liposomes by deepthi

Information about liposomes by deepthi

Published on July 31, 2014

Author: deepthibhaskar



LIPOSOMES: LIPOSOMES K.L.Deepthi , M.Pharm Assistant professor Pharmaceutical technology SVCP, Etcherla . contents: 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 7/31/2014 2 k.l.deepthi INTRODUCTION: 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. 7/31/2014 3 Definition: 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” 7/31/2014 4 PowerPoint Presentation: Phospholipid Bilayers are the core structure of liposome and cell membrane formations . Thus the structure of liposomes is similar to the structure of cell membranes.                       Liposome Cell Membrane 7/31/2014 5 k.l.deepthi Advantages with liposomes: 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 7/31/2014 6 Classification: Classification Classification based on size of liposomes Classification based on method of preparation Classification based on composition and in vivo application 7/31/2014 7 k.l.deepthi Classification based on size: 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 7/31/2014 8 k.l.deepthi Classification based on method of preparation: 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 . 7/31/2014 9 k.l.deepthi Classification based on specific properties: Classification based on specific properties Conventional Liposomes 7/31/2014 10 Long circulating liposomes (Stealth Technology) : Long circulating liposomes (Stealth Technology) PEG coating Low permeability liquid matrix and internal aqueous buffer system 7/31/2014 11 Targeted liposomes: Targeted liposomes Target specific ligands , such as antibodies, immunoglobulins , lectins and oligosaccharides attached to the surface 7/31/2014 12 Cationic Liposomes: Cationic Liposomes Cationic lipid component interact with negatively- charged DNA Results into Lipid –DNA Complexes 7/31/2014 13 Classes of Liposomes: Classes of Liposomes Conventional Long circulating Immuno Cationic 7/31/2014 14 k.l.deepthi Temperature sensitive liposome: Temperature sensitive liposome PH sensitive Liposomes 7/31/2014 15 MANUFACTURING OF LIPOSOMES: MANUFACTURING OF LIPOSOMES 7/31/2014 16 k.l.deepthi Materials used in preparation of liposomes: Materials used in preparation of liposomes Phospholipids cholesterol 7/31/2014 17 k.l.deepthi phospholipids: phospholipids Amphipathic molecules Helps in closed sealed vesicles Major structural components of membranes: Phosphotidylglycerides : phosphotidyl choline ( lecithine ) sphingolipids Property: Transition temperature: gel to liquid crystal (higher tem) Steps: 1. main transition 2. Pre transition(5 degrees below main transition) 7/31/2014 18 k.l.deepthi Structure of phospholipids: Structure of phospholipids 7/31/2014 19 k.l.deepthi PowerPoint Presentation: Cholesterol: It inserts into the membrane with its hydroxyl groups of cholesterol oriented towards the aqueous surface and aliphatic chain aligned parallel to the acyl chains in the center of the bilayers Increases separation b/w choline head groups and eliminates the normal electrostatic and hydrogen bonding interaction. Acts as fluidity buffer: Below tg –makes membrane less ordered Above tg – more ordered 7/31/2014 20 k.l.deepthi Issues to consider when selecting lipids.: Issues to consider when selecting lipids . Phase transition temperature Stability Charge Lipid mixtures Cholesterol Source 7/31/2014 21 k.l.deepthi Preparation of Liposomes: Preparation of Liposomes Mechanism of Vesicle Formation The budding theory The bilayer phospholipids theory 7/31/2014 22 k.l.deepthi The budding theory: 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 7/31/2014 23 k.l.deepthi The bilayer phospholipids theory: 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) 7/31/2014 24 Method of Liposome Preparation: Method of Liposome Preparation 7/31/2014 25 k.l.deepthi Conventional liposome preparation methods : Conventional liposome preparation methods Phospholipids Cholesterol Antioxidant Lipid component compounding Lipid solvent Pyrogen Ultrafilter yes No Filter Solvent removal Drug ,Salt Antioxidant Buffer WFI Filter Hydration Solvent recovery Extrusion Down sizing Free drug removal Prefilter Sterile filter Vial filling Free drug recovery Aseptic processing Lyophollization Seal / package 7/31/2014 26 k.l.deepthi PowerPoint Presentation: PREPARATION OF LIPOSOMES Numerous methods have been developed to meet different requirements. These can be divided into two categories: Those involving physical modification of existing bilayers Those involving generation of new bilayers by removal of a lipid solubilizing agent. 7/31/2014 27 k.l.deepthi PowerPoint Presentation: Multilamellar Vesicles Physical Methods. Simple "Hand-Shaken" MLV. MLV may be prepared from single-source natural or synthetic lipids, by suspending in a finely divided form in an aqueous solution maintained at a temperature greater than the Tc of the lipid. For unsaturated phospholipids such as egg and soy phosphatidylcholine (PC), which have Tc values below O 0 C, this is conveniently done at room temperature. 7/31/2014 28 k.l.deepthi PowerPoint Presentation: . 7/31/2014 29 k.l.deepthi PowerPoint Presentation: In this case it is essential that the different lipids be thoroughly mixed at the molecular level. This can be achieved by dissolving them in a common solvent such as a 2:1 (v/v) mixture of chloroform and methanol and then removing the solvent. This can be done using a rotary evaporator, where the lipid can be deposited as a thin film, which aids solvent removal and subsequent dispersion of the lipid. 7/31/2014 30 k.l.deepthi PowerPoint Presentation: Thin film hydration method for preparation of liposome using rotary evaporator 7/31/2014 31 k.l.deepthi PowerPoint Presentation: The disadvantages of this method is their low efficiency for incorporation of water-soluble solutes Reason: much of the volume is occupied by the internal lamellae and that the multilayers formed and sealed off with the majority of the lipid never having come into contact with the solute . Thus , in neutral liposomes , only a few percent of the starting material may become entrapped. 7/31/2014 32 k.l.deepthi PowerPoint Presentation: The encapsulation efficiency can be increased: by inclusion of a charged amphiphile , such as phosphatidyl glycerol or phosphatidic acid at a molar ratio of 10-20%, causes electrostatic repulsion between adjacent bilayers , leading to increased interlamellar separation , thus allowing more solute to be accommodated. 7/31/2014 33 k.l.deepthi PowerPoint Presentation: Dehydration/Rehydration Vesicles (DRV). designed to achieve high levels of entrapment. The intention of the DRV method is to maximize exposure of solute to the lipid before its final lamellar configuration has been fixed, so that the liposomes ultimately form around the solute. 7/31/2014 34 k.l.deepthi PowerPoint Presentation: This can be achieved by first preparing MLV in distilled water and then converting these to SUV so that the phospholipid achieves the highest possible level of dispersion within an aqueous phase. Thus when SUV are mixed with a solution of the material to be entrapped the majority of the amphiphile is directly exposed to the solute. Then, water is removed by freeze-drying, when a small amount of water is added with a large osmotic gradient between the internal and external phases leading to hyperosmotic inflation. 7/31/2014 35 k.l.deepthi PowerPoint Presentation: Steps for the manufacture of liposomes by the dehydration-rehydration method. 7/31/2014 36 k.l.deepthi PowerPoint Presentation: Small Unilamellar Vesicles Most of the commonly used methods for preparing SUV involve size-reduction of preexisting bilayers using ultrasonic irradiation by high-power probe sonication for seconds, in an inert atmosphere to prevent oxidative and by using a cooling bath to dissipate the large amounts of heat produced. A more gentle approach is to use bath sonication , Preparing SUV by sizing use ultrasonic irradiation 7/31/2014 37 k.l.deepthi PowerPoint Presentation: Preparing SUV by sizing use high pressure extrusion. High-pressure extrusion involves forcing multilamellar liposomes at high pressure through membranes having "straight-through," defined size pores. The liposomes have to deform to pass through the small pores, as a result of which lamellar fragments break away and reseal to form small vesicles of similar diameter to that of the pore. 7/31/2014 38 k.l.deepthi High pressure extrusion.: High pressure extrusion. 7/31/2014 39 PowerPoint Presentation: Repeated cycling through small-diameter pores at temperatures greater than the Tc of the lipid produces a homogeneous SUV. Advantage of the High-pressure extrusion method is that the disruptive effects of sonication are avoided. Liposome Extruders 7/31/2014 40 k.l.deepthi PowerPoint Presentation: Large Unilamellar Vesicles LUV’s single bilayer membrane (10-20 μ m) makes them well suited as model membrane systems whereas the large internal aqueous volume : lipid mass ratio means maximized efficiency of drug encapsulation. Methods for preparing LUV fall into two categories: 1. generation of new bilayers by removal of a lipid solubilizing agent, 2. physical modification of preformed bilayers . 7/31/2014 41 k.l.deepthi PowerPoint Presentation: For LUV preparation The solubilizing agents include detergents. 1. The lipid is initially dissolved by an aqueous solution of the detergent to form mixed lipid-detergent micelles, and the detergent is then removed by dialysis or gel chromatography. 2. Ionic detergents, such as cholate and deoxycholate or nonionic detergents such as Triton X 100 and have been used. 7/31/2014 42 k.l.deepthi PowerPoint Presentation: Removal of Organic Solvents. Solvent vaporization liposomes tend to be of a larger size range than those prepared by detergent removal. Three distinct types of process have been described, each involving addition of a solution of lipid in organic solvent, to an aqueous solution of the material to be encapsulated. Solvent Infusion Reverse Phase Evaporation. 7/31/2014 43 k.l.deepthi PowerPoint Presentation: Solvent Infusion . Solvent such as diethyl ether, petroleum ether, ethylmethyl ether, or diehlorofluoromethane containing dissolved lipid(s), is infused slowly into the aqueous phase, which is maintained at a temperature above the boiling point of the solvent so that bubbles are formed. 2. The lipid is deposited as unimellar liposomes . 3. High encapsulation efficiencies (up to 46%) were reported disadvantage is the need for exposure of the active ingredient to organic solvents, with the damage to labile materials such as proteins. 7/31/2014 44 k.l.deepthi Solvent injection method.: Solvent injection method. Vacuumpump Mix Gasket Ether/lipid solution Infusion pump Aqueous phase Mechanical drive Temperature Controlled bath 7/31/2014 45 k.l.deepthi PowerPoint Presentation: Reverse Phase Evaporation. Formation of a water-in-oil (diethyl ether) emulsion containing excess lipid. In the absence of cholesterol, these unilamellar vesicles have diameters in the range of 0.05-0.5 μ m, while with 50 mol % cholesterol, mean diameters are about 0.5 μ m. High encapsulation efficiencies of up 65% using hydrophilic solutes. 7/31/2014 46 k.l.deepthi Reverse phase evaporation technique.: Reverse phase evaporation technique . Lipid in solvent solution Two-phase system Water in oil emulsion Solvent removal Gel formation REV liposomes 7/31/2014 47 k.l.deepthi PowerPoint Presentation: When lipophilic drugs of appropriate structure are associated with liposonics by inclusion in the bilayer phase, the degree of "encapsulation" is dependent upon the saturation of the lipid phase with degrees of encapsulation of over 90%. Thus it is unnecessary to remove the unbound drug. However, in the case of water-soluble drugs, the encapsulated drug is only a fraction of the total drug used. Thus , it is required to remove the unbound drug from the drug-loaded liposomes in dispersion. REMOVAL OF UNBOUND DRUG 7/31/2014 48 k.l.deepthi PowerPoint Presentation: 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. A. Dialysis Dialysis is the simplest procedure used for the removal of the unbound drug, except when macromolecular compounds are involved . Liposome dispersion 7/31/2014 49 k.l.deepthi PowerPoint Presentation: Disadvantages: Dialysis is a slow process. Removal of over 95 % of the free drug require a minimum of 3 changes of the external medium over 10 to 24 hr at room temperature. Care is taken to balance the osmotic strengths of the liposomal dispersion and the dialyzing medium to avoid leakage of the encapsulated drug. 7/31/2014 50 k.l.deepthi PowerPoint Presentation: Centrifugation is an effective means of isolating liposomes from the free drug in the suspending medium. B. Centrifugation Two or more resuspension and centrifugation steps are included to effect a complete removal of the free drug. The centrifugal force required to pull liposomes down into a pellet is dependent upon the size of the liposomes . 7/31/2014 51 k.l.deepthi PowerPoint Presentation: Disadvantages: The use of refrigerated centrifuges operating at high speeds is energy intensive and expensive. It is essential to ensure that the osmotic strength of the resuspending medium is matched with that of original liposomal dispersion in order to avoid osmotic shock and rupture of liposomes . 7/31/2014 52 k.l.deepthi PowerPoint Presentation: Gel permeation chromatographic technique is used extensively both to separate liposomes from unbound drug and also to fractionate heterogeneous liposomal dispersions. Advantages: The technique is very effective and rapid at the laboraton level. C. Gel Filtration 7/31/2014 53 k.l.deepthi PowerPoint Presentation: Disadvantages: Gel filtration is expensive. Dilution of the liposomal dispersion with the eluting medium may necessitate another concentration step. Lipid losses on the column materials. 7/31/2014 54 k.l.deepthi REFERENCES: REFERENCES Controlled and novel drug delivery - N.K. JAIN Theory and practice in novel drug delivery system - S.P. VYAS

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