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resealed erythrocytes


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resealed erythrocytes

  1. 1. Targeted Drug Delivery System Resealed Erythrocytes
  2. 2. Learning objectives : <ul><li>Introduction </li></ul><ul><li>Basic features of erythrocytes </li></ul><ul><li>Source, fraction and isolation of erythrocytes </li></ul><ul><li>Methods of drug loading </li></ul><ul><li>In vitro characterization </li></ul><ul><li>Different forms of drug loaded RBCs </li></ul><ul><li>Applications </li></ul><ul><li>Novel systems </li></ul>
  3. 3. Blood Cells <ul><li>Blood cells are formed in the bone marrow. </li></ul><ul><li>All blood cells arise from the same bone marrow stem cells. </li></ul><ul><li>Stem cells are immortal - they never die (at least not until you do). </li></ul><ul><li>Stem cells are also undifferentiated - they have not yet developed into a particular cell type. </li></ul><ul><li>Stem cells are pluripotent - they have the potential to become any type of blood cell. </li></ul>
  4. 4. Blood Cells <ul><li>These immortal, undifferentiated, pluripotent stem cells give rise to erythrocytes, leukocytes and platelets. </li></ul><ul><li>different types of blood cells, Leukocytes, also known as white blood cells, are a group of related cell types that involved in immune function. Leukocytes include neutrophils, eosinophils, basophils, lymphocytes and monocytes. </li></ul>
  5. 6. Red blood c ells : <ul><li>Red blood c ells (RBC, erythrocytes) were discovered in 1658. </li></ul><ul><li>The prime function of these RBCs is to transport gases for respiratory process. </li></ul><ul><li>Erythrocytes are produced through a process called erythropoesis. </li></ul><ul><li>Erythrocytes are flexible in nature. </li></ul><ul><li>The carrier potential of these cells was first realized in early 1970. </li></ul>
  6. 7. Red Blood Cells (Erythrocytes, RBCs) Two million new RBCs are formed every second Diameter of RBC is 6-9 μ Composed of ~90% hemoglobin Each RBC contains ~270 million hemoglobin molecules RBCs have a life-span of ~120 days before they are removed by the spleen Typically 4-8 x 106 cells per ml in normal human blood 6-9 µ
  7. 8. Basic features of erythrocytes : Composition of erythrocytes :
  8. 9. <ul><li>Composition of erythrocytes : </li></ul><ul><li>The blood contains about 55% of fluid portion (plasma) and nearly 45% of corpuscles or formed elements. </li></ul><ul><li>The normal blood cells have extensile, elastic , bioconcave and non nucleated configuration with a diameter ranging from 6-9 µ and the thickness is nearly 1-2 µ. </li></ul><ul><li>Erythrocytes have a solid content of about 35% an rest of 65% being water. </li></ul>
  9. 10. <ul><li>Electrolyte composition of erythrocytes : </li></ul><ul><li>The electrolyte composition of the erythrocytes is although qualitatively similar to that of plasma however, quantitatively it differs from that of plasma. </li></ul><ul><li>The concentration of k + is more in erythrocytes and Na + in plasma. </li></ul><ul><li>The osmotic pressure of the interior of the erythrocytes is equal to that of the plasma and termed as isotonic.(0.9% NaCl) </li></ul><ul><li>Changes in the osmotic pressure of the medium surrounding the red blood cells changes the morphology of the cells. </li></ul>
  10. 11. <ul><li>Whole blood is composed of plasma (liquid), cells and platelets. </li></ul><ul><li>If blood is placed into a tube and centrifuged, the cells and the plasma will separate. </li></ul><ul><li>The erythrocytes, which are heavy, will pack into the bottom of the tube, the plasma will be at the top of the tube, and the leukocytes and platelets will form a thin layer (buffy coat) between the erythrocytes and the plasma. </li></ul><ul><li>Hematocrit value and erythrocytes sedimentation rate : </li></ul>
  11. 12. <ul><li>The hematocrit is defined as the percentage of whole blood made up of erythrocytes. </li></ul><ul><li>This value is determined by dividing the height of the erythrocytes by the total height of the blood in the tube and multiplying by 100. </li></ul>males.......... 40-50% females....... 38-45% athletes........ > 50%
  12. 13. Source, fractionation and isolation of erythrocytes : <ul><li>Different mammalian erythrocytes have been used for drug loading, resealing and subsequent use in drug and enzyme delivery. </li></ul><ul><li>E.g. mice, cattle, pigs, dogs, sheep, goats, monkeys, chicken, rats, and rabbits etc. </li></ul><ul><li>To isolate erythrocytes, blood is collected into the heparinized tubes by vein puncture. </li></ul><ul><li>EDTA or heparin can be used as anticoagulants agents. </li></ul>
  13. 14. Advantages : <ul><li>Biodegradable with no generation of toxic effect. </li></ul><ul><li>Relatively inert intracellular environment. </li></ul><ul><li>Wide variety of the drug can be entrapped. </li></ul><ul><li>Ability to circulate through out the body. </li></ul><ul><li>Their ability to target the organ of the RES. </li></ul><ul><li>The considerably uniform size and shape of the carrier. </li></ul><ul><li>The modification of pharmacokinetic and pharmacodynamic parameter of drug. </li></ul>
  14. 15. METHODS OF DRUG LOADING <ul><li>Irrespective of the method used, the optimal characteristic for the successful entrapment of the compound the drug to have </li></ul><ul><li>a considerable degree of water solubility </li></ul><ul><li>Resistance against degradation within erythrocytes </li></ul><ul><li>Lack of physical and chemical interaction with erythrocyte membrane </li></ul><ul><li>Well-defined pharmacokinetic and pharmacodynamic properties. </li></ul>
  15. 16. Electro encapsulation Drug loading in Resealed erythrocytes Membrane perturbation Hypo-osmotic lysis Lipid fusion endocytosis Osmotic-lysis Preswell method Dilution method
  16. 17. Hypotonic haemolysis and isotonic Resealing Methods : <ul><li>This method is based upon hypotonic lysis of cells in a solution containing the drug and enzyme. </li></ul><ul><li>The ghost populations so obtained are heterogeneous, the three type of the ghost can be distinguished </li></ul><ul><li>Type I : ghost which reseal immediately after haemolysis </li></ul><ul><li>Type II : ghost which reseal after reversal of haemolysis by addition of alkali ions </li></ul><ul><li>Type III : ghost which remain leaky under different experimental condition </li></ul>
  17. 18. <ul><li>Increase in volume initially leads to conversion of normal biconcave discocytes to spherocytes. </li></ul><ul><li>This swollen erythrocytes have little capability resist volume greater than 50 -75% of the initial volume. </li></ul><ul><li>The principle of using erythrocytes as a drug carriers is these ruptured membrane can be resealed by raising the salt concentration to its original levels and upon incubation the cells resume their normal biconcave shape and recover original permeability. </li></ul><ul><li>e.g. Erythrocytes are resealed on addition of sufficient 1.54 M KCL for isotonicity. </li></ul>
  18. 19. Loading by “ Red Cell Loader” <ul><li>Magnani and coworkers,1998 developed a novel method for the entrapment of nondiffusible drugs into human erythrocytes. </li></ul><ul><li>The method require as little as 50ml of blood. </li></ul><ul><li>The method is based on two sequential and controlled hypotonic dilutions of washed red blood cells followed by concentration with haemofilter. </li></ul><ul><li>Subsequent isotonic resealing of erythrocytes allow a 35-50% cell recovery and approximate 30% entrapment of added drug. </li></ul>
  19. 21. Dilution haemolysis <ul><li>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 become permeable to macromolecules and ions. </li></ul><ul><li>One volume of washed erythrocytes could be treated with 2-20 volumes of materials to be loaded in a hypotonic solution and for resealing incubation at 25.C in an isotonic solution. </li></ul><ul><li>e.g. enzymes such as ß -galactosides , ß -glucosides, asparginase and arginase. </li></ul>
  20. 22. Preswell Dilution Haemolysis <ul><li>The technique is based upon initial controlled swelling in a slightly hypotonic solution </li></ul><ul><li>This mixture is centrifuged at low ‘g’ value. </li></ul><ul><li>The cell fraction is brought to the lysis point by adding small portion of an solution of drug to be encapsulated. </li></ul><ul><li>The lysis point is detected by the disappearance of a distinct boundary between cell fraction and the supernatant upon centrifugation. </li></ul><ul><li>Add hypertonic solution and incubate at 37.C </li></ul><ul><li>e.g. propranolol, asparginase, methotrexate, insulin </li></ul>
  21. 23. Isotonic osmotic lysis <ul><li>Haemolysis can be achieved both by chemical and physical means. </li></ul><ul><li>If erythrocytes are incubated in isotonic solution of a substance with high membrane permeability, the solute will diffuse into the cells due to inwardly directed chemical potential gradient. </li></ul><ul><li>This will followed by water up take until osmotic equilibrium is restored. </li></ul><ul><li>The chemicals such as urea solution, polyethylene glycol and ammonium chloride. </li></ul>
  22. 24. Time consuming, impermeable to large molecules Better in vivo surveillance --- Isotonic osmotic lysis --- Good retention of cytoplasm 20-70% Preswell dilution Less Entrapment efficiency Fastest and simplest for low mol. Wt. 1-8% Dilution method Disadvantages Advantages %Loading Method
  23. 25. Electro-insertion / Electro-encapsulation : <ul><li>In 1977, Tsong and Kinosita suggested the use of transient electrolysis to generate desirable permeability for drug loading. </li></ul><ul><li>The erythrocyte membrane is opened by dielectric breakdown. Subsequently the pore can be resealed by incubation at 37.C in an isotonic medium. </li></ul><ul><li>The optimum intensity of an electric field is between 1-10kV/cm, and optimal discharge time is between 20-160 </li></ul>
  24. 26. <ul><li>The extent of pore formation depends upon the electrical field strength, pulse duration and ionic strength of the suspending medium. </li></ul><ul><li>Entrapment efficiency of this method is 35%, and life span of the resealed cells in circulation is same with that of normal cells. </li></ul><ul><li>Various compounds such as sucrose, urease, methotrexate, isoniazid, human glycoprotine, DNA fragment, etc can be entrapped within the erythrocytes by this method. </li></ul>
  25. 27. Chemical perturbation of membrane <ul><li>This method is based on the increase in membrane permeability of erythrocytes when the cells are exposed to certain chemicals. </li></ul><ul><li>In 1973, deuticke etal. Showed that the permeability of erythrocytic membrane increase upon exposure to polyene antibiotic such as amphotericin B. </li></ul><ul><li>This method induce irreversible destructive changes in the cell membrane. </li></ul><ul><li>The in-vivo survival of loaded erythrocytes by this method is found to be poor. </li></ul>
  26. 28. Lipid fusion <ul><li>Lipid vesicles containing drug can be directly fused with human erythrocytes . </li></ul><ul><li>Nicolau and gresonele,1979 used this technique for loading of inositol hexaphosphate into resealed erythrocytes. </li></ul><ul><li>The encapsulation efficiency by this method is low (1 %). </li></ul>
  27. 29. In-vitro characterization
  28. 31. Different forms of drug loaded RBCs <ul><li>Normally, more than 80% erythrocyte ghosts loaded with drug or enzyme appear as biconcave under electron microscope. </li></ul><ul><li>Less than 20% cells show abnormal morphology, and the rest appear as stomatocytes or spsherocytes or echinocytes, cells with different infoldings and other destroyed forms. </li></ul><ul><li>Some of the cells appear as pale or transparent in appearance and are referred to as “Erythrocytes ghosts” or “White resealable erythrocytes ghosts”. </li></ul>
  29. 32. <ul><li>Applications : </li></ul><ul><li>Erythrocytes as Drug / Enzyme carriers </li></ul><ul><li>Drug targeting </li></ul><ul><li>Thrombolytic Therapy </li></ul><ul><li>( Thromboembolism – Heparine ) </li></ul><ul><li>Delivery of Interleukins </li></ul><ul><li>(high dose toxicity and rapid clearance ) </li></ul><ul><li>Oxygen Defficiency Therapy </li></ul>
  30. 33. Pentamidine, Mycotoxine, HOmidium bromide Targeting to RES Albumin, Salbutamol, Tyrosine kinase circulating carriers Human recombinant interleukin-2 Immuno therapy Brinase, Aspirine, Heparine Thrombolytic activity ß-galactosidase, ß-fructo-furoodase, urease Enzyme deficiency, replacement therapy Drug / Enzyme / Macromolecule Application
  31. 34. Novel System <ul><li>Nanoerythrosomes : </li></ul><ul><li>An erythrocytes based new drug carrier, named nanoerythrosome has been developed which is prepared by extrusion of erythrocyte ghost to produce small vesicles having an average diameter of 100 nm. </li></ul><ul><li>Erythrosomes : </li></ul><ul><li>Erythrosome are specially engineered vesicular system in which chemically cross-linked human erythrocyte cytoskeletons are used as a support up on which bilayer is coated. </li></ul>