LIPOSOMES USED AS AN DRUG DELIVERY SYSTEMS

TARGETTED DREG DELIVERY SYSTEM

1. LIPOSOMES DRUG DELIVERY SYSTEM Presented By- ROHIT R.K.S.D college of pharmacy, Kaithal ( Hry ) M.Pharma 1 st year (Pharmaceutics)

2. INT R ODUCTION Liposomes are simple microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid molecule. Struc t u r all y , Lipo s o mes a r e c once n t r i c vesicles in which an b i l a y e r ed a q ue o u s v olu m e i s e n ti r ely enclosed b y a m e mb r an o u s l i pi d bil a y e r s main l y composed of natural or synthetic phospholipids. Liposomes is Greek words means ‘ Lipo ’ mean ‘Fat’ and ‘ Somes ’ mean ‘Body’. Liposomes were first produced in England in 1961 by Alec D. Bangham .

3. COMPOSITION OF LIPOSOMES There are number of components of liposomes however Lecithin (mixture of phospholipids) and cholesterol being main components . 1. Phospholipids are fatty substances the major structural components of cell wall & biological membranes. Phospholipids are amphipathic moieties with a hydrophilic head group and two hydrophobic tails. Two types of phospholipids exist – phosphodiglycerides and sphingolipids, together with their corresponding hydrolysis products. Phospholipids have phosphatidyl moiety ( tail ) with different head groups (choline, Etahnolamine, Serine) The most common phospholipid is phosphatidylcholine (PC) molecule.

4. Phosphatidylcholine has glycerol bridge links a pair of hydrophobic acyl hydrocarbon chains having 10-24 carbon atoms with a hydrophilic polar head group.

5. PHASE TRANSITION TEMPERATURE (Tc): 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 . 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 with decrease in bilayer thickness)

6. 2. Cholesterols Incorporation of sterols in liposome bilayer can bring about major changes in the preparation of these membranes. Cholesterol does not by itself form bilayer structure , it acts as fluidity buffer . It inserts into membrane with hydroxyl group oriented towards aqueous surface & aliphatic chain aligned parallel to acyl chains in the centre of bilayer. It can be incorporated into phospholipid membranes in very high Conc. upto 1:1 or even 2:1 molar ratios of PC. Cholesterol incorporation increases the separation between the choline head groups and eliminates the normal electrostatic and hydrogen-bonding interactions.

7. 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.

8. MECHANISM OF LIPOSOMES 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 bilayer sheets polar head groups face outwards into the aqueous medium, the lipidic chains turns inwards to avoid the water phase, giving rise to double layer. 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 These spheres are called as liposomes . Amphiphatic molecules other than PC from micellar structure in preference to bilayer sheet.

9. LIPOSOME

10. A D V AN T A G E S Liposomes increased efficacy and therapeutic index of drug. Liposomes increased stability via. encapsulation. Provide selective passive targeting to tumour tissues. Flexibility to couple with site-specific ligands to achieve active targeting. Improved pharmacokinetic effects (reduced elimination, increased circulation life time). Liposomes reduce the toxicity of the encapsulated agent. Facilitation of transport across membranes. Naturally occurring lipids are non-toxic & biodegradable.

11. DISADVANTAGES Low solubility in water. Production cost is high. Short half-life. Sometimes phospholipid undergoes oxidation and hydrolysis like reaction. Leakage and fusion of encapsulated drug / molecules. CLASSIFICATION On the basis of composition and applications CL (Conventional liposomes), Fusogenic liposomes, pH sensitive liposomes, Cationic liposomes, Immuno-liposomes. Long circulatory liposomes (stealth)

12. Liposome & cell interaction Liposomes interact 4 mechanisms Adsorption, Endocytosis, Fusion of cell with vesicle & Lipid exchange.

13. Method of liposome preparation & drug loading The general procedure involves 4 steps Preparation of lipid for hydration Hydration with agition Sizing for homogenous distribution of vesicles Removal of non-encapsulated material Drug loading may be active (after mfg.)/ passive (during mfg.)

14. Methods of liposome preparation These can be categorized on the basis of lipid dispersion. 1. Physical dispersion technique: Lipid film hydration by Hand-shaking (MLV), non-hand shaking (LUV) High shear homogenization/ Sonication Membrane extrusion Microfluidizer techniques for Micro emulsification French pressure cell Dried-reconstituted vesicles Fusion method 2. Solvent dispersion technique Ethanol Injection Ether injection Reverse phase evaporation vesicles Emulsion & Double emulsion vesicles. 3. Detergent solubilization technique

15. 1. Physical dispersion technique: Liposomes are produced when thin lipid films are hydrated and stacks of liquid crystalline bilayer become fluid and swell. The hydrated lipid sheets cutoff from lipid filmduring agitation and self close to form LMV. LMV particle size reduction is done by sonication/ extrusion. a) Lipid film hydration by Hand-shaking, non-hand shaking: Step-1: Preparation of film for hydration: Lipid + organic solvent (chloroform/ chloroform: methanol) 10-20mg lipid/ml  Mix thoroughly Solvent removal– Small volume (< 1ml)= dry N 2 /Argon stream, Large volume= Rotary evaporation  thin lipid film.

16. Lipid film is dried to eliminate residual organic solvent by placing in vacuum over night. Lipid film can also be prepared by freezing in dry ice/ dry ice- acetone/ alcohol bath. The frozen lipid cake is placed in vacuum pump and lyophilized until dry (1-3 days). Dry lipid films removed from vacuum, container closed tightly, taped and stored frozen. Step-2: Hydration of lipid film: The dried lipid film can be hydrated by addition of aqueous medium, followed by agitation (1 hr) and over night stand. Temperature of hydrating medium should be above “Tc”. Hydration media – buffer solution, saline, 5%dextrose, 10%sucrose. The obtained product- LMV suspension is downsized.

17. b) High shear homogenization/ Sonication:

18. DISADV:- Probe sonicator deliver high energy & heat leading to lipid degradation. It may release titanium particles in to lipid suspension which are to be removed. The lipid suspension of LMV clarifies to yield SUV of 15-50nm.

19. c) Membrane extrusion: Used for preparation of LUVs and MLVs. The size of liposomes is reduced by gently passing them through polycarbonate membrane filter of defined pore size at lower pressure Before extrusion LMV are disrupted by freeze-thaw cycles/ pre-filtering through large pore size (0.2-1 µm). Filter with 100nm pores yield LUV of 120-140nm .

20. d) Microfluidizer techniques for Micro emulsification “Micro Fluidizer” is used to prepare small ULV/ MLVs from concentrated lipid dispersion. The lipids are introduced into fluidizers, either as a dispersion of large MLVs or as a slurry of unhydrated lipids in organic medium. Microfluidizer pumps the fluid at very high pressure (10,000psi, 600-700 bar) through a 5um orifice . Then it is forced along defined micro channels, which direct two streams of fluid to collide together at right angles at a very high velocity, thereby affecting an efficient transfer of energy. The fluid collected of be recycled through the pump and interaction chamber untill vesicles of the spherical dimension are obtained. Adv: Samples with high % of lipids can be easily treated.

21. e) French pressure cell: French pressure cell is invented by ‟ Charles stacy French”. In this technique the large vesicles are converted to small vesicles under very high pressure. This technique yields uni or oligo lamellar liposomes of intermediate size ( 25-75nm in diameter depending on applied pressure). This liposomes are more stable as compared to sonicated liposomes. Suitable for drugs and compounds which degrade by ultrasonic radiations.

22. f) Dried-reconstituted vesicles: This method involves freeze drying the dispersion of empty SUV followed by rehydration with aqueous fluid, which have material to be entrapped. Useful for preparation of small uni-lamellar and oligo lamellar vesicles. ADV: High entrapment of water soluble components and bioactives. g) Fusion method: This method protects lipids and entrapped material from harmful physicochemical environment. Used for preparation of ULV in large quantities. Fusogenic agents are used for fusion of SUV for increasing entrapment efficiency. Alteration in pH increases surface charge density of lipid bilayer and brings on spontaneous vesiculation.

23. 2. Solvent dispersion technique: In this process lipid solution in organic phase is bought in contact with the aqeous phase including material to be entrapped inside the liposome. a) Ethanol injection: Suitable for incorporating hydrophobic & amphiphilic drugs in to liposomes. This is suitable for preparing small & large unilamellar vesicle. Lipid + ethanol  22-gauge fine needle, RAPIDLY  excess of saline/ aqueous medium. The rate of injection should be suitable to attain absolute & rapid mixing thus leads to equal distribution of phospholipids through the medium.

24. b) Ether injection: This was developed by Deamer & Bangham , similar to ethanol injection. Organic solution of lipid (ether + lipid)  narrow needle, SLOWLY  Aqueous phase. The lipid solution is added at the vaporizing temp. of organic phase. Lipid solution added very slowly for protection of sensitive lipids & material to be entrapped. DISADV:- low entrapment efficiency.

25. c) Aqueous medium + material to be entrapped  high vol. organic solution of lipid  Agitated mechanically  break aq. Medium to water droplets  W/O emulsion  Stabilized by phospholipid monolayer .

26. d) Double Emulsion Method: The organic solution, which already contains water droplets (W/O) is introduced in to excess aqueous medium followed by mechanical dispersion causing phase inversion (O/W). The (W/O/W) multi component vesicle is formed by double emulsion. Two aqueous compartments being separated from each other by two phospholipid monolayer. The hydrophobic surfaces of monolayers (tails) face each other, with a thin film of organic solvent. Removal of organic solvent results in formation of intermediated sized unilamellar vesicles.

27. e) Rapid solvent exchange method: Lipid solution in organic solvent is passed through an orifice of syringe by means of vacuum in to a tube containing aqueous buffer placed on vortex. The organic solvent vaporizes due to vacuum before contacting aqueous phase. The lipid mixture precipitates very quickly in aqueous buffer forming liposomes. f) Reverse phase evaporation: Mixture of 2 phases (Aq+Org) subjected to bath sonication. Droplets formed are dried to semisolid gel in rotary evaporator under reduced pressure. In this stage mono layer of phospholipids surround water compartment.

28. 4. Mechanical shaking with vortex shaker collapses few water droplets & lipid mono layers. 5. Collapsed lipid monolayers form outer layer for stable vesicles thus forming bilayer SUV .

29. 3. Detergent solublization technique: Detergents associate with the phospholipid molecules and serve to screen the hydrophobic portions of molecule from water. The structures formed as a result of this association is known as micelles. Up on removal of detergent, transition of mixed micelles occurs to form concentric bilayered vesicles. The concentration of detergent in water at which micelles instantly forms is called CMC . Liposome size and shape depend on chemical nature of detergent, concentration and other lipid involved. Methods to remove detergents : Dialysis & Column chromatography.

30. Hydrophilic nonbilayer- interacting low molecular weight compounds have low entrapment. This method is suitable for preparation of immuno- liposomes Sur f ac t a n t Micelle P h o s p h olip i d monolayer LIP O SO M E S

31. Characterization/ Evaluation of liposomes The liposomes prepared by various techniques are to be evaluated for their physical, chemical as well as biological properties. A) Physical Characterization: Entrapement efficiency Vesicle shape & morphology lamellarity Surface charge Phase transition behaviour Drug release B) Chemical Characterization: Phospholipid concentration Cholesterol concentration Lysolecithin concentration 4. Particle size & size distribution 4. Phospholipid peroxidation C) Biological Characterization: Sterility testing Pyrogenicity testing. Animal toxicity Phospholipid hydrolysis & Cholesterol autooxidation pH of liposomal dispersion. Osmolarity D) Stability testing:

32. A) PHYSICAL CHARACTERIZATION Characterization parameter Analytical methods / Instrument Entrapment efficiency Mini column centrifugation method– Sephadex column (cross linked dextran) Protamine aggregated method Vesicle shape and surface morphology Transmission electron microscopy, Freeze fracture electron microscopy Lamellarity Small angle X-ray scattering, 31 P-NMR, Freeze fracture electron microscopy Particle size & size distribution Light microscopy, Fluorescence microscopy, Transmission electron microscopy, Freeze fracture electron microscopy, Photon correlation spectroscopy, laser light scattering, gel permeation, gel exclusion and Zeta sizer. Surface charge Free-flow electrophoresis, Zeta potential measurements Phase transition behaviour DSC (differential scanning calorimetry) Drug release Diffusion cell, Dialysis tube.

33. B) CHEMICAL CHARACTERIZATION Cha r ac t eri za tion parameter Analytical methods / Instrument Phospholipid concentration (Lipid phosphorous content ) Barlett assay, stewart assay, TLC. Cholesterol concentration Cholesterol oxidase assay, Ferric perchlorate method Lysolecithin concentration (hydrolysis product of lecithin) Densitometer Phospholipid peroxidation UV absorbance, Iodometery, GLC. Phospholipid hydrolysis & Cholesterol autooxidation HPLC & TLC pH of liposomal dispersion pH meter Osmolarity Osmometer

34. C) BIOLOGICAL CHARACTERIZATION D) Stability of liposomes Stability is defined as capacity of particular formulation in a specific container/ closure system to remain with in physical, chemical, microbiological, therapeutic and toxicological specifications.

35. Stability testing of liposomes: Liposomal stability can be tested by storing under following six conditions. Highest & lowest temp. for 1 month. Room temp. for 12-24 months 2-3 freeze thaw cycles (20-25 o C) 60 cycles/ min in a reciprocating shaker for 24-48 hr 6-8 heat cool cycles (5-45 o C, 48 hrs at each temp.) Visual/ microscopic examination. After storage liposomes are valuated for vesicle size, shape, no. of vesicles/ cubic mm & residual drug content.

36. Applications of liposomes: Liposome as drug delivery vehicle Liposome as vaccine carrier Liposome in tumour therapy Liposome in gene delivery. Liposome as artificial blood surrogates Liposome as radio-pharmaceutical & radio- diagnostic carrier Liposome in cosmetics and dermatology Liposome in enzyme immobilization.

37. 1. Liposome as drug delivery vehicle: Liposomes enhance solublization of drugs (Amphotericibn-B, paclitaxel, Cyclosporin, Minoxidil). Provide protection to sensitive drug molecules (cystone arabinose, DNA, RNA, Ribozymes). Enhance intra cellular uptake (anti-cancer, anti-viral & anti-microbial agents). Alter pharmacokinetics & distribution of drugs. 2. Liposome as vaccine carrier: Liposomes potentiate both cell mediated & humoral immunity. Liposomal vaccines based on immunopotentiating reconstituted influenza virosome ( IRIV ) are prepared.

38. For immunopotentiation, immunomodulating agents like muramyl dipeptide, lipopolysaccharide & lipid can be incorporated in to liposome. Advantages: Non-toxic, bio-compatible & biodegradable. Incorporate adjuvants to provide strong immune response. Convert loaded non-immunogenic substance to immunogenic (Proliposome) Minimize & eliminate toxicity of toxic antigens & allergic reactions.

39. 3. Liposome in tumour therapy: Liposomes as drug carriers can be administered I.V route. If liposome is modified more hydrophilic, with lipids their circulation time in blood stream increases. These are called stealth liposomes , used as carriers for hydrophilic anti-cancer drugs (Doxorubicin, Mitoxantrone) In this form they can extravasate the tumour vascular endothelium.

40. 4. Liposome in gene delivery: The non-viral vector systems , are especially engineered liposome such as pH sensitive liposomes, cationic liposomes, fusogenic liposome, genosomes, lipoplex, and lipopolyplex have been extensively investigated for their gene delivery potential. Cationic liposomes deliver the content through membrane fusion, there by avoiding lysosomal and nucleolus degradation of DNA. pH sensitive liposomes use endosomal acidification for fusion with endosomal membarne. Genosomes are complex formulations of DNA with various cationic liposomes. Lipoplex aggregates with DNA to form large and heterogeneous particles. Lipopolyplex is composed of liposome + polycation + DNA.

41. 5. Liposome as artificial blood surrogates: Liposome encapsulated hemoglobin products can be used as artificial RBC . Sterically stabilized liposome bearing hemoglobin are better Oxygen carriers. These have low toxicity , less platelet activation& aggregation & less haemostatic generation. 6. Liposome as radio-pharmaceutical & radio-diagnostic carrier Liposomal radio-diagnostic applications include imaging of liver, spleen,brain, lymphatics, tumour, blood pool, cardiovascualr pathologies, visualization of inflammation, infection sites, bone marrow, eye vasculature. Liposome imaging agents are used for magnetic resonance, computed tomography & ultra sound imaging of tumours.

42. 7. Liposome in cosmetics and dermatology: Liposomes with essential oils provide an effective nourishing treatment that penetrates deeply in to the skin. Liposome based on anti-aging formulations (e.g. creams, lotions, gels and hydrogels) have been formulated and launched in the cosmetic market by L`oreal in 1986. Liposomal preparation reduce the roughness because of its interaction with corneocytes, the intracellular lipid resulting in skin softening and smoothing. Various liposome based products for facial and body care, make-up, mascara & foundation, haircare, sunscreen products & perfumes are in market.

43. 8. Enzyme immobilization: Liposomes can deliver enzymes to lysosomal system & other sites. β – glucosidase & α– glucosidase are loaded in liposomes for tretamnet of Gauchers & Pomps diseases respectively. Limitations of liposome technology 1. Stability 2. Sterilization 3. Encapsulation efficiency 4. Active targeting 5. Gene therapy 6. Lysosomal degradation