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definition,classification,method of prepration,marketed products


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LIPOSOMES Presented by: Abhinav Shahi M.Pharm ( P’ceutics ) II nd sem


PRINCIPLE Targeted Drug Delivery System( liposomes ) must supply drug directly (selectively) to the site(s) of action in a manner that provides maximum therapeutic activity through kinetics. It prevents degradation or inactivation during transmit to the target sites and protects the body from adverse reaction because of appropriate disposition.


Definition: When phospholipids are dispersed in water they spontaneously form closed structure with internal aqueous compartments bounded by phospholipid bilayer membranes , these are called liposomes . Hydrophilic (AQUEOUS CAVITY) Hydrophobic (PHOSPHOLIPID BILAYER)


Introduction Liposome was found by Alec Bangham of Babraham Institute in Cambridge, England in 1965. In 1990, drugs with liposome and Amphotericin B were approved by Ireland. In 1995 America F.D.A approved liposor doxodubicin . Liposome is a lipid vesicle suspending in the hydro-phase with a diameter around 0.0025~3.5um. The membrane of liposome is made of phospholipids, which have phosphoric acid sides to form the liposome bilayers .


COMPONENTS PHOSPHOLIPIDS: They are the major structural components of biological membranes. CHOLESTEROLS: It form bilayer structure by incorporating into phospholipid membrane in very high concentration up to 1:1 or 2:1. It acts as “fluidity buffer” 2 types of Phospholipids : 1.Phosphodiglycerides 2.Shingolipids


CLASSIFICATION Based on Structural Parameters Medium sized unilamellar Vesicles Large unilamellar vesicles ≥100nm Giant unilamellar vesicles ≥1um Multivesicular vesicles ≥1um Multilamellar large vesciles ≥0.5um Oligolamellar Vesicles 0.1 – 1um Unilamellar Vesicles(all size range) Small Unilamellar vesicles 20-100nm

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Based on Methods of Liposome Preparation Dehydration-Rehydration method Reverse Phase Evaporation Stable Plurilamellar MLV - REV Vesicles Extrusion Technique Frozen and thawed MLV Based upon Composition and Application Fusogenic pH sensitive Long Circulatory Cationic Immuno Conventional

Advantages :

Advantages Biocompatible, completely biodegradable, non-toxic, flexible , nonimmunogenic . Liposomes supply both a lipophilic environment and aqueous “milieu interne” in one system. Can protect encapsulated drug. Reduce exposure of sensitive tissues to toxic drugs. Alter the pharmacokinetic and pharmacodynamic property of drugs (reduced elimination, increased circulation life time). Flexibility to couple with site-specific ligands to achieve active targeting (Anticancer and Antimicrobial drugs). Liposomes can encapsulate both micro and macromolecules such as haemoglobin , erythropoeitin , interferon g etc. Can be formulated into multiple dosage forms.

Disadvantages :

Disadvantages Production cost is high. Leakage and fusion of encapsulated drug / molecules. Sometimes phospholipid undergoes oxidation and hydrolysis like reaction. Short half-life. Low solubility. Fewer stables.

Characterisation of liposomes :

Characterisation of liposomes 1. Physical Characterisation Characterization parameters - Analytical method/Instrument 1. Vesicle shape and surface morphology - Transmission electron microscopy, Freeze-fracture electron microscopy 2. Mean vesicle size and size distribution (submicron and micron range) - Dynamic light scattering, zetasizer , Photon correlation spectroscopy, laser light scattering, gel permeation and gel exclusion 3. Surface charge - Free-flow electrophoresis 4. Electrical surface potential and surface pH - Zetapotential measurements & pH sensitive probes 5. Lamellarity – Small angle X-ray scattering, 31 P-NMR, Freeze-fracture electron microscopy 6. Phase behavior - Freeze-fracture electron microscopy, Differential scanning colorimetery

2. Chemical Characterisation :

2. Chemical Characterisation Characterization parameters - Analytical method/Instrument . 1. Phospholipid concentration - Barlett assay, stewart assay, HPLC. 2. Cholesterol concentration - Cholesterol oxidase assay and HPLC. 3. Phopholipid peroxidation - UV absorbance, Iodometric and GLC. 4. Phospholipid hydrolysis,Cholesterol auto-oxidation - HPLC and TLC. 5. Osmolarity – Osmometer .

3. Biological Characterisation:

3. Biological Characterisation Characterization parameters - Analytical method/Instrument 1. Sterility - Aerobic or anaerobic cultures 2. Pyrogenicity - Limulus Amebocyte Lysate (LAL) test 3. Animal toxicity - Monitoring survival rates, histology and pathology

Basic steps in preparation :

Basic steps in preparation CHOLESTEROL LECITHIN CHARGE Dissolve in Organic Solvent Dring down lipid from organic solvent( Vaccum ) Dispersion of lipid in aqueous media (Hydration) Purification of resultant Liposomes Analysis of final product

Mechanism of liposome formation :


Mechanical dispersion methods :

Mechanical dispersion methods Lipids film hydration by HAND SHAKING,NON HAND SHAKING and FREEZE DRYING Micro-emulsification Sonication French Pressure Cell Membrane Extrusion Dried reconstituted vesicles Freeze-thawed liposomes


SOLVENT DISPERSION METHODS Ethanol injection Ether injection Double emulsion vesicles Reverse phase evaporation vesicles Stable plurilamellar vesicles


DETERGENT REMOVAL METHODS Detergent ( cholate , alkylglycoside , triton X-100) removal from mixed micelles by : Dialysis Column chromatography Dilution Reconstituted Sendai virus enveloped vesicles


THIN FILM HYDRATION USING HAND AND NON-HAND SHAKING METHODS Lipids are casted as stacks of film from their organic solution using flash rotary evaporator under reduced pressure(or by hand shaking) The casted film is dispersed in an aqueous medium. Upon hydration the lipids swell and peel off from the wall of the round bottom flask and vesiculate forming multilamellar vesicles(MLVs) PROLIPOSOMES: In order to increase the surface area of dried lipid film and to facilitate hydration, the lipid is dried over a finely divided particulate support, such as powdered NaCl or sorbitol or other polysaccharides. These dried lipid coated particulates are called Pro- Liposomes


MICRO EMULSIFICATION LIPOSOMES “Micro Fluidizer” is used to prepare small MLVs from Concentrated lipid dispersion The lipids can introduced into fluidizers, either as a dispersion of large MLVs or as a slurry of unhydrated lipids in organic medium. Microfluidizer pumps the fluid at very high pressure(10,000psi, 600-700 bar) through a 5um orifice. Then it is forced along defined micro channels, which direct two streams of fluid to collide together at right angles at a very high velocity, thereby affecting an efficient transfer of energy. The fluid collected can be recycled through the pump and interaction chamber until vesicles of the spherical dimension are obtained. After a single pass, the size of vesicles is reduced to a size 0.1 and 0.2um in diameter.


SONICATION SONICATION PROBE SONICATOR: The probe is employed for dispersions, which require high energy in a small volume(e.g., high concentration of lipids, or a viscous aqueous phase) BATH SONICATOR: The bath is more suitable for large volumes of diluted lipids. Method: Placing a test tube containing the dispersion in a bath sonicator and sonicating for 5-10min(1,00,000g) which yield a slightly hazy transparent solution. Using centrifugation to yield a clear SUV dispersion.


MEMBRANE EXTRUSION The technique can be used to process LUVs as well as MLVs. The size of liposomes is reduced by gently passing them through membrane filter of defined pore size achieved at much lower pressure(<100psi). In this process, the vesicles contents are exchanged with the dispersion medium during breaking and resealing of phospholipids bilayers as they pass through the polycarbonate membrane. The liposomes produced by this technique have been termed LUVETs. This techniques is most widely used method for SUV and LUV production for in vitro and in vivo studies.


FREEZE THAW SONICATION This method is based upon freezing of a unilamellar dispersion(SUV). Then thawing by standing at room temperature for 15min. Finally subjecting to a brief Sonication cycle which considerably reduces the permeability of the liposomes membrane. In order to prepare GIANT VESICLES of diameter between 10 and 50um, the freeze thaw technique has been modified to incorporate a dialysis step against hypo- osmolar buffer in the place of sonication . The method is simple, rapid and mild for entrapped solutes, and results in a high proportion of large unilamellar vesicles formation which are useful for study of membrane transport phenomenon.

pH induced vesiculation :

pH induced vesiculation This method is used to transform MLVs to LUVs using a change in the pH of the dispersion thus avoiding the use of Sonication and high-pressure application. Preformed MLVs (Prepared using hand shaking, freeze thawing) pH 2.5-3.0 exposed to high pH ie.,11.0 less than 2min (1M NaOH ) pH reduced by 0.1M HCl until pH 7.5 SUVs dispersion

Current liposomal drug preparations :

Current liposomal drug preparations 24 Anticancer Drugs Anti bacterial Antiviral DNA material Enzymes Radionuclide Fungicides Vaccines Malaria merozoite , Malaria sporozoite Hepatitis B antigen, Rabies virus glycoprotein Amphotericin B* Hexosaminidase A Glucocerebrosidase , Peroxidase Duanorubicin,Doxorubicin *, Epirubicin Methotrexate , Cisplatin *, Cytarabin Triclosan, Clindamycin hydrochloride, Ampicillin, peperacillin, rifamicin canal - CFTR*

Conclusions :

Conclusions Liposomes over the years have been investigated as the major drug delivery systems due to their flexibility to be tailored for varied desirable purposes. The flexibility in their behaviour can be exploited for the drug delivery through any route of administration and for any drug or material irrespective of its physicochemical properties. The uses of liposomes in the delivery of drugs and genes to tumour sites are promising and may serve as a handle for focus of future research.

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