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TARGETED DRUG DELIVERY SYSTEM By S.MYTHRI under the guidance of L.S.DANKI M.Pharm (Ph.D) :

TARGETED DRUG DELIVERY SYSTEM By S.MYTHRI under the guidance of L.S.DANKI M.Pharm ( P h.D )


INTRODUCTION: Liposomes are colloidal, lipid vesicles , normally composed of phospholipids that spontaneously form multilamellar, concentric, bilayer vesicles, with layers of aqueous media separating the lipid layers. Liposomes can improve drug loading, drug delivery and sustained release offering advantages over traditional dosage forms. Liposomes were first produced in England in 1961 by Alec D. Bangham.

Rationale and advantages of liposomes: :

Rationale and advantages of liposomes: The rationale of encapsulating a drug within liposomes is to prevent its rapid metabolism and its rapid removal from blood circulation after its administration so that the drugs from depot liposomes are ideally suited for drug delivery. Advantages : Liposomes are biocompatible due to their biodegradability, low toxicity and lack of immunogenicity. Improved solubility of lipophilic and amphiphilic drugs Passive targeting to the cells of the immune system When liposomes are coupled with antibodies serves as means of active targeting

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Sustained release system of systemically or locally administered liposomes. Site avoidance mechanism, liposomes do not dispose in organs like heart, kidney, brain and nervous system. This reduces cardiotoxicity , nephrotoxicity and neurotoxicity Protective action to plasma unstable drug site specific targeting Improved transfer of hydrophilic charged molecules such as antibiotics, plasmids and genes into cells Improved penetration into tissues dermally applied liposomal dosage forms.

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Provides selected passive targeting to tumour tissues Increased efficacy and therapeutic index Increased stability by encapsulation Reduction in toxicity of the encapsulated agent

Demerits: :

Demerits: Less stable Low solubility Short half life Oxidation and hydrolysis of phospholipids High cost of production Shelf life is too short Scale-up problems

Definition: :

Definition: “Liposomes are concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayer composed of natural or synthetic phospholipids .”

Liposome structure:

Liposome structure Liposomes are spherical vesicles with a phospholipid bilayer Hydrophilic Hydrophobic

Phospholipid structure:

Phospholipid structure

Liposome :




Microscopic view of liposome:

Microscopic view of liposome

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Based on structural parameters Multilamellar MLV Oligolamellar OLV Unilamellar UV Multivesicular MV Small unilamellar Medium unilamellar Large unilamellar Giant unilamellar

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Methods of preparation of li[posome METHODS OF LIPOSOME PREPARATIONS Passive loading technique Active loading technique Mechanical dispersion method Solvent dispersion method Detergent removal method Lipid film hydration Micro emulsification Sonication proliposomes Ethanol injection Ether injection Double emulsion vesicles column chromatography Dialysis

Mechanism of liposome formation:

Mechanism of liposome formation Liposomes are formed by open hydration of lipid molecules . Normally lipids are hydrated from dry state ( thin or thick lipid film, dried powders) and stacks of crystalline bilayers become fluid and swell in hydration condition. Then thin cylinders grow and upon agitation detach into self-closed large, multilamellar liposomes . Once the large particles are formed they can be broken by mechanical treatment into smaller bilayered fragments which close into smaller liposomes.

Mechanism of liposome formation:

Mechanism of liposome formation

General method of liposome preparation:

General method of liposome preparation Drying down lipids from organic solvent Dispersion of lipid in aqueous media Purification of resultant liposomes Analysis of final product


MECHANICAL DISPERSION METHOD -1.Thin film hydration In this method a 250 ml round bottom flask is taken containing organic solvent with lipids. Then this beaker is attached to a rotary evaporator and rotated at 60 rpm resulting in formation of stacks of lipids. Then the beaker containing stacks is dried using nitrogen for 15 min and then the casted film is dispersed in aqueous medium. This results in hydration of lipids which swell and peel of from the wall of flask resulting in formation of multilamellar vesicles.

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In this method, lipid mixture of different phospholipids and chloroform: methanol solvent mixture (2:1) is prepared first and then introduced into a 250 ml round bottom flask. This flask is attached in rotary evaporator at 60 rpm . The organic solvents are evaporated at about 30 o C or above the transition temperature of the lipids used. The rotation is continued for 15 minutes after the dry residue first appears.

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The evaporator is isolated from the vacuum source by closing the cap. Nitrogen is introduced into the evaporator and pressure at cylindrical head is raised until there is no pressure difference between inside and outside of the flask. The flask is removed from evaporator and fixed to lyophilizer to remove residual solvents.

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Hydration of lipids: A fter releasing the vacuum and removal from lyophilizer , the flask is flushed with nitrogen, 5 ml of saline phosphate buffer containing solute to be entrapped is added. The flask is attached to the evaporator again and rotated at room temperature and at 60 rpm or below. The flask is left for 30 min or until lipids has been removed from the wall of the flask and gives homogenous milky-white suspension free of visible particles. The suspension is allowed to stand for 2 hrs at room temperature above the transition temperature of the lipid inorder to complete swelling process to give MLVs.

2. Micro emulsification liposome:

2. Micro emulsification liposome Microfluidizer is used to prepare small MLVs. In this a lipid dispersion is placed in a microfluidizer pump which pumps the fluid at 600-700 bar pressure through a 5 µm orifice. Then this dispersion is forced along micro channels, which make two streams of fluid to collide with each other at right angles at a high velocity. Due to this transfer of energy takes place resulting in formation of multilamellar vesicles.

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The fluid collected can be recycled through the pump and interaction chamber until vesicles of the spherical dimensions are obtained. After a single pass the size of the vesicles is reduced to 0.1 to 0.2 mm in diameter. The exact size distribution depends on the nature of the components of the membrane and hydration medium. In addition to high rate of production this method has the advantage of being able to process samples with very high proportion of lipid. This process is very efficient for encapsulation of water soluble materials . Capture value of 70% is reported.

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Collision at right angle Vesicles of required dimension Reservoir of MLVs air Filter 5micrometer Separation into two streams

3. Sonicated unilamellar vesicles:

3. Sonicated unilamellar vesicles In this method MLVs are exposed to UV radiations to get small vesicles. There are two methods of sonication 1. bath sonicator 2. probe sonicator Probe is used for high concentrated lipids while bath is used for large volumes of diluted lipids. In probe a high energy is used which may result lipid degradation and also titanium particles may be released into dispersion.

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For these reasons bath sonicators are used for preparing MLVs. In this method dispersion is placed in a test tube which is placed In a sonicator Sonication is done for 5-10 min until a transparent solution appears. After sonication dispersion is placed in a plastic centrifugation tube and centrifuged for 30 min at 20 o C to get large MLVs and 3-4 hrs to get SUVs. After spinning, the tube is carefully removed from the rotary and with a Pasteur pipette, the liquid with top clear layer is decanted leaving the central opalescent layer. The top layer will be a pure suspension of SUVs.

4. Proliposomes :

4. Proliposomes This method is used to increase the surface area of dry lipid film and to facilitate instantaneous hydration. Lipid is dried over a finely divided solid support such as powdered sodium chloride or sorbitol or other polysaccharides. These dried lipid coated particulates swell upon adding water to form pro-liposomes. In this the support rapidly dissolves to give a suspension of MLVs. For preparing proliposomes Buchi rotary evaporator is employed.


SOLVENT DISPERSION METHOD -1.Ethanol injection In this method an ethanol solution of lipids is injected rapidly into an excess of saline or other aqueous medium , through a fine needle. The force of the injection is sufficient to achieve complete mixing so that ethanol is diluted instantaneously in water and phospholipid molecules are dispersed evenly in medium. This procedure yields high proportion of SUVs (25 nm).

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This is a simple method with low risk of degradation of sensitive lipids A major limitation is the solubility of lipids in ethanol.

2. Ether injection :

2. Ether injection This is a similar method as ethanol injection but contrasts in some respects. This involves injecting the immiscible organic solution very slowly into an aqueous phase through a narrow needle at the temperature of vaporizing the organic solvent. This method is used to treat sensitive lipids very gently. Disadvantage is the long time taken to produce a batch of liposomes.

3. Double emulsion vesicles:

3. Double emulsion vesicles In this method an organic solution containing water droplets is introduced into excess aqueous medium followed by mechanical dispersion. By this a multi-compartment vesicle is formed described as w/o/w system or double emulsion. These vesicles are suspended in a aqueous medium. These have a aqueous core, the two compartments being separated by pair of phospholipid monolayer. Organic solvent is evaporated using strong jet of nitrogen into double emulsion.


DETERGENT SOLUBILIZATION In this method phospholipids are brought into intimate contact with the aqueous phase using detergents which associate with phospholipid molecule and screen the hydrophobic portions of the molecule from water. The structure formed as a result is known as micelles. The concentration of detergent in water at which micelles just start to form is known as critical Micellar Concentration .

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Invariably in all methods, which employ detergent in the preparation of liposomes, the basic feature is to remove the detergent from preformed mixed micelles containing phospholipid, whereupon unilamellar vesicles form spontaneously. The detergent methods are not very efficient methods in terms of percentage entrapment values. But this is the best method for preparing liposomes with lipophilic proteins inserted into the membranes. Another special feature is the ability to vary size of the liposomes by precise control of the conditions of detergent removal.

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Three methods are employed for removal of detergent and transition of mixed micelles to concentric bilayered form. These include A. Dialysis B. Column chromatography C. Detergent adsorption using bio-beads.

1. Dialysis :

1. Dialysis In contrast to phospholipids, detergents are highly soluble in both aqueous and organic media. Upon lowering the concentration of detergent in the bulk aqueous phase, the molecules of detergent can be removed from mixed micelle by dialysis. Commonly used detergents are sodium cholate and sodium deoxycholate . Commercial version of dialysis system is LIPOREP.

2. Column chromatography:

2. Column chromatography Phospholipids in the form of either sonicated vesicles or as a dry film, at a molar ratio 2:1 with deoxy cholate form ULV of 100 nm on removal of deoxy cholate by Column chromatography. This can be achieved by passing the dispersion over a Sephadex G-25 column presaturated with lipids.

3. Detergent adsorption using bio-beads: :

3. Detergent adsorption using bio-beads: Detergent phospholipid mixture can form large unilamellar vesicles upon removal of non-ionic detergent (Triton X-100) using appropriate adsorbents for the detergent. The ability of bio-beads SM-2 to adsorb Triton X-100 selectively and rapidly makes them a suitable candidate for LUV preparation by detergent solubilization method.

Characterization of liposomes:

Characterization of liposomes Size and its distribution Surface charge Percent entrapment Lamellarity

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a. Size and its distribution The most precise method to determine size of liposome is electron microscopy as it permits to view each individual liposome. Freeze etch and freeze fracture microscopy techniques are used to study vesicle size and structure. Freeze etch is particularly used to measure small vesicle diameters.

b. Surface charge:

b. Surface charge A technique is developed that separates extruded vesicles on the basis of surface charge by electrophoresis on a cellulose acetate plate in a sodium borate buffer pH 8.8. Lipid samples are applied to plate and electrophoresis is carried at 4 O C on a flat bed apparatus for 30 min. The plate is dried and phospholipids are visualized by molybdenum blue reagent.

c. Percent entrapment:

c. Percent entrapment Two methods are used for this 1. protamine aggregation 2. mini column centrifugation In protamine aggragation liposome suspension is placed in conical glass centrifuge tube, 0.1 ml protamine solution is added and allowed to stand for 3 min. 30 ml saline is added and then tube is spun for 20 min. supernatant is removed and assayed for unentrapped compound

4. Lamellarity :

4. Lamellarity Average number of bilayers present are found by freeze electron microscopy and by NMR. In NMR technique a broadening agent such as manganese ions are added which interact with outer leaflet of bilayer. Thus a 50% reduction in NMR signal means it is unilamellar liposome and 25 % reduction indicates presence of 2 bilayers in the liposomes

Stability of liposomes:

Stability of liposomes Prevention of chemical degradation: Following precautions are to be taken to minimize degradation Start with freshly purified lipids and freshly distilled solvents Avoid procedure which involves high temperature Carry out manufacture in the absence of oxygen Deoxygenate aqueous solution with nitrogen Store all the liposome suspensions in inert atmosphere Include antioxidant as a component of lipid membrane.

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Freezing and lyophilization techniques are used to overcome stability problems by transforming liposomes into a solid or anhydrous form. Stability of liposomes can also be increased by cross-linking membrane component. This method increases mechanical strength of membrane.

Applications :

Applications 1. cancer chemotherapy: Liposomes are successfully used to entrap anticancer drugs. This increases circulation life time, protects from metabolic degradation. 2. liposomes as carrier of drug in oral treatment: Steroids used for arthritis can be incorporated into large MLVs. Alteration in blood glucose levels in diabetic animals was obtained by oral administration of liposome encapsulated insulin.

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3. liposomes for topical applications: Drugs like triamcilone, methotrexate, benzocaine, corticosteroids etc can be successfully incorporated as topical liposomes 4. liposomes for pulmonary delivery: In this inhalational devices like nebulizers are used to produce an aerosol of droplets containing liposomes.

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5. Leishmaniasis : In this parasitic disease antimonial drugs are used which are lethal at high concentrations as they damage heart, liver and kidney. Such drugs can be encapsulated in liposomes. 6. Cell biological applications: Liposomes are used to carry functional DNA and RNA molecules into cells. Liposomes are used to insert enzymatic cofactors and cyclic AMP into cells.

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7. Ophthalmic delivery: Drugs like idoxuridine, indoxol and carbochol are greater efficacy in the form of liposomes. Potential advantage of ophthalmic liposome is their intimate contact with corneal and conjuctival surfaces


REFERENCES Gilbert s Banker. Modern Pharmaceutics. 4 th edition. S.P. Vyas and R.K. Khar . Targeted and Controlled drug delivery. 1 st edition. N.K. Jain. Controlled and Novel drug delivery. 1 st edition. Y.W. Chien . Novel Drug Delivery Systems. Binghe Wany , Teruna Siahaan , Richard A Soltao . Drug Delivery Principles and Applications. Krishna RSM, Shivakumar HG, Gowda DV and Benerjee S. Nanoparticles: A Novel colloidal drug delivery system. Ind J Pharm Ed Res.2006; 40(1):15-9. Verma AM, Misra U, Mishra N and Bharadwaj P. Liposomes as carrier systems. Inpharma communiqué. 2009; 2(2):2009.

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