Double sided carbon tape was affixed on aluminum stubs. The powder sample was dispersed in the double distilled water and dispersion drop was put on the slide. Slide was allowed to dry and was placed on the aluminum stubs. The aluminum stubs were placed in the vacuum chamber of a scanning electron microscope (XL 30 ESEM with EDAX, Philips, The Netherlands). The samples were observed for morphological characterization using Inhibitors,research,lifescience,medical a gaseous secondary electron detector (XL 30, Philips, Eindhoven,

The Netherlands) with working pressure: 0.8Torr, acceleration voltage: 30.00KV. 2.2.5. Percentage of Drug Entrapment Efficiency and Percentage of Drug Loading The entrapment efficiency and drug loading of selected formulation were calculated by the AZD1480 following equation

[13]: % Drug  encapsulation  efficiency=Da−DsDa∗100,% Drug  loading=  Da−DsNa∗100, Inhibitors,research,lifescience,medical (1) where Da is the total amount of drug added in system, Ds is the amount of drug in supernatant after the centrifugation, and Na is the total amount of nanoparticles obtained. The amount Inhibitors,research,lifescience,medical of drug in supernatant was calculated from concentration values obtained from the calibration curve on spectrophotometric analysis of the samples at 475nm (Shimadzu UV 1800, Japan). 2.2.6. Statistical Analysis of Responses by Design Expert Design Expert 8.0.4. (Stat-Ease, Inc., USA) was used for the analysis of the effect of each variable on the designated response. The statistical significance of the difference in particle size, percentage of drug encapsulation, and percentage of drug loading was tested by one-way analysis of Inhibitors,research,lifescience,medical variance (ANOVA) using the following polynomial equation (2): Y=b0+b1X1+b2X2+b3X3+b1b2X1X2+b1b3X1X3+b2b3X2X3+b1b2b3X1X2X3, (2) where Y is the measured response, b0is the arithmetic mean response, b1 is the main effect of Chitosan concentration (X1), b2is the main effect of speed of homogenization (X2), andb3 is the main effect of TPP concentration (X3);b1b2,b1b3,b2b3,

andb1b2b3are the interactions of the main factors. The significant response polynomial equations generated Inhibitors,research,lifescience,medical by Design Expert were used to validate the statistical design. Quantitative and qualitative contributions of each variable on each of the responses were analyzed. Response surface plots were generated to visualize the simultaneous effect of each variable TCL on each response parameter. 2.2.7. Checkpoint Analysis A checkpoint analysis was performed to confirm the utility of the established polynomial equation in the preparation of rifampicin loaded Chitosan nanoparticles. Three checkpoint values of independent variables (X1, X2, and X3) were taken and the values of dependent variables were calculated by substituting the values in the respective polynomial equation (7). Rifampicin loaded Chitosan nanoparticles were prepared experimentally by taking the amounts of the independent variables (X1, X2, and X3). Each batch was prepared three times and mean values were determined.