Liposomes

Liposomes Introduction and preparation

1. LIPOSOMES Presenter: Dr Zoraiz Haider D14M127 Ahmad Nawaz D14M148 To: Dr. Fatima Rasool Punjab university college of pharmacy

2. INTRODUCTION Liposomes are microscopic, concentric bilayered vesicles, in which an aqueous volume is entirely enclosed by a membranous lipid layer, mainly composed of natural or synthetic phospholipids . The structural main components are Phospholipids Cholestrol

3. Phospholipids are amphipathic molecule . The tail portion consists of 2 fatty acid chains having, The head or polar portion consists of phosphoric acid bound to a water soluble molecule. PHOSPHOLIPIDS

4. Methods Of liposomal Preparation

5. Methods Of liposomal Preparation

6. 1- Mechanical Dispersion Method Passive Loading Technique

7. i . Hand Shaken MLV’s

8. ii. Non Shaking Vesicles

9. iii- Freez Drying Sonication method Freeze dry the lipid dissolved in a suitable organic solvent (prior to addition of aqueous media). The solvent choice depends on the freeze point which needs to above the temperature of the condenser lyophilizers . Tertiary butanol is considered to be most ideal solvent. After obtaining the freeze drying membrane lipid which is an expanded foam like structure,

10. DRV & FTS Method Than> In DRV(Dried reconstituted Vesicle) Method is rehydrated with water or saline contain material to be entrapped with rapid mixing above the phase transition temperature to give MLVs. In FTS(Freeze-Thaw) Method it is Thawed by allow it to stand at room temp for 15mins and finally sonicated . Sonicated reduces the permeability of liposome membrane . Uni or Oligo vesicles are formed.

11. Dried Reconstituted Vesicles(DRV)

12. iv- Micro emulsification method MICROEMULSIFICATION “ Microfluidizer ” is used to prepare small MLV’s from concentrated lipid dispersions/slurry/ unhydrated lipid in an organic medium. Pumping of the dispersion through a 5µm orifice at 10,000psi .(High pressure) It is forced along defined micro channels which direct two streams of fluids to colloid together at right angle at very high velocity, result in efficient transfer of Energy. After a single pass size reduce to 0.1 & 0.2 µm . Efficient for encapsulation of hydrophilic materials.

13. v- Sonication method The MLV dispersion in a Test tube is placed into a bath sonicator . And Sonicating for 5-10mins Controlling the temperature of the lipid dispersion is usually easier in this method . The tip of a probe sonicator is directly engrossed into the liposome dispersion. Supply high energy input to lipid dispersion and can cause lipid degradation due to overheating. At high energy level size of vesicles reduced with UltraSonic irradiation. The

14. Sonicators BATH SONICATOR Large volume of diluted lipids are processed. Less or no contamination PROBE SONICATOR Small volume of diluted lipids are processed. Chances of contamination

15. vi- French Pressure cell Is an equipment used to reduce the particle size of liposome by the use of high shear forces. The method involves the extrusion of MLV at 20,000 psi at 4°C through a small orifice . Passing the dispersion repeatedly through the press results in a progressive decrease in the mean particle diameter. D rawbacks are that, the temperature is difficult to achieve and the working volumes are relatively small, (about 50 mL maximum)

16. Principle of French Press Cell

17. vii- Membrane Extrusion method Liposomes passed through membrane of defined pore size. Lower pressure is required (<100 psi). LUVs as well as MLVs can be processed. Vesicle contents are exchanged with dispersion medium during breaking and resealing of phospholipid bilayers as they pass through the polycarbonate membrane. For high entrapment , the water soluble compounds should be present in suspending medium during the extrusion process

18. 2- Solvent Dispersion Method Passive Loading Technique

19. i . Ethanol Injection Method A lipid solution of ethanol is rapidly injected to a vast excess of buffer. The MLVs are immediately formed . The Drawback s of the method are that the population is heterogeneous (30-110 nm), liposomes are very dilute , It is difficult to remove all ethanol because it forms azeotrope with water and possibility of various biologically active macromolecules to inactivation in the presence of even low amounts of ethanol.

20. ii- Ether Injection Method A solution of lipids dissolved in diethyl ether or ether/methanol mixture is slowly injected to an aqueous solution of the material to be encapsulated at 55-65°C or under reduced pressure. The subsequent removal of ether under vacuum leads to the formation of liposomes . The D rawbacks of the method are population is heterogeneous (70-190 nm) and the exposure of compounds to be encapsulated to organic solvents or high temperature.

21. iii. Reverse phase Evaporation method First w/o emulsion is formed by brief sonication of a two phase system containing, phospholipids in organic solvent ( diethylether or isopropylether or mixture of isopropyl ether and chloroform ) and aqueous buffer . The organic solvents are removed under reduced pressure, resulting in the formation of a viscous gel . The liposomes are formed when residual solvent is removed by continued rotary evaporatio n under reduced pressure . High encapsulation efficiency up to 65% can be obtained in a medium of low ionic strength for example 0.01M NaCl .

22. 3- Detergent Removal methods Passive Loading Technique

23. Following methods are used for removal of detergents : Dialysis Column Chromatography Dilution 3- Detergent Removal methods

24. Active Loading Techniques

25. Active Loading Techniques Also known as Remote loading technique. Lipid bilayer membrane is impermeable to ions and polar molecules Weak acids and bases can be transported due to various transmembrane gradients i.e electric , electrochemical, pH,or specific salt gradients . This method involves loading of drug molecules in preformed vesicles using pH gradients and potential differences across liposomal membranes.

26. Steps in Active Loading Techniques Solute is neutralized by addition of acid or base so that its pH is neutral . Liposomes are made to have low internal pH . Neutral solute passes easily through bilayer membrane by diffusion . Charge acquired by solute inside liposome makes them unable to exit . As a result drug molecules are entrapped within the liposomes.

27. Active loading technique:

28. Characterization of Liposomes

29. There are three main types of characterization techniques of liposomes . Physical characterization Chemical characterization Biological characterization The liposomes prepared by various techniques has influence the behavior of liposomes in vivo. Characterization of Liposomes

30. PHYSICAL CHARACTERIZATION CHARACTERIZATION PARAMETERS Particle size Surface charge Percent drug encapsulated Phase behavior Drug release rate

31. PARTICLE SIZE Both particle size and particle size distribution of liposomes influence their physical stability . These can be determined by following methods: Laser light scattering Transmission electron microscopy

32. SURFACE CHARGE The passive, negative, neutral charge on the surface of the liposomes is due to head groups . The surface charge on the liposomes governs the kinetic and extent of distribution in vivo, as well as the interaction with the target cells. The method involved in the measurement of surface charge is based on free flow electrophoresis . It utilizes a cellulose acetate plate dipped in sodium borate buffer of pH 8.8. The liposomes get bifurcated depending on their surface charge. This technique can be used for determining heterogeneity of charges in the liposome suspension as well as to detect any impurities such as fatty acids.

33. PERCENT DRUG ENCAPSULATED Quantity of drug entrapped in the liposomes helps to estimate the behavior of the drug in the biological system. Liposomes are mixtures of encapsulated and un encapsulated 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 usinng suitable detergents. The methods used to separate free drug from the sample are; Mini column centrifugation method Protamine aggregated method

34. PHASE BEHAVIOR At transition temperature liposomes undergoes reversible phase transition The transition temperature is the indication of stability, permeability and also indicates the region of entrapment. It is done by DSC.

35. DRUG RELEASE RATE The rate of drug release from the liposomes can be determined by in vivo assays which helps to predict, the pharmacokinetics and bioavailability of the drug.

36. CHEMICAL CHARACTERIZATION Characterization parameters Phospholipid concentration Cholesterol concentration Phospholipid oxidation Phospholipid hydrolysis Cholesterol auto oxidation Anti oxidant degradation Osmolarity

37. PHOSPHOLIPID CONCENTRATION The phospholipid content can be determined directly by two assays ; Bartlett Assay. Steward Assay. BARTLETT ASSAY : Very sensitive method and produce results in the presence of even trace amounts of inorganic phosphate. Therefore, borosilicate glass tubes and double distilled water is used. STEWARD ASSAY : This assay overcomes the drawbacks of bartlett assay, but can not be used to mixture of unknown phospholipids.

38. CHOLESTEROL CONCENTRATION QUALITATIVE ANALYSIS : performed using a capillary column filled with fused silica. QUANTITAVE ANALYSIS : The sample is reacted with a reagent (containing ferric perchlorate , ethyl acetate and sulfuric acid) and the absorbance of purple colored complex is measured at 610nm .

39. PHOSPHOLIPID OXIDATION Free radical determination by UV, Iodometric method, GLC etc.

40. PHOSPHOLIPID HYDROLYSIS Phospholipids + hydrolysis = Lysolecithin One chain is lost by desterification , determined by HPLC

41. BIOLOGICAL CHARACTERIZATION Characterization parameters Sterility Pyrogenicity Animal toxicity

42. STERILITY Determined by using, aerobes or anaerobic cultures

43. PYROGENICITY Determined by limulus amoebocyte lysate (LAL) test.

44. ANIMAL TOXICITY By monitoring survival rates, histology and pathology .

45. Thanks