For TEM, a drop of diluted suspension of BSA-NPs was placed on the copper grid and the air-dried specimen was observed. For SEM, a drop of diluted

suspension was deposited on a silicon wafer. The air-dried sample was coated with gold and observed. RhB-BSA-NPs were observed by CLSM at an excitation wavelength of 555 nm and an emission wavelength of 580 nm. The BSA-NPs were dispersed in ultrapure water at a concentration of 0.1 mg/ml. The particle size and zeta potential determinations were performed by using a Malvern particle see more size analyzer (Zetasizer Nano-ZS, Malvern, UK). Drug loading capacity and encapsulation efficiency BSA-NPs (50 mg) were incubated with RhB (5 ~ 20 mg) for 2 h. After washing with ultrapure water, the supernatants were collected and analyzed for residual drug concentration by UV-vis analysis. The drug loading capacity and encapsulation efficiency were calculated as follows: Encapsulation efficiency (w / w%) = amount of RhB in BSA-NPs/RhB initially added × 100 find more In vitrodrug release behavior The assay was evaluated in a standard static diffusion cell at a speed of 100 rpm in a shaker at 37°C. The amount of RhB was evaluated using UV-vis spectrometer (560 nm). The amount of RhB released was evaluated at a series of time points, and the release curve was made accordingly. Cell biocompatibility assay Cells were seeded in 96-well plates

at a density of 1,000 cells/well. BSA-NPs with GA fixation (NP-GA) or heat denaturation (NP-H) were added to each well for a 24-h incubation. Cell viability was determined by CCK-8 assay. Untreated cells served as the control. The morphology of L929 cells in each group was also observed by using a phase contrast microscope. In vivoassay Guinea pigs were killed to sample the acoustic bullae (including the RWM). The acoustic bullae were placed in the solution of BSA-NPs and shaking for 30 min at 37°C. The air-dried specimens were observed by SEM. The penetration of RhB released from the RhB-BSA-NPs was evaluated by live images and microscopes. Guinea pigs were anaesthetized and the RWMs were exposed. The heat-denatured RhB-BSA-NPs and RhB dispersed in PBS were injected Urease slowly

into the bullae of the right and left ear, respectively. The left ear injected with RhB solution was the control. In vivo imaging system (Caliper IVIS imaging system, PerkinElmer, Waltham, MA, USA) was used to trace the particles at time points of 0 and 72 h. The RWM was then imaged by fluorescence microscopy and SEM to observe the distribution of RhB and BSA-NPs. Statistical analysis The statistical data was presented as the mean value and standard deviation. The analysis of t test was used in SPSS 12.0 to determine significant differences between groups, and P values less than 0.05 were considered statistically significant. Results and discussion Morphology of BSA-NPs BSA-NPs were prepared by the desolvation method in high yield (about 95%).