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Liposomes :

2 Liposomes Definition: Liposomes are simple microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid molecule. Structurally, liposomes are concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayers mainly composed of natural or synthetic phospholipids.

Advantages :

3 Advantages Provide selective passive targeting to tumour tissues (liposomal doxorubicin). Increased efficacy and therapeutic index. Increased stability via encapsulation. Reduction in toxicity of the encapsulated agent. Improved pharmacokinetic effects (reduced elimination, increased circulation life times). Flexibility to couple with site-specific ligand to achieve active targeting.


4 STRUCTURE AND COMPOSITION OF LIPOSOMES There are number of components of liposomes however phospholipids and cholesterol being main components. Phospholipids are the major structural components of biological membranes, where two types of phospholipids exist – phosphodiglycerides and sphingolipids , together with their corresponding hydrolysis products. The most common phospholipid is phosphatidylcholine (PC) molecule.

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5 Commonly used synthetic Phospholipids: DOPC = Dioleoyl Phosphatidylcholine DOPE = Dioleoyl phosphatidylethanolamine DSPC = Distearoyl phosphatidylcholine DSPE = Distearoyl phosphatidylethanolamine DLPC = Dilauryl phosphatidylcholine DMPC = Dimyristoyl phosphatidylcholine DLPE = Dilauryl phosphatidylethanolamine

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6 Cholesterols: Incorporation of sterols in liposome bilayer can bring about major changes in the preparation of these membranes. Cholesterol does not by itself form bilayer structure, but can be incorporated into phospholipid membranes in very high concentration unto 1:1 or even 2:1 molar ratios of PC. Cholesterol incorporation increases the separation between the cholin head groups and eliminates the normal electrostatic and hydrogen-bonding interactions.

Classification Based on structural parameters:

7 Classification Based on structural parameters Multivesicular vesicles->1 μm MV Giant unilamellar vesicles->1 μm GUV Large unilamellar vesicles->100nm LUV Medium sized unilamellar vesicles MUV Small unilamellar vesicles-20-100nm SUV Unilamellar vesicles (all in size) UV Oligolamellar vesicles- 0.1-1 μm OLV Multilamellar large vesicles- >0.5 μm MLV SPECIFICATIONS TYPE

Classification Based on the method of liposome preparation:

8 Classification Based on the method of liposome preparation TYPE SPECIFICATIONS REV Single or oligolamellar vesicles made by reverse-phase evaporation method MLV-REV Multilamellar vesicles made by reverse-phase evaporation method SPLV Stable plurilamellar vesicles FATMLV Frozen and thawed MLV VET Vesicles prepared by extrusion technique DRV Dehydration-rehydration method

Chemical Characterization :

9 Chemical Characterization Characterization parameters Analytical methods Phospholipid concentration Lipid phosphorous content using Barlett assay,HPLC Cholesterol concentration Cholesterol oxidase assay and HPLC Drug concentration Appropriate method given in monograph Phospholipid peroxidation UV absorbance, TBA,indometric and GLC Phospholipid hydrolysis HPLC and TLC Cholesterol auto-oxidation HPLC and TLC Anti-oxidant degradation HPLC and TLC pH pH meter osmolarity Osmometer

Physical Characterization :

10 Physical Characterization Characterization parameters Analytical methods Vesicle shape and surface morphology Transmission electron microscopy, freeze-fracture electron microscopy. Surface charge Free-flow electrophoresis. Lamellarity Small angle X-ray scattering, freeze-fracture electron microscopy, 31 P-NMR. Phase behavior Freeze-fracture electron microscopy, differential scanning calorimetry Percent capture/percent of free drug Minicolumn centrifugation, gel exclusion, ion-exchange chromatography, radiolabelling

Biological Characterization :

11 Biological Characterization Characterization parameters Analytical methods sterility Aerobic or anaerobic cultures pyrogenicity Limulus amebocyte lysate (LAL) test Animal toxicity Monitoring survival rates, histology and pathology


12 TECHNIQUES OF LIPOSOMES PREPARATION (A) physical dispersion a) Hand-shaken multilamellar vesicles (MLVs) b) Non-shaking vesicles c) Pro-liposomes d) Freeze drying (B) Processing of lipids hydrated by physical means a) Micro emulsification liposomes (MEL) b) Sonicated unilamellar vesicles (SUVs) c) French pressure cell liposomes d) Membrane extrusion liposomes e) Dried-reconstituted vesicles (DRVs) f) Freeze thaw sonication (FTS) g) pH induced vesiculation h) Calcium induced fusion

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13 (C ) Solvent dispersion methods Ethanol Injection Ether injection Water in organic phase Reverse phase evaporation vesicles Stable plurilamellar vesicles (SPVs) Double emulsion vesicles (D) DETERGENT SOLUBILIZATION

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14 All the methods of preparing liposomes involve three or four basic stages: Drying down lipids from organic solvent, Dispersion of lipids in aqueous media, Purification of resultant liposomes, and Analysis of final product.

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15 Physical dispersion : There are four basic method of physical dispersion. I.e. hand shaking, non-shaking, freeze dry and proliposomes. Hand-shaken multilamellar vesicles (MLVs ) :

Sonicated Unilamellar Vesicles (SUVs) ::

16 Sonicated Unilamellar Vesicles (SUVs) :

French pressure cell liposomes : :

17 French pressure cell liposomes : Type of liposomes : ULV or OLV Pressure :20000 or 40000 Sample volume : maximum 40ml Minimum 4ml Out flow : 0.5-1 ml / min.

Membrane extrusion liposomes ::

18 Membrane extrusion liposomes : Type of liposomes : MLV or LUV Pressure : 100 psi Type membranes : Tortuous path, Nucleation track (polycarbonate )

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19 Solvent dispersion methods: In this methods, lipids are first dissolved in an organic solution, which is then brought into contact with the aqueous phase containing material to be entrapped within the liposomes. Methods employing solvent dispersion fall into one of three categories. The organic solvent is miscible with the aqueous phase. The organic solvent is miscible with the aqueous phase, the latter being in a large excess. Organic solvent is in large excess, and is again immiscible

Ether injection &ethanol injection:

20 Ether injection &ethanol injection

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21 DETERGENT SOLUBILIZATION : In this method, the phospholipids are brought into intimate contact with the aqueous phase via the intermediary of 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 micelles, and can be composed of several hundred component molecules. Their shape and size depends on chemical nature of the detergent, the concentration and other lipids involved. As a general rule, membrane-solubilizing detergents have a higher affinity for phospholipid membranes than for the pure detergent micelles.



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23 Active loading method have following advantages over passive encapsulation techniques : A high encapsulating efficiency and capacity. A reduced leakage of the encapsulated compounds. “Bed side” loading of drug thus limiting loss of retention of drug by diffusion or chemical degradation during storage. Flexibility for the use of constitutive lipids, as drug is loaded after the formation of carrier units. Avoidance of biological compound during preparation steps in the dispersion thus reducing safety hazards. The transmembrane pH gradient can be developed using various method depending upon the nature of the drug be encapsulated. For amphipathic weak bases by active loading procedures such as using a proton gradient or an ammonium sulphate gradient or calcium acetate gradient.

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24 STABILITY OF LIPOSOMES The stability studies could be broadly covered under two main sections. The stability in vitro, which covers the stability aspects prior to the administration of the formulation and with regard to the stability of the constitutive lipids. The stability in vivo, which covers the stability aspects once the formulation, is administered via various routes to the biological fluids. These include stability aspects in blood (serum) if administered by systemic route or in gastrointestinal tract, if administered by oral or preoral routes.

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25 Stability in vitro Lipid oxidation an peroxidation Lipid hydrolysis Long term and accelerated stability Stability after systemic administration Stability in vivo after oral administration

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26 APPLICATION OF LIPOSOMES Liposomes as drug/protein delivery vehicles Controlled and sustained drug release in situ. Enhanced drug solubilization Altered pharmacokinetics and biodistribution Enzyme replacement therapy and lysosomal storage disorders Liposomes in antimicrobial, antifungal and antiviral therapy Liposomal drugs Liposomal biological response modifiers Liposomes in tumour therapy Carrier of small cytotoxic molecules Vehicle for macomolecules as cytokines o genes

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27 Liposomes in gene delivery Gene and antisense therapy Genetic vaccination Liposomes in immunology Immonoadjuvant Immunomodulator Immunodiagnosis Liposomes as artificial blood surrogates Liposomes as radiophamaceutical and radiodiagnostic cariers Liposomes in cosmetics an dermatology Liposomes in enzyme immobilization and bioreactor technology

Some liposomal formulation of Amphotericin B:

28 Some liposomal formulation of Amphotericin B System Target disease Brand name Product Liposomes (i.v) Systemic fungal infection, Visceral leishmaniasis AmBisome NeXstar, USA Liposomes (i.v) Systemic fungal infection Amphocil SEQUUS, USA Liposomes (i.v) Systemic fungal infection ABELECT The Liposome company, USA

Liposomes in gene therapy::

29 Liposomes in gene therapy: Type of vector Advantages Disadvantages Viral vector Relative high transfection efficiency Immunogenicity, presence of contaminants and safety Vector restricted size limitation for recombinant gene Unfavourable p’ceutical issue-large scale production, GMP, stability and cost Non-viral Favourable p’ceutical issue-large scale production, GMP, stability and cost Plasmid independent structure Low immunogenicity Opportunity for chemical/physical manipulation Relative low transfection efficiency

Various liposomal product in dermatology and cosmetics (launched or investigated):

30 Various liposomal product in dermatology and cosmetics (launched or investigated) Vesicular system Marketed by Liposomes and ingredients Capture TM Christian Dior Liposomes in gel with ingredients Plentitude TM L’Oreal Tanning agent in liposomes Dermosome TM Microfluidics Skin care, loaded liposomes Penta TM Pentapharm Humectant pentavitin R in liposomes Coatsome NC TM Nichiya liposomes Co Liposomes with humectant

Imaging modality and required concentration of diagnostic moieties::

31 Imaging modality and required concentration of diagnostic moieties: Imaging modality Diagnostic moiety Concentration  -scintigraphy Diagnostic radio-nucleus such as 111 In, 99m Tc, 67 Ga 10 -10 M Magnetic resonance(MR) Po-magnetic ions such as Gd, Mn and iron oxide 10 -4 M Computed tomography(CT) Iodine, Bromine an Barium 10 -2 M Ultrasound imaging or Ultrasonography(US) Gas (Air, Argon and Nitrogen)


32 COMMERCIAL MANUFACTUING OF LIPSOMAL DRUG NO Problems Remedies 1 Poor quality of the raw material mainly the phospholipids. High quality products with improved purification protocol and validated analytical techniques are available 2 Pay load is too low Use either lipophilic drug/lipophilic prodrug of hydrophilic drug or using active techniques. 3 Poor characterization of the physicochemical properties of the liposomes Quality control assay can be performed using sophisticated instruments and batch to batch variability can be checked.

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33 NO Problems Remedies 4 Shelf life is too short Improved by appropriate cryoprotectant and lyoprotectant and product can be successfully freeze dried. 5 Scale up related problems Scaling up can be improved by carefully selecting method of preparation, sterilization by autoclaving or membrane filtration coupled with aseptic processing and pyrogen removal using properly validated LAL test 6 Absence of any data on safety of these carrier systems on chronic use. By choosing candidate potent drugs with narrow therapeutic window the drug elated safety problems can be alleviated.

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34 Thank you

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