LIPOSOMES

liposomes

LIPOSOMES

Controlled and novel drug delivery system

INTRODUCTION: HISTORY

Ideal drug delivery system delivers drug at a specific rate dedicated by need of body over the period of treatment to the site of action. The targeted drug delivery system achieves the site specific delivery but is unable to control the release kinetic of drug in predictable manner.

1) PAUL EHRLICH in 1906 initiated development for targeted delivery.2) Number of carriers are utilized afterwards which are serum proteins by GREGORIADIS

in 1977, Immunoglobulin by GOLD BERG in 1983, Synthetic polymers by POST el.al in 1983, lipid vesicles(liposomes) by POZNANSKY & JULIANO in 1984.

3) Other carriers are microspheres, erythrocytes, niosomes, pharmacosomes, reverse micelles. Few drugs have reached the stage of clinical development.

What are Liposomes?

These carriers are biologically inert in nature, devoid of any antigenic, pyrogenic or allergic reactions and their components are used as the components of biological membrane. Drugs incorporated do not cause unfavorable side effects as well.”

Liposomes are simple microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid molecule”.

So the drug molecule can either be encapsulated in aqueous space or intercalated (coated) into lipid bilayer.

Advantages of liposomes :

Provides selective passive targeting to tumor tissues (liposomal doxorubicin). Increased efficacy and therapeutic index. Increased stability via encapsulation. Reduction in toxicity of the encapsulated agent. Site avoidance effect Improved pharmacokinetic effects ( reduced elimination, increased circulation life times) Flexibility to couple with site specific ligands to achieve active targeting.

1M.Phil Pharmaceutics semester 01

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Characteristics of Liposomes

Lipid bilayer nature (amphiphilic also called amphipathic nature): polar head covalently

attached to one or two hydrophobic hydrocarbon tails.

When these lipids exposed to an aqueous environment, interactions between themselves

(hydrophilic interactions between polar head groups and vander-waals interactions between

hydrocarbon chains and hydrogen bonding with water molecules) lead to formation of closed

bilayer.

LIPOSOME CLASSIFICATION BASED ON PHARMACEUTICAL AND THERAPEUTIC ASPECTS:

Classification:

Lamella: A Lamella is a flat plate like structure that appears during the formation of liposomes. The

Phospholipid bilayer first exists as a lamella before getting converted into spheres. Based on number of

lamellaes there are two types

Unilamellar Vesicle

Multilamellar Vesicles

Based upon composition and application:

Conventional liposomes(CL) Fusogenic liposomes(RSVE) Cationic liposomes Immuno-liposomes

Types: Specifications:

Based on structural parameters:MLVOLVUVSUVMUVLUVGUVMV

Multilamellar large vesicles>0.5µmOligolamellar vesicles>0.1-1µm Unilamellar vesicles(all size range)Small unilamellar vesicles 20-100nmMedium sized unilamellar vesiclesLarge unilamellar vesicles> 100nmGiant unilamellar vesicles> 1µmMultivesicular vesicles>1µm


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Based on method of liposome preparations:

REVMLV-REVSPLVFATMLVVETDRV

Single or oligolemellar vesicles made by reverse phase evaporation methodMultilamellar vesicles made by reverse phase evaporation methodStable plurilamellar vesiclesFrozen and thawed MLVVesicles prepared by extrusion techniqueDehydration-rehydration method

STRUCTURAL COMPONENTS OF LIPOSOMES:

Cholesterol Phospholipids( phosphodiglycerides, sphingolipids)

Phospholipids: Most common phospholipid is the phosphotidylcholine molecule. It is a molecule in which a glycerol bridge links a pair of hydrophobic acyl hydrocarbon chains with a hydrophilic polar head group phospho choline).

Head group Common name AbbreviationsO-CH2-CH2-N-3Methyl Choline PCO-CH2-CH2-NH3 Ethanolamine PEO-H Acid PA

Most common of them is PC also known as lecithin, can be derived from natural and synthetic sources. At various temperature lecithin can exist in different phases. At low temperature it will be in solid form, at high temperature lipid membrane passes from tightly ordered gel to liquid crystal phase.

Liposome membranes are semipermeable in that diffusion of molecules and ions acrozz membranes varies considerably for molecules with high solubility in both organic and


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aqueous media. A phospholipid membrane constitutes a very denser barrier, on other hand polar solutes such as glucose and higher molecular weight compounds pass across membrane very slowly. Smaller molecules with natural charge (e.g H2O and urea) can diffuse across quite rapidly while charged ions differ greatly in this behavior. Na+ and K+ ions diffuse very slowly. Proteins and metal ions diffuse at phase transition temperature.

Cholestrol: To make phospholipid hard and thick cholesterol is added (solidification). Cholesterol is added into phospholipid at ratio of 1:1 , 2:1 cholesterol to phosphotidyl choline.

Role of cholesterol in bilayer formation:

Acts as fluidity buffer After intercalation with phospholipid molecules alters the freedom of motion of carbon

molecules in the acyl chain Restricts the transformation of trans- to gauche- confirmations

Handling of liposomes: Standard composition of liposome is…

Egg lecithin: Cholesterol: Phophotidyl glycerol

These lipids can be stored either as solids or in organic solution at -20 to -70°C in order to reduce chances of oxidation. The solvent most widely used is a mixture of chloroform and methanol in a volume ratio of 2:1. Compounds which are sparingly soluble in either chloroform or methanol alone often dissolve readily in this 2:1 solvent mixture. Chloroform give rise to phosgene on standing (degraded product). This can be prevented by addition of 1% ethanol to stabilize the chloroform. All lipid solutions should be stored in dark in glass vessels with securely fastened ground glass stopper. Polypropylene containers may also be used. To reduce the possibility of oxidation of lipids nitrogen is commonly used.

Drying:

Large volume of organic solution of lipid is mostly dried in rotary evaporator fitted with a cooling coil and a thermostatically controlled water bath. Rapid evaporation of solvent is carried out by gentle warming (20-40°C) at reduced pressure (400-700mmHg).Rapid rotation of solvent containing flask increases the surface area for evaporation. If the concentration of lipid is particularly high it may be difficult to remove last traces of chloroform from lipid film. So it is recommended that after rotary evaporation, some further mean is employed to bring the residue to complete dryness. Attachment of flask to lyophilizer and overnight exposure to high vacuum is a good method.


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METHOD OF LIPOSOME PREPARATION:

All methods for preparing liposomes involve three or four basic stages:

1) Drying down lipids from the organic solvent.2) Dispersion of lipids in the aqueous media.3) Purification of resultant liposomes.4) Analysis of final product.

Steps:

1) Cholesterol, lecithin( charged)

2) Dissolved in organic solvent( chloroform and methanol)

3) Solution in organic solvent

4) Drying( solvent removed)

5) Thin film of liposomes( lipid bilayer is formed)

6) Dispersion in aqueous media

7) Hydration

8) Liposome suspension is formed


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Loading of Drug in Liposomes

Two methods are followed:

Passive loading Remote loading

Passive loading:

It involves the loading of entrapped agents before or during manufacture procedure.

Remote loading:

Certain types of compounds with ionisable groups, and those which display both lipid and water solubility, can be introduced into the liposomes after the formation of intact vesicles.

PHYSICAL DISPERSIONS OR MECHANICAL DISPERSION

There are four basic methods of physical dispersion

1. Hand shaking or rotary evaporator method.


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2. Non shaking3. Freeze drying4. Proliposomes

1. HAND SHAKING OR ROTARY EVAPORATOR METHOD:Lipid film hydration by hand shaking is most widely used physical dispersion method to prepare multi lamellar vesicle (MLVS).

Method of preparation:

1. Mixing 2. Drying3. Hydration of lipid

1. MixingLipid mixtures of different phospholipids and charge components in chloroform: methanol (2:1 v/v) solvent mixture is prepared and introduced in round bottom flask.

2. DryingIt is then attached to rotary evaporator and rotated at 60 rpm.Organic solvent is removed at 30 C or above the transition temperature of lipids within 15 minutes. Nitrogen is introduced in evaporator. Flask is removed form evaporator and Residual of organic solvent is removed with the help of lyophilizer.

3. Hydration of lipids:After drying the flask is flushed with nitrogen and 5 ml of saline phosphate buffer (containing solute to be entrapped) is added. The flask is again attached with the evaporator and rotated at room temperature and pressure at 60 rmp. The flask is left rotating for 30 minutes until lipids has been removed from the wall of the flask and has given homogenous milky white suspension free of visible particles.The suspension is allowed to stand for further 2 hours at room temperature or at the temperature above the transition temperature of the lipids in order to give the swelling process to give MLVS.


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2. NON SHAKING METHOD: (FOR LUVS)Same method as in hand shaking but care is taken over swelling process.

Hydration and swelling are carried out in two separate steps. Hydration is initiated by exposing the film with stream of water saturated nitrogen for 15 minutes which is followed by swelling in aqueous medium without shaking.

Drying:Solution of lipid in chloroform: methanol (2:1v/v) spread over flat bottom conical flask. Solution is evaporated at room temperature by the flow of nitrogen through the flask without any disturbance. After drying water saturated nitrogen is passed through the flask until opacity of lipid layer disappeared within 15 to 20 minute.

3. FREEZE DRYING: (MLVS)Lipoids dissolved in suitable organic solvent are freeze dried. Solvent added should be near the freeze point, which should be above the temperature of condense of freeze dried.Tertiary butyl alcohol is widely used solvent. Water or saline is added to lyophilized lipid with rapid mixing above the phase transition temperature to give MLVS.

4. PROLIPOSOMES:(MLVS)

What are proliposomes?In order the increase the surface area of dried lipid film and to facilitate instantaneous hydration the lipid is dried over finely divided particulate support such as powdered sodium chloride or sorbitol or other polysaccharides. These dried lipid coated particulates are called proliposomes.


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Pro liposomes form dispersions of MLV on adding water in to them where the support is rapidly dissolved and lipid film hydrates to form MLV.

ADVANTAGES:

1. This method over comes the stability problem of liposomes encounter during their storage as dispersion, dry or frozen form.

2. It is ideally suited for the preparations where the material to be entrapped incorporates in to lipid membrane.

3. It is suitable in the cases where 100 % entrapment of component is not required rather the stability is preferred.

PREPARATION OF PROLIPOSOMES:

Procedure: Buchi rotary evaporator:

1. Lipid solution in chloroform (60mg/ml) is prepared and sorbitol is introduced in flask.2. Fit the flask to the evaporator and rotate slowly so that powder tumbles gently off the

walls and ensure good mixing and solvent is evaporated.3. Flask is lowered in water bath at 50 to 55 C when a good vacuum has developed. An

aliquot of 5 ml of lipid solution is introduced in the flask via the solvent inlet tube.4. Solvent is absorbed completely by the powder. 5. Vacuum is released. Connect the flask to lyophilizer for drying.6. Powder is transferred to 10ml glass vial containing 600mg solid (100 mg lipid and 500mg

sorbitol) flushed with nitrogen sealed well and stored.

How to use?

For use introduce 10 ml of distilled water in vial mix on a whirl mixer for 30 sec or in a shaking water bath above Tc (phase transition temperature.

PROCESSING OF LIPIDS HYDRATED BY PHYSICAL MEANS

OR

OR MECHANICAL TREATMENT OF MLVS

A large number of methods have been developed to reduce their sizes and to convert liposomes of large size to smaller homogenous vesicle. Such techniques are

Microemulsification Sonication Extrusion French pressure cell


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Dried reconstituted vesicles (DRVS) Freeze thaw sonication method (FTS)1. MICRO ENCAPSULATION OR MICRO EMULSIFICATION:

MICROFLUDIZER

Microfludizer used for microemusification of liposomes. In this method micro fluidizer pump are used. It consists of following parts

Pump Interaction chamber Reservoir Filter

Procedure:

1. The lipids are introduced in the fludizer either as a suspension of lager MLVS or as a slurry of unhydrated lipid in organic medium.

2. Microfludizer pumps the fluid at a very high pressure (10,000 psi, 600 -700bar) through a 5micrometer orifice.

3. 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, therefore affecting an efficient transfer of energy.

4. The fluid collected can be recycled through the pump and interaction chamber until vesicles of spherical dimensions are obtained.

After a single cycle size of vesicles is reduced to a size of 0.1 and 0.2 micrometer in diameter.

Advantages:


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High rate of production Process the samples having a high proportion of lipids (20% or more by weight) This process is sufficient for encapsulation of water soluble drugs.

2. SONICATED UNILAMELLAR VESICLES (SUVS)

At a high energy level the size of vesicles is further reduced. There are two methods of sonication base on used of either probe or bath ultrasonic disintegrators.

1. Probe ultrasonic disintegrator:

Method:

Sonication of MLVS is achieved by placing the tip of probe sonicator in test tube containing the lipid dispersions and sonicationg for 5 to 10 minutes above the phase transition temperature. The lipid dispersions begin to clarify to yield slightly hazy transparent solution. These particles can be removed by centrifugation to yield clear SUV dispersion.

Centrifugation: Liposomal dispersion after sonication is placed in clear plastic walled ultracentrifuge tube. It is centrifuged at 100,000g (30 min at 20 C) to sediment titanium particles and large MLVS followed by high speed centrifugation (1, 59,000g for 3-4 hour)

After spinning tube is removed liquid at the top clear layer is decanted which contains pure dispersions of SUVS.

Use:

It is employed for the dispersions which require high energy in small volume i.e high concentration of lipids or viscous aqueous phase.

Disadvantage:

1. Probe tip sonicator supply a high energy in to liquid dispersions but suffer from overheating of liposomal dispersions resulting in lipid degradation.

2. Sonication tip also tend to release titanium particles in liposomal dispersions which must be removed by centrifugation.

1. Bath ultrasonic disintegrator:

Method:


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Sonication of MLVS dispersion is accomplished by placing the test tube containing lipid dispersions in bath sonicatorfor 5 to 10 minutes above the phase transition temperature.

Use:

Bath sonicators are used for suitable large volumes of diluted lipids. Widely used to prepare SUVS. It overcomes all the disadvantages associated with probe sonicator.

2. FRENCH PRESSURE CELL LIPOSOMES:

One of the first and very useful methods is the extrusion of preformed large liposomes in French pressure under very high pressure (Barenholt et al 1979, Hamilton et al 1980)

Advantages:

1. This technique yield rather uni or oligo lamellar liposomes of intermediate size (30 to 80 nm in diameter depending on applied pressure)


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2. Liposomes prepared by this method are less likely to suffer from structural defects and instabilities observed in sonaicated vesicles

3. Leakage of contents form vesicles is slower and lower than sonicated vesicles.4. It reduces the heterogeneity of population of liposomes obtained by detergent dialysis.

Draw backs: Initial high cost of press that consists of an electric hydraulic press and pressure cell.

Apparatus:

The heart of French pressure cell is pressure cell, manufactured in stainless steel and designed to resist up to 20,000 or even 40,000psi.

It consists of following parts:

Precision bored cylindrical cavity with small outlet orifice (valve) A bottom seal A piston Valve closure and outlet rubbing.

Method:

Liposomal suspension is filled in pressure chamber. The liquid sample is introduced in cavity. Height of piston is adjusted so that chamber is completely filled with the liquid.

Complete filling is important to prevent compression of trapped air which will result in uncontrollable splashing during extrusion.

Upon filling the bottom seal is inserted. Pressure is adjusted with hydraulic valves.


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At high force smaller liposomes are formed.

4 VESICLES PREPARED BY EXTRUSION TECHNIQUES: (VETS)

The membrane extrusion method the size of liposome is reduced by passing them through membrane filter of defined pore size. It can be achieved at lower pressure <100psi. Than required in French pressure cell.

Types of membrane filters:

1. Tortuous path type membrane2. Nucleation path type of membrane

1. Tortuous path type membrane:In this type random paths arise between cris crossed fibers. Liposomes which are larger than channels diameter get stuck when one tries to pass them. Fibers blocked easily because of convoluted nature of channels.

2. Nucleation path type membrane:In this type liposomes can pass easily through membrane even they are larger than pore diameter. The membrane consists of continuous sheet of polymer usually polycarbonate in which straight side pore holes of exact diameter are present so they offer much less resistance to the material passing through.

Uses:


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This technique is used to process LUVand MLV as well as to produce SUV and LUV due to their ease of production readily selectable diameter batch to batch reproducibility and freedom from solvent and surfactant contamination.

5. DRIED RECONTITUTED VESICLES (DRVS):

This method starts with freeze drying of a dispersion of empty SUV and then rehydrating it with aqueous fluid containing the material to be entrapped. This leads to a dispersion of lipid in finely divided form. liposomes obtained by this method are uni or oligo lamellar of the order of 1.0µm or less in diameter.

Reason of using freeze drying?

The step of freeze drying is used to freeze and lyophilized preformed SUVS dispersion rather than to dry a lipid from organic solvent this leads to organized membrane structure as compared to random matrix which on addition of water can rehydrate fuse and reseal to form vesicles with high capture efficiency.

Advantages:

1. High entrapment of water soluble drug.2. Use of mild conditions for loading and preparation of bioactive.

6. FREEZE THAW SONICATION (FTS):


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It’s an extension of DRVS method. This method is based upon freezing of a uni lamellar(mainly SUVs) dispersion and then thawing at room temperature for about 15 minutes and finely subjecting to sonication cycle.

Method:

1. Empty liposomes are prepared by sonication for about 15 to 30 sec.2. It is then followed by thawing.3. Liposomes are again subjected to sonication. This step reduces the permeability of

liposome membrane by accelerating the rate at which packing defects are eliminated.

Advantages:

It is simple rapid and mill for entrapped solute and result in high proportion of large unilamellar formation which is use full for study of membrane transport phenomenon.

Inclusion of some negatively charged lipid give higher entrapped volume (20µl/mg) as compared to (10µl/mg) for neutral lipid.

Disadvantages:

Neutral lipid cannot be subjected to freeze thaw sonaication. Sucrose divalent metal ions and high ionic strength salt solution cannot entrap.

2. SOLVENT DISPERSION METHOD:

PRINCIPLE:

Lipid first dissolve in organic solution Aqueous phase should mix with material to be entrapped within liposomes Lipid are then brought into contact with aqueous phase

METHOD INVOLVING SOLVENT DISPERSION:

1. Organic solvent miscible with aqueous phase2. Organic solvent immiscible with aqueous phase but aqueous phase is in excess.3. Organic solvent immiscible with aqueous phase but organic solvent is in excess.

TECHNIQUES:

1. ETHER INJECTION:

It involves injecting the immiscible organic solution very slowly into the aqueous phase. Method also treats sensitive lipids very gently and a little risk of causing


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oxidative degradation because ether is free from peroxides. For substances that degrade at high temperature fluorinated hydrocarbons are used.

2. ETHANOL INJECTION:

It involves injecting ethanol rapidly in aqueous phase. As a result of this ethanol dissolve and phospholipids molecules are dispersed. There is no need to increase the temperature as in the case with ether injection method.

3. WATER IN ORGANIC PHASE:

Liposomes are made in two steps

1. First the inner leaflet of lipd bilayer is form

2. Then the outer half is form

The common feature of this method Is formation of water in oil emulsion produced by introduction of small quantity of aqueous medium containing material to be entrapped intolarge volume of immiscible organic solution of lipid followed by mechanical agitation to break up the aqueous phase into microscopic water droplet


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4. DOUBLE EMULSION VESICLES:

The organic solution which already contains water droplet is introduced into excess of aqueous medium followed by mechanical agitation a multicompartment vesicles are obtained

It may be described as w/o/w system

The organic solvent is evaporated by strong jet of nitrogen in double emulsion. and final volume is adjusted by adding distilled water.

5. REVERSE PHASE EVAPORATION METHOD:

6. STABLE PLURILAMELLAR VESICLES:

In this method water in oil dispersion is prepared but drying process is accompanied by continuos bath sonication with stream of nitrogen.

3. DETERGENT SOLUBILIZATION:

In this method phospholipids are brought into contact with aqueous phase with the intermediary of detergent. The structure formed as a result of this is known as micelle and can be composed of several hundred components of molecules

Micelle containing components in addition to detergent is known as mixed micelles.


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PURIFICATION OF LIPOSOMES:

Gel Filtration Coloumn Chromatography Dialysis Centrifugation

CHARACTERIZATION OF LIPOSOMES:

Behaviour of liposomes in both physical and biological systems is goverened by the factors:

physical size membrane permeability percent entrapped solute chemical composition quantity of starting material percentage drug release


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CHEMICAL PROPERTIES

Quantitative determination of phspholipids; Phspholipids hydrolysis: Phspholipids oxidation: Cholestrol analysis

1-QUANTITATIVE DETERMINATION OF PHSPHOLIPIDS;

It is difficult to measure directly the phospholipids concentration since dried lipids often contain considerable quantities of residual solvents. So the most widely used methods are indirect, which includes


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Bartlett assay Stewart assay TCL (thin layer chromatography)A) Bartlett Assay:

phospholipids phosphorous in the sample is first hydrolyzed to inorganic phosphate, which is then treated with ammonium molybdate to form phosphomolybdis acid, which is then reduced by amino-naphtyl-sulphonic acid to a blue color compound. Its intensity is measured spectrophotometrically.

B) Stewart Assay;In this method first prepare the stdandard dilutions. Add lipids in chloroform and make different dilutions and then in each dilution add 0.1 M Ammonium ferrothiocyanate which result in formation of complex. Measure density at 485 nm and compare it with sample.

C) TLC:It is also employed for determining the purity and concentration of phospholipids.

2-PHSPHOLIPIDS HYDROLYSIS:

Major product of lecithin hydrolysis is lysolecithin. Phospholipids hydrolysis is measured by approximation of lysolecithin by HPLC.

3- PHSPHOLIPIDS OXIDATION:

In the absence of specific oxidant, oxidation takes place through free radical chain mechanism. Initiation step for this is removal of H-atom from fatty acid chain and it is brought about by

1. Electromagnatic radiation2. Transition metals

Techniques available for determining oxidation are

UV absorbance method TBA method Iodometric method GLC method

4) CHOLESTROL ANALYSIS:

It is qualitatively analyzed by capillary column of fused silica.

Quantitatively estimated by measuring absorbance of purple colour complex form by treating the sample with a combined reagent consisting of ethyl acetate, ferric perchlorate and sulfuric acid. Absorbance is measured at 610 nm.


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PHARMACOKINETICS OF LIPOSOMAL ENCAPSULATED DRUGS:

Most of the drug delivery system are designed to control one of the following parameters

1. Rate of input of drug into particular compartment2. Distribution and localization of drug in the body3. Rate of metabolism of drug

Liposomes used one possible approach to control the drug delivery.

Comparison of liposomes and polymeric devices?

1-Similarity: both are drug reservoir with an output controlled and limited by the permeability of membrane

2-Differences:

Liposomes give sustained effect from hrs-days while polymeric devices give effect from days to months

Drug disposition can be changed by liposomes but not by polymeric devices

CLEARANCE AND DISTRIBUTION OF LIPOSOMES IN VIVO

Liposomes injected into circulation are gradually sequestered into different organs and tissues. Distribution depend on two factors

Size of liposomes: smaller the size longer will be persistence in circulation and larger the size rapid will be clearance. That is why SUV’s persist in circulation for longer time and MLV’s are rapidly cleared

Surface charge of liposomes: SUV’s with positive and negative charge persist in circulation for longer time while SUV’s with negative charge are rapidly cleared.

After clearance from circulation liposomes are sequestered into Different tissues and organs. In case of MLV’s primary Site of uptake is Liver and Spleen. Out of MLV’s which are of larger size retained in capillaries of lungs Because of physical entrapment.

SUV’s have broader tissue distribution as compared to MLV’s however at the end they also accumulate in liver and spleen.

PHARMACODYNAMICS OF LIPOSOME ENCAPSULATED DRUG

Behaviour of drug is largely determined by the behavior of Carrier.Inshort encapsulated drugs will have following pharmacodynamic effects

1. Retardation of drug clearance from circulation


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2. High drug accumulation in tissues rich in reticulo endothelial cells especially liver and spleen

3. Retention of drug in tissues for longer time4. Protection of drug against metabolic degradation

STABILITY OF LIPOSOMES:

Liposomes stability is much and more severe. Liposomes are liable to fusion aggregation, leakage, hydrolysis and oxidation.

A) PREVENTION OF CHEMICAL DEGRADATION:

Following precautions should be followed

Use freshly purified lipids Use freshly distilled solvents Avoid the procedure which involve high temperature Store Liposomes in inert atmosphere Deoxygenate all aqueous solution with nitrogen Add an antioxidant as a component of lipid membrane e.g α-Toccopherol Add iron chelator. Slow down the hydrolysis by carrying the procedure at neutral pH Use the lipids which have ether linkage instead of ester linkage Enzymatic hydrolysis can be prevented by using sphingomyelins or phospholipid

derivatives.B) PREVENTION OF PHYSICAL DEGRADATION:

1. Leakage and fusion:

Leakage and fusion of vesicles occur as a result of packing defects in the membrane introduced during their manufacture. These irregularities can be dispensed by a process called annealing in which simply incubate the vesicles at a temp. High enough above the transition temp to allow differences in packing density b/w the opposite sides of the bilayer to equalize by trans-membrane flip-flop of lipid molecules.

SUV’s are more prone to fusion. They do so for relieving stress which arises from high curvature of membrane.

2-Aggregation and sedimentation

Aggregation of neutral liposomes is brought about by van der waals interaction and is more pronounced in larger vesicles because of inc. planarity of the membrane which allows the greater area of the membrane to come in contact with each other.


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TECNIQUES TO OVERCOME THE INSTABILITY:

1-Store liposome suspension at a temp. Away from transition temp.

2-Use more polar and hydrophilic solutes

3-Lyophilization: In this liposomes are converted into anhydrous or solid form, where chemical degradation is less likely to occur.

4-Cryo preservation: cryo protectants(liquid Nitrogen) are used. it is most successful method for liposomes.

APPLICATION OF LIPOSOMES:

Mode of action ApplicationIntracellular uptake Microbial disease Metal storage diseases Gene manipulation

Uptake by tumor cell Slow release of drug near the target area

Tumor near fixed macrophage, Macrophage activation to tumoricidal/microbicidal state

Circulating Reservoirs Blood surrogates

Facilitation of drug uptake by certain routes

Drug delivery to skin ,lungs.eyes,mucosal tissue

Avoidance of tissue sensitivity Cardiotoxicity of doxorubicin

The following are the few more properties which make liposomes applicable in various fields

Cell liposome interaction: SUV’s can interact with the cell in 4 ways;1. Stable adsorption: Association of intact vesicles cell surface.This association is

mediated by non-specific electrostatic,hydrophobic or other forces2. Endocytosis: It is the uptake of intact vesicles into endocytic vesicles and their

delivery to lysosomal apparatus.3. Fusion: It is the merging of the vesicle bilayer with the plasma membrane

bilayer,with the resultant release of vesicle contents into cytoplasm.4. Lipid exchange: It is the transfer of individual lipid molecules b/w vesicles and

cell surface,without the cell association with the aqueous vesicle content. Localized drug effects: Liposomes help in depositing the drug within selected sites or

selected cell types. This is because of their larger size and low degree of penetration in epithelial and connective tissue barrier.

Enhanced drug uptake: It occurs by vesicle cell fusion or via endocytosis. Molecules with different range of solubilities can be accommodated. Flexibility in structural characteristics.


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1-CANCER CHEMOTHERAPY AND NEOPLASIA:

Anticancer drugs are usually non selective and result into toxicity to normal cells.Liposomes have successfully been used for such drugs and it has following advantages;

1. Inc. circulation life time2. Protects the metabolic degradation of drug3. Altered the Tissue distribution of the drug with enhanced uptake in liver and spleen and

dec. uptake in brain myocardium and kidney.

2-LIPOSOMES AS CARRIER FOR VACCINES:

a) Liposomes as immunological adjuvants: Liposomes have adjuvant effect on protein antigen.Liposomes cab be effectively used as carrier for antigen. It has the following advantages;

1. A non-immunogenic substance can be converted into immunogenic2. Hydrophobic antigens can be reconstituted3. Small amounts may be suitable as immunogens.4. Multiple antigens may be incorporated into the single liposome.5. Adjuvants may be incorporated with antigen6. Longer duration of action may be achieved

b) Liposomes as carrier for antigens: Liposomes recognized as an efficient carrier for delivering biologically active material to specific cells. However on administration major fraction is uptaken by liver and spleen. This uptake can be retarded by Following approaches;

1. Rate of uptake of liposomes by RES is minimized by using small, neutral unilammelar vesicles having higher Tc and cholesterol.

2. By coating the surface of liposomes which make them less recognizable by RES.3. Coupling appropriate ligand on the liposome surface which can bind to the receptors on

the surface of target cells.

c) Liposomes as carrier of drug in oral treatment: Liposomes can be effectively used for delivering the drugs by oral route

1. Arthritis: Steroids are used in the treatment of arthritis but their effect is transient bcoz of rapid destruction in periphery. When encapsulated in liopsomes produces

Effect for longer duration Localized effect

2. Diabetes: Insulin encapsulated in liposome when administered to hyperglycemic animals via oral route, of glucose level was observed. Liposomes provided protection to insulin against the pepsin and pancretin.


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d) Liposomes for topical applications: Liposomes have great potential as topical drug delivery. Liopsomes based formulation of NSAIDS, local anesthetics, antimicrobials, and anti tumor drugs are effective in the treatment of respective disorders.

e) Leishmaniasis: It is a parasitic disease affecting millions of people. It become lethal if parasite invades into liver and if it is left untreated. Even many people die with the treatment, bcoz commonly prescribed drugs antimonials are related to arsenic are very toxic to Heart, liver and kidney. Their liposome encapsulation produces following advantages1-Toxicity is reduced2-Localized effect in liver is achieved.3-Amount of the drug needed is reduced

f) Lysosomal storage disease: These diseases mostly results from the genetically determined enzymatic deficits of particular lysosomal enzymes. So liposomes are suitable candidate for lysomotropis enzyme replacement. This includes Gaucher disease (β-Glucosidase deficiency) where site of pathological catabolite accumulation is RES and Pomp’s disease (α-glucosidase deficiency) where the primary affected tissues are liver and muscle.

References:


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