LIPOSOMES IN DRUG DELIVERY APPLICATIONS.

LIPOSOMAL DRUG DELIVERY

1. LIPOSOMES PRESENTED BY :- DHARMILA M. PHARM 2 ND SEM. M100400005 1

2. CONTENTS INTRODUCTION ADVANTAGES DISADVANTAGES CLASSIFICATION METHODS OF PREPARATION CHARACTERIZATION APPLICATIONS MARKETED FORMULATIONS 2

3. INTRODUCTION Discovered by Bangham and colleagues in 1965. Concentric bilayer vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayer composed of natural or synthetic phospholipids. Size :– 30 - 10,000 nm. They are formed by hydration of phospholipids. 3

4. A) Naturally occuring phospholipids :- Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine B) Synthetic phospholipids :- DOPC - Dioleoyl phosphatidylcholine DSPC - Distearoylphosphatidylcholine DMPC - dimyristoyl phosphatidylcholine DPPC - Dipalmitoyl phosphatidylcholine DOTAP - Dioleoyl trimethylammonium propane DOPG - Dioleoyl phosphatidylglycerol 4

5. ADVANTAGES Liposomes provide selective passive targeting to tumor tissues (liposomal doxorubicin). Increased efficacy & therapeutic index. Increased stability of the drug via encapsulation. Reduction in toxicity of encapsulated agent. Flexibility to couple with site-specific ligands to achieve active targeting. Liposomes are biocompatible. Completely biodegradable and non-toxic . Flexible and non immunogenic for systemic and non-systemic administrations . Improved pharmacokinetic effects ( reduced elimination, increased circulation life times ). Site avoidance effect. 5

6. DISADVANTAGES High Production cost Leakage and fusion of encapsulated drug / molecules Oxidation and hydrolysis of phospholipids Short half-life Low solubility Less stable 6

7. CLASSIFICATION A) Based on composition & application :- 7 LIPOSOME TYPE MAJOR APPLICATION Conventional liposomes Macrophage targeting, Local depot, Vaccination Long-circulating (stealth) liposomes Selective targeting to pathological areas Circulating microreservoir Immuno liposomes Specific targeting Cationic liposomes Gene delivery

8. B) Based on structural parameters :- 1. MLV ( multilamellar large vesicles ) >0.5 μm 2. OLV ( oligolamellar vesicles) 0.1–1 μ m 3. UV ( unilamellar vesicles) a) SUV (small) 20–40 nm b) MUV (medium 40-80 nm c) LUV (large) >100 nm d) GUV (giant) >1 μ m 4. MVV ( multivesicular vesicles) >1 μm 5. Double liposomes >1000 nm 8

9. C) Based on method of liposome preparation :- REV (single or oligolamellar vesicles) made by reverse-phase evaporation method MLV-REV ( multilamellar vesicles) made by the reverse-phase evaporation method SPLV (stable plurilamellar vesicles) FATMLV (frozen and thawed MLV) VET (vesicles prepared by extrusion methods) DRV (dehydration-rehydration vesicles) 9

10. 10

11. PASSIVE LOADING TECHNIQUES 11

12. 12 PASSIVE LOADING TECHNIQUES

13. MECHANICAL DISPERSION METHOD Lipid film hydration by hand shaking, non-hand shaking or freeze drying Micro-emulsification Sonication French pressure cell Membrane extrusion Dried reconstituted vesicles Freeze-thaw sonication method 13

14. SOLVENT DISPERSION METHOD Ethanol injection Ether injection Double emulsion vesicles Reverse phase evaporation vesicles Stable plurilamellar vesicles 14

15. DETERGENT REMOVAL METHOD Detergent removal from mixed micelles by:- Dialysis Column chromatography Detergent adsorption using bio-beads 15

16. Film hydration by hand shaking(MLVs), non-hand shaking(ULVs) methods 16 Organic solution of lipids Film formation using rotary evaporation under reduced pressure Dispersed the films in aqueous medium Lipids swell & peel off from wall of RBF & vesiculate Liposomes are formed with the efficacy of 30% Mechanical energy (for swelling) can be provided by:- Hand shaking (MLVs) Stream of water saturated nitrogen(LUVs)

17. 17 Freeze drying

18. Microemulsification Method Micro fluidizer prepare small MLVs from concentrated lipid suspension, pumps fluid at very high pressure (10000psi; 600-700 bar) through 5 µm screen Fluid forced through micro channels into two streams, which collide at right angle at very high velocity 18

19. Sonicated unilamellar Vesicles(SUVs) MLVs size reduced to small vesicles by ultrasonic irradiation Instruments used are Bath and probe ultrasonic disintegrators MLVs are sonicated for 5-10 mins and then centrifuged 19

20. French Pressure Cell It causes extrusion of preformed Large liposomes under very high pressure Liposomes are more stable, less structural defects and less content leakage; overcoming defects of sonication Yield uni -lamellar or oligo -lamellar Liposomes of intermediate size(30-80nm) 20

21. Membrane Extrusion Technique Liposome size reduced by passing through membrane filters (defined size) at lower pressure(100psig) Contents exchanged with dispersion medium during breaking and resealing, drug in medium for high entrapment Used to prepare SUVs, MUVs and LUVs 21

22. Dried-reconstituted Vesicles(DRVs) Empty SUVs are freeze dried for dispersion and then rehydrated by Aqueous Fluid containing Drug Organized membrane structure is formed which on rehydration form high capture efficiency liposomes 22

23. Freeze Thaw Sonication It is an extension of DRV method SUVs are frozen and thaw at room temperature for 15 mins and sonicated pH induced vesiculation pH change induce Transformation of MLVs to LUVs by increasing surface charge density of lipid bilayer MLVs exposed to pH 11.0(1 M NaOH,2mins);pH reduced by addition of 0.1 M HCl to pH 7.5 23

24. Calcium induced fusion SUVs made from negatively charged lipids on addition of Ca ++ fuse into cylindrical rolls Subsequent removal of Ca ++ by EDTA or Iron exchange or precipitation; form LUVs 24

25. Solvent Dispersion Methods Ethanol injection :- Lipid ethanol solution injected rapidly through a fine needle into excess of saline or aq. Medium Rate of injection causes mixing to disperse phospholipids evenly and yield SUVs(~25nm) Limitations:- Limited solubility of lipids in ethanol, volume of ethanol injected and difficult ethanol removal 25

26. b) Ether injection :- Immiscible organic phase injected very slowly into aq. Phase; at temperature to vaporize organic solvent Suitable for sensitive lipids but technique is time consuming and require careful control of needle For temperature sensitive fluorinated hydrocarbons 26

27. c) Rapid Solvent Exchange :- Lipid mixture quickly transferred between Pure solvent and aq. Buffer forming homogenous dispersion by precipitation of lipids in aq. Buffer Bulk solvent vaporizes before coming in contact with aqueous phase Preparation time is less i.e. <1min and have high entrapment volume 27

28. d) Reverse Phase Evaporation Vesicles :- It involve removal of solvent from emulsion to form a semisolid gel in a rotary evaporator Followed by vigorous mechanical shaking in vortex mixer to convert gel into homogenous fluid Solvent removed by dialysis to give unilamellar vesicles(0.5µm) with 50% encapsulation e) Stable Plurilamellar Vesicles(SPLVs) :- W/O phase with excess of lipid dried under sonication with intermittent nitrogen stream It give high entrapment efficiency 28

29. Detergent Solubilization Detergents are used for intimate contact between phospholipids and aq. Phase Initially detergent equilibrates in lipid and water phase At ‘critical detergent concentration’ unstable membrane form micelles; detergent saturated bilayer formed At still higher concentration mixed micelle (containing detergent and lipids) To remove detergent from bilayer follwing methods are used #Dialysis #Column Chromatography #Adsorption 29

30. ACTIVE LOADING TECHNIQUE 30

31. Ions and large hydrophilic molecules can not cross the bilayer ; transport of neutral and weakly hydrophobic molecules can be achieved by concentration gradient Weak acids and bases are transported by pH gradient and Potential difference Advantages:- High encapsulation efficiency Reduced leakage Flexibility in use of lipids Reduction in safety hazards Weak bases loaded using proton gradient or ammonium sulphate gradient; weak acids loaded using calcium acetate gradient 31

32. CHARACTERIZATION 32

33. Physical Characterization Vesicle shape and lamellarity :- Freeze-Fracture and Freeze-etch Electron Microscopy Can be used to study surface morphology (Topology) In this technique, Fracture plane passes through the vesicles, which are randomly positioned in frozen state. Thus, it may not pass through mid-plane and non-mid plane fracture may result in erroneous readings. Observed distribution profile depends on the distance of vesicle centre from the plane of fracture. Therefore, heterogenous populations require a careful monitoring. Quick freeze and deep etching techniques give much better lamellarity evaluation. After 5-mintues of etching, cross-fractured vesicles are clearly seen and the number of lamellae can readily be determined. 33

34. P 31 Nuclear Magnetic Resonance Analysis It is one of the most accurate and straightforward techniques for determination of lamellarity of liposomes . Monitors the phospholipid phosphorus signal intensity. Adding nonpermeable broadening agent such as Mn to external medium will decrease in intensity of intial P 31 NMR signal by an amount proportional to fraction of lipid exposed to external medium. 50% reduction in signal- Unilamellar , subsequent reductions indicate multilamellar vesicular preparation. 34

35. VESICLE SIZE & SIZE DISTRIBUTION OPTICAL MICROSCOPY Include methods like Bright-field, Phase Contrast Microscopy and Fluorescent Microscope. Useful in evaluating the vesicle size of large vesicles (>1 µm). Vesicular dispersion appropriately diluted are wet mounted on a haemocytometer and photographed with a phase contrast microscope. 35

36. NEGATIVE STAIN TRANSMISSION ELECTRON MICROSCOPY (TEM) This technique visualizes relatively electron transparent liposomes as bright areas against a dark background. Liposomes are embedded in this method in a thin film of electron-dense heavy metal (salt) stain. Negative stains used are ammonium molybdate , phosphotungstic acid or uranyl acetate. TEM facilitates estimation of liposome size range at the lower end of the frequency distribution. Mainly used for larger sizes or heterogenous populations of liposomes . DISADVANTAGE: Liposome morphology may be deformed. 36

37. CRYO-TRANSMISSION ELECTRON MICROSCOPY TECHNIQUE (CRYO-TEM) It is used to elucidate the surface morphology and size of vesicle. The method involves freeze fracturing of samples followed by their visualization using TEM. Thin sample films are prepared under controlled temperature (25 o C) and humidity conditions. Films are thereafter vitrified by quick freezing in liquid ethane and transferred to TEM analysis. 37

38. FREEZE-FRACTURE ELECTRON MICROSCOPY Mainly used to assess the surface features and lamellarity and to calculate true vesicle diameter. SURFACE CHARGE Two methods are used to assess the charge, Free-flowing electrophoresis Zeta potential measurement From the mobility of liposomal dispersion in a suitable buffer , the surface charge on the vesicle can be calculated. 38

39. ENCAPSULATION EFFICIENCY It describes the % of aqueous phase and hence the % of water soluble drug that becomes ultimately entrapped during preparation of liposomes . Usually expressed as %entrapment/mg lipid. Encapsulation efficiency is assessed using two techniques Minicolumn centrifugation method Protamine aggregation method. 39

40. MINICOLUMN CENTRIFUGATION METHOD :- The hydrated gel ( sephadex G-50) is filled in a barrel of 1mL syringe without plunger which is plugged with a whatmann filter pad. This barrel is rested in a centrifuged tube . This tube is spun at 2000 rpm for 3 mins to remove excess saline solution for gel. After centrifugation the gel column should be dried and have come away from the side of the barrel. Then, eluted saline is removed from the collection tube. Liposome suspension (0.2 ml undiluted) is applied drop wise to the top of the gel bed , and the column is spun at 2000rpm for 3 minutes to expel the void volume containing the liposomes into the centrifuge tube. The elute is then removed and set aside for assay. 40

41. PROTAMINE AGGREGATION METHOD :- The protamine aggregation method may be used for liposomes of any composition(both + and – vely charged particles). However, a preliminary test should be carried out before, to check that the solute material entrapped does not itself precipitate in the presence of protamine after release of liposomes . In this method liposome suspension (20mg/ml in normal saline) is placed in the conical glass centrifuge tube , 0.1 mL of protamine solution (10 mg/ml) is added with mixing, and allowed to stand for 3 min. 30 ml of saline is added and then the tube is spun for 20 min at 2000rpm at room temperature The supernatant is removed & assayed for free, untrapped compound by standard methods. The suspended pellet is resuspended in 0.6 mL of 10% triton X-100 and the material completely dissolved. The volume is made up to the desired value and then assayed for entrapped material by standard methods. 41

42. TRAPPED VOLUME :- Trapped volume is an important parameter that governs the encapsulation efficiency and morphology. The measurement of the quantity of aqueous buffer is the best way to calculate trapped volume. The internal or trapped volume is the aqueous volume entrapped per unit quantity of lipid and expressed as µl/µmol or µl/mg of total lipid. The best way to measure internal volume is to measure the quantity of water directly and this may be done by replacing the external medium(water, H 2 0) and with a spectroscopically inert fluid (deuterium oxide,D 2 O) and then measuring the water signal e.g using NMR. 42

43. Chemical Characteristics CHARACTERIZATION PARAMETERS ANALYTICAL METHODS/INSTRUMENTATION Phospholipid Concentration Lipid phosphorous content using Barlett assay/ Stewart assay, HPLC Cholesterol Concentration Cholesterol oxidase assay & HPLC Drug Concentration Appropriate methods given in monograph for individual drug(s) Phospholipid Peroxidation HPLC &TLC & fatty acid conc. Cholesterol Auto-oxidation HPLC & TLC Anti-oxidant degradation HPLC & TLC pH pH meter Osmolarity Osmometer Phospholipid Hydrolysis UV absorbance, TBA(for endoperoxidase ), idometric (for hydroperoxidase ) & GLC 43

44. BARLETT ASSAY :- In the Bartlett assay the phospholipid phosphorous in the sample is first hydrolyzed to inorganic phosphate . This is converted to phospho-molybidic acid by the addition of ammonium molybidate and phospho-molybidic acid and is quantitatively reduced to a blue colored compound by aminonaphthyl sulfonic acid . The intensity of the blue color is measured spectrohotometrically and is compared with the curve of standards to give phosphorus and hence phospholipid content. 44

45. STEWART ASSAY :- In this assay, the phospholipid forms a complex with ammonium ferrocyanate in organic solution . In this method, the standard curve is first prepared by adding ammonium ferrocyanate (0.1M) solution with different known concentrations of phoshpolipds in chloroform . Similarly, the samples are treated and optical density of these solution is measured at 485nm and the absorbance of samples compared with the standard curve of phospholipids to get the concentration. The advantage of this method is that the presence of inorganic phosphate does not interfere with the assay. 45

46. Biological Characterization Sterility testing (Aerobic or anaerobic cultures) Pyrogen testing {Limulus Amebocyte Lysate (LAL)} Animal toxicity (Monitoring survival rates, histology and pathology ) 46

47. APPLICATIONS Liposomes as drug/protein delivery vehicles :- Controlled and sustained drug release Enhanced drug solubility ( amphotericin B, minoxidil , paclitaxel , cyclosporin ) Altered pharmacokinetics & biodistribution (prolonged & sustained release) Protection of sensitive drug molecules (DNA, RNA, rivosome ) 47

48. 2) Liposomes as carrier to antigen :- Delivery of biologically active materials to specific cells but the major fraction is taken up by liver and spleen. This can be prevented by: By using small , unilamellar liposomes . By coating the liposomes which would render liposomes less recognizable by RES. Anticancer drugs are less selective due to which cause toxicity to the normal cells so with the help of liposomes Targeting of anticancer drugs can be done. 48

49. 3) Liposomes for pulmonary drug delivery system :- Liposomes are available in size ranges from 20nm to greater than 1 um diameter and of low toxicity hence can be used for drug delivery to respiratory tract. Studies on some liposome encapsulated drugs administered via pulmonary route 49 DRUG RESULTS Atropine glutathione Maintained much higher level of drug in the lung than solution form. Enriroxine Liposome encapsulated drug was observed to be 10-50 times less toxic to tissue culture cell than free drug

50. 4) Liposomes for ophthalmic drug delivery :- Liposomes are used for ocular drug delivery due to following advantages: These are biodegradable. These are non toxic. These have ability to intimately contact with the corneal and conjuctival surfaces thereby increases the probability of ocular drug absorption Protects the drug from metabolic enzymes present at the tear/corneal epithelium interface. 50 DRUG RESULT Idoxuridine Improved efficacy in the treatment of herpes simplex keratitis Inulin Absorption greatly enhanced

51. 5) Liposomes for topical application :- Liposomes are used in the preparation of semisolids for topical drug delivery. 51 DRUG RESULTS Triamcinolone In epidermis & dermis 4 times higher conc. than control ointment Benzocaine gel Shows prolonged anesthesia as compared to plain cream Hydrocortisone Higher conc. of drug in individual layer than control ointment.

52. 6) Liposomes as carrier in oral drug delivery :- Many drugs are unstable in the gut hence are given by making liposomes . Diabetes:-Liposomes are used in the oral delivery of insulin. Liposomes exerts protective action in gastric and intestinal areas and protect the drug from proteolytic enzymes. Hence the intestinal uptake of macromolecules increases. 52

53. 7) Cancer chemotherapy :- Anticancer drugs are less selective due to which cause toxicity to the normal cells so with the help of liposomes :- Targeting of anticancer drugs can be done. Increases the half life of anticancer drugs. Decrease the metabolic degradation of drugs. Eg . Of drugs includes: 6- Mercaptopurine , methotrexate , 5-fluorouracil 53

54. 8) Liposomes in gene therapy Gene and antisense therapy Genetic(DNA) vaccination(cholera toxin) 9) Liposomes in immunology Immunoadjuvant (Interleukin-6) Immunomodulator Immunodiagnosis 10) Liposomes as artificial blood surrogates 54

55. 11) Liposomes as radiopharmaceutical and radio diagnostic carriers. 12) Liposomes in cosmetics and dermatology. 13) Liposomes in enzyme immobilization and bioreactor technology. 55

56. 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 56

57. Marketed product Drug used Target diseases Company Novasome ® Smallpox vaccine Smallpox Novavax, USA Avian retrovirus vaccine Killed avian retrovirus Chicken pox Vineland lab, USA Epaxal –Berna Vaccine Inactivated hepatitis-A Virions Hepatitis A Swiss serum & vaccine institute, Switzerland Doxil ® Doxorubicin Hcl Refractory ovarian cancer ALZA, USA Evacet TM Doxorubicin Metastatic breast cancer The liposome company, USA VincaXome Vincristine Solid Tumours NeXstar , USA 57

58. REFERENCES “Targeted and controlled drug delivery” By Vyas and Khar “Controlled and Novel Drug Delivery” By N. K. Jain, 1 st edition, 2009,CBS Publishers & Distributors, New Delhi Sanjay S. Patel ; Liposome: A versatile platform for targeted delivery of drugs; pharmainfo.net . 58

59. THANK YOU 59