14 μg, indicating a good affinity Temsirolimus between the BSA and RhB. This was governed by Fickian diffusion due to the electrostatic interaction, which restricted the release of positively charged RhB from negatively charged BSA in vitro. In vitrocytocompatibility study In vitro experiment of BSA-NPs cross-linked with GA or denatured by heat against L929 cell lines were performed by CCK-8 to evaluate the cytocompatibility. As shown in Figure  3, cell mTOR target viability of NP-GA was significantly lower (P = 0.001) than that of the control possibly because the water wash in this study was only once. These results indicated that the NP-H had a better cytocompatibility

than the NP-GA. The slight cytotoxicity of NP-GA was in agreement see more with that reported by Speer [20]. There was no statistical difference between the NP-H (P = 0.114) and the control. Figure 3 Cytotoxity

evaluation of BSA-NPs fixed by GA or denatured by heat against L929 cells. Each value represents mean ± SD (n = 3) (**P < 0.01). The shape of L929 cells incubated with NP-H maintained high viability after the assay (Figure  4d) while round-shaped cells could be observed in the control and NP-GA groups (Figure  4b,c). This indicated that the addition of nontoxic NP-H might provide nutrition and promote cell proliferation due to the hydrophobic domain of such natural protein, just as the silk fibroin particles did [8]. But the nutrition property of BSA on cell proliferation cannot compensate the side effect of GA in the system, which explained the fact that most cells died with the addition of NP-GA Thalidomide (Figure  4c). The above findings disclosed that BSA was not only a soft material with good biocompatibility but also a nutrition provider. Further studies will focus on the assessment of BSA-NP drug delivery in the treatment of inner ear disorders. Figure 4 Morphology of L929 cells cultured with different conditions. L929 cells cultured in DMEM-10% FBS as the control (a), after performing CCK-8 assay (b), with the addition of NP-GA (c) and NP-H (d), are demonstrated respectively. All images

have an original magnification of × 200. In vivodistribution and drug delivery of BSA-NPs As for the good cytocompatibility, BSA-NPs with heat denaturation were loaded with RhB and used to evaluate the local drug delivery. Acoustic bullae of guinea pigs with entire RWM were isolated and injected with RhB-BSA-NP (right ear) and RhB solution (left ear). The live images were taken immediately (Figure  5a). Three days later, there was still obvious fluorescent signals with a larger area in the right ear (Figure  5b), which indicated that RhB-BSA-NPs was retained nearby the RWM and RhB possibly diffused into the Eustachian tube and the inner ear. We assumed that the BSA-NPs maybe useful for local drug delivery and controlled release.