resealed erythrocytes

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Resealed erythrocytes : 



CONTENTS Introduction Basic features of erythrocytes Drug carrying potential of erythrocytes Advantages of resealed erythrocytes as drug carrier Source and isolation of erythrocytes Methods of drug loading In vitro characterization Applications


INTRODUCTION Amongst various carriers explored for target oriented drug delivery, vesicular, microparticulate & cellular carriers meet several criteria rendering them useful in clinical applications. Leukocytes, platelets and erythrocytes have been proposed as cellular carrier systems. Among these, the erythrocytes have been the most investigated and have found to possess great potential in drug delivery. Erythrocytes have been the most extensively investigated and found to posses great potential in novel drug delivery . Until recently, the only biomedical use of erythrocytes were in fact in transfusion of medicine and in the preparation of blood products while with this new technology, drugs, chemicals or macromolecules can be loaded into erythrocytes, offering a further step towards optimal use of natural resource like blood.

Basic features of Erythrocytes : 

Basic features of Erythrocytes Composition of erythrocytes: The blood contains about 55% of fluid portion (plasma) and nearly 45% of corpuscles or formed elements (erythrocytes, leukocytes, thrombocytes). Erythrocytes are biconcave discs with an average diameter of 7.5 m, a thickness of 2.5 m in periphery, 1 m in the center, and a volume of 85–91 m3. They have a life span of about 100-120 days. Erythrocytes are produced as colorless nucleated cells from the bone marrow. The developing RBC has the capacity to synthesize hemoglobin.

Erythrocytes : 

Erythrocytes Erythro= red Cytes = cell Biconcave discs Filled with hemoglobin (Hb-33.67%), a protein that functions in gas transport. COMPOSITION Water-63% lipid-0.5% glucose-0.8% minerals- 0.7%

Drug carrying potential of Erythrocytes: : 

Drug carrying potential of Erythrocytes: The developing RBC has the capacity to synthesize haemoglobin, however adult RBCs do not have this capacity and serve as carrier for hemoglobin. The carrier potential of these cell was first realized in early 1970. Erythrocytes have been utilized as carriers for a wide range of bioactive components including drugs, enzymes, pesticides, DNA molecules and others.

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Properties as carrier :- 1. Biodegradability 2. Circulate throughout the circulatory system. 3. Large quantities of material can be encapsulated within them. 4. Can be utilized for organ targeting within RES. 5. A wide variety of bioactive agents can be encapsulated within them. 6. Erythrocytes are biocompatible cells are used in patients there is no possibility of triggered immunological response

Source, fractionation & isolation of erythrocytes : 

Source, fractionation & isolation of erythrocytes Source:- mice, cattle, pig, dog, sheep, goat, monkey, chicken, rat, rabbit & human. Blood can be collected by venipuncture or from orbital sinus in heparinized tube. Red blood cells can be harvested & centrifuged. Different centrifugal force & different buffer composition for different species is used. Fresh blood is used for loading of drugs.

Methods of drug loading : 

Methods of drug loading

1. Hypotonic Haemolysis and Isotonic Resealing Methods : 

1. Hypotonic Haemolysis and Isotonic Resealing Methods This method is based upon hypotonic lysis of cells in solution containing the drug/enzyme to be entrapped followed by restoration of tonicity to reseal them.

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Three types of ghosts can be distinguished: type1 ghosts which reseal immediately after haemolysis; type 2 ghosts which reseal after, reversal of haemolysis by addition of alkali ions; and type 3 ghosts which remain leaky under different experimental conditions. Erythrocytes have an exceptional capability for reversible shape changes

(i) Dilutional Haemolysis : 

(i) Dilutional Haemolysis Population of erythrocytes when exposed to hypotonic saline solution (0.4% NaCl), swells until it reaches a critical value of volume or pressure where membrane ruptures and becomes permeable to macromolecules and ions, therefore permitting the escape of cellular components.

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Dilutional Haemolysis RBC Membrane ruptured RBC Loaded RBC Resealed Loaded RBC 0.4% NaCl Hypotonic Drug Loading buffer Resealing buffer Incubation at 250c Efficiency  1-8% Enzymes delivery Hypotonic med Isotonic med. Washed

(ii) Preswell Dilutional Haemolysis : 

(ii) Preswell Dilutional Haemolysis The technique is based upon initial controlled swelling of erythrocytes without lysis by placing them in slightly hypotonic solution followed by centrifugation at low 'g' to take them up to point of lysis . Finally, the addition of small volume of drug solution to attain drug loaded resealed erythrocytes. RBC 0.6%w/v NaCl Swelled RBC Drug + Loading buffer 5 min incubation at 0 0c Loaded RBC Incubation at 25 0c Resealing Buffer Resealed RBC Efficiency  72%

(iii) Dialysis method : 

(iii) Dialysis method The major limitation of dilution procedure is low entrapment efficiency. It can be overcome by carrying out lysis and resealing within a dialysis tube. All the dialysis based methods works on common principle that 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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Dialysis method RBC Phosphate buffer + Placed in dialysis bag with air bubble Dialysis bag placed in 200ml of lysis buffer with mechanical rotator 2hrs. 4c. Drug Loading buffer Loaded RBC Dialysis bag placed in Resealing buffer with mechanical rotator 30 min 37c. Resealed RBC Efficiency  30-45%

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Isotonic osmotic lysis: In order to avoid disadvantages of hypotonic hemolysis, efforts were made to develop resealed under isotonic conditions. Hemolysis in isotonic conditions can be achieved both by physical and chemical means.



2.Electro-Insertion or Electro-Encapsulation : 

2.Electro-Insertion or Electro-Encapsulation This method is based on creating electrically induced permeability changes at high membrane potential differences. The components can be entrapped when an electric pulse of greater than a threshold voltage of 2kv/cm is applied for 20 μ sec. The potential difference across the membrane is built up either directly by inter and intracellular electrodes or indirectly by applying internal electric field to the cells.

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The extent of pore formation depends upon the electric field strength, pulse duration and ionic strength of suspending medium. Here large molecules (like bovine serum albumin) and ribonucleases can be loaded. These can be loaded in to the osmotic swelling of electrically perforated erythrocytes.

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Electro-insertion or Electro-encapsulation RBC 2.2 Kv Current for 20 micro sec At 250 C Pulsation medium + + Drug Loading suspension 3.7 Kv Current for 20 micro sec Isotonic NaCl Loaded RBC Resealing Buffer Resealed RBC Fig:- Electro-encapsulation Method

3. Endocytosis : 

3. Endocytosis First, one volume of washed packed erythrocytes are added with 9 volumes of buffer containing ATP, MgCl2 and CaCl2 to yield concentrations of 2.5 mM, 2.5 mM and 1 mM respectively and incubated at room temperature for 2 min. Resealing of erythrocyte membrane by the addition of NaCl to 154 mM followed by incubation for 2 min at 37oC. These resealed erythrocytes are washed in 5 mM imidazoleglycylglcine buffer, pH7.4 containing 154 mM NaCl. Third, entrapment of the material by allowing endocytosis following incubation of washed resealed cells with buffer containing material to be entrapped for 30 min at 37 oC.

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Entrapment By Endocytosis:- RBC Drug Suspension + Buffer containing ATP, MgCl2, and CaCl2 At 250 C Loaded RBC Resealing Buffer Resealed RBC Fig;- Entrapment By Endocytos Method

4. Membrane Perturbation : 

4. Membrane Perturbation This method is based upon the observation that the permeability of the erythrocytic membrane is increased, when it is exposed to some chemical agents. This allows small molecular weight substances to get entrapped. Antibiotics such as amphotericin B damage microorganisms by increasing the permeability of their membrane to metabolites and ions. This property could be exploited for loading of drug into erythrocytes. Amphotericin B interacts with the cholesterol of plasma membrane of eukaryotic cells causing change in permeability of the membrane.

5. Loading by Lipid Fusion : 

5. Loading by Lipid Fusion Lipid vesicles containing drug can be directly fused with human erythrocytes leading to exchange of lipid entrapped drug. This technique is used for loading of inositol hexaphosphate into erythrocytes for the increased oxygen carrying capacity. Encapsulation efficiency – 1%


IN VITRO CHARACTERIZATION Resealed erythrocytes after loading are characterized for following parameters 1. Drug Content Packed loaded erythrocytes are first deproteinized with acetonitrile and subjected to centrifugation at 2500 rpm for 10 min. The clear supernatant is analyzed for the drug content. 2. In vitro Drug and Haemoglobin Release Normal and loaded erythrocytes are incubated at 37± 2°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 µ Spectropore membrane filter.

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Percent haemoglobin can similarly be calculated at various time intervals at 540 run spectrophotometrically. Laser light scattering may also be used to evaluate haemoglobin content of individual resealed erythrocytes. Mean corpuscular [Haemoglobin (g/100ml) X 10 Haemoglobin = Erythrocyte count(per mm3) 3. Osmotic Fragility It is reliable parameter for in vitro evaluation of carrier erythrocytes with respect to shelf life, in vivo survival & effect of encapsulated substances. When RBC are exposed to solution of varying tonicities, this shape changes due to osmotic balance.

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To evaluate the effects of varying tonicities, drug loaded erythrocytes are incubated with saline solutions of different tonicities at 37±2oC for 10 min. The suspension after centrifugation for 15min, 2000 rpm is assayed for drug or haemoglobin release 4. Osmotic shock Osmotic shock describes 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 haemoglobin to varying degrees, which could be estimated spectrophotometrically.

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5. Turbulence Shock This parameter indicates the effects of shear force and pressure by which resealed erythrocyte formulations are injected, on the integrity of the loaded cell. Loaded erythrocytes are passed through a 23-guag hypodermic needle at a flow rate of 10 ml/min. After every pass, aliquot of the suspension is withdrawn and centrifuged at 300 g for 15 min, and haemoglobin content, leached out is estimated spectrophotometrically. 6. 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 surface features of loaded erythrocytes. Percent cell recovery can be determined by assessing the number of intact erythrocytes remaining per cubic mm with the help of haemocytometer.

Applications: : 

Applications: 1. Treatment of lysosomal storage disease: Resealed erythrocytes have been proposed to deliver lysosomal enzymes to lysosomes of the erythrophagocytic cells, thus resulting in replacement of the missing enzyme. Ex: β-glucoronidase, β-galactoronidase and β-glucosiade. 2. Treatment of Gaucher’s disease: Gaucher’s disease is due to accumulation of glucocerebroside from catabolised erythrocytes and leukocytes in spleen, liver and bone marrow. This disease was treated by encapsulating glucocerebroside in erythrocyte.

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3. Treatment of liver tumors: Anticancer agents like bleomycin, adriamycin, L-asparaginase, doxorobucin and methotrexate are encapsulated in erythrocyte to treat hepatic carcinomas. 4. Erythrocytes as circulating carriers: Various bioactive agents are encapsulated in erythrocytes for their slow release in circulation for treatment of parasitic diseases in cattle. Ex: homidium bromide is encapsulated in erythrocytes to treat trypanosomiasis.

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5. In enzyme delivery: To eliminate or minimize the problems related to immunologic responses and toxicity, encapsulated enzyme administration is suggested 6. Prevention of thromboembolism: Encapsulated heparin is liberated from circulating erythrocytes at the site of thrombus formation thus reducing the risk of further thrombus growth.

Example: : 

Example: Amikacin encapsulated in carrier erythrocytes Dexamethasone (Dexa) loaded resealed erythrocytes Fludarabine loaded resealed erythrocytes Antimalarial drugs loaded resealed erythrocytes.

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