by Hadziioannou and co-workers,97 this was an axisymmetrical needle/tubing device that could be prepared without the need for any microfabrication techniques (Figure 2–23 A). Within conventional planar microfluidic devices surface modification is usually required to prevent an inverse emulsion. The axisymmetrical setup overcame this requirement by avoiding direct contact of the dispersed phase with the channel walls.93 A simple T-junction and needle were used to introduce the dispersed phase into the continuous phase, both removing the need for microfabrication and avoiding the clogging problems that microchannels often suffer from. By varying the flow rates and solvent viscosities they were able to produce poly(methylmethacylate) (poly(MMA)) particles with a narrow distribution between 150-550 μm in diameter (Figure 2–23 B & C).97

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 2–23. A predictive approach of the influence of the operating parameters on the size of polymer particles synthesized in a simplified microfluidic system. A: Schematic of the microfluidic system for the synthesis of controlled-size polymer particles. The dispersed phase is injected via a thin needle positioned along the main axis. B: Effect of flow rate ratio of continuous and dispersed phases for two different continuous phase flow rates on average diameter, Dp, of polymer particles. C: Effect of flow ratio and continuous phase viscosity on the average diameter of polymer particles.97

2.5.1. Summary Microfluidic polymerisation techniques offer a highly controlled approach to the preparation of polymer particles. There are a number of different configurations available that allow particles to be produced with a very narrow size distribution (generally with polydispersities less than 2.5 %). Additionally, by introducing further co-flowing solvents it is possible to make particles with very well defined morphologies. However, there are a number of limitations relating to the preparation of polymer particles based on microfluidics. These include: the overall production rate of particles is low. Microfluidic devices cannot be ‘scaled up’ (although this

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

might be addressed though the combination of many microfluidic reactors in parallel). Additionally, microfabrication techniques are required to construct many microfluidic devices (those fabricated by soft lithography) making these approaches inaccessible for many researchers. Overall, microfluidics presents a versatile method for the small scale production of polymer particles with very narrow size distribution and desired morphology.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2.6. Aims of thesis The overall objective of this thesis was to synthesise and characterise enzyme responsive hydrogel particles functionalisation with peptide actuators. These functionalised particles were then to be applied to the triggered release of a macromolecule payload. Firstly, PEGA microparticles (µPEGA) were to be produced and characterised with the aim of establishing the optimum support of the enzyme responsive system. Previous approaches to modifying the properties of PEGA such as the introduction of

charged

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793

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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803

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

AwMDI1NDM4NjgwMDAy MzwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxlY3Ryb25pYy1yZXNvd XJjZS1udW0+MTAu MTAzOS9iNzE0NzUwYzwvZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+PGxhbm d1YWdlPkVuZ2xpc2g8 L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+ 57,58 in order to demonstrate enzyme responsive release of an entrapped payload. Peptide actuators with enhanced functionality were to be designed and incorporated into µPEGA, the enzyme responsive behaviour of this system could then be characterised and utilised for the triggered release of an entrapped macromolecule. Finally, based on the literature, a simplified microfluidic device97 was to be used produced and used to prepare PEGA particles with the smallest diameter possible and a very narrow size distribution.

808

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3 PEGA polymerisation, characterisation and enzyme responsive swelling through functionalisation with peptide actuators2 3.1. Abstract Within this chapter the polymerisation of PEGA hydrogel microparticles (µPEGA) with either neutral, positive or negative charge were demonstrated. The swelling of these different particles was characterised and neutral µPEGA were selected for further investigation. These particles had a mean diameter of 16 µm (similar to that of biological cells) and were compatible with different enzymes. Furthermore, it was demonstrated that enzyme catalysed reactions occur faster with these microparticles than with commercially available macrobeads which are typically 200-400 µm in diameter. μPEGA was then functionalised with peptide actuators. These particles demonstrated an enzyme specific increase in accessibility allowing fluorescently labelled dextran to diffuse into the particles. The pH responsive nature of the linear peptide actuators was shown and utilised for the physical entrapment of a macromolecule payload. Release of this payload was only possible when the functionalised particles were exposed to the target enzyme. However, at physiological ionic strength no response of the particles was observed due to electrostatic screening.

3.2. Introduction PEGA is a polymer hydrogel that was developed as a resin for solid phase peptide synthesis (SPPS). A particularly attractive property of this material is its compatibility with both aqueous and organic solvents. This allows for easy chemical modification through SPPS67 as well as compatibility with aqueous solutions containing biomolecules. There has been thorough research into using enzymes, specifically

proteases

to

catalyse

reactions

on

peptides

synthesised

on

PEGA.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5CYXNzbzwvQXV0aG9yPjxZZ WFyPjIwMDM8L1llYXI+PFJl 2 Published in part as: McDonald, T. O., Christensen, S., and Ulijn, R. V., Making peg-based microparticles for applications in biology and medicine. Mater. Res. Soc. Symp. Proc 1008E (T05), 18 (2007).

809

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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63,72,74

Several workers have shown that PEGA is accessible to small enzymes and that enzyme

activity

can

occur

inside

the

resin.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5NZWxkYWw8L0F1dGhvcj48W WVhcj4xOTk0PC9ZZWFyPjxS 825

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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or

out

of

the

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

V5d29yZD48a2V5d29y ZD5MSU5LRVI8L2tleXdvcmQ+PGtleXdvcmQ+U1BFQ0lGSUNJVFk8L2tleXdvc mQ+PGtleXdvcmQ+ Q0hFTUlTVFJZPC9rZXl3b3JkPjxrZXl3b3JkPkJFQURTPC9rZXl3b3JkPjxrZXl3b 3JkPkdFTkVS QVRJT048L2tleXdvcmQ+PGtleXdvcmQ+UkVTSU48L2tleXdvcmQ+PC9rZXl3b3 Jkcz48ZGF0ZXM+ PHllYXI+MjAwMjwveWVhcj48cHViLWRhdGVzPjxkYXRlPkF1ZzwvZGF0ZT48 L3B1Yi1kYXRlcz48 L2RhdGVzPjxpc2JuPjA5NDctNjUzOTwvaXNibj48YWNjZXNzaW9uLW51bT5J U0k6MDAwMTc3NjUy ODAwMDIxPC9hY2Nlc3Npb24tbnVtPjx3b3JrLXR5cGU+QXJ0aWNsZTwvd29ya y10eXBlPjx1cmxz PjxyZWxhdGVkLXVybHM+PHVybD4mbHQ7R28gdG8gSVNJJmd0OzovLzAwM DE3NzY1MjgwMDAyMTwv dXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48bGFuZ3VhZ2U+RW5nbGlzaDwvb GFuZ3VhZ2U+PC9y ZWNvcmQ+PC9DaXRlPjwvRW5kTm90ZT5= 63 Further details on the development and further investigation of enzyme reactions on PEGA polymers are outlined in section PEGA within the literature review. Currently, PEGA particles are commercially available between 200 - 400 µm in diameter, for convenience in solid phase chemistry applications.72 Depending on the biomedical applications smaller size ranges are desirable (such as injected into tissue (<200 μm), inhaled (<100 μm) or released into circulation (<10 μm).99 Smaller particles also offer an increase the rate of response due to their greater surface area to volume ratio.55 Additionally, microparticles allow for possible use in automated analysis of ‘on-bead’ libraries using a cell sorter. Therefore, PEGA microparticles were prepared that are smaller in size, thereby enhancing responses, but still conveniently handled for solid phase synthesis and analysis by fluorescence microscopy.100 Work carried out in the author’s MSc project investigated the effect of stirring speed and surfactant concentration on mean particle size.101 Numerous stimuli have been exploited in the development of responsive materials in a biomedical context including pH,102 temperature,9 ionic strength35 and small molecules (e.g. glucose).36 Bioresponsive materials21 are stimuli-responsive 848

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

surfaces, self-assembled structures or chemically crosslinked polymers that change their properties in response to biochemical recognition events, offering potential applications

in

biosensing,103

tissue

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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dGU+

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

materials is determined by a biorecognition moiety that, upon a recognition event, actuates structural changes in the material. In systems established using hydrogels there are three main categories of molecular actuation based on changes in: (i) crosslinking

density,

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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RW5kTm90ZT4A

11,27,28,41

(ii)

electrostatic

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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36,56,57

or

(iii)

conformation

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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to

trigger

molecular

actuation

in

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885

polymer

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald PjxkYXRlcz48eWVhcj4yMDA3PC95ZWFyPjwvZGF0ZXM+PGlzYm4+MTQzMy03ODUxPC9pc2JuPjxh Y2Nlc3Npb24tbnVtPklTSTowMDAyNTA5OTAwMDAwMjc8L2FjY2Vzc2lvbi1udW0+PHdvcmstdHlw ZT5BcnRpY2xlPC93b3JrLXR5cGU+PHVybHM+PHJlbGF0ZWQtdXJscz48dXJsPiZsdDtHbyB0byBJ U0kmZ3Q7Oi8vMDAwMjUwOTkwMDAwMDI3PC91cmw+PC9yZWxhdGVkLXVybHM+PC91cmxzPjxlbGVj dHJvbmljLXJlc291cmNlLW51bT4xMC4xMDAyL2FuaWUuMjAwNzAyNDM4PC9lbGVjdHJvbmljLXJl c291cmNlLW51bT48bGFuZ3VhZ2U+RW5nbGlzaDwvbGFuZ3VhZ2U+PC9yZWNvcmQ+PC9DaXRlPjwv PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5aaGFuZzwvQXV0aG9yPjxZZWFyPjIwMDc8L1llYXI+PFJl Y051bT45NzwvUmVjTnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+OTc8L3JlYy1udW1iZXI+PGZvcmVp Z24ta2V5cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJmYTl6ZHpzdmtzeDJwcmVyZTk4eGQ1MnFkZndm d3I5MHA1czIiPjk3PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG5hbWU9IkpvdXJuYWwg QXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcnM+PGF1dGhvcj5aaGFu ZywgUi48L2F1dGhvcj48YXV0aG9yPkJvd3llciwgQS48L2F1dGhvcj48YXV0aG9yPkVpc2VudGhh bCwgUi48L2F1dGhvcj48YXV0aG9yPkh1YmJsZSwgSi48L2F1dGhvcj48L2F1dGhvcnM+PC9jb250 cmlidXRvcnM+PHRpdGxlcz48dGl0bGU+QSBzbWFydCBtZW1icmFuZSBiYXNlZCBvbiBhbiBhbnRp Z2VuLXJlc3BvbnNpdmUgaHlyb2dlbDwvdGl0bGU+PHNlY29uZGFyeS10aXRsZT5CaW90ZWNobm9s b2d5IGFuZCBCaW9lbmdpbmVlcmluZzwvc2Vjb25kYXJ5LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2Rp Y2FsPjxmdWxsLXRpdGxlPkJpb3RlY2hub2xvZ3kgYW5kIEJpb2VuZ2luZWVyaW5nPC9mdWxsLXRp dGxlPjwvcGVyaW9kaWNhbD48cGFnZXM+OTc2LTk4NDwvcGFnZXM+PHZvbHVtZT45Nzwvdm9sdW1l PjxudW1iZXI+NDwvbnVtYmVyPjxkYXRlcz48eWVhcj4yMDA3PC95ZWFyPjxwdWItZGF0ZXM+PGRh dGU+SnVsIDE8L2RhdGU+PC9wdWItZGF0ZXM+PC9kYXRlcz48YWNjZXNzaW9uLW51bT5JU0k6MDAw MjQ3MTU4ODAwMDMwPC9hY2Nlc3Npb24tbnVtPjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD4mbHQ7 R28gdG8gSVNJJmd0OzovLzAwMDI0NzE1ODgwMDAzMDwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJs cz48L3JlY29yZD48L0NpdGU+PENpdGU+PEF1dGhvcj5QYXRlbDwvQXV0aG9yPjxZZWFyPjIwMDg8 L1llYXI+PFJlY051bT44MzwvUmVjTnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+ODM8L3JlYy1udW1i ZXI+PGZvcmVpZ24ta2V5cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJmYTl6ZHpzdmtzeDJwcmVyZTk4 eGQ1MnFkZndmd3I5MHA1czIiPjgzPC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG5hbWU9 IkpvdXJuYWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcnM+PGF1 dGhvcj5QYXRlbCwgSy48L2F1dGhvcj48YXV0aG9yPkFuZ2Vsb3MsIFMuPC9hdXRob3I+PGF1dGhv cj5EaWNodGVsLCBXLiBSLjwvYXV0aG9yPjxhdXRob3I+Q29za3VuLCBBLjwvYXV0aG9yPjxhdXRo b3I+WWFuZywgWS4gVy48L2F1dGhvcj48YXV0aG9yPlppbmssIEouIEkuPC9hdXRob3I+PGF1dGhv cj5TdG9kZGFydCwgSi4gRi48L2F1dGhvcj48L2F1dGhvcnM+PC9jb250cmlidXRvcnM+PHRpdGxl cz48dGl0bGU+RW56eW1lLXJlc3BvbnNpdmUgc25hcC10b3AgY292ZXJlZCBzaWxpY2EgbmFub2Nv bnRhaW5lcnM8L3RpdGxlPjxzZWNvbmRhcnktdGl0bGU+Sm91cm5hbCBvZiB0aGUgQW1lcmljYW4g Q2hlbWljYWwgU29jaWV0eTwvc2Vjb25kYXJ5LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2RpY2FsPjxm dWxsLXRpdGxlPkpvdXJuYWwgb2YgdGhlIEFtZXJpY2FuIENoZW1pY2FsIFNvY2lldHk8L2Z1bGwt

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald dGl0bGU+PGFiYnItMT5KLiBBbS4gQ2hlbS4gU29jLjwvYWJici0xPjwvcGVyaW9kaWNhbD48cGFn ZXM+MjM4Mi0yMzgzPC9wYWdlcz48dm9sdW1lPjEzMDwvdm9sdW1lPjxudW1iZXI+ODwvbnVtYmVy PjxkYXRlcz48eWVhcj4yMDA4PC95ZWFyPjxwdWItZGF0ZXM+PGRhdGU+RmViIDI3PC9kYXRlPjwv cHViLWRhdGVzPjwvZGF0ZXM+PGFjY2Vzc2lvbi1udW0+SVNJOjAwMDI1MzQwMDkwMDAwNTwvYWNj ZXNzaW9uLW51bT48dXJscz48cmVsYXRlZC11cmxzPjx1cmw+Jmx0O0dvIHRvIElTSSZndDs6Ly8w MDAyNTM0MDA5MDAwMDU8L3VybD48L3JlbGF0ZWQtdXJscz48L3VybHM+PC9yZWNvcmQ+PC9DaXRl PjxDaXRlPjxBdXRob3I+RHV4YnVyeTwvQXV0aG9yPjxZZWFyPjIwMDc8L1llYXI+PFJlY051bT4y OTI8L1JlY051bT48cmVjb3JkPjxyZWMtbnVtYmVyPjI5MjwvcmVjLW51bWJlcj48Zm9yZWlnbi1r ZXlzPjxrZXkgYXBwPSJFTiIgZGItaWQ9ImZhOXpkenN2a3N4MnByZXJlOTh4ZDUycWRmd2Z3cjkw cDVzMiI+MjkyPC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG5hbWU9IkpvdXJuYWwgQXJ0 aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcnM+PGF1dGhvcj5EdXhidXJ5 LCBDLiBKLjwvYXV0aG9yPjxhdXRob3I+SGlsa2VyLCBJLjwvYXV0aG9yPjxhdXRob3I+ZGUgV2ls ZGVtYW4sIFMuIE0uIEEuPC9hdXRob3I+PGF1dGhvcj5IZWlzZSwgQS48L2F1dGhvcj48L2F1dGhv cnM+PC9jb250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5EU00gUmVzICZhbXA7IFBhdGVudHMsIE5M LTYxNjAgTUQgR2VsZWVuLCBOZXRoZXJsYW5kcy4mI3hEO0hlaXNlLCBBLCBEU00gUmVzICZhbXA7 IFBhdGVudHMsIFBPIEJveCAxOCwgTkwtNjE2MCBNRCBHZWxlZW4sIE5ldGhlcmxhbmRzLiYjeEQ7 YW5kcmVhcy5oZWlzZUBkc20uY29tPC9hdXRoLWFkZHJlc3M+PHRpdGxlcz48dGl0bGU+RW56eW1l LXJlc3BvbnNpdmUgbWF0ZXJpYWxzOiBDaGlyYWxpdHkgdG8gcHJvZ3JhbSBwb2x5bWVyIHJlYWN0 aXZpdHk8L3RpdGxlPjxzZWNvbmRhcnktdGl0bGU+QW5nZXdhbmR0ZSBDaGVtaWUtSW50ZXJuYXRp b25hbCBFZGl0aW9uPC9zZWNvbmRhcnktdGl0bGU+PGFsdC10aXRsZT5Bbmdldy4gQ2hlbS4tSW50 LiBFZGl0LjwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2RpY2FsPjxmdWxsLXRpdGxlPkFuZ2V3 YW5kdGUgQ2hlbWllLUludGVybmF0aW9uYWwgRWRpdGlvbjwvZnVsbC10aXRsZT48YWJici0xPkFu Z2V3LiBDaGVtLi1JbnQuIEVkaXQuPC9hYmJyLTE+PC9wZXJpb2RpY2FsPjxhbHQtcGVyaW9kaWNh bD48ZnVsbC10aXRsZT5Bbmdld2FuZHRlIENoZW1pZS1JbnRlcm5hdGlvbmFsIEVkaXRpb248L2Z1 bGwtdGl0bGU+PGFiYnItMT5Bbmdldy4gQ2hlbS4tSW50LiBFZGl0LjwvYWJici0xPjwvYWx0LXBl cmlvZGljYWw+PHBhZ2VzPjg0NTItODQ1NDwvcGFnZXM+PHZvbHVtZT40Njwvdm9sdW1lPjxudW1i ZXI+NDQ8L251bWJlcj48a2V5d29yZHM+PGtleXdvcmQ+Y2hpcmFsaXR5PC9rZXl3b3JkPjxrZXl3 b3JkPmVuYW50aW9zZWxlY3Rpdml0eTwva2V5d29yZD48a2V5d29yZD5lbnp5bWUgY2F0YWx5c2lz PC9rZXl3b3JkPjxrZXl3b3JkPmVuenltZS1yZXNwb25zaXZlPC9rZXl3b3JkPjxrZXl3b3JkPm1h dGVyaWFsczwva2V5d29yZD48a2V5d29yZD5wb2x5bWVyaXphdGlvbjwva2V5d29yZD48a2V5d29y ZD5DQVRBTFlTSVM8L2tleXdvcmQ+PGtleXdvcmQ+UE9MWUVTVEVSUzwva2V5d29yZD48a2V5d29y ZD5SRURVQ1RJT048L2tleXdvcmQ+PGtleXdvcmQ+S0VUT05FUzwva2V5d29yZD48L2tleXdvcmRz PjxkYXRlcz48eWVhcj4yMDA3PC95ZWFyPjwvZGF0ZXM+PGlzYm4+MTQzMy03ODUxPC9pc2JuPjxh Y2Nlc3Npb24tbnVtPklTSTowMDAyNTA5OTAwMDAwMjc8L2FjY2Vzc2lvbi1udW0+PHdvcmstdHlw ZT5BcnRpY2xlPC93b3JrLXR5cGU+PHVybHM+PHJlbGF0ZWQtdXJscz48dXJsPiZsdDtHbyB0byBJ

888

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald U0kmZ3Q7Oi8vMDAwMjUwOTkwMDAwMDI3PC91cmw+PC9yZWxhdGVkLXVybHM+PC91cmxzPjxlbGVj dHJvbmljLXJlc291cmNlLW51bT4xMC4xMDAyL2FuaWUuMjAwNzAyNDM4PC9lbGVjdHJvbmljLXJl c291cmNlLW51bT48bGFuZ3VhZ2U+RW5nbGlzaDwvbGFuZ3VhZ2U+PC9yZWNvcmQ+PC9DaXRlPjwv

RW5kTm90ZT4A

51,104,105

Enzymes generally function under mild conditions and possess a high degree of selectivity. Also, they are vital to the function of all living systems, catalysing and controlling healthy and diseased biological processes. In particular proteases (enzymes that hydrolyse peptide bonds) have been shown to be specific markers in many disease states including cancers13 and chronic wounds.106 Most enzyme responsive systems that have been described make use of enzyme cleavable linkers which covalently attached the drug to a polymer whereby enzyme action

detaches

the

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PHB1Yi1kYXRlcz48 ZGF0ZT5NYXIgMTwvZGF0ZT48L3B1Yi1kYXRlcz48L2RhdGVzPjxhY2Nlc3Np b24tbnVtPklTSTow MDAyMzU1MjY1MDAwMDE8L2FjY2Vzc2lvbi1udW0+PHVybHM+PHJlbGF0Z WQtdXJscz48dXJsPiZs dDtHbyB0byBJU0kmZ3Q7Oi8vMDAwMjM1NTI2NTAwMDAxPC91cmw+PC9y ZWxhdGVkLXVybHM+PC91 cmxzPjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+AG== 107-109 An alternative approach through the development of peptide actuators has been shown by Ulijn and co-workers.

PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5UaG9ybnRvbjwvQXV0aG9yPjxZZWFyPjIwMDc8L1llYXI+

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895

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald aW9uLW51bT5JU0k6MDAwMjQ2NjU4MTAwMDE2PC9hY2Nlc3Npb24tbnVtPjx3b3JrLXR5cGU+QXJ0 aWNsZTwvd29yay10eXBlPjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD4mbHQ7R28gdG8gSVNJJmd0 OzovLzAwMDI0NjY1ODEwMDAxNjwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxlY3Ryb25p Yy1yZXNvdXJjZS1udW0+MTAuMTAwMi9hZG1hLjIwMDYwMTc4NDwvZWxlY3Ryb25pYy1yZXNvdXJj ZS1udW0+PGxhbmd1YWdlPkVuZ2xpc2g8L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48Q2l0ZT48 QXV0aG9yPlRob3JudG9uPC9BdXRob3I+PFllYXI+MjAwODwvWWVhcj48UmVjTnVtPjI3NTwvUmVj TnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+Mjc1PC9yZWMtbnVtYmVyPjxmb3JlaWduLWtleXM+PGtl eSBhcHA9IkVOIiBkYi1pZD0iZmE5emR6c3Zrc3gycHJlcmU5OHhkNTJxZGZ3ZndyOTBwNXMyIj4y NzU8L2tleT48L2ZvcmVpZ24ta2V5cz48cmVmLXR5cGUgbmFtZT0iSm91cm5hbCBBcnRpY2xlIj4x NzwvcmVmLXR5cGU+PGNvbnRyaWJ1dG9ycz48YXV0aG9ycz48YXV0aG9yPlRob3JudG9uLCBQLiBE LjwvYXV0aG9yPjxhdXRob3I+TWFydCwgUi4gSi48L2F1dGhvcj48YXV0aG9yPldlYmIsIFMuIEou PC9hdXRob3I+PGF1dGhvcj5VbGlqbiwgUi4gVi48L2F1dGhvcj48L2F1dGhvcnM+PC9jb250cmli dXRvcnM+PGF1dGgtYWRkcmVzcz5Vbml2IE1hbmNoZXN0ZXIsIFNjaCBNYXQsIE1hbmNoZXN0ZXIg TTEgN0ROLCBMYW5jcywgRW5nbGFuZC4gVW5pdiBNYW5jaGVzdGVyLCBNSUIsIE1hbmNoZXN0ZXIg TTEgN0ROLCBMYW5jcywgRW5nbGFuZC4mI3hEO1Rob3JudG9uLCBQRCwgVW5pdiBNYW5jaGVzdGVy LCBTY2ggTWF0LCBNYW5jaGVzdGVyIE0xIDdETiwgTGFuY3MsIEVuZ2xhbmQuJiN4RDtyZWluLnVs aWpuQG1hbmNoZXN0ZXIuYWMudWs8L2F1dGgtYWRkcmVzcz48dGl0bGVzPjx0aXRsZT5Fbnp5bWUt cmVzcG9uc2l2ZSBoeWRyb2dlbCBwYXJ0aWNsZXMgZm9yIHRoZSBjb250cm9sbGVkIHJlbGVhc2Ug b2YgcHJvdGVpbnM6IGRlc2lnbmluZyBwZXB0aWRlIGFjdHVhdG9ycyB0byBtYXRjaCBwYXlsb2Fk PC90aXRsZT48c2Vjb25kYXJ5LXRpdGxlPlNvZnQgTWF0dGVyPC9zZWNvbmRhcnktdGl0bGU+PGFs dC10aXRsZT5Tb2Z0IE1hdHRlcjwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2RpY2FsPjxmdWxs LXRpdGxlPlNvZnQgTWF0dGVyPC9mdWxsLXRpdGxlPjxhYmJyLTE+U29mdCBNYXR0ZXI8L2FiYnIt MT48L3BlcmlvZGljYWw+PGFsdC1wZXJpb2RpY2FsPjxmdWxsLXRpdGxlPlNvZnQgTWF0dGVyPC9m dWxsLXRpdGxlPjxhYmJyLTE+U29mdCBNYXR0ZXI8L2FiYnItMT48L2FsdC1wZXJpb2RpY2FsPjxw YWdlcz44MjEtODI3PC9wYWdlcz48dm9sdW1lPjQ8L3ZvbHVtZT48bnVtYmVyPjQ8L251bWJlcj48 a2V5d29yZHM+PGtleXdvcmQ+UE9MWU1FUiBUSEVSQVBFVVRJQ1M8L2tleXdvcmQ+PGtleXdvcmQ+ U01BUlQgQklPTUFURVJJQUxTPC9rZXl3b3JkPjxrZXl3b3JkPkRSVUctREVMSVZFUlk8L2tleXdv cmQ+PGtleXdvcmQ+TUlDUk9TQ09QWTwva2V5d29yZD48a2V5d29yZD5DT1BPTFlNRVI8L2tleXdv cmQ+PGtleXdvcmQ+TElCUkFSSUVTPC9rZXl3b3JkPjxrZXl3b3JkPlNVUFBPUlRTPC9rZXl3b3Jk PjxrZXl3b3JkPlBST0RSVUdTPC9rZXl3b3JkPjxrZXl3b3JkPkNBUlJJRVJTPC9rZXl3b3JkPjxr ZXl3b3JkPlNDSUVOQ0U8L2tleXdvcmQ+PC9rZXl3b3Jkcz48ZGF0ZXM+PHllYXI+MjAwODwveWVh cj48L2RhdGVzPjxpc2JuPjE3NDQtNjgzWDwvaXNibj48YWNjZXNzaW9uLW51bT5JU0k6MDAwMjU0 Mzg2ODAwMDIzPC9hY2Nlc3Npb24tbnVtPjx3b3JrLXR5cGU+QXJ0aWNsZTwvd29yay10eXBlPjx1 cmxzPjxyZWxhdGVkLXVybHM+PHVybD4mbHQ7R28gdG8gSVNJJmd0OzovLzAwMDI1NDM4NjgwMDAy MzwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+MTAu

896

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald MTAzOS9iNzE0NzUwYzwvZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+PGxhbmd1YWdlPkVuZ2xpc2g8 L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+ PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5UaG9ybnRvbjwvQXV0aG9yPjxZZWFyPjIwMDc8L1llYXI+ PFJlY051bT4xNjwvUmVjTnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+MTY8L3JlYy1udW1iZXI+PGZv cmVpZ24ta2V5cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJmYTl6ZHpzdmtzeDJwcmVyZTk4eGQ1MnFk Zndmd3I5MHA1czIiPjE2PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG5hbWU9IkpvdXJu YWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcnM+PGF1dGhvcj5U aG9ybnRvbiwgUC4gRC48L2F1dGhvcj48YXV0aG9yPk1hcnQsIFIuIEouPC9hdXRob3I+PGF1dGhv cj5VbGlqbiwgUi4gVi48L2F1dGhvcj48L2F1dGhvcnM+PC9jb250cmlidXRvcnM+PGF1dGgtYWRk cmVzcz5TY2ggTWF0LCBNYW5jaGVzdGVyIE0xIDdIUywgTGFuY3MsIEVuZ2xhbmQuIE1JQiwgTWFu Y2hlc3RlciBNMSA3SFMsIExhbmNzLCBFbmdsYW5kLiYjeEQ7VWxpam4sIFJWLCBTY2ggTWF0LCBH cm9zdmVub3IgU3QsIE1hbmNoZXN0ZXIgTTEgN0hTLCBMYW5jcywgRW5nbGFuZC4mI3hEO3JlaW4u dWxpam5AbWFuY2hlc3Rlci5hYy51azwvYXV0aC1hZGRyZXNzPjx0aXRsZXM+PHRpdGxlPkVuenlt ZS1yZXNwb25zaXZlIHBvbHltZXIgaHlkcm9nZWwgcGFydGljbGVzIGZvciBjb250cm9sbGVkIHJl bGVhc2U8L3RpdGxlPjxzZWNvbmRhcnktdGl0bGU+QWR2YW5jZWQgTWF0ZXJpYWxzPC9zZWNvbmRh cnktdGl0bGU+PGFsdC10aXRsZT5BZHYuIE1hdGVyLjwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZXJp b2RpY2FsPjxmdWxsLXRpdGxlPkFkdmFuY2VkIE1hdGVyaWFsczwvZnVsbC10aXRsZT48YWJici0x PkFkdi4gTWF0ZXIuPC9hYmJyLTE+PC9wZXJpb2RpY2FsPjxhbHQtcGVyaW9kaWNhbD48ZnVsbC10 aXRsZT5BZHZhbmNlZCBNYXRlcmlhbHM8L2Z1bGwtdGl0bGU+PGFiYnItMT5BZHYuIE1hdGVyLjwv YWJici0xPjwvYWx0LXBlcmlvZGljYWw+PHBhZ2VzPjEyNTItKzwvcGFnZXM+PHZvbHVtZT4xOTwv dm9sdW1lPjxudW1iZXI+OTwvbnVtYmVyPjxrZXl3b3Jkcz48a2V5d29yZD4yLVBIT1RPTiBNSUNS T1NDT1BZPC9rZXl3b3JkPjxrZXl3b3JkPlBFUFRJREUtU1lOVEhFU0lTPC9rZXl3b3JkPjxrZXl3 b3JkPlNPTElEIFNVUFBPUlRTPC9rZXl3b3JkPjxrZXl3b3JkPkRSVUctREVMSVZFUlk8L2tleXdv cmQ+PGtleXdvcmQ+UFJPVEVBU0U8L2tleXdvcmQ+PGtleXdvcmQ+QklPTUFURVJJQUxTPC9rZXl3 b3JkPjxrZXl3b3JkPlBST1RFSU5BU0VTPC9rZXl3b3JkPjxrZXl3b3JkPklOSElCSVRPUlM8L2tl eXdvcmQ+PGtleXdvcmQ+QklPTE9HWTwva2V5d29yZD48a2V5d29yZD5MSUJSQVJJRVM8L2tleXdv cmQ+PC9rZXl3b3Jkcz48ZGF0ZXM+PHllYXI+MjAwNzwveWVhcj48cHViLWRhdGVzPjxkYXRlPk1h eTwvZGF0ZT48L3B1Yi1kYXRlcz48L2RhdGVzPjxpc2JuPjA5MzUtOTY0ODwvaXNibj48YWNjZXNz aW9uLW51bT5JU0k6MDAwMjQ2NjU4MTAwMDE2PC9hY2Nlc3Npb24tbnVtPjx3b3JrLXR5cGU+QXJ0 aWNsZTwvd29yay10eXBlPjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD4mbHQ7R28gdG8gSVNJJmd0 OzovLzAwMDI0NjY1ODEwMDAxNjwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxlY3Ryb25p Yy1yZXNvdXJjZS1udW0+MTAuMTAwMi9hZG1hLjIwMDYwMTc4NDwvZWxlY3Ryb25pYy1yZXNvdXJj ZS1udW0+PGxhbmd1YWdlPkVuZ2xpc2g8L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48Q2l0ZT48 QXV0aG9yPlRob3JudG9uPC9BdXRob3I+PFllYXI+MjAwODwvWWVhcj48UmVjTnVtPjI3NTwvUmVj TnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+Mjc1PC9yZWMtbnVtYmVyPjxmb3JlaWduLWtleXM+PGtl

897

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald eSBhcHA9IkVOIiBkYi1pZD0iZmE5emR6c3Zrc3gycHJlcmU5OHhkNTJxZGZ3ZndyOTBwNXMyIj4y NzU8L2tleT48L2ZvcmVpZ24ta2V5cz48cmVmLXR5cGUgbmFtZT0iSm91cm5hbCBBcnRpY2xlIj4x NzwvcmVmLXR5cGU+PGNvbnRyaWJ1dG9ycz48YXV0aG9ycz48YXV0aG9yPlRob3JudG9uLCBQLiBE LjwvYXV0aG9yPjxhdXRob3I+TWFydCwgUi4gSi48L2F1dGhvcj48YXV0aG9yPldlYmIsIFMuIEou PC9hdXRob3I+PGF1dGhvcj5VbGlqbiwgUi4gVi48L2F1dGhvcj48L2F1dGhvcnM+PC9jb250cmli dXRvcnM+PGF1dGgtYWRkcmVzcz5Vbml2IE1hbmNoZXN0ZXIsIFNjaCBNYXQsIE1hbmNoZXN0ZXIg TTEgN0ROLCBMYW5jcywgRW5nbGFuZC4gVW5pdiBNYW5jaGVzdGVyLCBNSUIsIE1hbmNoZXN0ZXIg TTEgN0ROLCBMYW5jcywgRW5nbGFuZC4mI3hEO1Rob3JudG9uLCBQRCwgVW5pdiBNYW5jaGVzdGVy LCBTY2ggTWF0LCBNYW5jaGVzdGVyIE0xIDdETiwgTGFuY3MsIEVuZ2xhbmQuJiN4RDtyZWluLnVs aWpuQG1hbmNoZXN0ZXIuYWMudWs8L2F1dGgtYWRkcmVzcz48dGl0bGVzPjx0aXRsZT5Fbnp5bWUt cmVzcG9uc2l2ZSBoeWRyb2dlbCBwYXJ0aWNsZXMgZm9yIHRoZSBjb250cm9sbGVkIHJlbGVhc2Ug b2YgcHJvdGVpbnM6IGRlc2lnbmluZyBwZXB0aWRlIGFjdHVhdG9ycyB0byBtYXRjaCBwYXlsb2Fk PC90aXRsZT48c2Vjb25kYXJ5LXRpdGxlPlNvZnQgTWF0dGVyPC9zZWNvbmRhcnktdGl0bGU+PGFs dC10aXRsZT5Tb2Z0IE1hdHRlcjwvYWx0LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2RpY2FsPjxmdWxs LXRpdGxlPlNvZnQgTWF0dGVyPC9mdWxsLXRpdGxlPjxhYmJyLTE+U29mdCBNYXR0ZXI8L2FiYnIt MT48L3BlcmlvZGljYWw+PGFsdC1wZXJpb2RpY2FsPjxmdWxsLXRpdGxlPlNvZnQgTWF0dGVyPC9m dWxsLXRpdGxlPjxhYmJyLTE+U29mdCBNYXR0ZXI8L2FiYnItMT48L2FsdC1wZXJpb2RpY2FsPjxw YWdlcz44MjEtODI3PC9wYWdlcz48dm9sdW1lPjQ8L3ZvbHVtZT48bnVtYmVyPjQ8L251bWJlcj48 a2V5d29yZHM+PGtleXdvcmQ+UE9MWU1FUiBUSEVSQVBFVVRJQ1M8L2tleXdvcmQ+PGtleXdvcmQ+ U01BUlQgQklPTUFURVJJQUxTPC9rZXl3b3JkPjxrZXl3b3JkPkRSVUctREVMSVZFUlk8L2tleXdv cmQ+PGtleXdvcmQ+TUlDUk9TQ09QWTwva2V5d29yZD48a2V5d29yZD5DT1BPTFlNRVI8L2tleXdv cmQ+PGtleXdvcmQ+TElCUkFSSUVTPC9rZXl3b3JkPjxrZXl3b3JkPlNVUFBPUlRTPC9rZXl3b3Jk PjxrZXl3b3JkPlBST0RSVUdTPC9rZXl3b3JkPjxrZXl3b3JkPkNBUlJJRVJTPC9rZXl3b3JkPjxr ZXl3b3JkPlNDSUVOQ0U8L2tleXdvcmQ+PC9rZXl3b3Jkcz48ZGF0ZXM+PHllYXI+MjAwODwveWVh cj48L2RhdGVzPjxpc2JuPjE3NDQtNjgzWDwvaXNibj48YWNjZXNzaW9uLW51bT5JU0k6MDAwMjU0 Mzg2ODAwMDIzPC9hY2Nlc3Npb24tbnVtPjx3b3JrLXR5cGU+QXJ0aWNsZTwvd29yay10eXBlPjx1 cmxzPjxyZWxhdGVkLXVybHM+PHVybD4mbHQ7R28gdG8gSVNJJmd0OzovLzAwMDI1NDM4NjgwMDAy MzwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+MTAu MTAzOS9iNzE0NzUwYzwvZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+PGxhbmd1YWdlPkVuZ2xpc2g8 L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+ 57,58

These actuators constructed by SPPS on

PEGA, have the ability to release entrapped payloads in response to a specific enzyme by exploiting changes in electrostatic interactions resulting in an increase in the swelling of the polymer (shown schematically in Figure 3–1). This method of release offers advantages over pro-drug approaches: There is no need to covalently modify the drug molecules and drug release is not directly governed by enzyme 898

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

kinetics, i.e. instead of the enzymatic hydrolysis of one bond corresponding to the release of one drug molecule. This system allows for a variable number of payload molecules to be released upon enzymatically induced swelling. Using designed peptides allows the possibility to develop a range of different actuators using the varied functionalities that are offered by nature’s building blocks, for example the benefit of designing the actuator to match payload charge has previously been shown (described within section Electrostatic interactions between charges present in pendant actuators).58

Figure 3–1. Schematic of the enzyme responsive swelling of PEGA particles functionalised with linear peptide actuators. The linear peptide actuator is made up of 2 parts, a sensor and an actuator. The actuator consists of the 2 oppositely charged amino acids (a zwitterion) and the sensor is the amino acids between the charged residues. The sensor is hydrolysed by an enzyme with the correct specificity; therefore it is termed the enzyme cleavable peptide or ECP.

Therefore, the aims of this chapter were to: (i) Produce and characterise PEGA microparticles (µPEGA) and to investigate the effect of changing the chemical structure of PEGA on its swelling behaviour. (ii) Study the compatibility of µPEGA with enzyme activity using TPM. (iii) To establish the ideal experimental conditions to produce PEGA particles that offer the suitable properties (loading and particle size distribution) for use in the enzyme responsive release systems. (iv) Incorporate peptide actuators on µPEGA, (v) characterise the enzyme specific changes in accessibility of the functionalised particles, (vi) demonstrate the possibility of loading these particles with a payload and (vii) use enzymes to trigger specific release of the payload. 899

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3.3. Experimental 3.3.1. Materials All chemicals were used as supplied from Sigma with the exception of amino acids (Bachem)

and

O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium

hexafluorophosphate (HBTU) (AGTC Bioproducts Ltd). The enzymes used were thermolysin (EC 3.4.24.27), 36.5 U/mg and chymotrypsin (EC 3.4.21.1), 60 U/mg (both were supplied by Sigma). 3.3.2. Inverse suspension polymerisation μPEGA was prepared by inverse suspension polymerisation. A stainless steel baffleless reactor (250 ml) stirred with an anchor-style agitator was used for the polymerisation reactions. 3.14 g (3.5 mmol) of the PEGA800 macromonomers (a 2:1 (molar) mixture of acrylamide-PEG-acrylamide and amino-PEG-acrylamide) was dissolved in 10 ml of distilled water along with 0.156 g (2.2 mmol) of acrylamide (the chemical structures of the monomers are shown in Figure 3–2). This solution was then purged for 30 minutes with N2 gas. 50 ml of Isopar M (isoparaffin) was added to the reactor and was also purged for 30 minutes. The reactor was heated to 80°C. After 20 minutes of purging 0.16 ml (1.0 mmol) of N,N,N′,N′tetramethylethylenediamine (TEMED) was added to the oil phase, and 0.156 g (2.2 mmol) of acrylamide to the dissolved macromonomer solution. 0.164 g (0.47 mmol) of Span 20 (sorbitan monolaurate) was dissolved in the oil, which was stirred at 500 rpm for 30 seconds to ensure the surfactant was fully dispersed in the oil phase. 0.070 g (0.30 mmol) of ammonium persulfate (APS) was dissolved in the macromonomer solution, which was added to the oil phase in the reactor which was stirred at 2000 rpm for a further 30 minutes. The particles were washed with (3 x 50 ml) dichloromethane (DCM), (3 x 50 ml) Tetrahydrofuran (THF), (3 x 50 ml) methanol and (4 x 50 ml) distilled water (retained by centrifugation at each step). 3.3.3. Charged PEGA polymerisation Particles that incorporate permanent charges were prepared by substituting the acrylamide with either (3-trimethylammonium chloride) propyl acrylamide to produce positive particles or 1,1-dimethyl-2-(sulphonate) ethyl acrylamide to give negative particles (the chemical structures of these monomers are shown in Figure

900

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3–2). All remaining aspects of the polymerisation were the same as with the inverse suspension of neutral particles

Figure 3–2. Monomers used in the synthesis of PEGA and its charged variants. From the top down:

amino-PEG-acrylamide,

acrylamide-PEG-acrylamide,

acrylamide,

(3-

trimethylammonium chloride) propyl acrylamide and 1,1-dimethyl-2-(sulphonate) ethyl acrylamide.

3.3.4. Microscopy and particle size analysis An optical microscope was used to acquire images of the particles and environmental scanning electron microscopy (ESEM) images were taken on a FEI Quanta 200 ESEM, using ESEM low vac mode at 10.0 KV. Size distribution analysis of the microparticles was carried out using a Malvern Mastersizer particle size analyser. Deionised water was used to fill the small volume dispersion unit and the dispersion control unit set to 1500 rpm, the polymer particles were added until an obscuration value of above 10% was obtained. Mastersizer Microplus software version 2.18 was used to analyse the results and plot distribution graphs. 3.3.5. Solid phase peptide synthesis Peptides can be prepared synthetically in good yields, solid phase peptide synthesis (SPPS)68 is a popular synthetic method used to achieve this. Here, amine functionalised crosslinked polymers (resins) are reacted with amino acids (with 901

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

protected their amines protected) resulting in amide bond formation (shown schematically in Figure 3–3, while the chemical reactions used in this work can be seen in Figure 3–4). The removal of the protecting group allows for this process to be repeated in a step-wise process to build up the desired peptide.

Figure 3–3. Solid phase peptide synthesis scheme. A: A scheme of the step by step process in the synthesis of a peptide from protected amino acids. B: The key, (left) amines are present in PEGA particles, (right) Fmoc protected amino acid.

902

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 3–4. Chemical reactions involved in peptide synthesis. A: Formation of an amide bond. Firstly, the carboxyl of the amino acid/peptide reacts with O-(Benzotriazol-1-yl)-N,N,N′,N′tetramethyluronium

hexafluorophosphate

(HBTU)

in

the

presence

of

N,N-

diisopropylethylamine resulting in an activated ester. The amine of the free amino acid then reacts with the ester resulting in amide bond formation. B: The deprotection of the Fmoc protected amine group with piperidine.

3.3.5.1. Solid phase synthesis of dipeptides and enzyme treatment To quantify the loading of the microparticles Fmoc Amino acids (3 equiv.) were coupled to PEGA particles using DIC (di-isopropyl carbodiimide) (6 equiv.) and HOBt (hydroxybenzotriazole) (6 equiv.) in DMF. The first coupling was performed for 3 hours and the second overnight. A roller mixer at room temperature was used to agitate the solutions. Between steps the resin was washed extensively using 5 ml volumes of 5 x MeOH, 5 x 50 : 50 (v/v) DMF : MeOH and 5 x DMF. A 20 %

903

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

solution of piperidine in DMF was used for Fmoc deprotection for 2 hours. Enzyme reactions: 1.5 mg of thermolysin per 1 ml 0.01 M phosphate buffer solution of pH 7.5, reactions were at room temperature on a roller mixer. To determine how quickly the enzyme catalysed hydrolysis has occurred, a thermolysin solution was added to the Fmoc-A-A modified particles the reaction was then stopped with 0.1% trifluoroacetic acid (TFA) in deionised water at seven different times and analysed with high performance liquid chromatography (HPLC) to quantify any cleaved residues. 3.3.5.2. Solid phase peptide synthesis of peptide actuators Peptide actuators (Fmoc-DAAR-PEGA) were prepared by solid phase peptide synthesis using Fmoc protected amino acids. 8 equivalents of the amino acid and 7.8 equivalents of HBTU were dissolved in 2 ml of N,N-Dimethylformamide (DMF). 16 equivalents of N,N-Diisopropylethylamine (DIPEA) was added to this solution prior to its addition to the PEGA particles. The coupling reaction was left for 16 hours, and the Kaiser test26 was used to ensure complete coupling. Deprotection was achieved using 20 % piperidine in DMF for 2 hours. This cycle was repeated to build up the required peptide sequence with thorough washing between steps (5 x 5 ml methanol, 5 x 5 ml 1:1 methanol:DMF, 5 x 5 ml DMF). A solution 95 % Trifluoroacetic acid (TFA) 5 % water was used to remove the side chain protecting groups. 1.1.1. HPLC HPLC experiments were undertaken on a Dionex HPLC (P680 pump, ASI-100 Automated sample injector, Nucleosil 100-5-C18 column with a UVD170U detector), using a solvent ramp of 20 % ACN and 80 % water to 80 % ACN 20 % water over 30 minutes (0.1 % TFA was present in both phases) shown in Figure 8–3. Chromeleon 6.60 software was used for analysis. 1.1.1. Two-photon microscopy The distribution of amine groups and homogeneity of enzyme hydrolysis were assessed with TPM. Firstly, three differently modified batches of particles were prepared; unmodified, Fmoc-A-A (prepared with the previously described SPPS method) and Fmoc-A-A treated with thermolysin solution. 0.1 g of these particles were then placed in a filter column and rinsed with DMF (3 x 5 ml). 0.036 g (0.133 mmol) of dansyl chloride was dissolved in 2 ml of DMF and 25 μl (0.143 mmol) 904

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

DIPEA was then added to this solution. This coupling solution was then added to the PEGA particles and put on the blood rotator in the dark for 2 hours. The polymer was then rinsed with DMF (3 x 5 ml), ethanol (3 x 5 ml) and water (3 x 5 ml). TPM was used to take visual cross-sections of the particles to determine the distribution of free amine groups. A Ti:Sapphire laser was tuned to 770 nm and fluorescence from the sample was filtered using a 525/550 nm filter. 1.1.2. Determining particle swelling The neutral, positive and negative PEGA microparticles were swollen in water or buffer 0.1 M (pH 7.5) and centrifuged at 3000 rpm. 0.50 g of the hydrated polymers were weighed out and placed in a Gallenkamp vacuum oven at room temperature at 100 mbar. Every three hours the polymers were weighed, and when no further weight loss was recorded this mass was taken to be the dry mass. 1.1.1. Assessing accessibility To analyse the change in accessibility of the particles a 2 mg/ml solution of 40 kDa fluorescently labelled dextran was prepared in water. Approximately 50 mg of PEGA particles were added to these solutions. 10 minutes was left to allow diffusion into the hydrogel prior to imaging. The confocal microscope used was a Lecia sp2 AOBS. The laser was an Argon laser tuned to 488 nm, the fluorescence from the sample was filtered to leave 490-550 nm. 1.1.2. Entrapping payload The particles (0.1 g) were loaded with a 40 kDa FITC Dextran using the following method; 2 ml of solution of FITC dextran in 0.01 M HCl (10 mg/ml) was added to the particles for 30 minutes (pH 2.5). After this time 0.4 ml of 0.05 M NaOH was added dropwise (pH 7). The particles were then filtered and washed (2 x 5 ml water, 2 x 5 ml 0.18 M buffer, 2 x 5 ml methanol). 1.1.3. Fluorimetry The release of entrapped dextran was determined using the following method; loaded particles were treated in solution for 80 minutes, these samples were centrifuged and the supernatant taken off. A Jasco FP – 6500 Spectrofluorometer using an excitation wavelength of 490 mn was used to obtain the fluorescent intensity at 519 nm. Treated particles were filtered and imaged using fluorescence microscopy. 905

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1.1.1. Microscopy and determination of swelling Swelling measurements were obtained using a Zeiss Imager A1 microscope, a Leistungselektronik mbq 52 AC power source (Jena, Germany) equipped with a Canon Powershot G6 camera. For pH responsive swelling the diameter of a minimum of 300 particles was measure and the mean volume calculated. For enzyme responsive swelling individual particles were observed throughout the time course and ImageJ 1.38x analysis software was used to determine the change in particle diameter. An average V/Vo was determined from at least six representative particles, where V is the volume of the particle at time t and Vo is the volume of the particle immediately after exposure to enzyme solution. Enzyme solutions were made up at a concentration of 1 mg/ml and buffers were prepared by using the appropriate amounts of sodium phosphate dibasic heptahydrate and sodium phosphate monobasic.

906

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1.2. Results and Discussion 1.2.1. Production of µPEGA800 PEGA microparticles (µPEGA) were prepared by inverse suspension polymerisation using thermal initiators.67 The water soluble monomers were dispersed along with the initiator in the oil phase, which contained the co-initiator and the surfactant (Figure 3–5). Upon stirring monomer droplets were formed and undergo continuous break-up and coalescence, this leads to a dynamic equilibrium being established giving a stationary mean droplet size.110 The size and the distribution of the particles formed were controlled by the imposed shear field within the reactor and the stabilisation effect of the surfactant. Currently PEGA particles are commercially available in size ranges above 150 μm for ease of handling in SPPS. Depending on the biomedical applications smaller size ranges are desirable (such as injected into tissue (<200 μm), inhaled (<100 μm) or released into circulation (<10 μm).99 Smaller particles also offer the potential to give faster response times due to their greater surface area to volume ratio (as shown by Peppas and co-workers).55 In previous work, the stirring speed and the surfactant concentration were varied in order to find the optimum conditions to produce microparticles.101 There are three monomers used in the preparation of PEGA; aminoacrylamido PEG, bisacrylamido PEG and acrylamide.

The amino functionalised monomer imparts the amine

functional group or ‘handle’ on a flexible chain allowing easy modification by solid phase peptide synthesis (SPPS). The bisacrylamide PEG was the crosslinking agent in the polymerisation while the acrylamide acts as a spacer to separate the PEG based monomers. The PEG macro-monomers used had an average molecular weight of 800 g/mole, the molar mass of the PEG chain in the resulting cross-linked polyacrylamide-graft-PEG co-polymer. This structure leads to PEGA’s well defined molecular

weight

cut-off,

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907

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

GFuZ3VhZ2U+PC9y

ZWNvcmQ+PC9DaXRlPjwvRW5kTm90ZT5=

63

with

molecules above this unable to diffuse in or out of the polymer matrix.

Figure 3–5. Polymerisation of PEGA particles by inverse suspension polymerisation. The monomers; acrylamide, aminoacrylamido PEG and bisacrylamido PEG along with the initiator APS were dissolved in water and dispersed in an oil phase containing surfactant and coinitiator. Upon stirring, monomer droplets are formed and undergo continuous break-up and coalescence and a dynamic equilibrium is established giving a stationary mean particle size.

1.2.1.1. Particle morphology and size characterisation of µPEGA In order to produce μPEGA with the desired size distribution a stirring speed 2000 rpm was employed with 3.28 mg/ml of surfactant in the oil phase. These particles had a mean diameter of 15 µm. Further investigation was carried out on these particles, including analysis of the size distribution, determining the loading capacity and its homogeneity throughout the particles as well as their compatibility with enzymes. Study of the particles by optical microscopy (Figure 3–6 A) showed that the microparticles are separate spherical entities, with no obvious surface detail or texture on the sub-micron scale between the range of about 5-25 µm. The data provided by particle size analysis (Figure 3–6 B) gave a mean particle diameter of 15 µm with a polydispersity ((standard deviation/mean particle diameter) x 100) of 43 %. Images of the particles obtained by environmental scanning electron microscopy (ESEM) (Figure 3–6 C & B) concurred with the optical microscopy.

912

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

The ESEM images showed that the majority of particles have diameters between 110 µm. This difference in size measurement is due to the low pressure within the ESEM chamber resulting in removal of water from the particles (hence reducing the particle diameters). Individual spherical PEGA microparticles with a mean diameter of 15 µm and polydispersity of 43 % were produced as a result of the combination of shear forces and stabilisation effect of surfactant on the droplets within the reactor.

Figure 3–6. A: Optical micrograph of microparticles is water. B: Size distribution of microparticles in water obtained by particle size analyser. C and D: ESEM micrographs of PEGA microparticles. (Scale bar in all figures is 20 µm).

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald RWRpbmJ1cmdoIEVIOSAzSkosIE1pZGxvdGhpYW4sIFNjb3RsYW5kLiBVbml2IFRyaWVz dGUsIERp cGFydGltZW50byBTY2kgRmFybWFjZXV0LCBJLTM0MTI3IFRyaWVzdGUsIEl0YWx5LiB Qb2x5bWVy IExhYnMgTHRkLCBDaHVyY2ggU3RyZXR0b24gU1k2IDZBWCwgU2hyb3BzLCBFbmdsY W5kLiYjeEQ7 RmxpdHNjaCwgU0wsIFVuaXYgRWRpbmJ1cmdoLCBTY2ggQ2hlbSwgS2luZ3MgQmxkZyx XIE1haW5z IFJkLCBFZGluYnVyZ2ggRUg5IDNKSiwgTWlkbG90aGlhbiwgU2NvdGxhbmQuJiN4RDtzb GYwMUBz dGFmZm1haWwuZWQuYWMudWs8L2F1dGgtYWRkcmVzcz48dGl0bGVzPjx0aXRsZT5Jb nRyb2R1Y3Rp b24gb2YgcGVybWFuZW50bHkgY2hhcmdlZCBncm91cHMgaW50byBQRUdBIHJlc2lucyBsZ WFkcyB0 byBpbXByb3ZlZCBiaW90cmFuc2Zvcm1hdGlvbnMgb24gc29saWQgc3VwcG9ydDwvdGl0bG U+PHNl Y29uZGFyeS10aXRsZT5UZXRyYWhlZHJvbjwvc2Vjb25kYXJ5LXRpdGxlPjxhbHQtdGl0bG U+VGV0 cmFoZWRyb248L2FsdC10aXRsZT48L3RpdGxlcz48cGVyaW9kaWNhbD48ZnVsbC10aXRsZ T5UZXRy YWhlZHJvbjwvZnVsbC10aXRsZT48YWJici0xPlRldHJhaGVkcm9uPC9hYmJyLTE+PC9wZ XJpb2Rp Y2FsPjxhbHQtcGVyaW9kaWNhbD48ZnVsbC10aXRsZT5UZXRyYWhlZHJvbjwvZnVsbC10 aXRsZT48 YWJici0xPlRldHJhaGVkcm9uPC9hYmJyLTE+PC9hbHQtcGVyaW9kaWNhbD48cGFnZXM +NTg5LTU5 NDwvcGFnZXM+PHZvbHVtZT42MDwvdm9sdW1lPjxudW1iZXI+MzwvbnVtYmVyPjxrZXl 3b3Jkcz48 a2V5d29yZD5zb2xpZCBwaGFzZSBjaGVtaXN0cnk8L2tleXdvcmQ+PGtleXdvcmQ+UEVHQT wva2V5 d29yZD48a2V5d29yZD5jaGFyZ2VkIFBFR0E8L2tleXdvcmQ+PGtleXdvcmQ+cGVuaWNpbG xpbiBH IGFtaWRhc2U8L2tleXdvcmQ+PGtleXdvcmQ+dGhlcm1vbHlzaW48L2tleXdvcmQ+PGtleXdvc mQ+ Q09NQklOQVRPUklBTCBMSUJSQVJJRVM8L2tleXdvcmQ+PGtleXdvcmQ+UFJPVEVDVE lORyBHUk9V UDwva2V5d29yZD48a2V5d29yZD5NSUNST1NDT1BZPC9rZXl3b3JkPjxrZXl3b3JkPkNIRU 1JU1RS WTwva2V5d29yZD48a2V5d29yZD5MSU5LRVI8L2tleXdvcmQ+PGtleXdvcmQ+QVNTQVk8 L2tleXdv cmQ+PC9rZXl3b3Jkcz48ZGF0ZXM+PHllYXI+MjAwNDwveWVhcj48cHViLWRhdGVzPjxk YXRlPkph

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald bjwvZGF0ZT48L3B1Yi1kYXRlcz48L2RhdGVzPjxpc2JuPjAwNDAtNDAyMDwvaXNibj48Y WNjZXNz aW9uLW51bT5JU0k6MDAwMTg4MjI1NzAwMDEyPC9hY2Nlc3Npb24tbnVtPjx3b3JrLXR5 cGU+QXJ0 aWNsZTwvd29yay10eXBlPjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD4mbHQ7R28gdG8g SVNJJmd0 OzovLzAwMDE4ODIyNTcwMDAxMjwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxl Y3Ryb25p Yy1yZXNvdXJjZS1udW0+MTAuMTAxNi9qLnRldC4yMDAzLjEwLjEyNTwvZWxlY3Ryb25 pYy1yZXNv dXJjZS1udW0+PGxhbmd1YWdlPkVuZ2xpc2g8L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT 48L0Vu ZE5vdGU+ PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5CYXNzbzwvQXV0aG9yPjxZZWFyPjIwMDQ8L1l lYXI+PFJl Y051bT4yNTY8L1JlY051bT48cmVjb3JkPjxyZWMtbnVtYmVyPjI1NjwvcmVjLW51bWJlcj4 8Zm9y ZWlnbi1rZXlzPjxrZXkgYXBwPSJFTiIgZGItaWQ9ImZhOXpkenN2a3N4MnByZXJlOTh4Z DUycWRm d2Z3cjkwcDVzMiI+MjU2PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG5hbWU9Ik pvdXJu YWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcnM+PGF1dG hvcj5C YXNzbywgQS48L2F1dGhvcj48YXV0aG9yPlVsaWpuLCBSLiBWLjwvYXV0aG9yPjxhdXRob 3I+Rmxp dHNjaCwgUy4gTC48L2F1dGhvcj48YXV0aG9yPk1hcmdldHRzLCBHLjwvYXV0aG9yPjxhd XRob3I+ QnJhemVuZGFsZSwgSS48L2F1dGhvcj48YXV0aG9yPkViZXJ0LCBDLjwvYXV0aG9yPjxhd XRob3I+ RGUgTWFydGluLCBMLjwvYXV0aG9yPjxhdXRob3I+TGluZGEsIFAuPC9hdXRob3I+PGF1 dGhvcj5W ZXJkZWxsaSwgUy48L2F1dGhvcj48YXV0aG9yPkdhcmRvc3NpLCBMLjwvYXV0aG9yPjwv YXV0aG9y cz48L2NvbnRyaWJ1dG9ycz48YXV0aC1hZGRyZXNzPlVuaXYgRWRpbmJ1cmdoLCBTY2gg Q2hlbSwg RWRpbmJ1cmdoIEVIOSAzSkosIE1pZGxvdGhpYW4sIFNjb3RsYW5kLiBVbml2IFRyaWVz dGUsIERp cGFydGltZW50byBTY2kgRmFybWFjZXV0LCBJLTM0MTI3IFRyaWVzdGUsIEl0YWx5LiB Qb2x5bWVy IExhYnMgTHRkLCBDaHVyY2ggU3RyZXR0b24gU1k2IDZBWCwgU2hyb3BzLCBFbmdsY W5kLiYjeEQ7 RmxpdHNjaCwgU0wsIFVuaXYgRWRpbmJ1cmdoLCBTY2ggQ2hlbSwgS2luZ3MgQmxkZyx XIE1haW5z

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald IFJkLCBFZGluYnVyZ2ggRUg5IDNKSiwgTWlkbG90aGlhbiwgU2NvdGxhbmQuJiN4RDtzb GYwMUBz dGFmZm1haWwuZWQuYWMudWs8L2F1dGgtYWRkcmVzcz48dGl0bGVzPjx0aXRsZT5Jb nRyb2R1Y3Rp b24gb2YgcGVybWFuZW50bHkgY2hhcmdlZCBncm91cHMgaW50byBQRUdBIHJlc2lucyBsZ WFkcyB0 byBpbXByb3ZlZCBiaW90cmFuc2Zvcm1hdGlvbnMgb24gc29saWQgc3VwcG9ydDwvdGl0bG U+PHNl Y29uZGFyeS10aXRsZT5UZXRyYWhlZHJvbjwvc2Vjb25kYXJ5LXRpdGxlPjxhbHQtdGl0bG U+VGV0 cmFoZWRyb248L2FsdC10aXRsZT48L3RpdGxlcz48cGVyaW9kaWNhbD48ZnVsbC10aXRsZ T5UZXRy YWhlZHJvbjwvZnVsbC10aXRsZT48YWJici0xPlRldHJhaGVkcm9uPC9hYmJyLTE+PC9wZ XJpb2Rp Y2FsPjxhbHQtcGVyaW9kaWNhbD48ZnVsbC10aXRsZT5UZXRyYWhlZHJvbjwvZnVsbC10 aXRsZT48 YWJici0xPlRldHJhaGVkcm9uPC9hYmJyLTE+PC9hbHQtcGVyaW9kaWNhbD48cGFnZXM +NTg5LTU5 NDwvcGFnZXM+PHZvbHVtZT42MDwvdm9sdW1lPjxudW1iZXI+MzwvbnVtYmVyPjxrZXl 3b3Jkcz48 a2V5d29yZD5zb2xpZCBwaGFzZSBjaGVtaXN0cnk8L2tleXdvcmQ+PGtleXdvcmQ+UEVHQT wva2V5 d29yZD48a2V5d29yZD5jaGFyZ2VkIFBFR0E8L2tleXdvcmQ+PGtleXdvcmQ+cGVuaWNpbG xpbiBH IGFtaWRhc2U8L2tleXdvcmQ+PGtleXdvcmQ+dGhlcm1vbHlzaW48L2tleXdvcmQ+PGtleXdvc mQ+ Q09NQklOQVRPUklBTCBMSUJSQVJJRVM8L2tleXdvcmQ+PGtleXdvcmQ+UFJPVEVDVE lORyBHUk9V UDwva2V5d29yZD48a2V5d29yZD5NSUNST1NDT1BZPC9rZXl3b3JkPjxrZXl3b3JkPkNIRU 1JU1RS WTwva2V5d29yZD48a2V5d29yZD5MSU5LRVI8L2tleXdvcmQ+PGtleXdvcmQ+QVNTQVk8 L2tleXdv cmQ+PC9rZXl3b3Jkcz48ZGF0ZXM+PHllYXI+MjAwNDwveWVhcj48cHViLWRhdGVzPjxk YXRlPkph bjwvZGF0ZT48L3B1Yi1kYXRlcz48L2RhdGVzPjxpc2JuPjAwNDAtNDAyMDwvaXNibj48Y WNjZXNz aW9uLW51bT5JU0k6MDAwMTg4MjI1NzAwMDEyPC9hY2Nlc3Npb24tbnVtPjx3b3JrLXR5 cGU+QXJ0 aWNsZTwvd29yay10eXBlPjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD4mbHQ7R28gdG8g SVNJJmd0 OzovLzAwMDE4ODIyNTcwMDAxMjwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxl Y3Ryb25p

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald Yy1yZXNvdXJjZS1udW0+MTAuMTAxNi9qLnRldC4yMDAzLjEwLjEyNTwvZWxlY3Ryb25 pYy1yZXNv dXJjZS1udW0+PGxhbmd1YWdlPkVuZ2xpc2g8L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT 48L0Vu ZE5vdGU+ 75

1.2.2.1.1 Particle morphology and size characterisation of µPEGA+ The inverse suspension polymerisation of the PEGA monomers with the acrylamide substituted with (3-trimethylammonium chloride) propyl acrylamide produced PEGA+ particles.74 Optical and ESEM microscopy images (Figure 3–8) show the positively charged particles produced are highly aggregated with a narrow size distribution. Based on optical micrograph analysis the particle diameter were approximately 10 μm, whilst analysis of the ESEM images indicates the particles had a diameter of approximately 8 μm. Much like the neutral PEGA800 particles this was because the polymer is not fully hydrated within the ESEM chamber. When the pressure within the ESEM chamber was further lowered there was an appearance of a textured surface, this was water being drawn out of the hydrogel onto the surface prior to evaporating (Figure 3–8 D). The positively charged particles have no apparent surface features or texture on the sub-micron level. The aggregation of the particles meant that determining a size distribution using the particle size analyser did not yield any quantative information. The PEGA+ particles had an aggregated structure which may have be due to incomplete polymerisation during stirring leading to coalescence of the partially polymerised monomer droplets upon settling. It is possible that the negatively charged initiator (ammonium persulfate) cancelled the electrostatic repulsion between the positively charged polymerising monomer droplets. This may have removed the electrostatic stabilisation within the system allowing the partially polymerised particles to aggregate during the sticky period of the reaction.111

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 3–8. PEGA+ microparticles. A & B: ESEM micrograph of positively charged PEGA particles (scale bar in A is 50 μm and in B is 10 μm). C: Optical micrograph of positively charged PEGA particles (scale bar is 15 μm). D: ESEM micrograph of positively charge PEGA particles, arrows indicate examples of water droplet forming on the surface as the pressure is reduced (scale bar is 5 μm).

1.2.2.1.2 Particle morphology and size characterisation of µPEGABy carrying out the inverse suspension polymerisation reaction with 1,1-dimethyl-2(sulphonate) ethyl acrylamide replacing the acrylamide (with all other conditions kept the same) PEGA- was prepared.74 Optical microscopy of these negative microparticles (Figure 3–9) produced images similar to μPEGA although with a slightly larger and more polydisperse. Unlike the PEGA+ there was no aggregation visible. Data produced from the particle size analyser agrees with this comparison (a mean diameter of 20 μm with a polydispersity of 59 % compared to 15 μm and 43 % respectively for μPEGA). It is likely that the different between PEGA800 and PEGAis due to batch-to-batch variation. PEGA- particles were produced with morphology similar to that of neutral PEGA.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 3–9. Right: Optical micrographs PEGA-, scale bar is 20 μm. Left: Size distribution of PEGA-.

1.2.2.1.3 Comparison of swelling the different types of PEGA The swelling properties of a hydrogel are dependent on the ability of the material to absorb water. This ability is determined of a number of structural factors such as: crosslinking density, hydrophilicity of the polymer and the number of polar/ionisable groups. Greater swelling may be advantageous as in certain cases, greater swelling of the network results in increased mesh size of the polymer and therefore accessibility. The uptake of water and 0.1 M buffer (pH 7.4) into the particles was determined for dry particles from each of the three different PEGA polymers. This was achieved by measuring the dry and hydrated masses of the particles, providing information about polymer’s interactions with water (hydrophilicity) and neighbouring polymer chains (Figure 3–10).

937

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 3–10. The uptake of water by the three different types of dry PEGA microparticles and the effect of ionic strength on the swelling of the three different types of PEGA microparticles. Blue; uptake of water, red; uptake of 0.1 M buffer.

The positively charged particles gave the highest values for uptake of water because of the electrostatic repulsion between chains resulted in an osmotic driving force drawing more water into the polymer. The swelling of negative PEGA was much less than seen for PEGA+, this is likely due to interactions between the amines present (positively charged at pH below 7.5 in a test solution (water) which has a pH of 6.5) and the negatively charged acrylamide. This finding is in agreement with published work in which it was shown that PEGA- swelled less than PEGA+.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5CYXNzbzwvQXV0aG9yPjxZZ WFyPjIwMDQ8L1llYXI+PFJl Y051bT4xMTwvUmVjTnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+MTE8L3JlYy1u dW1iZXI+PGZvcmVp Z24ta2V5cz48a2V5IGFwcD0iRU4iIGRiLWlkPSIyMHAwcjJkcDlmOXRkMmUyc mE5dnIycG5zMHp4 dndwczB4NWUiPjExPC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG5hb WU9IkpvdXJuYWwg QXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcnM+PG F1dGhvcj5CYXNz

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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dGU+AG==

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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dGU+AG==

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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1.2.3. Further characterisation of µPEGA µPEGA was selected as the most promising polymer for the incorporation of the enzyme responsive functionalisation. PEGA+ and PEGA- offered greater swelling and have been demonstrated in the literature to have higher accessibilities than neutral μPEGA. An important criterion for the material selection is accessibility; it

947

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

must high enough to allow the target enzyme to diffuse into the particles while being low enough to entrap the payload molecules. Neutral µPEGA offered this, as well a simpler base to understand the effect of peptide actuator design of particle swelling. Therefore, further characterisation was carried out on µPEGA.

1.2.3.1. Amine

characterisation

by two-photon

microscopy

and

enzyme

compatibility The ease with which PEGA could be chemically modified with amino acids or peptides by SPPS and compatibility with aqueous environments makes it a particularly interesting material. This capacity for functionalisation is determined by the loading; the amount of amines per unit mass of polymer, usually quoted in mmol/g. In order to access this property, µPEGA was functionalised with an Fmoc protected amino acid using standard DIC/HOBT chemistry was undertaken. Within SPPS, piperidine is used to remove the Fmoc protecting group. These Fmoc groups were quantified by HPLC against Fmoc standards and the loading was found to be 0.37 mmol/g of (dry) µPEGA. This is in close agreement with the declared loading of commercially available PEGA800 macrobeads (0.4 mmol/g). Furthermore, the distribution of the amine groups was analysed by TPM.79 Here, the amine groups within the particles were reacted dansyl and the spacial distribution of the fluorophore determined. In TPM the sample is irradiated with a laser with a wavelength approximately twice that of the excitation length wavelength of the fluorophore. This means that excitation can only occur when two photons are absorbed simultaneously. This is extremely unlikely and therefore only occurs at very high photon density, the focal point of the laser beam. This method of imaging is well established for the characterisation of PEGA particles as the long wavelength of the infrared excitation laser gives good penetration into solid objects allowing for optical

cross-sections

within

the

z

plane

of

the

particles

to

be

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948

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

VT1JFU0NFTlQgUFJP UEVSVElFUzwva2V5d29yZD48a2V5d29yZD4yLVBIT1RPTiBNSUNST1NDT1B ZPC9rZXl3b3JkPjxr ZXl3b3JkPkNZU1RFSU5FIFBST1RFQVNFPC9rZXl3b3JkPjxrZXl3b3JkPlNJVE UgRElTVFJJQlVU SU9OPC9rZXl3b3JkPjxrZXl3b3JkPlBFUFRJREUgTElCUkFSWTwva2V5d29yZD 48a2V5d29yZD5T UEVDSUZJQ0lUWTwva2V5d29yZD48L2tleXdvcmRzPjxkYXRlcz48eWVhcj4y MDA3PC95ZWFyPjxw dWItZGF0ZXM+PGRhdGU+SnVuPC9kYXRlPjwvcHViLWRhdGVzPjwvZGF0Z XM+PGlzYm4+MTYxNS00 MTUwPC9pc2JuPjxhY2Nlc3Npb24tbnVtPklTSTowMDAyNDczMzg4MDAwMD Y8L2FjY2Vzc2lvbi1u dW0+PHdvcmstdHlwZT5BcnRpY2xlPC93b3JrLXR5cGU+PHVybHM+PHJlbGF0 ZWQtdXJscz48dXJs PiZsdDtHbyB0byBJU0kmZ3Q7Oi8vMDAwMjQ3MzM4ODAwMDA2PC91cmw+ PC9yZWxhdGVkLXVybHM+ PC91cmxzPjxlbGVjdHJvbmljLXJlc291cmNlLW51bT4xMC4xMDAyL2Fkc2MuM jAwNzAwMDQ0PC9l bGVjdHJvbmljLXJlc291cmNlLW51bT48bGFuZ3VhZ2U+RW5nbGlzaDwvbGFuZ 3VhZ2U+PC9yZWNv cmQ+PC9DaXRlPjwvRW5kTm90ZT4A 79,82 The TPM analysis of the µPEGA are shown in Figure 3–11. Here, an optical cross-section obtained was at the equatorial plane (the widest point) and fluorescence indicates where amines were present. Firstly, unmodified µPEGA were treated with dansyl chloride and imaged. From Figure 3–11 A it is clear that the free amine groups are homogeneously distributed throughout the particles. Other microparticles were functionalised with the protected dipeptide; Fmoc-AA and some then treated with dansyl chloride and imaged (Figure 3–11 B), it can be observed that 90 % of the amine groups have been functionalised with the protected dipeptide. The enzyme compatibility of µPEGA was then assessed. Here, Fmoc-AA functionalised particles were also exposed to a solution of the enzyme thermolysin for 12 hours before being reacted with dansyl chloride and imaged. Thermolysin, a protease which has been used previously on PEGA particles was used as the model enzyme. 957

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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PjwvQ2l0ZT48L0VuZE5vdGU+AG==

62,63,75,85

Thermolysin is a proteolytic enzyme with a well-known specificity for hydrophobic amino acids in the P’1 and little specificity for the P1 position.112 Additionally, thermolysin

is

known

to

be

active

on

peptides

linked

to

PEGA

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

WN0cm9uaWMtcmVzb3Vy Y2UtbnVtPjxsYW5ndWFnZT5FbmdsaXNoPC9sYW5ndWFnZT48L3JlY29yZD48 L0NpdGU+PENpdGU+ PEF1dGhvcj5UaG9ybnRvbjwvQXV0aG9yPjxZZWFyPjIwMDU8L1llYXI+PFJlY0 51bT4xOTwvUmVj TnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+MTk8L3JlYy1udW1iZXI+PGZvcmVp Z24ta2V5cz48a2V5 IGFwcD0iRU4iIGRiLWlkPSIyMHAwcjJkcDlmOXRkMmUycmE5dnIycG5zMHp4 dndwczB4NWUiPjE5 PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG5hbWU9IkpvdXJuYWwgQ XJ0aWNsZSI+MTc8 L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcnM+PGF1dGhvcj5UaG9ybnRv biwgUC4gRC48 L2F1dGhvcj48YXV0aG9yPk1jQ29ubmVsbCwgRy48L2F1dGhvcj48YXV0aG9yPl VsaWpuLCBSLiBW LjwvYXV0aG9yPjwvYXV0aG9ycz48L2NvbnRyaWJ1dG9ycz48dGl0bGVzPjx0aX RsZT5Fbnp5bWUg cmVzcG9uc2l2ZSBwb2x5bWVyIGh5ZHJvZ2VsIGJlYWRzPC90aXRsZT48c2Vjb 25kYXJ5LXRpdGxl PkNoZW1pY2FsIENvbW11bmljYXRpb25zPC9zZWNvbmRhcnktdGl0bGU+PC90 aXRsZXM+PHBlcmlv ZGljYWw+PGZ1bGwtdGl0bGU+Q2hlbWljYWwgQ29tbXVuaWNhdGlvbnM8L2Z 1bGwtdGl0bGU+PGFi YnItMT5DaGVtLiBDb21tdW4uPC9hYmJyLTE+PC9wZXJpb2RpY2FsPjxwYWdl cz41OTEzLTU5MTU8 L3BhZ2VzPjxudW1iZXI+NDc8L251bWJlcj48ZGF0ZXM+PHllYXI+MjAwNTwv eWVhcj48L2RhdGVz PjxhY2Nlc3Npb24tbnVtPklTSTowMDAyMzM3NzU2MDAwMjI8L2FjY2Vzc2lvb i1udW0+PHVybHM+ PHJlbGF0ZWQtdXJscz48dXJsPiZsdDtHbyB0byBJU0kmZ3Q7Oi8vMDAwMjMz Nzc1NjAwMDIyPC91 cmw+PC9yZWxhdGVkLXVybHM+PC91cmxzPjwvcmVjb3JkPjwvQ2l0ZT48L0V uZE5vdGU+ 62,79. Figure 3–11 C shows that ~70 % of the original number of amine groups available (due to the cleavage of the peptide bond) and that enzyme action occurs evenly throughout the interior of the particles. This result agrees with 976

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

HPLC results, in that >100 minutes the enzyme cleaves a maximum of around 70 % of the peptide bonds.

Figure 3–11. Assessment of the distribution of the amines within μPEGA by TPM. A: Schematic of the process used to determine the homogeneity of free amine groups and enzyme activity using dansyl labelling. Three different batches of particles were prepared: a) unmodified and labelled with dansyl-chloride, (b) functionalised with Fmoc-AA then labelled with dansyl-chloride and (c) functionalised with Fmoc-AA then treated with thermolysin and labelled with dansyl-chloride (scale bar is 15 μm). B: Two photon micrographs (TPM) of representative microparticles for each of the different treatments. C: Cross-section of the intensity of fluorescence in each of the 3 microparticles

1.2.3.2. Comparison of enzymatic hydrolysis within µPEGA800 and macroparticles Enzyme hydrolysis is the process that initiates a responsive change in particle swelling (when peptide actuator functionality has been incorporated). Therefore, the rate at which enzyme hydrolysis occurs within the PEGA particles was the main factor determining the response time of these functionalised particles. In order to

977

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

assess how quickly the enzyme catalysed hydrolysis occurs, a thermolysin solution was added to Fmoc-AA modified particles. The reaction was then stopped with 0.1% TFA at different times and analysed with HPLC to quantify any cleaved residues. The Figure 3–12 shows that a maximum hydrolysis of the dipeptide was attained after approximately 110 minutes for µPEGA. This experiment was repeated on larger (200-400 µm diameter) commercially available PEGA800 macroparticles, where maximum hydrolysis was found after 150 minutes with an initial rate three times slower than the enzyme reaction on the microparticles. Other workers79 have also shown that on larger commercially available particles maximum hydrolysis was obtained after a much longer time, typically ~ 4 hours.

Figure 3–12. Comparison of time dependence of enzyme reactions on ♦ (blue): µPEGA and ■ (red): commercially available macroparticles (curves are guides to the eye).

1.1.1. Functionalisation with peptide actuators Enzyme responsiveness was incorporated into PEGA microparticles through SPPS. The peptide actuator functionality is shown schematically in Figure 3–1. These peptide actuators are made up of four amino acids and which can be divided into two sections based on their role; in the centre of the peptide actuator is an uncharged dipeptide (shown in green) this is the sensing element the enzyme cleavable peptide (ECP), while at each end of the peptide actuator is an oppositely charged amino acid

978

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

(making the peptide actuator a zwitterion). If a protease with specificity for the ECP is present it hydrolyses the amide bond in the ECP and only the positively charged half of the peptide actuator remains covalently attached to the polymer. Additionally, dependant on the pH of the surrounding solution (if below pH 7.5) the amine on the remaining half of the ECP may become protonated and therefore positively charged. Within the polymer network these neighbouring cationic fragments of the peptide actuators electrostatically repel one another resulting in an increase in the swelling of the polymer particles. These peptide actuators have already been shown on macroparticles (this is covered in more detail in the preceding literature review, within section Electrostatic interactions between charges present in pendant actuators). By making use of the faster rates of complete hydrolysis observed on microparticles it should be possible to demonstrate a faster enzyme responsive system. 1.1.1.1. Enzyme responsive increase in accessibility Enzymes are highly specific and in order to determine whether this specificity could be used to for selective release, the change in accessibility of the microparticles was measured after exposure to three different enzymes. µPEGA was firstly functionalised with peptide actuator with three different ECPs, these were AA, GG and FF. The change in accessibility upon enzyme treatment was then determined by immersing the particles in a solution containing FITC labelled dextrans of known molecular weight, then using confocal microscopy to visualise the location of fluorescence. Confocal microscopy allows fluorescent cross-sectional images of the particles to be obtained. This technique can have problems with quenching occurring in the centre of hydrogel particles79 however, because of the small size of the microparticles quenching was not observed and the technique is suitable for this purpose. The three proteolytic enzymes tested were Thermolysin, Chymotrypsin and Elastase. Each of these enzymes has a different specificity (i.e. the composition of the peptides that they will hydrolyse), details of these specificities and the molecular weight of the enzymes is shown in Table 3–1. Thermolysin, a thermostable metalloproteinase

produced

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979

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5ZYXN1a2F3YTwvQXV0aG9yPjxZZWF yPjIwMDc8L1llYXI+ PFJlY051bT4zNzI8L1JlY051bT48cmVjb3JkPjxyZWMtbnVtYmVyPjM3MjwvcmVjL W51bWJlcj48 Zm9yZWlnbi1rZXlzPjxrZXkgYXBwPSJFTiIgZGItaWQ9ImZhOXpkenN2a3N4MnBy ZXJlOTh4ZDUy cWRmd2Z3cjkwcDVzMiI+MzcyPC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eX BlIG5hbWU9Ikpv dXJuYWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhv cnM+PGF1dGhv cj5ZYXN1a2F3YSwgSy48L2F1dGhvcj48YXV0aG9yPkt1c2FubywgTS48L2F1dGhvc j48YXV0aG9y Pklub3V5ZSwgSy48L2F1dGhvcj48L2F1dGhvcnM+PC9jb250cmlidXRvcnM+PGF1 dGgtYWRkcmVz cz5LeW90byBVbml2LCBHcmFkIFNjaCBBZ3IsIERpdiBGb29kIFNjaSAmYW1wOyB iaW90ZWNobm9s LCBLeW90byA2MDYsIEphcGFuLiYjeEQ7SW5vdXllLCBLLCBLeW90byBVbml2LC BHcmFkIFNjaCBB Z3IsIERpdiBGb29kIFNjaSAmYW1wOyBiaW90ZWNobm9sLCBLeW90byA2MDYsIE phcGFuLiYjeEQ7 aW5vdXllQGthaXMua3lvdG8tdS5hYy5qcDwvYXV0aC1hZGRyZXNzPjx0aXRsZXM +PHRpdGxlPkEg bmV3IG1ldGhvZCBmb3IgdGhlIGV4dHJhY2VsbHVsYXIgcHJvZHVjdGlvbiBvZiByZ WNvbWJpbmFu dCB0aGVybW9seXNpbiBieSBjby1leHByZXNzaW5nIHRoZSBtYXR1cmUgc2VxdWV uY2UgYW5kIHBy by1zZXF1ZW5jZSBpbiBFc2NoZXJpY2hpYSBjb2xpPC90aXRsZT48c2Vjb25kYXJ5L XRpdGxlPlBy b3RlaW4gRW5naW5lZXJpbmcgRGVzaWduICZhbXA7IFNlbGVjdGlvbjwvc2Vjb25k YXJ5LXRpdGxl PjxhbHQtdGl0bGU+UHJvdGVpbiBFbmcuIERlcy4gU2VsLjwvYWx0LXRpdGxlPjw vdGl0bGVzPjxw 982

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

ZXJpb2RpY2FsPjxmdWxsLXRpdGxlPlByb3RlaW4gRW5naW5lZXJpbmcgRGVzaWd uICZhbXA7IFNl bGVjdGlvbjwvZnVsbC10aXRsZT48YWJici0xPlByb3RlaW4gRW5nLiBEZXMuIFNlb C48L2FiYnIt MT48L3BlcmlvZGljYWw+PGFsdC1wZXJpb2RpY2FsPjxmdWxsLXRpdGxlPlByb3R laW4gRW5naW5l ZXJpbmcgRGVzaWduICZhbXA7IFNlbGVjdGlvbjwvZnVsbC10aXRsZT48YWJici0xP lByb3RlaW4g RW5nLiBEZXMuIFNlbC48L2FiYnItMT48L2FsdC1wZXJpb2RpY2FsPjxwYWdlcz4z NzUtMzgzPC9w YWdlcz48dm9sdW1lPjIwPC92b2x1bWU+PG51bWJlcj44PC9udW1iZXI+PGtleXdv cmRzPjxrZXl3 b3JkPmF1dG9jYXRhbHl0aWMgZGlnZXN0aW9uIGFjdGl2aXR5PC9rZXl3b3JkPjxr ZXl3b3JkPkVz Y2hlcmljaGlhIGNvbGk8L2tleXdvcmQ+PGtleXdvcmQ+bWV0YWxsb3Byb3RlaW5hc 2U8L2tleXdv cmQ+PGtleXdvcmQ+cHJvLXNlcXVlbmNlPC9rZXl3b3JkPjxrZXl3b3JkPnRoZXJtb 2x5c2luPC9r ZXl3b3JkPjxrZXl3b3JkPlNJVEUtRElSRUNURUQgTVVUQUdFTkVTSVM8L2tleXd vcmQ+PGtleXdv cmQ+TkVVVFJBTCBQUk9URUFTRTwva2V5d29yZD48a2V5d29yZD5CQUNJTEx VUy1TVUJUSUxJUzwv a2V5d29yZD48a2V5d29yZD5JTlRSQU1PTEVDVUxBUiBDSEFQRVJPTkVTPC9r ZXl3b3JkPjxrZXl3 b3JkPlJFTUFSS0FCTEUgQUNUSVZBVElPTjwva2V5d29yZD48a2V5d29yZD5DQ VRBTFlUSUMtQUNU SVZJVFk8L2tleXdvcmQ+PGtleXdvcmQ+Q1JZU1RBTC1TVFJVQ1RVUkU8L2tleX dvcmQ+PGtleXdv cmQ+RU5aWU1FPC9rZXl3b3JkPjxrZXl3b3JkPlNBTFRTPC9rZXl3b3JkPjxrZXl3b 3JkPlNUQUJJ TElUWTwva2V5d29yZD48L2tleXdvcmRzPjxkYXRlcz48eWVhcj4yMDA3PC95ZWFy PjxwdWItZGF0 ZXM+PGRhdGU+QXVnPC9kYXRlPjwvcHViLWRhdGVzPjwvZGF0ZXM+PGlzYm 4+MTc0MS0wMTI2PC9p c2JuPjxhY2Nlc3Npb24tbnVtPklTSTowMDAyNTAxOTc5MDAwMDI8L2FjY2Vzc2lv 983

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

bi1udW0+PHdv cmstdHlwZT5BcnRpY2xlPC93b3JrLXR5cGU+PHVybHM+PHJlbGF0ZWQtdXJscz 48dXJsPiZsdDtH byB0byBJU0kmZ3Q7Oi8vMDAwMjUwMTk3OTAwMDAyPC91cmw+PC9yZWxhd GVkLXVybHM+PC91cmxz PjxlbGVjdHJvbmljLXJlc291cmNlLW51bT4xMC4xMDkzL3Byb3RlaW4vZ3ptMDMx PC9lbGVjdHJv bmljLXJlc291cmNlLW51bT48bGFuZ3VhZ2U+RW5nbGlzaDwvbGFuZ3VhZ2U+P C9yZWNvcmQ+PC9D aXRlPjwvRW5kTm90ZT5= 113 has a well-known specificity for hydrophobic amino acids in the P’1 and little specificity for the P1 position.112 Chymotrypsin, an enzyme found in the digestive systems of mammals was chosen due to its complementary specificity towards substrates with aromatic groups or to a lesser extent, hydrophobic amino acids with bulky side chains in the P1 position with little specificity for the amino acid in P’1.114 Finally, Elastase was included due to its clinically relevance in its involvement in breaking down the extracellular matrix (ECM); it preferentially cleaves peptide bonds between amino acids with small uncharged side chains (alanine or valine in the P1).114 Table 3–1. Specificity of the enzymes used towards amino acids (AA) in the substrate and their molecular weight.

Enzyme Specificity

Thermolysin Hydrophobic AA in

Chymotrypsin Aromatic/Hydrophobic

Elastase Small uncharged

P’1

AA in P1

AA in P1 and P’1

35

25

25

Molecular Weight (kDa) Table 3–2 shows the relative cleavages of each ECP for each enzyme, in which 100% cleavage was taken as the maximum peak area detected on HPLC for that ECP (therefore discounting the dipeptides at sites inaccessible to the enzymes). As expected, thermolysin was found to give the highest cleavages for both the AA and FF enzyme cleavable peptides due to its specificity for hydrophobic residues. Some hydrolysis also occurred for ECP the composed of GG indicating that the enzyme is somewhat unspecific. Chymotrypsin would be expected to cleave the FF ECP more than the other ECPs, however, the comparatively high cleavage of the peptide actuator with GG as the ECP was not expected (due to the hydrophilic nature of

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

glycine). Standard chymotrypsin is known to contain trypsin impurities, as it is derived from the proteolytic cleavage of chymotrypsinogen by trypsin.115 Trypsin has been shown to cleave peptides when hydrophobic residues or lysine or arginine are in the P’1 position (except when praline is in the P1),116 therefore, the higher observed value of hydrolysis for the GG ECP may be due to some cleavage occurring between the ECP and arginine. Elastase was observed to give high percentage cleavages for the AA ECP due to its preference for cleaving peptide bonds between amino acids with small uncharged side chains. Table 3–2. Values for HPLC relative enzyme cleavage for each ECP.

ECP

Thermolysin

Chymotrypsin

Elastase

Ala-Ala (AA) Gly-Gly (GG) Phe-Phe (FF)

100% 9% 100%

4% 20% 23%

99% 6% 14%

When the peptide actuator was intact (the peptide actuators were zwitterions) the accessibility was determined to be less than 40 kDa. The 40 kDa fluorescently labelled dextran was not able enter the particles. This was because the net charge of the peptide actuators was neutral. However, if the ECP was cleaved then there was a switch from uncharged to positively charged resulting in an increase in swelling of the particles and therefore mesh size of the network. This made the polymer network more accessible allowing the 40 kDa dextran to diffuse into the microparticles. This process is shown schematically in Figure 3–13. The confocal micrographs of representative microparticles observed in the enzyme specificity investigation are provided in Figure 3–14. It can noted that a sufficient increase in accessibility to allow the FITC dextran to enter the particles is only seen for thermolysin treatment of AA and FF, while elastase only initiated a response with AA as the ECP. HPLC was used to provide quantitative information about the hydrolysis of the peptide actuators (Table 3–2) showing that there was still some hydrolysis of the peptide actuators for all ECPs. However, the hydrolysis (up to 23 %) was insufficient to trigger an increase in accessibility.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 3–13. A schematic of enzyme responsive microparticles illustrating both successful (top) and unsuccessful (bottom) cleavage of the ECP resulting in a change of accessibility to a 40 kDa FITC labelled dextran.

Figure 3–14. Confocal microscopy images of representative microparticles in an aqueous solution of 40 kDa fluorescently labelled dextran. Scale bar is 20 µm.

1.1.1.2. Demonstration of the encapsulation of a payload In order to be able to release a payload it must first be encapsulated. To achieve this the pH responsive nature of the peptide actuators was utilised. The charges within the peptide actuator are due to the acidic and basic amino acid side groups. By lowering the pH below the pKa of the carboxylate side group it becomes protonated,

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

changing the net charge of the peptide actuator from neutral to positive. This results in electrostatically induced swelling leading to an increase in accessibility. Through the use of confocal microscopy to observe the spatial confinement of a FITC labelled 40 kDa dextran the loading and encapsulation process was demonstrated (Figure 3–15). At pH 7 fluorescence was not observed within the particles indicating that the dextran was not able to diffuse into the polymer. Upon lowering the pH to 3 the 40 kDa FITC dextran was able to diffuse into the particles. The apparent decrease in the fluorescence at pH 3 is due to the pH dependence of the fluorophore efficiency. By returning the pH to around pH 7 by the addition of base the dextran remained in the particles. After repeated washing with water, fluorescence remained inside the particles.

Figure 3–15. The pH responsive loading of the 40 kDa FITC labelled dextran (1 mg/ml) into the PEGA particles, gain of images varied. (A) Particles in a solution of the dextran in water. (B) Particles in a solution of dextran in dilute HCL at pH 3, purple colour represents detector saturation. (C) Particles after being lowered to pH 3, neutralised to pH 7 and washed several times with water. Scale bar is 15 µm.

1.1.1.3. Enzyme specific release Utilising the method described in section Demonstration of the encapsulation of a payload the peptide actuator functionalised particles were loaded with 40 kDa FITC dextran and washed. The particles were then treated with either thermolysin or chymotrypsin in water. Thermolysin has specificity for the ECP (AA) in the peptide sequence therefore it should cleave the sequence triggering an increase in swelling and thus release. The fluorescent intensity of the solution surrounding the particles was then measured (after 85 minutes), determining whether the FITC labelled dextran has been released. The result showed there was a dramatic difference between the amount of release for thermolysin and chymotrypsin with approximately 15 times more release occurs when treated with thermolysin compared to chymotrypsin. This is due to the specific enzymatically hydrolysis of the ECP that only occurs with thermolysin. 987

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1.1.1.4. Enzyme responsive increase in swelling; effect of ionic strength The responsive nature of the functionalised particles could also be assessed by monitoring the change in size of individual particles when treated with different enzymes. Figure 3–16 shows that when the particles were treated with thermolysin dissolved in water there is an increase in volume of ≈ 60 % after 15 minutes. Additionally, we observe the specificity of the response as chymotrypsin did not trigger an increase in swelling. However, if the linear peptide actuator functionalised µPEGA was treated with thermolysin in a solution at ionic strength of 0.18 M no change in swelling was observed. This was a result of the electrostatic screening of the charges on the peptide actuators by the ions within the solution. This finding has also been observed on peptide actuator-functionalised PEGA macroparticles by other researchers.58

Figure 3–16. Change in volume of individual particles over time with different treatments: ♦ (blue): Thermolysin in water. ■ (red): Chymotrypsin in water. ▲ (green): thermolysin in buffer at an ionic strength of 0.18 M.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1.1. Conclusions Three different types of PEGA microparticles have been prepared. PEGA hydrogels incorporating charges within the polymeric backbone displayed increased swelling in water due to the electrostatic repulsion resulting in a greater uptake of water. Within a buffer solution 0.1 M all PEGA hydrogels had similar swelling ratios due to the screening of some of the longer range electrostatic interactions within the polymers. Neutral µPEGA was chosen for further investigation these cell-sized particles had a mean diameter of 15 µm and a polydispersity of 43 %. The distribution of amines was homogeneous throughout the particles. It was shown that enzyme action on coupled peptides was also homogeneous. These microparticles gave rise to faster enzymatic hydrolysis than commercially available large PEGA particles (macroparticles) due to the greater surface area to volume ratio of smaller particles. When µPEGA was functionalised with peptides actuators an enzyme specific response through the increase in the accessibility was observed. The pH responsiveness of these particles has been demonstrated by the loading of the particles with a fluorescently labelled macromolecule. Enzyme specific release of this payload was possible. However, at physiological ionic strength no enzyme triggered swelling of the functionalised particles was observed. This limitation will be addressed in the following chapter with the design of branched peptide actuators.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2 Designing new peptide actuators for improved enzyme responsive behaviour3 2.1. Abstract This chapter describes the functionalised μPEGA with branched peptide actuators. These peptide actuators provided enhanced charge density and responded through an increase in swelling to the target enzyme at physiological ionic strength. Analysis of enzymatic activity revealed that the target enzyme (thermolysin) could access the core of particles when linear peptides are used, while access was restricted to the surface when using branched actuators due to electrostatic interactions. These responsive μPEGA particles were then loaded with a fluorescent labelled dextran by application of a sequential pH change. The macromolecule payload could be selectively released at physiological ionic strength when exposed to the target enzyme.

2.2. Introduction 1.1.1. Branched peptide actuators As demonstrated at the end of Chapter PEGA polymerisation, characterisation and enzyme responsive swelling through functionalisation with peptide actuators2 and earlier systems from the Ulijn and co-workers,58 responsive systems based on electrostatic actuation using peptide actuators are inherently sensitive to the ionic strength of the solution. This limitation meant that they were unable to function in solutions with physiological ionic strength due to electrostatic screening.58 In this chapter this problem is addressed through the design of branched peptide actuators with enhanced charge density. The inability of the linear peptide actuator to respond at physiological ionic strength was related to the electrostatic screening of neighbouring charges.58 For charge induced swelling to occur the distance between the charges must be less than the Debye length. At physiological ionic strength (0.15 M) the Debye length is 0.8 nm.

3 This chapter has been published as: Mcdonald, T.O., Qu, H., Saunders, B.R. and

Ulijn, R.V.,

Branched Peptide Actuators for Enzyme Responsive Hydrogel Particles. Soft Matter. (2009).

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

By increasing the space occupied by the actuator we aimed to reduce the distance between neighbouring charges to less than 0.8 nm. The approach used to was to incorporate the di-amino functionalised amino acid lysine (K) to act as a branch point thus causing the peptide actuator to occupy more space (Figure 4–1). Each arm consists of three parts: i) a di-glycine (G) spacer to enable enzyme access, ii) oppositely charged actuation amino acids and which are iii) separated by an enzyme cleavable peptide (ECP) sequence which serves as the sensing element. By matching the ECP to the specificity of a target protease, the material may be programmed to respond exclusively to a target enzyme. Enzymatic hydrolysis of the ECP leads to release of anionic fragments and conversion of the branched zwitterionic peptide actuator to cationic groups remaining covalently bonded to the polymer (Figure 4– 1). These neighbouring cationic groups induce swelling and an increase in the mesh size within the polymer which can be exploited in triggered release of pre-entrapped payload molecules. The objectives within this chapter were to: (i) synthesise and characterise branched peptide actuators on µPEGA, (ii) investigate the enzyme response behaviour of the functionalised particles and (iii) utilise these particles for triggered enzyme of a payload at physiological ionic strength.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 4–1. Schematic of enzyme responsive branched peptide actuator. Left; peptide actuator, Right; upon cleavage of peptide by an enzyme only the cationic groups remain attached. These charged peptide fragments then electrostatically repel one another leading to an increase in swelling of the polymer.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1.1. Materials and methods 1.1.1. Materials All chemicals were used as supplied from Sigma with the exception of amino acids (Bachem), PEGA macromonomers (Versamatrix), Isopar M (Multisol) and N,N,N′,N ′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium

hexafluorophosphate

(HBTU)

(AGTC Bioproducts Ltd). The enzymes used were thermolysin (EC 3.4.24.27), 36.5 U/mg and chymotrypsin (EC 3.4.21.1), 60 U/mg. 1.1.2. Inverse suspension polymerisation and polymer characterisation A stainless steel baffleless reactor (250 ml) stirred with an anchor-style agitator was used for the polymerisation reaction. 3.14 g (3.4 mmol) of the PEGA 800 macromonomers (2:1 ratio of acrylamide-PEG-acrylamide to amino-PEGacrylamide) and 0.156 g (2.2 mmol) of acrylamide were dissolved in 10 ml of distilled water and purged for 30 minutes with N gas. 50 ml of Isopar M 2

(isoparaffin) was added to the reactor and was also purged for 30 minutes. The reactor was heated to 70°C. After 20 minutes of purging 0.16 ml (1.0 mmol) of N,N,N′,N′- tetramethylethylenediamine (TEMED) was added to the oil phase. 0.164 g (0.47 mmol) of Span 20 (sorbitan monolaurate) was dissolved in the oil, which was stirred at 500 rpm for 30 seconds to ensure the surfactant was fully dispersed in the oil phase. 0.070 g (0.30 mmol) of ammonium persulfate (APS) was dissolved in the macromonomer solution, which was added to the oil phase in the reactor and stirred at 2000 rpm for a further 30 minutes. The particles were washed with (3 x 50 ml) dichloromethane (DCM), (3 x 50 ml) Tetrahydrofuran (THF), (3 x 50 ml) methanol and (4 x 50 ml) distilled water. Particle size distribution and mean particle diameter were determined using a Malvern Mastersizer particle size analyser, Mastersizer Microplus software version 2.18 was used to analyse the results. Environmental scanning electron microscopy (ESEM) images were taken on a FEI Quanta 200 ESEM, using low vac mode at 10.0 KV. 1.1.3. Solid phase peptide synthesis Peptide actuators (linear: Fmoc-DAAR-PEGA) and branched: (Fmoc-DAARGG)2K-PEGA) were prepared by solid phase peptide synthesis using Fmoc protected amino acids. 8 equivalents of the amino acid and 7.8 equivalents of HBTU were 993

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

dissolved in 2 ml of N,N-Dimethylformamide (DMF). 16 equivalents of N,NDiisopropylethylamine (DIPEA) was added to this solution prior to its addition to 0.1 g of µPEGA. Coupling was left for 16 hours, and Kaiser test117 was used to check for complete coupling. Deprotection was achieved using 20 % piperidine in DMF for 2 hours. This procedure was repeated to build up the peptide sequence with thorough washing between steps (5 x 5 ml methanol, 5 x 5 ml 1:1 methanol:DMF, 5 x 5 ml DMF). A solution of 95 % Trifluoroacetic acid (TFA) and 5 % water was used to remove the amino acid side chain protecting groups. 1.1.4. Microscopy and determination of swelling Swelling measurements and fluorescent images were obtained using a Zeiss Imager A1 microscope, a Leistungselektronik mbq 52 AC power source (Jena, Germany) equipped with an HBO 50 mercury lamp and Canon Powershot G6 camera. A Zeiss filter set 09 (excitation 450 - 490 nm, emission 515 + nm) was used to visualise FITC. For pH responsive swelling peptide actuator functionalised PEGA particles were immersed in either water (CHROMASOLV plus for HPLC) or 0.01 M HCl (pH 2.5) and images obtained using the microscope. ImageJ 1.38x analysis software was used to determine the change in particle diameter. For pH responsive swelling the diameter of a minimum of 300 particles was measure and the mean volume calculated. For enzyme responsive swelling individual particles were observed throughout the timecourse and an average V/Vo was determined from at least six representative particles, where V is the volume of the particle at time t and Vo is the volume of the particle immediately after exposure to enzyme solution. Enzyme solutions were made up at a concentration of 1 mg/ml and buffers were prepared by using the appropriate amounts of sodium phosphate dibasic heptahydrate and sodium phosphate monobasic. 1.1.5. Two-photon microscopy µPEGA functionalised with either linear or branched peptide actuators was exposed to an aqueous thermolysin solution (1 mg/ml) for 40 minutes. The particles were then washed with (5 x 1 ml) Acetonitrile (AcN):H2O (50:50) containing 0.1 % TFA and then (5 x 1 ml) DMF. 6 equivalents of dansyl-Cl were dissolved in 2 ml DMF along with 10 equivalents DIPEA this solution was added to the particles and incubated in the dark at room temperature for 2 hours. The dansyl-labelled particles were washed with (3 x 1 ml) DMF and (3 x 1 ml) water. Two-photon microscopy

994

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

images of the particles in water were obtained on a Leica TCS SP2 inverted microscope. ImageJ software was used to produce the surface plots of intensity. The intensities at the centre, the outside (1 µm from the edge of the particle) and the average intensity across the particle were determined for 15 particles functionalised with either the linear or branched peptide actuators. 1.1.6. Entrapping payload The particles (0.1 g) were loaded with a 40 kDa FITC Dextran using the following method; 2 ml of solution of FITC dextran in 0.01 M HCl (10 mg/ml) was added to the particles for 30 minutes (pH 2.5). After this time 0.4 ml of 0.05 M NaOH was added dropwise (pH 7). The particles were then filtered and washed (2 x 5 ml water, 2 x 5 ml 0.18 M buffer, 2 x 5 ml methanol). 1.1.7. Release measurements The release of entrapped dextran was determined using the following method; loaded particles were treated in solution for 80 minutes, these samples were centrifuged

and

the

supernatant

was

removed.

A

Jasco

FP

–

6500

Spectrofluorometer using an excitation wavelength of 490 nm was used to obtain the fluorescent intensity at 519 nm. Treated particles were filtered and imaged using fluorescence microscopy. 1.1.8. HPLC and LCMS HPLC experiments were undertaken on a Dionex HPLC (P680 pump, ASI-100 Automated sample injector, Nucleosil 100-5-C18 column with a UVD170U detector), using a solvent ramp of 20 % ACN and 80 % water to 80 % ACN 20 % water over 30 minutes (0.1 % TFA was present in both phases). Chromeleon 6.60 software was used for analysis. All LCMS analyses were carried out on a reversephase Luna C18(2), 250 x 2mm, 5 micron column (Phenomenex). The LC-MS instrument was an Agilent 1100 Series HPLC, coupled to an Agilent 1956B Mass Detector. The solvent ramp of 90 % water 10 % ACN to 15 % water 85 % AcN over 14 minutes was used in all analyses; the flow rate was set at 1.0 mL min-1. Mass detection was set to analyse in SCAN mode with electrospray ionisation.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1.2. Results and discussion 1.2.1. Microparticle characterisation and peptide functionalisation µPEGA was produced (described in detail in Chapter PEGA polymerisation, characterisation and enzyme responsive swelling through functionalisation with peptide actuators2) and peptide functionalisation was achieved through solid phase peptide synthesis using Fmoc protected amino acids. Use of the Kaiser test and the quantification of Fmoc removed at each deprotection step indicated high yield peptide formation with over 90 % of the initial loading achieved for the final step (Figure 4–2).

Figure 4–2. HPLC quantification of Fmoc removed after each coupling step for linear and branched peptide actuators.

The charged structure of the peptide actuators can be indirectly determined by examining the pH responsive swelling of the peptide actuators. The charged state of these peptide actuators is pH dependant, as the amino acid side chains have pKa values of 4.4 and 12.0 for aspartic acid (D) and arginine (R) respectively. Therefore, if the pH was lowered below 4.4, the previously anionic carboxylate groups became protonated giving the peptide actuators a net positive charge. Electrostatic repulsion between neighbouring positively charged actuators led to an increase in swelling of the particles. This can be seen in Figure 4–3 where at pH 2.5 there was a 30 % increase in volume for particles functionalised with linear peptide actuator while a

996

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

57 % increase in volume was noted with branched peptide actuators. This difference can be attributed to the higher density present in the branched actuator.

Figure 4–3. pH responsive swelling behaviour of peptide actuators on PEGA microparticles (average of at a minimum of 300 particles). Student’s t-test shows a difference is significant at 98 %.

1.2.2. Actuator design and responsiveness of peptide functionalised particles The responsive increase in particle swelling is dictated by the extent of electrostatic repulsion between neighbouring charged groups. Figure 4–4 A shows the enzyme responsive swelling of PEGA microparticles functionalised with linear peptide actuators in water. The distance between charged groups in linear actuators (FmocDAAR-PEGA) is greater than the Debye length at 0.15 M (0.8 nm) and therefore did not give rise to sufficient electrostatic repulsion to cause swelling. The branched design of the new actuator occupies more space than the linear actuator resulting in the cationic groups being closer together. Figure 4–4 B shows that when the particles functionalised with the branched actuator are treated with thermolysin (from Bacillus thermoproteolyticus rokko E.C. 3.4.24.27) at an ionic strength of 0.18 M an increase in volume is observed reaching a maximum of ~1.3 V/Vo after 20 minutes. This response is exclusively observed upon treatment with thermolysin. This enzyme has a relatively broad specificity preferring hydrophobic residues in the P1’ position.112 While treatment with α-chymotrypsin from bovine pancreas (E.C.

997

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

232.671.2), which has a preference for large hydrophobic residues in the P1 position,114 did not give rise to a change in swelling (visualised in Figure 4–4 C). Indeed, HPLC and LCMS analysis of the solutions obtained after enzyme treatment shows that ECP cleavage occurs exclusively with thermolysin (Figure 4–5 A). Furthermore, LCMS data after enzyme treatment shows two main peaks consisting of fragments of the desired peptide sequence (Figure 4–5 B & C). Therefore, functionalisation of µPEGA with the branched peptide actuator has achieved enzyme specific response at physiological ionic strength. Next, the ionic strength dependence of the branched actuators (Figure 4–6) was investigated by determining the final increase in swelling. It was found that, due to the enzyme triggered increased charge density maximum swelling (~1.3 V/V o) was observed at and above physiological ionic strength (0.15 M)118,119 up to 0.3 M. This corresponds to a Debye length of 0.55 nm. Presumably, at higher ionic strengths the mobile ions in solution begin to screen the static peptide charges effectively resulting in a decrease in the amplitude of the response. This is most pronounced at an ionic strength of about 0.45 M where V/Vo ≈ 1.15. As expected, chymotrypsin did not initiate a response at any ionic strength tested. To conclude this section, the branched peptide actuator functionalised particles demonstrate specific enzyme responsive at physiological ionic strength and above.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 4–4. Enzyme responsive swelling behaviour of peptide actuator functionalised µPEGA at pH 7.

A: Swelling of particles functionalised with the linear peptide actuator; ●:

Thermolysin in water, ¯ : Thermolysin at ionic strength 0.18 M. B: Swelling of particles functionalised with the branched peptide actuator; ¯: Thermolysin at ionic strength 0.18 M, r: Thermolysin at ionic strength 0.76 M, ¢: Chymotrypsin in water. C: Swelling of individual particles treated with either Thermolysin or Chymotrypsin at 0.18 M ionic strength (circle shows original size, scale bars are 15µm).

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 4–5. HPLC and MS analysis of enzyme hydrolysis of branched peptide actuators. A: HPLC traces for thermolysin (blue) and chymotrypsin (pink) treated particles modified with branched peptide actuators. Absorbance at 254 nm. The ratio of peak areas is 3:1 (1st peak : 2nd peak). B: Mass spectra for thermolysin treated particles modified with branched peptide actuators peak at 22.1 minutes. C: Mass spectra for thermolysin treated particles modified with branched peptide actuators peak at 24.8 minutes. D: Key of peptide fragments found.

Figure 4–6. Effect of ionic strength on the maximal enzyme responsive swelling of branched peptide actuator functionalised µPEGA at pH 7 (after 40 minutes) ¯ :Thermolysin , r: No enzyme, ¢: Chymotrypsin. Dashed line indicates physiological ionic strength.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1.2.3. Characterisation of enzyme action on peptide actuators Enzymatic hydrolysis of the peptide actuators produces polymer-bond peptide fragments with free amines (see Figure 4–1). The distribution of amines can be examined through labelling with dansyl which allows the location of enzyme hydrolysis within the particles to be investigated (as also previously described in section

Amine

characterisation

by

two-photon

microscopy

and

enzyme

compatibility). TPM was used to assess the distribution of fluorophores (dansyl) within PEGA particles functionalised with either linear or branched peptide actuators after 40 minutes thermolysin treatment. As seen in Figure 4–7 A & B enzyme cleavage of the linear peptide actuators on PEGA particles was homogeneous on the micrometer scale, while branched peptide actuator functionalised particles had greater cleavage in the outer regions of the particles. The pI

of

thermolysin

is

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

dvcmQ+R0VORTwv a2V5d29yZD48a2V5d29yZD5TVEVBUk9USEVSTU9QSElMVVM8L2tleXdvcm Q+PGtleXdvcmQ+SU5I SUJJVE9SUzwva2V5d29yZD48a2V5d29yZD5CSU5ESU5HPC9rZXl3b3JkPjwva2 V5d29yZHM+PGRh dGVzPjx5ZWFyPjE5OTY8L3llYXI+PHB1Yi1kYXRlcz48ZGF0ZT5KdW48L2Rh dGU+PC9wdWItZGF0 ZXM+PC9kYXRlcz48aXNibj4xMzgxLTExNzc8L2lzYm4+PGFjY2Vzc2lvbi1udW 0+SVNJOkExOTk2 VVUzMTQwMDAxNTwvYWNjZXNzaW9uLW51bT48d29yay10eXBlPkFydGljb GU8L3dvcmstdHlwZT48 dXJscz48cmVsYXRlZC11cmxzPjx1cmw+Jmx0O0dvIHRvIElTSSZndDs6Ly9BMT k5NlVVMzE0MDAw MTU8L3VybD48L3JlbGF0ZWQtdXJscz48L3VybHM+PGxhbmd1YWdlPkVuZ2x pc2g8L2xhbmd1YWdl

PjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+AG==

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

QUdBTUlIQVJBLEtBTkFHQVdBIDIyOSxKQVBBTi4gVE9TT0ggQ09SUCxCSU 9URUNITk9MIFJFUyBM QUIsQVlBU0UsS0FOQUdBV0EgMjUyLEpBUEFOLjwvYXV0aC1hZGRyZXNzP jx0aXRsZXM+PHRpdGxl PkVmZmVjdCBvZiBhIGNoYXJnZWQgcmVzaWR1ZSBhdCB0aGUgMjEzdGggc 2l0ZSBvZiB0aGVybW9s eXNpbiBvbiB0aGUgZW56eW1hdGljIGFjdGl2aXR5PC90aXRsZT48c2Vjb25kYX J5LXRpdGxlPkpv dXJuYWwgb2YgTW9sZWN1bGFyIENhdGFseXNpcyBCLUVuenltYXRpYzwvc2 Vjb25kYXJ5LXRpdGxl PjxhbHQtdGl0bGU+Si4gTW9sLiBDYXRhbC4gQi1Fbnp5bS48L2FsdC10aXRsZT 48L3RpdGxlcz48 cGVyaW9kaWNhbD48ZnVsbC10aXRsZT5Kb3VybmFsIG9mIE1vbGVjdWxhciB DYXRhbHlzaXMgQi1F bnp5bWF0aWM8L2Z1bGwtdGl0bGU+PGFiYnItMT5KLiBNb2wuIENhdGFsLiBC LUVuenltLjwvYWJi ci0xPjwvcGVyaW9kaWNhbD48YWx0LXBlcmlvZGljYWw+PGZ1bGwtdGl0bGU +Sm91cm5hbCBvZiBN b2xlY3VsYXIgQ2F0YWx5c2lzIEItRW56eW1hdGljPC9mdWxsLXRpdGxlPjxhYm JyLTE+Si4gTW9s LiBDYXRhbC4gQi1Fbnp5bS48L2FiYnItMT48L2FsdC1wZXJpb2RpY2FsPjxwY Wdlcz4xOTEtMTk5 PC9wYWdlcz48dm9sdW1lPjE8L3ZvbHVtZT48bnVtYmVyPjMtNjwvbnVtYmVyP jxrZXl3b3Jkcz48 a2V5d29yZD50aGVybW9seXNpbjwva2V5d29yZD48a2V5d29yZD5hY3Rpdml0e Twva2V5d29yZD48 a2V5d29yZD5lbGVjdHJvc3RhdGljIHBvdGVudGlhbDwva2V5d29yZD48a2V5d29 yZD5vcHRpbXVt IHBIPC9rZXl3b3JkPjxrZXl3b3JkPnBJPC9rZXl3b3JkPjxrZXl3b3JkPlNVQlRJTEl TIE5FVVRS QUwgUFJPVEVBU0U8L2tleXdvcmQ+PGtleXdvcmQ+QU1JTk8tQUNJRCBTRV FVRU5DRTwva2V5d29y ZD48a2V5d29yZD5PUEVOIFJFQURJTkcgRlJBTUU8L2tleXdvcmQ+PGtleXdvc mQ+QkFDSUxMVVMt U1VCVElMSVM8L2tleXdvcmQ+PGtleXdvcmQ+RUxFQ1RST1NUQVRJQyBQT 1004

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1RFTlRJQUxTPC9rZXl3 b3JkPjxrZXl3b3JkPk5VQ0xFT1RJREUtU0VRVUVOQ0U8L2tleXdvcmQ+PGtleX dvcmQ+R0VORTwv a2V5d29yZD48a2V5d29yZD5TVEVBUk9USEVSTU9QSElMVVM8L2tleXdvcm Q+PGtleXdvcmQ+SU5I SUJJVE9SUzwva2V5d29yZD48a2V5d29yZD5CSU5ESU5HPC9rZXl3b3JkPjwva2 V5d29yZHM+PGRh dGVzPjx5ZWFyPjE5OTY8L3llYXI+PHB1Yi1kYXRlcz48ZGF0ZT5KdW48L2Rh dGU+PC9wdWItZGF0 ZXM+PC9kYXRlcz48aXNibj4xMzgxLTExNzc8L2lzYm4+PGFjY2Vzc2lvbi1udW 0+SVNJOkExOTk2 VVUzMTQwMDAxNTwvYWNjZXNzaW9uLW51bT48d29yay10eXBlPkFydGljb GU8L3dvcmstdHlwZT48 dXJscz48cmVsYXRlZC11cmxzPjx1cmw+Jmx0O0dvIHRvIElTSSZndDs6Ly9BMT k5NlVVMzE0MDAw MTU8L3VybD48L3JlbGF0ZWQtdXJscz48L3VybHM+PGxhbmd1YWdlPkVuZ2x pc2g8L2xhbmd1YWdl PjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+AG== 120 and the protein was therefore negatively charged at neutral pH. It is likely that electrostatic attraction between the cationic fragments on the particle formed as a result the enzymatic hydrolysis of the ECP and the anionic enzyme lead to enzyme retention. It is expected that this interaction would to be stronger for branched actuators (double the positive charge). Therefore, enzyme diffusion may be slower in particles containing branched actuators (i.e. enzymes are held in place after enzymatic hydrolysis). Indeed, similar electrostatic retention has been observed previously.58 Linear peptide actuators have been shown in water to produce a maximal V/Vo of 1.6 (Figure 4–4 A) while the maximal swelling for the branched peptide actuators observed was 1.35 (Figure 4–4 B). The heterogeneous enzymatic cleavage apparent for the branched peptide actuators explains the reduced maximum swelling observed when compared to the linear peptide actuator.

1005

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 4–7. Comparison of thermolysin action on both linear and branched peptide actuators on µPEGA. A: Two-photon micrographs of representative particles labelled with dansyl (scale bar is 15 μm). B: Surface plot of two-photon images indicating qualitatively the fluorescent intensities of the particles. C: Quantification of fluorescent intensities of dansyl labelled, enzyme treated particles functionalised with either linear or branched peptide actuators.

1.2.4. Enzyme triggered release The applicability of branched peptide actuator functionalised particles to enzymatically triggered release a payload was assessed. Firstly, a payload was entrapped within the particles. This was achieved by utilising the pH responsiveness of the peptide actuators.58 Here, fluorescein isothiocyanate (FITC) dextran (40 kDa) was the payload macromolecule. By lowering the pH of the payload solution to pH 2.5 the aspartic acid residue side chain is protonated (while the R side chain remains protonated) making the net charge of the peptide actuator positive, resulting in an 1006

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

increase in the swelling (Figure 4–3 B) and thus mesh size of the polymer microparticles. The pH switch therefore allowed the 40 kDa dextran to diffuse into the particle. By returning the system to pH 7 the D side chain carboxyl group again becomes ionised. In this case the net charge of the peptide actuator becomes zero and the repulsive forces are removed. The polymer particle deswells and entraps the dextran. Figure 4–8 A shows a fluorescent micrograph of these particles after washing demonstrating that the FITC labelled dextran was entrapped within the particles. In Figure 4–8 B & C the particles are treated with buffer and chymotrypsin respectively (both at 0.18 M ionic strength), because no actuation had occurred the majority of the FITC dextran remained entrapped with some leakage apparent. It is likely that some of the dextran was not completely entrapped within the particles resulting in leakage.

Figure 4–8. µPEGA particles functionalised with the branched peptide actuator at pH 7 (scale bars are 100 μm). A: After loading with FITC labelled 40 kDa dextran and washing. B: After loading and 80 min treatment with Thermolysin. C: After loading and 80 min treatment with Chymotrypsin. D : After loading and 80 min treatment with buffer E: mmoles of FITC labelled dextran released per g of particles after 80 min treatment. (all experiments were carried out at an ionic strength of 0.18 M)

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

When the particles were treated with thermolysin (Figure 4–8 D) the increase in swelling (and corresponding increased pore size) allowed the dextran to diffuse out of the particles, resulting in a reduction of fluorescence of the particles. By measuring the fluoroscence of the solution surrounding the particles it was possible to measure the amount of FITC dextran released (Figure 4–8 E). When the particles were exposed to either buffer alone or chymotrypsin a small amount of leakage was observed (~ 0.02 mg dextran/mg particles). While treatment with thermolysin resulted in a 350 % increase in dextran release (Figure 4–8 E). Hence the enzyme triggered increase in swelling and accessibility was responsible for the greater release of the entrapped macromolecules. To summarise, by exploiting the enzyme responsive swelling it was possible to release an entrapped macromolecule in the presence of the target enzyme. Electrostatically induced swelling occurs upon cleavage of the ECP within the peptide actuator at physiological ionic strength.

1.3. Conclusions Branched peptide actuators have been synthesised and incorporated into μPEGA. These functionalised particles were capable of specifically responding to selected enzymes at a variety of ionic strengths. Through the physical entrapment of a macromolecule payload within the functionalised particles it was possible to obtain payload release at physiological ionic strength in response to target enzyme. This system may have applications in a number of areas including drug delivery and automated bio-sensing of ‘on-bead’ libraries.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

2 Microfluidic preparation of low polydispersity PEGA particles 1.1. Abstract This chapter details the preparation of PEGA particles by a simplified microfluidic setup assembled using a needle and tubing. The size of the particles produced was determined by the surfactant concentration and the relative flow rates of the dispersed and continuous phases. The minimum particle size obtained was limited by the diameter of the needle (335 μm). The development of a flow-focussing setup allowed for the production of smaller particles down to 100 μm in diameter. Optimum conditions were determined allowing for the production of 4000 particles a minute with a mean diameter of 160 μm.

1.2. Introduction This chapter describes the development of microfluidic devices for the preparation of near monodisperse4 polymer particles. Monodisperse particles present a number of advantages over polydisperse samples: the sample of particles is well defined, any reaction occurring on or in the particles occurs at the same rate. Additionally, any change in swelling or size is easy to determine without the need for a large sample size. Microfluidic systems offer the potential to produce particles with a very narrow distribution because the size each monomer droplet formed is determined by the conditions at the mixing point. As each droplet is formed under constant conditions the size of the droplets are the same. The introduction of a polymerisation method, normally UV initiation results in the production of polymer particles. In these systems there are a number of variables that control the mixing process and therefore ultimately determined particle size. The following work aims to find the optimum conditions to produce near monodisperse µPEGA. These particles would be better defined and more homogenous than the samples prepared by inverse suspension polymerisation in Chapter PEGA polymerisation, characterisation and enzyme 4 According to the standards of the National Institute of Standards and Technology (NIST): “a particle distribution may be considered monodisperse if at least 90% of the distribution lies within 5% of the median size” (Particle Size Characterization, Special Publication 960–961, January 2001).

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

responsive swelling through functionalisation with peptide actuators2. The very narrow size distribution would offer the potential for rapid screening with an automated cell sorter. Using this technique it could be possible to rapidly screen for enzyme

action

on

µPEGA

using

peptide

libraries.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5SZW5pbDwvQXV0aG9yPjxZ ZWFyPjE5OTg8L1llYXI+PFJl Y051bT41ODwvUmVjTnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+NTg8L3JlYy1u dW1iZXI+PGZvcmVp Z24ta2V5cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJmYTl6ZHpzdmtzeDJwcmVyZTk 4eGQ1MnFkZndm d3I5MHA1czIiPjU4PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG5hbW U9IkpvdXJuYWwg QXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcnM+PG F1dGhvcj5SZW5p bCwgTS48L2F1dGhvcj48YXV0aG9yPkZlcnJlcmFzLCBNLjwvYXV0aG9yPjxhdX Rob3I+RGVsYWlz c2UsIEouIE0uPC9hdXRob3I+PGF1dGhvcj5Gb2dlZCwgTi4gVC48L2F1dGhvcj48 YXV0aG9yPk1l bGRhbCwgTS48L2F1dGhvcj48L2F1dGhvcnM+PC9jb250cmlidXRvcnM+PHRpd Gxlcz48dGl0bGU+ UEVHQSBzdXBwb3J0cyBmb3IgY29tYmluYXRvcmlhbCBwZXB0aWRlIHN5bn RoZXNpcyBhbmQgc29s aWQtcGhhc2UgZW56eW1hdGljIGxpYnJhcnkgYXNzYXlzPC90aXRsZT48c2Vjb2 5kYXJ5LXRpdGxl PkpvdXJuYWwgb2YgUGVwdGlkZSBTY2llbmNlPC9zZWNvbmRhcnktdGl0bGU +PC90aXRsZXM+PHBl cmlvZGljYWw+PGZ1bGwtdGl0bGU+Sm91cm5hbCBvZiBQZXB0aWRlIFNjaWV uY2U8L2Z1bGwtdGl0 bGU+PC9wZXJpb2RpY2FsPjxwYWdlcz4xOTUtMjEwPC9wYWdlcz48dm9sdW1l PjQ8L3ZvbHVtZT48 bnVtYmVyPjM8L251bWJlcj48ZGF0ZXM+PHllYXI+MTk5ODwveWVhcj48cHVi LWRhdGVzPjxkYXRl Pk1heTwvZGF0ZT48L3B1Yi1kYXRlcz48L2RhdGVzPjxhY2Nlc3Npb24tbnVtPkl TSTowMDAwNzM5 ODUxMDAwMDM8L2FjY2Vzc2lvbi1udW0+PHVybHM+PHJlbGF0ZWQtdXJscz 1010

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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planar

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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ZT48L0VuZE5vdGU+AG== 1046

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Nzg1MTwvaXNibj48 YWNjZXNzaW9uLW51bT5JU0k6MDAwMjMwMDk2ODAwMDAxPC9hY2Nlc3 Npb24tbnVtPjx3b3JrLXR5 cGU+Q29ycmVjdGlvbjwvd29yay10eXBlPjx1cmxzPjxyZWxhdGVkLXVybHM+P HVybD4mbHQ7R28g dG8gSVNJJmd0OzovLzAwMDIzMDA5NjgwMDAwMTwvdXJsPjwvcmVsYXRl ZC11cmxzPjwvdXJscz48 ZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+MTAuMTAwMi9hbmllLjIwMDQ2MjI yNjwvZWxlY3Ryb25p Yy1yZXNvdXJjZS1udW0+PGxhbmd1YWdlPkVuZ2xpc2g8L2xhbmd1YWdlPjwvc mVjb3JkPjwvQ2l0 ZT48L0VuZE5vdGU+AG== 91,95,122 There are a number of limitations associated with these devices: Usually a clean room is required for device production. Surface treatment of the channel walls can be needed to prevent an inverse emulsion forming. Additionally it is often easy for the microchannels to become blocked by solid polymer. Simplified microfluidics setups have been shown that do not require no specialist fabrications techniques. These typically make use of needles

and/or

tubing

to

create

near

monodisperse

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

= 92,97 These publications have been covered in more detail in the preceding literature review (Section Microfluidic polymerisation of particles). The research in this section is based on a paper by Hadziioannou and co-workers in which the dispersed phase is introduced to the continuous phase via a needle positioned along the main axis of the tubing.97 The axisymmetric setup of the device prevents the need of surface modification because the dispersed phase droplets do not come into contact with the channel walls.93 While any blockages in the tubing due to polymer build up can simply be rectified through a fast and low cost replacement of that section of tubing. In this chapter a microfluidic device as described by Hadziioannou (shown schematically in Figure 5–1) was used to produce PEGA particles.

Figure 5–1. Schematic of microfluidic setup for the synthesis of controlled-size polymer particles. The dispersed phase (containing the monomer) and the continuous phase are delivered by syringe pumps. The dispersed phase is introduced to the co-flowing continuous phase the centre of the PTFE tubing by means of a 26 gauge needle.

The objectives of this chapter were to: (i) Optimise the conditions to manufacture particles with the smallest diameter possible with a very narrow size distribution. (ii) Develop strategies to produce smaller particles, through the use of a simplified flowfocusing setup.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1.3. Materials and methods 1.3.1. Microfluidic system Figure 5–1 shows a schematic of the microfluidic system. A stainless steel needle blunt tip needle (26 gauge) internal diameter (ID) 240 μm was inserted into a Tjunction (Swaglok T-junction SS-100-3). The needle tips exits the T-junction in the centre of the polytetrafluoroethylene (PTFE) tubing. Two syringe pumps (New Era Pump Systems Inc. NE-1000) were used to deliver the continuous and dispersed phases at a specific flow rate. The two phases were carried in PTFE tubing with an internal diameter (ID) of 1.6 mm. The dispersed phase was injected through the needle while the continuous phase was injected perpendicular to the main axis of the T-junction. The outlet tubing was also PTFE with ID 1.6 mm tubing with a length of 120 cm. The exit of the needle was located in the centre of the outlet tubing. The outlet tubing entered a UV box where it was exposed to UV light (320-390 nm wavelength) from a Dymax model 5000 Flood using a Dymax 400 W power source. Residence time within the UV box was 30-240 seconds depending on the flow rates. Particles were collected at the end of the outlet in a water bath. 1.3.2. Flow focussing setup A modified T-junction was used to produce smaller droplets. Here, a 26 G needle was inserted though a Fisher Tubing connector T connector Nylon 1/16in Masterflex entering a 21 G needle. The internal needle ends approximately 2/3 of the way down the external needle (Figure 5–7). Two syringe pumps were used to deliver the continuous and dispersed phases at a specific flow rate. The two phases were carried in PTFE tubing with an ID of 1.6 mm. The dispersed phase was injected through the central needle while the continuous phase was injected perpendicular to the main axis of the T-junction and flowed through the outer needle. The outlet tubing was also PTFE ID 1.6 mm tubing with a length of 30 cm. The exit of the outer needle was located in the centre of the outlet tubing. The outlet tubing entered a UV box where it was exposed to UV light (320-390 nm wavelength) from a Dymax model 5000 Flood using a Dymax 400 W power source. Residence time within the UV box was 30-240 seconds depending on the flow rates. Particles were collected at the end of the outlet in a water bath.

1061

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1.3.3. Monomer and continuous phase preparation All materials were used as supplied from Sigma-Aldrich with the exception of PEGA macromonomers (Versamatrix), photoinitiator (Ciba) and Isopar M (Multisol) The dispersed phase consisted of an aqueous solution (10 ml ultrapure water) containing 3 g (3.34 mmol) PEGA macromonomers, 0.15 g (2.11 mmol) acrylamide and 0.10 ml darocure 1173 photoinitiator. 0.05 ml of red food colouring (Supercook) was included in early experiments to give greater contrast to the monomer droplets. The density of the solution was 1.008 g/ml. The viscosity solution as measured using a Hydramotion Viscolite 700HP model VL700-T15HP was 4.8 cP at 20 ˚C. The continuous phase consisted of Isopar M (viscosity of 2.1 cP) in which the surfactant Span 20 (Sorbitan monolaurate) was dissolved, the amount of surfactant was varied. The free radical polymerisation of the dispersed phase droplets led to the formation of insoluble particles, these particles were collected in a water bath at room temperature. Silicone oil (20 cSt) was used without surfactant (viscosity of 20 cP). 1.3.4. Particle size measurement The polymerised particles were collected at the exit of the outlet tube and washed (2 x 10 ml DCM, 2 x 10 ml THF, 2 x 10 ml methanol and 4 x 10 ml water). The diameter of the particles (fully swollen in water) was measured using an optical microscope (a Zeiss Imager A1 microscope and Canon Powershot G6 camera). Particles above 1500 µm in diameter were photographed without use of the microscope over grid marker paper. ImageJ was used to determine the mean diameter of the particles, due to the sometimes asymmetrical shape of the particles the cross sectional area of each individual particle was measured and a mean diameter then determined from it. The mean diameter and standard deviation were obtained by measuring the diameter of at least 30 particles.

1062

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1.1. Results and discussion 1.1.1. Microfluidic system Droplet formation in microfluidic systems is determined by the balance between the shear forces imparted on the forming droplet by the co-flowing continuous phase and the interfacial energy between the two phases. These hydrodynamic conditions within the microfluidic device are usually described by two dimensionless numbers; the Reynolds number and the capillary number.95 The Reynolds number is defined in Equation 1 and the capillary number in Equation 2. Where ρ and µ are the density and viscosity of the liquid, respectively, D is the diameter of the outlet tubing, V is the

velocity

of

the

liquid

and

γ

is

the

interfacial

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1063

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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AG==

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

8L3RpdGxlcz48 cGVyaW9kaWNhbD48ZnVsbC10aXRsZT5MYW5nbXVpcjwvZnVsbC10aXRsZT 48YWJici0xPkxhbmdt dWlyPC9hYmJyLTE+PC9wZXJpb2RpY2FsPjxhbHQtcGVyaW9kaWNhbD48ZnV sbC10aXRsZT5MYW5n bXVpcjwvZnVsbC10aXRsZT48YWJici0xPkxhbmdtdWlyPC9hYmJyLTE+PC9hb HQtcGVyaW9kaWNh bD48cGFnZXM+Nzc0NS03NzUwPC9wYWdlcz48dm9sdW1lPjIzPC92b2x1bWU+ PG51bWJlcj4xNDwv bnVtYmVyPjxrZXl3b3Jkcz48a2V5d29yZD5FTVBMT1lJTkcgTUlDUk9DSEFOTk VMIEVNVUxTSUZJ Q0FUSU9OPC9rZXl3b3JkPjxrZXl3b3JkPkRST1BMRVQgRk9STUFUSU9OPC9r ZXl3b3JkPjxrZXl3 b3JkPklOVEVSRkFDSUFMLVRFTlNJT048L2tleXdvcmQ+PGtleXdvcmQ+R0VO RVJBVElPTjwva2V5 d29yZD48a2V5d29yZD5ERVZJQ0U8L2tleXdvcmQ+PGtleXdvcmQ+RkxPVzwva 2V5d29yZD48a2V5 d29yZD5NSUNST1BBUlRJQ0xFUzwva2V5d29yZD48a2V5d29yZD5SRUFDVE9 SUzwva2V5d29yZD48 a2V5d29yZD5NSUNST1JFQUNUT1JTPC9rZXl3b3JkPjxrZXl3b3JkPk1JQ1JPU1 BIRVJFUzwva2V5 d29yZD48L2tleXdvcmRzPjxkYXRlcz48eWVhcj4yMDA3PC95ZWFyPjxwdWItZ GF0ZXM+PGRhdGU+ SnVsPC9kYXRlPjwvcHViLWRhdGVzPjwvZGF0ZXM+PGlzYm4+MDc0My03N DYzPC9pc2JuPjxhY2Nl c3Npb24tbnVtPklTSTowMDAyNDc0ODcyMDAwNTM8L2FjY2Vzc2lvbi1udW0+ PHdvcmstdHlwZT5B cnRpY2xlPC93b3JrLXR5cGU+PHVybHM+PHJlbGF0ZWQtdXJscz48dXJsPiZsdD tHbyB0byBJU0km Z3Q7Oi8vMDAwMjQ3NDg3MjAwMDUzPC91cmw+PC9yZWxhdGVkLXVybH M+PC91cmxzPjxlbGVjdHJv bmljLXJlc291cmNlLW51bT4xMC4xMDIxL2xhMDYzMjg5czwvZWxlY3Ryb25p Yy1yZXNvdXJjZS1u dW0+PGxhbmd1YWdlPkVuZ2xpc2g8L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0Z T48L0VuZE5vdGU+ AG== 95,97 It has been shown that increasing the value of the 1070

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

capillary number for the dispersed phase (Cad) produces smaller droplets.95 Therefore, by increasing the velocity or viscosity of the continuous phase or decreasing the interfacial tension smaller droplets should be produced. Re ≡ ρDV/µ Equation 1. Reynolds number

Ca ≡ µV/γ Equation 2. Capillary number.

1.1.1.1. Variation of particle size with flow rate ratios and surfactant concentration Initial experiments produced droplets with diameters equal to that of the tubing (Figure 5–2 A & B). Increasing Qc/Qd (where Qc and Qd are the flow rates of the continuous and dispersed phases respectively), did not lead to a significant decrease in droplet size and the particles formed (once washed and fully swollen in water) had diameters greater than 1800 μm (Figure 5–2). This result suggested that the interfacial energy between the aqueous monomer solution and organic continuous phase (isoparaffin) was relatively high. Indeed, the interfacial tension of linear alkanes with boiling points in the range of Isopar M (190 - 260 ˚C) have been shown to be 53.1 - 54.5 mN/m.123 Higher interfacial tensions result in higher capillary pressures, (the pressure difference between inside and outside of a droplet) as given by the Young-Laplace (Equation 3). Δp = γ(1/R1 + 1/R2) Equation 3. Young-Laplace equation

Where Δp is the pressure difference across the liquid-liquid interface, γ is the surface tension and R1 and R2 and the principal radius of curvature.124 This means that larger droplets have a lower capillary pressure and are therefore more thermodynamically favourable than smaller droplets. The highest calculated Cad for the experimental results shown in Figure 5–2 was ~ 3 x 10-5. This setup did not lead to control of particle size, as presumably it was not possible to overcome the interfacial forces with shear forces.

1071

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 5–2. A: Effect of Qc/Qd on mean particle diameter with no surfactant in the continuous phase, Qc= 2 ml/min. B: Photograph of PEGA particles produced using a Qc/Qd of 10 and a Qc of 2 ml/min (scale bar is 3500 µm).

The addition of surfactant to the continuous phase results in reduced interfacial energy due to the surfactant molecules sitting at the interface. The use of surfactants is

well

established

within

conventional

dispersion

type

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

c2Vjb25kYXJ5LXRp dGxlPjxhbHQtdGl0bGU+Si4gQ29sbG9pZCBJbnRlcmZhY2UgU2NpLjwvYWx0L XRpdGxlPjwvdGl0 bGVzPjxwZXJpb2RpY2FsPjxmdWxsLXRpdGxlPkpvdXJuYWwgb2YgQ29sbG9p ZCBhbmQgSW50ZXJm YWNlIFNjaWVuY2U8L2Z1bGwtdGl0bGU+PGFiYnItMT5KLiBDb2xsb2lkIElud GVyZmFjZSBTY2ku PC9hYmJyLTE+PC9wZXJpb2RpY2FsPjxhbHQtcGVyaW9kaWNhbD48ZnVsbC1 0aXRsZT5Kb3VybmFs IG9mIENvbGxvaWQgYW5kIEludGVyZmFjZSBTY2llbmNlPC9mdWxsLXRpdGx lPjxhYmJyLTE+Si4g Q29sbG9pZCBJbnRlcmZhY2UgU2NpLjwvYWJici0xPjwvYWx0LXBlcmlvZGljY Ww+PHBhZ2VzPjI3 Mi0yNzY8L3BhZ2VzPjx2b2x1bWU+MjQwPC92b2x1bWU+PG51bWJlcj4xPC9u dW1iZXI+PGtleXdv cmRzPjxrZXl3b3JkPmludGVyZmFjaWFsIHRlbnNpb248L2tleXdvcmQ+PGtleXdv cmQ+c29yYml0 YW4gbW9ub2VzdGVyczwva2V5d29yZD48a2V5d29yZD53YXRlci1vaWwgaW50 ZXJmYWNlPC9rZXl3 b3JkPjxrZXl3b3JkPmNtYzwva2V5d29yZD48a2V5d29yZD5FTVVMU0lPTiBTVE FCSUxJVFk8L2tl eXdvcmQ+PGtleXdvcmQ+Tk9OQVFVRU9VUyBQSEFTRTwva2V5d29yZD48a2 V5d29yZD5HVUVSQkVU IEFMQ09IT0xTPC9rZXl3b3JkPjxrZXl3b3JkPkJSQU5DSEVEPC9rZXl3b3JkPjxrZ Xl3b3JkPlNV TEZBVEU8L2tleXdvcmQ+PGtleXdvcmQ+VFJJVE9OIFgtMTAwPC9rZXl3b3JkP jxrZXl3b3JkPk1P REVMLURSVUdTPC9rZXl3b3JkPjxrZXl3b3JkPlRFTlNJT048L2tleXdvcmQ+PGt leXdvcmQ+U1lT VEVNUzwva2V5d29yZD48a2V5d29yZD5UUkFOU1BPUlQ8L2tleXdvcmQ+PGtl eXdvcmQ+UFJFU1NV UkU8L2tleXdvcmQ+PC9rZXl3b3Jkcz48ZGF0ZXM+PHllYXI+MjAwMTwveWV hcj48cHViLWRhdGVz PjxkYXRlPkF1ZzwvZGF0ZT48L3B1Yi1kYXRlcz48L2RhdGVzPjxpc2JuPjAwMj EtOTc5NzwvaXNi 1084

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

bj48YWNjZXNzaW9uLW51bT5JU0k6MDAwMTcwMTExOTAwMDMyPC9hY2 Nlc3Npb24tbnVtPjx3b3Jr LXR5cGU+QXJ0aWNsZTwvd29yay10eXBlPjx1cmxzPjxyZWxhdGVkLXVybHM +PHVybD4mbHQ7R28g dG8gSVNJJmd0OzovLzAwMDE3MDExMTkwMDAzMjwvdXJsPjwvcmVsYXRl ZC11cmxzPjwvdXJscz48 ZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+MTAuMTAwNi9qY2lzLjIwMDEuNz YxMjwvZWxlY3Ryb25p Yy1yZXNvdXJjZS1udW0+PGxhbmd1YWdlPkVuZ2xpc2g8L2xhbmd1YWdlPjwvc mVjb3JkPjwvQ2l0 ZT48L0VuZE5vdGU+ 126 The effect of including surfactant in the continuous phase was dramatic (Figure 5–3 A & B), even at the lowest Qc/Qd (10) and lowest surfactant concentration (0.01 g/ml (three times the concentration used in the inverse suspension polymerisation)) there was a large reduction in mean particle diameter (625 µm compared to 2105 µm when no surfactant is present). The lowered interfacial energy allowed the shear forces to influenced droplet size. As Qc/Qd was increased particle size decreased reaching a plateau in mean particle diameter of about 450 µm at flow ratio of 200. Slightly smaller particles (422 µm) were obtained at very high values of Qc/Qd. This relationship between mean particle diameter and Qc/Qd was seen as surfactant concentration was increased (further lowering the interfacial forces increasing the dominance of shear forces), albeit with smaller particles produced at higher surfactant concentrations. Upon the addition of surfactant the value for Cad was greatly increased to ~ 9 x 10-5. Surfactant was removed from the outside of the polymerisation particles by repeated washing in solvents with a range of polar/non-polar characters.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 5–3. Effect of surfactant concentration on particle size. A: Effect of Qc/Qd and surfactant concentration (♦ (blue): 0.01 g/ml, ■ (red): 0.04 g/ml, ▲ (green): 0.08 g/ml) on mean particle diameter, Qc= 2 ml/min. B: Effect of increasing surfactant concentration on mean particle diameter at varying values of Qc/Qd. C: Effect of increasing surfactant concentration on mean particle diameter at varying values of Qc/Qd excluding 0 g/ml surfactant concentration.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

At a Qc/Qd of 10 the mean diameter of particles produced is very similar independent of whether 0.04 g/ml or 0.08 g/ml of surfactant was used, indicating that the minimal particle size at that flow rate had been obtained. However, as the shear forces are increased particle diameter is also decreased. A surfactant concentration of 0.08 g/ml and Qc/Qd of 1000 offered the smallest particles for this setup (335 µm). Typically upon the polymerisation of a monomer droplet the resulting polymer particle has a smaller volume. This corresponds to the increase density of the polymer

verses

the

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cGFnZXM+Nzc0NS03NzUwPC9wYWdlcz48dm9sdW1lPjIzPC92b2x1bWU+PG51 bWJlcj4xNDwvbnVt YmVyPjxrZXl3b3Jkcz48a2V5d29yZD5FTVBMT1lJTkcgTUlDUk9DSEFOTkVMI EVNVUxTSUZJQ0FU SU9OPC9rZXl3b3JkPjxrZXl3b3JkPkRST1BMRVQgRk9STUFUSU9OPC9rZXl3b 3JkPjxrZXl3b3Jk PklOVEVSRkFDSUFMLVRFTlNJT048L2tleXdvcmQ+PGtleXdvcmQ+R0VORVJ BVElPTjwva2V5d29y ZD48a2V5d29yZD5ERVZJQ0U8L2tleXdvcmQ+PGtleXdvcmQ+RkxPVzwva2V5d 29yZD48a2V5d29y ZD5NSUNST1BBUlRJQ0xFUzwva2V5d29yZD48a2V5d29yZD5SRUFDVE9SUz wva2V5d29yZD48a2V5 d29yZD5NSUNST1JFQUNUT1JTPC9rZXl3b3JkPjxrZXl3b3JkPk1JQ1JPU1BIRVJ FUzwva2V5d29y ZD48L2tleXdvcmRzPjxkYXRlcz48eWVhcj4yMDA3PC95ZWFyPjxwdWItZGF0Z XM+PGRhdGU+SnVs PC9kYXRlPjwvcHViLWRhdGVzPjwvZGF0ZXM+PGlzYm4+MDc0My03NDYzP C9pc2JuPjxhY2Nlc3Np b24tbnVtPklTSTowMDAyNDc0ODcyMDAwNTM8L2FjY2Vzc2lvbi1udW0+PHd vcmstdHlwZT5BcnRp Y2xlPC93b3JrLXR5cGU+PHVybHM+PHJlbGF0ZWQtdXJscz48dXJsPiZsdDtHby B0byBJU0kmZ3Q7 Oi8vMDAwMjQ3NDg3MjAwMDUzPC91cmw+PC9yZWxhdGVkLXVybHM+PC 91cmxzPjxlbGVjdHJvbmlj LXJlc291cmNlLW51bT4xMC4xMDIxL2xhMDYzMjg5czwvZWxlY3Ryb25pYy1y ZXNvdXJjZS1udW0+ PGxhbmd1YWdlPkVuZ2xpc2g8L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48Q2 l0ZT48QXV0aG9y Plh1PC9BdXRob3I+PFllYXI+MjAwNTwvWWVhcj48UmVjTnVtPjIyNjwvUmVj TnVtPjxyZWNvcmQ+ PHJlYy1udW1iZXI+MjI2PC9yZWMtbnVtYmVyPjxmb3JlaWduLWtleXM+PGtleS BhcHA9IkVOIiBk Yi1pZD0iZmE5emR6c3Zrc3gycHJlcmU5OHhkNTJxZGZ3ZndyOTBwNXMyIj4y MjY8L2tleT48L2Zv cmVpZ24ta2V5cz48cmVmLXR5cGUgbmFtZT0iSm91cm5hbCBBcnRpY2xlIj4xN 1092

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

zwvcmVmLXR5cGU+ PGNvbnRyaWJ1dG9ycz48YXV0aG9ycz48YXV0aG9yPlh1LCBTLjwvYXV0aG9y PjxhdXRob3I+Tmll LCBaLjwvYXV0aG9yPjxhdXRob3I+U2VvLCBNLjwvYXV0aG9yPjxhdXRob3I+ TGV3aXMsIFAuPC9h dXRob3I+PGF1dGhvcj5LdW1hY2hldmEsIEUuPC9hdXRob3I+PGF1dGhvcj5TdG 9uZSwgSC4gQS48 L2F1dGhvcj48YXV0aG9yPkdhcnN0ZWNraSwgUC48L2F1dGhvcj48YXV0aG9yP ldlaWJlbCwgRC4g Qi48L2F1dGhvcj48YXV0aG9yPkdpdGxpbiwgSS48L2F1dGhvcj48YXV0aG9yPld oaXRlc2lkZXMs IEcuIE0uPC9hdXRob3I+PC9hdXRob3JzPjwvY29udHJpYnV0b3JzPjx0aXRsZXM +PHRpdGxlPkdl bmVyYXRpb24gb2YgbW9ub2Rpc3BlcnNlIHBhcnRpY2xlcyBieSB1c2luZyBtaW Nyb2ZsdWlkaWNz OiBDb250cm9sIG92ZXIgc2l6ZSwgc2hhcGUsIGFuZCBjb21wb3NpdGlvbiAodm9s IDQ0LCBwZyA3 MjQsIDIwMDUpPC90aXRsZT48c2Vjb25kYXJ5LXRpdGxlPkFuZ2V3YW5kdGU gQ2hlbWllLUludGVy bmF0aW9uYWwgRWRpdGlvbjwvc2Vjb25kYXJ5LXRpdGxlPjxhbHQtdGl0bGU+ QW5nZXcuIENoZW0u LUludC4gRWRpdC48L2FsdC10aXRsZT48L3RpdGxlcz48cGVyaW9kaWNhbD48 ZnVsbC10aXRsZT5B bmdld2FuZHRlIENoZW1pZS1JbnRlcm5hdGlvbmFsIEVkaXRpb248L2Z1bGwtdGl 0bGU+PGFiYnIt MT5Bbmdldy4gQ2hlbS4tSW50LiBFZGl0LjwvYWJici0xPjwvcGVyaW9kaWNhb D48YWx0LXBlcmlv ZGljYWw+PGZ1bGwtdGl0bGU+QW5nZXdhbmR0ZSBDaGVtaWUtSW50ZXJuY XRpb25hbCBFZGl0aW9u PC9mdWxsLXRpdGxlPjxhYmJyLTE+QW5nZXcuIENoZW0uLUludC4gRWRpdC 48L2FiYnItMT48L2Fs dC1wZXJpb2RpY2FsPjxwYWdlcz4zNzk5LTM3OTk8L3BhZ2VzPjx2b2x1bWU+ NDQ8L3ZvbHVtZT48 bnVtYmVyPjI1PC9udW1iZXI+PGtleXdvcmRzPjxrZXl3b3JkPk1JQ1JPUEFSVElD TEVTPC9rZXl3 1093

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

b3JkPjwva2V5d29yZHM+PGRhdGVzPjx5ZWFyPjIwMDU8L3llYXI+PC9kYXRlc z48aXNibj4xNDMz LTc4NTE8L2lzYm4+PGFjY2Vzc2lvbi1udW0+SVNJOjAwMDIzMDA5NjgwMD AwMTwvYWNjZXNzaW9u LW51bT48d29yay10eXBlPkNvcnJlY3Rpb248L3dvcmstdHlwZT48dXJscz48cmVs YXRlZC11cmxz Pjx1cmw+Jmx0O0dvIHRvIElTSSZndDs6Ly8wMDAyMzAwOTY4MDAwMDE8L 3VybD48L3JlbGF0ZWQt dXJscz48L3VybHM+PGVsZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjEwLjEwMDI vYW5pZS4yMDA0NjIy MjY8L2VsZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjxsYW5ndWFnZT5FbmdsaX NoPC9sYW5ndWFnZT48 L3JlY29yZD48L0NpdGU+PC9FbmROb3RlPgB= 91,97 With this system the dispersed phase was an aqueous solution of the monomers. This was done to maintain a comparable method to the inverse suspension polymerisation. It was found that monomer droplets formed at the diameter of the tubing (≈1600 µm) initially give PEGA particles of similar diameter (if collected in oil) which swelled after washing in water to give much larger particles (≈2100 µm). The size of particular importance was that of the hydrated particles as further applications of the polymer would be in aqueous environments. Further investigation of swelling this behaviour was undertaken. Particles were initially collected in oil then measured then washed and measured again in water. Using this method it was determined that on average fully swollen particles had diameter 35 % (± 3 %) larger than initial monomer droplets.

Considering this information a

diameter of 335 µm which was found to be the lower limit for particle size corresponds to droplet diameter of 248 µm, very close to the internal diameter of the 26 gauge needle (240 µm). Earlier work using a similar setup determined that the smallest droplets that could be produced had diameters just above that of the needle.97 Surfactant concentrations above (0.08 g/ml) were not assessed because above this concentration the surfactant was not completely soluble. In summary, the introduction of surfactant to the continuous phase lowered the interfacial tension resulting in the production of smaller particles. While increasing Qc/Qd led to greater shear forces during droplet formation resulting in smaller droplets and therefore particles, the lower limit in particle size was due to the droplets formed at the needle diameter.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1.1.1.2. Effect of increasing total flow rate on particle size

Figure 5–4. A: Effect of total flow rate (at constant Qc/Qd) on mean particle diameter. B: Effect of Qc/Qd and total flow rate (♦ (blue): 2 ml/min, ■ (red): 4 ml/min) on mean particle diameter, 0.08 g/ml surfactant concentration.

Very high Qc/Qd ratios did not offer efficient particle production; the rate of particles was slow and the process was wasteful with a large amount of oil is used per particle produced. Higher surfactant concentrations might have shown slightly reduced diameters at lower flow ratios however, at concentrations higher than 0.08 g/ml the surfactant did not fully dissolve in the oil. Another approach was to increase the 1095

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

shear forces by increasing the total flow rate, a preliminary investigation into the effect of total flow rate (at a constant Qc/Qd) on mean particle diameter showed as flow rate was increased there was slight size decrease ( A). However, flow rates higher than 4 ml/min did not lead to completely cured particles. Using a Qc of 4 ml/min over a range of Qc/Qd produced particles of only slightly reduced diameter compared to 2 ml/min (Figure 5–4 B). Hadziioannou and co-workers produced similar findings showing that similar values of Qc/Qd led to similar diameters independently of the total flow, Qc + Qd.97

1.1.1.3. Effect of flow ratios and surfactant concentration on polydispersity The polydispersity5 of the particles increases when there is variation between droplet to droplet formation or when coalescence occurs prior to polymerisation. In order to determine whether the variables that were changed within the microfluidic setup produced measureable trends in the polydispersity of the particles the data shown in Figure 5–5 was obtained. At the highest and lowest surfactant concentrations (0.01 g/ml and 0.08 g/ml) used there was a tendency for the polydispersity to increase at higher values of Qc/Qd. While using 0.04 g/ml produced particles with polydispersities in the range of 2-3 % over the range of Qc/Qd values tested. The overall variation of polydispersity was found to be small (between 1.4 - 4.3 %).

1.1.1.4. Variation of particle production rates with the conditions assessed In determining the optimum conditions with which to produce PEGA particles the production rate was assessed (Figure 5–6). Lower values of Qc/Qd led to higher production rates (due to higher flow rate of the continuous phase) while higher surfactant concentrations offered higher production rates as a greater number of smaller particles were produced from the same volume of monomer solution (dispersed phase).

5 Defined as the standard deviation in the particle diameter divided by the mean particle diameter.91

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Figure 5–5. Effect of Qc/Qd and surfactant concentration (♦: 0.01 g/ml ■: 0.04 g/ml ▲: 0.08 g/ml) on polydispersity (Qc=2 ml/min).

Figure 5–6. Effect of Qc/Qd and surfactant concentration on particle production rate (♦ (blue): 0.01 g/ml ■ (red): 0.04 g/ml ▲ (green): 0.08 g/ml) (Qc=2 ml/min).

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

1.1.2. Using a simplified microfluidic flow-focussing device to produce polymer particles The minimum size of particles of particles obtained using the microfluidic setup described up to this point was limited to the diameter of the needle from which the dispersed phase was introduced into the continuous phase. To overcome this minimum size limitation an adjustment to the microfluidic setup was made (Figure 5–7). Flow-focussing (FF) is a technique that allows for the formation of droplets smaller than the orifice of the dispersed phase of the device by focussing the two phases through a second orifice, this leads to greater shear forces. This technique is well described within the literature for planar type microfluidic devices and also for glass capillariesPEVuZE5vdGU+PENpdGU+PEF1dGhvcj5HYXJzdGVja2k8L0F1dGhvc j48WWVhcj4yMDA0PC9ZZWFy PjxSZWNOdW0+MjwvUmVjTnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+Mjwvcm VjLW51bWJlcj48Zm9y ZWlnbi1rZXlzPjxrZXkgYXBwPSdFTicgZGItaWQ9J2ZhOXpkenN2a3N4MnByZ XJlOTh4ZDUycWRm d2Z3cjkwcDVzMic+Mjwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYtdHlwZSBuY W1lPSdKb3VybmFs IEFydGljbGUnPjE3PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxhdXRob3JzPjxh dXRob3I+R2Fy c3RlY2tpLCBQLjwvYXV0aG9yPjxhdXRob3I+R2l0bGluLCBJLjwvYXV0aG9yPj xhdXRob3I+RGlM dXppbywgVy48L2F1dGhvcj48YXV0aG9yPldoaXRlc2lkZXMsIEcuIE0uPC9hdXR ob3I+PGF1dGhv cj5LdW1hY2hldmEsIEUuPC9hdXRob3I+PGF1dGhvcj5TdG9uZSwgSC4gQS48L2 F1dGhvcj48L2F1 dGhvcnM+PC9jb250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5IYXJ2YXJkIFVua XYsIERlcHQgQ2hl bSAmYW1wOyBCaW9sIENoZW0sIENhbWJyaWRnZSwgTUEgMDIxMzggVVN BLiBVbml2IFRvcm9udG8s IERlcHQgQ2hlbSwgVG9yb250bywgT04gTTVTIDFBMSwgQ2FuYWRhLiBIYXJ 2YXJkIFVuaXYsIERp diBFbmduICZhbXA7IEFwcGwgU2NpLCBDYW1icmlkZ2UsIE1BIDAyMTM4IF VTQS4mI3hEO0dhcnN0 1098

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

yb25pYy1yZXNvdXJj ZS1udW0+PGxhbmd1YWdlPkVuZ2xpc2g8L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ 2l0ZT48Q2l0ZT48 QXV0aG9yPlRha2V1Y2hpPC9BdXRob3I+PFllYXI+MjAwNTwvWWVhcj48Um VjTnVtPjEwMjwvUmVj TnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+MTAyPC9yZWMtbnVtYmVyPjxmb3J laWduLWtleXM+PGtl eSBhcHA9IkVOIiBkYi1pZD0iZmE5emR6c3Zrc3gycHJlcmU5OHhkNTJxZGZ3Zn dyOTBwNXMyIj4x MDI8L2tleT48L2ZvcmVpZ24ta2V5cz48cmVmLXR5cGUgbmFtZT0iSm91cm5hb CBBcnRpY2xlIj4x NzwvcmVmLXR5cGU+PGNvbnRyaWJ1dG9ycz48YXV0aG9ycz48YXV0aG9yPl Rha2V1Y2hpLCBTLjwv YXV0aG9yPjxhdXRob3I+R2Fyc3RlY2tpLCBQLjwvYXV0aG9yPjxhdXRob3I+V 2VpYmVsLCBELiBC LjwvYXV0aG9yPjxhdXRob3I+V2hpdGVzaWRlcywgRy4gTS48L2F1dGhvcj48L2 F1dGhvcnM+PC9j b250cmlidXRvcnM+PHRpdGxlcz48dGl0bGU+QW4gYXhpc3ltbWV0cmljIGZsb3c tZm9jdXNpbmcg bWljcm9mbHVpZGljIGRldmljZTwvdGl0bGU+PHNlY29uZGFyeS10aXRsZT5BZ HZhbmNlZCBNYXRl cmlhbHM8L3NlY29uZGFyeS10aXRsZT48L3RpdGxlcz48cGVyaW9kaWNhbD48 ZnVsbC10aXRsZT5B ZHZhbmNlZCBNYXRlcmlhbHM8L2Z1bGwtdGl0bGU+PGFiYnItMT5BZHYuIE1 hdGVyLjwvYWJici0x PjwvcGVyaW9kaWNhbD48cGFnZXM+MTA2Ny0rPC9wYWdlcz48dm9sdW1lPj E3PC92b2x1bWU+PG51 bWJlcj44PC9udW1iZXI+PGRhdGVzPjx5ZWFyPjIwMDU8L3llYXI+PHB1Yi1kY XRlcz48ZGF0ZT5B cHIgMTg8L2RhdGU+PC9wdWItZGF0ZXM+PC9kYXRlcz48YWNjZXNzaW9uL W51bT5JU0k6MDAwMjI4 NjAxNTAwMDI2PC9hY2Nlc3Npb24tbnVtPjx1cmxzPjxyZWxhdGVkLXVybHM+ PHVybD4mbHQ7R28g dG8gSVNJJmd0OzovLzAwMDIyODYwMTUwMDAyNjwvdXJsPjwvcmVsYXRl ZC11cmxzPjwvdXJscz48 1108

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

L3JlY29yZD48L0NpdGU+PENpdGU+PEF1dGhvcj5TaGFoPC9BdXRob3I+PFllY XI+MjAwODwvWWVh cj48UmVjTnVtPjEwMTwvUmVjTnVtPjxyZWNvcmQ+PHJlYy1udW1iZXI+MTA xPC9yZWMtbnVtYmVy Pjxmb3JlaWduLWtleXM+PGtleSBhcHA9IkVOIiBkYi1pZD0iZmE5emR6c3Zrc3g ycHJlcmU5OHhk NTJxZGZ3ZndyOTBwNXMyIj4xMDE8L2tleT48L2ZvcmVpZ24ta2V5cz48cmVm LXR5cGUgbmFtZT0i Sm91cm5hbCBBcnRpY2xlIj4xNzwvcmVmLXR5cGU+PGNvbnRyaWJ1dG9ycz48 YXV0aG9ycz48YXV0 aG9yPlNoYWgsIFIuIEsuPC9hdXRob3I+PGF1dGhvcj5TaHVtLCBILiBDLjwvYX V0aG9yPjxhdXRo b3I+Um93YXQsIEEuIEMuPC9hdXRob3I+PGF1dGhvcj5MZWUsIEQuPC9hdXRo b3I+PGF1dGhvcj5B Z3Jlc3RpLCBKLiBKLjwvYXV0aG9yPjxhdXRob3I+VXRhZGEsIEEuIFMuPC9hd XRob3I+PGF1dGhv cj5DaHUsIEwuIFkuPC9hdXRob3I+PGF1dGhvcj5LaW0sIEouIFcuPC9hdXRob3I+ PGF1dGhvcj5G ZXJuYW5kZXotTmlldmVzLCBBLjwvYXV0aG9yPjxhdXRob3I+TWFydGluZXos IEMuIEouPC9hdXRo b3I+PGF1dGhvcj5XZWl0eiwgRC4gQS48L2F1dGhvcj48L2F1dGhvcnM+PC9jb25 0cmlidXRvcnM+ PHRpdGxlcz48dGl0bGU+RGVzaWduZXIgZW11bHNpb25zIHVzaW5nIG1pY3Jv Zmx1aWRpY3M8L3Rp dGxlPjxzZWNvbmRhcnktdGl0bGU+TWF0ZXJpYWxzIFRvZGF5PC9zZWNvbmR hcnktdGl0bGU+PC90 aXRsZXM+PHBlcmlvZGljYWw+PGZ1bGwtdGl0bGU+TWF0ZXJpYWxzIFRvZ GF5PC9mdWxsLXRpdGxl PjwvcGVyaW9kaWNhbD48cGFnZXM+MTgtMjc8L3BhZ2VzPjx2b2x1bWU+MT E8L3ZvbHVtZT48bnVt YmVyPjQ8L251bWJlcj48ZGF0ZXM+PHllYXI+MjAwODwveWVhcj48cHViLW RhdGVzPjxkYXRlPkFw cjwvZGF0ZT48L3B1Yi1kYXRlcz48L2RhdGVzPjxhY2Nlc3Npb24tbnVtPklTSTo wMDAyNTQ2OTE5 MDAwMTg8L2FjY2Vzc2lvbi1udW0+PHVybHM+PHJlbGF0ZWQtdXJscz48dXJs 1109

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

PiZsdDtHbyB0byBJ U0kmZ3Q7Oi8vMDAwMjU0NjkxOTAwMDE4PC91cmw+PC9yZWxhdGVkLXV ybHM+PC91cmxzPjwvcmVj b3JkPjwvQ2l0ZT48L0VuZE5vdGU+ 93,94,122,127, but to our knowledge this is the first time that this process has been shown simply using needles. Therefore a FF type design was developed for use at the mixing point of our microfluidic system as shown schematically in Figure 5–7.

Figure 5–7. Schematic representation of the ‘flow-focussing’ device. The internal needle fits within the external needle.

1.1.2.1. Orientation of device The shear forces at the point of mixing between the two phases is controlled by the relative flow rates of the two phases. By reducing the diameter of the outlet at the end of the needle (from which the continuous phase flows) the shear forces are increased due to the greater velocity of the liquids. This led to small droplets being produced at a high rate however, the high production rate of droplets often lead to the coalescence of the droplets. The density of oil phase was 0.79 g/cm3 while the density of the monomer solution was 1.00 g/cm3. This led to the monomer droplets settling on the bottom of the PTFE tubing. Additionally, this difference in density meant that the orientation of setup up was especially important because the speed of the monomer droplets was determined by both the flow rate of the continuous phase and gravity. If the flow direction opposed gravity the monomer droplets would cluster upon leaving the external needle, this resulted in a polydisperse sample with a larger mean diameter as some of the droplets coalesced before being polymerised. If the flow direction and gravity were in the same direction the distance between the

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

monomer droplets was greater than when the device was horizontal (i.e. flow direction was at 90˚ to gravity) (Figure 5–8 A & B), however, because the PTFE tubing had to pass through the UV box in a relatively horizontal position coalescence of droplets also occurred where the tubing was bent from vertical to horizontal. This bend led to a reduction in the speed of the monomer droplets, greatly reducing their separation from neighbouring droplets causing coalescence. The optimum setup was found to be a constant slightly ‘downhill’ gradient (20˚ from horizontal) from the needle to the end of the outlet tubing. The slight slope led to a slight increase in the separation between monomer droplets at the tip of the needle preventing coalescence (except at very high monomer flow rates). The constant gradient removed the problem of coalescence due to the changing of droplet speed (and therefore separation) caused by a change in the angle of the tubing. The FF design lead to the production of smaller droplets that the earlier microfluidic setup, however the high production rate of droplets and the difference in density meant that the orientation of the device had to be adjusted to minimise coalescence of droplets. Figure 5–8. Effect device orientation on droplet and resulting particle formation. A: Schematic representation of effect of gravity and flow direction on droplet formation (left; the flow of gravity opposes gravity, right; flow direction and gravity are in the same direction. B: Optical micrographs of particles produced (left; when flow opposes gravity, right; when flow is with gravity) scale bar is 110 µm.

1.1.2.2. Optimising conditions with the FF device Using the optimised device orientation, a range of flow rates were tested to determine the conditions for the production of monodisperse particles (Figure 5–9 A). At high values of Qd polydisperse particles were polymerised as a result of insufficient droplet separation leading to coalescence of droplets. While values of Qc above 1.5 ml/min produced particles that were not fully polymerised. A narrow range of conditions were found to give near monodisperse particles, these conditions were investigated to determine the variation of particle size with Qc/Qd (Figure 5–9 B) using 0.08 g/ml of surfactant in the continuous phase. As Q c/Qd was increased particle size decreased until an apparent minimum diameter of approximately 100 µm at Qc/Qd values of 375 and above. The effect of surfactant concentration was not tested for the FF setup as the highest concentration (0.08 g/ml) had already been 1111

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

established to give the smallest particle diameters with the earlier microfluidic setup. The optimised flow focussing setup produced particles below the limit of the earlier microfluidic setup down to a minimum of 100 µm in diameter. However, it offered a less robust production with near monodisperse particles only found under a narrow range of conditions.

Figure 5–9. Development of flow-focussing setup. A: Particle morphology as a function of the flow rate of the dispersed phase (Qd) and continuous phase (Qc) where ■; polydisperse particles (polydispersity > 8 %), ♦; near monodisperse particles (polydispersity < 8 %) and ▲; particles did not fully polymerise. B: Effect of Qc/Qd on mean particle diameter using flow-focussing.

1.1.2.3. Effect of changing oil phase The shear forces exerted on the disperse phase are determined by the velocities and the viscosities of the two phases. Up to this point only the velocities have been varied, therefore, in order to achieve smaller particle sizes the continuous phase was changed to oil with higher viscosity. Silicone oil was chosen as it was obtainable in a range of different viscosities additionally it had a density of 0.95 g/ml which 1112

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

would reduce the problems associated with the difference in density found for Isopar M. Initial polymerisations using silicone oil (with a viscosity of 20 cP) as the continuous phase produced either polydisperse or highly aggregated particles (Figure 5–10).

Figure 5–10. Optical micrographs of PEGA particles produced using FF device with silicone oil (50 cSt viscosity) at a variety of conditions (clockwise from top right: Qc/Qd = 100 with Qc = 0.5, Qc/Qd = 100 with Qc = 0.1, Qc/Qd = 450 with Qc = 0.25, and top left Qc/Qd = 650 with Qc = 0.1. Scale bar is 200 µm.

Particles with a narrow size distribution were not formed at any of the conditions investigated. At high total flow (Qc + Qd) rates polydisperse particles were formed, while at lower flow rates aggregated particles were produced (Figure 5–10). The aggregated particles appear to be of a relatively narrow size distribution with a mean diameter of approximately 60 µm. Presumably, droplets underwent coalescence prior to polymerisation at higher flow rates while at lower flow rate droplets began to polymerise before coming into contact with one another resulting a mass of aggregated particles. An increase in surfactant concentration within the continuous phase was investigated however it was still not possible to produce individual particles. Coalescence occurred as a result of slight variations in the speed of movement of the dispersed droplets causing the separation between droplets to become insufficient. It is likely that this is an intrinsic limitation of microfluidic 1113

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

devices fabricated from flexible tubing. This problem is not reported in planar ‘chip’ type designs. As a result of these findings silicone oil was not further investigated as the continuous phase. 1.1.3. Optimum conditions for particle production The aim of this work was to produce particles with the smallest possible diameter with a very narrow size distribution. However, the rate of particle production was slower at higher values Qc/Qd. With these factors in mind the optimum conditions used in the FF design were: 0.08 g/ml of surfactant in the oil phase, a Qc/Qd of 175 and a Qc of 1.5 ml/min. 1.1.3.1. Particle size distribution Optimum conditions (described above) were used to produce approximately one gram of polymer. These particles were further analysed by optical microscopy (Figure 5–11 A) and a size distribution obtained (Figure 5–11 B). The majority of the polymer volume was in the size range (150-170 μm) a low volume of smaller satellite were also observed. The mean particle diameter was 161 μm with a polydispersity of 3.3 %. The rate of particle production was 4000 particles min-1.

Figure 5–11. PEGA Particles produced under the optimum conditions. A: Optical microscopy of particle (scale bar is 160 μm). B: Particle size distribution.

1.1.4. Morphology of particles The particles produced by this technique often had an elliptical morphology with aspect ratios128 ranging from 1.006-1.098. While Hadziioannou and co-workers97 did not report this; no images of the particles that were produced were shown in the paper. Within this work the formation of these asymmetrical particles seems to be a 1114

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

result of the difference in density of the water (dispersed phase) and the oil (continuous phase). This led to the monomer droplets descending and flowing along the bottom of PTFE tubing. This contact with the tubing wall would have lead to asymmetrical flow around the droplets.

Figure 5–12. The typically slightly asymmetric shape of particles produced by microfluidics each image is of particles produced under different conditions. Scale bar is 200 µm.

1.1. Conclusions This work provides an overview of using simplified microfluidic devices to produce PEGA particles. The creation of the device required no specialist fabrication methods and could be assembled using readily available lab supplies. Surfactant was required to obtain control of the particle size by varying Qc/Qd, upon increasing Qc/Qd smaller particles were obtained. Additionally, increasing the surfactant concentration led to a reduction to the particle size. A minimum particle diameter of 335 µm was determined. A flow-focussing setup was devised to overcome this limitation and allowed for the production of smaller particles with a diameter of 99 µm and a polydispersity of 3.2 %. This low value for polydispersity was a dramatic improvement compared to the particles prepared by inverse suspension polymerisation (43 %).

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2 Conclusions This thesis describes the design, synthesis and application of enzyme responsive hydrogel particles through the use and development of peptide actuators. Initially, PEGA hydrogel microparticles (μPEGA) were produced as the support for the enzyme responsive system based on peptide actuators. These particles had a homogeneous distribution of amines and were used as the chemical ‘handle’ from which peptides could be synthesised. Enzymatic hydrolysis on the coupled peptides was shown to be homogeneous throughout the particles. Additionally, μPEGA was found to give rise to faster enzyme hydrolysis than commercially available large PEGA particles (macroparticles) due to reduced diffusion distance. μPEGA was then functionalised with linear peptide actuators. These zwitterionic molecules were shown to actuate changes in the accessibility of the crosslinked PEGA particles. This occurred through a switch in the overall charge balance of the polymer network (from neutral to cationic) upon enzymatic hydrolysis of the enzyme cleavage peptide (ECP) within the peptide actuator. The resulting electrostatic repulsion between neighbouring peptide actuator fragments led to an increase in the mesh size of the polymer network. By selecting the composition of the ECP to match an enzyme’s specificity it was possible respond to different enzymes. The pH responsiveness of the linear actuator functionalised particles was demonstrated. This behaviour was utilised for the loading of the particles with a fluorescently labelled macromolecule. Enzyme specific release of this payload was then possible. However, at physiological ionic strength no enzyme triggered swelling of the functionalised particles was observed. This was a result of the mobile ions in solution screening out the interactions between the peptide actuators. This limitation was addressed with the design of branched peptide actuators. Branched peptide actuators consisted of two linear actuators using the amino acid lysine as the branch point. This structure was built up from each of the amines within μPEGA. Upon enzyme hydrolysis there was double the charge density within particles (compared to when using linear peptide actuators) and the distance between charged groups was likely reduced. Therefore, particles functionalised with

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

branched peptide actuators capable of specifically responding to enzymes at physiological ionic strength (by overcoming electrostatic screening). Much like the linear peptide actuator functionalised particles this system capable for physically entrapping a macromolecular payload, was could be released only in response to a defined enzyme at physiological ionic strength. The final experimental chapter provided an overview of using simplified microfluidic devices to produce PEGA particles. Rather than requiring specific microfabrication techniques it was possible to produce the device using a needle and tubing. It was found that by varying the surfactant concentration and the flow rates of the continuous and dispersed phase particles of different sizes were obtained. Increasing the surfactant concentration or increasing the ratio of continuous to dispersed phase flow rates led to smaller particles. The minimum particle diameter possible was found to be determined by the diameter of the needle. Therefore, a flow-focussing setup was devised to overcome this limitation and allowed for the production of smaller particles.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

3 Future work The most obvious future work at this stage would be using PEGA particles produced by microfluidics as the basis of the peptide actuator system. These particles with their narrow size distribution would allow for the kinetics of both enzyme and pH responsive swelling behaviour to be characterised in greater depth. As narrow size distribution of these PEGA particles would allow for a relatively small number of particles to be measured in order to obtain the change in swelling. It would also be of interest to use microfluidics to prepare polymer particles crosslinked by enzyme cleavable peptides. This would be possible by employing a strategy similar to Moore and co-workers.11 Monomers capable disulphide transfer reactions to thiols would be synthesised (this has been carried out details are contained in appendix section Synthesis of activated disulfide-methacrylamide monomer. These could then be conjugated to peptides containing two cysteine residues resulting in a peptide crosslinker. A mixture of monomer and peptide crosslinker could then be polymerised into particles using the microfluidic device. Much like the enzyme responsive particles using peptide actuators, these particles would also allow for enzyme responsive release of a macromolecule (by tuning the crosslinking density). However, if required these peptide crosslinked particles could be formulated so that they undergo complete dissolution upon enzyme action (along with release of the payload). Additionally, it may be possible to combine an enzyme cleavable peptide crosslinker and peptide actuators into the same polymer. These systems could potentially have greater amplitudes of response or by using different peptide substrates in the crosslinker and ECP only response to a solution containing two different target enzymes. Rapid and high throughput analysis of the response of the functionalised particles may be possible if monodisperse PEGA particles of around 20 μm in diameter could be

produced

(using

a

microfluidic

chip

or

possibly

by

membrane

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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129,130).

These particles could then be passed through an automated cell sorter (flow cytometry)131. Using a split and mix method it would be possible to prepare PEGA particles functionalised with a variety of different ECP compositions. If these particles were then exposed to an enzyme and then sorted by size it would be possible to screen for effective substrates for the enzyme. By designing the peptide actuator and tuning the molecular weight cut-off of the functionalised particles it may be possible to use enzyme hydrolysis of the ECP to trigger a deswelling of the particles. This offers the potential for the specific entrapment of the enzyme responsible for hydrolysing the ECP and additionally, might allow for removal of a desired protease from a complex solution. Ultimately, there are a number of steps that need to be overcome in order to use particles functionalised with peptide actuators as drug delivery vehicles: Depending on target site and method of introduction to the body (i.e. into circulation or injected directly into the tissue) particles of smaller size may need to be produced. Emulsion132

or

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Tm90ZT4A

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Tm90ZT4A

133,134

may

offer

this

possibility.

Additionally, the particles must respond only to the target enzyme (i.e. the marker for the disease to be treated), to achieve this goal the ECP must be a substrate of that enzyme. This would be assessed by synthesising the ECP as the substrate of a known disease specific protease, then exposing these particles to that enzyme in an appropriate complex mixture. Finally, the ultimate destiny of the particles within the body must be determined. If the particles are not able to be cleared from the body then some form possible degradation must be introduced into the polymer.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

4 Appendices 4.1. Background 4.1.1. Polymers Polymers have been used as structural materials within nature since life began with substances such as DNA, polysaccharides and peptides playing crucial roles in animal and plant life. The term polymer is defined from the Greek words poly and meros, meaning many parts. In the strictest sense a polymer is a substance composed of molecules consisting of one or more types of atoms linked to each other by primarily, usually covalent bonds.87,135 Polymers are created through the linking together of the small monomer molecules through chemical reactions known as polymerisation reactions. There are two main types of polymerisation reaction; addition or chain-growth polymerisation in which, typically a vinyl monomer (CH2=CHX) is attacked by an initiator to yield an active centre that can then attack another vinyl monomer linking them together by covalent bonds. Condensation or step-growth is the second type of polymerisation, here, monomers have different functional groups (e.g. A-A + B-B → A-a-b-B or A-B + A-B → A-b-a-B) that react together in a condensation type reaction to form larger molecules consisting of covalent bonds. Individual polymer chains can be linked together through the introduction of a difunctional (or multi-functional) monomer or crosslinker and high levels of crosslinking result in the formation of a three-dimensional network that is insoluble. 4.1.1.1. Peptides and proteins Peptides are polymers of amino acids. These substances exhibit a wide range of differing biological properties allowing them to act as; antibiotics, hormones, food additives, poisons or pain-killers. Peptides are formed by a condensation reaction between a carboxylic acid and an (primary) amine forming a peptide bond. Essentially peptide and proteins are the same at the molecular level. The term protein is used to refer to large molecules typically containing at least fifty amino acids with a well defined three-dimensional structure. There are twenty amino acids that are encoded by DNA, each amino acid has the same generalised structure but 1133

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

with a different side group. These twenty amino acids offer a wide range of functionalities that can be incorporated into peptides (Figure 8–1). This gives as the possibly of preparing a huge number of different peptides, for example, for a pentapeptide there are 3,200,000 possible combinations.136 Within this thesis amino acids will be referred to either by their full name or the 1-letter abbreviation shown in Figure 8–1.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald Figure 8–1. The structures of the twenty DNA encoded amino acids. The single letter abbreviation is indicated with the brackets.

4.1.2. Two-photon microscopy Two-photon microscopy (TPM)78 is a fluorescent microscopy technique traditionally used in the fluorescent imaging of biological cells. In this technique the sample is irradiated with a laser with a wavelength approximately twice that of the excitation length wavelength of the fluorophore. This means that excitation can only occur when two photons are absorbed simultaneously. This is extremely unlikely and therefore only occurs at very high photon density; the focal point of the laser beam. TPM has a number of advantages over the similar technique, confocal microscopy (in which the sample is irradiated with ultraviolet light with a wavelength equal to that of the excitation wavelength of the fluorophore) seen in Figure 8–2. The use of a longer wavelength from the laser for excitation allows for deeper penetration into the sample. Additionally, because excitation of the fluorophore only occurs at the focal point the problem of photobleaching is greatly reduced.131

Figure 8–2. Jablonski energy diagram showing a comparison of the excitation of a fluorophore with a single photon (confocal microscopy) and two photons (two-photon microscopy).

4.1.3. Fluorescence resonance energy transfer Fluorescence (or Förster) resonance energy transfer (FRET) microscopy is a technique used for quantifying the distance between two different fluorophores.

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

cmVzb3VyY2UtbnVtPjxs YW5ndWFnZT5FbmdsaXNoPC9sYW5ndWFnZT48L3JlY29yZD48L0NpdGU+PC 9FbmROb3RlPgB= 137,138 FRET involves the transfer of energy from a fluorescent donor in its excited state to another excitable moiety, the acceptor, by a non-radiative dipole-dipole interaction In order for FRET to occur the distance between the donor and the acceptor must be small (1-10 nm) and results in a decrease in donor emission and an increase in acceptor emission. FRET allows the determination of whether there is a close association between the donor and the acceptor. This technique has been used to assess (amongst others) calcium ion concentration,139 protein-protein colocalisation140 and enzyme hydrolysis.141

4.2. Supplementary data 4.2.1. HPLC solvent gradient

Figure 8–3. HPLC solvent gradient used in analytical runs. Concentration of buffer B over the length of a HPLC run.

4.2.2. Synthesis of activated disulfide-methacrylamide monomer An activated disulfide-methacrylamide monomer was synthesised with the aim of preparing enzyme responsive particles using on peptide crosslinkers based on the work of Moore and co-workers.11 This monomer, N-[2-(2-pyridyldithio)]ethyl methacrylamide (PDTEMA) (Figure 8–4 was initially described by Ruffner and coworkers for conjugation to oligonucleotides and oligopeptides.142 The procedure

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

described in this paper was followed, resulting in the successful synthesis of the monomer (as determined by NMR, Figure 8–5).

Figure 8–4. Synthesis of PDTEMA.142

Figure 8–5. Characterisation of PDTEMA by NMR. 1H NMR (CDCl3, 200 MHz): δ (ppm) 7.0-8.6 (m, 5H, Ar-H and -NH), 5.809 (s, 1H, one of d, CH2), 5.361 (s, 1H, one of dCH2), 3.603 (m, 2H, -CH2NR), 2.960 (t, 2H, -S-CH2-), 2.002 (d, 3H, -CH3, the split of this peak is due to the tautomerization of the adjacent double bond).

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Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

5 References 1

Ratner, B. D. and Bryant, S. J., Biomaterials: Where we have been and where we are going. Annu. Rev. Biomed. Eng. 6, 41-75 (2004).

2

Peppas, N. A. and Langer, R., New challenges in biomaterials. Science 263 (5154), 1715-1720 (1994).

3

Stauffer, R. N., 10-year follow-up-study of total hip-replacement - with particular reference to roentgenographic loosening of the components. J. Bone Joint Surg.-Am. Vol. 64 (7), 983-990 (1982).

4

Conrad, H. J., Seong, W. J., and Pesun, G. J., Current ceramic materials and systems with clinical recommendations: A systematic review. J. Prosthet. Dent. 98 (5), 389-404 (2007).

5

Langer, R., Drug delivery and targeting. Nature 392 (6679), 5-10 (1998).

6

Refojo, M. F., Current status of biomaterials in ophthalmology. Surv. Ophthalmol. 26 (5), 257-265 (1982).

7

Ratner, B.D., in Biomaterials science: An introduction to materials in medicine, edited by AS Hoffman BD Ratner, FJ Schoen, JE Lemons (Academic Press, San Diego, California, 1996), pp. 1-10.

8

Langer, R. and Tirrell, D. A., Designing materials for biology and medicine. Nature 428 (6982), 487-492 (2004).

9

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