The data reported were particle size, evaluated as the intensity obtained from three repeat measurements, and the polydispersity index [20]. Before measurement of zeta potential, nanoparticulate dispersions were diluted with MLN8237 chemical structure filtered

1mM NaCl solution (Millex-HV Filter, 0.45 μm, Millipore, Billerica, MA, USA) up to a count rate of 100 to 1000Kcps. All measurements were performed in triplicate. Evaluation of Entrapment Efficiency of NPs. Entrapment efficiency of DRZ loaded NPs was determined according to the previously reported method [20]. For the determination of the entrapment efficiency, the NPs were first separated from the aqueous suspension medium by ultracentrifugation Inhibitors,research,lifescience,medical at 40,000rpm for 30min using Optima Max XP ultracentrifuge (Beckman Coulter, USA). The entrapment efficiency was determined in triplicate

indirectly by analyzing the amount of free DRZ in supernatant. The free DRZ in supernatant was quantified by validated UV spectrophotometric method at 254nm. The entrapment efficiency of DRZ NPs was Inhibitors,research,lifescience,medical calculated as follows: Entrapment  efficiency=[Total  amount  of  DRZ  loaded−Free  DRZ  in  supernatant]Total  amount  of  DRZ  loaded  ×100. (3) In Vitro Drug Release of NPs. The in Inhibitors,research,lifescience,medical vitro drug release profiles of optimized, lyophilized, and sterilized DRZ loaded NPs were determined in 50mL simulated tear fluid, pH 7.4 (STF) using dialysis bags (Himedia Laboratories, India) at 37°C under magnetic stirring. At predetermined time intervals, 5mL aliquots were withdrawn from the medium and analyzed for DRZ Inhibitors,research,lifescience,medical by validated UV spectrometry. To determine the release behavior of free drug, in vitro study of DRZ solution (2% w/v) was also performed as control experiment [20]. Studies were performed in triplicates and data was analyzed for release kinetics. Inhibitors,research,lifescience,medical In Vitro Mucoadhesion of NPs. The binding efficiency of mucin to NPs was determined by mixing 2mL of mucin (625μg/mL) with the same volume of NPs. NPs were previously ultra centrifuged at 40,000rpm for 30min and resuspended in distilled water. After incubation at 37°C for 30min, the samples

were ultracentrifuged at 40,000rpm for 30min. The concentration of free mucin in the supernatant was determined at 555nm by periodic acid/Schiff (PAS) colorimetric method [21, 22]. The mucin binding efficiency of NPs was calculated from the following equation: Mucin  binding  efficiency  (%)=[(C0−Cs)C0]×100, out (4) where C0 is the initial concentration of mucin used for incubation and Cs is the concentration of free mucin in the supernatant. FT-IR Spectroscopy and DSC Analysis of NPs. IR spectra and DSC thermograms of lyophilized OCM-CS and CS NPs (without cryoprotectant) were obtained to study any possible interaction and to characterize the thermal behavior between polymers and DRZ. Morphological Characterization of NPs. Transmission electron microscopy (TEM) was used to examine the morphology of the OCM-CSNs.