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.