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  2. 2. Liposomes Definition: Liposomes are simple microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid molecule. Structurally, liposomes are concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayers mainly composed of natural or synthetic phospholipids. 2
  3. 3. Advantages Provide selective passive targeting to tumour tissues (liposomal doxorubicin). Increased efficacy and therapeutic index. Increased stability via encapsulation. Reduction in toxicity of the encapsulated agent. Improved pharmacokinetic effects (reduced elimination, increased circulation life times). Flexibility to couple with site-specific ligand to achieve active targeting. 3
  4. 4. STRUCTURE AND COMPOSITION OF LIPOSOMES There are number of components of liposomes however phospholipids and cholesterol being main components. Phospholipids are the major structural components of biological membranes, where two types of phospholipids exist – phosphodiglycerides and sphingolipids, together with their corresponding hydrolysis products. The most common phospholipid is phosphatidylcholine (PC) molecule. 4
  5. 5.  Commonly used synthetic Phospholipids: DOPC = Dioleoyl Phosphatidylcholine DOPE = Dioleoyl phosphatidylethanolamine DSPC = Distearoyl phosphatidylcholine DSPE = Distearoyl phosphatidylethanolamine DLPC = Dilauryl phosphatidylcholine DMPC = Dimyristoyl phosphatidylcholine DLPE = Dilauryl phosphatidylethanolamine 5
  6. 6.  Cholesterols: Incorporation of sterols in liposome bilayer can bring about major changes in the preparation of these membranes. Cholesterol does not by itself form bilayer structure, but can be incorporated into phospholipid membranes in very high concentration unto 1:1 or even 2:1 molar ratios of PC. Cholesterol incorporation increases the separation between the cholin head groups and eliminates the normal electrostatic and hydrogen-bonding interactions. 6
  7. 7. Classification Based on structural parametersTYPE SPECIFICATIONSMLV Multilamellar large vesicles- >0.5 μmOLV Oligolamellar vesicles- 0.1-1 μmUV Unilamellar vesicles (all in size)SUV Small unilamellar vesicles-20-100nmMUV Medium sized unilamellar vesiclesLUV Large unilamellar vesicles->100nmGUV Giant unilamellar vesicles->1 μmMV Multivesicular vesicles->1 μm 7
  8. 8. Classification Based on the method of liposome preparationTYPE SPECIFICATIONSREV Single or oligolamellar vesicles made by reverse-phase evaporation methodMLV-REV Multilamellar vesicles made by reverse- phase evaporation methodSPLV Stable plurilamellar vesiclesFATMLV Frozen and thawed MLVVET Vesicles prepared by extrusion techniqueDRV Dehydration-rehydration method 8
  9. 9. Chemical CharacterizationCharacterization parameters Analytical methodsPhospholipid concentration Lipid phosphorous content using Barlett assay,HPLCCholesterol concentration Cholesterol oxidase assay and HPLCDrug concentration Appropriate method given in monographPhospholipid peroxidation UV absorbance, TBA,indometric and GLCPhospholipid hydrolysis HPLC and TLCCholesterol auto-oxidation HPLC and TLCAnti-oxidant degradation HPLC and TLCpH pH meter 9osmolarity Osmometer
  10. 10. Physical CharacterizationCharacterization parameters Analytical methodsVesicle shape and surface Transmission electronmorphology microscopy, freeze-fracture electron microscopy.Surface charge Free-flow electrophoresis.Lamellarity Small angle X-ray scattering, freeze-fracture electron microscopy, 31P-NMR.Phase behavior Freeze-fracture electron microscopy, differential scanning calorimetryPercent capture/percent of free Minicolumn centrifugation, geldrug exclusion, ion-exchange 10 chromatography, radiolabelling
  11. 11. Biological CharacterizationCharacterization Analytical methodsparameterssterility Aerobic or anaerobic culturespyrogenicity Limulus amebocyte lysate (LAL) testAnimal toxicity Monitoring survival rates, histology and pathology 11
  12. 12. TECHNIQUES OF LIPOSOMES PREPARATION(A) physical dispersion a) Hand-shaken multilamellar vesicles (MLVs) b) Non-shaking vesicles c) Pro-liposomes d) Freeze drying(B) Processing of lipids hydrated by physical means a) Micro emulsification liposomes (MEL) b) Sonicated unilamellar vesicles (SUVs) c) French pressure cell liposomes d) Membrane extrusion liposomes e) Dried-reconstituted vesicles (DRVs) f) Freeze thaw sonication (FTS) g) pH induced vesiculation h) Calcium induced fusion 12
  13. 13. (C ) Solvent dispersion methods Ethanol Injection Ether injection Water in organic phase Reverse phase evaporation vesicles Stable plurilamellar vesicles (SPVs) Double emulsion vesicles(D) DETERGENT SOLUBILIZATION 13
  14. 14. All the methods of preparing liposomes involve three or four basicstages: •Drying down lipids from organic solvent, •Dispersion of lipids in aqueous media, •Purification of resultant liposomes, and •Analysis of final product. 14
  15. 15. Physical dispersion:There are four basic method of physical dispersion. I.e. hand shaking,non-shaking, freeze dry and proliposomes. Hand-shaken multilamellar vesicles (MLVs) : 15
  16. 16. Sonicated Unilamellar Vesicles (SUVs) : 16
  17. 17. French pressure cell liposomes : Type of liposomes : ULV or OLV Pressure :20000 or 40000 Sample volume : maximum 40ml Minimum 4ml Out flow : 0.5-1 ml / min. 17
  18. 18. Membrane extrusion liposomes : Type of liposomes : MLV or LUV Pressure : 100 psi Type membranes : Tortuous path, Nucleation track (polycarbonate ) 18
  19. 19.  Solvent dispersion methods: In this methods, lipids are first dissolved in an organic solution, which is then brought into contact with the aqueous phase containing material to be entrapped within the liposomes. Methods employing solvent dispersion fall into one of three categories.  The organic solvent is miscible with the aqueous phase.  The organic solvent is miscible with the aqueous phase, the latter being in a large excess.  Organic solvent is in large excess, and is again immiscible 19
  20. 20. Ether injection &ethanol injection 20
  21. 21.  DETERGENT SOLUBILIZATION : In this method, the phospholipids are brought into intimate contact with the aqueous phase via the intermediary of detergents, which associate with phospholipid molecules and serve to screen the hydrophobic portions of the molecule from water. The structures formed as a result of this association are known as micelles, and can be composed of several hundred component molecules. Their shape and size depends on chemical nature of the detergent, the concentration and other lipids involved. As a general rule, membrane-solubilizing detergents have a higher affinity for phospholipid membranes than for the pure detergent micelles. 21
  23. 23.  Active loading method have following advantages over passive encapsulation techniques:  A high encapsulating efficiency and capacity.  A reduced leakage of the encapsulated compounds.  “Bed side” loading of drug thus limiting loss of retention of drug by diffusion or chemical degradation during storage.  Flexibility for the use of constitutive lipids, as drug is loaded after the formation of carrier units.  Avoidance of biological compound during preparation steps in the dispersion thus reducing safety hazards.  The transmembrane pH gradient can be developed using various method depending upon the nature of the drug be encapsulated.  For amphipathic weak bases by active loading procedures such as using a proton gradient or an ammonium sulphate gradient or calcium acetate gradient. 23
  24. 24.  STABILITY OF LIPOSOMES The stability studies could be broadly covered under two main sections.1. The stability in vitro, which covers the stability aspects prior to the administration of the formulation and with regard to the stability of the constitutive lipids.2. The stability in vivo, which covers the stability aspects once the formulation, is administered via various routes to the biological fluids. These include stability aspects in blood (serum) if administered by systemic route or in gastrointestinal tract, if administered by oral or preoral routes. 24
  25. 25. Stability in vitro  Lipid oxidation an peroxidation  Lipid hydrolysis  Long term and accelerated stability Stability after systemic administration Stability in vivo after oral administration 25
  26. 26.  APPLICATION OF LIPOSOMES Liposomes as drug/protein delivery vehicles  Controlled and sustained drug release in situ.  Enhanced drug solubilization  Altered pharmacokinetics and biodistribution  Enzyme replacement therapy and lysosomal storage disorders Liposomes in antimicrobial, antifungal and antiviral therapy  Liposomal drugs  Liposomal biological response modifiers Liposomes in tumour therapy  Carrier of small cytotoxic molecules  Vehicle for macomolecules as cytokines o genes 26
  27. 27.  Liposomes in gene delivery  Gene and antisense therapy  Genetic vaccination Liposomes in immunology  Immonoadjuvant  Immunomodulator  Immunodiagnosis Liposomes as artificial blood surrogates Liposomes as radiophamaceutical and radiodiagnostic cariers Liposomes in cosmetics an dermatology Liposomes in enzyme immobilization and bioreactor technology 27
  28. 28. Some liposomal formulation of Amphotericin BSystem Target disease Brand name ProductLiposomes Systemic fungal AmBisome NeXstar, USA (i.v) infection, Visceral leishmaniasisLiposomes Systemic fungal Amphocil SEQUUS, USA (i.v) infectionLiposomes Systemic fungal ABELECT The Liposome (i.v) infection company, USA 28
  29. 29. Liposomes in gene therapy:Type of Advantages Disadvantages vector Viral • Relative high transfection • Immunogenicity, presence of vector efficiency contaminants and safety • Vector restricted size limitation for recombinant gene • Unfavourable p’ceutical issue- large scale production, GMP, stability and costNon-viral • Favourable p’ceutical issue- • Relative low transfection large scale production, GMP, efficiency stability and cost • Plasmid independent structure • Low immunogenicity • Opportunity for chemical/physical manipulation 29
  30. 30. Various liposomal product in dermatology and cosmetics (launched or investigated) Vesicular Marketed by Liposomes and system ingredients CaptureTM Christian Dior Liposomes in gel with ingredients PlentitudeTM L’Oreal Tanning agent in liposomesDermosomeTM Microfluidics Skin care, loaded liposomes PentaTM Pentapharm Humectant pentavitin R in liposomesCoatsome NCTM Nichiya liposomes Liposomes with humectant Co 30
  31. 31. Imaging modality and required concentration of diagnostic moieties:Imaging modality Diagnostic moiety Concentrat ionγ-scintigraphy Diagnostic radio-nucleus 10-10M such as 111In, 99mTc, 67GaMagnetic Po-magnetic ions such as 10-4M resonance(MR) Gd, Mn and iron oxideComputed Iodine, Bromine an Barium 10-2M tomography(CT)Ultrasound imaging Gas (Air, Argon and or Nitrogen) Ultrasonography( US) 31
  32. 32. COMMERCIAL MANUFACTUING OF LIPSOMAL DRUGNO Problems Remedies1 Poor quality of the High quality products with improved raw material purification protocol and validated mainly the analytical techniques are available phospholipids.2 Pay load is too Use either lipophilic drug/lipophilic low prodrug of hydrophilic drug or using active techniques.3 Poor Quality control assay can be performed characterization of using sophisticated instruments and batch the to batch variability can be checked. physicochemical properties of the liposomes 32
  33. 33. NO Problems Remedies4 Shelf life is too Improved by appropriate cryoprotectant short and lyoprotectant and product can be successfully freeze dried.5 Scale up related Scaling up can be improved by problems carefully selecting method of preparation, sterilization by autoclaving or membrane filtration coupled with aseptic processing and pyrogen removal using properly validated LAL test6 Absence of any By choosing candidate potent drugs data on safety of with narrow therapeutic window the these carrier drug elated safety problems can be systems on alleviated. chronic use. 33
  34. 34. 34