RESEALED EYTHROCYTES: RESEALED EYTHROCYTES PRESENTATION BY: MOHAMMED ABDUL KAREEM B.PHARMACY MESCO COLLEGE OF PHARMACY INTRODUCTION: INTRODUCTION Erythrocytes, the most abundant cells in the human body extensively studied for their potential carrier capabilities for the delivery of drugs. Such drug-loaded carrier erythrocytes are prepared simply by collecting blood samples from the organism of interest, separating erythrocytes from plasma, entrapping drug in the erythrocytes, and resealing the resultant cellular carriers. Hence, these carriers are called resealed erythrocytes. ERYTHROCYTES: ERYTHROCYTES Erythrocytes are most abundant cells in human body (5.4 -4.4 million cells/mm 3 blood ). Biconcave in shape and flexible in nature & carries O 2 from lungs to the tissues . They have a life span of 120 days and are reproduced ( erythropoiesis ) in the bone marrow under the regulation of erythropoietin. Erythrocytes have a solid content of about 35% most of which is Hb and rest 65% being water. ESSENTIAL PROPERTIES: Appropriate size & shape. Biocompatible & minimum toxic side effects. Minimum leakage before target site is achieved. Should be able to carry broad spectrum of drugs. Appreciable stability during storage period Should have sufficient space & should carry adequate amounts of drugs. ESSENTIAL PROPERTIES SOURCE AND ISOLATION: SOURCE AND ISOLATION SOURCE - Various types of mammalian erythrocytes have been used for drug delivery, including erythrocytes of mice, cattle, dogs, sheep, goats, monkeys, chicken, rats, and rabbits. ORGANISM OF INTEREST ISOLATION:: ISOLATION : Blood is collected in heparinized tubes by venipuncture. Fresh blood is used for loading purposes because the encapsulation efficiency of is higher than that of the aged blood. Erythrocytes are then harvested, The blood is centrifuged at 2500 rpm for 5 min at 4 ±1 0 C . Packed cells washed three times with phosphate buffer saline (pH=7.4). Suspended in buffer solutions at various hematocrit values as desired and are often stored in acid–citrate–dextrose buffer at 4 0 C for as long as 48 hrs before use . METHODS OF DRUG LOADING: METHODS OF DRUG LOADING METHODS OF DRUG LOADING: METHODS OF DRUG LOADING Hypotonic hemolysis. Hypotonic dilution. Hypotonic preswelling. Hypotonic dialysis. Use of red cell loader. Isotonic osmotic lysis. Chemical perturbation of the membrane. Electro-insertion or electroencapsulation. Entrapment by endocytosis. Loading by lipid fusion. HYPOTONIC HEMOLYSIS: HYPOTONIC HEMOLYSIS HYPOTONIC HEMOLYSIS:: HYPOTONIC HEMOLYSIS: RUPTURING & FILLING Erythrocytes to undergo reversible swelling in a hypotonic solution. An increase in volume leads to an initial change in the shape from biconcave to spherical. The cells can maintain their integrity up to a tonicity of 150mosm/kg above which the membrane ruptures, releasing the cellular contents . HYPOTONIC HEMOLYSIS:: HYPOTONIC HEMOLYSIS: PowerPoint Presentation: Membrane ruptured RBC Loaded RBC Resealed Loaded RBC 0.4% NaCl Hypotonic Drug Loading buffer Resealing buffer Incubation at 25 0 c HYPOTONIC HEMOLYSIS: RBC Chemicals – Urea, Polyethylene, Polypropylene, and NH4Cl HYPOTONIC HEMOLYSIS:: HYPOTONIC HEMOLYSIS: These ruptured erythrocytes as drug carriers is based on the fact that the ruptured membranes can be resealed by restoring isotonic conditions. Upon incubation at 25 o c the cells resume their original biconcave shape and recover original impermeability. HYPOTONIC DILUTION: HYPOTONIC DILUTION HYPOTONIC DILUTION: HYPOTONIC DILUTION In this method, a volume of packed erythrocytes is diluted with 2–20 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. HYPOTONIC DILUTION: HYPOTONIC DILUTION Compounds that can be encapsulated are enzymes such as -galactosidase and -glucosidase , asparginase, and arginase, salbutamol. RBC Hypertonic / Drug solution Membrane ruptured RBC Isotonic solution Resealed erythrocytes HYPOTONIC PRESWELLING: HYPOTONIC PRESWELLING HYPOTONIC PRESWELLING: HYPOTONIC PRESWELLING The technique is based upon initial controlled swelling in a hypotonic buffered solution. This mixture is centrifuged at low g values. The supernatant is discarded and the cell fraction is brought to the lysis point by adding 100–120 L portions of an aqueous solution of the drug to be encapsulated. The mixture is centrifuged between the drug-addition steps. The lysis point is detected by the disappearance of a distinct boundary between the cell fraction and the supernatant upon centrifugation. The tonicity of a cell mixture is restored at the lysis point by adding a calculated amount of hypertonic buffer. Then, the cell suspension is incubated at 37 C to reanneal the resealed erythrocytes. PowerPoint Presentation: Loaded RBC Resealed Loaded RBC Loading buffer Resealing buffer Incubation at 25 0 c HYPOTONIC PRESWELLING 0.6%w/v NaCl 5 min incubation at 0 0 c Swelled RBC RBC Drugs: Propranolol, Asparginase, Methotrexate, Insulin , Metronidazole , Levothyroxine, Isoniazid. ISOTONIC OSMOTICS LYSIS: ISOTONIC OSMOTICS LYSIS ISOTONIC OSMOTICS LYSIS: This method, also known as the osmotic pulse method, involves isotonic hemolysis that is achieved by physical or chemical means. The isotonic solutions may or may not be isoionic. If erythrocytes are incubated in solutions of a substance with high membrane permeability, the solute will diffuse into the cells because of the concentration gradient. This process is followed by an influx of water to maintain osmotic equilibrium. ISOTONIC OSMOTICS LYSIS ISOTONIC OSMOTICS LYSIS: ISOTONIC OSMOTICS LYSIS RBC Physical/chemical rupturing Isotonic buffer Drug Isotonically ruptured RBC Resealed RBC loaded RBC Incubation at 25 o c Compounds encapsulated are – Urea, polyethylene, polypropylene, and NH4Cl Entrapment By Endocytosis:- : Entrapment By Endocytosis:- Entrapment By Endocytosis:- : Entrapment By Endocytosis:- RBC Drug Suspension + Buffer containing ATP, MgCl 2 , and CaCl 2 At 25 0 C Loaded RBC Resealing Buffer Resealed RBC drugs : primaquine ,quinolines, vinblastine, chlorpromazine, phenothiazines, propranolol, vitamin A. PowerPoint Presentation: Fig:- Entrapment By Endocytosis Method FIG: ENTRAPMENT BY ENDOCYTOSIS: FIG: ENTRAPMENT BY ENDOCYTOSIS DRUG DRUG ENGULFED HYPOTONIC DIALYSIS: HYPOTONIC DIALYSIS PowerPoint Presentation: Hypotonic Dialysis In the process, an isotonic, buffered suspension of erythrocytes with a hematocrit value of 70–80 is prepared and placed in a conventional dialysis tube immersed in 10–20 volumes of a hypotonic buffer. The medium is agitated slowly for 2 h. The tonicity of the dialysis tube is restored by directly adding a calculated amount of a hypertonic buffer to the surrounding medium or by replacing the surrounding medium by isotonic buffer. The drug to be loaded can be added by dissolving the drug in isotonic cell suspending buffer inside a dialysis bag at the beginning of the experiment. Hypotonic Dialysis: Hypotonic Dialysis 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 Dialysis bag placed in Resealing buffer with mechanical rotator 30 min 37c. Resealed RBC DRUGS: gentamicin, pentamidine, interlukin-2 , desferroxamine and recombinant erythropoietin. PowerPoint Presentation: Hypotonic Dialysis Electro-insertion or Electro-encapsulation: Electro-insertion or Electro-encapsulation PowerPoint Presentation: This method is based on the observation that electrical shock brings about irreversible changes in an erythrocyte membrane. The use of transient electrolysis to generate desirable membrane permeability for drug loading. The erythrocyte membrane is opened by a dielectric breakdown. Subsequently, the pores can be resealed by incubation at 37 O C in an isotonic medium. Electro-insertion or Electro-encapsulation Electro-insertion or Electro-encapsulation: Electro-insertion or Electro-encapsulation RBC 2.2 Kv Current for 20 micro sec At 25 0 C Pulsation medium + + Drug Loading suspension 3.7 Kv Current for 20 micro sec Isotonic NaCl Loaded RBC Resealing Buffer Resealed RBC DRUGS : Urease , Methotrexate , isoniazid , human glycophorin , DNA fragments, and latex particles of diameter 0.2 m. PowerPoint Presentation: Electro-insertion or Electro-encapsulation Fig:- Electro-encapsulation Method ENTRAPMENT BY RED CELL LOADER: ENTRAPMENT BY RED CELL LOADER ENTRAPMENT BY RED CELL LOADER: ENTRAPMENT BY RED CELL LOADER It is a novel method for entrapment of nondiffusible 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 35–50% 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. RED CELL LOADER: RED CELL LOADER RED CELL LOADER Chemical perturbation method: Chemical perturbation method Chemical perturbation method: Chemical perturbation method “THIS METHOD IS BASED ON THE FACT THAT THE PERMEABILITY OF THE ERYTHROCYTES INCREASES ON EXPOSURE TO CERTAIN CHEMICAL AGENTS .” However, these methods induce irreversible destructive changes in the cell membrane and hence are not very popular. RBC Amphotericin B Drug RBC with increased permeability Resealing Buffer Resealed RBC ROUTES OF ADMINISTRATION: ROUTES OF ADMINISTRATION Routes of administration include: Intravenous (most common). subcutaneous. Intraperitoneal. Intranasal . MECHANISM OF DRUG RELEASE: MECHANISM OF DRUG RELEASE There are mainly three ways for a drug to efflux out from erythrocyte carriers. Phagocytosis. Diffusion through the membrane of the cell. Using a specific transport system. INVITRO CHARACTERISATION OF RESEALED ERYTHROCYTES: INVITRO CHARACTERISATION OF RESEALED ERYTHROCYTES APPLICATIONS: APPLICATIONS 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 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 APPLICATIONS: APPLICATIONS Enzyme replacement therapy : ß galactosidase, ß-fructo-furonodase, urease. Thrombolytic activity : Brinase, Aspirin, Heparin. Immuno therapy : Human recombinant Interleukin-2 Circulating carriers : Albumin, Salbutamol, Tyrosine kinase, Prednisolone Targeting to RES : Pentamidine, Mycotoxine, Homidium bromide. Iron overload : Desferroxamine Chemotherapy: Rubomucin, Methotrexate , Doxorubicin. Novel Systems : Novel Systems Nanoerythrosomes : An erythrocytes 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. Daunorubicin (DNR) was covalently conjugated to the nEryt (nEryt-DNR) using glutaraldehyde as homobifunctional linking arm. This led to a complex that is more active than free DNR both in vitro and in vivo. Daunorubicin (DNR) conjugated to these nanoerythrosomes has a higher antineoplastic index than the free drug. Erythrosomes.: 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 form macromolecular drugs. CONCLUSION: CONCLUSION 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. REFERENCE:: REFERENCE: R. Green and K.J.Widder, Methods in Enzymology (Academic Press, San Diego, 1987), p. 149. C. Ropars, M. Chassaigne, and C.Nicoulau, Advances in the BioSciences, (Pergamon Press, Oxford, 1987), p. 67. D.A. Lewis and H.O. Alpar, “Therapeutic Possibilities of Drugs Encapsulated in Erythrocytes,” Int. J. Pharm. 22, 137–146 (1984). U. Zimmermann, Cellular Drug-Carrier Systems and Their Possible Targeting In Targeted Drugs, EP Goldberg, Ed. (John Wiley & Sons, New York, 1983), pp. 153–200. S. Jain and N.K. Jain, “Engineered Erythrocytes as a Drug Delivery S.P. Vyas and V.K. Dixit, Pharmaceutical Biotechnology 1 (CBS Publishers & Distributors, New Delhi, 655.(1999).