LIPOSOMES AND NANOTECHNOLOGY

1. SEMINAR ON SEMINAR ON …….. LIPOSOMES ANDNANOTECHNOLOGY PRESENTED BY: MAHESH DABHI Department Of Pharmaceutics and Pharmaceutical Technology, L.M. College of Pharmacy.

2. List of contents • Introduction: • Advantages with use of liposomes as drug delivery system. • Classification • Manufacturing of liposomes • Liposome characterization and control • Stability consideration for liposomal formulations • Regulatory science of liposome drug products • Drug release from liposomes • Applications • Recent innovations • Approved liposome products • References

3. INTRODUCTION • The preparation of liposomes, with entrapped solutes, was demonstrated for the first time in 1965 by Prof. A.D. Bangham of the United Kingdom.

4. Definition • “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”

5. Advantages with liposomes • Suitable for delivery of hydrophobic, hydrophilic and amphipatic drugs and agents • Chemically and physically well characterized entities • Biocompatible • Suitable for controlled release • Suitable to give localized action in particular tissues. • Suitable to administer via various routes

6. Classification • Classification based on size of liposomes • Classification based on method of preparation • Classification based on composition and in vivo application

7. Classification based on size Small unilamellar vesicles Medium sized unilamellar vesicles Large unilamellar vesicles Giant unilamellar vesicles Unilamellar vesicles Oligolamellar vesicles Multilamellar large vesicles Multivesicular vesicles

8. Classification based on method of preparation • Vesicles prepared by extrusion method. • Vesicles prepared by French press. • Vesicles prepared by fusion. • Vesicles prepared by reverse phase evaporation. • Frozen and thawed MLV. • Dehydration and rehydration vesicles. • Stable plurilamellar vesicles.

9. Classification based on specific properties • Conventional Liposomes

10. Long circulating liposomes (Stealth Technology) • PEG coating • Low permeability liquid matrix and internal aqueous buffer system

11. Targeted liposomes • Target specific ligands, such as antibodies, immunoglobulins, lectins and oligosaccharides attached to the surface

12. Cationic Liposomes • Cationic lipid component interact with negatively- charged DNA • Results into Lipid –DNA Complexes

13. Temperature sensitive liposome PH sensitive Liposomes

14. MANUFACTURING OF LIPOSOMES

15. Materials used in preparation of liposomes • Phospholipids • Synthetic Phospholipids • Glycerolipids • Sphingolipids • Glycosphingolipids • Steroids • Polymeric material • Charge-inducing lipids

16. Structure of phospholipids

17. Issues to consider when selecting lipids. • Phase transition temperature • Stability • Charge • Lipid mixtures • Cholesterol • Source

18. Preparation of Liposomes Mechanism of Vesicle Formation • The budding theory • The bilayer phospholipids theory

19. 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

20. 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)

21. Method of Liposome Preparation

22. Pyrogen Conventional liposome preparation methods Drug ,Salt Antioxidant Buffer WFI Phospholipids Cholesterol Antioxidant Lipid component compounding Lipid solvent yes Ultrafilter Filter No Filter Solvent recovery Solvent removal Hydration Extrusion Down sizing Free drug recovery Free drug removal Prefilter Sterile filter Vial filling Lyophollization Seal / package Aseptic processing

23. Method for preparation of (SUV) • Sonication • High shear fragmentation • Solvent injection method • Injection of water immiscible solvent. • Ether infusion. • Fluorocarbon injection. • Injection of water miscible solvent. • Ethanol injection • Modified ethanol injection method

24. High shear fragmentation Piston Cell body Rubber-O-ring Pressure relief valve Aqueous samples Closure plug Outlet Fig. French pressure cell

25. Mechanical drive Vacuumpump Mix Solvent injection method. Ether/lipid solution Infusion pump Gasket Aqueous phase Temperature Controlled bath

26. Large and Intermediate sized unilamellar vesicles. • Methods used to prepare LUV and IUV • Detergent dialysis • Water in oil emulsion technique • Freeze thaw cycling • Slow swelling in non electrolytes • Dehydration followed by rehydration • Dilution or dialysis of lipids in the presence of chaotropic ions.

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

28. High pressure extrusion.

29. Methods for controlling liposome size • Fractionation • Centrifugation • Size exclusion chromatography • Homogenization • Capillary pore membrane extrusion • Ceramic extrusion

30. Size Number of lamellae Stability Charge Preparation Raw materials Protection Head group Protecting agents Hydration methods Degree of saturation Presence of sterols Sizing method Liposome characterization and control Liposomes Characterized by Determined by Classified by

31. Physical characterization parameters • Mean size and size distribution • Number of lamellae • Osmotic behavior and entrapped volume • Internal distribution of drug • Structural and motional behavior of lipids • Electrical surface potential & Surface PH

32. Stability consideration • Stabilization aspect for physical instability of liposomes • Chemical stability • Biological stability of liposomes

33. Regulatory aspects • Safety concerns: liposome formulation • Lipid toxicity (RBC lysis) • Presence of protein and lipoprotein for natural lipids • Residual solvent • Overload of RES • Particle size • (tail above 1 um) - Blockage of capillaries • Size affects RES uptake and tissue targeting • Stability: shelf-live and in vivo • Dose dumping (via protein binding) • Sterility

34. Drug release from liposomes • The lipid bilayer of the liposome can fuse with other bilayers (e.g. cell membrane) thus delivering the liposome contents.

35. Liposome Performance – In Vitro Release and Stability • In vitro drug release from liposomal systems was determined using dialysis sacks. • Release test for a targeted liposome would need to show that liposome is stable until uptake at the site.

36. Factors affecting release of drug • Solvents • pH • Temperature • Agitation • Enzymes • Cell culture • Sink conditions • Volume • Sampling interval

37. Applications • Liposomes as Protein Carriers in Immunology • Oral Drug Delivery • Site Specific Delivery • Sustained or Controlled Delivery • Gene Therapeutics

38. Applications

39. Innovations in vesicular drug delivery systems • Provesicles in drug delivery systems • Provesicles in drug delivery systems • Proliposomes :- • Dry granular liposomes • Mixed micellar proliposomes • Protransferosomes

40. Characterization of provesicular system • Morphology • Angle of repose • Size and size distribution • Rate of hydration • Entrapment efficiency • Degree of deformability and permeability measurement • In vitro release rate • In vivo fate and pharmacokinetic

41. Lipopolyplexes • A combination of DNA, polymers and liposomes • This method has resulted in better gene transfer and lower toxicity as compare to cationic liposomes

42. Transferosomes • Modified liposomes developed to increase the transdermal permeation of drug • Deformability is achieved by using surface active agent in proper ratio • Concentration of surfactant is very crucial

43. Ethosomes • Composed of phospholipids & alcohol ( ethanol or IPA) • Sometimes polyols or glycols in relatively high concentration & water • Better membrane permeability

44. Discomes • Virosomes • Emulsomes

45. Cochleates • Cochleates are cigar-like microstructures • Consist of a series of lipid bilayers which are formed as a result of the condensation of small unilamellar negatively charged liposomes.

46. Depofoam technology • Depofoam particles include hundred of bilayer enclosed aqueous compound. • Formed by first emulsifying a mixture of an aq phase containing the compound to be encapsulated & an organic phase containing lipid. Niosomes • Nonionic surfactant vesicles(NSV) • Niosomes are formed from the self assembly of non-ionic amphiphiles in aqueous media resulting in closed bilayer.

47. Preparation of Liposomes by dry film method • Lipids and drug dissolved in CHCl3 and evaporated to form thin film • Film is hydrated with buffer solution • Sonicated to form large unilamellar vesicles

48. Preparation of Liposomes by dry film method Thin film Rotary evaporation Lipid + drug + CHCl3 Sonication LUV

49. Approved liposome products marketed in US Approved lipid complex products Ambelcet Amphotericin B Alza corporation Amphotec Amphotericin B Elan corporation

50. NANOTECHNOLOGY INDRUG DELIVERY