ERYTHROSOMES Pinank V. Pandya BABARIA INSTITUTE OF PHARMACY, VADODARA
ERYTHROSOMES
Pinank V. PandyaBABARIA INSTITUTE OF PHARMACY, VADODARA
INTRODUCTION
What are erythrosomes?• They are specially engineered vesicular systems
in which chemically cross-linked human erythrocyte cytoskeleton are as a support on which a lipid bilayer is coated.
• This can be achieved by modified procedure normally adopted for reverse phase evaporation.
• They are proposed as useful encapsulation system particularly for macromolecular drugs.
HISTORY
• Erythrocytes (RBCs) were discovered in 1658.• The carrier potential of these cells was first
realized in early 1970s.
They are difficult to engineer for a non RES target, but recent innovation based on biophysically devices may render them suitable for targeting to organs other than RES. (Juliano,1980; DeLoach et al, 1991; Vyas & Jain, 1994)
Why erythrosome?
The desirable properties, which substantiate stability of RBCs as drug carrier are:-
• Biodegradability• Circulate through circulatory system.• Large volume of material can be encapsulated.• can be utilized for organ targeting with in RES.• A wide variety of bioactive agents can be
encapsulated within them.• Biocompatibility.
METHOD OF PREPARATION
ISOLATION OF ERYTHROCYTES :
-SOURCE :- Different mammalian RBCs have been exploited for drug loading. Majority of them constructed for RBCs of mice, cattle, pigs, dogs, sheep, goats, monkeys, chicken, rats & rabbits.
-ISOLATION:- Blood is collected in heparinized tubes by vein puncture (EDTA or heparin can be used as anti coagulant). Fresh RBCs are preffered because of their higher encapsulation efficiency.
• RBCs are harvested & washed by centrifugation. According to source centrifugal force & washing fluid is changed.
-General conditions:-• Centrifugation-2000 RPM for 5 min at 4*C• Buffer solution(washing fluid):• NaCl -14 mmol/L • KCl -16 mmol
• MgCl2 -4 mmol
• CaCl2 -2 mmol
Tris -5 mmol
RBC s are often stored in acid-citrate-dextrose buffer at 4*C upto 48h prior to use.
METHOD OF DRUG LOADING :
Mostly hypotonic lysis of cell in a solution containing drug/enzyme to be entrapped followed by restoration of tonicity to reseal them serves as a loading procedure.
other techniques such as electrical breakdown, endocytosis, chemical purturbation of membrane & lipid erythrocyte fusion have also been utilised.
SHELF & STORAGE STABILITY OF ERYTHROSOMES
It is a major challenge in the their practical utility as DDS.
• Lewis & alpar, 1984 have reported that encapsulated product & carrier both exhibit satisfactory shelf stability, when stored in Hank’s balanced salt solution (HBBS)at 4*C for 2 weeks.
• Similar results were obtained by suspending cells in oxygenated HBBS containing 1% soft bloom gelatin.
• The cells are well recovered after liquefying the gel by placing the tube in water bath at 37*C followed by centrifugation.
• Under clinical condition standard blood bags may be used for both encapsulation & storage.
• The procedure would be to used group ‘O’(universal donor) cells by preswell or dialysis technique.
• Another method utilized for storage has been the cryopreservation of RBCs in liquid nitrogen.
IN VIVO SURVIVAL AND IMMUNOLOGICAL CONSEQUENCES• A bimodel type of survival kinetic is observed :
A rapid loss of cell during first 24 h followed much slower release after word.
• The early loss that accounts for 15% of total population represents the cells that are severly damaged during drug loading.
• The inter subject & inter species variation from different sources require ajudicial analysis &use of the source before experiment is designed.
• There three general modes of efflux of loaded drug from erythrosome.
-phagocytosis
-diffusion
-specific transport mechanism• The phagocytosis occurs within RES.• The degree of cross linking determines
whether spleen or liver will preferentially remove the cell.
• It is observed that erythrocyte carriers constructed from analogous sources do not elicit an immunological consequences.
BIOMEDICAL APPLICATIONS OF ERYTHROSOMES
(1) Erythrosomes as carriers of enzymes:-
-ideal carriers of enzymes in inherited metabolic diseases.
-enzymes that are usually carried are
catalase , urease , uricase , invertase, arginase , asparaginase , glucuronidase , galactosidase etc.
e.g. alcohol dehydrogenase & acetaldehyde dehydrogenase encapsulated in human RBCs used in vivo for complete metabolism of ethanol.
(2) Erythrosomes as carrier of drugs
-slow and sustained release of drugs in treatment of paracytic disease.
-erythrosomes serves as ideal carrier for antineoplastic agents like bleomycin, actinomycin-D, adriamycin or cytosin arabinoside.
-vitamins & steroids have also been encapsulated in erythrosomes.
(3) As carriers of proteins & macromolecules
-Bird et al.1983 proposed erythrocytes as carrier for insulin for its sustain release.
-erythrosomes may serve as cellular sustain delivery system for in vivo administration of recombinant human erythropoeitin.
-RBCs coated with recombinant interlukin-2 (rIL-2) are reported to provide sustain delivery so as to allow low and non toxic concentrations of IL-2 in circulation.
(4) Drug targeting
-osmotically loaded ehrocytes can act as drug carrier in systemic circulation.
-chemically surface modified erythrocytes are targeted to organ of the mononuclear phagocytic system /reticular endothelial system because changes incorporated in membrane that are recognized by macrophage cell.
Treatment of paracytic disease
-paracytic disease in which paracyte reside in RES have been effectively treated with erythrosomes.
-Pentamidine primaquine phosphate & metronidazole have been successfully utilized for treatment of leishmaniasis, malaria, extra intestinal amoebiasis on exp. Laboratory models.
• Targeting in areas other than RES using:
-paramagnetic particle loaded RBCs.
-photosensitive element loaded RBCs.
-ultra sound waves.
-target specific immunoglobulin attached to RBCs.
(5)In oxygen deficiency therapy erythrocytes are used in case of oxygen
deficiency where an improved oxygen supply is required as in following cases:
-high altitude condition (where oxygen partial pressure is low)
-small number of alveoli -increased resistance to oxygen diffusion in lungs -increased radio sensitivity in of radiation sensitive tumors -liver mediated detoxification process where an increased oxygen supply is
required.
- Inositol hexaphosphate (IHP, phytic acid) loaded in RBCs binds irreversibly to hemoglobin & reduces its oxygen binding, thus releasing same in capillaries.
(6) Erythrosomes in cell biological application microinjection of macromolecules into cultured
cells using erythrocyte ghosts :
-using either Sendai virus (haemagglutinating virus of
Japan, HVJ) or PEG mediated fusion, & then macromolecules are transferred efficiently from cytoplasm to nucleus of their expression.
-erythrocyte ghost cell fusion (microinjection) method mediated HVJ is relatively easier, & can be used for microinjection in many cells at the same time.
(7) Cell biological application
-fragment A of diphtheria toxin (which on entering cytoplasm inhibits peptide chain elongation in translation &causing cell death) can be introduced in to target cell by erythrocyte ghost cell fusion.
FUTURE PERSPECTIVE
• A large amount of valuable work is needed so as to utilize the potential of erythrocytes in passive as well as active targeting of drugs.
• Diseases like cancer can surely find its cure
• Genetic engineering aspects can be coupled to give newer dimension to the existing drug cellular concept.