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Targeted Drug Delivery System


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LIPOSOMES Target Drug Delivery System:



LIPOSOMES Introduction Principle Definition Components Classification Modes of Liposomes Classes of Liposomes Advantage Disadvantage General Preparation Mechanism of Liposomes Methods of Preparations Route of Administration Evaluation Stability Application Current Marketed Drugs


INTRODUCTION Liposomes were first described by British haematologist Dr Alec D Bangham and colleagues in 1961 (published 1964), at the Babraham Institute, in Cambridge and subsequently became the most extensively explored drug delivery system.


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 The name liposome is derived from two Greek words: ' LIPO ' meaning fat and ' Soma ' meaning body. Liposomes are simple Microscopic Vesicles in which an aqueous volume is entirely enclosed by a membrane composed of a lipid molecules. Structurally liposomes are concentric bilayed vesicles in which an aqueous volume is entirely enclosed by a membraneous lipid bilayer mainly composed by natural or synthetic phospholipids.


LIPOSOMES Hydrophilic Hydrophobic


phospholipids Polar head group Three Carbon Glycerol


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

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Based upon Composition and Application Fusogenic pH sensitive Long Circulatory Cationic Immuno Conventional

Modes of Liposome/Cell Interaction :

Modes of Liposome/Cell Interaction Adsorption Endocytosis Fusion Lipid


CLASSES OF LIPOSOMES Conventional Long Circulating Immuno Cationic


POLYMERS USED IN LIPOSOMES PREPARATION Polymer properties to be considered :- Low immunogenic. structural versatility. Improves liposomal stability. Easy to associate with liposome surface. pH-Sensitive Polymer –Liposome system pH-sensitive synthetic polymers used:- Poly-L-lysine & Poly (His) – Positively charged at low pH Interact with negatively charged membranes & promotes fusogenic properties. PAMAM – Shows pH-dependent membrane destabilization & thus used for cytoplasmic gene delivery.

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N- isopropylacrylamide - Provide Temp-responsive properties. STEALTH LIPOSOMES POLYETHYLENE GLYCOL – Hydrophilic, Flexible ↑ Repulsive forces at surface & thus ↓ interaction & plasma protein adsorption. POLOXAMER - POLOXAMINES-


Advantages Increased efficacy and therapeutic index Increased stability of encapsulated drug Reduction in toxicity of the encapsulated agents Site avoidance effect(avoids non-target tissue) Allows the sustained delivery of drugs with low therapeutic index by altering their Pharmacokinetics pattern They facilitate site specific drug uptake and delivery Flexibility to couple with site-specific ligands to achieve active targeting Provides selective passive targeting to tumor tissues(Doxorubicin)


Disadvantages Their loading capacity is quite weak Allergic reactions are possible Poor encapsulation of drugs could be limitation Presence of Phospholipids and Proteins, adversely effect their release kinetics, shelf life and stability Poor viability to commercial scale production

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:

Mechanism of liposome formation Dry Lipid Film/Lipid cake hydration Swelling Agitation Extrusion LUV Large,MLV Sonication /Homogenization (Sonic and Mechanica energy) SUV

Methods of liposomes:


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



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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 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


FRENCH PRESSURE CELL LIPOSOMES This techniques yields rather “uni or oligo lamellar liposomes” of intermediate size of 30-80 nm in diameter depending on the applied pressure. Dispersion of MLVs can be converted to SUVs by passage through a small orifice under high pressure. MLV dispersion are placed in the French pressure cell and extruded at about 20,000psi at 45˙C By multiple extrusion i.e.., 4.5 passed about 95% of MLVs get converted into SUVs which can be determined by size exclusion chromatography.


FRENCH PRESSURE CELL Pistion Cell body Aqueous Sample outlet


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.


DRIED-RECONSTITUTED VESICLES Liposomes obtained by this method are usually “uni or oligo lamellar” of the order of 1.0um or less in diameter. SUVs in aqueous phase SUVs with solutes to be entrapped Freeze dried membrane Solutes in uni lamellar vesicles Solutes in uni or oligo lamellar vesicles FST method DRV method Rehydration Film stacks dispersion Aqueous phase Thawing Sonication (15-30 sec)


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:

p H 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


CALCIUM INDUCED FUSION Calcium induced fusion method is principally based upon the concept of aggregation and fusion of acidic phospholipid vesicles in the presence of calcium. Lipids is dried down and suspended in sonication buffer( NaCl 0.385g, hisditine 31.0mg,Tris-base 24.2mg, EDTA 3.72mg, water 100ml,pH 7.4) and sonicated to prepare small liposomes. The large liposomes and lipid particles are removed by centrifugation at 100,000g. Equimolar proportion of Calcium solution(CaCl 2 ) is added to Phospholipids in the supernatant, resulting in white precipitate, incubated for 60min at 37 ˙C and the pellets are separated

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A white flocculent precipitate is formed which incubated for 60mins at 37˙C and the precipitate(pellet) is separated by spinning the contents at 3000g for 20min at room temperature. The pellet is resuspended in buffer saline containing the material to be entrapped and incubated at 37˙C for 10mins. The addition of EDTA to the pellet suspension with mixing, results in the formation of a cloudy dispersion, which clears rapidly and incubated for 15mins at 37˙C and further 15mins at room temperature. Finally, the Ca/EDTA complex is removed by dialysis overnight against a litre of phosphate saline buffer. The method has the advantage that it does not expose lipids or entrapped materials to deleterious chemical or physical conditions.


COCHLEATE METHOD Small unilamellar vesicles Negatively charged lipids (Phosphatidylserine PS) addition of Ca ++ Cylindrical rolls (Cochleate cylinders) removal of Ca ++ by EDTA or ion exchange or Precipitation Large unilamellar vesicles

Ethanol injection:

Ethanol injection An ethanol solution of lipids is injected rapidly through a fine needle into an excess of saline or other aqueous medium. The rate of injection is usually sufficient to achieve complete mixing .so that the ethanol is diluted almost instantaneously in water and phospho-lipid molecules are dispersed evenly throughout the medium. This procedure yields a high proportion of SUVs(~25nm). The vesicles of 100nm size may be obtained by little modification in this method, i.e.., by varying the concentration of lipid in ethanol or by changing the rate of injection of ethanol solution in preheated aqueous solution.

Ether injection:

Ether injection It involves injecting the immiscible organic solution very slowly into an aqueous phase through a narrow needle at the temperature of vapourizing the organic solvent. This method may also treat sensitive lipids very gently. The efficiency of encapsulation is relatively low because substances are degraded at elevated temperature(60˙C),then the fluorinated hydrocarbons( Freons ) may be used instead of ether.

De-emulsification methods:

De-emulsification methods This method requires two steps for preparation of liposomes * inner leaflet of the bilayer * outer half The common feature of this method is the formation of “Water in oil” emulsion Method: Small quantity of large volume of Aqueous medium + immiscible organic containing material solution of lipids aqueous phase into Stabilization microscopic water droplets Mechanical agitation

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STABILIZATION: The droplets are stabilized by the presence of phospholipids monolayer at the interface. The size of the droplets is determined by the intensity of mechanical energy used to form the emulsion and amount of lipid relative to the volume of aqueous phase. Each droplet requires a complete monolayer of phospholipid covering its surface in order to prevent the possible coalescence with other droplets. There are number of methods used to prepare droplets such as Double Emulsion Vesicles Rapid solvent exchange vesicles Sonication methods

Double emulsion vesicles:

Double emulsion vesicles In this method, the outer half of the liposome membrane is created at a second interface between two phases by emulsification of an organic solution in water . If the organic solution, which already contain water droplet is introduced into excess aqueous medium by mechanical dispersion, Multi compartment vesicles are obtained. The ordered dispersion so obtained is described as a W/O/W system or a Double emulsion. These vesicles with aqueous core are suspended in aqueous medium, the two aqueous compartments being separated from each other by a pair of phospholipids monolayers whose hydrophobic surfaces face each other across a thin film of organic solvents intermediate sized unilamellar vesicles.

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DOUBLE EMULSION PREPARATION : Injecting Dispersion of micro- droplets + hot aqueous solution of Tris -buffer 22-gauge hypodermic needle vigorous stirring Organic solvent is evaporated using strong jet of nitrogen + volume is adjusted with distilled water centrifuge 20 ˙C-30min 37,000g DOUBLE EMULSION lipid aggregates

Rapid solvent exchange vesicles:

Rapid solvent exchange vesicles Lipid mixture + orifice of blue tipped syringe vaccum Pure Organic Solvent Pure Aqueous Environment Environment Solvent vaporizes lipid mixture precipitates in aqueous buffer


STABLE PLURILAMELLAR VESICLES Sonication method: Preparation of water-in-organic phase dispersion with an excess of lipid Drying under continued bath sonication with an intermitted stream of nitrogen Redistribution and equilibration of aqueous solvent and solute occurs in between the various bilayers in each plurilamellar vesicle.

Detergent depletion (removal) :

Detergent depletion (removal) The Phospholipids are brought into intimate contact with the aqueous phase via detergents, which associate with phospholipid molecules and serve to screen the hydrophobic portions of the molecule from water. The structures formed as a result of this association are known as Miscelles and the concentration of detergent in water at which micelles just start to form is known as Critical Miscelle Concentration (CMC). Aqueous phase + Lipid phase Excess lipids W/O emulsion Minimal lipids MLVs LUVs vortexing vortexing

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Micelles containing components in addition to the detergents (or composed of 2 or more detergents) are known as Mixed micelles. As the detergent concentration is increased, the micelles are reduced in size. again it is usually found that a high concentration is advantageous for solubilizing membrane phospholipids. There are methods are applied for the removal of detergent. They are - Dialysis - Column chromatography - Use of Biobeads.


dialysis In contrast to phospholipids, detergents are highly soluble in both aqueous and organic media and there is an equilibrium between the detergent molecules n the water phase and in the lipid environment of the micelles. Upon lowering the concentration of the detergent in the bulk aqueous phase, the molecules of the detergents can be removed by dialysis. E.g., of detergent: bile salts sodium cholate and sodium deoxycholate and synthetic detergents such as octylglucoside. Dialysis: Egg PC + sodium cholate (2:1) vesicles(100nm) Trade name-LIPOREP dialysis

Column chromatography:

Column chromatography Phospholipids + deoxycholate (sonicated vesicles 2:1 or as a dry film) removal of deoxycholate by Column chromatography (Sephadex G-25) Unilamellar vesicles (100 nm)

Detergent adsorption using bio-beads:

Detergent adsorption using bio-beads Detergent (non-ionic)/phospholipids mixtures can form LUVs by removal of non-ionic detergent(Triton X-100) using appropriate adsorbents for the detergent. E.g. Casted lipid film + 0.5-1.0% Triton X-100 + washed bio-beads(0.3g/ml of dispersion) and rocked for about 2hrs at 4±1 ˙C gives LUVs

Active loading (remote):

Active loading (remote) Loading drug molecules into preformed liposomes using concentration gradients. The membrane from lipid bilayer is in general impermeable to ions and large hydrophilic molecules. Ions transport can be regulated by the ionophores while permeation of neutral and weakly hydrophobic molecules can be controlled by concentration gradients. The transmembrane pH gradient can be developed using various methods depending upon the nature of the drug to be encapsulated. * For amphipathic weak bases by remote loading procedures such as using a proton gradient or an ammonium sulphate gradient. * For amphipathic weak acids by remote loading procedures using a calcium acetate gradient.

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The pH gradient is created by preparing liposomes with a low pH inside and outside the vesicles, followed by the addition of the base to the extraliposomal. Usually, a two step process generates this pH imbalance and remote loading: 1 st , the vesicles are prepared in a low pH solution this is followed by addition of base to the extraliposomal medium. At the low pH side, the molecules are predominately protonated, which lowers the concentration of the drug in the unprotonated form, and thus promote the diffusion of more drug molecules at the low pH side of the bilayer, then exchange of external medium by gel exclusion chromatography with a neutral solution. e.g., Doxorubicin, Adriamycin, Vincristine, peptides and insulin


evaluation Physical Chemical Biological Size, Purity safety Shape potency Suitability Surface features of formulation Lamellarity Phase behavior Drug release profile

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VESICLE SHAPE, SURFACE AND LAMELLARITY : Freeze –fracture and freeze-etch Electron microscopy 31 P Nuclear Magnetic Resonance analysis VESICLE SIZE AND SIZE DISTRIBUTION : Optical Micrscopy Negative Stain Transmission Electron Microscopy(TEM) Cryo -TEM Freeze –fracture Electron microscopy Scanning Electron Microscopy (SEM) Diffraction and Scattering Techniques Hydrodynamic Techniques Field-Flow-Fractionation (FFF) Gel Permeation Ultracentrifuge

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SURFACE CHARGE : Free-Flow Electrophoresis Zeta potential measurement CHEMICAL CHARACTERS : Phospholipids: Barlett assay Stewart assay Thin layer chromatography Cholesterols: Cholesterol oxidase assay Ferric perchlorate method Gas Liquid chromatography

Stability of liposomes:

Stability of liposomes Stability In Vitro : Lipid oxidation and Peroxidation Lipid hydrolysis Long Term and Accelerated Stability Stability after Systemic Administration Stability In Vivo: Stability after oral Administration


ROUTES OF ADMINISTRATION Suspended in an aqueous suspension form, the liposomes are ready for application They could be applied topically to the eyes, to wounds, and to burns. They could be injected subcutaneously or intramuscularly or introduced into the peritoneal cavity through injection or infusion. Aerosols of drug-encapsulating liposomes (regular and surface modified) that would be useful for pulmonary applications.


APPLICATIONS 1.Liposomes as drug/Protein delivery vehicles Controlled and Sustained drug release in site Enhanced drug solubilization Altered Pharmacokinetics and Bio distribution Enzyme replacement therapy and Lysosomal storage 2.Liposomes in Anti Microbial, Anti-Fungal(Lung therapeutics) and Anti-Viral(anti-HIV)therapy Liposomal drugs Liposomal biological response modifiers

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3.Liposomes in Tumor Therapy Carrier of small cytotoxic molecules Vehicle for macromolecules as cytokines or gene 4.Liposomes in Gene delivery Gene and Antisense therapy Genetic (DNA) vaccination 5.Liposomes in Immunology Immunoadjuvant Immunomodulator Immunodiagnosis

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6. Liposomes as Artificial blood surrogates 7. Liposomes as Radiopharmaceutical and Radio diagnostic carriers 8. Liposomes in Cosmetics and Dermatology 9. Liposomes in Enzymes immobilization and bioreactor technology 10. In the biodetoxification of drugs by injecting empty liposomes with a transmembrane pH gradient. In this case the vesicles act as sinks to scavenge the drug in the blood circulation and prevent its toxic effect. 11. liposomes can be used as carriers for the delivery of dyes to textiles, pesticides to plants, enzymes and nutritional supplements to foods, and cosmetics to the skin. 12. Liposomes are also used as outer shells of some micro bubble contrast agents used in contrast-enhanced ultrasound

Liposomes in the pharmaceutical industry:

Liposomes in the pharmaceutical industry Liposome Utility Current Applications Disease States Treated Solubilization Amphotericin B, minoxidil Fungal infections Site-Avoidance Amphotericin B – reduced nephrotoxicity , Fungal infections, cancer doxorubicin – decreased cardiotoxicity Sustained-Release Systemic antineoplastic drugs, hormones, Cancer, biotherapeutics corticosteroids, drug depot in the lungs Drug protection Cytosine arabinoside , interleukins Cancer, etc. RES Targeting Immunomodulators , vaccines, antimalarials , Cancer, MAI, tropical macophage -located diseases parasites Specific Targeting Cells bearing specific antigens Wide therapeutic applicability Extravasation Leaky vasculature of tumours , inflammations, Cancer, bacterial infections infection Accumulation Prostaglandins Cardiovascular diseases Enhanced Penetration Topical vehicles Dermatology Drug Depot Lungs, sub- cutaneous , intra-muscular, ocular Wide therapeutic applicability

List of clinically approved liposomal drugs:

List of clinically approved liposomal drugs Name Trade name Company Indication Amphotericin B Abelcet Enzon Fungal infection Amphotericin B Ambisome Gilead Sciences Fungal and infections Cytarabine Depocyt Pacira Malignant lymphomatous meningitis

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Name Trade name Company Indication Daunorubicin DaunoXome Gilead Sciences HIV-related Kaposi’s sarcoma Doxorubicin Myocet Zeneus Breast cancer IRIV vaccine Epaxal Berna Biotech Hepatitis A IRIV vaccine Inflexal V ” Influenza PEG doxorubicin Doxil / Caelyx Ortho Biotech, Ovarian Schering-Plough cancer

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REFERENCES: Target & Controlled Drug Delivery – Novel carrier systems by S.P.VYAS and R.K.KHAR Controlled and Novel Drug Delivery Systems by K.JAIN and N.K.JAIN WIKIPEDIA Textbook of Industrial Pharmacy by SHOBA RANI HIREMATH

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