Liposome ppt

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LIPOSOME

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Liposome:

Liposome Presented by : Riteksha Patel

Contents:

Contents Introduction Mechanism of liposome formation Classification Biological fate of liposome Methods of preparation Characterization Advantages & Disadvantages Applications 2

Introduction:

Introduction Liposomes are simple microscopic, concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids. Discovered in 1960’s by Bangham and coworkers. The structural main components are phospholipids and cholesterol 3

Lets take a look at liposome:

Lets take a look at liposome 4

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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. 5

Phospholipids:

Phospholipids 6

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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. 7

Cholesterol alignment between phospholipid bilayer:

Cholesterol alignment between phospholipid bilayer 8

Mechanism of liposome formation:

Mechanism of liposome formation 9

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Vesicles are formed by hydrophobic effect. 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. 10

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CPP = v/ lc Ap = Ahp / Ap Where: v = hydrophobic group volume lc = hydrophobic group length Ap = cross sectional area of hydrophilic head group Ahp = cross sectional area of hydrophobic group. 11

Classification:

Classification 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 12

Different types of liposomes:

Different types of liposomes 13

Biological fate of liposome:

Biological fate of liposome 14

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Method of liposome preparation:

Method of liposome preparation Physical dispersion method:- Hand shaking MLVs Non-shaking LUVs Freeze drying Pro-liposomes To reduce liposome size: Micro emulsification Membrane extrusion Ultrasonication French pressure cell To increase liposome size: Dried reconstituted vesicle Freeze thawing Induction of vesiculation by P H change 17

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Solvent dispersion method : Ethanol injection Ether injection Water organic phase: A) Double emulsion method B) Reverse phase evaporation C) Stable plurilamellar vesicles Detergent solubilization : 18

Hand shaken MLV’s:

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) 19

Rotary Evaporator :

Rotary Evaporator 20

Non Shaking vesicles:

Non Shaking vesicles Lipid + solvent 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 21

Pro liposome:

Pro liposome 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 22

Freeze Drying:

Freeze Drying Lipid + Solvent ( Tertiary butanol) Freeze drying Add Aqueous phase / Saline containing drug Rapid mixing above phase transition temperature MLVs 23

Micro emulsification liposome (MEL):

Micro emulsification liposome (MEL) 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. 24

Microfluidizer:

Microfluidizer 25

Sonicated unilamellar vesicles:

Sonicated unilamellar vesicles 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 26

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French Pressure Cell:

French Pressure Cell 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. 28

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Membrane extrusion liposomes:

Membrane extrusion liposomes In this technique vesicle contents are exchanged with dispersion medium during breaking & resealing of phosphate lipid bilayer as they pass through polycarbonate membrane. Less pressure is required here (> 100 psi), as compare to French pressure cell. Use to process MLVs and LUVs. Two types of membrane one is Tortuous ( zigzag) and another is Nucleation trach ( vertically parallel). 30

To increase size of liposome: Freeze thaw sonication:

To increase size of liposome: Freeze thaw sonication SUV in aqueous phase + Solute Freeze drying FTS method, thawing = melting Sonication ( 15-30 sec) Solutes in unilamellar vesicle 31

Dried reconstituted vesicle:

Dried reconstituted vesicle SUV in aqueous phase + Solute Freeze drying DRV method: Rehydration, film stacks dispersed in aqueous phase Solute in uni or oligo lamellar vesicles. 32

PH induced vesiculation :

P H induced vesiculation 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 P H brings about an increase in surface charge density of lipid bilayer, which induces spontaneous vesiculation 33

Solvent dispersion method: Ethanol injection:

Solvent dispersion method: Ethanol injection Lipid + ethanol solution in the syringe Inject rapidly In the aqueous phase Small unilamellar vesicles 34

Ether injection:

Ether injection Lipid + ether solution in the syringe Inject slowly In the aqueous phase ( On heated water bath, 60ºc) Large unilamellar vesicles 35

Water organic phase: Double emulsion:

Water organic phase: Double emulsion Organic solution + Lipid + Aqueous phase Emulsion (W/O) Hot aqueous solution of buffer Multi compartment vesicle W/O/W (double emulsion) LUVs 36

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Reverse phase evaporation: (MLV, LUV) :

Reverse phase evaporation: (MLV, LUV) 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”. 38

Stable plurilamellar vesicle (SPLVs):

Stable plurilamellar vesicle (SPLVs) 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. The internal SPLV is different from that of MLV-REVs, in that they lack a large aqueous core. The internal environment of both the vesicle is different from each other. Detergent dispersion: Phospholipids & aqueous phase comes in contact with the help of detergent 39

Characterization of Liposome: Physical:

Characterization of Liposome: Physical 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

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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 41

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Chemical characterization: Quantitative determination of phospholipids Phospholipid hydrolysis Phospholipid oxidation 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 42

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Phospholipid hydrolysis: 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

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