Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Liposomes- A Novel Drug Delivery System

45,802 views

Published on

Liposomes- A Novel Drug Delivery System

Published in: Healthcare
  • Login to see the comments

Liposomes- A Novel Drug Delivery System

  1. 1. Liposomes- A Novel Drug Delivery System From – Miss Snehal K. Dhobale M-Pharm ( Pharmaceutics)
  2. 2. What are Liposomes??? • Liposomes are concentric bilayered vesicles in which an aqueous core is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids. • Size range: 25nm-5000nm • Liposomes consist of Cholesterol, Phospholipid and drug molecule. 2
  3. 3. A simple view of liposomes “Liposomes are microscopic spheres made from fatty materials, predominantly phospholipids. “made up of one or more concentric lipid bilayers, and range in size from 50 nanometers to several micrometers in diameter 3
  4. 4. Advantages with liposomes Suitable for delivery of hydrophobic, hydrophilic and amphipatic drugs and agents Liposomes increases efficacy and therapeutic index of drug (actinomycin-D)  Liposome increased stability via encapsulation Suitable for controlled release Suitable to give localized action in particular tissues. Suitable to administer via various routes Liposomes help reduce the exposure of sensitive 4 tissues to toxic drugs
  5. 5. Disadvantages of liposomes Production cost is high. Leakage and fusion of encapsulated drug / molecules. Sometimes phospholipid undergoes oxidation and hydrolysis like reaction. Short half-life. Low solubility. 5
  6. 6. Components of liposomes The structural components of liposomes include: A. Phospholipids B. cholesterol
  7. 7. PHOSPHOLIPID • Major component of biological cell membrane Phospholipid Hydrophobic tail 2 fatty acid chain containing 10-20 carbon atoms 0-6 double bond in each chain Hydrophillic head or polar head Phosphoric acid bound to water soluble molecule 7
  8. 8. Commonly used other Phospholipids Natural phospholipid  PC- phosphatidyl choline  PE- phosphatidyl ethanolamine  PS –phosphatidyl serine Synthetic phosholipid  DOPC = Dioleoyl Phosphatidylcholine  DOPE = Dioleoyl phosphatidyl ethanolamine  DSPC = Distearoyl phosphatidyl choline  DSPE = Distearoyl phosphatidyl ethanolamine  DLPC = Dilauryl phosphatidyl choline 8
  9. 9. Phosphatidylcholine PC • The most common phospholipid use is phosphatidylcholine PC • Phosphatidylcholine is amphipathic molecule containing i. A hydrophillic polar head group phosphocholine ii. A glycerol bridge iii. A pair of hydrophobic acyl hydrocarbon chain 9
  10. 10. Cholesterol: • 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. 10
  11. 11. Role of cholesterol in bilayer formation:  Cholesterol act as fluidity buffer  After intercalation with phospholipid molecules alter the freedom of motion of carbon molecules in the acyl Chain  Restricts the transformations of trans to gauche Conformations.  Incorporated into phospholipid membrane upto 1:1 or 2:1 of cholesterol to PC. 11
  12. 12. Preparation of Liposomes • Mechanism of Vesicle Formation A. The budding theory B. The bilayer phospholipids theory 12
  13. 13. The budding theory – Stress induced hydration of phospholipids – Organization in to lamellar arrays – Results in to budding of lipid bilayer leading to down sizing SUV OLV 13
  14. 14. The bilayer phospholipids theory • Liposomes are formed when thin lipid films are hydrated • The hydrated lipid sheets detach during agitation and self-close to form large, multilamellar vesicles (LMV) 14
  15. 15. Modes of liposomes/cell interaction 1. Endocytosis 2. Adsorption 3. fusion 4. Lipid transfer 15
  16. 16. Factors affecting release of drug • Solvents • pH • Temperature • Agitation • Enzymes • Cell culture • Volume Drug release from liposomes • The lipid bilayer of the liposome can fuse with other bilayers (e.g. cell membrane) thus delivering the liposome contents. 16
  17. 17. CLASSIFICATION OF LIPOSOMES Structure Method of preparation Composition and application Conventional liposome Speciality liposome 17
  18. 18. 1. Classification of liposomes based on Structure Unilamellar (UV)-all size range Small Unilamellar(SUV) [20-100nm] Medium Unilamellar (MUV) Large Unilamellar (LUV) [>100nm] Giant Unilamellar (GUV) [>1μm] Multi Lamellar Vesicles (MLV) [0.5nm] Oligolamellar Vesicles (OLV) Multi Vesicular (MV) [>1μm] 18
  19. 19. 2. Classification of liposomes Based on Method of Preparation Single or oligo lamellar vesicle made by reverse phase evaporation method (REV’s) Multi lamellar vesicle made by reverse phase evaporation method (MLV-REV Stable pluri lamellar vesicle (SPLV) Frozen and thawed multi lamellar vesicle (FATMLV) Vesicle prepared by extrusion technique (VET) Dehydration- Rehydration method (DRV) Dehydration- Rehydration method (DRV) 19
  20. 20. 3. Classification of liposomes Based on Composition and Application Type of liposome Abbreviation Composition Conventional liposome CL Neutral of negatively charge phospholipids and cholesterol Fusogenic liposome RSVE Reconstituted sendai virus enveops pH sensitive liposomes - Phospholipids such as PER or DOPE Cationic liposome - Cationic lipid with DOPE Long circulatory liposome LCL Neutral high temp, cholesterol and 5-10% PEG, DSP Immune liposome IL CL or LCL with attached monoclonal antibody or recognition sequences 20
  21. 21. 4. Classification of Liposomes Based Upon Conventional Liposome 1). Stabilize natural lecithin (PC) mixtures 2). Synthetic identical, chain phospholipids 3). Glycolipids containing liposome 21
  22. 22. 5.)Classification of Liposomes based upon Speciality 5.) Liposomes Bipolar Fatty Acids Antibody directed Liposome Methyl/Methylene x- linked liposome Lipoprotein coated liposome Carbohydrate coated liposome Muiltiple Encapsulated Liposome 22
  23. 23. Methods of liposome preparation Passive loading techniques Active loading techniques Mechanical disp ersion methods Solvent dispersi on methods Detergent removal technique  LIPID FILM HYDRATION  BY HAND SHAKING,  FREEZE DRYING  NON HAND SHAKING  MICRO EMULSIFICATION  SONICATION  FRENCH PRESSURE CELL  MEMBRANE EXTRUSON  DRIED RECONSTITUTED VESICLES  ETHANOL INJECTION  ETHER INJECTION  DOUBLE EMULSION  REVERSE PHASE EVAPORATION 1. Detergent removal from mixed vesicles by  DIALYSIS  Column Chromatography  DILUTION
  24. 24. General Method Of Liposome Preparation 1 • Cholesterol + Lecithin + Charge and Dissolve in organic solvent 2 • Drying down lipid from organic solvent( Vaccum ) • Dispersion of lipid in aqueous media (Hydration 3 • Purification of resultant Liposomes • Analysis of final product 24
  25. 25. 1. Mechanical dispersion method Lipid dissolve in organic solvent/co-solvent Remove organic solvent under vacuum Film deposition Solid lipid mixture is hydrated by using aqueous buffer Lipid spontaneously swell & Hydrate Liposome 25
  26. 26. 1) Lipid Hydration Method A.) By hand shaking method vesiculate to form Multi lamellar vesicles(MLVs) Upon hydration lipids swell and peel out from RB flask Then film is treated with aqueous medium Lipids form stacks of film from organic solution 26
  27. 27. 1) Lipid Hydration Method B.) NON-HAND SHAKING METHOD The procedure differs from hand shaken method wherein it uses a stream of nitrogen to provide agitation rather than rotationary movements.  Here the lipid film is exposed to water saturated nitrogen for 15-20 min Milky suspension centrifugation LUV 27
  28. 28. 1) Lipid Hydration Method C.) FREEZE DRYING • Another method of dispersing the lipid in a finely divided form, prior to addition of aqueous media, is to freeze dry the lipid dissolved in a suitable organic solvent. • The solvent choice depends on the freeze point which needs to above the temperature of the condenser lyophilizers. Tertiary butanol is considered to be most ideal solvent. • After obtaining the dry lipid which is an expanded foam like structure, water or saline can be added with rapid mixing above the phase transition temperature to give MLVs. 28
  29. 29. 2.) MICRO EMULSIFICATION • This method is provided for preparing small lipid vesicles in commercial quantities by microemulsifying lipid compositions using very high shear forces generated in a homogenizing apparatus operated at high pressures at a selected temperature. • At least 20 circulations (approximately 10 minutes) but not greater than 200 circulations (100 minutes) are sufficient to produce a micro emulsion of small vesicles suitable for biological application. 29
  30. 30. 3.) SONICATION a)Probe sonication  The tip of a sonicator is directly engrossed into the liposome dispersion.  The energy input into lipid dispersion is very high in this method.  The coupling of energy at the tip results in local hotness; therefore, the vessel must be engrossed into a water/ice bath. b)Bath sonication  The liposome dispersion in a cylinder is placed into a bath sonicator.  Controlling the temperature of the lipid dispersion is usually easier in this method, in contrast to sonication by dispersal directly using the tip. 30
  31. 31. 4.) FRENCH PRESSURE CELL • The method involves the extrusion of MLV at 20,000 psi at 4°C through a small orifice. • The method has several advantages over sonication method. • The method is simple rapid, reproducible and involves gentle handling of unstable materials. • The resulting liposomes are somewhat larger than sonicated SUVs. • The drawbacks of the method are that the temperature is difficult to achieve and the working volumes are relatively small (about 50 mL maximum). 31
  32. 32. 5.) MEMBRANE EXTRUSON  Liposomes passed through membrane of defined pore size. Lower pressure is required (<100 psi).  LUVs as well as MLVs can be processed.  Vesicle contents are exchanged with dispersion medium during breaking and resealing of phospholipid bilayers as they pass through the polycarbonate membrane.  For high entrapment, the water soluble compounds should be present in suspending medium during the extrusion process. 32
  33. 33. 6.) DRIED RECONSTITUTED VESICLES SUV’s in SUV’s with DRV Freeze dried aqueous solute to be membrane phase entrapped Solutes with oligo and uni lamellar Freeze membranes drying Rehydration 33
  34. 34. B] Solvent dispersion methods • Ethanol injection –SUV • Ether injection -LUV • Reverse phase evaporation vesicle –LUV • Double emulsion vesicles Stable plurilamellar vesicles 34
  35. 35. 1.) Ethanol Injection Method • A lipid solution of ethanol is rapidly injected to a vast excess of buffer. The MLVs are immediately formed. • The drawbacks of the method are that the population is heterogeneous (30-110 nm), liposomes are very dilute, it is difficult to remove all ethanol because it forms azeotrope with water and the possibility of various biologically active macromolecules to inactivation in the presence of even low amounts of ethanol. 35
  36. 36. 2.) Ether Infusion Method • A solution of lipids dissolved in diethyl ether or ether/methanol mixture is slowly injected to an aqueous solution of the material to be encapsulated at 55-65°C or under reduced pressure. • The subsequent removal of ether under vacuum leads to the formation of liposomes. • This method is used to treat sensitive lipids very gently. 36
  37. 37. Double emulsification • In this process, an active ingredient is first dissolved in an aqueous phase (w1) which is then emulsified in an organic solvent of a polymer to make a primary w1/o emulsion. • This primary emulsion is further mixed in an emulsifier-containing aqueous solution (w2) to make a w1/o/w2 double emulsion. • The removal of the solvent leaves microspheres in the aqueous continuous phase, making it possible to collect them by filtering or centrifuging. 37
  38. 38. 3.) Reverse-phase evaporation Lipid organic solvent and aqueous solution are i. mixed, ii. sonicated, iii. formation of w/o emulsion, iv. evaporate to remove the organic solvent Lipids form a phospholipid bilayer on vigorous shaking , water droplets collapse and formation of LUV’s takes place. 38
  39. 39. Reverse phase evaporation technique. Lipid in solvent solution Two-phase system Water in oil emulsion Gel formation Solvent removal REV liposomes Conti… 39
  40. 40. C. Detergent removal method • The micellar dispersion is then subjected to one of the following methods to remove the detergent: I. DIALYSIS :- Detergents with high CMC (10-20 mM ) are used so that their removal is facilitated e.g. bile salts- sodium cholate and sodium deoxycholate , or synthetic detergents like octileglucoside . II. COLUMN CHROMATOGRAPHY :- By passing dispersion over a Sephadex G-25 column. 40
  41. 41. i.) Dialysis Method Dialysis is the simplest procedure used for the removal of the unbound drug, except when macromolecular compounds are involved Advantages: Dialysis Technique requiring no complicated or expensive equipment. Dialysis is effective in removing nearly all of the free drug with a sufficient number of changes of the dialyzing medium 41
  42. 42. ii.) Column Chromatography • Phospholipid in the form of either sonicated vesicle or as a dry film, at a molar ratio of 2:1 with deoxycholate form unilamellar vesicles of 100nm on removal of deoxycholate by column chromatography 42
  43. 43. B. Active loading technique AFTER DRYING IN PROCESS FILM/CAKE OF LIPID IS FROM STACKS OF LIPID BILAYER FORM SWELLING IN FLUID SHEET IS SELF CLOSE LOADING OF DRUG ON pH- GRADIENT TECHNIQUE FORMATION OF BILAYER (LIPOSOMES) IF DRUG  Two steps process generates this pH imbalance and active (remote) loading. 1) Vesicles are prepared in low pH solution, thus generating low pH within the liposomal interiors 2) followed by addition of the base to extraliposomal medium.  Basic compounds, carrying amino groups are relatively lipophipic at high pH and hydrophilic at low pH. 43
  44. 44. 1.) Lyophilization • Freeze-drying (lyophilization) involves the removal of water from products in the frozen state at extremely low pressures. • The process is generally used to dry products and are thermo labile and would be destroyed by heat-drying. • The technique has a great potential as a method to solve long term stability problems with respect to liposomal stability. • It is exposed that leakage of entrapped materials may take place during the process of freeze- drying and on reconstitution. 44
  45. 45. 2.) Pro-liposomes: lipid Dried over lipid Finely divided particulate support like powdered NACL/ sorbital  To increase the surface area of dried lipid film and to facilitate continuous hydration and lipid is dried over the finally divided particulate support i.e.- NaCl, Sorbitol, or other polysaccharides. These dried lipid coated particulates are called as proliposomes  Proliposomes form dispersion of MLVs on addition of water, where support is rapidly dissolved and lipid film hydrate to form MLVs 45
  46. 46. Uses of liposomes • Chelation therapy for treatment of heavy metal poisoning • Enzyme replacement • Diagnostic imaging of tumors • Study of membranes • In gene delivery. • As drug delivery carriers. • Enzyme replacement therapy. • Liposomes in antiviral/anti microbial therapy. • In multi drug resistance. • In tumour therapy. • In immunology. • In cosmetology 46
  47. 47. Applications • Liposomes are successfully used to entrap anticancer drugs. This increases circulation life time, protects from metabolic degradation. 1.Cancer chemotherapy • 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. 2.Liposomes as carrier of drug in oral treatment • Drugs like triamcilone, methotrexate, benzocaine, corticosteroids etc can be successfully incorporated as topical liposome 3.Liposomes for topical applications 4. Liposomes for pulmonary delivery Inhalational devices like nebulizers are use to produce an aerosol of droplets containing liposomes. 47
  48. 48. DNA delivery of Genes by Liposomes  Cheaper than viruses  No immune response  Especially good for in-lung delivery (cystic fibrosis)  100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector 48
  49. 49. Lipofection 49
  50. 50. Liposomes could serve as tumor specific vehicles (even without special targeting) Liposomes better penetrate into tissues with disrupted endothelial lining 50
  51. 51. Characterization of liposomes • Shape, size and its distribution • Surface charge • Percentage drug entrapment • Entrapped volume • Lamellarity • Phase behavior of liposome • Percentage drug release 51
  52. 52. References 1) ‘Controlled and Novel Drug Delivery’, “JAIN N.K.’’, CBS Publisher And Distributors.Page No.307-321. 2) ‘Targeted and controlled drug delivery, Novel carrier Systems’ , “VYAS S.P. and KHAR R.K.’’, CBS Publishers Page no.181 -195. 3) A.Chonn,P.R.Cullis,“Recent advances in liposome technologies and their applications for systemic gene delivery”,Advanced Drug Delivery Reviews 4) Liposomes preparation methods by Mohammad riaz ,Pakistan Journal of Pharmaceutical Sciences Vol.19(1), January 1996, pp.65-77 5) Liposome- as drug carriers-International Journal of Pharmacy & life sciences- Himanshu Anwekar*, Sitasharan Patel and A.K Singhai 6) http://www.avantilipids.com 7) http://www. Mssm.edu/medicine/thrombosis/phosphol.html 8) Garrett, R. and Grisham C. Biochemistry, 2nd ed. Saunders Colleges Publishing. New York (1999). 264 9) "Liposomes." www.collabo.com/liposom0.htm 10) Sharma Vijay K1*, Liposomes: Present Prospective and Future Challenges,International Journal Of Current Pharmaceutical Review And Research, oct 2010,vol1, issue 2,6-16 11) Himanshu Anwekar*, Liposome- as drug carriers, International Journal Of Pharmacy & Life Sciences, Vol.2, Issue 7: July: 2011, 945-951 52
  53. 53. 53

×