Introduction Into Nanomedicine Get Your Feet Wet

Nanomedicine Review Alexandria faculty of medicine

NanoTechnology students Group

Abstract The past few years have seen numerous breakthroughs in Nanotechnology which had its impact on different fields of scientific research . A few of these breakthroughs proved to be very promising from a medical point of view . The term " Nanomedicine " describes these applications and the various ways in which Nanotechnology can be used medically

In this report we will discuss a few of the current applications and the different methods of disease prevention , diagnosis and treatment utilizing these applications . We then will discuss in more detail new modalities for " Cancer Management by Selective conjugation to HER-2 receptors " as an example of employing Nanomedicine for disease management . The choice of Cancer is owed to the fact that it is one of the most common diseases of our time , one that has proven to very difficult to diagnose and cure , and the traditional treatment modalities are very invasive , induced very intolerable side effects and do not guarantee complete cure . The proposed management employes : Quantum Dots and Dendrimers for diagnosis ; photothermal ablation of Gold Nanoshells and Targeted Drugdelivering for treatment , that resulted in very selective and specific diagnosing techniques and very promising treatment results and

prognosis , all of which are thoroughly explained in this presentation. The details of these management modalities were obtained from various research Additionally we will discuss some of the limitations, implications , ethical issues, and possibilities of Toxicology induced by Nanomedicine in general

June 11, 09

Alexandria Faculty of medicine

NANOTECHNOLOGY GROUP • •

ALI AL NOWAEM

HESHAM GHONEIM

• • •

KARIM ISMAIL

MOMEN ZALABANY PASSENT MAGED

Special thanks to

Prof Dr. Mahmoud Al-Zalabany Dean

Prof. Dr Ahmed Osman Vice Dean

Prof. Dr. Sedeak Abd-Alsalam Vice Dean

Dr. Tatjana Paunesku,

Feinberg School of Medicine Dr. Tamer Refat Alexandria faculty of medicine For any Questions and Information Do not hesitate to contact us at

[email protected]

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INDEX

1 Brief History ............................................................. 4 2 Introduction ............................................................. 5 2.1 Definition 2.2 Nanomaterials 2.3 Nanodevices

3 Nanotools .................................................................. 6 3.1 Nanopores……………………………………… 3.2 PEG 3.3 FRET 3.4 Qdots 3.5 NanoShells 3.6 Dendrimers 3.4.1 Moleculer Structure 3.4.2 Branches 3.4.3 InternalCavities

4 Treatment ................................................................. 10 4.1 Smart Drugs Trojan horse Passive targeting Active targeting 4.2 Nanoshell assisted Photoablation

5 Diagnosis ................................................................ 13 5.1 Imaging ………………………………………………………………

5.2 Laboratory Diagnosis ………………………………………………

6 Limitations .............................................................. 17 Nanotoxicity 7 References................................................................ 19

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NANOTECHNOLOGY STORY !

Nanotechnology story began, when we at last could visualize and manipulate particles at molecular scale! As there was first era that humans used there naked eye to study objects, then came the Era of Microscopes where microscopes were used to visualize smaller objects, and then came the era of E.M where we were able to study Tiny objects and particles as cells and intra cellar organelles, and then came the era of STM and Atomic force microscope where we were able at last to visualize and manipulate atoms at its very small nanoscale size !.

The immerge of the nanotechnology as a science filed is owned to 2 facts,, 1st is that nanotech enabled us to not only visualize but also manipulate object scanned. 2nd and most important, is that at this very small size, particles did not behave the same as it did at its micro or bulky size, and that what needed a science to immerge and explain , study and utilize these particles unique changing properties at this nano size .

1 BRIEF HISTORY

Nanotechnology and nanoscience started in the early 1980s with two major developments; the birth of Cluster Science and the invention of the Scanning Tunneling Microscope (STM). IBM Fellow ‘Don Eigler’ was the first to accurately manipulate individual atoms on a surface, using the STM to spell out "I-B-M" by positioning 35 Xenon atoms on a Nickel surface

1)

(

Nanomedicine: Has always been side to side with nanotechnology since the very beginning, In 1999 Robert

Freitas assembled an impressive array of ingenious ideas that Derive from ongoing developments through his book on Nanomedicine, In April 2006, the journal Nature Materials

estimated that 130 nanotechnology-based drugs and delivery systems were being developed worldwide.

With the advance in Molecular Nanotechnology and the consequent advance in Nanomedicine, researchers began to consider employing these technologies to develop new techniques to manage diseases that are currently very difficult to manage. Of these diseases, Cancer is a priority, as it is one of the leading causes of death in the modern world, with rapidly rising morbidity and mortality rates, as shown in the figure below.

This discovery opened the door for visualizing and manipulating materials at the molecular scale Six years later the Atomic Force Microscope (AFM) was invented in 1986, providing even better resoultion and higher control.

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2 INTRODUCTION TO NANOMEDICINE AND NANODEVICES Nanotechnology is defined as Research and technology development at the atomic, molecular and macromolecular levels in the length scale of approximately 1 – 100 nm range, to provide a fundamental understanding of phenomena and materials at the nanoscale and to create and use structures, devices and systems that have novel properties and functions because of their small and/or intermediate size Since the development of STM and Atomic force microscope, the field of Nanotechnology at last became possible, researches started to develop many tools that will help them build up a useful Nanodevice. These Nanodevices are manufactured by either bottom up approach: (involves assembling structures atom-

by-atom or molecule-by-molecule, traditionally used in Nanomedicine) or Top-down approach (involve breaking down of macros, rarely used in Nanomedicine).

nano-tools and unique phenomena’s to create a multi-purpose particle very handy in biomedical fields.

In the approach of making this real, lots of nanotools and backbones have been synthesized.

NANOMATERIALS Are materials used in nanotechnologies which are either Fullerenes or Nanoparticles: Fullerenes are allotropes of carbon which are graphene sheets rolled into tubes or spheres, to form carbon Nanotubes with their unique mechanical and electrical properties as heat resistance and superconductivity Nanoparticles: are of particular interest for their unique electrical, magnetic, optical, chemical and even mechanical properties, in such particles quantum-size effects are observed such as quantum confinement in semiconductor particles, surface Plasmon resonance in some metal particles and superparamagnetism in magnetic materials.

NANODEVICES

Nanodevices are not as sophisticated as some people might think, however Nanodevices are much simpler polymer backbone structure (<100nm) attached to some nano-tools that make the device smarter and even more versatile so it can do what brute force could not.

Another definition is the aim of Nanodevices is to make the management of human health threats easier by bottom-up constructing a molecule using

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Förster Resonance Energy Transfer

3 NANO-TOOLS 3.1) NANOPORES

Nanopores are small holes in a surface that will allow particles through depending on their size and voltage. In 1997 Desai and Ferrari constructed a cell containing like chambers with polycrystalline walls full of Nanopores 20nm in diameter –at this size, only small molecules as oxygen and nutrients can pass while large IG and graft rejecting viruses are blocked, Inside these chambers, rat pancreatic cells were inserted away and safe from Immune responses, as so the cells remained healthy secreting insulin in response to blood glucose change. This concept would be of a great value in management of any hormone or Enzyme deficient disease as encapsulated Gland cells could be used to replace Normal nonfunctioning cells.

is a mechanism describing energy transfer between two chromophores. In which there is a distancedependent interaction two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon, stating that Donor and acceptor molecules must be in close proximity (typically 10<100 nm) and The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor Applications :

Since FRET is distant-dependant, it is a powerful reporter on the separation of two fluorophores

This has been applied to many fields to detect certain chemical reactions, like HIV(figure) or cell apoptosis sensors.

Picture from :http://www.invitrogen.com Example:

3.2) PEG

Poly(ethylene glycol)

the covalent coupling of a PEG structure to another molecule, and is a process which can be used for enhanced drug characteristics, as immune stealth, renal clearance. example : peginterferon alpha-2a for treatment of cancer In clinical practice, a once-weekly dosage of peginterferon alpha-2a was tested in a randomized prospective trial with a dosage of interferon alpha-2a three times per week. In a study population of 531 patients, PEG-interferon alpha-2a was more effective as a single weekly than the standard tri-weekly therapy with interferon alpha-2a.

3.3) FRET

Utilizing caspase-3 enzyme -which is activated early in the apoptosis process-. As it cleaves valine and aspartic acid which changes the distance between donor and acceptor molecules leading to fluorescence and apoptosis sensing

3.4 QUANTUM DOTS Quantum Dots are Nanocrystal particles with Quantum confinement properties, they can be excited to fluorescence with different wavelengths of Electromagnetic radiation. The resulting fluorescent color can be adjusted by changing the Qdot’s size and composition.

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This process requires some insight to Quantum mechanics that govern the behavior of electrons in particles of the nanoscale size. As a particle’s size decreases from bulk to nanoscale, its properties change rapidly, till the particles size reaches a value of

the magnitude of its Exciton Bohr radius, the bandgap increases, requiring more energy for electrons to shift from the valency band to the conduction band creating electron holes in the valency band. As electrons always move back from the conduction band to the valency band, they emmit energy in the form of Electromagnetic radiation, essentially the same amount of energy obtained to cross the bandgap.

This allows unpreceded control over the colors emitted from Quantum Dots, simply by altering their size, they will form colors that will not blend together or fade out with time as presented by conventional dyes. Figure below shows Quantum Dots Vs conventional dyes where Quantum Dots did not fade while conventional dyes did

Another very powerful aspect of Quantum Dots is that they can be attached to various Biomolecules , enabling them to act as highly sensitive probes or detectors, they can be used to target certain proteins in the blood , which would provide a very specific and selective rapid screening tool for different diseases , or designed to attach to tumor cells only among other healthy cells creating a novel tumor diagnosis tool, which provides with unpreceded ease and accuracy many details about tumors including stage , size and progression . Therefore, as the particles size decreases, a blue shift in illumination is observed due to the increased energy of emitted photons resulting from electrons crossing the band gap back to the valence band . In more common terms, the larger the particle, the “redder“ will it fluoresce when excited by an external stimulus as the larger the particle the the smaller the band gap ,the less energy it will need to move between bands.

Future researches are being aimed at tagging Qdots to multiple intra and extra-cellular particles to allow pathologists to study firsthand the various stages and changes induced by certain diseases, providing a massive leap forward in pathology and new disease management regimens.

3.5 NANOSHELLS

Nanoshells are thin metallic shells of a size that ranges from a few to a few hundred nano meters, they are first synthesized from a dielectric core (Glass or Silica beads) covered with a thin metallic shell (usually Gold) named Core-Shell Nanoparticles . The core is later degraded forming Nanoshells.

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terminal branches or inside its internal cavities as many nano-tools as your device may need.

Their small size allows them to absorb and scatter light at different wavelengths, depending on their size and their surface plasmonic resonance . Gold’s plasmonic resonance gives it a large photon capture cross section . To this is owed the use of Gold as the element of choice to cover the Nanoshells, as its plasmon resonance is ~520 nm which can be shifted to Near InfraRed with excitation wavelengths of about 800-1200 nm, which is extremely useful as body tissues are mostly relatively transparent to Near Infrared light . Gold’s plasmonic resonance gives it a large photon capture cross section . Their optical properties are very sensitive to aggregation, as the Nanoshells cluster, their absorption spectra Red shifts, and absorption at the original peak is markedly reduced . This can be utilized in various imaging and detection techniques.

MANUFACTURING

Dendrimers Are produced in an accumulative (iterative) sequence of reactions , in which each additional interaction leads to a higher generation and doubling The number of active sites as so as the molecular weight and molecule size the two main methods of synthesis for dendrimers are the divergent and convergent methods

The most valuable application though is the utilization of Nanoshells’ extinction properties to induce local heating of Nanoshells with NIR lasers to produce photothermal ablation only in the cells to which the Nanoshells are attached. This process can be applied to tumor cells producing a very rapid and accurate tumor ablation tool.

3.6 DENDRIMERS

SYNTHESIS:

Are large complex man-made molecule with a very well-defined chemical structure and a perfectly spherical shape with a highly branched 3 dimensional architecture composed of core, branching units ,internal cavities -formed in between the branching units- and terminal groups. It is due to this highly branching architecture that Dendrimers are widely recognized as the most versatile, compositionally and structurally controlled nanoscale building blocks available as It provides critically needed nanoscale building blocks suitable for the development of high performance bottom-up designed devices by hosting on its

There is two main methods of dendrimers synthesis, divergent and convergent. In 1980 first dendrimers was built using divergent technique, where chosen core as EDA, chemical steps was started as 1) Core ( Nh2—Nh2)) + Michael addition (Ch3OO) resulted in half generation -G0.5-, 2) Additional amidation with another EDA resulted in G0 with N~N core and 4 terminals O=C-NH

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This Divergent require many iterative reactions which may lead to imperfect shape and many side reactions, and so Convergent technique immerged which aims to avoid the disadvantages of divergent method by creating the Dendron branches then attaching them to the desired core

Branches: Dendrimers’ physical and chemical properties can be strongly influenced by the nature of their surface groups, Dendrimers terminated with hydrophilic groups are freely soluble in polar solvents and vice versa , This can be of great help increasing drug bioavaliblilty, also these branches conjugate various types of molecules, These molecules can be classified into ::: • Protective (PEG, carboxylate, hydroxyl, pyrrolidone, Acetamide) • Solubility modifiers • Active receptor targeting (Folic acid, CHO, IgG/fab fragment , VEGF) • Nanodecoys( Specific protein to exploit the viral Tropism as in Vivagel for HIV-1 by starpharma , sialic acid to competitively inhibit Influenza Virus) • Imaging tags.

Internal Cavities: One of the most important properties of dendrimers is the possibility to encapsulate guest molecules by entrapping them in their internal cavity by H bond , ionic or van der Waals forces , then forming a shell at the terminal group to stably encapsulate the particle inside the dendrimers awaiting their controlled release, This shell might be L-phenylalanine which will release its content upon hydrolysis , or any other switch nano-tool for even more control. “targeted encapsulated methotrexate delivery improved the cytotoxic response of the cells to 100fold over free drug “ Dendrimers as a particle Dendrimers can be very useful in creating a multipurpose NanoDevice, as it can combine as many tools as needed, dendrimers can encapsulate a drug, releasing it on specific stimuli, report cell apoptosis by the use of FRET phenomenon (fluorescence resonance energy transfer) and diagnose the disease Current Dendritic products. Product Vivagel

Application Vaginal Gel for preventing HIV(Nanodecoy) Stratus CS Cardiac Marker

Company Starpharma

SuperFect Gene Transfection Alert Ticket Anthrax Detection

Dade Behring Qiagen US army R.L

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only one functioning end group to avoid protein cross-linking during conjugation  linking chemistry between the protein and polymer is itself biocompatible  synthesis that allows reproduction with regard to site-specific protein modification III. Polyplex: This type of polymer is designed to compete with its viral counterpart, as adeno viruses which is an excellent vector for delivering nucleic acids into cells IV. Polymer-drug : elegant example of a “multifunctional” nano-device, polymer provides biocompatibility, and selectivity ,while linker attach drug with polymer V. Micelles : polymeric analogue of the liposome and carries its drug payload on the “inside” of its hydrophobic-cored micelle, This micelle have great advantage hence there self assembly in solvent and ability to transport non water soluble drugs PLUS their ability to be targeted 

4) CANCER MANAGEMENT TREATMENT

4.1) SMART DRUGS, TROJAN HORSES AND TARGETED NANOPARTICLES Smart drugs: refers to drugs that are only medically active in specific circumstances, as Enzyme-activated drugs, which were first developed in the 1980s and are still under active investigation.

TROJAN HORSE : Trojan horses (drug carriers) started in early 1955 when researchers first used the water-soluble polymer PVP (polyvinyl pyrrolidone) as a drug depot for the biologically active primary amine mescalin, since then lots of polymers have been developed for this aim, these polymers where classified into 5 classes that discus the nature of the carrier.

Targeted Nanoparticles:

Are nanoparticles that can be targeted to a specific location, This can be by either Passive or Active

PASSIVE TARGETING : is nano dimensionally

I. Sequestrants Polymer (WelChol™) : act to isolate compounds systemically within an organism. This macro-molecule is an orally administered compound with a polymer backbone and hydrophobic side-arms that have a high affinity for glyocholic acid bile salts. The action of binding the bile salts in the gastrointestinal tract and their ultimate excretion causes the liver in turn to create more bile salts requiring more cholesterol, which it derives from LDLcholesterol that circulates in the bloodstream.

mediated (size mediated) via EPR (enhanced permeability and retention) effect involving primary tumour vascularization or organ-specific targeting. EPR in Tumor cells is owned to its rapidly dividing cells that constantly require increasing resources and blood supply and employ surrounding cells to provide these resources. They have the ability to secrete Vasoactive Endothelial Growth Factor (VEGF) which induces local neo-vascularisation providing better blood supplies. These newly formed vessels are more permeable than normal vessels i.e. leaky vessels, and their lymphatic drainage is less developed than normal tissues. This causes particles injected into the bloodstream to accumulate at tumor sites .

II. Polymer-protein Conjugates: The proteins as peptides and antibodies due to elicitation of immune response to these proteins, 3 conditions must be met

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Breast cancer tissue shows Aurimune™ Drug deposition (black dots) after passive targeting .

Aurimune drug by Cytimmune, A First-in-class nanomedical passive drug targeting was developed in “ Cytimmune , where Colloidal Gold attached to PEG, TNF and Anticancer drug has been used . By customizing the drug molecule’s size to 27nm , allowing it to primarily exit the circulation through leaky, newly formed vasculature of tumor sites and not through normal blood vessels fenestrations, while TNF and PEG-Thiol allows the therapeutic payload travels safely through the blood stream avoiding immune detection and preferentially delivered to the site of disease.

As mentioned earlier, Silicon Core Nanoshells are able to absorb and scatter certain wavelengths of light depending on their size and core to shell diameter ratio. This property of Nanoshells is what allows them to be “ tuned “ to absorb specific wavelengths, in this case, between 700-900 nm, which falls in the Near Infra Red region (NIR) of the Electromagnetic spectrum. Optically tuned Nanoshells will absorb NIR lasers and they generate heat that will be immediately lethal to the cells to which they are attached. The process starts by binding Poly(ethylene glycol) or PEG molecules to the Nanoshells surface which acts as an “ Immunostealth “ molecule , or in other terms, suppresses the immune reaction to the Nanoshells preventing their removal by the immune system, therefore enhancing their biocompatibility. Nanoshells can be targeted to tumor cells by using both modalities of targeting, (active and passive). Active targeting is a more specific process; it depends on the antigenic structures of tumor cells which differs from normal antigenic structure of normal cells. The process requires identification of an antigen that is over expressed on tumor cell surface , which in this procedure is the Her-2 receptor.

ACTIVE TARGETING : Receptor-mediated cellspecific targeting involving receptor-specific targeting groups, example in NAPT

4.2 ) NANOSHELLS ASSISTED PHOTOTHERMAL THERAPY (NAPT) Nanoshells Assisted Photo-thermal Therapy (NAPT) is a newly proposed cancer therapy that promises better prognosis and is much more tolerable than current modalities and is much less invasive.

HER-2 (Human Epidermal growth factor Receptor 2), also known as ErbB-2, is a member of the Epidermal Growth Factor (EGF) receptor family. Although Her-2 has no known legends, i.e. Her-2 is an orphan receptor, it is known to form heterodimers with ErbB-1, ErbB-3, and ErbB-4 which plays an important role in normal growth signals. It is over expressed on ~30% of breast cancers and its presence was correlated to poor cancer prognosis. Anti-Her-2 antibodies can be conjugated to PEG molecules before these are attached to the Nanoshells, so when the Anti-Her-2-PEG complex is added to the Nanoshells the resulting particle will consist of Nanoshells conjugated with PEG immunostealth molecules and Her-2 targeting antibodies. These

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Nanoshells will accumulate on cells where Her-2 is over expressed . This is the process of Active targeting.

After the Nanoshells where attached to the cancer cells, by active or passive targeting pathways, the process of Photothermal Ablation can be initiated. Cells are then exposed to Near InfraRed laser for a period of time depending on the extent of the tumor, the Nanoshells will absorb and scatter light at its peak extinction wavelength, the scattered rays can be used for accurate 3-dimensional imaging of the tumor, while the absorbed rays will cause local heating at the Nanoshells, with consecutive marked heating of all cells attached them, and no significant heating of cells that are not attached to the Nanoshells, providing a very rapid and selective method of tumor ablation.

Experiments were performed in vitro using Her-2 positive SKBR 3 cancer cells mixed with normal Human Dermal Fibroblasts , the Nanoshells only attached to the Her-2 positive cells, and under NIR laser only these cells were destroyed leaving the Fibroblasts viable after laser ablation . Similar experiments were performed in vivo on mice with Her-2 positive breast cancers and the procedure came back with astounding results. All mice treated with Nanoshells showed complete cancer resorption within 10 days of exposure to NIR laser. The mice were further monitored for 90 days and no tumor regrowth was observed. Nanoshells Assisted Photothermal ablation could very well be the future cancer therapy in use providing excellent prognosis with a non-invasive procedure.

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5 DIAGNOSIS 5.1 IMAGING

Improving the field of Medical Imaging is one of the cardinal directions towards which Nanomedicine is aimed . Researchers are always in search of better imaging techniques that provide higher specificity , accuracy and faster disease diagnosis . Some new Nanomedical imaging techniques have been under development in recent years, employing special properties of Nanoparticles and tools to provide results with depth that has not been yet achieved .

DENDRIMER-BASED MRI CONTRAST AGENTS Historically, the first in vivo diagnostic imaging applications using dendrimer-based MRI contrast agents were demonstrated in the early 1990s by In contrast with the commercial small-molecule agent (Magnevist®, Schering, AG), the dendrimer-based reagents exhibited blood pool properties and extraordinary relaxivity values when chelated Gadolinium groups (Magnevist®) were attached to PAMAM dendrimer surfaces.

These generation-dependent, dramatic enhancements of MRI contrast properties were some of the first examples of a ‘Dendritic effect’. the work by Kobayashi and Brechbiel (7) extended this concept by demonstrating the efficacy of ‘size-mediated targeting’, using the discrete dendrimer generation sizes for in vivo imaging of primary tumors via the EPR effect 1. 2.

Designing ‘organ-specific diagnostic imaging modalities’ Defining size-dependent mammalian excretion routes (i.e. urinary compared with bile pathways).

Each of these ‘passive targeting’ modalities was based on appropriate use of dendrimer scaffolding dimensions for presenting the MRI imaging moieties

QUANTUM DOTS AND IMAGING As mentioned earlier , Quantum Dots are small Nanoparticles produced from semiconductor materials as cadmium sulfide, cadmium telluride or cadmium selenide . The fact that Quantum Dots have a size range that varies from a few to a few hundred nanometers enable them to fluoresce in different colours of spectrum depending on their size and composition. QDs can be used for the study of live cell singlemolecule dynamics, monitoring of Intracellular protein–protein interactions, disease detection in deeper tissues, detection of cancer/ tumor cells based on selective binding of bioconjugated QDs to known cancer biomarkers, and much more

The figure above shows green Qdots conjugated to streptavidin , the image shows F-actins of fixed fibroblasts.

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Functional cellular imaging around This shows human living cancer cells incubated with red fluorescent CdSe ZnS Qdots conjugated to TAT peptides, their intracellular aggregation pattern suggests their presence inside intracellular vesicles .

single molecules has been reported, taking advantage of the enhanced second harmonic signal by antibody conjugated gold nanospheres (Peleg et al 1999). Many other studies have been reported which employed photothermal interference contrast (Cognet et al 2003), AFM (Yang et al 2005), dark-field imaging (Loo et al 2005a; Dunn and Spudich 2007), reflectance imaging (Sokolov et al 2003; Nitin et al 2007b), as well as fluorescence and scanning electron microscopy (de la Fuente et al 2006; Shi et al 2007a). Even a two-photon luminescence imaging of cancer cells has been achieved using nanorods (Durr et al2007). It was noticed that the signal intensity of gold nanorod-labelled tumor cells was three times brighter than the two-photon autofluorescence emission from unlabeled cancer cells. Furthermore fluorescent dyes have been conjugated to gold nanoparticles for fluorescence imaging of cells, upon additional modification with certain targeting ligands (Nitin et al 2007a).

NANOPARTICLES AND CELL IMAGING: One of the most important advantages of nanoparicles is their use in cellular imaging. The versatile optical properties of gold nanoparticles have enabled optical imaging of cells with a wide variety of optical contrast

Photobleaching or blinking has always been a problem while using other flurophores for imaging which has been greatly reduced using gold nano particles for imaging (Yao et al 2005;Li et al 2007b) despite the fact that the optical intensity of gold nanoparticles may not be as strong as other fluorescent dyes or quantum dots. Gold nanorods have been reported for cell imaging using techniques such as dark field light SPR scattering (Oyelere et al 2007) and photoacoustic imaging (Li et al 2007a). Photoacoustic tomography (PAT) is a hybrid imaging modality that uses light to rapidly heat elements within the tissue, which results in photoacoustic waves (generated by thermoelastic expansion) that can be detected with an

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ultrasonic transducer. A significant enhancement of the imaging contrast was achieved using NIR-absorbing nanoparticles due to more substantial differences in optical absorption, Photoacoustic flow cytometry was also developed for real-time detection of circulating cells labeled with gold nanorods in the vasculature of mouse ear (Zharov et al 2006a). The threshold sensitivity was estimated to be one cancer cell in the background of 107 normal blood cells. However, the amount of gold nanorods per tumor cell was not reported. Cell imaging using gold nanoparticles serves as a proofof-principle for their potential applications in live animals or cancer patients. However In vivo targeted cancer imaging using nanoparticles has rarely been achieved (Sipkins et al 1998; Gao et al 2004; Cai et al 2006, 2007a), and even fewer exhibited tumor targeting efficacy that is sufficient for potential molecular imaging or molecular therapy applications in the clinical setting (Liu et al 2007b). Recently, in vivo imaging using gold nanoparticles as contrast agents has been reported.

In vivo imaging: Many paramagnetic nanoparticles have been used for magnetic resonance (MR) imaging, both preclinically and clinically (de Roos et al 1988; Thorek et al 2006). Recently,Au3Cu1 nanoshells were reported to be capable of enhancing the contrast of blood vessels in vivo, However, due to the low sensitivity of MR imaging, a dose-dependent toxic effect of the nanoshells was observed: 17% of the mice died at a dose of 20 mg/kg.

Gold nanosphere quenched probes were shown to enable visual monitoring of the activities of both proteases and protease inhibitors in vitro and in vivo (Lee et al 2008). This technique can also be applied to other proteases by using the appropriate peptide substrate as the spacer. Raman spectroscopy is the most promising imaging technique for gold nanoparticle-based contrast agents. The Raman spectra and Raman images of methylene blue molecules adsorbed as a single layer on gold

nanospheres were found useful for studying the plasmon properties (Laurent et al 2005). Later, antibody conjugated gold nanorods were reported to give a Raman spectrum that is greatly enhanced, sharpened, and polarized. But it should be taken in consideration that Raman imaging was only tested on cells but not living tissues. Recently, in vivo cell targeting was reported using Raman spectroscopy and SERS nanoparticles, the small molecule Raman reporters (such as fluorescent dyes) were stabilized by thiolated PEG and gave large optical enhancements (Qian et al 2008).When conjugated to tumor-targeting ligands, the conjugated SERS nanoparticles were able to target tumor markers such as epidermal growth factor receptor (EGFR) on human cancer cells. Other studies reported that SERS nanoparticles composed of a gold core and coated with silica was used for Raman imaging in vivo. Multiplexed in vivo Raman imaging using SERS nanoparticles. Copyright © 2008, PNAS. Adapted with permission from Keren S, Zavaleta C, Cheng Z, et al. 2008.Noninvasive molecular imaging of small living subjects using Raman spectroscopy. Proc Natl Acad Sci U S A, 105:5844–9

Raman imaging holds significant potential as a strategy for biomedical imaging of living subjects. However, one has to keep in mind that optical imaging in mice cannot be directly scaled up to in vivo imaging in human applications due to the limited tissue penetration of optical signal. NIR optical imaging devices for detecting and diagnosing breast cancer have been tested in patients and the initial results are encouraging (Taroni et al 2004; Intes 2005). Multiple SERS nanoparticles with different absorption wavelengths in the NIR region, which can allow for multiplexed imaging of many tumor markers simultaneously if efficient targeting can be achieved, may have signifi cant potential clinical applications.

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5.2 LABORATORY DIAGNOSIS

Cantilevers where produced from semiconductor materials using advanced nanolithographic techniques to form a multi-beam device , antibodies attached to its surface are selected to bind to specific proteins or molecules producing a change in surface tension and other forces affecting the cantilever’s shape, manifested by a deflection in one of the beams . As the cantilever’s shape changes, its physical properties, eg Conduction, changes as well, such a change can be detected by laboratory technicians, providing a very rapid laboratory diagnosis tool for fast screening of diseases as cancers and viral infections by detection of even the smallest amounts of target molecules or proteins produced by tumor cells or viruses .

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6) LIMITATIONS

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Nanometals:

Nanotoxicity It’s the study of pathological and toxicological effects that are unique to the size of the nanomaterials ,which are not observed on bulk materials, their toxicity is a Multi disciplinary area concerning effects related to their small size, larger surface area , nature, solubility, agglutination, higher chemical and biological activity . The large number of variables influencing toxicity implies that it is difficult to generalize health risks associated with exposure to nanomaterials . each new material must be assessed individually and all material properties must be taken into account . I. II. III. IV.

V.

Nanosize: as it has been proved that some metaloxides as(zinc oxide) produce different effects according to their size. Shape: carbon Nanotubes which resemble very thin pins can penetrate lungs sensitive areas if inhaled Extremely large specific surface area Chemistry: according to their nature, they produce different effects as (parallel sizes of cAg(gold)and cAu(silver) induce significantly different toxic profiles, with the former being toxic and the latter being inert in all exposed sizes). Production of Reactive Oxygen Species

Another threat is owed to their small size, they enter the body ingested ,inhaled, or through skin and they are able to cross membranes and pass to unreachable areas by larger particles unnoticed by immune cells , also their ability to move freely from their site of injection to distant sites reaching blood, lung and brain producing a multisystem effect that is not limited to a certain organ toxicity. Also due to their large surface area they can absorb to macromolecules (proteins) forming a nanoparticlesprotein complex which is freely mobile with higher accessibility and enhanced degradation thus altering their normal function. Studies are being managed to track their behavior in human body to detect the exact health hazards.

Toxicity of Nanoparticles:

Metal

Human lung epithelial cells:

Zebra fish

Copper oxide

highest toxic , Clear risk

toxic effects on fish larvae and embryo

Iron oxide

little DNA damage and were non-toxic

-

Zinc oxide

more damage than that of Iron-oxide.

-

Titanium dioxide

little DNA damage.

No toxic effect at all

Nanoshells : Regarding gold nothing has yet been established on health hazards caused by its nano particles used in Nanoshells’ manufacturing, and a study on zebra fish treated with silver and gold resulted that gold was completely inert with no pathognomonic effects at all size and levels on the contrary to silver. Carbon Nanotubes: They are shown to produce a tissue reaction similar to asbestos in lung tissue which puts them in line with asbestos’ carcinogenic effects in ( Mesothelioma ) also their shaping as very thin pins penetrating the lining endothelium of the lung produces pro-inflammatory effects. Carbon Nanotubes cause DNA damage at low levels on human lung endothelial cells, their inhalation chronically poses a threat for those working in its manufacture so general restrictions on the manufacturing and preventive measurements should be taken strictly concerning the worker and their handlers. *a study on mice stated that aspiration of single-walled carbon Nanotubes (SWNT) elicited an unusual inflammatory response in the lungs of exposed mice with a very early switch from the acute inflammatory phase to fibrogenic events resulting in pulmonary deposition of collagen and elastin. This was accompanied by a characteristic change in the production and release of pro-inflammatory to antiinflammatory profibrogenic cytokines, decline in pulmonary function, and enhanced susceptibility to infection. Fullerenes: They are suspected to cause toxicity at low levels but it’s not been established yet, although it’s thought that their toxic effect results from tetrahydrofuran used in

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preparing the 30 nm–100 nm particles of C60 , as so removing The TetraHydroFuran from the C60 particle results in a marked decrease of its toxicity.

Frank Chen, a biologist at the Lawrence Berkeley National Labs in California, US, believes that quantum effects play an important role in the interaction between nanomaterials and the molecular machinery within cells. And now he has built a device for testing these effects on gene expression. The device works by measuring how various biological pathways associated with inflammation and cell death are triggered when biological cells are exposed to nanomaterials. In tests, Chen says the method works well and provides a reliable way to measure the toxicity of nanomaterials that would otherwise go unnoticed, and to work out how the material may produce damage.

Skin is unique because it is a potential route for exposure to nanoparticles and also provides an environment within the avascular epidermis where particles could potentially lodge and not be susceptible to removal by phagocytosis. The skin is a primary route of potential exposure to toxicants, including novel nanoparticles.

Effects on Various Organs neurological: Nanoparticles may take two possible routes to the Nervous System, an olfactory pathway by inhalation, and across the blood brain barrier which is no match for their nanoscale size .Acute toxicity may be unlikely,except in extreme circumstances, but chronic toxicity might occur. Some evidence comes from studies on mice with a deletion of the gene for apolipoprotein (Apo) E; this gene is often mutated in Alzheimer’s disease,and these mice have been shown to manifest increased levels of oxidative stress which is critical in nervous tissue, causing cellular damage, apoptosis or necrosis. Pulmonary: Inhalation of engineered nanoparticles may have adverse pulmonary effects arises from the fact that, nanoparticles exhibit a high deposition in the conducting and respiratory zones of the lung The recognition of nanoparticles by alveolar macrophages is dependent on particle type, i.e., quantum dots and nanogold particles are rapidly phagocytized by alveolar macrophages while Single walled carbon nanotubes are not. Once deposited, some nanoparticles may escape clearance by alveolar macrophages and enter the alveolar interstitium.There is some evidence that nanoparticles can migrate from the alveolar interstitium to the pulmonary capillary blood and translocate to systemic organs. Dermatological:

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Archut, A., Azzellini, G.C., Balzani, V., Cola, L.D. & Vögtle, F. (1998) Toward photoswitchable dendritic hosts. Interaction between azobenzene-functionalized dendrimers and eosin. J. Am. Chem. Soc.) Dendrimers, Tecto-Dendrimers, 3-D Dendrimers,” Center for Biologic Nanotechnology, University of Michigan (2003) Nanoparticulates as drug carriers By V. P. Torchilin” L.F .T ietze and T.Feuerstein, Aust. J. Chem. 56, 841 (2003). Dendrimers as multi-purpose nanodevices for oncology drug delivery and diagnostic imaging D.A. Tomalia*†1, L.A. Reyna* and S. Svenson* Kobayashi, H. and Brechbiel, M.W. (2003) Mol. Imaging 2, 1–10 Current Status of Nanomedicine and Medical Nanorobotics Robert A. Freitas, Jr. Institute for Molecular Manufacturing, Palo Alto, California, USA Robert Berry DENDRITIC NANOTECHNOLOGIES, INC. Mt. Pleasant, MI 48858 D.A. Tomalia*†1, L.A. Reyna* and S. Svenson* *Dendritic Nanotechnologies Inc., 2625 Denison Drive, Mt. Pleasant, MI 48858, U.S.A., and †Department of Chemistry, Central Michigan University,Mt. Pleasant, MI 48859, U.S.A. Dendrimer Chemistry. Fritz Vgtle, Gabriele Richardt and Nicole Werner Copyright © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32066-0 Dendrimers: properties and applications Barbara Klajnert_ and Maria Bryszewska Department of General Biophysics, University of £ódŸ, £ódŸ, Poland Received: 13 December, 2000; revised: 9 February, 2001; accepted: 8 March, 2001 “Dynamics of Bengal Rose Encapsulated in the Meijer Dendrimer Box”J. Am. Chem. Soc. 1997, 119, 7458-7462 DENDRIMER-BASED NANOMEDICINE ISBN-13 978981 4241-04-5 Nano the essentials-understanding nanotechnology 0-07154829-7. Nanoparticulate Drug Delivery SystemsI SB N‑10: 0‑8493‑9073‑7 The Dendritic Box: Shape-Selective Liberation of Encapsulated Guests J. Am. Chem. SOC. 1995,117, 44174418 Nanoshell-Assisted Cancer Therapy: Targeted Photothermal Tumor Ablation By Amanda Raley Lowery Rice university UMI Number: 3256716 NANOMEDICINE DESIGN AND APPLICATIONS Of MAGNETIC NANOMATERIALS, NANOSENSORS AND NANOSYSTEMS ISBN 978-0-470-03351-7 http://www.evidenttech.com http://www.cytimmune.com Self-Assembled Nanomaterials II ISBN 978-3-540-85104-2 Nanophotothermolysis of cancer cells Google® Wikipedia®

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