ERYTHROCYTES AS CARRIERS Presented by : Srinivas Dinakar ,
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Blood is withdrawn from cardiac/splenic puncture( in small animals) and through veins (in large animals) in a syringe containing a drop of anti coagulant.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 ±1 0 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 4 o C until used.
The following methods have been employed for drug entrapment in erythrocytes:
Hypo- osmotic lysis method
Isotonic osmosis lysis method
Electrical breakdown method
Membrane perturbation method
Normal transport method
Lipid fusion method
In vitro characterization
The in vitro characterizations are pivotal to ensure their in vivo performance and therapeutic benefits.
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:
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.
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.
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:
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 to RES organs
Surface modification with antibodies
Surface modification with Glutaraldehyde
Surface modification involving sulphydryls
Drug targeting to Liver
Enzyme deficiency/replacement therapy
Treatment of liver tumors
Treatment of parasitic diseases
Removal of RES Iron Overload
Targeting to sites other than RES- rich organs
Magnet-responsive Erythrocyte Ghosts
Ultrasound mediated Delivery of Erythrocytes loaded drugs