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Resealed Erythrocytes Venki
Resealed Erythrocytes Venki
SEMINAR BY :: R. Venkatesh
10DG1SO314
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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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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 NONDIFFUSIBLE (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 BODYS 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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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 RETICULOENDOTHELIAL SYSTEM (RES) AND SERVING AS CIRCULATORY BIOVECTORS 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.
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THEIR BIOCOMPATIBILITY.
THEIR BIODEGRADABILITY WITH NO GENERATION OF TOXIC PRODUCTS. CONSIDERABLY UNIFORM SIZE AND SHAPE OF THE CARRIER DEGRADATION OF LOADED ENDOGENOUS CHEMICALS. DRUG AND ITS INACTIVATION IS PREVENTED BY
WIDE VARIETY OF CHEMICALS CAN BE ENTRAPPED. PREVENTION OF UNDESIRED IMMUNE RESPONSE AGAINST THE LOADED DRUG.
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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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ADVANTAGES:
EXCELLENT IN-VIVO PERFORMANCE.
DIS-ADVANTAGE:
TIME CONSUMING AND COST FACTOR ARE THE LIMITATIONS HERE.
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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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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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DIALYSIS METHOD
PRINCIPLE :
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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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 CONCs OF 2.5 mM, 2.5mM AND 1mM RESPECTIVELY. INCUBATE THIS FOR 2 mins AT ROOM TEMPERATURE.
2) RESEALING OF ERYTHROCYTE MEMBRANE BY ADDITION OF NaCl TO 154 mM, FOLLOWED BY INCUBATION FOR 2 mins AT 370 C THESE RESEALED R.B.Cs 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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IN-VITRO CHARACTERIZATION
CHARACTERIZATION PARAMETERS LOADING ARE CHARACTERIZED FOR THE FOLOWING ANALYTICAL METHODS RESEALED ERYTHROCYTES AFTER PARAMETERS. SHAPE AND SURFACE MORPHOLOGY TRANSMISSION ELECTRON MICROSCOPY, SCANNING ELECTRON MICROSCOPY.
AEROBIC / ANAEROBIC CULTURES. RABBIT FEVER RESPONSE TEST OR L.A.L TEST. Page 38
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.
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 10mins. THE SUSPENSION AFTER CENTRIFUGATION AT 30 g FOR 15 mins 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 15mins FOLLOWED BY CENTRIFUGATION AT 3000 rpm FOR 15 mins, 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 15mins, AND HAEMOGLOBIN CONTENT, LEACHED OUT ARE ESTIMATED SPECTOPHOTOMETRICALLY.
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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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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 (nErytDNR) 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.
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