liposomes

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A Seminar on “Role of Liposome in Novel Drug Delivery Systems”:

Prepared By :- Kaushik Patel Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur A Seminar on “Role of L iposome in Novel Drug Delivery Systems”

-:Contents of Seminar :-:

1) Introduction 2) Advantages 3) Mechanism of Liposome Formation 4) Classification of Liposome Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Contents of Seminar :-

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 5) Methods of Preparation of Liposome 6) Characterization of Liposome 7) Stability Protocols 8) Therapeutic Application of Liposome

-: Introduction :-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Introduction :- # Definition :- Liposomes are small biocompatible and Biodegradable lipid vesicles composed of Uni lamellar or Multi lamellar phospholipid bilayers surrounding aqueous compartment. Their Biophysical properties, such as size, surface charge, lipid composition, and amount of cholesterol controls the distribution, tissue uptake and drug delivery.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Basically Liposomes are resultant of self assembly of phospholipid in an aqueous core. The lipid soluble drug will be enclosed in the phospholipid bilayer and the water soluble drug will be enclosed in aqueous core. Liposomes are spherical in shape and they are mainly used in the drug delivery in Tumor or cancer. In Recent research liposomes are mainly “Brain Targeting Drug Delivery Systems” in Brain cancer.

-: Advantages :-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Advantages :- Easy to construction. Non Toxic and Biodregadable & Biocompatible. Increased efficacy and therapeutic index. Provides both targetting active and passive. Does not accumulate in heart and so there is no cardiotoxicity .

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Improve pharmacokinetic effect and decrease many kinds of toxicities. Prevent oxidation of the drug. Increase the stability of the drug….. i.e , Protein stabilisation (Encapsulation).

Mechanism :- Liposome Formation:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Mechanism :- Liposome Formation As liposomes are made up of phospholipids, they are amhpipathic in nature and have ability to binds both aqueous and polar moiety. They have polar head and non polar tail. The polar end is mainly phosphoric acid and it will bound to water soluble molecule.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur In aqueous medium the molecules in self-assembled structure are oriented in such way that the polar portion of the molecule remain in contact with in polar environment and at same time shields the non polar part. However, in aqueous mixtures these molecules are able to form various phases, some of them are stable and other remains in metastable form.

-: Parameters affecting :-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Parameters affecting :- The large free energy difference difference between the aqueous and hydrophilic environment promotes the bilayer structure in order to achieve the lowest free energy level. The driving force for the bilayer configuration of liposomes is the hydrophobic interaction coupled with amphiphilic nature of the principle phospholipid molecules.

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Department of Pharmaceutics, NIMS Institute of Pharmacy, NIMS University, Jaipur Liposomes are formed when the thin films are hydrated and stacks of liquid crystalline bilayers become fluid and swells. Once these vesicle get formed, a change in vesicle shape and morphology required energy input in the form of …. Sonic energy to get SUVs and mechanical energy to get LUVs.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

-: Role Of Cholesterol :-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Role Of Cholesterol :- Acts as a fluidity buffer. Restricting the transformation of trans to gauche. After interaction with phospholipid molecules alters the freedom of motion.

-: Classification :-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Classification :- Type Specification Based on Structure a) MLV b) OLV c) UV d) SUV e) MUV f) LUV g) GUV h) MV  Multi lamellar vesicles---- >0.5 micron  Oligo lamellar vesicles--- 0.1-1micron  Uni lamellar vesicles --- all sizes  Small uni lamellar vesicles--- 20-100 nm  Medium uni lamellar vesicles  Large uni lamellar vesicles --- > 100 micron  Giant uni lamellar vesicles---- > 1 micron  Multi vesicular vesicles ---- > 1 micron

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 2) Based on method of preparation a) REV b) MLV REV c) SPLV d) FAT MLV e) DRY f) VET  Single / oligo lamellar vesicles made by ‘Reverse phase evaporation method.  Multi lamellar vesicles made by ‘Reverse phase evaporation method. Stable pluri lamellar vesicles (100nm- 2 micron). Frozen and Thawed multi lamellar vesicles. Dehydration vesicles method.  Vesicles prepared by extrusion technique.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

-: Methods of Preparation :-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Methods of Preparation :- Passive Targeting Active Loading Technique T echnique Mechanical Solvent Detergent Dispersion Dispersion Removal

-: Passive targeting techniques :-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Passive targeting techniques :- Mechanical dispersion :- 1) Lipid film hydration by hand shaking. 2) Non Hand shaking and freeze drying. 3) Micro emulsification. 4) Sonication. 5) French pressure cell technique. 6) Membrane Extrusion technique. 7) Dried reconstituted vesicles. 8) Thawed liposomes.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur B) Solvent Dispersion :- 1) Ethanol injection. 2) Ether injection. 3) Double emulsion vesicles. 4) Reverse phase evaporation vesicles. 5) Stable pluri lamellar vesicles C) Detergent Removal Methods :- 1) Dialysis. 2) Column chromatography. 3) Detergent adsorption method.

-: Active Loading Techniques :-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Active Loading Techniques :-

-: Passive targeting techniques :-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Passive targeting techniques :- -: Mechanical Dispersion Methods :- 1)Lipid Film Hydration by hand shaking and non hand shaking:- In this methods, the lipids are casted as stacks of film from their organic solution using ‘Flash rotatory evaporator’ or ‘hand shaking’

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur The film formation will be takes place and the film will be dried in presence of reduced Nitrogen. After the film stacks are dispersed in aqueous phase. Upon hydration the liquid will swell and peel off from wall of round bottom flask and vesiculate forming multi lamellar vesicles(MLVs). L iposomes stored under the nitrogen umbrella store.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 2) Sonication :- This is the method in which Multi lamellar vesicles are transformed to the small uni lamellar vesicles. The ultra sonic irradiation is provided to the MLVs to get the SUVs. There are two methods are used. a) Probe sonication method. b) Bath sonication method.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur The probe is employed for dispersion, which requires high energy in small volume(e.g. high conc. of lipids or a viscous aqueous phase) while is more suitable for large volumes of diluted liquid Probe tip sonicator provides high energy input to the liquid dispersion but suffer from over heating of liposomal dispersion causing lipid degradation.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur sonication tip also release titanium into the liposome dispersion which will be removed from the it by centrifugation prior to use. Due to above reason most widely the bath sonicators are used. Sonication of MLVs is accomplished by placing dispersion into the the bath sonicator or placing tip fo probe sonicator into the test tube of dispersion.(5-10 min.)

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur After sonication applied the resultant dispersion is centrifuged and according to diagram the SUVs will stay on the top and the small MLVs and aggregated lipids will get settled down. The top layer constitutes pure dispersion of SUVs with varying diameter as size is influenced by composition and concentration, temperature, sonication, volume and sonication tuning.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 3) Frech pressure cell :- This method is having the mechanism of high pressure. This method will give the either uni - or oligo - lamellar liposomes of intermediate size(30-80nm) these liposomes are more stable compared to the sonicated liposomes.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur This method is having some drawbacks are that … initial high cost for the press and the pressure cell. liposomes prepared by this method having less structural defects unlike sonicated liposomes

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 4) Micro emulsification Liposomes :- ‘Micro fluidizer’ is used to prepare small MLVs from concentrated Lipid dispersion. Micro fluidizer pumps the fluid at very at very high pressure (10,000 psi), through a 5 micrometer orifice. Then, it is forced along defined micro channels which direct two streams of fluid to collide together at the right angles at a very high velocity, there by affecting an efficient transfer of energy.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur The lipids can be introduced into the fluidizer, either as large MLVs or as the slurry of un hydrated lipid in organic medium. The fluid collected can be recycled through the the pump and interaction chamber until vesicles of spherical dimensions are obtained.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 5) Membrane Extrusion technique :- In membrane extrusion technique, the sizes of liposomes is reduced by gently passing them through membrane filter of defined pore size. This can be achieved at very low pressure unlike the ‘ F rench pressure cell’(<100psi).

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Extrusion technique is used mainly used to prepare SUVs and LUVs for in vitro and in vivo studies. The liposomal dispersion is passed through the polycarbonate filter to get LUVs and SUVs.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 6) Dried Reconstituted vesicles(DRVs) :- This method starts with freeze drying of a dispersion of empty SUVs. After freeze drying the freeze dried membrane is obtained. Then these freeze dried SUVs are rehydrated with the use of aqueous fluid containing the material to be entrapped. This leads to formation of the solutes in uni - or oligo - lamellar vesicles.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 7) Freeze Thaw Sonication :- This method starts with freeze drying of a dispersion of empty SUVs. After freeze drying the freeze dried membrane is obtained.Then the thawing is done at the room temperature for 15 min. and finally subjected to brief sonication cycle. Thus, the process ruptures and refuses SUVs during which the solute equilibrates between inside and outside and liposome fuse and increase markedly in size.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

-: Solvent Dispersion Techniques:-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 1) Ether and Ethanol Injection :- Ethanol Injection :- This method has been reported as alternatives used for preparation of SUVs without sonication. An ethanol solution of lipids is injected rapidly through a fine needle into an excess of saline or other aqueous medium. -: Solvent Dispersion Techniques:-

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur This procedure yields a high proportion of SUVs , although lipid aggregates and larger vesicles may form if the mixing is not thorough enogh . This method is very simple and very less risk for the degradation of sensitive lipids and up to 100 nm size can be obtained

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Ether Injection :- This method is also similar to the Ethanol injection method. It is somewhat differ from Ethanol injection. It involves the injecting the immiscible organic solution very slowly into an aqueous phase through a narrow needle at the temperature of vaporizing the organic solvent according to diagram.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur This method may also treat sensitive liquids very gently. It has little risk of causing oxidative degradation due to ether.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 2 ) Rapid Solvent Exchange Vesicles(RSEVs ) :- It is the recent method of preparation of the liposomes. This method is especially designed to form compositionally homogenous dispersion by sudden precipitation of a lipid mixture in an aqueous buffer.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Phospholipid/cholesterol dispersion turns to be free of artifactual crystals when prepared by this method. This method involves passing of organic solution of lipids through the orifice of blue tipped syringe under vacuum into a tube containing Aq. Buffer.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur The tube is mounted on vortex and bulk solvents will vaporize and removed within second before coming in contact with aqueous environment, while lipid rapidly precipitates in aqueous buffer. This process does not require highly volatile solvent. The liposome preparation by this method is very fast and liposomes will be formed within a minute.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 3) De-Emulsification Method :- This method of preparation of liposomes require two steps. First the inner leaflet of bilayer and then outer half. The common feature of this method is to prepare ‘water in oil’ emulsion by introduction of a small quantity of aqueous medium containing material to be entrapped into large volume of organic solution of lipid.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur This was followed by mechanical agitation to break up aqueous phase into microscopic water droplets. These droplets are stabilized by presence of phospholipid monolayer at the interface.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 4) Double Emulsion Vesicles :- In this method, the outer half of the liposome membrane is created at a second interphase between two phases by emulsification of an organic solution in water.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur If the organic solution, which already contains water droplet, is introduced into excess aqueous medium followed by mechanical dispersion, multi compartment vesicles are obtained. The ordered dispersion so obtained is described as W/O/W system.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 5) Reverse Phase Evaporation Vesicles :- In this method the removal of solvent from emulsion takes place by evaporation. The droplets are formed by bath sonicator and than emulsion is dried down to semi solid gel in rotary evaporator under reduced pressure.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur At this step monolayers of phospholipids surrounding each water compartment are closely opposed by each other. The next step is to bring about the collapse of a certain proportion of the water droplets by vigorous shaking by using mechanical vortex mixer.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Then the lipid monolayer which enclosed the collapsed vesicle is contributed to adjacent intact vesicle to form the outer leaflet of bilayer of large unilamellar liposomes. The vesicles formed are unilamellar and are having diameter of 0.5 micrometer. T he encapsulation is found to be 50%.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur 6) Stable Plurilamellar Vesicles :- The method of pluri lamellar vesicle formation involves preparation of water in organic phase dispersion with excess of lipid followed by drying under continuous bath sonication with an intermittent stream of nitrogen.

-: Detergent Removal Methods :-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Detergent Removal Methods :- Dialysis. Column chromatography. Detergent Adsorption:-  Detergent/Phospholipids mixtures can form large unilamellar vesicles upon removal of non ionic detergent using appropriate adsorbents for the detergent.

-: Active Loading Techniques:-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Active Loading Techniques:-

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Active loading techniques are having following advantages over passive loading techniques. A high encapsulation efficiency and capacity. A reduced leakage of encapsulated compounds ‘Bed Side’ loading of drugs thus loss of retention of drugs by diffusion or chemical degradation during storage. Reduction in hazards

-: Locus of Drugs in Liposomes :-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Locus of Drugs in Liposomes :-

Removal of un entrapped drugs:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Removal of un entrapped drugs It is important to estimate the amount of drug encapsulated within liposome. This is easier in case of MLVs compared to LUVs and SUVs. MLVs due to their large size, will be settle down and in pellet form when they will be centrifuged at high speed, while non-encapsulated drug remains supernatent .

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur LUVs and SUVs are estimated using following methods :- Dialysis. Minimum centrifugation. Protamine aggregation. Gel chromatography. Filtration techniques.

Characterization of Liposomes:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Characterization of Liposomes Chemical Analytical method Phospholipid concentration Cholesterol concentration Phospholipid peroxidation Phospholipid hydrolysis pH Cholesterol auto oxidation HPLC HPLC UV & GLC HPLC & TLC pH meter HPLC & TLC

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Physical Analytical method Vesicle shape Vesicle size Lamellarity Drug release TEM TEM & Gel exclusion X-ray scattering Dialysis

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Biological Analytical method Sterility Pyrogenicity Animal toxicity Aerobic and un aerobic cultures. Rabbit fever test and LAL test. Histology and pathology.

Stability Protocols:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Stability Protocols

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

-: Application :-:

Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur -: Application :- In Chemo therapy and neoplasia :- Anti cancer drugs are non selective so, there are more chances of toxicity to normal cell. So, drugs incorporated in liposome will helpful for target of drug to neoplastic region…because liposome acts as depot . They are bio degradable and non toxic

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Carriers for vaccines. Decreases systemic toxicities of drugs like doxorubicin. They are identical with lipid bio membrane due to phospholipids used in preparation so, more absorption of drug and also prolonged duration. E.g., Steroids , Insulin Used in occular drug delivery systems.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur Used for topical application like anti microbial, anti infective, anti tumor, anti septic, NSAIDs It can carry non penetrating material like genetic molecules so, helpful in genetic manipulation. Treatment of Leishmaniasis :- Drugs used are toxic to heart, liver, kidney but safe when incorporated in liposomes. Flexible in surface characteristic.

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Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur THANK YOU THANK YOU THANK YOU THANK YOU THANK TOU THANK YOU THANK YOU THANK YOU THANK YOU

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