Oral Controlled Release System of Propranolol - Dissertation Example

?CHAPTER 2: METHODOLOGY METHODOLOGY Ionotropic gelation method Ionotropic gelation is based on the ability of polyelectrolytes such as gellan gum to cross link in the presence of counter ions, like Calcium (Ca2+) and Zinc (Zn3+) ions, to form hydrophilic hydrogel beads (gelispheres). These beads swell when exposed to biological fluids, thereby gradually releasing the drug inside. Gellan gum is a bacterial exopolysaccharide prepared by aerobic submerged fermentation of Sphingomones Eloda. Gellan gum, being a natural polymer, has an advantage as a drug delivery system because it induces less adverse reactions compared to the synthetic ones. It contains anionic moieties on its chemical structure, combines with the counter cations to induce gelation by cross linking. This polymer is unique in that when a concentrated solution of gellan gum is exposed to high temperatures, gelation is induced. Gradually decreasing the temperature allows the random coils of its structure to form organized double helices, which promote the formation of ordered junction zones. Measurement of PPN To allow determination of PPN concentration from absorbance measurements, a calibration curve was made by getting the absorbance of standard solutions with known concentrations. The resulting mathematical equation relating PPN concentration with its absorbance at 289 nm should help in determining the PPN concentration of the solutions to be obtained in the next parts of the experiments. Preparation of standard solutions First, the stock solution was prepared by diluting 10mg of PPN in 50ml of 2% EDTA solution. 1ml of stock solution was mixed with 9 ml of 2% EDTA solution. Subsequent dilutions were made based on the following table. No Total 1 1ml of Stock 2 1ml of 2%EDTA solution 2ml 2 1ml of Stock 2 4ml of 2%EDTA solution 5ml 3 1ml of Stock 2 9ml of 2%EDTA solution 10ml 4 1ml of number (3) 1ml of 2%EDTA solution 2ml 5 1ml of number (3) 4ml of 2%EDTA solution 5ml 6 1ml of number (3) 9ml of 2%EDTA solution 10ml UV Spectrophotometry The solutions then underwent UV Spectrophotometry in order to measure their absorbance of light at 289 nm. Since PPN readily absorbs light at this wavelength, the absorbed light increases with increasing amounts of PPN present in the solution. This is why UV Spectrophotometry is used to measure the concentration of a solution. Briefly, each dilution placed on a clear cuvette was loaded into the spectrophotometer (fig. 1), which will apply 289 nm light on the solution, while subsequently measuring the liquid’s absorbance. Fig. 1 UV-160A used to measure the absorbance at 289nm. After measuring the absorbance of each solutions at 289 nm using UV106, the points were plotted, with the concentration at x-axis, and the absorbance at y-axis. The equation that plots the curve was then obtained. Preparation of PPN-MAS complex dispersion Suspension of 8% (w/v) MAS was prepared using hot, sterile double deionised water (50 ml in 60?C) and cooled prior to use to room temperature. Then, 4.7, 9.4, and 18.8, volumes of this suspension were each added with 25 ml of the 1% w/v PPN double deionised to produce MAS concentrations of 0.75, 1.5%, or 3% (w/v), respectively. Finally, PPN was allowed to adsorb onto each of the MAS solutions by incubating them in a sterile environment at room temperature for 24 hours. Preparation of GG dispersions with PPN-MAS complexes To add in the delivery system, 0.5g GG was gently added to 50ml of sterile double deionised water. It was then heated to 55?C to allow the gellan gum to be hydrated. After cooling the dispersed GG in room temperature, the different PPN-MAS complexes of various MAS concentrations, which were prepared in the previous section, were centrifuged for 15mins using Centrifuge 5702 (fig. 2). This was done to remove uncoupled PPN and MAS (in the supernatant) from PPN-MAS complexes (in the solid layer). After pouring off the supernatant into a separate beaker, the solid layer of each was mixed with the GG dispersion at constant stirrer, 500rpm, until a homogenous dispersion was formed. These were used for the preparation of the beads. The set-up is shown at fig. 3. Fig. 2. Centrifuge 5702 was sed to centrifuge the PPN-MAS complex. Fig. 3. The set up during the gellan gum solution preparation. Determination of PPN adsorbed onto MAS This was done to compare the different concentrations of MAS in terms of the amount of PPN adsorbed. The supernatant collected from each solution in the procedure above was poured through a 0.45 µm Nylon filter membrane. The absorbance of each of the filtered supernatant at 289 nm was measured using the Shimadzu UV160A UV–vis spectrophotometer. The concentration was subsequently calculated using the measured absorbance on the calibration curve. The amount of PPN adsorbed onto the MAS was calculated as the difference between the amount of PPN added and the amount of PPN in the supernatant. The preparation with the greatest amount of adsorption would be the one prepared for characterization studies. Preparation of beads Using a Sp200 slow infusion syringe pump (fig. 4 and fig. 5), 20 ml of the optimal GG- PPN-MAS formulation, which was prepared in the previous section, was slowly added to 25 ml 1.0% (w/v) calcium chloride solution and 25 ml 1.0% zinc chloride with gentle agitation (300rpm). After soaking the beads in this solution for 4 min., they were washed with 20ml sterile, double deionised water, and dried for 24 h at 40?C. Fig. 4. Luer Syringe Needle used to make the beads+ 20ml syringe Fig. 5 Sp200 syringe Pump used when the beads prepared at pressure speed of 1.0. GG-MAS beads without the drug were also prepared using the steps above, in order to have a reference point for the subsequent comparative studies conducted. Characterization of the oral controlled release system Determination of drug entrapment efficiency To free the formulation from any cations such as Ca2+ and Zn3+, a mortar and pestle were used to granulate the beads, and then 50 mg of granulated beads was mixed with 25 ml of 2% solution of ethylenediaminetetraacetic acid (EDTA). This was incubated for a day at room temperature at constant stirrer. The solutions were then filtered using 0.45 µm Nylon filter membranes with 5ml syringe (fig. 6). The PPN concentration of the filtrate was measured using the UV-vis spectrophotometer, which read the absorbance at 289 nm. The proportion of actual to theoretical beads drug composition was referred to in order to determine the entrapment efficiency. A value close to one indicates efficient encapsulation of PPN. Fig. 6. Filtered used (nylon 0.45um) Scanning electron microscopy (SEM) SEM is an imaging technique whereby electrons are ejected onto the surface of a solid specimen. The result of the high-energy electron-sample interactions, such as secondary electrons, backscattered electrons, photons, visible light, and heat, are recorded and interpreted to provide information regarding the sample’s texture, chemical composition, as well as crystalline and chemical structure (Swapp, 2012). In this experiment, SEM was used to examine the surface and matrix structure of gellan gum macrobeads. A sample of beads was prepared by coating it with a gold layer in a vacuum evaporator. This was done to increase its conductivity. The sample as then placed on a stub that was introduced into the microscope. Determination of release kinetics Differential scanning Calorimetry (DSC) DSC is a technique used to measure enthalpy changes resulting from physical and chemical changes of a material over a range of temperature. Briefly, energy is introduced simultaneously into a sample cell containing the PPN-loaded gellan bead, and reference cells containing either the blank gellan bead or PPN only. Across temperatures, the heat flow values in all cells were measured, and the data was summarized in a plot called thermogram. A rise in the heat flow indicates an exothermic response, while a decrease implies an endothermic response. Since the changes observed are only in terms of the PPN’s phase, an endothermic peak shows at which point the material has evaporated. Comparing the thermograms from sample and reference cells would reveal whether the addition of gellan gum to PPN would result to a prolonged stability of PPN, as indicated by a higher temperature of endothermic peak when compared to that of PPN alone. In this experiment, Mettler DSC (fig. 7), was employed to analyse the DSC sample curves of 5-10 mg. The measurements were carried out under nitrogen (N2) at flow rate of 50ml/min, temperatures from 25°C to 500°C, and heating rate of 5°C per min. Fig. 7 DSC machine In vitro drug release studies To characterize the release and subsequent absorption of PPN from the GG beads, in comparison with the naked GG and PPN, an USP dissolution apparatus was used. This was done by obtaining an aliquot from the dissolution medium over time, and subsequently assaying for their concentrations. For the formulation to be effective, the GG should allow a sustained PPN release, indicated by increasing cumulative concentration over time. Particularly, the UPS Dissolution apparatus 1 (fig. 8) was used for this experiment’s drug release study. With the amount of drug containing 20 mg PPN in 250 ml dissolution medium (0.1 M HCl in phosphate buffer, pH = 6.8), the baskets were rotated at a rate of 50 rev/minute. The temperature was 37°±0.5°C. After the machine automatically obtained aliquots over time, the amount of PPN released was also analyzed automatically at a wavelength of 289 nm. Fig. 8. USP dissolution test which used to see the release of drug from the beads at two different medium( buffer pH6.8 and Acid 0.1HCl) References Kedzierewicz, F., Lombry, C., Rios, R., Hoffman, M., and Maincent, P.. 1999. Effect of the formulation on the in-vitro release of propranolol from gellan beads. International Journal of Pharmaceutics, 178(1), pp. 129-136. Patil, P., Chavanke, D., and Wagh, M., 2012. A Review of Ionotropic Gelation Method: Novel Approach for Gastroretentive Gelispheres. International Journal of Pharmacy and Pharmaceutical Sciences, 4(suppl 4), pp. 27-32. Read More