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RESEALED ERYTHROCYTES

BY- DEEPAK S. DERE

INTRODUCTION  Amongst

various carriers explored for target oriented drug delivery, vesicular, microparticulate & cellular carriers meet several criteria rendering them useful in clinical applications.  Erythrocytes have been the most extensively investigated and found to posses great potential in novel drug delivery .

 ERYTHROCYTES

ARE LOADED WITH DRUG/ENZYMES & PROVIDE TARGET DRUG DILIVARY SYSTEM  ERYTHROCYTE GHOSTS: RBC WITHOUT HAEMOGLOBIN

DRUG CARRYING POTENTIAL OF ERYTHROCYTES  The

developing RBC has capacity to synthesize haemoglobin, however, adult RBCs do not have this capacity and serve as carriers for haemoglobin.  The carrier potentials of these cells was first realized in early 1970.  Drug which are normally unable to penetrate the membrane, should be made to transverse the membrane without causing any irreversible changes in the membrane structure and permeability.

 Cells

must be able to release the entrapped drug in a controlled manner upon reaching the desired target.  The processing of drug entrapment requires a reversible and transient permeability change in the membrane, which can be achieved by various physical and chemical means.

 The

desirable properties, which substantiate the suitability of red blood cells(erythrocytes)as drug carriers are –  1.Biodegradability  2.Circulate throughout the circulatory system.  3.Large quantities of material can be encapsulated within them.  4.Can be utilized for organ targeting within RES.  5.A wide variety of bioactive agents can be encapsulated within them.  6.Erythrocytes are biocompatible cells are used in patients there is no possibility of triggered immunological response.

BASIC FEATURES OF ERYTHROCYTES  Composition

of Erythrocytes Normal blood cells have extensile, elastic, Biconcave and non-nucleated configuration with a diameter ranging from 6-9 u with a mean diameter of 7.5 u. the thickness is nearly 1u in the center with an increase thickness near the periphery. They have a life span of about 100-120 days Erythrocytes have solid content of about 35% most of which is haemoglobin which remains tightly bound to the stroma of the cell membrane.

Electrolyte Composition of Erythrocytes  The

concentration of K+ and Na+ differ in that the former is more in erythrocytes and the later in plasma.  The osmotic pressure of the interior of the erythrocytes is equal to that of plasma and termed as isotonic(normally equivalent to the osmotic pressure of 0.9% Nacl, commonly known as normal or physiological saline).

Haematocrit value and erythrocyte Sedimentation Rate  The

blood volume in normal individuals is about 7%(for male) and -6.5%(female) of body weight respectively  The haematocrit is the percent volume occupied by the cells and is determined by simple centrifugation of the blood.  The cells constitute about 45%(for male”) and 41%(for female) of the total volume. This is referred to as normal haematocrit or in modern terminology as volume of packed red cells(VPRC).

Source , fractionation and isolation of erythrocytes  Different

mammalian erythrocytes have been exploited for drug loading,resealing and subsequent use in drug and enzyme delivery  To isolate erythrocytes, blood is collected in to heparinized tubes by venipuncture. EDTA or heparin can be used as an anticoagulant. Whole blood from horse,sheep goat,dog,rabbit is easily collected through venipuncture .  Fresh whole blood in this sense is defined as any blood collected and immediately chilled to 4 degC. and stored for not more than 2 days

VARIOUS CONDITIONS AND CENTRIFUGAL FORCE USED FOR THE ISOLATION OF ERYTHROCYTES. SPECIES

WASHING BUFFER

CENTRIFUGAL FORCE (g)

MOUSE

10 mmol KH2PO4/Na2HPO4, pH7.0; 5 mmol adenosine; 5mmol MgCl2;10mmol glucose

100-500

HUMAN

154 mmol NaCl or 10 mmol KH2PO4,pH7.0; 2mmol MgCl2; 10 mmol glucose 10 mmol KH2PO4 /Na2HPO4, pH7.0 15 mmol KH2PO4 /Na2HPO4, pH 7.0; 5 mmol MgCl2; 10mmol glucose 10-15 mmol KH2PO4/ Na2HPO4, pH7.0; 2 mmol MgCl2; 10 mmol glucose 10 mmol KH2PO4/Na2HPO4, pH7.0

<500

10 mmol KH2PO4/Na2HPO4, pH7.0 2 mmol MgCl2 ; 10mmol glucose 10 mmol KH2PO4/Na2HPO4, pH7.0

1000

10 mmol KH2PO4/Na2HPO4, pH7.0; 5 mmol MgCl2

500-1000

RABBIT DOG

COW

GOAT HORSE

PIG SHEEP

500-1000 500-1000

1000

500-1000

500-1000

M E T H O D S O F D R U G L O A D IN G D R U G L O A D IN G IN R E S E A L E D E R Y T H R O C Y T E S M E M B R A N E P E R T U R B A T IO N E L E C T R O E N C A P S U L A T IO N D IL U T IO N M E T H O D

H Y P O -O S M O T IC L Y S IS

L IP ID F U S IO N E N D O C Y T O S IS

D IA L Y S IS M E T H O D P R E S W E L L M E T H O D O S M O T IC M E T H O D

1. Hypotonic Haemolysis and Isotonic Resealing Methods  This

method is based upon hypotonic lysis of cells in solution containing the drug/enzyme to be entrapped followed by restoration of tonicity to reseal them.

 Three

types of ghosts can be distinguished: type1 ghosts which reseal immediately after haemolysis; type 2 ghosts which reseal after, reversal of haemolysis by addition of alkali ions; and type 3 ghosts which remain leaky under different experimental conditions.  Erythrocytes have an exceptional capability for reversible shape changes with or without accompanying volume changes and for reversible deformation under distension (stress)

 The

cells become spheres as they accommodate additional volume with a fixed surface area. However, these swollen erythrocytes have little capacity to resist volume greater than 50-75% of the initial volume and when placed in solutions less than about 150mOsm/kg (corresponding to about 0.4% w/v NaCl), the membrane ruptures, permitting escape of the Cellular components  These ruptured membranes can be resealed by raising the salt concentration to its original (isotonic) levels and upon incubation, the resealed erythrocytes resume their normal biconcave shape and recover their normal impermeability to both macromolecules and ions.

 (i)Dilutional

Haemolysis Population of erythrocytes when exposed to hypotonic saline solution (0.4% NaCl), swells until it reaches a critical value of volume or pressure where membrane ruptures and becomes permeable to macromolecules and ions, therefore permitting the escape of cellular components .

 (ii)

Preswell Dilutional Haemolysis

The technique is based upon initial controlled swelling of erythrocytes without lysis by placing them in slightly hypotonic solution followed by centrifugation at low 'g' to take them up to point of lysis . Finally, the addition of small volume of drug solution to attain drug loaded resealed erythrocytes.

 (iii)

Isotonic Osmotic Lysis Haemolysis in isotonic solutions can be achieved both by chemical and physical means. If erythrocytes are incubated in solutions of a substance with high transerythrocytic membrane permeability (i.e., small reflection coefficient as defined by the thermodynamics of irreversible processes) the solute will diffuse into the cells due to inwardly directed chemical potential gradient. This will be followed by water uptake until osmotic equilibrium is restored.

Several methods for dialysis based loading of erythrocytes are reported but all take advantage of the common principle that the semipermeable dialysis membrane maximizes the intracellular:extracellular -volume ratio for macromolecules during lysis and resealing, but also allows for free flow of small ions, responsible for lysis and resealing of the erythrocytes.

 (iv)Dialysis:

COMPARISION OF VARIOUS HYPOOSMOTIC LYSIS METHOD: METHOD

Dilution method

Dialysis

Preswell dilution

Isotonic osmotic lysis

%LOADING

ADVANTAGES

DISADVANTAGES

1-8%

Fastest & simplest especially for low molecular weight drugs

Entrapment efficiency is very less (1-8%)

30-45%

Better in vitro survival of membrane due to lesser ionic load

Time consuming; heterogeneous size distribution of resealed erythrocytes

20-70%

Good retention of cytoplasm constituents & good survival in vivo.

-

-

Better in vivo surveillance

Impermeable only to large molecules , process is time consuming

2.Electro-insertion or Electroencapsulation  The erythrocyte membrane could be opened by dielectric breakdown and subsequently the pores can be resealed by incubation at 37 deg.C in osmotically balanced medium.. The method is based on creating electrically induced permeability changes at high membrane potential differences.  The components can be entrapped when electric pulse of greater than a threshold voltage of 2kV /cm is applied for 20 u sec

 Loading

by Electric Cell Fusion In this method, the molecules are first loaded into erythrocyte ghosts. These ghosts are then caused to adhere to target cells. Electric pulses are applied to induce fusion of ghost with subsequent release of the encapsulated molecule.

3.Loading by Chemical Perturbation of Membrane This method is based upon the observation that the permeability of the erythrocytic membrane is increased, when it is exposed to some chemical agents. Amphotericin B, a polyene antifungal antibiotic, damages microorganism by increasing permeability of their membranes to metabolites and ions.

4.Loading by Lipid Fusion  Lipid

vesicles containing drug can be directly fused with human erythrocytes leading to exchange of lipid entrapped drug  Nicolau and Gresonele, 1979 used this technique for loading of inositol hexaphosphate into erythrocytes for the increased oxygen carrying capacity.

STEPS IN RESEALING

IN VITRO CHARACTERIZATION  



Resealed erythrocytes after loading are characterized for following parameters Drug Content Packed loaded erythrocytes (0.5 ml) are first deproteinized with acetonitrile (2.0 ml) and subjected to centrifugation at 2500 rpm for 10 min. The clear supernatant is analyzed for the drug content. In vitro Drug and Haemoglobin Release Normal and loaded erythrocytes are incubated at 37+/- 2°C in phosphate buffer saline (pH 7.4) at 50% haematocrit in a metabolic rotating wheel incubator bath. Periodically, the samples are withdrawn with the help of a hypodermic syringe fitted with a 0.8 u Spectropore membrane filter.

 Percent

haemoglobin can similarly be calculated at various time intervals at 540 run spectrophotometrically.  Laser light scattering may also be used to evaluate haemoglobin content of individual resealed erythrocytes.

Mean corpuscular [Haemoglobin (g/100ml) X 10 Haemoglobin = Erythrocyte count(per mm3)

 Osmotic

Fragility When red blood cells are exposed to solutions of varying tonicities their shape changes (swell in hypotonic and shrink in hypertonic environments) due to osmotic imbalance.  Osmotic Shock Osmotic shock describes a sudden (and not tapering) exposure of drug loaded erythrocytes to an environment, which is far from isotonic to evaluate the ability of resealed erythrocytes to withstand the stress and maintain their integrity as well as appearance.





Turbulence Shock The parameter indicates the effects of shear force and pressure by which resealed erythrocytes formulations are injected, on the integrity of the loaded cells Loaded erythrocytes (10% haematocrit, 5 ml) are passed through a 23-gauge hypodermic needle at a flow rate of 10 ml/min . After every pass, aliquote of the suspension is withdrawn and centrifuged at 300 G for 15 min, and haemoglobin content, leached out are estimated spectrophotometrically. Morphology and Percent Cellular Recovery Phase-contrast optical microscopy, transmission electron microscopy and scanning electron microscopy are the microscopic methods used to evaluate the shape, size and the surface features of the loaded erythrocytes.

DIFFERENT FORMS OF DRUG LOADED RED BLOOD CELLS 



Normally, more than 80% of the erythrocyte ghosts loaded with drugs or enzymes appear as biconcave disks (discocytes) when they are observed under electron microscope. Less than 20% cells show abnormal morphology. The rest appear as stomatocytes or spherocytes On swelling, the cells get converted from diskocytes to spherocytes (sphere shaped structures) and thus get compromised with a lower ratio of surface features (surface area to surface volume). Further increase in hypotonicity may lead to the formation of echinocytes and cells with different infoldings and other damaged forms

SHELF AND STORAGE STABILITY OF RESEALED ERYTHROCYTE  Storage

of resealed erythrocytes places a major challenge in their practical utility as drug delivery system.  encapsulated product and carrier both exhibit satisfactory self-stability when stored in Hank's Balanced Salt Solution (HBSS) at 4°C for 2 weeks, Similar results were obtained by suspending cells in oxygenated HBSS containing 1 % soft bloom gelatin.

IN VIVO SURVIVAL AND IMMUNOLOGICAL CONSEQUENCES  There

are three general modes of efflux of loaded contents from resealed erythrocytes: Phagocytosis, Diffusion or specific transport mechanisms once placed in vivo.  Phagocytosis occurs within the reticuloendothelial system.  During loading process, some antigenic impurities may get entrapped resulting in immunological manifestations.

INFLUENCE OF MEMBRANOLYTIC AND MEMBRANOTROPIC SUBSTRATES  Substances

which are either utilized in cases of drug loading or chemical cross--linking to render a specific surface property to the resealed erythrocytes can be categorized as follows : • Membrane active substrates • Membrane lytic substrates  On the other hand, some drugs like Daunomycin, Amphotericin B and Primaquine affect the erythrocytes in different way altogether. Amphotericin B, a polyene antifungal antibiotic, damages microorganism by increasing permeability of their membranes to metabolites and ions.

VARIOUS APPLICATIONS OF RESEALED ERYTHROCYTES APPLICATION

DRUG/ENZYME/ MACROMOLECULES

Enzyme deficiency,& Enzyme replacement Therapy

B-galactosidase,B-fructofuronodase, Urease ,Glucose-6-phosphate dehydrogenase,corticol-2-phosphate

Thrombolytic activity

Brinase,Aspirin,Heparin

Iron overload chemotherapy

Desferroxamine Rubomycin,Methotrexate, L-asparginase,Doxorubicin, Daunomycin,Cytosine,Arabinoside Human recombinant interleukin-2

Immuno therapy Circulating carriers

Albumin,Prednisolone, Salbutamol, Tyrosine kinase,Phosphotriesterase.

Circulating Bioreacters

Arginase,Uricase,Luciferase, Acetaldehyde dehydrogenase.

Targeting to RES

Pentamidine,Mycotoxin,Imidocarb Dipropionate,Homidium bromide.

Targeting to other than RES

Daunomycin,Methotrexate, Diclofenac sodium.

DRUG TARGETING 

  

A drug delivery should ideally be site-specific and target oriented in order to exhibit maximal therapeutic index and minimum side and toxic effects. Drug Targeting to RES Organs Drug Targeting to Liver TARGETING TO SITES OTHER THAN RES-RICH ORGAN

 Drug

Targeting to RES Organs  The damaged erythrocytes are quickly removed from circulation by phagocytic Kupffer cells located in liver and spleen.  Surface Modification with Antibodies  Surface Modification with Glutaraldehyde  Surface Modification-involving Carbohydrates  Surface Modification with Sulphydrysis

 Drug

Targeting to Liver

 Enzyme

Deficiency/Replacement Therapy  Treatment of LiverTumours  Treatment of Parasitic diseases  Removal of Toxic Agents

 TARGETING

TO SITES OTHER THAN RESRICH ORGAN Resealed erythrocytes have the ability to deliver a drug or enzyme to the macrophage-rich organs, this is unfortunately the premium limitation of this delivery system.  Magnet-responsive Erythrocyte Ghosts Encapsulation of small paramagnetic particles into erythrocytes might allow their localization to a particular location under the influence of external magnetic field. Recently, the loading of ferrofluids (colloidal suspension of magnetite ,Fe3O4) in erythrocytes has been reported in a study

 Delivery  Delivery

of Antiviral Agents

of Azidothymidine Derivative  Delivery of Deoxycytidine Derivatives

 Macrophage

Activation  Thrombolytic Therapy  Oxygen Deficiency Therapy

Resealed Erythrocytes in Cell Biological Applications 

Microinjection of Macromolecules into Cultured cells using erythrocyte ghosts This method has certain advantages and disadvantages as listed below:

 Advantages ·    

It can be employed for quantitative injection of material into

cells. · It does not require any special apparatus and/or technique. · Simultaneous introduction of materials into a large number of cells is possible. · It permits introduction of materials into cells in suspension culture. · The damage to the cells is minimal.

Disadvantages · Co-introduction of the erythrocyte membranes, viral envelopes, viral RNA and residual haemoglobin may have unpredicted effects on the cells. · A comparatively larger amount of test material is desired than that for the microcapillary method · Direct injection into the cell nucleus is not feasible. 

IN VIVO ACCELERATED ACETALDEHYDE METABOLISM USING ACETALDEHYDE DEHYDROGENASE-LOADED ERYTHROCYTES 

AcDH-overloaded mouse red blood cells from donor animals were also injected intraperitoneally into compatible recipients and 80 to 85% of these were found to enter into circulation within 24 hr and to circulate with a half-life of 6–7.3 days (normal half-life 11 days). AcDH-overloaded erythrocytes can perform in vitro and in vivo as bioreactors improving alcohol and acetaldehyde metabolism, and suggest that administration of these cells to alcoholic patients could be of value in restoring to normal, or improving, alcohol and acetaldehyde metabolism.

“Golden Eggs in novel drug delivery systems”  Numerous

applications have been proposed for the use of resealed erythrocytes as carrier for drugs, enzyme replacement therapy etc.  The commercial medical applications of carrier erythrocytes are currently being tested in Europe by a recently formed company that is developing products for human use.  The International Society For the Use of Resealed erythrocytes(ISURE),through its biannual meetings provides an excellent forum for exchange of information to the scientists in this exiting & rewarding field of research.

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