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  1. 1. BYPRIYANKA ODELA (M pharm 1st yr)Department of pharmaceutics
  2. 2.  Principle Definition Introduction Biological fate of liposomes Advantages and limitations Advancements in liposomes Stealth liposomes Pegylation Composition of liposomes Mechanism of liposome formation Classification of liposomes Methods of preparation Locus of drugs in liposomes Characterisation of liposomes Modes of liposome/cell interaction Stability of liposomes Storage of liposomes Pharmacokinetics of liposomes Uses of liposomes Therapeutic applications List of marketed products 1 Conclusion
  3. 3. Targeted Drug Delivery System( liposomes )must supply drug directly (selectively) to thesite(s) of action in a manner that providesmaximum therapeutic activity through kinetics. It prevents degradation or inactivation duringtransmit to the target sites and protects thebody from adverse reaction because ofinappropriate disposition. 2
  4. 4. The name liposome is derived from two Greekwords: Lipos meaning fat and Soma meaningbody. liposomes are concentric bilayered vesicles inwhich an aqueous volume is entirely enclosedby a membranous lipid bilayer mainlycomposed of phospholipids. 3
  5. 5. Liposome was found by Alec Bangham ofBabraham Institute in Cambridge, England in1965 In 1990, drugs with liposome and AmphotericinB were approved by Ireland.In 1995 U.S.F.D.A approved liposor doxorubicin . Liposome is a lipid vesicle suspending in theaqueous phase with a diameter around0.0025~3.5um. The membrane of liposome ismade of phospholipids, which have phosphoricacid sides to form the liposome bilayers . 4
  6. 6. Liposomes in Macrophages engulf Taken by RES liposomes(endocytosis) blood stream Membrane of phago lysosyme also contains protonpumps whichRelease of the drug decrease pH of the Phagosome+lysosome into cytosol phagolysosyme n the =phagolysosyme enzymes phospholipases destruct the liposomal membrane 5
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  8. 8. Biocompatible, completely biodegradable, non-toxic, flexible, nonimmunogenic .Provides selective passive targetting to tumours. Liposomes supply both a lipophilic environment andaqueous in one system. Can protect encapsulated drug.Increased efficacy and therapeutic index Reduce exposure of sensitive tissues to toxic drugs. Alter the pharmacokinetic and pharmacodynamicproperty of drugs (reduced elimination, increasedcirculation life time ).Flexibility to couple with site-specific ligands to achieveactive targeting ( Anticancer and Antimicrobial drugs).Liposomes can encapsulate both micro andmacromolecules such as haemoglobin , erythropoeitin, interferon g etc . Can be formulated into multiple dosage forms. 7
  9. 9. Production cost is high Leakage and fusion of encapsulated drug /molecules. Sometimes phospholipid undergoes oxidationand hydrolysis like reactionShort half-life Low solubilityFast elimination from the blood and localisationin reticuloendothelial system primarily kupffercells of liver. 8
  10. 10. Ethosomes : efficient at delivering to theskin.composed of soya phosphotidylcholine + 30%ethanol. Immunoliposomes : modified with antibodies.Niosomes : SUV’s made from nonionic surfactants.Proliposomes : dry, free flowing particles thatimmediately form a liposomal dispersion on contactwith water. Stealth liposomes : coating of liposomes withPEG(hydrophilic polymer) to improve Stability &lengthens their half life in circulation.PEG coatinginhibits recognition by RES system. 9
  11. 11. hydrophilicCoating liposomes with PEG reduces the rate of uptake bymacrophages(stealth effect) and leads to prolonged presence ofliposomes in the circulation and consequently provides ampletime for these liposomes to escape from the circulation throughleaky endothelium.Liposomes can be composed of naturally derived phospholipidswith mixed lipid chains coated or stabilised by polymers of PEG.Ex:doxorubicin loaded liposome in market as DOXIL orCAELYX,treatment of solid tumours. 10
  12. 12. NATURAL POLYMERS:-polyhydroxyethyl l-asparagines coated-PEG coated-H-PG-PEG coated-dope coated Biocompatible synthetic polymers:-polyvinyl pyrolidone-polyaniline-polyacrylamide-poly (2- methyl 2- oxazoline) 11
  13. 13. Process of construction of stealth liposomeswith PEG studding the outside membrane iscalled as pegylation.Increase in mole% of PEG on surface ofliposomes by 4 to 10% signifantly increasedcirculation time invivo from 200-1000 minutes. 12
  14. 14. Conventional Long circulating Immuno Cationic 24
  15. 15. General representation of phospholipid 13
  16. 16. The most common natural phospholipid is the phosphatidylcholine (PC ).Naturally occurring phospholipids used in liposomes are: Phosphatidylcholine Phosphatidylethanolamine PhosphatidylserineSynthetic phospholipids used in the liposomes are: Dioleoyl phosphatidylcholine Disteroyl phosphatidylcholine Dioleoyl phosphatidylethanolamine Distearoyl phosphatidylethanolamine 14
  17. 17. Phosphatidylcholine is an amphipathic molecule in which exists – a hydrophilic polar head group, phosphocholine . – a glycerol bridge – a pair of hydrophobic acyl hydrocarbon chains 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 with other amphipathic molecules such as detergents, In that they form-Bilayer sheets- not micellar structures 15
  18. 18. Cholesterol: Stabilizes the Membrane Steroid lipid Interdigitates between phospholipids i.e. below Tc , it makes membrane less ordered & above Tc more ordered. 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 bilayerRole 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. 16
  20. 20. Based on structural parameters MVV-MultiMLV-Multilamellar OLV-Oligolamellar UV-Unilamellar vesicularvesicles(>0.5um) vesicles(0.1-1um) vesicles vesicles(>1um) MUV-Medium SUV-Small LUV-Large GUV-Gaint unilamellar unilamellar unilamellar unilamellar vesicles vesicles(20- vesicles(>100nm) vesicles(>1um) 100nm) 19
  21. 21. Lamella: A Lamella is a flat plate like structure thatappears during the formation of liposomes. ThePhospholipid bilayer first exists as a lamella beforegetting convered into spheres. 20
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  28. 28. PHYSICAL DISPERSION OR MECHANICALDISPERSION METHOD:There are four basic methods of physicaldispersion :1)Hand shaken method.2)Non shaking method.3)Pro – liposomes .4) Freeze drying . 27
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  31. 31. The procedure differs from hand shaken method inthat it uses a stream of nitrogen to provide agitationrather than the rotationary movements.Here the lipid film is exposed to water - saturatednitrogen(15 – 20min).After Hydration, lipid is swelled by addition of bulkfluid. 10-20ml of 0.2M sucrose in distilledwater(degassed) is introduced.The flask is flushed with nitrogen, sealed and allowedto stand for 2 hrs at 37 degrees celsius . Afterswelling, the vesicles are harvested by swirling thecontents of the flask gently, to yield amilky‐suspension Centrifugation LUV 30
  32. 32. To increase the surface area of dried lipid film &to facilitate instantaneous hydration. Dried over Finely divided particulate LIPID support like powdered PROLIPOSOMES NaCl/sorbitol/polysacchari des PROLIPOSOMES WATER Dispersion of MLV’s 31
  33. 33. Another method of dispersing the lipid in afinely divided form, prior to addition of aq.Medium is to freeze dry the lipid dissolved in asuitable organic solvent. Tertiary butanol us considered to be the mostideal solvent.After obtaining the dry lipid which is aexpanded foam like structure, water or salinecan be added with rapid mixing above thephase tranisition temperature to give MLVs. 32
  34. 34. 1) Micro Emulsification liposomes (MEL)2)Sonicated unilamellar vesicles (SUVs)3) French Pressure Cell Liposomes .4) Membrane extrusion Liposomes5)Dried reconstituted vesicles(DRVs)6)Freeze thaw sonification (FTS)7)pH induced vesiculation8) Calcium Induced fusion 33
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  40. 40. The transient change in pH brings about anincrease in surface charge of the lipid bilayerwhich induces spontaneous vesiculation .COCHLEATE METHOD : LUV’s Removal of calcium by Cylindrical EDTA rolls(cochleate cylinders) Addition of Ca++ ions SUV made from phosphatidyl 39 choline
  41. 41. • In this method lipids are first dissolved in an organic solution, which is then brought into contact with the aqueous phase containing materials to be entrapped within the liposome.• At the interface between the organic and aqueous media ,the phospholipids align themselves into a monolayer which form the basis for half of the bilayer of the liposome. 40
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  43. 43. Generally the liposome is made up in 2 steps: 1 st the inner leaflet of the bilayer . Then the outer half. Aq medium contng matrl to be entrapped Methods to prepare the droplets: 1)Double emulsion vesicles Add to immiscible org sol of lipid 2) Reverse phase evaporation vesicles 3)Sonication methods Mechanical agitation42 Microscopic water droplets
  44. 44. In this method, the outer portion of liposomemembrane is created at a second Interface b/wtwo phases by emulsification of an organic solnin water. w/o Excess aq w/o/w Removal of Unilamellar emulsion medium emulsion solvent vesicles 43
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  46. 46. 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.At 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 45
  47. 47. 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 . 46
  48. 48. Liposomes with low internal pHSolute bearing no charge atneutral pH Neutral solute passes easily through bilayer membrane by diffusion Charge aquired by solute inside liposomes makes 47 them unable to exit
  49. 49. pH gradient is created by preparing liposomes with low internalpH. 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 . 48
  50. 50. The following have beem successfully encapsulated:Weak bases such as Doxorubicin Adriamycin Vincristine Short modified peptides and insulin 49
  51. 51. HYDROPHILIC (DOXORUBICIN)Low entrapmentLeakageHydrolytic degradation LIPOPHILIC (CYCLOSPORINE) High entrapment Low leakage Chemical stability AMPIPHILIC (VINBLASTIN) High entrapment Rapid leakage BIPHASIC INSOLUBLE (ALLOPURINOL, 6- MERCAPTOPURINE) Poor loading and entrapment 50
  52. 52. CHARACTERISATION OF LIPOSOMESCHARACTERISATION PARAMETERS ANALYTICAL METHOD/INSTRUMENT1. Vesicle shape and surface morphology Transmission electron microscopy, Freeze-fracture electron microscopy2. Mean vesicle size and size distribution Photon correlation spectroscopy, laser(submicron and micron range) light scattering, gel permeation and gel exclusion3. Surface charge Free-flow electrophoresis4. Electrical surface potential and surface Zetapotential measurementspH5. Lamellarity Small angle X-ray scattering, 31 P-NMR, Freeze-fracture electron microscopy6. Phase behavior Freeze-fracture electron microscopy, Differential scanning calorimetery7. Percent of free drug/ percent capture Minicolumn centrifugation, ion-exchange chromatography, radiolabelling8. Drug release Diffusion cell/ dialysis 51
  53. 53. CHARACTERISATION PARAMETERS ANALYTICAL METHOD/INSTRUMENT1. Phospholipid concentration Barlett assay, stewart assay, HPLC2. Cholesterol concentration Cholesterol oxidase assay and HPLC3. Phopholipid peroxidation UV absorbance4. Phospholipid hydrolysis Cholesterol auto-oxidation. HPLC and TLC5. Osmolarity Osmometer 52
  54. 54. CHARACTERISATION PARAMETER ANALYTICAL METHOD/INSTRUMENT1. Sterility Aerobic or anaerobic cultures2. Pyrogenicity Limulus Amebocyte Lysate (LAL) test3. Animal toxicity Monitoring survival rates, histology and pathology 53
  55. 55. Adsorption EndocytosisFusion Lipid transfer 54
  56. 56.  Stability invitro . Lipid oxidation Lipid peroxidation Lipid hydrolysis Long term & accelerated stability Stability invivoStability after systemic administration.Stability after oral administration. 55
  57. 57. STORAGE CONDITIONS Liposomes are packed in 1-2 ml sterile vials and stored at 4 C and room temperature. The samples stored at 4 C exhibited activity in transfection showed no aggregation or precipitation. The samples stored at RT still showed some transfection activity.Liposome dispersions are potentially prone to hydrolytic degradation and leakage. Hence, it is desirable to freeze dry the suspension to a powder and store in this dried form. The powder can be reconstituted to an aqueous suspension immediately before use. By doing so SUVs may be converted to MLVs dispersion upon rehydration. Addition of a carbohydrate (trehalose) during freeze drying prevents fusion and leakage of the vesicles. 56
  58. 58. Stability of liposomes mainly depend onstructure of lipids and its amount.Examples:1)DOXIL: stable for more than 18 months inliquid state without lyophilisation due topegylated nature.2)AMBISOME: liposomal preparation ofAmphotericine available as lyophilisedcake.After reconstitution not stable for morethan a day. 57
  59. 59. Pharmacokinetics of liposomes mainly deals with timecourse of absorption, distribution and degradation ofliposomal carriers invivo.Pharmacokinetic information can be used to interpretthe differences in pharmacological effect of theliposome entrapped drug and free drug andsubsequently can be exploited for dose regimen.Liposomes can alter both tissue distribution and rate ofclearance of the drug as they are affected bypharmacokinetic parameters of the carrier.Bioavailabilty in case of liposomal carriers can bedefined as the amount of free drug that is able toescape the carriers and thus available for redistributionto neighbouring tissue. 58
  60. 60.  Protection of drug from metabolism and inactivation in plasma. Reduced volume of distribution and hence decrease in non specific localisation. Higher therapeutic index. Decrease in amount and type of non specific toxicity. Increase in concentration of drug at target site. 59
  61. 61.  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 60
  62. 62. THERAPEUTIC APPLICATIONS OF LIPOSOMESDRUG ROUTE OF APPLICATION TARGETED DISEASES ADMINISTRATIONAmphotericin B Oral delivery Ergosterol membrane Mycotic infectionInsulin Oral,ocular,pulmonary Decrease glucose level Diabetic mellitus And transdermalKetoprofen Ocular delivary Cyclooxygenase enzyme inhibitor Pain muscle conditionPentoxyfyllin Pulmonary delivery phosphodiesterase AsthamaTobramycin Pulmonary delivery Protein synthesis inhibitor Pseudomonas infection,aeroginosaSalbutamol Pulmonary delivery ß2-adrenoceptor antagonist AsthamaCytarabin Pulmonary delivery DNA-polymerase inhibition Acute leukameiasBenzocaine Transdermal Inhibition of nerve impulse from Ulcer on mucous surface sensory nerves with painKetaconazole Transdermal Inhibit ergosterol membrane Candida albicansLevanogesterol Transdermal Rhamnose receptor skin disorderhydroxyzine Transdermal H1-receptor antagonist Urtecaria,allergic skin diseaseIbuprofen Oral delivery Chaemoceptor,free ending Rheumatoid arthritistriamcilonone Ocular delivery,Transdermal Inhibition of prostaglandin Anti-inflammatory 61
  63. 63. NAME TRADE NAME COMPANY INDICATIONLiposomal Abelcet Enzon Fungal infectionsamphotericin BLiposomal Ambisome Gilead Sciences Fungal and protozoal infectionsamphotericin BLiposomal cytarabine Depocyt Pacira (formerly Malignant lymphomatous meningitis SkyePharma)Liposomal DaunoXome Gilead Sciences HIV-related Kaposi’s sarcomadaunorubicinLiposomal doxorubicin Myocet Zeneus Combination therapy with cyclophosphamide in metastatic breast cancerLiposomal IRIV vaccine Epaxal Berna Biotech Hepatitis ALiposomal IRIV vaccine Inflexal V Berna Biotech InfluenzaLiposomal morphine DepoDur SkyePharma, Endo Postsurgical analgesiaLiposomal verteporfin Visudyne QLT, Novartis Age-related macular degeneration, pathologic myopia, ocular histoplasmosisLiposome-PEG Doxil/Caelyx Ortho Biotech, HIV-related Kaposi’s sarcoma, metastaticdoxorubicin Schering-Plough breast cancer, metastatic ovarian cancerMicellular estradiol Estrasorb Novavax Menopausal therapy 62
  64. 64. MARKETED DRUG USED TARGET DISEASE COMPANYPRODUCTDoxil Doxorubicin Kaposis sarcoma SEQUUS,USAAmphotec Amphotericin B Fungal infections SEQUUS,USA leishmaniasisFungizone Amphotericin B Fungal infections Bristol squibb Leishmaniasis netherlandVentus Prostaglandin –E1 Systemic inflammatory The liposome disease company USATopex Br Terbutaline sulphate asthma Ozone ,USADepocyt cytarabine Cancer therapy Skye pharm,USANovasome Small pox vaccine Small pox Novavax,USAAvian retrovirus Killed avian retro virus Chicken pox Vineland lab,USAvaccineVincasome vincristine Solid tumours Nextar, USA 63
  65. 65. Several methods of preparing liposomes wereidentified, which could influence the particlestructure, degree of drug entrapment and leakage of theliposomes. It was also identified that there are improved pharmacokineticproperties with liposomal drugs compared to the free drugs. Furthermore, liposomes are tools for drug targeting in certainbiomedical situations (e.g., cancer) and for reducing theincidence of dose related drug toxicity. Instability of the preparations is a problem, which is yet tobe overcome before full commercialisation of the processcan be realised. 64
  66. 66.  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 . platform-targeted-delivery-drugs “Liposomes preparation methods” a review by Mohammad riaz in Pakistan journal of pharmaceutical sciences “A review on liposomes” by venkateshwarlu in Research Journal of Pharmaceutical, Biological and Chemical Sciences “Stealth liposomes” a review by Kataria Sahil in IJRAP
  67. 67. PRESENTED BYPRIYANKA ODELADepartment of pharmaceuticsM –PHARM 1st