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About Authors: Varun Raj Vemula*1, Swathi Thakkalapally2, Chaitanya Kumari Bairi3 1 DepartmentofPharmaceutical Chemistry, Vikas College of Pharmacy, Jangaon, Warangal. Affiliated to Kakatiya University. 2 Department of Pharmaceutics, Care College of Pharmacy, Warangal. Affiliated to Kakatiya University. 3 Department of Pharmaceutics, TallaPadmavathi College of pharmacy, Warangal. Affiliated to Kakatiya University. Abstract: Carrier erythrocytes have been evaluated in thousands of drug administration in humans proving safety and efficacy of the treatments. Carrier erythrocytes, resealed erythrocytes loaded by a drug or other therapeutic agents, have been exploited extensively in recent years for both temporally and spatially controlled delivery of a wide variety of drugs and other bioactive agents owing to their remarkable degree of biocompatibility, biodegradability and a series of other potential advantages. Biopharmaceuticals, therapeutically significant peptides and proteins, nucleic acid-based biologicals, antigens and vaccines, are among the recently focused pharmaceuticals for being delivered using carrier erythrocytes. In this review article, the potential applications of erythrocytes in drug delivery have been reviewed with a particular stress on the studies and laboratory experiences on successful erythrocyte loading and characterization of the different classes of biopharmaceuticals. INTRODUCTION: Erythrocytes, the most abundant cells in the human body, have potential carrier capabilities for the delivery of drugs. Erythrocytes are biocompatible, biodegradable, possess very long circulation half lives and can be loaded with a variety of chemically and biologically active compounds using various chemical and physical methods. Application of erythrocytes as promising slow drug release or site-targeted delivery systems for a variety of bioactive agents from different fields of therapy has gained a remarkable degree of interest in recent years. Biopharmaceuticals are among the most widely exploited candidates for being delivered to the host body using these cellular carriers. In this review, the potential applications of erythrocytes in drug delivery have been highlighted. [1-3]

ISOLATION OF ERYTHROCYTES: Various types of mammalian erythrocytes have been used for drug delivery, including erythrocytes of mice, cattle, pigs, dogs, sheep, goats, monkeys, chicken, rats, and rabbits. To isolate erythrocytes, blood is collected in heparinized tubes by venipuncture. Fresh whole blood is typically used for loading purposes because the encapsulation efficiency of the erythrocytes isolated from fresh blood is higher than that of the aged blood.[4] To isolate erythrocytes, blood is collected in heparinized tubes by venipuncture.Fresh whole blood is typically used for loading purposes because the encapsulation efficiency of the erythrocytes isolated from fresh blood is higher than that of the aged blood. Fresh whole blood is the blood that is collected and immediately chilled to 40c and stored for less than two days. The erythrocytes are then harvested and washed by centrifugation. The washed cells are suspended in buffer solutions at various hematocrit values as desired and are often stored in acidcitratedextrose buffer at 40 c as long as 48 h before use. Jain and Vyas have described a well-established protocol for the isolation of erythrocytes. The loading of drugs in erythrocytes was reported separately by Ihler et al. and Zimmermann. In 1979, the term carrier erythrocytes were coined to describe drug-loaded erythrocytes. [5] [6] Advantages and disadvantages of erythrocytes in drug delivery Advantages Some of the most important advantages encouraging the use of erythrocytes in drug delivery include: [6-15] 1. A remarkable degree of biocompatibility, particularly when the autologous cells are used for drug loading. 2. Complete biodegradability and the lack of toxic product(s) resulting from the carrier biodegradation. 3. Avoidance of any undesired immune responses against the encapsulated drug. 4. Considerable protection of the organism against the toxic effects of the encapsulated drug, e.g. antineoplasms. 5. Remarkably longer life-span of the carrier erythrocytes in circulation in comparison to the synthetic carriers. In the optimum condition of the loading procedure, the life-span of the resulting carrier cells may be comparable to that of the normal erythrocytes. 6. An easily controllable life-span within a wide range from minutes to months. 7. Desirable size range and the considerably uniform size and shape. 8. Protection of the loaded compound from inactivation by the endogenous factors.

9. Possibility of targeted drug delivery to the RES organs. 10. Relatively inert intracellular environment. 11. Availability of knowledge, techniques, and facilities for handling, transfusion, and working with erythrocytes. 12. Possibility of ideal zero-order kinetics of drug release. 13. Wide variety of compounds with the capability of being entrapped within the erythrocytes. 14. Possibility of loading a relatively high amount of drug in a small volume of erythrocytes, which, in turn, assures the dose sufficiency in clinical as well as animal studies using a limited volume of erythrocyte samples. 15. Modification of the pharmacokinetic and pharmacodynamic parameters of the drug. 16. Remarkable decrease in concentration fluctuations in steady state in comparison to the conventional methods of drug administration, which is a common advantage for most of the novel drug delivery systems. 17. Considerable increase in drug dosing intervals with drug concentration in the safe and effective level for a relatively long time. 18. Possibility of decreasing drug side effects. Drawbacks: The use of erythrocytes as carrier systems also presents some disadvantages, which can be summarized as follows: [16-20] 1. The major problem encountered in the use of biodegradable materials or natural cells as drug carriers is that they are removed in vivo by the RES as result of modification that occurred during loading procedure in cells. This, although expands the capability to drug targeting to RES, seriously limits their life-span as long-circulating drug carriers in circulation and, in some cases, may pose toxicological problems. 2. The rapid leakage of certain encapsulated substances from the loaded erythrocytes. 3. Several molecules may alter the physiology of the erythrocyte. 4. Given that they are carriers of biological origin, encapsulated erythrocytes may present some inherent variations in their loading and characteristics compared to other carrier systems. 5. The storage of the loaded erythrocytes is a further problem provided that there are viable cells and need to survive in circulation for a long time upon re-entry to the host body. Conditioning carrier cells in isotonic buffers containing all essential nutrients, as well as in low temperatures, the addition of nucleosides or chelators, lyophilization with glycerol or gel immobilization have all been exploited to overcome this problem. 6. Possible contamination due to the origin of the blood, the equipment used and the loading environment. Rigorous controls are required accordingly for the collection and handling of the erythrocytes.

METHODS OF DRUG LOADING: Several methods can be used to load drugs or other bioactive compounds in erythrocytes, including physical (e.g., electrical pulse method) osmosisbased systems, and chemical methods (e.g., chemical perturbation of the erythrocytes membrane).the following are types of drug loading: Hypotonic hemolysis, hypotonic dilution, hypotonic preswelling, isotonic osmotic lysis, Chemical perturbation of the membrane. Electro-insertion or electron capsulation, Entrapment by endocytosis, loading by electric cell fusion, loading by lipid fusion. [21] OSMOTIC SHOCK: For 0.5 study, erythrocyte suspension (1 ml, 10%) was diluted & centrifuge at 3000 rpm for 15 minute. The supernatant was estimated for % Hb release spectrophotometrically. TURBULENCE SHOCK: It is the measure of simulating distribution of loaded cells during injection. In this drug loaded cells are passed through a 23 gauge hypodermic at a flow rate of 10 ml/min which is comparable to the flow rate of blood. It is followed by collecting of an aliquot and centrifugation sample is estimated. Drug loaded erythrocytes appears to be less resistant to turbulence, probably indicating destruction of cells upon shaking. ERYTHROCYTE SEDIMENTATION RATE (ESR): It is an estimate of the suspension stability of RBC in plasma and is related to the number and size of the red cells and to relative concentration of plasma protein, especially fibrinogen and , globulins. This test is performed by determining the rate of sedimentation of blood cells in a standard tube. Normal blood ESR is 0 to 15 mm/hr. higher rate is indication of active but obscure disease processes. Use of red cell loader: Magnani et al. developed a novel method for entrapment of non diffusible drugs into erythrocytes. They developed a piece of equipment called a red cell loader. With as little as 50 mL of a blood sample, different biologically active compounds were entrapped into erythrocytes within a period of 2 h at room temperature under blood banking conditions. The process is based on two sequential hypotonic dilutions of washed erythrocytes followed by concentration with a hemofilter and an isotonic resealing of the cells. There was 30% drug loading with 3550% cell recovery. The processed erythrocytes had normal survival in vivo. The same cells could be used for targeting by improving their recognition by tissue macrophages. [22]

Hypotonic dilution: Hypotonic dilution was the first method investigated for the encapsulation of chemicals into erythrocytes and is the simplest and fastest. In this method, a volume of packed erythrocytes is diluted with 220 volumes of aqueous solution of a drug. The solution tonicity is then restored by adding a hypertonic buffer. The resultant mixture is then centrifuged, the supernatant is discarded, and the pellet is washed with isotonic buffer solution. [23] The major drawbacks of this method include low entrapment efficiency and a considerable loss of hemoglobin and other cell components. This reduces the circulation half life of the loaded cells. These cells are readily phagocytosed by RES macrophages and hence can be used for targeting RES organs. Hypotonic dilution is used for loading enzymes such as galactosidase and glucosidase, asparginase and arginase, as well as bronchodilators such as salbutamol. [24] [25-26] Chemical perturbation of the membrane: This method is based on the increase in membrane permeability of erythrocytes when the cells are exposed to certain chemicals. In 1973, Deuticke et al. showed that the permeability of erythrocytic membrane increases upon exposure to polyene antibiotic such as amphotericin B. In 1980, this method was used successfully by Kitao and Hattori to entrap the antineoplastic drug daunomycin in human and mouse erythrocytes. Lin et al. used halothane for the same purpose. However, these methods induce irreversible destructive changes in the cell membrane and hence are not very popular. [27-29] Entrapment by endocytosis: This methodwas reported by Schrier et al. in 1975. Endocytosis involves the additionof one volume of washed packed erythrocytesto nine volumes of buffer containing2.5 mM ATP, 2.5 mM MgCl2, and1mM CaCl2, followed by incubation for2 min at room temperature. The porescreated by this method are resealed byusing 154 mM of NaCl and incubationat 370cfor 2 min. The entrapment ofmaterial occurs by endocytosis. The vesiclemembrane separates endocytosed materialfrom cytoplasm thus protecting itfrom the erythrocytes and vice-versa. Thevarious candidates entrapped by thismethod include primaquine and related8aminoquinolines, vinblastine, chlorpromazineand related phenothiazines,hydrocortisone, propranolol and tetracaine. [30-32] Loading by electric cell fusion: This method involves the initial loading of drug molecules into erythrocyte ghosts followed by adhesion of these cells to target cells. The fusion is accentuated by the application of an electric pulse, which causes the release of an entrapped molecule. An example of this method is loading a cell-

specific monoclonal antibody into an erythrocyte ghost. An antibody against a specific surface protein of target cells can be chemically cross-linked to drug-loaded cells that would direct these cells to desired cells. [33-34] Loading by lipid fusion: Lipid vesicles containing a drug can be directly fused to human erythrocytes, which lead to an exchange with a lipid-entrapped drug. [35] This technique was used for entrapping inositol mono phosphate to improve the oxygen carrying capacity of cells. However, the entrapment efficiency of this method is very low (1%). 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 and 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 by layer. It is greatest for a molecule with high lipid solubility. DELIVERY STRATEGIES As mentioned earlier, there are two major strategies in the delivery of drugs using erythrocytes as carriers which include intravenous slow drug release strategy and target gene delivery. Intravenous slow drug release strategy: The normal life-span of an erythrocyte in systemic circulation is about 120 days. As mentioned as an advantage, in the optimum conditions of the loading procedure, the life-span of the resulting carrier cells may be comparable to that of the normal erythrocytes. [36] Erythrocytes have been used as circulating intravenous slow-release carriers for the delivery of antineoplasms, antiparasitics, antiretroviral agents, vitamins, steroids, antibiotics and cardiovascular drugs among others. [37-42] A series of mechanisms have been proposed for drug release in circulation from carrier erythrocytes, including passive diffusion out of the loaded cells into circulation, specialized membrane-associated carriers, phagocytosis of the carrier cells by the macrophages of RES and, then, depletion of the drug into circulation, accumulation of the drug in RES upon lysis of the carrier and slow release from this system into circulation, accumulation of the carrier erythrocytes in lymphatic nodes following subcutaneous injection of the cells

and drug release upon hemolysis in this sites, and, finally, hemolysis in the injection sites. [43] Targeted drug delivery: RES or non-RES targeting is another important strategy using erythrocytes as carriers. RES targeting: It is a well-known fact that, in physiologic conditions, as a result of the gradual inactivation of the metabolic pathways of the erythrocyte by aging, the cell membrane loses its natural integrity, flexibility and chemical composition. These changes, in turn, finally result in the destruction of these cells upon passage through the spleen. The other effective site for the destruction of the aged or abnormal erythrocytes is the macrophages of the RES including peritoneal macrophages, hepatic Kupffer cells and alveolar macrophages of the lung, peripheral blood monocytes, and vascular endothelial cells. We know that aging and a series of other factors (e.g., stress during non-gentle loading methods) make the erythrocytes recognizable by the phagocyting macrophages via changing the chemical composition of the erythrocyte membrane, i.e., the phospholipids component. Therefore, a considerable fraction of carrier erythrocytes that have undergone some degrees of structural changes during the loading procedure will be trapped by the RES organs, mainly the liver and spleen, within a short time period after re-injection. [44-45] A series of approaches have been evaluated to improve RES targeting using carrier erythrocytes. In one of these approaches, the drug-loaded erythrocytes have been exposed to membrane stabilizing agents. This may increase the targeting index of the erythrocytes to RES via decreasing the deformability of these cells. [46] [47] Non-RES targeting: Recently, carrier erythrocytes have been used to target organs outside the RES. The various approaches include: * Co-encapsulation of paramagnetic particles or photosensitive agents in erythrocytes along with the drug to be targeted; * Application of ultrasound waves; * Site-specific antibody attachment to erythrocyte membrane. Chiarantini et al. have reported in vitro targeting of erythrocytes to cytotoxic T-cells by coupling them to Thy-1.2 monoclonal antibody. [48] Price et al. reported the delivery of colloidal particles and erythrocytes to tissue through micro vessel ruptures created by targeted micro bubble destruction with ultrasound. In another study, the differential response of photosensitized young and old erythrocytes to photodynamic activation has been studied by Rollan. [49-50]

APPLICATIONS OF RESEALED ERYTHROCYTES Resealed erythrocytes have several possible applications in various fields of human and veterinary medicine. Such cells could be used as circulating carriers to disseminate a drug within a prolonged period of time in circulation or in target-specific organs, including the liver, spleen, and lymph nodes. A majority of the drug delivery studies using drug-loaded erythrocytes are in the preclinical phase. In a few clinical studies, successful results were obtained. [51-53] Slow drug release: Erythrocytes have been used as circulating depots for the sustained delivery of antineoplastics, antiparasitics, veterinary antiamoebics, vitamins, steroids, antibiotics and cardiovascular drugs. The various mechanisms proposed for drug release include [54] * Passive diffusion * Specialized membrane associated carrier transport * Phagocytosis of resealed cells by macrophages of RES, subsequent accumulation of drug into the macrophage interior, followed by slow release. * Accumulation of erythrocytes in lymph nodes upon subcutaneous administration followed by hemolysis to release the drug. [55-57] Routes of administration include intravenous, which is the most common, followed by subcutaneous, intraperitoneal, intranasal, and oral. Studies regarding the improved efficacy of various drugs given in this form in animal models have been reported. Examples include an enhancement in antiinflammatory effect of corticosteroids in experimentally inflamed rats, increase in half life of isoniazid and levothyroxine. [58] Targeting the liver: Enzyme deficiency/replacement therapy: Many metabolic disorders related to deficient or missing enzymes can be treated by injecting these enzymes. However, the problems of exogenous enzyme therapy include a shorter circulation half life of enzymes, allergic reactions, and toxic manifestations. [59] Treatment of hepatic tumors: Hepatic tumors are one of the most prevalent types of cancer. Antineoplastic drugs such as methotrexate, bleomycin has been successfully delivered by erythrocytes. Agents such as daunorubicin diffuse rapidly from the cells upon loading and hence pose a problem. This problem can be overcome by covalently linking daunorubicin to the erythrocytic membrane using gluteraldehyde as a spacer. The resealed erythrocytes loaded with carboplatin show localization in liver. [60] [61]

Treatment of parasitic diseases: The ability of resealed erythrocytes to selectively accumulate within RES organs make them useful tool during the delivery of antiparasitic agents. Parasitic diseases that involve harboring parasites in the RES organs can be successfully controlled by this method. Results were favorable in studies involving animal models for erythrocytes loaded with antimalarial, antileishmanial and antiamoebic drugs. [62] Removal of RES iron overload: Desferrioxamine-loaded erythrocytes have been used to treat excess iron accumulated because of multiple transfusions to thalassemic patients. Targeting this drug to the RES is very beneficial because the aged erythrocytes are destroyed in RES organs, which results in an accumulation of iron in these organs. [63] Removal of toxic agents: Cannon et al. reported inhibition of cyanide intoxication with murine carrier erythrocytes containing bovine rhodanase and sodium thiosulfate. Antagonization of organophosphorus intoxication by resealed erythrocytes containing a recombinant phosphodiestrase also has been reported. [64] [65] Delivery of antiviral agents: Several reports have been cited in the literature about antiviral agents entrapped in resealed erythrocytes for effective delivery and targeting. Because most antiviral drugs are nucleotides or nucleoside analogs, their entrapment and exit through the membrane needs careful consideration. Nucleosides are rapidly transported across the membrane whereas nucleotides are not and thus exhibiting prolonged release profiles. The release of nucleotides requires conversion of these moieties to purine or pyrimidine bases. Resealed erythrocytes have been used to deliver deoxycytidine derivatives, recombinant herpes simplex virus type 1 (HSV-1) glycoprotein B, azidothymidine derivatives, azathioprene, acyclovir, and fludarabine phosphate. [66-68] Enzyme therapy: Enzymes are widely used in clinical practice as replacement therapies to treat diseases associated with their deficiency (e.g., Gauchers disease, galactosuria), degradation of toxic compounds secondary to some kind of poisoning (cyanide, organophosphorus), and as drugs. The problems involved in the direct injection of enzymes into the body have been cited. One method to overcome these problems is the use of enzyme-loaded erythrocytes. These cells then release enzymes into circulation upon hemolysis act as a circulating bioreactors in which substrates enter into the

cell, interact with enzymes, and generate products or accumulate enzymes in RES upon hemolysis for future catalysis. [69] [70] The most important application of resealed erythrocytes in enzyme therapy is that of asparginase loading for the treatment of pediatric neoplasm. This enzyme degrades aspargine, an amino acid vital for cells. This treatment prevents remission of pediatric acute lymphocytic leukemia. There are reports of improved intensity and duration of action in animal models as well as humans. Other enzymes used for loading resealed erythrocytes include urease, galactose-1-phosphate uridyl transferase, uricase, and acetaldehyde dehydrogenase. [71] Improvement in oxygen delivery to tissues: Hemoglobin is the protein responsible for the oxygen-carrying capacity of erythrocytes. Under normal conditions, 95% of hemoglobin is saturated with oxygen in the lungs, whereas under physiologic conditions in peripheral blood stream only 25% of oxygenated hemoglobin becomes deoxygenated. Thus, the major fraction of oxygen bound to hemoglobin is recirculated with venous blood to the lungs. The use of this bound fraction has been suggested for the treatment of oxygen deficiency. 2, 3-Diphosphoglycerate (2, 3-DPG) is a natural effector of hemoglobin. The binding affinity of hemoglobin for oxygen changes reversibly with changes in intracellular concentration of 2, 3-DPG. This compensates for changes in the oxygen pressure outside of the body, as the affinity of 2, 3-DPG to oxygen is much higher than that of hemoglobin. [72] Microinjection of macromolecules: Biological functions of macromolecules such as DNA, RNA, and proteins are exploited for various cell biological applications. Hence, various methods are used to entrap these macromolecules into cultured cells (e.g., microinjection). A relatively simple structure and a lack of complex cellular components (e.g., nucleus) in erythrocytes make them good candidates for the entrapment of macromolecules. [73] In microinjection, erythrocytes are used as microsyringes for injection to the host cells. The microinjection process involves culturing host eukaryotic cells in vitro. The cells are coated with fusogenic agent and then suspended with erythrocytes loaded with the compound of interest in an isotonic medium. Sendai virus (hemagglutinating virus of Japan, HVJ) or its glycoproteins or polyethylene glycol have been used as fusogenic agents. The fusogen causes fusion of co-suspended erythrocytes and eukaryotic cells. Thus, the contents of resealed erythrocytes and the compound of interest are transferred to host cell. This procedure has been used to microinject DNA fragments, proteins, nucleic acids to various eukaryotic cells. [74]

Advantages of this method include quantitative injection of materials into cells, simultaneous introduction of several materials into a large number of cells, minimal damage to the cell, avoidance of degradation effects of lysosomal enzymes, and simplicity of the technique. Disadvantages include a need for a larger size of fused cells, thus making them amenable to RES clearance, adverse effects of fusogens, and unpredictable effects on cell resulting from the co- introduction of various components. Hence, this method is limited to mainly cell biological applications rather than drug delivery. [75] [76] Other applications of resealed erythrocytes include * surface modification with antibodies * surface modification with gluteraldehyde * surface modification with carbohydrates such as sialic acid * entrapment of paramagnetic particles along with the drug * entrapment of photosensitive material * antibody attachment to erythrocyte membrane to get specificity of action NOVEL APPROACHES: Erythrosomes:These are specially engineered vesicular systems that are chemically cross-linked to human erythrocytes support upon which a lipid bilayer is coated. This process is achieved by modifying a reverse-phase evaporation technique. These vesicles have been proposed as useful encapsulation systems for macromolecular drugs. [77] [78] Nanoerythrosomes:These are prepared by extrusion of erythrocyte ghosts to produce small vesicles with an average diameter of 100 nm. Daunorubicin was covalently conjugated to nanoerythrosomes using gluteraldehyde spacer. This complex was more active than free daunorubicin alone. [79] [80] CONCLUSION: During the past decade, numerous applications have been proposed for the use of resealed erythrocytes as carrier for drugs, enzyme replacement therapy etc. The use of resealed erythrocytes looks promising for a safe and sure delivery of various drugs for passive and active targeting. However, the concept needs further optimization to become a routine drug delivery system. The same concept also can be extended to the delivery of biopharmaceuticals and much remains to be explored regarding the potential of resealed erythrocytes. For the present, it is concluded that erythrocyte carriers are golden eggs in novel drug delivery systems considering their tremendous potential.Most of the studies in this area are in the in vitro phase and the ongoing projects worldwide remain to step into preclinical

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