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Liposome Contents 2 Introduction Mechanism of liposome formation Classification Biological fate of liposome Methods of preparation Characterization Advantages & Disadvantages Applications 3 Introduction Lipo – fat or lipid and some-body Liposomes are simple micro particulate drug carrier consisting of one or more concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids. When Phospholipid come in contact with water they formed spherical structure enclosing aqueous compartment. Discovered in 1960’s by Bangham and coworkers. The structural main components are phospholipids and cholesterol 4 Lets take a look at liposome 5 Phospholipids are amphipathic molecule i.e. having affinity for both aqueous & polar moieties, as they have a hydrophobic tail & hydrophilic head. The tail portion consist of 2 fatty acid chains having 10-24 carbon atoms & 0-6 double bonds in each chain. The head or polar portion consist of phosphoric acid bound to a water soluble molecule. 6 Phospholipids 7 8 Cholesterol by itself do not form a bilayer structure, it acts as fluidity buffer. That means below phase transition temperature it makes the membrane less ordered & slightly more permeable while above phase transition temperature it makes the membrane more ordered & stable. It inserts into membrane with hydroxyl group oriented towards aqueous surface & aliphatic chain aligned parallel to acyl chains in the centre of bilayer. Cholesterol alignment between phospholipid bilayer 9 Mechanism of liposome formation 10 Vesicles are formed by hydrophobic effect. 11 Ratio of hydrophilic & hydrophobic moieties. CPP ( Critical packing parameter) If CPP value is less than 0.5 than liposomes are formed by hydrophobic effect. If CPP value is more than 0.5 than liposomes are formed by hydrophilic effect. If CPP value is between 0.5-1.0 than the liposomes are formed by surfactant effect. CPP = v/ lc Ap = Ahp / Ap 12 Where: v = hydrophobic group volume lc = hydrophobic group length Ap = cross sectional area of hydrophilic head group Ahp = cross sectional area of hydrophobic group. Classification 13 On the basis of structural parameters: Multilamellar vesicles (> 0.5 um) MLV Oligolamellar vesicles (0.1-1 um) OLV Unilamellar vesicles (all size range) UV Small unilamellar vesicles (20-100 nm) SUV Medium sized unilamellar vesicles MUV Large unilamellar vesicles (> 100 um) LUV Giant unilamellar vesicles (>1 um) GUV Multi vesicular vesicles (>1 um) MVV On the basis of liposome preparation: Vesicles prepared by reverse phase evaporation method REV Multi lamellar vesicle by REV MLV-REV Stable plurilamellar vesicle SPLV Frozen & thawed MLV FATMLV Vesicles prepared by extrusion techniques VET Dried reconstituted vesicles DRV Different types of liposomes 14 Biological fate of liposome 15 Liposomes in blood stream Taken by reticulo-endothelial system Macrophages engulf liposomes ( endocytosis) Phagosome + lysosyme = phagolysosyme Membrane of phagolysosyme have proton pumps which decrease PH of phagolysosyme & the enzymes phospholipase destruct the liposomal membrane 16 17 Method of liposome preparation 18 Physical dispersion method:1. Hand shaking MLVs 2. Non-shaking LUVs 3. Freeze drying 4. Pro-liposomes To reduce liposome size: 1. Micro emulsification 2. Membrane extrusion 3. Ultrasonication 4. French pressure cell To increase liposome size: 1. Dried reconstituted vesicle 2. Freeze thawing 3. Induction of vesiculation by PH change Solvent dispersion method : 19 1. Ethanol injection 2. Ether injection 3. Water organic phase: A) Double emulsion method B) Reverse phase evaporation C) Stable plurilamellar vesicles Detergent solubilization : Hand shaken MLV’s Lipids + solvent ( chloroform: Methanol) ( In 250 ml RBF) Evaporate for 15 min above phase transition temperature (Flush with nitrogen) Till residues dry Add 5 ml buffer containing material to be entrapped Rotate flask at room temp, at 60 RPM for 30 min until lipid removes from wall of RBF Milky white dispersion (stand for 2 hours to get MLV) 2 Rotary Evaporator 21 Non Shaking vesicles Lipid + solvent 22 Evaporate at room temperature by flow of nitrogen for drying Add water saturated nitrogen until opacity disappears Add bulk fluid (drug) & 10-20 ml 0.2M sucrose solution to swell (Flush again with nitrogen) Stand for 2 hrs at 37º c, do not disturb for 2 hrs (Swirl to yield milky dispersion ) Centrifuge at 12000 rpm for 10 min at room temp (MLV on surface is removed) To remaining fluid add iso-osmolar glucose solution ( centrifuge at 12000 rpm) LUV is formed Pro liposome 23 Sorbitol / Nacl ( increase surface area of lipid film) + 5ml lipid solution ( fitted to evaporator ) (Evaporation) Again add lipid solution Dry the content using Lyophilizer ( freeze dryer) (Stand over night at room temp) Flushed with nitrogen for drying properly MLVs Freeze Drying 24 Lipid + Solvent ( Tertiary butanol) Freeze drying Add Aqueous phase / Saline containing drug Rapid mixing above phase transition temperature MLVs Micro emulsification liposome (MEL) 25 MEL is prepared by the “Micro fluidizer”, which pumps fluid at very high pressure (10,000 psi) through a 5 um orifice. Then, it is forced along defined micro channels, which direct two streams of fluid to colloid together at right angle at very high velocity. After a single pass, size reduced to a size 0.1& 0.2 um in diameter. Microfluidizer 26 Sonicated unilamellar vesicles 27 MLV in test tube Sonicate for 5-10 min above phase transition temp Filter & centrifuge at 100000 rpm for 30 min at 20º c Decant top layer to get Sonicated unilamellar vesicles BATH SONICATOR PROBE SONICATOR French Pressure Cell 28 French pressure cell is invented by ‘Charles stacy French’. In this technique the large vesicles are converted to small vesicles under very high pressure. This technique yields uni or oligo lamellar liposomes of intermediate size (30-80 nm in diameter depending on applied pressure). This liposomes are more stable as compared to sonicated liposomes. 29 To increase size of liposome: Freeze thaw sonication 30 SUV in aqueous phase + Solute Freeze drying FTS method, thawing = melting Sonication ( 15-30 sec) Solutes in unilamellar vesicle Dried reconstituted vesicle 31 SUV in aqueous phase + Solute Freeze drying DRV method: Rehydration, film stacks dispersed in aqueous phase Solute in uni or oligo lamellar vesicles. PH induced vesiculation 32 MLVs or LUVs ( PH 2.5-3) Add 1 M NaoH ( less than 2 min) PH rises to 11 Now add 0.1 M Hcl PH moves down to 7.5 SUV Change in PH brings about an increase in surface charge density of lipid bilayer, which induces spontaneous vesiculation Solvent dispersion method: Ethanol injection 33 Lipid + ethanol solution in the syringe Inject rapidly In the aqueous phase Small unilamellar vesicles Ether injection 34 Lipid + ether solution in the syringe Inject slowly In the aqueous phase ( On heated water bath, 60ºc) Large unilamellar vesicles Water organic phase: Double emulsion 35 Organic solution + Lipid + Aqueous phase Emulsion (W/O) Hot aqueous solution of buffer Multi compartment vesicle W/O/W (double emulsion) LUVs 36 Reverse phase evaporation: (MLV, LUV) 37 Emulsion Evaporation under reduced pressure, rotary evaporator Semi solid gel Shake to get LUVs “Lipid monolayer which enclosed the collapsed vesicle, is contributed to adjacent intact vesicle to form the outer leaflet of bilayer of LUV”. Stable plurilamellar vesicle (SPLVs) 38 It involves preparation of water in organic phase dispersion with an excess of lipid followed by drying under continued bath sonication with stream of nitrogen. Detergent dispersion: Phospholipids & aqueous phase comes in contact with the help of detergent Characterization of Liposome: Physical 39 Vesicle shape & lamellarity ( No. of bilayers): Sample + 31p NMR + Mangnese (affect signal intensity) If intensity is decrease by 50% = unilamellar vesicle are formed If intensity is decrease by more intensity = MLVs are formed Freeze fracture electron microscopy. Vesicle Size: Determined by: Light microscopy Fluorescent microscopy Electron microscopy: SEM, TEM Laser light scattering Gel permeation Ultracentrifugation 40 Surface charge: Determined by Electrophoresis Drug release: Dissolution Entrapped volume: (water content is determined) Water is replaced with deuterium oxide & is analyzed by NMR Encapsulation efficiency: Protamine aggregation method: Liposome + Protamine = Precipitation Centrifuge (2000 rpm), remove supernatant Liposome pellet + Trixon x-100 (surface breaker) The encapsulation efficiency can be determined (Analytically) Mini column centrifugation Chemical characterization: 41 1. Quantitative determination of phospholipids 2. Phospholipid hydrolysis 3. Phospholipid oxidation 4. Cholesterol analysis Phospholipid determination: (Bartlett assay) Phospholipid phosphorous + Hydrolysis= Inorganic phosphate. Inorganic phosphate +ammonium molybdate= phospho molybdic acid phospho molybdic acid + Amino naphthyl sulfonic acid= reduced to blue color whose intensity is measured & compared with standard Phospholipid hydrolysis: 42 Phospholipids + Hydrolysis= Lysolecithin One chain is lost by desterification Determined by HPLC Phospholipid oxidation: Free radical determination by UV, iodometric method, GLC etc. Cholesterol analysis: Cholesterol + Iron + Reagent (Ferric per chlorate, ethyl acetate & Sulfuric acid= Purple complex, which is determined at 610 nm. 43 THANK YOU -PHARMA STREET