liposomes and niosomes

1. Liposomes & niosomes DEPARTMENT OF PHARMACEUTICS S.R.R COLLEGE OF PHARMACEUTICAL SCIENCES ELAKATHURTHY(M),VALBHAPUR(V),KARIMNAGAR Presented by; AITHA SWETHA M.PHARMACY 1 ST YEAR 2 ND SEM

2. Contents:

3. LIPOSOMES 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. Liposomes were first produced in England in 1961 by Alec D. Bangham . The size of a liposome ranges from some 20 nm up to several micrometers 1

4. Structure Of Liposome Hydrophillic head Hydrophobic tail The lipid moecules are usually phospholipids- amphipathic moieties with a hydrophilic head group and two hydrophobic tails. Liposome = Phospholipid + cholesterol 2

5. 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 . Flexibility to couple with site-specific  ligands  to achieve  active targeting. Advantages of liposomes : 3

6. Disadvantages of liposomes : Production cost is high. Leakage and fusion of encapsulated drug / molecules. Sometimes phospholipid undergoes oxidation and hydrolysis like reaction. Short half-life. Low solubility . 4

7. H 2 O Layer Polar Lipids ( Phospholipid ) Water Soluble ingredients (Drugs, Nutrients & vitamins ) Lipid Soluble ingredients ( Drugs,Nutrients & vitamins ) Cross-section of liposomes : 5

8. components of liposomes : The structural components of liposomes include: A. Phospholipids B. cholesterol 6

9. A. General representation of phospholipids: 7

10. Phospholipids Phosphatidylcholine - natural Amphipathic molecule Hydrophilic polar head- Phosphoric acid bound to water soluble molecule. Glyceryl bridge Hydrophobic tail- 2 fatty acid chain containing 10-24 carbon atoms and 0-6 double bond in each chain. The amphipathic molecule self organise in ordered supramolecular structure when confronted (meet face to face) with solvent. 8

11. The most common natural phospholipid is the phospatidylcholine (PC ). Naturally occurring phospholipids used are : PC: Phosphatidylcholine . PE: Phosphatidylethanolamine . PS: Phosphatidylserine Synthetic phospholipids used are: DOPC: Dioleoyl phosphatidylcholine DSPC: Disteroyl phosphatidylcholine DOPE: Dioleoyl phosphatidylethanolamine DSPE: Distearoyl phosphatidylethanolamine Polar Head Groups Three carbon glycerol 9

12. Molecular geometry on structure of amphiphillic aggregates: 10

13. Molecules of PC are not soluble in water. In aqueous media they align themselves closely in planar  bilayer  sheets  in order to minimize the  unfavorable action between the bulk aqueous  phase and the long hydrocarbon fatty chain.  Such unfavorable interactions are completely eliminated when the sheets fold on themselves to form closed sealed vesicles 11

14. At various temperatures, phospholipid membranes can exist in different phases. The transition from one phase to another can be detected by technique like micro calorimetry . What exactly happens during phase transition? Tightly ordered At elevated temperature   liquid crystal phase gel state ( lipid membrane) (movement is higher) This is due to the fatty acid chain adopting a new conformation other than all trans straight chain configuration, such as gauche configuration state( phenomenon- chain tilt ) PHASE TRANSITION TEMPERATURE 12

15. B. Cholesterol: Cholesterol stabilizes the Membrane Steroid lipid Interdigitates between phospholipids. i.e. below Tc , it makes membrane less ordered & above Tc more ordered. Being an amphipathic molecule, cholesterol inserts into the membrane with its hydroxyl group of cholesterol oriented towards the aqueous surface and aliphatic chain aligned parallel to the acyl chains in the center of the bilayer . 13

16. Role of cholesterol in bilayer formation: Cholesterol act as fluidity buffer After intercalation with phospholipid molecules alter the freedom of motion of carbon molecules in the acyl Chain Restricts the transformations of trans to gauche Conformations. Incorporated into phospholipid membrane upto 1:1 or 2:1 of cholesterol to PC. 14

17. Mechanism of liposome formation: 15

18. Classification of liposome : Classification of liposome Structural parameters Method of preparation Composition and application 16

19. Types of vesicles based on lamella Lamella : 17

20. Based on structural  parameters MLV Multilamellar Large vesicles (>0.5 um) OLV oligolamellar vesicles (>0.1-1.0 um) UV   Unilamellar Vesicles  MVV Multivesicularvesicles (> 1.0 UM) MUV GUV >1um SUV 20-100nm LUV >100nm A. Structural parameters: 18

21. B. Based on method of preparation: 19

22. Based on composition and application: 20

23. Loading of the entrapped agents before/ during the manufacture procedure. Certain types of compounds with ionizable groups & those with both lipid & water solubility can be Introduced into liposomes after the formation of intact vesicles. MethodS of Liposome Preparation 21

24. LIPID FILM HYDRATION BY HAND SHAKING,FREEZE DRYING OR NON HAND SHAKING MICRO EMULSIFICATION SONICATION FRENCH PRESSURE CELL MEMBRANE EXTRUSON DRIED RECONSTITUTED VESICLES ETHANOL INJECTION ETHER INJECTION DOUBLE EMULSION REVERSE PHASE VAPOURATION VESICLES STABLE PLURI LAMELLER VESICLES DETERGENT REMOVAL FORM MIXED MICELLES BY DIALYSIS CHROMATIGRALPY DIFFUSION VESICLES LIKE …. RECONSTITUTED & SANDAI VIRUS ENVELO PE Methods of liposome preparation Passive loading techniques Active loading techniques Mechanical dispersion  methods Solvent dispersion  methods Detergent removal technique 22

25. General Method Of Liposome Preparation: 23

26. 1. Mechanical dispersion method: 24

27.   There are four basic methods of physical/mechanical dispersion : Hand shaken method. Non shaking method. Pro – liposomes . Freeze drying .   25

28. Lipid film hydration by hand shaking method: L ipids form stacks of film from organic solution (FE/HS) Then film is treated with aqueous medium Upon hydration lipids swell and peel out from RB flask vesiculate to form Multi lamellar vesicles(MLVs) 26

29. Pro- liposomes : To increase the surface area of dried lipid film & to facilitate instantaneous hydration. lipid Dried over lipid Finely divided particulate support like powdered NACL/ sorbital Pro - liposomes Pro- liposomes water Dispersion of MLV’S This Method overcome the stability problem. 27

30. Micro Emulsification liposomes (MEL) Sonicated unilamellar vesicles (SUVs) French Pressure Cell Liposomes . Membrane extrusion Liposomes Dried reconstituted vesicles(DRVs) Freeze thaw sonification (FTS) pH induced vesiculation Cochleate method. Processing of the lipids hydrated by physical means or the mechanical treatments of MLVs : 28

31. Sonicated unilamellar vesicles : The exposure of MLVs to ultrasonic irradation for producing small vesicles. Probe sonicator Bath sonicator Used for dispersions large volume require high of dilute lipids energy in small volumes Sonication MLVs hazy transparent 5-10 min solution centrifugation 30 min clear SUV Dispersion. 29

32. Micro emulsification liposomes : Micro fluidizer 30

33. French pressure cell liposomes : Extrusion of preformed large liposomes in french press under very high pressure . uni or oligo lamellar liposomes of intermediate size (30-80nm ) . Advantages Less leakage and more stable liposomes are formed compared to sonicated forms 31

34. Vesicles prepared by extrusion technique : The size of liposomes is reduced by gently passing them through polycarbonate membrane filter of defined pore size at lower pressure Used for preparation of LUVs and MLVs 32

35. Dried reconstituted vesicles& freeze thaw sonication method 33

36. pH induced vesiculation : The transient change in pH brings about an increase in surface charge of the lipid bilayer which induces spontaneous vesiculation . MLVs LUVs 34

37. Cochleate method: Cochleates 35

38. Note:- Organic solvent miscible with aqueous phase Solvent dispersion methods: 36

39. Solvent dispersion methods: ETHANOL INJECTION/ETHER INJECTION: 37

40. De-Emulsification method: Generally the liposome is made up in 2 steps: 1 st the inner leaflet of the bilayer . Then the outer half. Methods to prepare the droplets: ~Double emulsion vesicles ~Reverse phase evaporation vesicles ~ Sonication methods 38

41. Reverse phase evapouration method: 39

42. Note:- Liposome size and shape depend on chemical nature of detergent, concentration and other lipid involved Below CMC, detergent molecules exist in free soln. As the concentration is increased, micelles are formed .  DETERGENT SOLUBILISATIOIN methods Methods to remove detergents: Dialysis Column chromatography. 40

43. Active/remote loading technique: The lipid bilayer membrane is impermeable to ions & hydrophilic molecules. But, Permeation of hydrophobic molecules can be controlled by concentration gradients. Some weak acids or bases can be transported due to various transmembrane gradients Electrical gradients. Ionic(pH) gradients. Chemical potential gradients. Weak amphipathic bases accumulate in aq phase of lipid vesicles in response to difference in pH b/w Inside & outside of liposomes   41

44. pH gradient is created by preparing liposomes with low internal pH. Addtn of base to extraliposomal medium. [Basic compds ( lipophilic (non ionic) at high pH & hydrophilic(ionic) at low pH)] Lipophilic (UNPROTONATED) drug diffuse through the bilayer At low pH side, the molecules are predominantly protonated . Exchange of external medium by gel extrusion chromatorapghy with neutral solution . Weak bases like doxorubicine , adriamycin and vincristine are encapsulated. Solute bearing no charge at neutral pH Liposomes with low internal pH Neutral solute passes easily through bilayer membrane by diffusion Charge aquired by solute inside liposomes makes them unable to exit 42

45. Locus of drugs in liposomes : Hydrophilic (DOXORUBICIN) Low entrapment Leakage Hydrolytic degradation Lipophilic (CYCLOSPORINE) High entrapment Low leakage Chemical stability Ampiphilic (VINBLASTIN) High entrapment Rapid leakage Biphasic insoluble (ALLOPURINOL, 6-MERCAPTOPURINE) Poor loading & entrapment 43

46. Characterization of liposomes : PHYSICAL CHARACTERISATION Vesicles size/shape/morphology Surface -charge/electrical potential Phase behaviour / lamellarity Drug release % capture /free drug CHEMICA L CHARACTERISATION Phospholipids /lipid concentration Drug concentration PH / Osmomolality Antioxidant degradation Phospholipids / cholesterols – peroxidation /oxidation/hydrolysis BIOLOGICAL CHARACTERISATION Sterility Pyrogenisity Animal toxicity Plasma Stability : 44

47. 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) 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 5. Lamellarity Small angle X-ray scattering, 31 P-NMR, Freeze-fracture electron microscopy 6. Phase behavior Freeze-fracture electron microscopy, Differential scanning calorimetery 7. Percent of free drug/ percent capture Minicolumn centrifugation, ion-exchange chromatography, radio labelling 8. Drug release Diffusion cell/ dialysis 1.PHYSICAL CHARACTERIZATION: 45

48. 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 4. Phospholipid hydrolysis, Cholesterol auto-oxidation. HPLC and TLC 5. Osmolarity Osmomete 2. CHEMICAL CHARACTERIZATION: 46

49. 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 3. BIOLOGICAL CHARACTERIZATION: STABILITY OF LIPOSOMES: Stability invitro . ~ Lipid oxidation ~ Lipid peroxidation ~ Long term & accelerated stability Stability after systemic administration. 47

50. Modes of liposomes /cell interaction: 1. Endocytosis 2. Adsorption 3. fusion 4. Lipid transfer 48

51. Encapsulation volume/Trapped volume Volume of aqueous solution entrapped in liposomes per mole of PL (µL/µmol PL) Encapsulation Efficiency Assessed by mini column centrifugation method & protamine aggregation method. protamine aggregation method used for neutral and negetively charged liposomes . Liposome dispersion can be precipitated with protamine solution and subsequent centrifugation at 2000RPM. By analysing the material in super natent & in liposome pellet ( after disrupting liposomal pellet with 0.6 ml of 10% triton x-100 ). The encapsulation efficiency of entrapped material can be estimated. % Encapsulation Drug entrapped in liposomes x 100 Total drug added Encapsulation of drugs in liposomes : 49

52. In gene delivery. As drug delivery carriers. Enzyme replacement therapy. Chelation therapy for treatment of heavy metal poisoning. Liposomes in antiviral/anti microbial therapy. In multi drug resistance. In tumour therapy. In immunology. In cosmetology USES OF LIPOSOMES : 50

53. DNA delivery of Genes by Liposomes Cheaper than viruses No immune response Especially good for in-lung delivery (cystic fibrosis) 100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector 51

54. Lipofection 52

55. Liposomes could serve as tumor specific vehicles (even without special targeting) Liposomes better penetrate into tissues with disrupted endothelial lining 53

56. DRUG ROUTE OF ADMINISTRATION APPLICATION TARGETED DISEASES Amphotericin B Oral delivery Ergosterol membrane Mycotic infection Insulin Oral,ocular,pulmonary And transdermal Decrease glucose level Diabetic mellitus Ketoprofen Ocular delivary Cyclooxygenase enzyme inhibitor Pain muscle condition Pentoxyfyllin Pulmonary delivery phosphodiesterase Asthama Tobramycin Pulmonary delivery Protein synthesis inhibitor Pseudomonas infection,aeroginosa Salbutamol Pulmonary delivery ß 2 -adrenoceptor antagonist Asthama Cytarabin Pulmonary delivery DNA-polymerase inhibition Acute leukameias Benzocaine Transdermal Inhibition of nerve impulse from sensory nerves Ulcer on mucous surface with pain Ketaconazole Transdermal Inhibit ergosterol membrane Candida albicans Levanogesterol Transdermal Rhamnose receptor skin disorder hydroxyzine Transdermal H1-receptor antagonist Urtecaria,allergic skin disease Ibuprofen Oral delivery Chaemoceptor,free ending Rheumatoid arthritis triamcilonone Ocular delivery,Transdermal Inhibition of prostaglandin Anti-inflammatory Therapeutic application of liposomes : 54

57. NAME TRADE NAME COMPANY INDICATION Liposomal amphotericin B Abelcet Enzon Fungal infections Liposomal amphotericin B Ambisome Gilead Sciences Fungal and protozoal infections Liposomal cytarabine Depocyt Pacira (formerly SkyePharma ) Malignant lymphomatous meningitis Liposomal daunorubicin DaunoXome Gilead Sciences HIV-related Kaposi’s sarcoma Liposomal doxorubicin Myocet Zeneus Combination therapy with cyclophosphamide in metastatic breast cancer Liposomal IRIV vaccine Epaxal Berna Biotech Hepatitis A Liposomal IRIV vaccine Inflexal V Berna Biotech Influenza Liposomal morphine DepoDur SkyePharma , Endo Postsurgical analgesia Liposomal verteporfin Visudyne QLT, Novartis Age-related macular degeneration, pathologic myopia, ocular histoplasmosis Liposome-PEG doxorubicin Doxil / Caelyx Ortho Biotech, Schering-Plough HIV-related Kaposi’s sarcoma, metastatic breast cancer, metastatic ovarian cancer Micellular estradiol Estrasorb Novavax Menopausal therapy List of marketed products : 55

58. summary: liposomes  are concentric  bilayered vesicles in which an aqueous volume is entirely enclosed  by a  membraneous lipid  bilayer Liposomes are one of the unique drug delivery system, in controlling and targeting drug delivery. Components of liposomes include phospholipid and cholesterol. Method of preparation of liposomes include active loading technique and passive loading technique. Passive loading techniques include solvent mechanical dispersion, solvent dispersion & detergent solubilisation Characterization of liposomes include physical,chemical and biological. 56

59. NIOSOMES

60. 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. 57

61. Structure of niosomes : Head part ( hydrophillic ) Tail part (hydrophobic) Drug molecules Phospholipids 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. 58

62. 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 : 59

63. Disadvantages of Niosomes : Aggregation Fusion Leaking of entrapped drug Hydrolysis of encapsulated drugs which limiting the shelf life of the dispersion. 60

64. Small Unilamellar Vesicle (SUV) Large Unilamellar Vesicle (LUV) Multilamellar Vesicle (MLV) Typical Size Ranges: SLV: 20-50 nm – MLV:100-1000 nm Classification of niosomes 61

65. 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 : 62

66. Factors Affecting Niosomes Formation Factors affecting niosomes formation Non-ionic surfactant nature Membrane additives Nature of encapsulated drug Surfactants and lipid levels Hydration Temperature alkyl group chain length : C 12 -C 18 Span surfactants with HLB values 4 and 8 Cholesterol: Prevent vesicle aggregation. Dicetyl phosphate: - ve charge surfactant/lipid ratio: 10-30 mM Shud be above the gel to liquid phase transition temperature of the system 63

67. Prediction of vesicle forming ability is not a simply a matter of HLB CPP = v /l c a where v - hydrophobic group volume, l c - critical hydrophobic group length and a - area of the hydrophilic head group CPP between 0.5 and 1 likely to form vesicles. < 0.5 (indicating a large contribution from the hydrophilic head group area) is said to give spherical micelles. >1 (indicating a large contribution from the hydrophobic group volume) should produce inverted micelles. Concept of Critical Packing Parameter 64

68. Sl. No. Liposomes Niosomes 1. Vesicles made up of concentric bilayer of phospholipids Vesicles made up of surfactants with or without incorporation of cholesterol. 2. Size ranges from 10-3000nm Size ranges from 10-100nm 3. Comparatively expensive Inexpensive 4. Special storage condition are required No such special requirement 5. Phospholipids used are unstable Non-ionic surfactants are stable 6. Comparatively more toxic Less toxic Comparisition between liposomes & niosomes : 65

69. Hand Shaking method Reverse phase evaporation technique Ether Injection method Multiple membrane extrusion method Bubble method Sonication From Proniosomes Methods of Niosome preparation: 66

70. Hand shaking method: Surfactant & cholesterol (150µmole) solution is dissloved in 10ml ether in round bottom flask Rotary evaporator Ether is evaporated under vacuum at room temperature hydration Surfactant swells and peeled off into a film like lipids swollen amphiphiles fold to form vesicles. Rotary evaporator 67

71. Reverse phase evaporation technique : Surfactant is dissolved in chloroform ond 0.25 volume of PBS buffer is emulsified to get a W/O emulsion. sonicated chloroform is evaporated under reduced pressure. The lipid or surfactant forms a gel first and hydrates to form vesicles. Free drug ( unentrapped ) is generally removed by dialysis. sonication : Surfactant + cholesterol mixture is dispersed in 2 ml aqueous phase in vial Mixture is sonicated for 3 min at 60°C using titanium probe sonicator Unilamellar niosomes 68

72. Ether injection Method: Surfactant : cholesterol (150µmole) solution is dissloved in ether Slowly injected into preheated 4.0ml aqueous phase maintained at 60 c through a 14 gauge needle Vaporization of ether leads to formation of single layered vesicles. formation of a bilayer sheet, which eventually folds on itself to form sealed unilamellar vesicles. 14 guage needle 69

73. Multiple membrane extrusion Method: Mixture of surfactant, cholesterol and dicetyl phosphate in chloroform is made into thin film by evaporation The film is hydrated with aqueous drug solution and the resultant suspension extruded through polycarbonate membranes 70

74. Bubble method: RBF as bubbling unit with three necks in water bath . Reflux , thermometer and nitrogen supply by three necks Cholesterol+ Surfactant dispersed in buffer pH 7.4 at 70°C Above dispersion is homogenized for 15 sec and then bubbled with nitrogen gas at 70°C to get niosomes It is novel technique for the one step preparation of liposomes and niosomes without the use of organic solvents. 71

75. proniosomes : Bubble Method Formation of niosomes from proniosomes : It is prepared by coating water-soluble carrier such as sorbitol with surfactant. The result of the coating process is a dry formulation. In which each water-soluble particle is covered with a thin film of dry surfactant. This preparation is termed “ Proniosomes ”. 72

76. Separation of unentrapped drug Dialysis Centrifugation Gel filtration Separation of unentrapped drug: Dialyzed in a dialysis tubing against phosphate buffer or normal saline The unentrapped drug is removed by gel filtration of niosomal dispersion through a Sephadex-G-50 column and elution with phosphate buffered saline The niosomal suspension is centrifuged and the supernatant is separated. The pellet is washed and then resuspended to obtain a niosomal suspension free from unentrapped drug. Centrifuser Gel Filtration 73

77. Characterization of Niosomes a) Size, Shape and Morphology Freeze Fracture Electron Microscopy:- Visualize the vesicular structure of surfactant based vesicles. Photon Correlation spectroscopy :- Determine mean diameter of the vesicles. Electron Microscopy :- Morphological studies of vesicles. b) Entrapment efficiency After preparing niosomal dispersion, unentrapped drug is separated by dialysis and the drug remained entrapped in niosomes is determined by complete vesicle disruption using 50% n- propanol or 0.1% Triton X-100 and analysing the resultant solution by appropriate assay method for the drug. c) Vesicle Suface Charge Determined by measurement of electrophoretic mobility and expressed in expressed in terms of zeta potential d) In vitro studies 74

78. Applications of Niosomes 75

79. Marketed product: Lancôme has come out with a variety of anti-ageing products which are based on noisome formulations. L’Oreal is also conducting research on anti-ageing cosmetic products. 76

80. Summary : Niosomes provide incorporating the drug into for a better targeting of the drug at appropriate tissue destination .   They presents a structure similar to liposome and hence they can represent alternative vesicular systems with respect to liposomes Niosomes are thoughts to be better candidates drug delivery as compared to liposomes due to various factors like cost, stability etc. Various type of drug deliveries can be possible using niosomes like targeting, ophthalmic, topical, parenteral etc.   77

81. References: S.P. Vyas And R.K. Khar,targeted & Controlled Drug Delivery, liposomes, 173-279. 2. Mohammad Riaz , Liposomes :Preparation Methods, Pakistan Journal Of Pharmaceutical Sciences , January 1996,Vol.19(1),65-77. 3. Sharma Vijay K1*, Liposomes : Present Prospective and Future Challenges, International Journal Of Current Pharmaceutical Review And Research , oct 2010,vol1, issue 2,6-16 4. Himanshu Anwekar *, Liposome- as drug carriers , International Journal Of Pharmacy & Life Sciences, Vol.2, Issue 7: July: 2011, 945-951 78

82. 5. Madhav Nvs * And Saini A, Niosomes : A Novel Drug Delivery System, International Journal Of Research In Pharmacy And Chemistry , 2011, 1(3),498-511. 6. Lohumi Ashutosh , Rawat Suman , A Novel Drug Delivery System: Niosomes Review, Journal Of Drug Delivery & Therapeutics ; 2012, 2(5), 129-135. 7. Pawar Sd *, Pawar Rg , Niosome : An Unique Drug Delivery System, International journal Of Pharmacy, Biology and Allied Sciences , April, 2012, 1(3): 406-416. 8. Rajesh Z. Mujoriya , Niosomal Drug Delivery System – A Review, International Journal Of Applied Pharmaceutics , Vol 3, Issue 3, 2011,7-10. 79

83. Success in life mostly depends on the power of ‘CONCENTRATION’ --- Swami Vivekananda