LIPOSOMES PPT 9-11-12

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LIPOSOMES:

LIPOSOMES AISSMS College of Pharmacy,Pune-1 By Nikhil.S.Shrisunder Department of Pharmaceutics Guided by Mr.J.W.Gajbe Department of Pharmaceutics 1 1/6/2013

CONTENTS:

CONTENTS DEFINITION INTRODUCTION CLASSIFICATION STRUCTURE COMPONENTS MODES OF LIPOSOME/CELL INTERACTION PREPARATION OF LIPOSOMES CHARACTERIZATION OF LIPOSOMES STABILITY APPLICATIONS RECENT ADVANCES IN LIPOSOME PREPARATIONS MARKETED PRODUCTS REFERENCES 2 1/6/2013

LIPOSOMES:

Liposomes are cocentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids. LIPOSOMES 3 1/6/2013

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Liposomes were discovered about 40 years ago by Alec Bingham. Liposomes can be produced from cholesterols, non toxic surfactants,sphingolipids,glycolipids,long chain fatty acids & even membrane proteins. Liposomes are the drug carrier loaded with great variety of molecules such as small drug molecules, proteins, nucleotides & even plasmids. Considerable progress was made during 1970s and 1980s in the field of liposome stability leading to long circulation times of liposomes . 4 1/6/2013

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STRUCTURAL COMPONENTS 5 1/6/2013

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PHOSPHOLIPID 6 1/6/2013

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Phospholipids are major structural components of biological membranes in human body, where 2 types of phospholipids exist i.e. phosphodiglycerides & sphingolipids . Each phospholipid molecule has 3 major parts, 1 head & 2 tails. Head is made from 3 molecular components: choline , phosphate & glycerol which is hydrophilic.Each tail with a long chain EFA which are hydrophobic. Most commonly used phospholipids – PC an amphipathic molecule with a hydrophilic polar head group, phosphocholine . PC, also known as “lecithin”, can be derived from natural and synthetic sources. 7 1/6/2013

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The lipid bi-layer used in the liposomes are usually made of phospholipids and cholesterol. Following are the n aturally occurring phospholipids used in liposomes are: Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS). S ynthetic phospholipids used in the liposomes are: dioleoyl phosphatidylcholine (DOPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE). 8 1/6/2013

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FORMATION OF LIPOSOME 9 When phospholipids are dispersed in water, they spontaneously form closed structure with internal aqueous environment bounded by phospholipid bilayer membranes, this vesicular system is called as liposome. 1/6/2013

MODES OF LIPOSOME/CELL INTERACTION:

MODES OF LIPOSOME/CELL INTERACTION 10 1/6/2013

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ADVANTAGES 1. Liposomes are biocompatible & biodegradable 2. They can entrap hydrophilic pharmaceutical agents in their internal water compartment & hydrophobic pharmaceutical agents into the membrane 3. Liposome act as sustained release depots( Propranolol,Cyclosporin ) 4. Increased efficacy & therapeutic index of drugs( Actinomycin -D) 5. It can be administered through various routes 6. It has lack of antigenic properties 7. It acts as drug reservoir 11 1/6/2013

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DISADVANTAGES 1. Less stability 2. Low solubility 3. Problem of targeting to various tissues due to their large size 4. Short half life 5. Phospholipids may undergo oxidation and hydrolysis 6. Leakage of encapsulated drug / molecules may occur 7. High cost of production 8. Allergic reactions may occur due to liposomal constituents 12 1/6/2013

CLASSIFICATION:

CLASSIFICATION Liposomes are classified on the basis of: STRUCTURE METHOD OF PREPARATION COMPOSITION & APPLICATION 13 1/6/2013

1.BASED ON STRUCTURE:

1.BASED ON STRUCTURE VESICLE TYPE ABBREVIATION DIAMETER SIZE No. of LIPID BILAYER Unilamellar vesicle UV All size range One Small unilamellar vesicle SUV 20-100 nm One Medium unilamellar vesicle MUV >100 nm One Large unilamellar vesicle LUV >1000 nm One Giant unilamellar vesicle GUV >1 μ m One Oligolamellar vesicle OLV 0.1-1 μ m ~5 Multilamellar vesicle MLV >0.5 μ m 5-25 Multivesicular vesicle MV >1 μ m Multicompartmental structure 14 1/6/2013

2.METHOD OF PREPARATION:

2.METHOD OF PREPARATION PASSIVE LOADING ACTIVE LOADING (REMOTE LOADING) MECHANICAL DISPERSION METHOD SOLVENT DISPERSION METHOD DETERGENT REMOVAL METHOD 15 1/6/2013

MECHANICAL DISPERSION METHODS:

MECHANICAL DISPERSION METHODS 1.HAND SHAKING & NON SHAKING 2.PROLIPOSOME 3.SONICATION 4.FRENCH PRESSURE CELL 5.DRIED RECONSTITUTED VESICLES 6.FREEZE-THAW TECHNIQUE 7.MEMBRANE EXTRUSION 8.MICROEMULSIFICATION 16 1/6/2013

SOLVENT DISPERSION METHODS:

SOLVENT DISPERSION METHODS 1.ETHANOL INJECTION 2.ETHER INJECTION 3.DOUBLE EMULSION VESICLES 4.REVERSE PHASE EVAPORATION VESICLES 5.STABLE PLURILAMELLAR VESICLES DETERGENT REMOVAL METHODS 1.DETERGENT(CHOLATE,TRITON X 100) REMOVAL FROM MIXED MICELLES BY DIALYSIS COLUMN CHROMATOGRAPHY DILUTION 17 1/6/2013

3.COMPOSITION & APPLICATIONS:

3.COMPOSITION & APPLICATIONS TYPE OF LIPOSOME ABBREVIATION COMPOSITION CONVENTIONAL LIPOSOME CL Neutral/negatively charged phospholipids & cholesterol FUSOGENIC LIPOSOMES RSVE Reconstituted Sendai virus envelopes. pH SENSITIVE LIPOSOMES -- PE/DOPE with OA CATIONIC LIPOSOMES -- Cationic lipids with DOPE. LONG(STEALTH) CIRCULATORY LIPOSOMES LCL Cholesterol, 5-10% PEG-DSPE. IMMUNO-LIPOSOMES -- CL/LCL with attached Mab . 18 1/6/2013

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MECHANICAL DISPERSION METHOD HAND SHAKING METHOD(MLV’s) 20 1/6/2013

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NON SHAKING METHOD(LUV’S ) 2 Steps: 1.Hydration 2.Swelling Hydration is initiated by exposing the dried film to stream of saturated water. Nitrogen for 15 min, followed by swelling in aqueous medium without shaking. 21 1/6/2013

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PROLIPOSOMES(MLV’s) Drying of lipid on finely divided suuport ( NaCl ) Surface area increases instantaneous hydration addition of water lipid swells formation of MLV’s. 22 1/6/2013

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SONICATION: 2TYPES: 1.BATH SONICATOR 2.PROBE SONICATOR 23 1/6/2013

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BATH SONICATOR PROBE SONICATOR 1.Large volume of diluted lipids are processed. 1.Small volume of diluted lipids are processed. 2.Less or no contamination. 2.Chances of contamination. At high energy levels,average size of vesicles is further reduced. Exposure of MLV’s to ultrasonic irradiations is the most widely used method for producing small vesicles. As chances of contamination are likely to occur in probe sonicator,bath sonicator is widely used. 24 1/6/2013

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FRENCH PRESSURE CELLS(ULV/OLV) Method developed by Barenholtz & Hamilton et al. Very useful method in which extrusion of preformed large liposomes in a French Pressure under very high pressure is carried out . This technique yields ULV’s/OLV’s of intermediate size(30-80nm/depending upon applied pressure). Liposomes are more stable. Free from structural defects. Leakage problem is also less. However it has high production cost. 25 1/6/2013

VESICLES PREPARED BY EXTRUSION METHOD:

VESICLES PREPARED BY EXTRUSION METHOD Size of liposomes is reduced by gently passing thru’ membrane filter (polycarbonate) of defined pore size. Pressure applied is low(‹100 psig). Used to process LUV’s & MLV’s. Widely used method for SUV & LUV production for in vivo & in vitro studies. Ease of production. Free from surface contamination. Batch-batch reproducibility. 26 1/6/2013

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DRIED RECONSTITUTED VESICLES Empty SUV dispersion freeze dry rehydrate containing materials to be entrapped formation of ULV’s/OLV’s Size obtained is in the range 1.0µm or less in diameter. High entrapment of water soluble components. 27 1/6/2013

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FREEZE THAW SONICATION(FTS) Freeze SUV dispersion thaw at room temperature for 15 min sonicate rupture of SUV’s occur Formation of liposomes 28 1/6/2013

MICROEMULSIFICATION:

MICROEMULSIFICATION “ Microfluidizer ” is used to prepare small MLV’s from concentrated lipid dispersions/slurry/ unhydrated lipid in an organic medium . Pumping of the dispersion thru’ a 5µm orifice at 10,000psig. Affecting an efficient transfer of energy is the basic principle. Has high rate of production. Can process samples with very high proportion of lipids. Efficient for encapsulation of hydrophilic materials. 29 1/6/2013

MICROFLUIDIZER:

MICROFLUIDIZER 30 1/6/2013

SOLVENT DISPERSION METHODS:

SOLVENT DISPERSION METHODS ETHANOL INJECTION METHOD Lipids ethanol rapidly inject through a fine needle saline buffer containing materials to be entrapped dissolution of ethanol formation of SUV’s. 31 1/6/2013

ETHANOL INJECTION:

ETHANOL INJECTION 32 1/6/2013

ETHER INJECTION METHOD:

ETHER INJECTION METHOD Lipid ether slowly injecting through a narrow needle vapourize temperature at 60˚C production of SUV’s. Less risk of oxidation as ether is free of peroxides. Low efficiency. Long time needed for production. 33 1/6/2013

DOUBLE EMULSION VESICLES:

DOUBLE EMULSION VESICLES Micro-droplet dispersion rapidly injecting using 22 guage needle in hot aqueous solution of Triss buffer with vigorous stirring strong jet of N₂ evaporate the solvent formation of double emulsion vesicles. Formation of w/o/w emulsion occurs,hence the name. 34 1/6/2013

REVERSE PHASE EVAPORATION VESICLES:

REVERSE PHASE EVAPORATION VESICLES Lipid organic solvent aqueous solution mix sonicate formation of w/o emulsion evaporate to remove the organic solvent Lipids form a phospholipid bilayer vigorous shaking water droplets collapse formation of LUV’s. 35 1/6/2013

CHARACTERIZATION:

CHARACTERIZATION 1. PHYSICAL PROPERTIES : PARTICLE SIZE ANALYSIS- Laser light scattering, transmission electron microscopy determines the particle size & its distribution. SURFACE CHARGE- Free-flow electrophoresis on a cellulose acetate plate in a sodium borate buffer pH 8.8 & a zeta potential measurement. The samples are applied to plate & electrophoresis is carried out at 4˚C for 30 min. The plate is dried and phospholipids are visualised by the molybdenum blue reagent. The liposomes get bifurcated based on the surface charge . 36 1/6/2013

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PERCENT DRUG ENTRAPMENT- This can be determined by ‘PROTAMINE AGGREGATE’ & ‘MINICOLUMN CENTRIFUGATION method . Expressed as %entrapment/mg lipid. PHASE BEHAVIOUR- Liposomes at transition temperature undergo reversible phase transition i.e the polar head groups in gel state become disordered to form the liquid crystalline state which can be determined by DSC. LAMELLARITY- Freeze-fracture electron microscopy & freeze-etch electron microscopy & P-NMR method. 37 1/6/2013

2.CHEMICAL PROPERTIES::

2.CHEMICAL PROPERTIES: DETERMINATION OF PHOSPHOLIPIDS- Determined by Barlett assay & Steward assay . CHOLESTEROL ANALYSIS- Qualitative or quantitative . Capillary column filled with fused silica for qualitative analysis & for quantitative analysis sample is reacted with reagent and measuring the absorbance of purple colored complex. Reagents used areFerric perchlorate,ethyl acetate & sulphuric acid. 3.BIOLOGICAL PROPERTIES: PURITY & POTENCY. 38 1/6/2013

STABILITY OF LIPOSOMES:

STABILITY OF LIPOSOMES A.PREVENTION OF CHEMICAL DEGRADATION: 1.Start with freshly purified lipids & freshly distilled solvents. 2.Avoid procedure which involving high temperature. 3.Carry out manufacturing in the absence of oxygen. 4.Deoxygenate aqueous solution with nitrogen. 5.Store liposome suspension in an inert atmosphere. 6.Include an antioxidant as a component. 7.Iron chelater is used to prevent initiation of free radical chain reaction. 39 1/6/2013

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B.PREVENTION OF PHYSICAL DEGRADATION: 1.‘ ANNEALING ‘ is best method to control physical degradation i.e incubating the liposomes at a temperature high enough above the phase transition temperature to allow differences in packing density between opposite sides of the bilayers to equalize by trans membrane flip-flop . 2. The stability of liposomes may also be increased by cross linking membrane component covalently using Gluteraldehyde fixation, osmification or polymerization of alkyne containing phospholipids. These methods increases mechanical strength of the membrane & render them less susceptible to disruption. 40 1/6/2013

APPLICATIONS:

APPLICATIONS 1.The therapeutic value of liposomes as drug carriers, particularly for anticancer, antifungal, and antibacterial agents. 2.As anticancer , cytotoxic drugs like 6-MP,Cytarabine,5- FU,alkylating agents . 3.As vaccine adjuvants i.e.when administered by IM route,they slowly release the antigens and accumulate in lymph nodes. 4.In opthalmic drug delivery systems,Idoxuridine used in acute & chronic keratitis . 41 1/6/2013

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5. Passive targeting to the cells of the immune system, especially cells of reticuloendothelial system(RES).Ex antimonials , Amphotericin B, porphyrins & also vaccines,immunomodulators or ( immuno ) supressors . 6. Sustained release system of systemically or locally administered liposomes.Ex doxorubicin, cytosine arabinose , cortisones, biological proteins or peptides such as vasopressin. 7. Site specific targeting: in certain cases liposomes with surface attached ligands can bind to target cells (‘key and lock’ mechanism). Ex antineos,anti infectors & antiinflammatory drugs. 8. Improved transfer of hydrophilic, charged molecules like chelators , antibiotics,plasmids & genes into the cells. 42 1/6/2013

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43 MARKETED PREPARATIONS 1/6/2013

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LIPOSOME FOR DRUG DELIVERY :

LIPOSOME FOR DRUG DELIVERY 45 1/6/2013

RECENT ADVANCES & ON GOING CLINICAL TRIALS :

RECENT ADVANCES & ON GOING CLINICAL TRIALS Antigens as Liposomal Preparation Applications Cholera toxin = Enhanced Ab * level Diphtheria toxoid = Superior immunoadjuvant Herpes simplex virus = Enhanced Ab level Hepatitis B virus = Higher Ab response Bacterial polysaccharides = Superior immunoadjuvants Tetanus toxoids = Increased Ab titre Influenza subunit antigen = Intranasal, protects animal from virus Carbohydrate antigen = Increased Ab titre in salivary gland 46 1/6/2013

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Liposomal Preparation of Amphotericin B: Liposome (Am Bisome ™) Amphotericin B = Systemic fungal infection used in Visceral lieshmaniaisis Liposome ( Amphocil ™) Amphotericin B = Systemic fungal infection Liposome (ABLC™) Amphotericin B = Systemic fungal infection 47 1/6/2013

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Liposomal Preparation for Infective Disease: Pentamidine = Leishmaniasis Anamycin = Leishmaniasis Asiaticoside = Tuberculosis and Leprosy Rifampicin = Tuberculosis Amphotericin B = Meningitis,Leishmaniasis , Candidiasis . Praziquantal = Macrophage activation Sparfloxacin = M. avium Gentamycin = Staphylococcal pneumonias 48 1/6/2013

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Intravenous Liposomal Antibiotics / Anti- Neoplastics Preparation Drug Targeted Site- Liposome ( Doxil ™) Doxorubicin = Kaposi’ sarcoma Liposome (EVACT ™) = Metastatic breast cancer Liposome( DaunoXome ™) Daunosome = Advanced Kaposi’ sarcoma,small cell lung cancer, leukaemia & solid tumour . Liposome ( VincaXome ™) Vincristine = Solid tumour 49 1/6/2013

REFERENCES:

REFERENCES Targeted & Controlled Drug Delivery Novel Carrier System by S.P.Vyas & R.K.Khar,Reprint Edition(2007),CBS PUBLISHERS & DISTRIBUTORS NEW DELHI:173-243 A.Chonn,P.R.Cullis,“Recent advances in liposome technologies and their applications for systemic gene delivery”,Advanced Drug Delivery Reviews 30(1998): 73-83 D.D.Lasic,“Application of Liposomes ”, ELSEVIER;Volume 1,Edited by R.Lipowsky and E.Sackmann:499-502 50 1/6/2013

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A.Samad,Y.Sultana and M.Aqil,Liposomal Drug Delivery Systems: An Update Review ,Current Drug Delivery, 2007 ,4, 297-305 www.pharmatutor.com (2-11-12) 51 1/6/2013

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52 THANKYOU THANKYOU 1/6/2013

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