Liposome Evaluation

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Presensted by : Sajesh Kuruvilla Joseph, M.Pharm(p ceutics) Grace College of Pharmacy

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They are simply vesicles or bags in which an aqueous volume is entirely enclosed by a membrane composed of lipid (fat) molecules, usually phospholipids

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The behavior of liposomes in both physical and biological systems is governed byy Physical

size y Membrane permeability y Percent entrapped solutes y Chemical composition y Quantity and purity of the starting material

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Therefore, the liposomes are characterized for physical attributes: y Shape, size and its distribution y Surface Charge y Percent Capture (entrapment) y Entrapped Volume y Lamellarity y Phase behavior of liposomes y Drug release and Chemical Compositions: -estimation of phospholipids - phospholipid oxidation - analysis of cholesterol4

1) Size and its distributionElectron microscopy- most precise method, y allows to view each individual liposome, y time consuming

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Light microscopy: has been utilized to examine the gross size distribution of large vesicles fluorescent microscopy: If the bilayers are having fluorescent hydrophilic probes

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photomicroscope (Nikon Model UFX-II)

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Negative stain electron microscopy: To obtain an estimate of the lower end of the size distribution. y A technical difficulty in obtaining good negative stains of liposomes is the spreading of the vesicles on the carbon-coated grid. y Treating the grid with 0.1mg/mL solution of bacitracin or coating the support films with silica by the evaporation of silicon monoxide, usually permits a satisfactory spreading of liposomes for negative staining.8

y The freeze etch microscopy is particularly

suitable for the measurement of small vesicle diameters. y For populations of large size vesicles freeze fracture technique can yield a representative morphological view of the liposome and has been useful for examining the morphologi cal changes that can occur in the bilayer surface as the phospholipids pass through the gel-liquid-crystalline transition, or through the lamellar hexagonal transition.9

TEM images of ciprofl oxacin liposomes. (a) 7:4 MLVs; (b) 7:2 MLVs; (c) -7:4 REVs, (d) -7:2 REVs

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Photomicrograph of ciprofloxacin liposomes. (a) 7:4 MLVs; (b) 7:2 MLVs; (c) 7:4 REVs; (d) 7:2 REVs

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Laser light scattering (quasi-elastic laser light scattering) method y very simple and rapid to perform y disadvantage of measuring an average property of the bulk of the liposomes. y As biomolecules or a distribution of biomolecules diffuse around the laser-beam coherence area, light scattered from them overlaps and interferes with the transmission of the laser light. A high-sensitivity detector can then record the time-varying signal caused by scattered light and compare it to the constant signal emitted when no molecules are present. This process is known as dynamic light scattering (DLS), or quasielastic light scattering12

y Small particles or biomolecules diffuse quickly,

causing rapid fluctuations of the scattered light. Larger particles diffuse slowly, resulting in less frequent fluctuations in the intensity of the scattered light. y Since small particles diffuse more rapidly than large particles, the rate of fluctuation of scattered light intensity varies accordingly.

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y Thus by measuring the rate of fluctuation of

the scattered light, the translational diffusion coefficient (D) can be measured.y D= kT/ 6 Rh y D = Translational Diffusion Coefficient y K = Boltzmann s Constant y T = Absolute Temperature y = Solvent Viscosity y Rh = mean hydrodynamic radius14

Particle size analyzer (Malvern 2000SM)15

-Gel Permeationy Exclusion chromatography on large pure gels

was introduced to separate SUVs from radial MLVs.y A thin layer chromatography system using

agarose beads has been introduced as a convenient, fast technique for obtaining a rough estimation of the size distribution of a liposome preparation.16

2) Surface Chargey A technique has been developed that separates extruded vesicles on the basis of their surface charge by

electrophoresis on cellulose acetate plate in a sodium borate buffer pH 8.8. y The lipid samples (5 nmoles) are applied to the plate and electrophoresis is carried out at 4 degrees celsius on a flat bed apparatus for 30 mins. y The plate is dried and the phospholipids are visualized by the molybdenum blue reagent. y This sensitive assay should prove valuable for examining the charge heterogeneity in liposome preparation for following fusion between two populations of vesicles with different charge and for determining Percent capture 17 (entrapment)

3)Percent capture (entrapment)In general two methods may be usedy Mini column centrifugation y Protamine aggregation % of encapsulated drug substance = 100 % of free drug substance Mini Column Centrifugation method y In this method, the hydrated gel (sephadex G-50) is filled in a barrel of 1mL syringe without plunger which is plugged with a whatman GF/B filter pad. y This barrel is rested in a centrifuge tube. This tube is spun at 2000rpm for 3 mins to remove excess saline solution from the gel. y Liposome suspension (0.2mL undiluted) is applied dropwise to the top of the gel bed,and the column is spun at 2000 rpm for 3 min. y The elute is then removed and set aside for assay.18

The Protamine Aggregation Method y liposome suspension (20mg/ml in normal saline) is placed in conical glass centrifuge tube, 0.1mL of protamine solution (10mg/ml) is added with mixing , and allowed to stand for 3 min. y 30 mL of saline is added and then the tube is spun for 20min at 2000rpm at room temperature. y The supernatant is removed and assayed for free, untrapped compound by standard methods. y The suspended pellet is resuspended in 0.6mL of 10% triton X- 100 and material completely dissolved. y The volume is made up to the desired volume and then assayed.19

4)The entrapped volumey liposomes prepared in aqueous solution consisting of ordinary

water are spun at high centrifugal force (200,000g for 6 hours) y to give a tight pellet, from which the supernatant is decanted off to remove every drop of excess fluid (including some liposome, if necessary). y The pellet is then resuspended in deuterium oxide (D2O). The permeability of the membrane to water is such that D2O and H2O equilibrate very rapidly throughout the whole of the volume of the medium. y A small aliquot is removed for quantification of phosphlipid and the remainder is used to obtain an NMR scan of H2O, the peak height of which can be related to concentration by comparison with standards containing known amount of H2O and D2O.

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5) Lamellarityy The average number of bilayers present in a liposome can be found by freeze

electron microscopy and by NMR. y 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. y 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.21

6) Phase Behavior of LiposomesThe hydrocarbon chains of the phospholipid undergo a transformation from an ordered (gel) state to a more disordered fluid (liquid crystalline) state.

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y These changes have been documented by

freeze fracture electron microscopy, but most easily demonstrated by differential scanning calorimetery. y The phase transition temperature (Tc) is a function of phospholipid content of the bilayers. y The Tc can give good clues regarding Liposomal stability, permeability and whether drug is entrapped in the bilayers or the aqueous compartment.23

7) Drug Releasey The mechanism of drug release from the liposomes

can be assessed by the use of a well calibrated in vitro diffusion cell. y Percent drug diffused =CrVr/CdVd

In vitro drug released from Nimesulide Loaded Liposomes

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CHEMICAL PROPERTIESy Quantitative Determination of Phospholipids y Phospholipid Hydrolysis y Phospholipid Oxidatin y Cholesterol Analysis a) Quantitative Determination of Phospholipids The phospholipids are measured either using Bartlett assay or Stewart Assay y Bartlett Assay y phospholipid phosphorous 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.

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y Stewart assayy In Stewart assay, the phospholipid forms a complex with y y

y

y

ammonium ferrothiocyanate in organic solution. The advantage of this method is that the presence of inorganic phosphate does not interfere with the assay. The standard curve is first prepared by adding ammonium ferrothiocyanate (0.1M) solution with different known concentrations of phospholipids in chloroform 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. TLC method may also be employed for determining the purity and the concentration of lipids.26

Paper chromatogram of liposomal formulations. (SL- soyalecithin)

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b) Phospholipid Hydrolysisy The major product of lecithin hydrolysis is lysolecithin. 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. y Unfortunately many natural phospholipids have fatty acids which are unsaturated and therefore, absorb to different extent in the 1- and 2- position.28

c) Phospholipid Oxidationy Oxidation of the fatty acids of phospholipids

in the absence of specific oxidants occurs via a free radical chain mechanism. y A number of techniques are available for determining the oxidation of phospholipids at different stages ie UV absorbance method, iodometric method(for hydroperoxides) and GLC method d) Cholesterol Analysis y Cholesterol is quantitatively estimated 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 610nm29

y Physical stability and drug retention study y For this liposomes were sealed in 20-mL glass

vials and stored in refrigerator at 4C for a period of 3 months. y Samples from each liposomal formulation were withdrawn at definite time intervals and the residual amount of the drug in the vesicles was determined after separation from unentrapped drug as described previously under the separation of free drug and entrapment efficiency.30

Referencesy Theory and Practice in Novel Drug Delivery System, y y y y y y y

S.P.Vyas Page no: 164-174 Controlled and Novel Drug Delivery, N.K.Jain Page No: 321-328 2. http://noopurmandrek.files.wordpress.com/2010/0 9/lipo1.jpg (accessed on 15-04-2011) 3. www.nanobiotec.iqm.unicamp.br/download/liposo mas-3.ppt accessed on 15-04-2011) 4. http://www.unimagdeburg.de/imos/mea_sen/img/pictures/Lipo.jpg (accessed on 15-04-2011) 5. http://www.nanolifenutra.com/images/image_lipo some_01.jpg (accessed on 15-04-2011) 6. http://www.azonano.com/work/bFgW9FjRw248U 2PRC2In_files/image002.gif (accessed on 15-04-2011) 7. http://upload.wikimedia.org/wikipedia/en/2/28/Li posome.jpg (accessed on 15-04-2011)31