ERYTHROCYTES AS CARRIERS

Presented by: Srinivas Dinakar

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Introduction• Erythrocytes (RBCs) contain oxygen carrying

protein hemoglobin, which is a pigment that gives whole blood its red color.

• A healthy adult male has about 4.5 million RBCs/µl of blood, and a healthy adult female has about 4.8 million.

• Because matured RBCs have no nucleus, all their internal space is available for oxygen transport.

• As they lack mitochondria and generate ATP anaerobically, they do not use up any oxygen they transport.

Isolation of erythrocytes

• Blood is collected into heparinized tubes by venipuncture.

• Blood is withdrawn from cardiac/splenic puncture( in small animals) and through veins (in large animals) in a syringe containing a drop of anti coagulant.

• The whole blood is centrifuged at 2500 rpm for 5 min at 4±10 C in a refrigerated centrifuge.

• The serum and buffy coats are carefully removed and packed cells washed three times with phosphate buffer saline (pH=7.4).

• The washed erythrocytes are diluted with PBS and stored at 4oC until used.

Entrapment methods

• The following methods have been employed for drug entrapment in erythrocytes:– Hypo- osmotic lysis method

Dilution methodDialysis methodPreswell methodIsotonic osmosis lysis method

– Electrical breakdown method– Endocytosis method– Membrane perturbation method– Normal transport method– Lipid fusion method

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In vitro characterization

• The in vitro characterizations are pivotal to ensure their in vivo performance and therapeutic benefits.

• Drug content: packed loaded erythrocytes (0.5ml) are first

deproteinized with acetonitrile(2ml) and subjected to centrifugation at 2500 rpm for 10 min. The clear supernatant is analyzed for the drug content using specified estimation methodology for entrapped drug.

• In vitro drug and hemoglobin release:

• In vitro drug and hemoglobin release:

• Osmotic fragility:simulates and mimics the bio-environmental conditions that are encountered on in vivo administration, in vitro handling and the effect of loaded contents on the survival rates of the erythrocytes.

• Osmotic shock:A sudden 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. Incubating the resealed erythrocytes with distilled water for 15 min followed by centrifugation at 3000 rpm for 15 min, may cause the release of hemoglobin to varying degrees, which could be estimated spectrophotometrically.

• Turbulence shock:

The effect 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 10ml/min.After every pass, aliquot of the suspension is withdrawn and centrifuged at 2000 rpm for 10 min, and hemoglobin 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 RBCs

• 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 or echinocytes, cells with different infoldings and other abnormal or destroyed forms.

• On swelling, the cells get converted from discocytes to spherocytes and thus get compromised with a lower ratio of surface features. Further increase in hypotonicity may lead to the formation of echinocytes and cells with different infoldings and other damaged forms.

• Erythrocytes on hemolysis and washing with large volumes of hypotonic medium, loose nearly all their hemoglobin and on releasing the resultant cells appear as pale or transparent in appearance and are referred to as “erythrocytes ghosts”.

In vivo survival

&immunological consequences

• A rapid loss of cells during first 24 hrs followed by much slower loss afterwards.

• The first phase represents the cells that are severally damaged during the drug loading procedures.

• The second phase has a half life of the orders of weeks for different mammalian erythrocytes.

• Resealed erythrocytes prepared from RBCs of chicken, rats and rabbits exhibited relatively poor circulation profile as compared against unloaded normal erythrocytes.

Release characteristics of loaded drugs

• There are mainly three ways for a drug to efflux out from the erythrocyte carriers:

– Phagocytosis– Diffusion through the membrane of the cells– Using a specific transport system.

• RBCs are normally removed from circulation by the process of phagocytosis.

• The degree of cross-linking determines whether liver or spleen will preferentially remove the cells

• Carrier erythrocytes following heat treatment or antibody cross-linking are quickly removed from the circulation by phagocytic cells located mainly in liver and spleen.

• The rate of diffusion depends upon the rate at which a particular molecule penetrates through a lipid bilayer. It is greatest for a molecule with high lipid solubility.

•Many susbtances enter cells by a specific membrane protein system because the carriers are proteins with many properties analogous to that of enzymes, including specificity.•Erythrocytes carrier have the potential of releasing encapsulated substance following zero-order kinetics. By incorporating polymers to erythrocytes, the release pattern may be modified. •The drug however could be released from macrophages after phagocytosis if the linkage is susceptible to lysosomal enzymes.•If the drug is encapsulated in a random population of erythrocytes, then a constant fraction of the cells will be removed each day and a constant amount of drug will be made available each day.

Applications of released erythrocytes

Erythrocytes as drug/ enzyme carriers:• Erythrocytes as carriers for enzymes.• Erythrocytes as carriers for drugs.• Erythrocytes as carriers for proteins and

macromolecules. Drug targeting:

• Drug targeting to RES organsSurface modification with antibodiesSurface modification with GlutaraldehydeSurface modification involving

sulphydryls

• Drug targeting to LiverEnzyme deficiency/replacement therapyTreatment of liver tumorsTreatment of parasitic diseasesRemoval of RES Iron Overload

• Targeting to sites other than RES- rich organsMagnet-responsive Erythrocyte GhostsPhotosensitized ErythrocytesUltrasound mediated Delivery of Erythrocytes

loaded drugsAntibody Anchored Erythrocytes

(Immunoerythrocytes).

Erythrocytes as Circulating Bioreactors• Delivery of Antiviral Agents.

Recent developments NOVEL SYSTEMS

• NanoerythrosomesAn erythrocyte based new drug carrier, named nanoerythrosome has been developed which is prepared by extrusion of erythrocyte ghosts to produce small vesicles having an average diameter of 100 nm.

• ErythrosomesErythrosomes are specifically engineered vesicular systems in which chemically cross-linked human erythrocyte cytoskeletons are used as a support upon a lipid bilayer is coated.

Future perspectives

• A large amount of valuable work is needed so as to utilize the potentials of erythrocytes in passive as well as active targeting of drugs.

• Diseases like cancer could surely find its cure.

• Genetic engineering aspects can be coupled to give a newer dimension to the existing cellular drug carrier concept.

Conclusion

• Numerous applications have been proposed for the use of resealed erythrocytes as carrier for drugs, enzyme replacement therapy etc.

• Resealed erythrocytes technology will remain an active arena for the further research.

• Erythrocytes based delivery system with their ability to provide controlled and site-specific drug delivery will revolutionize disease management.

• For the present it is concluded that erythrocyte carriers are “GOLDEN EGGS IN NOVEL DRUG DELIVERY SYSTEMS” considering their tremendous potential.

References

• Controlled and Novel Drug Delivery, edited by N.K Jain, CBS Publishers, Pg. no.256-281.

• Targeted & Controlled Drug Delivery Novel Carrier Systems, by S.P Vyas and R.K Khar, CBS Publishers, Pg.no.387-413.

• INTERNET URL.