LIPOSOMES - Novel drug delivery system NDDS

overall explanation on the Liposomes i.e. novel drug delivery system
lipid formulations
liposome preparation in India
Evaluation of Liposomes
reference text book added in the last slide.
Methods of preparation of liposome

1. LIPOSOMES Presented by Ayushi p Jain M.Pharmacy 1

2. CONTENTS Introduction Advantages Disadvantages Mechanism of lipid formation Classification of lipids Methods of preparation of liposomes Materials used in preparation of liposomes Incorporation of drugs in liposomes Physical characterization Chemical characterization Biological characterization Evaluation of liposomes Application of liposomes Liposomal preparation available in India 2

3. INTRODUCTION The word liposome is derived from Greek word “lipos” meaning fat and “soma” meaning body . Discovered in 1961 by British hematologist Dr. Alec D Bangham published in 1964 at the Babraham Institute, when he and R. W. Horne were testing the Institute’s new electron microscope by adding negative stain to dry phospholipids. 3

4. Liposomes are simple microscopic, concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid molecule mainly composed of natural or synthetic phospholipids and cholesterol being the main ingredient. The structural main components are Phospholipids & Cholesterol . Size range : 25nm-5000nm The lipid molecules are usually phospholipids – amphipathic moieties with a hydrophilic head & hydrophobic tails. On addition of excess water, such lipid moieties spontaneously originate to give the most thermodynamically stable conformation. In which polar head groups, face outwards into the aqueous medium, & the lipid chains turns inwards to avoid the water phase, giving rise to double layer or bilayer lamellar structures. 4

5. The type of phospholipids includes phosglycerides and sphingolipids together with their hydrolysis products. Phospholipids form closed fluid-filled spheres when mixed with water & they are arranged in bilayer, which form closed vesicle Liposomes have two standard forms 1. Unilamellar vesicles : ULV’S consisting of a single bilayer surrounding an entirely fluid core. They are characterized as being small (SUL’S) or large (LIV’S) 2. Multilamellar vesicles : MLU’S are made up of several lipid bilayers separated by fluid These are suitable for encapsulation of a variety of substances including drugs Disadv :- low encapsulation capacity 5

6. Advantages Liposomes are biocompatible, completely biodegradable, non-toxic in nature. They are suitable for delivery of hydrophobic, amphipathic and hydrophilic drugs. They protect the encapsulated drug from external environment. They reduce toxicity and increase stability-Since therapeutic activity of chemotherapeutic agent can be improved through liposome encapsulation. This reduces deleterious effects that are observed at concentration similar to / lower than those required for maximum therapeutic activity. It reduces exposure of sensitive tissue to toxic drugs. 6

7. Increase efficacy & therapeutic index. pharmacokinetic effects i.e. decrease elimination & increase circulation. Flexibility i.e. site specific achieve targeting. Passive targeting for tumour tissues. Liposomes can carry water soluble drugs in their aqueous phase & lipid soluble drugs in lipid bilayer. Non antigenic in nature. Maintenance of in vivo drug level over prolonged period of time 7

8. Disadvantages The production cost is high. Leakage and fusion of encapsulated drug/molecules can occur. It has short half life - In reticuloendothelial system, particularly the Kupffer cells in the liver remove liposomes from the circulation. Chemically unstable because of their predisposition to oxidative degradation. Slow penetration into tumors. Batch to batch variation. once administered cannot be removed. Dose dumping 8

9. Mechanism of lipid formation In aqueous media phospholipids as they are not soluble align themselves closely in planar bilayer sheets or lipid cakes which is thermodynamically stable In which polar head groups face outwards into the aqueous medium & the lipidic chains turns inwards to avoid the water phase giving rise to double layer or bilayer This structure is also called as lamella For the liposomes to be formed upon further hydration , the lipid cakes ( lamella ) swells eventually they curve to form a closed vesicles in the form of spheres This spheres is called as liposomes 9

10. Molecular geometry & liposome formation ( israelachvili hypothesis ) Vesicles are formed by hydrophobic effect governed by self assemblages critical packing parameter (cpp) It depends on the ratio of hydrophilicity & hydrophobicity of lipids as well as mesogen molecular geometry The symmetry of lipid self assembly & liquid crystalline phase formation show strong dependence on the molecular shape of mesogen/ amphiphiles The different shapes & volumes constructing different phases are characterized by dimensionless cpp Cpp = v/ lcAp = Ahp / Ap Where V - hydrophobic group volume Lc - hydrophobic group length 10

11. Ap - cross sectional area of hydrophilic head group Ahp - cross sectional area of hydrophobic group If cpp value Is less than 0.5 than liposomes are formed by hydrophobic effect Is more than 0.5 than liposomes are formed by hydrophilic effect Is between 0.5 than liposomes are formed by surfactant effect 11

12. Classification of liposomes based on size and lamellarity TYPES SIZE Multilamellar large vesicles(MLV) (>0.5µm) Oligolamellar vesicles(OLV) 0.1-1 µm Unilamellar vesicles(ULV) All size range Small unilamellar vesicles(SUV) 20-100 nm Medium sized unilamellar vesicles(MUV) _ Large unilamellar vesicles(LUV) >100 nm Giant unilamellar vesicles(GU) >0.1 µm Multivesicular vesicles(MVV) usually >1 µm 12

13. Classification of liposomes based on method of preparation TYPES OF LIPOSOMES METHOD OF PREPARATION REV(Reverse Evaporation vesicles) Single or Oligolemellar vesicles made by reverse osmosis MLV-REV Multilamellar vesicles made by Reverse Phase Evaporation method SPLV Stable Plurilamellar vesicles FATMLV Frozen and Thawed MLV VET Vesicles prepared by Extrusion method DRV Vesicles prepared by Dehydration-Rehydration method 13

14. Classification of liposomes based on composition & application Types of liposomes Composition & application Conventional liposomes (CL) Natural or negatively charges phospholipids & cholesterol Fusogenic liposomes (RSVE ) Reconstituted sandal virus envelopes PH sensitive liposomes Phospholipids such as PE or DOPE with either CHEMS or OA Cationic liposomes Cationic lipids with DOPE Long circulatory (stealth) liposomes (LCL) Neutral high Tc , cholesterol & 5-10% of PEG-DSPE or GMI Immunoliposomes CL or LCL with attached monoclonal antibody or recognition sequence 14

15. Method of liposomes preparation Passive Loading Techniques 1. Mechanical dispersion methods Active loading technique 3. Detergent removal methods 2. Solvent dispersion methods Lipid film hydration by hand shaking, non hand shaking or freeze drying Micro emulsification Sonication French pressure cell Membrane extrusion Dried reconstituted vesicles Freeze thawed liposomes Ethanol injection Ether injection Double emulsion vesicles Reverse phase evaporation vesicles Stable plurilamellar vesicles Dialysis Column chromatography Dilution Reconstituted Sendai virus enveloped vesicles 15

16. Materials used in preparation of liposomes 16

17. Natural phospholipids Glycerol containing phospholipids are most commonly used 2. Synthetic phospholipids 3. Sphingolipids Derived from aliphatic aminoalcohol sphingosine which is backbone Most abundant is sphingomylin which is similar to phospholipids, they are zwitter ions at pH & readily form bilayer structure in aqueous media Saturated fatty acid Unsaturated fatty acid Palmitic acid Palmitoleic acid Stearic acid Lionliec acid Synthetic saturated Synthetic unsaturated Dipalmytoyl phosphatidyl choline Dioleoyl phosphatidyl choline Dipalmitoyl phosphatidyl serine Dioleoyl phosphatidyl glycerol 17

18. 4. Glycosphingolipids Gangliosides , a second class of sphingolipid – grey material – brain – human being – used in liposome preparation – layer of surface charged groups 5. Steroids Cholestrol & its derivatives used & its inclusion in liposomal membranes has three recognised effects increasing fluidity or viscosity reducing the permeability of the membrane to water soluble molecules stabilizing the membrane in presence of plasma hence used in IV administration liposomes without cholesterol are known to interact with plasma protein such as albumin transferring & macro globulins. These proteins tend to extract build phospholipid from liposomes thereby depleting the outer monolayer of the vesicles leading to physical instability 18

19. 6. Polymeric materials Synthetic phospholipids with diacetylenic groups in the hydrocarbon undergo polymerization when exposed to UV light. Liposomes are formed using this material & resultant vesicles are exposed to UV light leading to the formulation of permeability barriers to drugs entrapped in their aqueous compartments 7. Charge inducing substances To impart surface charge & their by helps in preventing aggregation Diacyglycerol , starylane & diacetylphosphate can be either a negative or a positive surface charge to these structures. 19

20. GENERAL METHOD OF PREPARATION Liposomes are mainly manufactured using various procedures in which the water soluble (hydrophilic) materials are entrapped by using aqueous solution of these materials as hydrating fluid or by the addition of drug/ drug solution at some stage during manufacture of liposomes. The lipid soluble (lipophilic) materials are solubilized in the organic solution of the constitutive lipid and then evaporated to a dry drug containing lipid film followed by its hydration. These methods involve loading of the entrapped agents before or during the manufacturing procedure (Passive loading). However, certain type of compounds with ionizable groups, and those which display both lipid and water solubility, can be introduced into the liposomes after the formation of intact vesicles (remote loading). 20

21. dissolve in organic solvent Drying Dispersion (hydration) cholestrol lecithin charge Solution in organic solvent Thin film Liposome suspension 21

22. MECHANICAL DISPERSION METHODS Preparation of liposomes by lipid film hydration When preparing liposomes with mixed lipid composition, the lipids must be dissolved and mixed in organic solvent to assure a homogeneous mixture of lipids. Usually this process is carried out using chloroform or chloroform: methanol mixture. The purpose is to obtain a clear lipid solution for complete mixing of lipids. Typically lipid solutions are prepared at 10–20 mg lipid/ml of organic solvent, although higher concentrations may be used if the lipid solubility and mixing are acceptable. Once the lipids are thoroughly mixed in the organic solvent, the solvent is removed to yield a lipid film. For small volumes of organic solvent (<1ml), the solvent is evaporated by using dry nitrogen or argon stream in a fume hood. For larger volumes, the organic solvent should be removed by rotary evaporation yielding a thin lipid film on the sides of round bottom flask. The lipid film is thoroughly dried to remove residual organic solvent by placing the vial or flask on a vacuum pump overnight. 22

23. If the use of chloroform is objectionable, an alternative is to dissolve the lipids in tertiary butanol or cyclohexane. The lipid solution is transferred to containers and frozen by placing the containers on a block of dry ice or swirling the container in a dry ice-acetone or alcohol (ethanol or methanol) bath. Care should be taken when using the bath procedure that, the container can withstand sudden temperature changes without cracking. After complete freezing, the frozen lipid cake is placed on a vacuum pump and lyophilized until dry (1–3 days depending on volume). The thickness of the lipid cake should not be more than the diameter of the container being used for lyophilization. Dry lipid films or cake can be removed from the vacuum pump; the container should be closed tightly, tapped and stored frozen until ready to hydrate. 23

24. Hand shaking method Lipids mixture + solvent (phospholipids & charge components ) + ( chloroform: methanol, 2:1V/V ) in 250ml RBF The flask is attached to rotary evaporator & rotated at 60 RPM isolated with vacuum Evaporate the organic solvent at 30 above transition temperature of lipid Till residue 1 st appears + nitrogen Pressure is increased gradually until equilibrium flask is removed from evaporator Remove residual solvents   24

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26. Hydration of lipid Release vacuum flush with nitrogen + 5ml saline phosphate buffer +drug solution Flask is attached to evaporator rotated at room temp & pressure at speed 60rpm flush with nitrogen Flask is rotated at 30 - lipid Milky white suspension ( stand for 2hrs ) for swelling process MLV’S formed   26

27. The amount of the aqueous buffer ( or drug solution ) entrapped within the internal compartments of the MLV depends on the time allowed for hydration condition of agitation thickness of the film 27

28. Micro emulsification “micro-fluidizer” is used to prepare small MLV’S from concentrated lipid dispersion Lipids introduced as large MLV’S or slurry of unhydrated lipid in organic solvent Micro fluidizer pumps fluid at very high pressure (10000 psi, 600-700 bar) through 5µm orifice Then it is forced along defined micro channels, which direct two streams of fluid to collide together at 90° at a very high velocity, there by affecting an efficient transfer of energy The fluid collected of be recycled through the pump & interaction chamber until vesicles of the spherical dimensions are obtained After a single pass, the size of vesicles is reduced to a size of 0.1 & 0.2µm in diameter 28

29. Micro Emulsification 29

30. Sonication SUV’S are produced from MLV’S by exposing to ultrasonic irradiation Achieved by two methods Probe sonicator Bath sonicator the tip of the sonicator is directly engrossed into the liposome dispersion The energy input is very high Disadv : 1. tips release titanium particles into dispersion which is removed by centrifugation. 2. The coupling at the tip results in hotness hence vessel must be engrossed into a water/ice bath.3. Lipid degradation Lipid dispersion in a cylinder is placed into a bath sonicator Adv : 1. Controlling the temperature is easy. 2. no lipid degradation 3. no release of titanium particles More suitable for large volume of diluted lipids 30

31. MLV dispersion in a test tube placed in a bath sonicator Sonicate for 5-10mins above the transition temperature of the lipid hazy transparent solution Filter & centrifuge at 10000rpm for 30mins at 20 Decant the top clear layer to get sonicated unilamellar vesicles (SUV’S)   31

32. French press or high pressure exclusion French pressure cell is invented by ‘Charles Stacy French’ This technique yields Uni or Oligolamellar liposomes Mainly used for reduction of particles size by high shear forces Dispersion of MLV’S can be converted to SUV’S by passing through a small orifice under high pressure MLV dispersion is placed in the French press & extruded at about 20000 psi at 40 Adv :- more stable liposomes Disadv :- this high shear homogenizers need over 10ml of liposome dispersion to operate properly, this minimum threshold quantity excludes their use in high cost small scale lab operations   32

33. Membrane extrusion In this technique, the vesicle contents are exchanged with the dispersion medium during breaking & resealing of phospholipid bilayers as they pass through the polycarbamate membrane. In this method, the size of liposomes is reduced by gently passing them through membrane filter of defined pore size Two types of membrane used are Tortuous ( zigzag ) Nucleation trach ( vertically parallel ) Achieved at low pressure ( >100 psi ) as compared to French pressure cell Used to process LUV’S & MLV’S 33

34. Dried reconstituted vesicles SUV in aqueous phase + solute Freeze drying Rehydration, film stacks dispersed in aqueous phase Solute in uni or oligo lamellar vesicles 34

35. Freeze thawed liposomes done to increase size of liposomes SUV dispersion Freeze drying Thawing by standing at room temperature for 15 mins Sonication cycle (15-30 sec ) process ruptures & refuses SUV’S Solute equilibrates Liposomes themselves fuse & increase markedly in size 35

36. Dried reconstituted vesicles & Freeze thawed liposomes 36

37. Solvent dispersion method In this method lipid is first dissolved in an organic solution, which is then brought into contact with an aqueous phase containing materials to be entrapped within the liposomes Ethanol injection method Lipid + ethanol injected in aqueous phase precipitating lipids rapid injection SUV’S formed ( as small as 2mm ) Disadv :- slower injection results in larger vesicles. Low solubility in ethanol limiting liposomal concentration Removal of ethanol from lipoidal bilayer is difficult A low encapsulation efficiency 37

38. Ether injection Solution of lipid + diethylether using syringe type infusion by pump at 55-65 Injected slowly in aqueous phase encapsulation Evaporation at high temperature or reduced pressure - organic solvents Single layer vesicles formed ( 50-200nm )   38

39. Ether and Ethanol Injection 39

40. Double emulsion vesicles Also known for water organic phase Organic soln + lipid + aqueous phase Emulsion (W/O) Hot aqueous soln of buffer Multi component vesicle W/O/W ( double emulsion) -solvent by filtering / centrifuge LUV’S 40

41. Double Emulsion Method 41

42. Reverse phase evaporation method Aqueous material + phospholipid solvent mixture sonicated Emulsion -water Lipid paste formed (semisolid gel ) solvent evaporation Liposome suspension vigorous shaking LUV’S or MLV’S 42

43. Adv :- lipid monolayer which enclosed the collapse vesicles is contributed to adjacent intact vesicle to form the outer leaflet of bilayer of LUV Increased ratio of aqueous space to lipid & can encapsulate a higher percentage of initial aqueous phase Disadv :- non encapsulation of macromolecules such as proteins which get Denaturated by organic solvents 43

44. Stable plurilamellar vesicles It involves preparation of water in organic phase dispersion with an excess of lipid followed by drying under continued bath sonication with stream of nitrogen The internal SPLV are different from that of MLV-REVs In that they lack a large aqueous core Internal environment is different 44

45. Detergent removal method Detergents associated with the phospholipid molecules and serve to screen the hydrophobic portion of a molecule from water The structures formed as a result of the association is called micelles As the detergent is removed the micelles become richer in phospholipids & finally coalesce to form single bilayer vesicles Three stage model of interaction for detergents with lipid bilayers :- Stage 1 – at low concentration detergents equilibrates between vesicular lipid & water phase Stage 2 – after reaching a critical detergent concentration, membrane structure tends to unstable & transforms gradually in to micelles Stage 3 – all lipids exists in mixed micelle form 45

46. Adv :- First choice for incorporating membrane (lipoproteins into liposomes) Liposome dispersions with narrow particle size distributions ( Can be obtained Technique can also be used for the preparation of immunoliposomes (for lung endothelia ) Disadv :- Low encapsulation efficiency for hydrophilic non bilayer interacting low molecular weight compounds It is difficult to remove last traces of detergents once liposomes is formed High cost & high quality   46

47. Three methods are applied for removal of detergents Dialysis Removal of detergent from mixed micelles by lowering the concentration of detergent in bulk aqueous phase Eg : sodium cholate , octylglucoside Simplest procedure Adv :- Requires no expensive equipment's No /less complications Effective in removing nearly all free drug with a sufficient number of changes in dialyzing medium 47

48. Column chromatography Removal of detergent is achieved by passing the dispersion over a sephedex G-25 column pre-saturated with constitutive lipids & pre-equilibrated with hydrating buffer Ex :- deoxy cholate 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 48

49. Active loading Proliposomes Sorbitol / Nacl ( increase SA of lipid film ) + 5ml of lipid solution evaporation Add lipid solution dry Freeze dying using lyophilizer stand overnight at room temp Flushed with nitrogen drying MLV’S 49

50. Freeze drying Lipid + solvent (tertiary butanol) Freeze drying Add aqueous phase / saline containing drug Rapid mixing above transition temp MLV’S 50

51. NON shaking vesicles Lipid + solvent flow of nitrogen Evaporate at room temp for drying Add water saturated nitrogen until opacity disappears Add drug +10-20ml sucrose soln to swell flush again with N2 Stand for 2hrs at 37 dnd for 2hrs swirl to yield milky dispersion Centrifuge at 12000rpm for 10mins at RT MLV on surface is removed Remaining fluid add iso-osmolar glucose soln Centrifuge LUV   51

52. It is simple hydration of a lipid with an aqueous solution of drug Formation of liposomes passively entraps dissolved drugs in the intracellular spaces especially encapsulating a small volume Useful for water soluble drugs Drug dissolved along with phospholipid in a suitable organic solvent Either dried first or added directly to the aqueous phase & residual solvent is removed under vaccum Acyl group of phospholipid provides a solubilizing environment for drug Used for drugs such as weak acids which exist in both charged/uncharged form depending on the pH Such drug added to aqueous phase in the uncharged state to permeate into liposomes through their lipid bilayers Incorporation of drugs into liposomes 52

53. Physical characterization Parameter Characterixation method Vesicle shape and surface morphology Transmission electron microscopy Mean vesicle size and size distribution Dynamic light scattering , zetasizer, photon correlation spectroscopy, laser light scattering, gel permeation & gel exclusion Surface charge Free flow electrophoresis Electrical surface potential & surface charge Zeta potential measurements & pH sensitive probes Percent of free drug / percent capture Minicolumn centrifugation, ion exchange chromatography radiolabelling Drug release Diffusion cell dialysis lamellarity X-ray scattering, freeze fracture electron microscopy Phase behaviour Freeze fracture electron microscopy DSC 53

54. Chemical characterization parameter Characterization method Phospholipid concentration Barlett assay, stewart assay, HPLC Cholesterol concentration Cholesterol oxidase assay HPLC Phospholipid peroxidation Uv absorbance iodometric & GLC Phospholipid hydrolysis cholesterol auto oxidation , anti oxidation degradation HPLC & TLC osmolality Osmometer pH pH meter 54

55. Biological characterization Parameter Characterization method sterility Aerobic & anaerobic culture pyrogenicity Limulus amebocyte lysate (LAL) test Animal toxicity Monitoring survival rates, histology & pathology 55

56. Evaluation of liposomes Physical properties Chemical properties 1.Size & distribution 2.Surface charge 3.Percent captive 4.Entrapped volume 5.Phase behavior of liposomes 6.Drug release 1Quantitative detection of phospholipids 2.cholestrol analysis 3.Phospholipid hydrolysis &oxidation 56

57. Particle size Both particle size & particle size distribution is determined by following methods Laser light scattering Transmission electron microscopy Gel permeation 57

58. Surface charge The positive, negative or neutral charge on the surface charge of the liposomes is due to the composition of the head groups The surface charge of liposomes governs the kinetic & extent of distribution invivo , as well as interaction with the target cells The method involved in measurement of surface charge is based on free flow electrophoresis of MLVs It utilises a cellulose acetate plate dipped in sodium borate buffer of pH 8.8 About 5N moles of lipid samples are applied on the plate, which is then subjected to electrophoresis for 30mins The liposomes get bifurcated depending upon surface charge This technique can be used for determining the heterogeneity of charges in the liposome suspension as well as to detect any impurities such as fatty acids 58

59. Percent drug encapsulated Quantity of drug entrapped in the liposomes helps to estimate the behaviour of the drug in biological system Liposomes are mixture of encapsulated & uncapsulated drug fractions The % of drug encapsulation is done by first separating the free drug fraction from encapsulated drug fraction The encapsulated fraction is then made to leak off the liposome into aqueous solution using suitable detergents The method used to separate the free drug from the sample R : a) Mini column centrifugation method b) Protamine aggregated method 59

60. Trapped volume Usually determined experimentally by dispersing lipid in an aqueous medium containing non-permeable radioactive solute such as or inulin The trapped volume of lipid preparations is usually expressed as the trapped volume per lipid & can vary from 0.5µL for some MLV & SUV systems to as much as 30µL for certain LUV system Phase behavior At transition temperature liposomes undergo reversible phase transition The transition temperature is the indication of stability permeability & also indicates the region of drug entrapment Done by DSC   60

61. Drug release rate The rate of drug release from the liposomes can be determined by invivo assays which helps to predict the pharmacokinetics & bioavailability of the drug. However invivo studies are found to be more complete. Liposome encapsulating the tracer INSULIN are employed for the study. This Insulin is preferred, as it is released only in the ECF & undergoes rapid renal excretion of the phase tracer coupled to the degradation rate constant 0 the tracer released from the liposomes   61

62. Determination of phospholipids Determined by two methods Bartlett assay Steward assay This method of determining the phospholipid is very sensitive & may produce erroneous results in the presence of even trace amounts of inorganic phosphates. Therefore, borosilicate glass tubes & double distilled water is used . Initially the phosphorous present in the lipid bilayer of the sample is hydrolysed to inorganic phosphate. The ammonium molybdate is added to convert inorganic phosphate to phosphomolybdic acid ( PMA ). The sample is then treated with amminophthyl sulphonic acid to quantitatatively reduced the PMA to a blue – colored compound. The intensity of the blue color produced can be measured by spectrophotometric means & the value is plotted on the standard curve to obtain the content of phospholipids This assay overcomes the dreawbakcs of Bartlett assay, but cannot be used to mixture of unknown phospholipids. A standard curve is prepared bytreating known concentration of phospholipids in chloroform with 0.1m solution of ammonium ferrothiocyonate reagent The sample are also treated with the same reagent & the optical density is determined at 485nm The absorbance of the sample can be plotted on th standard curve to obtain the concentration of phospholipids 62

63. Cholestrol analysis Qualitative analysis Performed using a capillary column filled with fused silica B) Quantitative analysis The sample is reacted with reagent (containing ferric perchlorate, ethyl acetate & sulphuric acid ) and the absorbance of purple coloured complex is measured at 610nm. Lamellarity The lamellarity of liposomes made from different lipids or preparation procedure varies widely. The average number of bilayer present in the liposome can be found by Freeze-fracture electron microscopy & 𝑃31- NMR Electron microscopy X-ray scattering 63

64. Stability of liposomes Liposomes are basically stable structures since they represent the favourable thermodynamic state of phospholipid in water. Liposomes stablity is a complex issue. One can distinguish between physical, chemical & biological stability of liposomes Physical stability It indicates mostly the constancy of size distribution & the ratio of lipid to active agent of liposomes, which indicates the stability of encapsulation or binding the active agents. At high charges of cationic liposomes, these vesicles can be stabled at 4 for years if properly sterilized.   64

65. Chemical stability It involves oxidation & hydrolysis of lipids. Hydrolysis detaches / hydrophobic bonds, especially if they are attached via an ester bond, ether bonds are rather stable. Oxidation is more likely due to presence of unsaturated chains. The presence of antioxidants can reduce the degradation. Chemical degradation of these liposomes can be prevented by the following Freshly purified & prepare solvents must be used in the preparation Avoid procedures which involve high temperature Manufacturing of liposomes in absence of oxygen Storing liposomal preparations in an inert atmosphere Including antioxidants as a component of lipid membranes 65

66. Biological stability It is rather limited A simple test of plasma incubation is often reveals extensive leakage & aggregation. This is especially true for cationic liposomes. Cationic liposomes because of negatively charged surfaces & colloidal particles in biological systems are least stable invivo . Typically these liposomes are absorbed upon IV administration in seconds 66

67. Application of liposomes used in treatment of 1.systemic fungal infections Ex liposomal amphotericin 2. malignant tumors in leukaemia Ex :- doxorubicin 3. enzyme deficiency diseases 4. Leishmaniasis 5. Rheumatoid arthritis liposomes are used as carriers for both water & lipid soluble agents. Substances entrapped in liposomes include enzymes, glycolipids, immunoglobulins , monoclonal antibodies, antigens, antifungals, antigens, vaccines & chromosomes 67

68. liposomal insulin is expensive than that available, but that trauma of repeated injection can be avoided. metal toxicity Liposomal EDTA is used in heavy metal poisoning. The encapsulated drug for more effective then the free drug in enhancing the removal of plutonium from the tissues & in promoting its excretion liposomal desferroxamine , an iron chelator is used in the treatment of chronic hemosiderosis high antibody response was observed when liposomes were used as imunoadjuvants along with antigens, diphtheria toxoid, tetanus toxoid, cholera toxin, herps simplex virus. 68

69. Liposomes preparation available in India Amphoject Amphi gel Sporotar Aditracin Adrosal Cardia Dobicin Doxocare inj Doxiglan Doxilyd Peg- doxorub Doxolem Oncodox – cipla Pelodox Oncordia – sun pharma lipodox inj -sun pharma 69

70. THANKYOU 70

71. References 1. S.P.Vyas & R.K.Khar , Controlled Drug Delivery – concepts and advances, Vallabh Prakashan , New Delhi, First edition 2002. 2. N.K.Jain , Controlled and Novel Drug Delivery, CBS Publishers & Distributors, New Delhi, First edition 1997 (reprint in 2011). 71