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RESEALED ERYTHROCYTES

SEMINAR BY :: R. Venkatesh 10DG1SO314

TEEGALA KRISHNA REDDY COLLEGE OF PHARMACY

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CONTENTS• INTRODUCTION.• BASIC FEATURES OF ERYTHROCYTES.• DRUG CARRYING POTENTIAL OF ERYTHROCYTES.• ADVANTAGES AND LIMITATIONS OF RESEALED ERYTHROCYTES AS DRUG CARRIERS.• DESIRABLE PROPERTIES OF DRUGS BEING INCORPORATED IN THE RED BLOOD CELLS.• ROUTES OF ADMINISTRATION.• SOURCE OF ERYTHROCYTES.• FRACTIONATION AND ISOLATION OF ERYTHROCYTES.• METHODS OF DRUG LOADING.• RELEASE CHARACTERISTICS OF LOADED DRUG.• INVITRO CHARACTERISATION.• IN VIVO CHARACTERISATION .• SHELF AND STORAGE OF RESEALED ERYTHROCYTES.• APPLICATIONS.• CONCLUSION.

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BASIC FEATURES OF ERYTHROCYTES DISCOVERED IN 1658. NORMAL BLOOD CELLS HAVE

EXTENSILE ,ELASTIC BICONCAVE , AND NON NUCLEATED CONFIGURATION.

DIAMETER = 6 - 9 microns. MEAN DIAMETER = 7.5 microns. THICKNESS IN THE CENTRE =

1 microns. THICKNESS AT THE PERIPHERY = 2

microns.

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COMPOSITION

THE BLOOD CONTAINS 55% OF FLUID PORTION (PLASMA) AND NEARLY 45% OF CORPUSCLES OR FORMED ELEMENTS.

THE FLUID PORTION CONTAINS A LARGE NUMBER OF ORGANIC AND INORGANIC SUBSTANCES IN SOLUTION, WHICH MAY BE DIFFUSIBLE ( ELECTROLYTES, ANABOLIC AND CATABOLIC SUBSTANCES FORMED DURING METABOLISM) AND NON-DIFFUSIBLE (PROTEINS).

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THE FORMED ELEMENT OR CELLULAR PORTION OF THE BLOOD CONSISTS OF ERYTHROCYTES ( RED BLOOD CELLS ) , LEUKOCYTES ( WHITE BLOOD CELLS ) AND THROMBOCYTES (PLATELETS).

THE PLASMA CONSTITUENTS HELP IN MAINTAINIG THE ISOTONICITY AND MORPHOLOGY OF THE RED BLOOD CELLS.

THE PRIME FUNCTION OF THESE R.B.Cs IS TO TRANSPORT GASES FOR RESPIRATORY PROCESSES.

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HEALTHY ADULT MALE = 5.0 millions / cmm.

HEALTHY ADULT FEMALE = 4.5 millions / cmm.

LIFE SPAN ABOUT 100-120 DAYS

DEVELOPING R.B.C HAS THE CAPACITY TO SYNTHESIZE HAEMOGLOBIN.

ADULT R.B.Cs DO NOT HAVE THIS CAPACITY AND SERVE AS CARRIERS FOR HAEMOGLOBIN.

NON NUCLEATED DO NOT HAVE THE MACHINERY TO SYNTHESIZE NEW CARBOHYDRATES, PROTEINS, AND LIPIDS

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DEFINITION

DRUG LOADING IN BODY’S OWN ERYTHROCYTES WHEN USED TO SERVE AS CONTROLLED DRUG DELIVERY SYSTEMS.

HAEMATOCRIT VALUE : % VOLUME OCCUPIED BY CELLS. THIS IS DONE BY CENTRIFUGATION IN THE PRESENCE OF ANTICOAGULANTS. THE BLOOD VOLUME IN NORMAL INDIVIDUALS IS ABOUT 7% (for male) AND

6.5% (for females) OF BODY WEIGHT RESPECTIVELY. NORMALLY 45% (males) AND 41% (females) OF THE TOTAL VOLUME.(also called

as VOLUME OF PACKED RED CELLS)

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ELECTROLYTE COMPOSITION OF ERYTHROCYTES QUALITATIVELY SIMILAR TO PLASMA HOWEVER, QUANTITATIVELY IT DIFFERS. K+ MORE IN ERYTHROCYTES. OSMOTIC PRESSURE IN R.B.C IS EQUAL TO THAT OF PLASMA. CHANGE IN OSMOTIC PRESSURE MAY LEAD TO CHANGE IN SIZE , SHAPE AND TONICITY

OF THE CELLS. HYPOTONICITY = R.B.Cs SWELLS AND BURST. HYPERTONICITY = R.B.Cs SHRINK BY MAKING THE MEDIUM ISOTONIC , THEY REGAIN THEIR SHAPE AND MORPHOLOGY.

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DRUG CARRYING POTENTIAL OF ERYTHROCYTES THE CARRIER POTENTIAL OF RBC’s WAS FIRST REALIZED IN EARLY 1970. THE USE OF CELLS AS DRUG DELIVERY SYSTEMS REQUIRES THE DRUG

(ENZYMES) WHICH ARE NORMALLY UNABLE TO PERMEATE THE MEMBRANE, SHOULD BE MADE TO TRNSVERSE THE MEMBRANE, WITHOUT CAUSING ANY IRREVERSIBLE CHANGES IN THE MEMBRANE STRUCTURE AND PERMEABILITY.

THE CELLS MUST BE ABLE TO RELEASE THE ENTRAPPED DRUG IN A CONTROLLED MANNER UPON REACHING THE DESIRED TARGET.

ONCE LOADED, THESE CELLS CAN BE USED EITHER FOR CARRYING THE DRUG TO DESIRED SITE OR THEY THEMSELVES MAY FUNCTION AS CIRCULATING BIOREACTOR.

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ERYTHROCYTES HAVE BEEN PROPOSED AND RECENTLY UTILISED AS CARRIERS FOR A WIDE RANGE OF BIO-ACTIVE COMPPONENTS INCLUDIND DRUGS, ENZYMES, PESTICIDES AND OTHERS.

THEIR CAPABILITY FOR PREVENTION OF PREMATURE DEGRADATION OR INACTIVATION, SLOW RELEASE PROFILE OF LOADED DRUGS, TARGETING POTENTIAL TO RETICULO-ENDOTHELIAL SYSTEM (RES) AND SERVING AS CIRCULATORY BIO-VECTORS FOR ENZYMES MAKE THEM VERSATILE CARRIERS IN PHARMACEUTICAL RESEARCH AND DEVELOPMENT.

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DESIRABLE FEATURES

BIODEGRADABILITY.

CIRCULATE THROUGHOUT THE CIRCULATING SYSTEM.

LARGE QUANTITIES OF MATERIAL CAN BE ENCAPSULATED WITHIN SMALL VOLUME OF CELLS.

CAN BE UTILIZED FOR ORGAN TARGETING WITHIN RES.

A WIDE VARIETY OF BIOACTIVE AGENTS CAN BE ENCAPSULATED WITHIN THEM.

ERYTHROCYTES ARE BIOCOMPATIBLE PROVIDED THAT COMPATIBLE CELLS ARE USED IN PATIENTS THERE IS NO POSSIBILITY OF TRIGGERED IMMUNOLOGICAL RESPONSE.

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ADVANTAGES OF RESEALED ERYHTROCYTES

• THEIR BIOCOMPATIBILITY.

• THEIR BIODEGRADABILITY WITH NO GENERATION OF TOXIC PRODUCTS.

• CONSIDERABLY UNIFORM SIZE AND SHAPE OF THE CARRIER

• DEGRADATION OF LOADED DRUG AND ITS INACTIVATION IS PREVENTED BY ENDOGENOUS CHEMICALS.

• WIDE VARIETY OF CHEMICALS CAN BE ENTRAPPED.

• PREVENTION OF UNDESIRED IMMUNE RESPONSE AGAINST THE LOADED DRUG.

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LIMITATIONS OF RESEALED ERYTHROCYTES

POSSIBILITY OF DRUG LEAKAGE FROM CELLS DUE TO RUPTURED MEMBRANES.

DRUG MOLECULES MAY ALTER THE PHYSIOLOGY OF THE RED BLOOD CELLS.

DOSE DUMPING MAY OCCUR.

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DESIRABLE PROPERTIES OF DRUGS BEING INCORPORATED IN THE RED BLOOD CELLS

SHOULD BE HIGHLY WATER SOLUBLE.

SHOULD BE RESISTANT AGAINST DEGRADATION WITHIN THE ERYTHROCYTES.

SHOULD BE NON-TOXIC TO THE ERYTHROCYTIC MEMBRANE.

SHOULD EXIHIBIT WELL DEFINED PHARMACO KINETIC AND PHARMACODYNAMIC PROPERTIES.

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ROUTES OF ADMINISTRATION

INTRAVENOUS – MOST COMMON ROUTE.

SUBCUTANEOUS ROUTE.

INTRAPERITONIAL ROUTE.

INTRA NASAL ROUTE.

ORAL ROUTE.

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SOURCES OF ERYTHROCYTES SOURCES INCLUDE MICE, RATS, CATTLE, PIGS, DOGS, SHEEP, GOATS, MONKEYS,

CHICKEN, RABBITS, HORSE, COWS AND HUMANS. BLOOD IS COLLECTED IN HEPARINIZED TUBES THROUGH VENIPUNCTURE EXCEPT

IN MICE (ORBITAL SINUS OR HEART). IN MICE EITHER THE ORBITAL SINUS OR HEART IS USED AS THE COLLECTION

SITE. FRESH BLOOD SHOWS MORE ACTIVITY THAN AGED BLOOD. FRESH BLOOD IN SENSE IS DEFINED AS ANY BLOOD COLLECTED AND

IMMEDIATELY CHILLED TO 40 C AND STORED FOR NOT MORE THAN 2 DAYS.

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FRACTIONATION AND ISOLATION OF ERYTHROCYTES

PREPARATION OF WASHING BUFFER OR BUFFER SOLUTION A 10-15 mmol phosphate buffer having a pH of 7.0-7.4 containing 144 mmol NaCl, 5

mmol MgCl2 and 10mM glucose is adequate for most species.

PROCEDURE After recovering the blood from venipuncture or heart and admixing with heparin, it is

centrifuged at 2000 g for 5min at 4 +/- 1 0C.

This helps in separation of plasma and buffy coat . Packed erythrocytes so obtained are washed with buffer solution .The erythrocytes recovered after proper washing periods are adjusted with buffer solutions at different haematocrit values as desired. These erythrocytes are often stored in acid-citrate-dextrose buffer at 40 C upto 48hr prior to use.

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METHODS OF DRUG LOADING THE POTENTIAL USE OF ERYTHROCYTES DEPENDS ON THEIR ABILITY TO ENCAPSULATE

EXOGENOUS ENZYMES OR OTHER SUBSTANCE IN THEM.

MOSTLY HYPOTONIC LYSIS OF CELLS IN A SOLUTION CONTAINING THE DRUG /ENZYME TO BE ENTRAPPED FOLLOWED BY RESTORATION OF TONICITY TO RESEAL THEM SERVES AS A LOADING PROCEDURE.

BUT OTHER TECHNIQUES SUCH AS ELECTRICAL BREAKDOWN, ENDOCYTOSIS, CHEMICAL PERTUTBATION OF MEMBRANE AND LIPID-ERYTHROCYTE FUSION HAVE ALSO BEEN UTILIZED.

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METHODS OF DRUG LOADING.

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LOADING BY CHEMICAL PERTURBATION OF MEMBRANE(DRUG MEDIATED LOADING)

THIS METHOD IS BASED ON THE OBSERVATION THAT

PERMEABILITY OF ELECTROLYTIC MEMBRANE IS INCREASED WHEN EXPOSED TO SOME CHEMICAL AGENTS.

THIS ALLOWS LOW MOLECULAR WEIGHT SUBSTANCES TO GET ENTRAPPED.

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LOADING BY CHEMICAL PERTURBATION OF MEMBRANE(DRUG MEDIATED LOADING)

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ELECTO INSERTION OR ELECTRO-ENCAPSULATION THIS METHOD IS BASED ON CREATING ELECTRICALLY INDUCED PERMEABILITY

CHANGES AT HIGH MEMBRANE POTENTIAL DIFFERENCES. R.B.C MEMBRANE CAN BE OPENED BY DIELECTRIC BREAKDOWN AND

SUBSEQUENTLY PORES CAN BE RESEALED BY INCUBATION AT 370 C IN OSMOTICALLY BALANCED MEDIUM.

ELECTRIC BREAKDOWN IS EVIDENT WHEN THE MEMBRANE IS POLARIZED FOR MICRO SECONDS USING VARIED VOLTAGE VALUES.

THE COMPONENTS CAN BE ENTRAPPRD WHEN AN ELECTRIC IMPULSE OF GREATER THAN A THRESHOLD VOLTAGE OF 2 Kv/cm IS APPLIED FOR 20 μ SEC.

THE POTENTIAL DIFFERENCE ACROSS THE MEMBRANE IS BUILT UP DIRECTLY BY INTER- AND INTRACELLULAR ELECTRODES OR INDIRECTLY BY APPLYING INTERNAL ELECTRIC FIELD TO THE CELLS.

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ADVANTAGES:

EXCELLENT IN-VIVO PERFORMANCE.

NORMAL HAEMOGLOBIN PROPERTIES WERE RETAINED.

USED TO INSERT PROTEINS INTO ERYTHROCYTE MEMBRANES.

SUSTAINED RELEASE OF ENTRAPPED DRUG.

DIS-ADVANTAGE:

TIME CONSUMING AND COST FACTOR ARE THE LIMITATIONS HERE.

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ELECTRO INSERTION OR ELECTRO-ENCAPSULATION

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ELECTO INSERTION OR ELECTRO-ENCAPSULATION

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DILUTIONAL HAEMOLYSIS RAPID AND SIMPLEST METHOD.

ESPECIALLLY FOR LOW MOLECULAR WEIGHT DRUGS.

% LOADING OR ENTRAPMENT EFFICIENCY = 1-8 %

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PRESWELL DILUTIONAL HAEMOLYSIS TECHNIQUE IS BASED ON AN 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, ADDITION OF SMALL VOLUME OF DRUG SOLUTION TO ATTAIN DRUG LOADED RESEALED ERYTHROCYTES.

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ISOTONIC OSMOTIC LYSIS

HAEMOLYSIS IN ISOTONIC SOLUTION CAN BE ACHIEVED BOTH BY CHEMICAL & PHYSICAL MEANS .

IF ERYTHROCYTES ARE INCUBATED IN SOLUTIONS OF SUBSTANCES WITH HIGH TRANS-ERYTHROCYTIC MEMBRANE PERMEABILITY, THE SOLUTE WILL DIFFUSE INTO THE CELLS DUE TO INWARDLY DIRECTED CHEMICAL POTENTIAL GRADIENT.

THIS IS FOLLOWED BY WATER UPTAKE UNTIL OSMOTIC EQUILIBRIUM IS RESTORED.

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METHODS BASED ON THIS MECHANISM :

CONVENTIONAL / CLASSICAL HAEMOLYSIS IN ISOTONIC UREA SOLUTIONS.

POLY ETHYLENE INDUCED HAEMOLYSIS.

AMMONIUM CHLORIDE INDUCED HAEMOLYSIS.

PEG INDUCED HAEMOLYSIS TECHNIQUE

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DIALYSIS METHODPRINCIPLE :

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.

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LOADING BY LIPID FUSION LIPID VESICLES CONTAINING DRUG CAN BE DIRECTLY FUSED WITH HUMAN

ERYTHROCYTES LEADING TO EXCHANGE OF LIPID ENTRAPPED DRUG.

THE METHOD, HOWEVER GIVES VERY LOW ENCAPSULATION EFFICIENCY (1%)

EXAMPLE : LOADING OF INOSITOL HEXAPHOSPHATE.

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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 TARGET CELLS WITH SUBSEQUENT RELEASE OF THE ENCAPSULATED MOLECULE.

THIS LOADING CAN BE EXEMPLIFIED WITH THE LOADING OF CELL SPECIFIC MONOCLONAL ANTIBODY TO ERYTHROCYTE GHOSTS.

AN ANTIBODY AGAINST A SPECIFIC SURFACE PROTEIN OF THE TARGET CELLS CAN BE CHEMICALLY CROSS-LINKED TO DRUG LOADED GHOSTS. THE ANTIBODY WILL DIRECT TO THE TARGET CELL FOR ADHESION.

ELECTRIC CELL FUSION THEN ALLOWS INJECTION OF THESE DRUGS INTO THE TARGET CELLS.

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ENTRAPMENT BY ENDOCYTOSIS ENTRAPMENT IN ERYTHROCYTE GHOSTS BY ENDOCYTOSIS INVOLVES 3 STEPS.

1) 1 VOLUME OF WASHED PACKED ERYTHROCYTES + 9 VOLUME OF BUFFER CONTAINING ATP, MgCl2 AND CaCl2 TO YIELD FINAL CONC’s OF 2.5 mM, 2.5mM AND 1mM RESPECTIVELY.INCUBATE THIS FOR 2 min’s AT ROOM TEMPERATURE.

2) RESEALING OF ERYTHROCYTE MEMBRANE BY ADDITION OF NaCl TO 154 mM, FOLLOWED BY INCUBATION FOR 2 min’s AT 370 CTHESE RESEALED R.B.C’s ARE WASHED IN 5mM IMIDAZOLE-GLYCYL-GLYCINE BUFFER.

3) ENTRAPMENT OF MATERIAL BY ALLOWING ENDOCYTOSIS FOLLOWING INCUBATION OF WASHED RESEALED CELLS WITH BUFFER CONTAINING THE MATERIAL TO BE ENTRAPED FOR 30 min AT 370 C.

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RELEASE CHARACTERISTICS OF LOADED DRUGS

THERE ARE MAINLY 3 WAYS FOR A DRUG TO EFFLUX OUT FROM THE ERYTHROCYTE CARRIERS :

• PHAGOCYTOSIS.

• DIFFUSION THROUGH THE MEMBRANE OF THE CELLS .

• USING A SPECIFIC TRANSPORT SYSTEM.

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IN-VITRO CHARACTERIZATION

RESEALED ERYTHROCYTES AFTER LOADING ARE CHARACTERIZED FOR THE FOLOWING PARAMETERS.

CHARACTERIZATION PARAMETERS ANALYTICAL METHODS SHAPE AND SURFACE MORPHOLOGY

VESICLE SIZE & SIZE DISTRIBUTION

% ENCAPSULATION

% CELL RECOVERY

ERYTHROCYTE SEDIMENTATION RATE.

STERILITY

PYROGENICITY

TRANSMISSION ELECTRON MICROSCOPY, SCANNING ELECTRON MICROSCOPY.

TRANSMISSION ELECTRON MICROSCOPY & OPTICAL MICROSCOPY.

DE-PROTEINIZATION OF CELL MEMBRANE AND ASSAY FOR RELEASED DRUG OR RADIO-LABELLED MARKERS.

HAEMATOLOGICAL ANALYZER; NEUBEUR’S CHAMBER

E.S.R APPARATUS.

AEROBIC / ANAEROBIC CULTURES.

RABBIT FEVER RESPONSE TEST OR L.A.L TEST.

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IN-VITRO CHARACTERIZATION• DRUG CONTENT PACKED LOADED ERYTHROCYTES (0.5ml) ARE FIRST DEPROTEINIZED WITH ACETO NITRILE

(2.0 ml) AND SUBJECTED TO CENTRIFUGATION AT 2500 rpm FOR 10 min .THE CLEAR SUPERNATANT IS ANALYZED FOR DRUG CONTENT.

• IN-VITRO DRUG AND HAEMOGLOBIN RELEASE NORMAL AND LOADED ERYTHROCYTES ARE INCUBATED AT 37+/-2o 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 MICRON SPECTRPORE MEMBRANE FILTER.

% HAEMOGLOBIN CAN SIMILARLY BE CALCULATED AT VARIOUS TIME INTERVALS AT 540nm SPECTROPHOTOMETRICALLY.

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LASER LIGHT SCATTERING MAY ALSO BE USED TO EVALUATE HAEMOGLOBIN CONTENT OF INDIVIDUAL RESEALED ERYTHROCYTES.

MEAN CORPUSCULAR = HAEMOGLOBIN (g/100ml) * 10 HAEMOGLOBIN ERYTHROCYTE COUNT(per mm3)• OSMOTIC FRAGILITY IT IS A RELIABLE PARAMETER TO EVALUATE THE EFFECTS OF VARYING TONICITIES,

DRUG LOADED ERYTHROCYTES ARE INCUBATED WITH SALINE SOLUTIONS OF DIFFERENT TONICITIES AT 37+2 / 37-2 o C FOR 10min’s.

THE SUSPENSION AFTER CENTRIFUGATION AT 30 g FOR 15 min’s IS ASSAYED FOR DRUG AND / OR HAEMOGLOBIN RELEASE.

• OSMOTIC SHOCK DESCRIBES A SUDDEN EXPOSURE OF DRUG LOADED ERYTHROCYES 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.

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INCUBATING THE RESEALED ERYTHROCYTES WITH DISTILLED WATER FOR 15min’s FOLLOWED BY CENTRIFUGATION AT 3000 rpm FOR 15 min’s, MAY CAUSE THE RELEASE OF HAEMOGLOBIN TO VARYING DEGREES, WHICH COULD BE ESTIMATED SPECTROPHOTOMETRICALLY.

• 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 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 300G FOR 15min’s, AND HAEMOGLOBIN CONTENT, LEACHED OUT ARE ESTIMATED SPECTOPHOTOMETRICALLY.

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• MORPHOLOGY AND PERCENTAGE CELLULAR RECOVERY

PHASE-CONTRAST N 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.

PERCENTAGE CELL RECOVERY CAN BE DETERMINED BY ASSESSING THE NUMBER OF INTACT ERYTHROCYTES REMAINING PER CUBIC mm WITH THE HELP OF A HAEMOCYTOMETER.

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IN-VIVO CHARACTERIZATION

A BIMODAL TYPE OF SURVIVAL KINETICS IS OBSERVED - A RAPID LOSS OF CELLS DURING FIRST 24 HOURS FOLLOWED BY MUCH SLOWER LOSS AFTERWARDS.

THE EARLY LOSS POSSIBILITY ACCOUNTS FOR REMOVAL OF NEARLY 15% OF TOTAL CELL POPULATION, THIS REPRESENTS THE CELLS SEVERELY DAMAGE DURING DRUG LOADING PROCEDURES.

THE ERYTHROCYTE CARRIERS CONSTRUCTED OF RED BLOOD CELLS OF MICE, CATTLE, PIGS, DOGS, SHEEP, GOATS AND MONEKYS EXHIBIT COMPARABLE CIRCULATION PROFILE.

RESEALED ERYTHROCYTES PREPARED FROM RED BLOOD CELLS OF CHICKEN, RATS AND RABBITS, EXHIBIT RELATIVELY POOR CIRCULATION PROFILE AS COMPARED AGAINST UNLOADED NORMAL ERYTHROCYTES.

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SHELF AND STORAGE OF RESEALED ERYTHROCYTES

RESEALED ERYTHROCYTES EXHIBIT SATISFACTORY SELF STABILITY WHEN STORED IN HANK’S BALANCED SALT SOLUTION (HBSS) AT 4o C FOR 2 WEEKS.

UNDER CLINICAL CONDITIONS STANDARD BLOOD BAGS MAY BE USED FOR BOTH, ENCAPSULATION AND STORAGE.

ANOTHER METHOD UTILIZED FOR STORAGE HAS BEEN THE CRYO PRESERVATION OF ERYTHROCYTES IN LIQUID NITROGEN TEMPERATURES.

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APPLICATIONS ERYTHROCYTES AS CARRIERS FOR ENZYMES. ERYTHROCYTES AS CARRIERS FOR DRUGS. ERYTHROCYTES AS CARRIERS FOR PROTEINS AND MACRO MOLECULES. DRUG TARGETING:

DRUG TARGETING TO RES ORGANS.SURFACE MODIFICATION WITH ANTIBODIES.SURFACE MODIFICATION WITH GLUTARALDEHYDE.

DRUG TARGETING TO LIVER ENZYMES DEFICIENCY OR REPLACEMENT THERAPY.TREATMENT OF LIVER TUMORS.

TARGETING TO SITES OTHER THAN RES-RICH ORGANS.MAGNET-RESPONSIVE ERYTHROCYTE GHOSTS.

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ERYTHROCYTES AS CIRCULATING BIOREACTORS.

DELIVERY OF ANTIVIRAL AGENTS.DELIVERY OF FLUDARABINE PHOSPHATE.

MACROPHAGE ACTIVATION.

THROMBOLYTIC THERAPY.

DELIVERY OF INTERLEUKINS.

OXYGEN DEFICIENCY THERAPY.

CELL BIOLOGICAL APPLICATIONS - MICRO-INJECTION OF MACRO-MOLECULES INTO CULTURED CELLS USING ERYTHROCYTE GHOSTS.

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NOVAL SYSTEMS• NANO-ERYTHROSOMES

IT IS PREPARED BY EXTRUSION OF ERYTHROCYTE GHOSTS TO PRODUCE SMALL VESICLES HAVING AN AVG DIAMETER OF 100 nm.

DAUNORUBICIN (DNR) WAS COVALENTLY CONJUGATED TO THE nEryt (nEryt-DNR) USING GLUTARALDEHYDE AS HOMO-BIFUNCTIONAL LINKING ARM. THIS LED TO A COMPLEX THAT IS MORE ACTIVE THAN FREE DNR BOTH INVIVO & IN VITRO.

DAUNORUBICIN CONJUGATED TO THESE NANOERYTHROSOMES HAS A HIGHER ANTI-NEOPLASTIC INDEX THAN THE FREE DRUG.

MOREOVER, SINCE NANOERYTHROSOMES ARE PARTICLES, PHAGOCYTOSIS MAY BE INVOLVED IN THEIR MECHANISM OF POTENTIATION.

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• ERYTHROSOMES

ERYTHROSOMES ARE SPECIALLY ENGINEERED VESICULAR SYSTEMS IN WHICH CHEMICALLY CROSS-LINKED HUMAN ERYTHROCYTE CYTOSKELETONS ARE USED AS A SUPPORT UPON WHICH A LIPID BILAYER IS COATED.

THIS CAN BE ACHIEVED BY A MODIFIED PEOCEDURE NORMALLY ADOPTED FOR REVERSE PHASE EVAPORATION.

ERYTHROSOMES ARE PROPOSED AS USEFUL ENCAPSULATION SYSTEMS FOR DRUG DELIVERY PARTICULARLY FOR MACRO-MOLECULAR DRUGS.

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CONCLUSION LARGE AMOUNT OF WORK IS NEEDED TO UTILISE THE POTENTIALS OF ERYTHROCYTES

IN TARGETED DRUG DELIVERY SYSTEMS.

DISEASES LIKE CANCER COULD SURELY FIND ITS CURE.

GENETIC ENGINEERING ASPECTS CAN BE COUPLED TO GIVE A NEWER DIMENSIONS TO EXISTING CELLULAR DRUG CARRIER CONCEPT.

DEVELOPMENT OF NANOSOMES.

DEVELOPMENT OF ERYTHROSOMES.

THE USE OF ERYTHROCYTES LOOKS TO BE PROMISING, FOR THE SAFE , SECURE AND SURED DELIVERY OF DRUGS.

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REFERENCE TARGETED AND CONTROLLED DRUG DELIVERY. NOVEL

CARRIER SYSTEMS. BY S.P.VYAS AND R.K.KHAR.

MODERN PHARMACEUTICS BY GILBERT S. BANKER AND CHRISTOPHER T.RHODE.

CONTROLLED DRUG DELIVERY BY N.K.JAIN.

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