Liposomes & Niosomes.pptx

introduction to liposomes, niosomes

1. LIPOSOMES Dr.RLC Sasidhar Associate Professor, CHPS

2. Liposomes are simple microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid molecule. Structurally, liposomes are concentric bilayered vesicles in which an aqueous volume is entirely enclosed  by a membraneous lipid bilayer mainly composed of natural or synthetic phospholipids.

3. Hydrophobic Hydrophilic cavity

4. H 2 O Layer Polar Lipids (Phospholipid) Water Soluble ingredients (Drugs, Nutrients & vitamins) Lipid Soluble ingredients (Drugs,Nutrients & vitamins) Cross-section of liposomes :

5. ADVANTAGES OF LIPOSOMES Provides selective passive targeting to tumor  tissues (liposomal doxorubicin) Increased efficacy and therapeutic index Reduction in toxicity of the encapsulated agent Site avoidance effect (avoids non-target tissues) Improved pharmacokinetic effects (reduced  elimination increased circulation life times) Flexibility to couple with site specific ligands to  achieve active targeting

6. DISADVANTAGES The development of liposomes at industrial level is difficult due to its physiological and physicochemical instability. They aggregate and fuse together upon prolonged storage disturbing the reproducibility. They are prone to degradation by oxidation and hydrolysis. 6

7. STRUCTURAL COMPONENTS OF LIPOSOMES The main components of liposomes are PHOSPHOLIPIDS CHOLESTEROL

8. PHOSPHOLIPIDS Phospholipids are the major structural component of biological membranes such as  the cell  membrane. TWO TYPES OF  PHOSPHOLIPIDS  PHOSPHOGLYCERIDES SPHINGOLIPIDS

9. Some Other Commonly Used  Phospholipids Naturally occurring phospholipids: -PC : Phosphatidylcholine – PE : Phosphatidylethanolamine – PS : Phosphatidylserine Synthetic phospholipids: -DOPC : Dioleoylphosphatidylcholine – DSPC : Distearoylphosphatidylcholine

10. CHOLESTEROL Incorporation of sterols in liposome bilayer  brings about major changes in the preparation of these membranes. Cholesterol by it self does not form a bilayer structure. However, cholesterol 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 temperature it makes  the membrane more ordered and stable.

11. CLASSIFICATION : Based on Structural Parameters: Multi-laminar vesicles (MLV): made up of series of concentric bi-layer of lipid enclosing a small internal volume with size range > 0.5um. b. Oligolamelar vesicles (OLV): constitutes 2 to 10 bi layer of lipids surrounding a large internal volume with size range of 0.1 – 1um.

12. c. Unilamellar vesicle (ULV): single layer of lipids. Based on the size of the single layer they are further divide into the following types with in ULV as Small unilaminar vesicle: size of 20 to 40 nm Medium unilaminar vesicle: size of 40 to 80 nm Large unilaminar vesicle: size of 100 to 1000 nm Gaint unilaminar vesicle: size of more than 1000 nm d. Multivesicular Vesicle (MV): constitutes for multiple vesicles and size range >1um.

13. Mechanical dispersion methods Lipid film hydration by hand shaking,non-hand shaking or freeze drying Microemulsification Sonication French pressure cell Membrane extrusion Dried reconstituted vesicles Freeze-thawed liposomes Solvent dispersion methods Ethanol injection Ether injection Double emulsion vesicles Reverse phase evaporation vesicles Stable plurilamellar vesicles Detergent removal methods Dialysis Column chromatography Dilution Reconstituted Sendai Virus enveloped vesicles Method of Preparation of Liposomes Passive Loading Techniques

14. HAND SHAKING METHOD:

15. Sonicated unilamellar vesicles (SUVs)

16. 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.

17. Solvent Dispersion methods Ethanol injection method Ether injection method

18. In ether injection method a solution of lipids is dissolved in ether or diethyl ether/methanol mixture which is slowly injected to an aqueous solution of the material to be capsulated. The subsequent removal of the organic solvent under reduced pressure leads to the formation of liposomes . In ethanol injection method the ethanolic lipid solution is rapidly injected to a vast excess of preheated distilled water.

19. CHARACTERISATION OF LIPOSOMES: 1. Physical Characterization 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) 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 Zetapotential measurements & pH sensitive probes 5. Phase behavior Freeze-fracture electron microscopy, Differential scanning colorimetery 6. Percent of free drug/ percent capture Minicolumn centrifugation, ion-exchange chromatography,  radiolabelling 7. Drug release Diffusion cell/ dialysis

20. 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, Iodometric  and GLC 4. Phospholipid hydrolysis, Cholesterol auto-oxidation. HPLC and TLC 5. Osmolarity Osmometer

21. 3. 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

22. 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 ALEC TM Dry protein free powder of DPPC-PG Expanding lung diseases in babies Britannia Pharm, UK Topex-Br Terbutaline sulphate Asthma Ozone, USA Depocyt Cytarabine Cancer therapy Skye Pharm, USA Novasome ® Smallpox vaccine Smallpox Novavax, USA VincaXome Vincristine Solid Tumours NeXstar, USA

23. NIOSOMES

24. introduction Niosomes are non-ionic surfactant based unilamellar or multilamellar bilayer vesicles up on hydration of non ionic surfactants with or without incorporation cholesterol . The niosomes are very small, and microscopic in size. Their size lies in the nanometric scale. Niosomes are a novel drug delivery system, in which the medication is encapsulated in a vesicle. Both hydrophilic & lipophilic drugs ,entrap either in the aqueous layer or in vesicular membrane made of lipid materials.

25. Structure of niosomes : Head part (hydrophillic) Tail part (hydrophobic) Drug molecules Polar heads facing hydrophilic region Hydrophobic drugs localized in the hydrophobic lamellae Hydrophilic drugs located in aqueous regions encapsulated These vesicular systems are similar to liposomes that can be used as carriers of amphiphilic and lipophilic drugs. It is less toxic and improves the therapeutic index of drug by restricting its action to target cells.

26. They are osmotically active and stable. They increase the stability of the entrapped drug. The vesicle suspension being water based offers greater patient compliance over oil based systems Since the structure of the niosome offers place to accommodate hydrophilic, lipophilic as well as ampiphilic drug moieties, they can be used for a variety of drugs. The vesicles can act as a depot to release the drug slowly and of controlled release. Biodegradable, non-immunogenic and biocompatible. Advantages of niosomes :

27. Disadvantages of Niosomes : Aggregation Fusion Leaking of entrapped drug Hydrolysis of encapsulated drugs which limiting the shelf life of the dispersion.

28. Cholesterol and Non ionic surfactants are the two major components used for the preparation of niosomes. Cholesterol provides rigidity and proper shape. The surfactants play a major role in the formation of niosomes. non-ionic surfactants like spans(span 20,40,60,85,80), tweens (tween 20,40,60,80) are generally used for the preparation of Niosomes. Few other surfactants that are reported to form niosomes are as follows : Ether linked surfactant Di-alkyl chain surfactant Ester linked Sorbitan Esters Poly-sorbates Components of niosomes :

29. Hand shaking Method Sonication Ether injection Ethanol Injection Method of Preparation of Liposomes

30. HAND SHAKING METHOD:

31. The mixture of vesicles forming ingredients like surfactant and cholesterol are dissolved in a volatile organic solvent (diethyl ether, chloroform or methanol) in a round bottom flask. The organic solvent is removed at room temperature (20°C) using rotary evaporator leaving a thin layer of solid mixture deposited on the wall of the flask. The dried surfactant film can be rehydrated with aqueous phase at 0-60°C with gentle agitation. This process forms typical multilamellar niosomes . Hand shaking Method

32. Sonicated unilamellar vesicles (SUVs)

33. In the sonication-mediated procedure, niosomes were prepared by distributing the surfactant cholesterol combination in water phase that contains the drug in flax. The mixture is subjected to probe sonication or bath sonicator for 3 minutes at 60°C until formation of multilamellar vesicles

34. Solvent Dispersion methods Ethanol injection method Ether injection method

35. The mix of surfactant, cholesterol and drug, is dissolved in diethyl ether and over a gauze needle injected gradually into an aqueous phase. The ether solution is evaporated by rotary evaporator above the boiling point of the organic solvent. Vaporization of ether leads to formation of single layered vesicles.

36. 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) 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 Zetapotential measurements & pH sensitive probes 5. Phase behavior Freeze-fracture electron microscopy, Differential scanning colorimetery 6. Percent of free drug/ percent capture Minicolumn centrifugation, ion-exchange chromatography,  radiolabelling 7. Drug release Diffusion cell/ dialysis

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