liposomes

LIPOSOMES: 

LIPOSOMES Presented by S L priyanka ratnala , M pharm 2 nd year, Sri sai aditya institute of pharmaceutical sciences and research.

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overview

Definition : : 

Definition : liposome's are concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayer mainly composed of phospholipids.

Introduction :: 

Introduction : Liposome was found by Alec Bangham of Babraham Institute in Cambridge, England in 1965. In 1990, drugs with liposome and Amphotericin B were approved by Ireland. In 1995 America F.D.A approved liposor doxorubicin . Liposome is a lipid vesicle suspending in the hydro-phase with a diameter around 0.0025~3.5um. The membrane of liposome is made of phospholipids, which have phosphoric acid sides to form the liposome bilayers .

Liposome Structure :: 

Liposome Structure : Aqueous cavity Phospholipid bilayer

Advantages :: 

Advantages : Biocompatible, completely biodegradable, non-toxic, flexible, nonimmunogenic . Liposomes supply both a lipophilic environment and aqueous “milieu interne ” in one system. Can protect encapsulated drug. Reduce exposure of sensitive tissues to toxic drugs. Alter the pharmacokinetic and pharmacodynamic property of drugs (reduced elimination, increased circulation life time ). Flexibility to couple with site-specific ligands to achieve active targeting ( Anticancer and Antimicrobial drugs). Liposomes can encapsulate both micro and macromolecules such as haemoglobin , erythropoeitin , interferon g etc . Can be formulated into multiple dosage forms.

Disadvantages :: 

Disadvantages : Production cost is high Leakage and fusion of encapsulated drug / molecules. Sometimes phospholipid undergoes oxidation and hydrolysis like reaction Short half-life Low solubility

Composition of liposomes: A. Phospholipids: 

Composition of liposomes : A. Phospholipids General representation of phospholipids

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The most common natural phospholipid is the phospatidylcholine (PC ). Naturally occurring phospholipids used in liposomes are: Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine Synthetic phospholipids used in the liposomes are: Dioleoyl phosphatidylcholine Disteroyl phosphatidylcholine Dioleoyl phosphatidylethanolamine Distearoyl phosphatidylethanolamine

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phosphatidylcholine (PC) • Phosphatidylcholine is an amphipathic molecule in which exists – a hydrophilic polar head group, phosphocholine . – a glycerol bridge – a pair of hydrophobic acyl hydrocarbon chains

Mechanism In short,this is what happens: 

Mechanism In short,this is what happens Mechanism of liposome formation & subsequent processing to generate various types of vesicles

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Molecules of PC are not soluble in water. In aq media they align themselves in planar bilayer sheets inorder to minimize the Unfavorable action b/w the bulk aq phase & long hydrocarbon fatty chain. PC molecules contrast markedly with other amphipathic molecules such as detergents, In that they form Bilayer sheets not micellar structures

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Phase transition temperature: At various temperatures, phospholipid membranes can exist in different phases. The transition from one phase to another can be detected by technique like micro calorimetry . What exactly happens during phase transition? ( lipid membrane) Tightly ordered gel state At elevated temperature liquid crystal phase (movement is higher) This is due to the fatty acid chain adopting a new conformation other than all trans straight chain configuration, such as gauche configuration state( phenomenon- chain tilt )

B. Cholesterol: fluidity buffer : 

B. Cholesterol: fluidity buffer Being an amphipathic molecule, cholesterol inserts into the membrane with its hydroxyl group of cholesterol oriented towards the aqueous surface and aliphatic chain aligned parallel to the acyl chains in the center of the bilayer . i.e. below Tc , it makes membrane less ordered & above Tc more ordered. Cholesterol: Stabilizes the Membrane Steroid lipid Interdigitates between phospholipids

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Role of cholesterol in bilayer formation: Restricts the transformations of trans to gauche Conformations. Incorporated into phospholipid membrane upto 1:1 or 2:1 of cholesterol to PC.

Types of Liposome's: : 

Types of Liposome's:

Classification :: 

Classification :

Classes of Liposomes:: 

Classes of Liposomes : Conventiona l Long circulating Immuno Cationic Unilammellar PEG coated liposomes

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Classification of some commonly known lipid vesicles according to their structures and/or preparation Ethosomes : efficient at delivering to the skin.composed of soya phosphotidylcholine + 30% ethanol. Immunoliposomes : modified with antibodies. Niosomes : SUV’s made from nonionic surfactants. Proliposomes : dry, free flowing particles that immediately form a liposomal dispersion on contact with water. Stealth liposomes : coating of liposomes with PEG(hydrophilic polymer) to improve Stability & lengthens their half life in circulation.PEG coating inhibits recognition by RES system.

Lamella:: 

Lamella: 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 convered into spheres. Based on number of lamella’s there are two types: 1. Unilamellar Vesicles 2. Multilamellar Vesicles

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Preparation of liposomes Loading of the entrapped agents before/ during the manufacture procedure. Certain types of compounds with ionizable groups & those with both lipid & water solubility can be Introduced into liposomes after the formation of intact vesicles.

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Lipid film hydrationby hand /non hand shaking Micro emulsification Sonication French pressure cell Membrane extrusion Dried reconstituted vesicles Freeze thawed liposomes Ethanol injection Ether injection Double emulsion vesicles Stable plurilamellar vesicles Detergent removal from mixed micelles by Dialysis Column chromatography Adsorption using bio- beads

General Method of Liposome Preparation:: 

General Method of Liposome Preparation:

1. Physical Dispersion/mechanical dispersion methods:: 

1. Physical Dispersion/mechanical dispersion methods: There are four basic methods of physical dispersion : Hand shaken method. Non shaking method. Pro – liposomes . Freeze drying . 1) Passive loading

Hand Shaking Method:: 

Hand Shaking Method:

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Rotary flash evaporator

Non Shaking Method: 

Non Shaking Method The procedure differs from hand shaken method in that it uses a stream of nitrogen to provide agitation rather than the rotationary movements. Here the lipid film is exposed to water - saturated nitrogen(15 – 20min). After Hydration, lipid is swelled by addition of bulk fluid. 10-20ml of 0.2M sucrose in distilled water(degassed) is introduced. The flask is flushed with nitrogen, sealed and allowed to stand for 2 hrs at 37 degrees celsius . After swelling, the vesicles are harvested by swirling the contents of the flask gently, to yield a milky‐suspension. Centrifugation luv’s .

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Pro- liposomes To increase the surface area of dried lipid film & to facilitate instantaneous hydration. Finely divided particulate support like powdered NACL lipid Dried onto Pro- liposomes Pro- liposomes water Dispersion of MLV’S

Freeze Drying Method:: 

Freeze Drying Method: Another method of dispersing the lipid in a finely divided form, prior to addition of aq. Medium is to freeze dry the lipid dissolved in a suitable organic solvent. Tertiary butanol us considered to be the most ideal solvent. After obtaining the dry lipid which is a expanded foam like structure, water or saline can be added with rapid mixing above the phase tranisition temperature to give MLVs.

Processing of the lipids hydrated by physical means or the mechanical treatments of MLVs : : 

Processing of the lipids hydrated by physical means or the mechanical treatments of MLVs : Micro Emulsification liposomes (MEL) Sonicated unilamellar vesicles (SUVs) French Pressure Cell Liposomes . Membrane extrusion Liposomes Dried reconstituted vesicles(DRVs) Freeze thaw sonification (FTS) pH induced vesiculation Calcium Induced fusion

Micro Emulsification Liposomes (MEL): : 

Micro Emulsification Liposomes (MEL):

Sonicated Unilamellar Vesicles: : 

Sonicated Unilamellar Vesicles:

Membrane Extrusion Liposomes: : 

Membrane Extrusion Liposomes :

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French pressure cell liposomes Extrusion of preformed large liposomes in a french press under high pressure Uni or oligo lammellar liposomes Advantages: More stable & less leakage of contents compared to sonicated liposomes .

Dried Reconstituted Vesicles (DRV) and Freeze Thaw Sonication (FTS):: 

Dried Reconstituted Vesicles (DRV) and Freeze Thaw Sonication (FTS):

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pH induced vesiculation The transient change in pH brings about an increase in surface charge of the lipid bilayer which induces spontaneous vesiculation .

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COCHLEATE METHOD LUV’s

Ethanol / Ether Injection Method: 

Ethanol / Ether Injection Method Solvent dispersion methods

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De- emulsification methods Generally the liposome is made up in 2 steps: 1 st the inner leaflet of the bilayer . Then the outer half. Methods to prepare the droplets: Double emulsion vesicles Reverse phase evaporation vesicles Sonication methods

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DOUBLE EMULSION VESICLES In this method, the outer portion of liposome membrane is created at a second Interface b/w two phases by emulsification of an organic soln in water.

Reverse Phase Evaporation Vesicles: : 

Reverse Phase Evaporation Vesicles:

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Detergent solubilisation : The phospholipids are brought into contact with aq phase via the detergent,which associate with phospholipid molecules.The structures formed as a result of this association are MICELLES. Below CMC,detergent molecules exist in free soln. As the concentration is increased,micelles are formed. A three stage model of interaction for detergents with lipid bilayers : At low concn,detergent equilibrates b/w vesicular lipid and water phase. After reaching CMC, membrane tends to be unstable & transforms into micelles At stage three, all lipid exists in mixed micelle form. In all the methods,the basic feature is to remove the detergent from preformed Mixed vesicles contng phopholipid,whereupon unilamellar vesicles form spontaneously. Methods to remove detergents : Dialysis Column chromatography Use of bio-beads

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2) Remote or active loading: The lipid bilayer membrane is impermeable to ions & hydrophilic molecules. But, Permeation of hydrophobic molecules can be controlled by concentration gradients. Some weak acids or bases can be transported due to various transmembrane gradients Electrical gradients. Ionic(pH) gradients. Chemical potential gradients. Weak amphipathic bases accumulate in aq phase of lipid vesicles in response to difference in pH b/w Inside & outside of liposomes .

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pH gradient is created by preparing liposomes with low internal pH. Addtn of base to extraliposomal medium. [Basic compds ( lipophilic (non ionic) at high pH & hydrophilic(ionic) at low pH)] Lipophilic (UNPROTONATED) drug diffuse through the bilayer . At low pH side, the molecules are predominantly protonated .

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Use of remote/ active loading : The following have beem successfully encapsulated: Weak bases such as Doxorubicin Adriamycin Vincristine Short modified peptides and insulin

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Locus of drugs in liposomes : Hydrophilic Low entrapment Leakage Hydrolytic degradation Lipophilic High entrapment Low leakage Chemical stability Ampiphilic High entrapment Rapid leakage Biphasic insoluble Poor loading & entrapment

Characterisation of liposomes: 1. Physical Characterisation:: 

Characterisation of liposomes : 1. Physical Characterisation : Characterization parameters Analytical method/Instrument 1. Vesicle shape and surface morphology Transmission electron microscopy, Freeze-fracture electron microscopy 2. Mean vesicle size and size distribution (submicron and micron range) Photon correlation spectroscopy, laser light scattering, gel permeation and gel exclusion 3. Surface charge Free-flow electrophoresis 4. Electrical surface potential and surface pH Zetapotential measurements 5. Lamellarity Small angle X-ray scattering, 31 P-NMR, Freeze-fracture electron microscopy 6. Phase behavior Freeze-fracture electron microscopy, Differential scanning calorimetery 7. Percent of free drug/ percent capture Minicolumn centrifugation, ion-exchange chromatography, radiolabelling 8. Drug release Diffusion cell/ dialysis

2. Chemical Characterisation:: 

2. Chemical Characterisation : Characterization parameters Analytical method/Instrument 1. Phospholipid concentration Barlett assay, stewart assay, HPLC 2. Cholesterol concentration Cholesterol oxidase assay and HPLC 3. Phopholipid peroxidation UV absorbance 4. Phospholipid hydrolysis, Cholesterol auto-oxidation. HPLC and TLC 5. Osmolarity Osmometer

2. Biological Characterisation:: 

2. Biological Characterisation : Characterization parameters Analytical method/Instrument 1. Sterility Aerobic or anaerobic cultures 2. Pyrogenicity Limulus Amebocyte Lysate (LAL) test 3. Animal toxicity Monitoring survival rates, histology and pathology

Modes of Liposome/Cell Interaction : 

Modes of Liposome/Cell Interaction Adsorption Endocytosis Fusion Lipid transfer

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STABILITY OF LIPOSOMES: Stability invitro . Lipid oxidation Lipid peroxidation Long term & accelerated stability Stability after systemic administration.

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Use of liposomes : As drug delivery carriers. Enzyme replacement therapy. Chelation therapy for treatment of heavy metal poisoning. Liposomes in antiviral/anti microbial therapy. In multi drug resistance. In tumour therapy. In gene delivery. In immunology. In cosmetology

Therapeutic applications of liposomes: 

Therapeutic applications of liposomes Drug Route of administration Application Targeted Diseases Amphotericin -B Oral delivery Ergosterol membrane Mycotic infection Insulin Oral, Ocular, Pulmonary and Transdermal delivery Decreaase glucose level Diabetic mellitus Ketoprofen Ocular delivery Cyclo-oxygenase enzyme inhibitor Pain muscle condition Pentoxyfylline Pulmonary delivery Phosphodiesterase Asthma Tobramycin Pulmonary delivery Protein synthesis inhibitor Pseudomonas infection, aeruginosa Salbutamol Pulmonary delivery β 2 - adrenoceptor antagonist Asthma Cytarabin Pulmonary delivery DNA-polymerase inhibition Acute-leukemias Benzocain e Transdermal Inhibition of nerve impulse from sensory nerves ulcer on mucous surface with pain Ketoconazole Transdermal Inhibit ergosterol membrane Candida- albican’s Levonogesterol Transdermal Rhamnose receptor Skin disorder Hydroxyzine Transdermal H 1 - receptor antagonist Urtecaria, allergic skin disorder Ibuprofen Oral delivery Chemoreceptor, free nerve ending Rheumatoid arthritis Triamcinolone Ocular delivery Transdermal Inhibition of prostaglandin Anti-inflammatory

List of marketed products: 

List of marketed products Marketed product Drug used Target diseases Company Doxil TM or Caelyx TM Doxorubicin Kaposi’s sarcoma SEQUUS, USA DaunoXome TM Daunorubicin Kaposi’s sarcoma, breast & lung cancer NeXstar, USA Amphotec TM Amphotericin-B fungal infections, Leishmaniasis SEQUUS, USA Fungizone® Amphotericin-B fungal infections, Leishmaniasis Bristol-squibb, Netherland VENTUS TM Prostaglandin-E 1 Systemic inflammatory diseases The liposome company, USA Topex-Br Terbutaline sulphate Asthma Ozone, USA Depocyt Cytarabine Cancer therapy Skye Pharm, USA Novasome ® Smallpox vaccine Smallpox Novavax, USA Avian retrovirus vaccine Killed avian retrovirus Chicken pox Vineland lab, USA Doxil ® Doxorubicin Hcl Refractory ovarian cancer ALZA, USA Evacet TM Doxorubicin Metastatic breast cancer The liposome company, USA VincaXome Vincristine Solid Tumours NeXstar , USA

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Name Trade name Company Indication Liposomal amphotericin B Abelcet Enzon Fungal infections Liposomal amphotericin B Ambisome Gilead Sciences Fungal and protozoal infections Liposomal cytarabine Depocyt Pacira (formerly SkyePharma) Malignant lymphomatous meningitis Liposomal daunorubicin DaunoXome Gilead Sciences HIV-related Kaposi’s sarcoma Liposomal doxorubicin Myocet Zeneus Combination therapy with cyclophosphamide in metastatic breast cancer Liposomal IRIV vaccine Epaxal Berna Biotech Hepatitis A Liposomal IRIV vaccine Inflexal V Berna Biotech Influenza Liposomal morphine DepoDur SkyePharma , Endo Postsurgical analgesia Liposomal verteporfin Visudyne QLT, Novartis Age-related macular degeneration, pathologic myopia, ocular histoplasmosis Liposome-PEG doxorubicin Doxil / Caelyx Ortho Biotech , Schering-Plough HIV-related Kaposi’s sarcoma, metastatic breast cancer, metastatic ovarian cancer Micellular estradiol Estrasorb Novavax Menopausal therapy

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Liposomal Formulations in market Phospholipid:AmB ratio AmB Lipid

References :: 

References : Target and Controlled Drug delivery – Novel Carrier Systems by S.P. Vyas and R.K. Khar . Controlled and Novel Drug Delivery Systems by Sanjay K. Jain and N.K.Jain . http://noopurmandrek.files.wordpress.com/2010/09/lipo1.jpg www.nanobiotec.iqm.unicamp.br/download/liposomas-3.ppt http://www.uni-magdeburg.de/imos/mea_sen/img/pictures/Lipo.jpg http://www.nanolifenutra.com/images/image_liposome_01.jpg http://www.azonano.com/work/bFgW9FjRw248U2PRC2In_files/image00 2.gif http://upload.wikimedia.org/wikipedia/en/2/28/Liposome.jpg http://www.pharmaxchange.info Biopharmaceutics and pharmacokinetics a treatise by D.M. Brahmankar and sunil B. jaiswal (first edition reprint 2005) page.no - 360-361. “ Liposomes preparation methods” a review by Mohammad riaz in Pakistan journal of pharmaceutical sciences vol.19(1), January 1996, pp.65-77. http://www.pharmainfo.net/reviews/liposome-versatile-platform-targeted-delivery-drugs

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