Formulation Optimization and In Vivo Evaluation of Gamma Oryzanol Loaded Chitosan Nanoparticles

Abstract

The present research work is aimed at the development and optimization of gamma oryzanol loaded chitosan nanoparticles and to study its antihyperlipidemic and anticancer activities in vivo using animal model and cell line respectively. Drugs characterization and its compatibility with excipients were studied using FTIR. Gamma oryzanol chitosan nanoparticles were prepared by two methods, ionic gelation and coacervation phase separation using sodium tripolyphosphate and glutaraldehyde as crosslinking agent respectively. Effect of various parameters such as chitosan and TPP mass ratio, drug to polymer ratio, stirring speed and acetic acid concentration have been taken into consideration while preparing the nanoparticles. 23 full factorial design was applied for the optimization of final formulation by taking drug: polymer (X1), polymer: crosslinking agent ratio (X2) and stirring speed (X3) as independent variables. Optimized batch was then freeze dried and liquid and freezed dried nanoparticles were kept for stability studies. The selected batch was evaluated for % in vitro drug release in gastric media and PBS 7.4, in vitro permeation studies, zeta potential and particle size, mucoadhesion, TEM analysis, in vivo antihyperlipidemic study in poloxamer-407 induced hyperlipidemia acute animal model and cell line study on HEP-3P human carcinoma cell line using MTT assay. FTIR spectra indicated that drug was pure and compatible with all selected excipients. Ionic gelation method was selected over coacervation phase separation method for further optimization due to the low toxicity, absence of organic solvent and above of all, better entrapment efficiency and required particle size. From factorial batches, batch having composition of factor X1 (1:2), X2 (2:1) and X3 (600) showed 141 nm average particle size, 64.4% drug entrapment efficiency and 13.2, 43.4 & 59.6 % drug release at 2 hr, 12 hr and 24 hr respectively was selected as optimized batch. Furthermore, optimized batch showed 6.45 mV zeta potential, 83% mucoadhesion. TEM images showed discrete, evenly distributed spherical particles inside the polymer matrix. Formulation showed less drug release in the gastric media as compared to the intestinal media. However, ~ 100% drug release was achieved in 36 hrs in PBS 7.4 from the drug loaded chitosan nanoparticels. Freeze dried nanoparticles of scaled up batch showed good storage stability up to 3 months when kept under refrigerated conditions. FTIR analysis indicated the presence of crosslinking between the chitosan and TPP along with the entrapment of the drug. In vivo antihyperlipidemic study, g-OZ loaded CS NP has showed improvement in serum lipid profile (TC, TG, LDL, VLDL, HDL) along with the decrease in the coronary risk factors and oxidative stress. Formulation was also found to prolong the APTT and PT time along with the less endothelial and lipid core damage seen on histopathological examination showing its positive effect in decreasing the risk of artherothrombotic events in the body. Cell line study showed that g-oryzanol loaded CS NP showed 83.34% cell inhibition in 48 hrs which showed its therapeutic potential. The optimized g-OZ loaded CS NP found to have potential therapeutic effect in the prevention of antihyperlipidemia and cancer.