LIPOSOMES CHARACTERIZATION AND STABILITY: LIPOSOMES CHARACTERIZATION AND STABILITY Presented by - Mr. Ashwin Arun Shinde M Pharm I st year Roll no-14 Semester- II (Pharmaceutics) CONTENT: CONTENT INTRODUCTION CHARACTERIZATION OF LIPOSOMES 1) PHYSICAL PROPERTIES 2) CHEMICAL COMPOSITIONS 3) BIOLOGICAL PARAMETERS STABILITY STUDIES REFERENCE INTRODUCTION: INTRODUCTION Liposomes are simple microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of a lipid molecule. Structurally , liposomes are concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids. CHARACTERIZATION OF LIPOSOMES: CHARACTERIZATION OF LIPOSOMES PHYSICAL PROPERTIES CHEMICAL COMPOSITIONS BIOLOGICAL PARAMETERS PHYSICAL PROPERTIES: PHYSICAL PROPERTIES Size and its Distribution Surface Charge Percent Capture (entrapment) Entrapped Volume Lamellarity Phase behaviour of liposomes Release kinetics of encapsulated drug from liposomes SIZE AND ITS DISTRIBUTION: SIZE AND ITS DISTRIBUTION Transmission electron microscopy Laser light scattering X-ray scattering Freeze Fracture Electron Microscopy Negative Stain Electron Microscopy Confocal Laser Light Scanning Microscopy Scanning Electron Microscopy Transmission electron microscopy: Transmission electron microscopy The microstructure of colloidal system can visualized with high magnification power of electron microscope. Fluid vesicle dispersions can be characterized using cryo-TEM. A disadvantage of cryo-TEM is the difficulty in establishing the size classification of vesicles. LASER LIGHT SCATTERING MICROSCOPY: LASER LIGHT SCATTERING MICROSCOPY Laser light diffraction is applied for particles >1μm in size and refer to the proportionality between the intensity of diffraction and square of the particle diameter. For particles <200nm in size and refer to proportionality between the scattering intensity and to the sixth potency of the particle diameter. Most of the liposomes consist of particles <200nm to 1μm. X-ray scattering: X-ray scattering Small angle x-ray diffraction is most appropriate technique for exact determination of the distance of interlayer spacing of liquid systems. The inter layer spacing d can be calculated as bragg’s equation÷ d= n /2sin γ This is especially useful for colloidal dispersion . FREEZE FACTURE ELECTRON MICROSCOPY: FREEZE FACTURE ELECTRON MICROSCOPY The freeze-fracture/freeze etch technique starts with rapid freezing of a cell. Then the frozen cells are cleaved along a fracture plane. This fracture plane is in between the leaflets of the lipid bilayer, The two fractured sections are then coated with heavy metal (etched) and a replica is made of their surfaces. This replica is then viewed in an electron microscope . NEGATIVE STAIN ELECTRON MICROSCOPY: NEGATIVE STAIN ELECTRON MICROSCOPY Negative stain electron microscopy visualizes electron transparent liposomes as bright areas against a dark background. Negative stains used in the TEM analysis is ammonium molybdate. CONFOCAL LASER LIGHT SCANNING MICROSCOPY: CONFOCAL LASER LIGHT SCANNING MICROSCOPY Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. It enables the reconstruction of three-dimensional structures from the obtained images . Technique for obtaining high- resolution optical images with depth selectivity i.e. Penetration and Permeation Studies. SCANNING ELECTRON MICROSCOPY: SCANNING ELECTRON MICROSCOPY A scanning electron microscope ( SEM ) is a type of electron microscope that images a sample by scanning it with a high-energy beam of electrons in a f aster scan pattern. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface composition and other properties such as electrical conductivity. SURFACE CHARGE: SURFACE CHARGE Depending on head group composition of the lipid and pH forms, liposomes may bear a negative, neutral, positive charge on their surface. Its normally characterized in term of voltage at particle surface, the surface potential rather than charge density. A zeta potential value of -40 to -50 mV has been found to be sufficient to provide good stability. PERCENTAGE ENTRAPMENT: PERCENTAGE ENTRAPMENT Various method are employed to separate the free drug from the entrapped material . Two method are used ÷ 1) mini column centrifugation 2) protamine aggregation ENTRAPPED VOLUME: ENTRAPPED VOLUME The percent drug encapsulated in liposomes can also be determined by entrapped volume per lipid weight varies for Multilamellar Vesicles and Small Unilamellar vesicles. The trapped volume is determined by dispersing lipid in an aqueous medium containing a radioactive solute. Carboxyfluorescein is also used as a marker to determine the volume of entrapped water per mole of phospholipid . LAMELLARITY: LAMELLARITY The average number of bilayers present in a liposome can be found by freeze electron microscopy and by 31P‐NMR. In the latter technique, the signals are recorded before and after the addition of broadening agent such as manganese ions which interact with the outer leaflet of the outermost bilayers . Thus , a 50% reduction in NMR signal means that the liposome preparation is unilamellar and a 25% reduction in the intensity of the original NMR signal means that there are 2 bilayers in the liposome. PHASE BEHAVIOUR OF LIPOSOMES: PHASE BEHAVIOUR OF LIPOSOMES An important feature of lipid membrane is the existence of a temperature dependent, reversible phase transition, where the hydrocarbon chains of the phospholipid undergo a transformation from an ordered (gel) state to a more disordered fluid (liquid crystalline ) state . These changes have been documented by freeze fracture electron microscopy , but most easily demonstrated by differential scanning calorimetery. The physical state of the bilayers profoundly affects the permeability , leakage rates and overall stability of the liposomes . PowerPoint Presentation: CONTD… The phase transition temperature (Tc) can give good clues regarding liposomal stability, permeability and whether drug is entrapped in the bilayers or the aqueous compartment. LIPOSOMES KINETICES OF ENCAPSULATED DRUG FROM LIPOSOME: LIPOSOMES KINETICES OF ENCAPSULATED DRUG FROM LIPOSOME The in vitro release of drug from conventional and PEGylated liposomes was determined by dialysis method. After reconstituting the freeze dried liposomes in 10ml phosphate buffer solution (PBS)( pH 7.4), an aliquote of each liposomal dispersion was placed in dialysis tube Then , dialysis tube was immersed in a beaker containing 200 ml of release medium , and stirred with magnetic stirrer at 150 rpm to maintain sink condition. CONTD…: CONTD… The sample (5ml) were taken at predetermined time intervals from release medium and replaced by same volume of fresh medium. Concentration of drug was determined after filtering the samples through 0.22µm syringe filter and were assayed UV spectrophotometrically at 268 nm. CHEMICAL PROPERTIES: CHEMICAL PROPERTIES Quantitative Determination of Phospholipids Phospholipid Hydrolysis Phospholipid Oxidation Cholesterol Analysis QUANTITATIVE DETERMINATION OF PHOSPHOLIPID: QUANTITATIVE DETERMINATION OF PHOSPHOLIPID Consequently the method most widely used for determination of phospholipid is an indirect one in which the phosphate content of the sample is first measured. The phospholipids are measured either using- 1) Bartlett assay 2) Stewart Assay BARTLETT ASSAY: BARTLETT ASSAY In the Bartlett assay the phospholipid phosphorous in the sample is first hydrolyzed to inorganic phosphate . This is converted to phospho‐molybdic acid by the addition of ammonium molybdate and phospho‐molybdic acid is quantitatively reduced to a blue colored compound by amino‐naphthyl‐sulfonic acid . The intensity of the blue color is measured spectrophotometrically and is compared with the curve of standards to give phosphorous and hence phospholipid content. STEWART ASSAY: STEWART ASSAY In Stewart assay, the phospholipid forms a complex with ammonium ferrothiocyanate in organic solution. The advantage of this method is that the presence of inorganic phosphate does not interfere with the assay. In this method, the standard curve is first prepared by adding ammonium ferrothiocyanate (0.1M) solution with different known concentrations of phospholipids in chloroform. Similarly, the samples are treated and optical density of these solutions is measured at 485nm and the absorbance of samples compared with the standard curve of phospholipids to get the concentration. CONTD…: CONTD… TLC method may also be employed for determining the purity and the concentration of lipids . If the compound is pure it should run as a single spot in all elution solvents . Phospholipids which have undergone extensive degradation can be observed as a long smear with a tail trailing to the origin, compared with the pure material which runs as a one clearly defined spot. PHOSPHOLIPID OXIDATION: PHOSPHOLIPID OXIDATION Oxidation of the fatty acids of phospholipids in the absence of specific oxidants occurs via a free radical chain mechanism . The initiation step is abstraction of a hydrogen atom from the lipid chain that can occur most commonly as a result of exposure to electro‐magnetic radiation or trace amount of contamination with the transition metal ions. A number of techniques are available for determining the oxidation of phospholipids at different stages i.e., UV absorbance method, and GLC method. PHOSPHOLIPID HYROLYSIS: PHOSPHOLIPID HYROLYSIS Ideally, estimation of phospholipid hydrolysis by quantitation of lysolecithin could be carried out by HPLC where the column outflow can be monitored continuously by UV absorbance to obtain a quantitative record of the eluted components. It is difficult to relate peak height accurately to absolute quantities of lysophosphatidyl choline (LPC), since one does not know the absorbance of the fatty acid species that have been retained on the glycerol bridge. CHOLESTEROL ANALYSIS: CHOLESTEROL ANALYSIS Cholesterol is qualitatively analyzed using capillary column of flexible fused silica whereas it is quantitatively estimated (in the range of 0‐8ug) by measuring the absorbance of purple complex produced with iron upon reaction with a combined reagent containing ferric perchlorate, ethyl acetate and sulfuric acid at 610nm. BIOLOGICAL PARAMETERS: BIOLOGICAL PARAMETERS Sterility test Blood toxicity in animals HPLC analysis STERILITY TEST: STERILITY TEST In order to ensure the sterility of finished products, the optimized formulations were subjected to sterility test. The sterile formulations were incubated with different culture media like Fluid thioglycolate medium for anaerobic/ aerobic bacteria, Soybean casein digest for fungi, Nutrient agar for bacillus subtilis , Maconkeys agar for E-coli , Potato dextrose agar for Candida albicans , and Mannitol salt agar for Staphelococcous aureus . The sterility test was performed by spread plate method. Same media for positive control with specific organisms and negative control without any inoculation was incubated for 14 days and results were noted. ANIMAL TOXICITY: ANIMAL TOXICITY The modification of blood biochemical indexes was evaluated to measure the blood toxicity index of free drug and drug encapsulated in conventional as well as PEGylated liposomes. Four groups each containing 3 albino rats was treated i.v. with 0.5 ml of drug, GEM-2 & PGEM-3 formulations (5 mg/kg) every three days for 30 days. Then blood samples were collected via ocular vein plexus immediately frozen on addition of anticoagulant. Different blood parameters were then measured by biochemical auto analyzer (Type 7170, Hitachi, Japan). The blood samples obtained by healthy albino rats were used as control HPLC ANALYSIS: HPLC ANALYSIS From, retro orbital plexus the blood samples were directly collected in micro cups containing 200 µl tri chlor acetic acid and 50µl of glacial acetic acid were added to decrease hydrogen bonding between nucleotide and proteins. Acetonitrile (1ml) was added to plasma samples, the mixture was vortexed and centrifuged for 15 min at 4°C. The supernant was separated in glass tube and again two washing of acetonitrile was given to extract the drug from plasma samples. The combined supernatant was filtered through 0.22 µm syringe filter and was injected into HPLC. Analysis was carried out using a RP-HPLC system (Jasco PU-2080, intelligent HPLC pump) STABILITY: STABILITY Stability in vitro Long term and accelerated stability Stability in vivo after oral administration STABILITY IN VITRO: STABILITY IN VITRO Stability in vitro mainly covers the chemical stability of constitutive lipid under various accelerated or long-term storage condition. 1) Lipid oxidation and peroxidation 2) lipid hydrolysis LIPID OXIDATION AND PEROXIDATION: LIPID OXIDATION AND PEROXIDATION Most of the procedure used to measure lipid peroxidation either based on disappearance of unsaturated fatty acid or appearance of conjugated dines. Latter technique is now well established as it accompanied by increase UV absorption in 230 to 260nm range LIPID HYDROLYSIS: LIPID HYDROLYSIS Formation of lyso-phospholipid from phospholipid is chemical instability of lipid. Lyso-PC is usually analyzed by phospholipid extraction followed by separation of PC and lyso-PC by TLC . LONG TREM ACCELERATED STABILITY: LONG TREM ACCELERATED STABILITY Stability test commonly stress the system to the limit . Typical example of stress test include exposure of product to high temperature and large gravitational forces >25 o c which alter the nature of interfacial films. If liposomes dispersion is partially frozen and then thawed ice crystals nucleate and grow at the expense of water. Liposomes are pressed against ice crystals at high pressure if crystal grow to a size greater than void space lead to instability. IN VIVO ORAL ADMINISTRATION: IN VIVO ORAL ADMINISTRATION The stability of different liposomal constituent was tested under gastrointestinal condition. 1) low pH and pepsin for the gastric environment, 2) bile salts and lipases from the intestine PowerPoint Presentation: conclusion REFERENCES: REFERENCES Target & Controlled Drug Delivery – Novel Carrier Systems by S. P. Vyas & R. K. Khar Controlled and Novel Drug Delivery Systems, Chapter 15‐ Liposomes as drug carriers by Sanjay K. Jain and N. K. Jain www. science direct .com Mayhew, E, Malson, D, Rustum Y,and Szoka,F Euro, J, cancer Clin Martin s.r, Biopolymer 19, 713-721 Mayer L.D, Bally MB, Hope M.S and Cullis, PR 1980 Chem.