The samples were then subjected to centrifugation at 9000▒rpm for 10▒min and the supernatant used to determine the concentration of soluble

protein. Next, 1▒ml of 6▒M urea was added to the pellet to dissolve the water-insoluble protein fraction. In all cases, a clear solution without noticeable light scattering was obtained and used to determine the concentration of aggregated protein by measuring the UV absorbance at 280▒nm and by BCA assay at 562▒nm. The encapsulation efficiency was calculated from the actual and theoretical loading of protein in the nanospheres. The experiments were performed in triplicate, the results averaged, and the standard deviations calculated to highlight the reproducibility of the experiments. To find more determine Bortezomib the enzyme activity after encapsulation, ethyl acetate was used to dissolve PLGA because it does not cause enzyme inactivation in the process [27]. Activity of a-chymotrypsin was determined using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrate [28]. The reaction was carried out in 1▒ml of 0.1▒M Tris-HCl buffer containing 0.05▒mg/ml enzyme, 0.35▒mM substrate, and 10▒mM CaCl2 at pH 7.8 and our data for a-chymotrypsin as purchased are comparable to those reported [28]. The activity of 0.01▒mg/ml

lysozyme was determined by measuring the decrease in turbidity at 450▒nm of a 0.015% (w/v) suspension of Micrococcus lysodeikticus cells in 1▒ml of 66▒mM potassium phosphate buffer at pH 6.2 and 25▒°C as described by us [ 29]. The peroxidase-like activity of Cyt-c which is not a natural enzyme was obtained as described [ 30]. Briefly, the reaction was followed at 415▒nm using 0.25▒ml of 0.01▒mg/ml Cyt-c, 0.2▒ml of 300▒mM H2O2, and 0.55▒ml of 0.05▒mM 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) in 20▒mM potassium phosphate buffer

at pH 7. The data obtained by us for commercial Cyt-c are comparable to those reported in the literature [ 30]. The activity was obtained by plotting the time-dependent absorbance changes vs. time. The linear portions of the graphs at less than 10% substrate conversion were used to obtain the initial velocities (V0). In all cases the specific activity (mM of substrate converted into product per min and per mg of protein) was calculated. The experiments were performed in triplicate and the results averaged and the standard Methocarbamol deviations calculated. In vitro release studies were conducted as described by us in the past [[26], [27], [28] and [29]]. In brief, nanospheres (30▒mg) were placed in 1▒ml of 10▒mM phosphate buffered saline (PBS) at pH 7.3 and incubated at 37▒°C. At pre-determined times (typically every 24▒h) the supernatant was removed after a short centrifugation. The concentration of the released protein in the supernatant was determined by absorbance measurement at 280▒nm (the absorbance was corrected by the very small absorbance produced by degrading empty PLGA nanospheres).