Liposomes

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Presented by , K .Vinod kumar M.Pharmacy (pharmaceutics). Guided by , Mrs. Samyuktha , M.Pharmacy, Department of pharmaceutics . ST.ANN’S COLLEGE OF PHARMACY, CHIRALA LIPOSOMES - as micro-particulate drug carriers

What are liposomes ?:

What are liposomes ? Liposome is Greek word - lipo means “ fat ” - soma means “ body ” These are the micro-particulate drug carriers consisting of one or more concentric spheres of lipid bilayer separated by aqueous buffer compartments. When phospholipids are dispersed in water they spontaneously form closed structures with internal aqueous compartments bounded by phospholipid bilayer membranes. Their diameter ranges from 80 nm to 100 u m

General structure of liposome:

General structure of liposome . Lipid bilayer

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When phospholipids dispersed in aqueous phase , hydration of polar head groups of lipid results in heterogenous mixture structure, referred to as vesicles ,contain multiple lipid bilayer which are referred to as Multilamellar vesicles Sonication of the liposomes results in there size reduction to vesicles containing only a single bilayer with diameter 25 nm to 50 nm are referred to as Unilamellar vesicles. Large unilamellar vesicles exhibit size range of 100 – 500 nm.

Structure of the phospholipid molecule:

Structure of the phospholipid molecule .

Mechanisms by which liposomes act :-:

Mechanisms by which liposomes act :- There are several mechanisms by which liposomes act which are as follows : Liposomes attaches to plasma membrane and appears to fuse with them,releasing their content into cell. Liposomes are taken up by cell in some cases and their phospholipids are incorporated into plasma membrane by which drug trapped inside is released. In case of phagocyte cell, the liposomes are taken up, the phospholipid walls are acted upon by organelles called lysosomes and the drug is released.

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Outside of the cell. Liposome Inside of cell. Inside of cell. Drug releasing into cell Drug content released into the cell Cell membrane Cell membrane Phospholipids of liposome are incorporated into cell membrane

Materials used in liposome preparation :-:

Materials used in liposome preparation :- PHOSPHOLIPIDS Glycerol containing phospholipids are commonly used component of liposome formulation. The most abundant glycerol phosphotides in plants and animals are phosphotidyl choline(lecithin) and phosphotidyl ethanol amine(cephaline). These two are major structural components of most biological membranes. Fatty acids are also important constituent of glycerol phosphotides(triglycerides of fat cells).

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2. SPHINGOLIPIDS They contain sphingosine (or) a related base as their structural backbone. They contain 3 characteristic building blocks:- - A molecule of fatty acid - A molecule of sphingosine & - Head groups that can vary from simple alcohol as choline to very complex carbohydrates. The most abundant sphingolipid in higher animals is sphingomyelin.

3. GLYCOSPHINGOLIPIDS:

3. GLYCOSPHINGOLIPIDS These are found mainly in grey matter of brain tissue of higher animals and are used in minor component. These are included in liposomes formulation to provide a layer of surface charged groups. STEROLS Cholesterol & its derivatives are quite often included as components of liposomal membrane.

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Cholesterol can be added to bilayer mixture for the following purposes :- - Acts as a fluidity buffer. - Acts as intercalator with phospholipid molecules. - Decreases the permeability of membrane to water soluble molecules. Liposomes without cholesterol are known to interact rapidly with plasma proteins like albumin , transferin and macroglobulins which lead to physical instability of liposomes.

5. SYNTHETIC PHOSPHOLIPIDS:

5. SYNTHETIC PHOSPHOLIPIDS Saturated phospholipids include dipalmitoyl phosphatidyl choline(DPPC) , dipalmitoyl phosphatidyl ethanol amine(DPPE) , dipalmitoyl phosphatidyl serine(DPPS) . Unsaturated phospholipids have also been used which include dioleo-phosphatidyl choline(DOPC) , dioleo-phosphatidyl glycerol(DOPG)

6. POLYMERIC MATERIALS:

6. POLYMERIC MATERIALS A large variety of polymerizable lipids which can form vesicles , has been synthesized. These include lipids containing conjugated diene , methacrylate , and thiol groups as polymerizable moieties.

Special consideration for selection of lipids :-:

Special consideration for selection of lipids :- For non-irritant topical formulations :- -- non- ionic liposomes. For localized infection :- -- ionic liposomes. For other systemic applications :- -- liposomes with covalently attached polymer. For nucleic acid complexation :- -- cationic liposomes that contain large fraction of neutral lipids.

Preparation of liposomes:

Preparation of liposomes The three different strategies for the preparation of liposomes include :- 1. Mechanical method 2. Method based on replacement of organic solvent 3. Methods based on size transformation or fusion of preformed vesicle

1. MECHANICAL METHOD:

1. MECHANICAL METHOD FILM METHOD:- Thin lipid film + organic solvent Solvent removed by film deposition under vacuum. After all the solvent is removed. Solid-lipid mixture hydrated using aqueous buffer. Heterogenous Multilamellar vesicle(1 u m diameter) Lipids swell spontaneously.

Limitation of film method:-:

Limitation of film method:- Encapsulation efficiency is low. b) ULTRASONIC METHOD:- It is used for separation of small unilamellar vesicles with diameter 15 – 25u m. Ultrasonication of an aqueous dispersion of phospholipids is done by 2 types of sonicators :- probe sonicator - for small volume requiring high energy. bath sonicator - for large volume.

2. METHODS BASED ON REPLACEMENT OF ORGANIC SOLVENTS :-:

2. METHODS BASED ON REPLACEMENT OF ORGANIC SOLVENTS :- REVERSE PHASE EVAPORATION:- This method is used for preparation of large unilamellar and oligolamellar vesicles. It has the ability to encapsulate large macro -molecules of with high efficiency. Diethyl ether and isopropyl ether are the solvents generally used.

Procedure for reverse phase evaporation:-:

Procedure for reverse phase evaporation:- . Lipid mixture is placed in round bottom flask. Solvent removed under reduced pressure by rotary evaporator. Purged with nitrogen & lipids are re-dissolved in organic phase. Non-encapsulated material is removed. Solvent is removed from emulsion by evaporation under reduced pressure. Liposomes formed are called reverse phase evaporation vesicles.

b) ETHER VAPORIZATION METHOD::

b) ETHER VAPORIZATION METHOD: There are 2 methods according to the solvents used:- Ethanol injection method:- lipid is injected rapidly through fine needle into an excess of aqueous medium. Ether injection method:- Lipid is injected slowly through fine needle into an excess of aqueous medium.

3. METHODS BASED ON SIZE TRANSFORMATION OR FUSION OF PREFORMED VESICLE :-:

3. METHODS BASED ON SIZE TRANSFORMATION OR FUSION OF PREFORMED VESICLE :- THE DEHYDRATION - REHYDRATION METHOD:- EMPTY BUFFER CONTAINING SUV’S. REHYDRATE WITH AQ. FLUID CONTAINING MATERIAL TO BE ENTRAPPED. VESICLES FORMED ARE THEN REHYDRATED. DISPERSION OF SOLID LIPIDS IN FINELY SUBDIVIDED FORM. LIPOSOMES OBTAINED ARE OLIGOLAMELLAR VESICLES. SUBJECTED TO DRYING

b) FREEZE THAW EXTRUSION METHOD :-:

b) FREEZE THAW EXTRUSION METHOD :- . Liposomes formed by film method. Vortexed with the solute to be entrapped until entire film is suspended Subjected to two cycles of freeze thaw & vortexing the sample is extruded 3 times. Formed MLV’s are frozen in luke warm water & vortexed again. Ruptures and defuses SUV’s & liposomes fuse and increase in size forming LUVET Subjected to 6 freeze thaw cycles & addition 8 extrusions This method is mainly used for encapsulation of proteins.

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METHODS FOR CONTROLLING THE PARTICLE SIZE & SIZE DISTRIBUTION OF LIPOSOMES FRACTIONATION HOMOGENIZATION CAPILLARY PORE MEMBRANE EXTRUSION

1. FRACTIONATION:-:

1. FRACTIONATION:- CENTRIFUGATION: Liposomes sediment in a centrifugal field at a rate that is dependent on their size and density. Disadvantage liposomes smaller than about 0.5 u m tend to require high forces and long spinning times SIZE EXCLUSION CHROMATOGRAPHY : It is particularly useful for separation of small unilamellar vesicles from large structures.

2. HOMOGENIZATION::

2. HOMOGENIZATION: By using high pressure homogenizers like micro fluidizer particle size can be reduced. 3. CAPILLARY PORE MEMBRANE EXTRUSION : It is wide spread , simple technique for production of defined & narrow sized liposomes. The extrusion of heterogenous population of fairly large liposomes through polycarbonate membranes under reduced pressure gives uniform particle sized liposomes

Characterization of liposomes:

Characterization of liposomes a) Factors affecting drug entrapment: - Partition coefficient of drug. - Solubility of drug. - Lipid quantity used. - Internal volume of liposome. - Drug concentration(it should not reach the saturation limit).

b) Internal volume and encapsulation efficiency:- :

b) Internal volume and encapsulation efficiency:- These two parameters are used to describe entrapment of water soluble drugs in aqueous compartment of liposome. These parameters depend on liposomal content , lipid concentration , method of preparation and drug used. Incorporation of charged lipids into lipids increases the volume of aqueous compartment by separating bilayer due to charge repulsion.

CHARACTERIZATION OF LIPOSOMES WITH THEIR QUALITY CONTROL ASSAYS a) BIOLOGICAL CHARACTERIZATION :

CHARACTERIZATION OF LIPOSOMES WITH THEIR QUALITY CONTROL ASSAYS a) BIOLOGICAL CHARACTERIZATION CHARACTERIZATION PARAMETERS INSTRUMENT FOR ANALYSIS STERILITY AEROBIC AND ANEROBIC CULTURE PYROGENICITY RABBIT FEVER RESPONSE ANIMAL TOXICITY MONITORING SURVIVAL RATS

b) CHEMICAL CHARACTERIZATION:

b) CHEMICAL CHARACTERIZATION CHARACTERIZATION PARAMETERS INSTRUMENT FOR ANALYSIS PHOSPHOLIPID CONCENTRATION HPLC/BARRLET ASSAY CHOLESTEROL CONCENTRATION HPLC/CHOLESTEROL OXIDE ASSAY DRUG CONCENTRATION ASSAY METHOD ANTI-OXIDANT DEGRADATION HPLC/TLC P H PH METER OSMOLARITY OSMOMETER PHOSPHOLIPIDS HYDROLYSIS HPLC/TLC CHOLESTEROL OXIDATION HPLC/TLC PHOSPHOLIPIDS OXIDATION UV ABSORBENCE

c) PHYSICAL CHARACTERIZATION:

c) PHYSICAL CHARACTERIZATION CHARACTERIZATION PARAMETER INSTRUMENT FOR ANALYSIS VESICLE SHAPE & SURFACE MORPHOLOGY TEM & SEM VESICLE SIZE AND SIZE DISTRIBUTION DYNAMIC LIGHT SCATTERING & TEM SURFACE CHARGE FREE FLOW ELECTROPHORESIS ELECTRICAL SURFACE POTENTIAL & SURFACE P H ZETA POTENTIAL MEASUREMENT AND P H SENSITIVE PROBES LAMELLARITY P 32 NMR PERCENT CAPTURE MINI COLUMN CENTRIFUGATION & GEL EXCLUSION DRUG RELEASE DIFFUSE CELL / DIALYSIS

APPLICATIONS OF LIPOSOMES:

APPLICATIONS OF LIPOSOMES LIPOSOMES IN RESPIRATORY DRUG DELIVERY SYSTEM : ISONIAZID & RIFAMPICIN - improved the effect of drugs for tuberculosis. CYCLOSPORINS - preferentially absorbed by lungs & show sustained release LIPOSOMES AS VACCINE ADJUVANTS: RABIES GLYCOPROTEINS - interleukin 2 enhancement CHOLERA TOXIN - enhanced antibody level

LIPOSOMES FOR BRAIN TARGETING:

LIPOSOMES FOR BRAIN TARGETING Addition of sulphatide group to liposome composition increases their ability to cross blood-brain barrier. Liposomes coated with mannose was found to reach brain tissue easily. LIPOSOMES AS ANTI-INFECTIVE AGENTS Active targeting approach - pentamidin and anamycin for leishmaniasis Passive targeting approach - gentamycin for staphylococcal pneumoniasis

LIPOSOMES IN TUMOUR THERAPY:

LIPOSOMES IN TUMOUR THERAPY DOXARUBICIN - For the treatment of refractory tumour and breast cancer. VINCRISTINE - for the treatment of solid tumour.

Conclusion :

Conclusion During the last years, liposomes as pharmaceutical drug carriers have received a lot of attention. The new developments in liposomes is a continuous process which helps for better targeting with less toxicity. Some commercially available liposomes in market include MIKASOME, DAUNAXOME, VERTEPORFIN.

REFERENCES:

REFERENCES Wikipedia.org Island net.com Bentham science publishers ltd. Journals Allen, Theresa M. "Liposomal Drug Formulations: Rationale for Development and What We Can Expect for the Future." Drugs 56: 747-756, 1998. Janknegt , Robert. "Liposomal and Lipid Formulations of Amphotericin B." Clinical Pharmacokinetics. Kim, Anna et al. "Pharmacodynamics of insulin in polyethylene glycol-coated liposomes." International Journal of Pharmaceutics. 180: 75-81, 1999. Ranade , Vasant V. "Drug Delivery Systems: Site-Specific Drug Delivery Using Liposomes as Carriers." Pharmacology. 29: 685-694, 1989. Storm, Gert and Daan J.A. Crommelin . " Liposomes:quo vadi ?" PSTT 1: 19-31, 1998.

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