Chapter on Liposomes

Chapter on Liposomes

1. A SEMINAR ON LIPOSOMES IN DRUG DELIVERY 1 By:Rajesh L. Dumpala ( B.Pharm , M. Pharm.) PhD. ( Pursuing) Research Scientist, Alembic Research Centre. Vadodara E.Mail :[email protected]

2. Outlines Introduction Structural components Rationale , Advantages and Disadvantages Types of liposomes Methods of preparation of liposomes Characterization of liposomes Pharmacokinetic & Pharmacodynamic of liposome encapsulated drugs Stability of liposomes Recent advances Applications Marketed formulations of liposome products References 2

3. Introduction Liposomes were first described by British haematologist Dr. Alec D Bangham in 1961 (published 1964), at the Babraham institute, Cambridge. The name liposome is derived from two Greek words 'Lipid' meaning fat and 'Soma' meaning body. Definitions : Bangham et al .,1965 : Simple microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid molecule. Weiner N. et al .,1989 : A s a microstructure consisting of one or more concentric spheres of lipid bilayer separated by water or aqueous buffer compartments. 3

4. The drug molecules can encapsulated in aqueous space or intercalated into the lipid bilayer The particle size of liposomes ranges from 20 nm to 10 μm in diameter. Pharmaceutical researchers use the tools of biophysics in evaluating liposomal dosage forms. Liposomes have covered predominantly medical, albeit some non-medical areas like bioreactors, catalysts, cosmetics and ecology. Structure of liposome 4

5. Structural components Main components :- Phospholipids & Cholesterols The major structural components of biological membranes such as the cell membrane Two types :- Phosphoglycerides and Sphingolipids . Phospholipids 5

6. The most common phospholipid used phosphotidylcholine (PC). PC is an amphipathic molecule in which exists, 6

7. Cholesterol can be incorporated in very high concentration upto 1:1 or even 2:1 molar ratios of cholesterol to PC. It acts as a fluidity buffer, i.e. below the phase transition temperature, it makes the membrane less ordered and slightly more permeable; while above the phase transition temp. it makes the membrane more ordered and stable. Cholesterol inserts the membrane with its hydroxyl groups oriented towards the aqueous surface and aliphatic chain aligned parallel to the acyl chians in the center of the bilayer . Cholesterols 7

8. Rationale , Advantages &Disadvantages Why use liposomes??? Protection Duration Duration Direction Amplification Internalization 8

9. Advantages Provide controlled /sustained release drug delivery Provide targeted / site specific drug delivery Biodegradable and biocompatible Non ionic, non toxic and flexible Site avoidance effect Improved P/K effects (reduced elimination , circulation life times) Enhanced drug solubility Can carry both lipid and water soluble drugs 9

10. Improve drug and protein stability Improve efficacy and therapeutic index of drugs Can be administered through various routes Can incorporate micro and macro molecules Disadvantages Short half life Less stability of liposomes Phospholipid undergoes oxidation,hydrolysis,leakage High production cost 10

11. Types of liposomes Based on composition & application Based on pharmaceutical aspects 11

12. Based on composition & application 12

13. 13

14. Based on pharmaceutical aspects 14

15. Small Unilamellar Vesicle (SUV) Large Unilamellar Vesicle (LUV) Multilamellar Vesicle (MLV) (20-100nm )  100nm  0.5  m 15

16. Methods of preparation of liposomes MLVs Liposomes : a) Lipid hydration method b) Solvent spherule method LUVs Liposomes : a) Freeze drying method b) Micro emulsification / micro fluidization method c) Membrane extrusion method d) Reversed phase evaporation method e) Freeze thaw method f) Calcium-induced fusion method SUVs Liposomes : a) Sonication method b) French pressure method c) pH induced vesiculation 16

17. g) Solvent injection methods - Ethanol injection method and - Ether infusion method h) Detergent removal method Giant Liposomes : Multivesicular Liposomes : 17

18. Common stages of all methods of preparation of liposomes Cholesterol + Lecithin + Charge d issolve in organic solvent Solution in organic solvent Drying Thin film Dispersion (Hydration) Liposome suspension 18

19. Lipid Hydration Method ( using rotary evaporator) 19

20. Solvent Spherule method Small spherule of volatile hydrophobic solvent in which lipids are has been dissolved, are dispersed in aqueous solution. MLVs are formed when controlled evaporation of organic solvent occurred in a water bath. Advantage : Homogeneous size distribution. 20

21. Sonication method Two method of sonication : using Probe or Bath ultrasonic disintegrator Using Probe : For suspensions which require high energy in a small volume Disadv . : contamination of preparation with metal ,can lead to degradation of lipid . Using Bath : For large volume of dilute lipids where may not necessary to reach the vesicle size limit. Finally ,they are purified into the SUVs by ultracentrifugation. 21

22. French pressure method Extrusion of MLV at 2000 psi at 40 °C through a small orifice Adv :- - simple, rapid, reproducible and gentle handling of unstable materials. - possibility of leakage of vesicle contents from liposome is less than sonication method. Aqueous samples Piston Cell body Rubber-O-ring Closure plug Pressure relief valve Outlet Fig. French pressure cell 22

23. pH induced vesiculation ULVs are prepared from MLVs without sonication or high pressure application It is carried out by simply changing the pH (electrostatic phenomena) Dry film of lipids using rotary evaporator Hydration with minimum qty of water at RT Dispersion by six times freeze thawing cycles (15°C - 5°C) add 1M NaOH rapidly with mixing Reduces pH by 0.1M Hcl until pH : 7.5 obtained. 23

24. Freeze drying method Finely divided form of lipid dissoved in suitable organic solvent is to freeze dry , prior to addition aqueous media. Best solvent :- tertiary butanol (solvent choice depends on the freeze point which needs to above temp. of the condenser lyophilizers .) After obtaining the dry lipid which is an expanded foam like structure, water / saline can be added with rapid mixing above the phase transition temp. to give MLVs. 24

25. Microfluidization method Adv : - Excellent size reduction upto 0.2 mm - High rate of production - For encapsulation of water soluble materials due to high proportion of lipid . 25

26. Membrane extrusion method LUV can be prepared by passing under nitrogen through polycarbonate membrane filters of defined pore size. Done under moderate pressures (100-250 psi) Before experiment , Integrity test is carried out. Bubble point test : This test relies on the fact that after membrane has been wetted, the surface tension between water and air is such that air can not pass through the membrane until sufficient pressure has been reached to overcome that surface tension. 26

27. Lipid in solvent solution Two-phase system Water in oil emulsion Solvent removal Gel formation REV liposomes Reversed phase evaporation 27

28. Freeze thaw method In this method, rapid freezing and slowly thawing process is used to rupture and re-fuse SUVs during which the solute equilibrates between inside and outside, and the liposomes themselves fuse and increase in markedly in size. The formation of unilamellar vesicles due to the fusion of SUV during the processes of freezing and or thawing. Adv. : - High encapsulation efficiency (20-30 %) 28

29. Solvent injection methods Solution of lipids in diethyl ether :methanol mixture is slowly injected to aq. solution of materials to be encapsulated at 55-65°C Subsequent removal of ether under vacuum leads to the formation of liposomes. Drawbacks : - Heterogeneous size (70 – 190 nm). - exposure of compounds to organic solvents or high temperature. TB Vacuum pump Mix Gasket Ether/lipid solution Mechanical drive Infusion pump Aqueous phase 29

30. Detergent removal method The detergents at their critical micelles concentration are used to solubilize lipids. As the detergent is removed, the micelles become progressively richer in phospholipid and finally combine to form LUVs. Adv. : - Excellent reproducibilty and homogeneous size distribution. Drawbacks. : - Retention of traces of detergent within the liposomes 30

31. Giant liposomes : Procedure : Dialysis of a methanol solution of PC in presence of methylglucoside detergent against aq. Solution containing up to 1M NaCl Liposomes range : 10 – 100 mm . Multivesicular liposomes : The w/o emulsion was converted to organic solvent spherules by the addition of emulsion to across solution. The evaporation of organic solvent resultes in formation of multivesicular liposomes Size range : 5.6 – 29 pm Material to be encapsulated : Glucose, EDTA, human DNA . 31

32. Characterization of liposomes Physical characterization : Parameters Analytical methods/Instruments 1. Vesicles shape & surface morphology Transmission electron microscopy, Freeze-fracture electron microscopy 2. Mean vesicle size and size distribution (submicron and micron range) Dynamic light scattering, zetasizer, Photon correlation spectroscopy, laser light scattering, gel permeation and gel exclusion 3. Surface charge Free-flow electrophoresis 4. Electrical surface potential and surface pH Zeta potential measurements & pH sensitive probes 5. Lamellarity Small angle X-ray scattering, 31 P-NMR, Freeze-fracture electron microscopy 32

33. Parameters Analytical methods/Instruments 6. Phase behavior Freeze-fracture electron microscopy, Differential scanning colorimetery 7. Percent of free drug/ percent capture Minicolumn centrifugation, ion-exchange chromatography,  radiolabelling 8. Drug release Diffusion cell/ dialysis Chemical characterization Parameters Analytical methods/Instruments 1. Phospholipid concentration Barlett assay, stewart assay, HPLC 2. Cholesterol concentration Cholesterol oxidase assay and HPLC 3. Phopholipid peroxidation UV absorbance, Iodometric   and GLC 4. Phospholipid hydrolysis, Cholesterol auto-oxidation HPLC and TLC 5. Osmolarity Osmometer 33

34. Biological characterization Parameters Analytical methods/Instruments 1. Sterility Aerobic or anaerobic cultures 2. Pyrogenicity Limulus Amebocyte Lysate (LAL) test 3. Animal toxicity Monitoring survival rates, histology and pathology 34

35. P/K & P/D of liposome encapsulated drug Systems are designed to control following parameters: Rate of input of drug into particular body compartment Distribution & localization of drug in to body Persistence or rate of metabolism of drug Clearance and distribution of liposome in vivo : Two major determinants of liposome clearance Size : SUVs persist in the circulation for longer periods than large MLVs of same composition Also depends on homogeneou s in size or heterogeneous in size 35

36. Charge : SUVs with + and – charge retained longer time whereas small negative vesicles are rapidly cleared After clearance from circulation, they are sequestered in various tissues and organs MLVs :- mainly in liver & spleen (due to taken up presence of hepatic and spleenic reticuloendothelial cells rather than blood capillary) - Lung (due to physical entrapment of liposomes in the capillary beds of this organ) SUVs :- broader tissue distribution 36

37. Pharmacodynamics of liposome encapsulated drug: Pharmacodynamic effects Retardation of drug clearance from the circulation High drug accumulation in tissues rich in reticuloendothelial cells, especially liver and spleen Retention of drug in tissue for large periods Protection of drug against metabolic degradation and elimination Localized drug delivery primarily for cancer therapy 37

38. Stability of liposomes Chemical degradation Liposomal phospholipid oxidation and hydrolysis Prevention :- Start with freshly purified lipids & distilled solvents Avoid procedure which involves high temp. Carry out manufacturing in absence of oxygen Deoxygenate aqueous solution with nitrogen Store all liposome suspensions in inert atmosphere Include anti-oxidants as a component of the lipid membrane Saturated lipid reduces level of oxidizable lipid in membrane 38

39. Physical degradation leakage and fusion of vesicles Preventions: - SUVs (prone to fusion) is stored at temp away from the Tc . Avoid high conc. of metal ions for liposome having negative charge in the membrane and use of metal ion chelater in the suspending buffer. High molar ratio of cholesterol is most stable with regarded to leakage of solute for large polar or ionic molecule and low MW lipophilic compound. Freezing / lyophillization / cryopreservation – most suitable. 39

40. Proliposomes : Proliposomes are the products which are mixed with water phase containing drug before use, liposomes formed automatically and load the drug. Three types – Dry granular, protransferosomes and mixed micellar proliposomes Transferosomes Modified liposomes developed to increase the transdermal permeation of drug. Deformability is achieved by using surface active agent in proper ratio Recent advances in liposomes 40

41. Ethosomes Ethosomal system is a vesicular system composed mainly of phospholipids & alcohol (ethanol or IPA, sometimes polyols; glycol) in relatively high concentration & water. Better membrane permeability. 41

42. Cochleates Cochleates are cigar-like microstructures that consist of a series of lipid bilayers which are formed as a result of the condensation of small unilamellar negatively charged liposomes. In the presence of calcium, the small phosphatidylserine (PS) liposomes fuse and form large sheets. 42

43. Applications Cancer therapy and neoplasia Liposomes as carriers for vaccines - as immunological adjuvants - as carriers of antigens - as carriers of drug in oral treatment - for topical application - for pulmonary delivery Lysosomal storage diseases Opthalmic delivery of drug Metal storage diseases Cell biological application 43

44. Liposome based pharmaceuticals in market or in clinical trials 44

45. References Controlled and novel drug delivery systems, by Sanjay K. Jain and N.K. Jain http//en.wikipedia.org/wiki/liposome Mohammad Riaz , ‘Liposomes preparation methods’, pakistan jouranal of pharmaceutical science. www.pharmainfo.com(Sanjay S. Patel, Liposome: A versatile platform for targeted delivery of drugs, vol. 4, issue-5,2006. 45

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