Hailong Zhang, Wei Zhang, Yong Zhou, Yuhua Jiang, Shupeng Li

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Dual Functional Mesoporous Silicon Nanoparticles Enhance the Radiosensitivity of VPA in Glioblastoma Hailong Zhang, Wei Zhang, Yong Zhou, Yuhua Jiang, Shupeng Li Translational Oncology Volume 10, Issue 2, Pages 229-240 (April 2017) DOI: 10.1016/j.tranon.2016.12.011 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Characterization of MSNs and VPA releasing profiling. (A) SEM image of MSNs. (B) TEM image of MSNs. (C) FTIR spectra of MSNs: nonfunctionalized (control), benzimidazole functionalized (BI-MSNs), amine functionalized (NH2/BI-MSNs), and folate grafted MSNs (FA/BI-MSNs). (D) Releasing profiling of beta-CD and VPA from FA/BI-MSNs in different condition. Release of VPA salt was monitored using HPLC-MS with positive mode. (E and F) Confocal microscopy images of HEK-293 (FR−, E) and C6 (FR+, F) cells after treatment with 10 μg/ml of MSN@FITC for 12hours. Images are taken and merged under green channel from FITC (488/520±10 nm) and blue channel from Hoechst 33258 (340/461 nm±10 nm). For HEK-293 cells where FR is not expressed, minimal unspecific binding of MSN@FITC could be found (E). For C6 cells expressing FRs, massive folic-MSN@FITC binding was observed (F). Translational Oncology 2017 10, 229-240DOI: (10.1016/j.tranon.2016.12.011) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Dual-function MSN showed specific binding and intracellular releasing. Confocal microscopy images of HEK-293 (FR−, A, C, E, G) and C6 (FR+, B, D, F, H) cells treated with doxorubicin-MSN@FITC (C to F) for 12hours. Images are taken and merged under green channel from FITC (488/520±10 nm), blue channel from Hoechst 33258 (340/461 nm±10 nm), and red channel from doxorubicin (470/585±10 nm). For HEK-293 cells where FRs were not expressed, minimal unspecific binding of MSN@FITC could be found. For C6 cells expressing FRs, massive folic-MSN@FITC binding (B, D, F, H) and intracellular releasing of doxorubucin could be observed (F). Translational Oncology 2017 10, 229-240DOI: (10.1016/j.tranon.2016.12.011) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Effect of VPA, MSNs, and VPA-MSNs on C6 and U87 glioma cell viability. C6 and U87 gliomas were treated with various concentrations of VPA, MSNs, and VPA-MSNs for 12 or 24hours, and cell viability were examined with MTT assay. Cell viability was calculated relative to untreated controls. Translational Oncology 2017 10, 229-240DOI: (10.1016/j.tranon.2016.12.011) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Flow cytometry of C6 and U87 cell apoptosis without irradiation. Flow cytometric analysis of cell apoptotic levels by PI and FITC–Annexin V fluorescence (A and B). The calculated apoptotic rates were shown in histograms (C and D). Without irradiation, no significant apoptosis was observed under VPA and MSNs treatment in both C6 and U87 cells. No distinguishable changes of apoptotic rate can be acquired from the calculation. There was also no significant apoptosis under VPA-MSNs treatment in pH 6.0 compared with VPA-MSNs treatment or the control in PBS (pH 7.4). Translational Oncology 2017 10, 229-240DOI: (10.1016/j.tranon.2016.12.011) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Flow cytometry of C6 and U87 cell apoptosis with 4- and 8-Gy radiation. (A) Cells were exposed to 4-Gy irradiation. A total of 100 μg/ml of nanoparticles caused increases in the apoptosis compared to the VPA group. A total of 100 μg/ml of nanoparticles treatment increased the apoptotic rate of C6, and in pH 6 conditions, the apoptotic rate was higher than in normal PH conditions. (B) Apoptotic rate was further increased with exposure to 8-Gy irradiation. (C and D). Histograms show changes of the apoptotic rate. With 4- and 8-Gy radiation, significant changes were observed in PH6 VPA-MSNs group compared with the VPA group or the control (*P<.05, **P<.01). Translational Oncology 2017 10, 229-240DOI: (10.1016/j.tranon.2016.12.011) Copyright © 2017 The Authors Terms and Conditions

Figure 6 The colony formation assay of C6 cells. The IR-induced clonogenic survival was significantly reduced under VPA-MSNs treatment compared to cells treated with MSNs or VPA alone. Furthermore, the remarkable decrease in the number of cells was observed under acid condition (pH 6.0) (*P<.05, **P<.01). Translational Oncology 2017 10, 229-240DOI: (10.1016/j.tranon.2016.12.011) Copyright © 2017 The Authors Terms and Conditions

Figure 7 Clonogenic survival after VPA, nanoparticles, and IR exposure. (A) Clonogenic assay of C6 cells treated with VPA/or MSNs combined with 8-Gy IR; photographs of Petri dishes in a representative experiment are shown. (B) Percentage inhibition of colony formation as shown in A. Values represent the mean from three independent experiments. Error bars indicate 1 SD. Translational Oncology 2017 10, 229-240DOI: (10.1016/j.tranon.2016.12.011) Copyright © 2017 The Authors Terms and Conditions

Figure 8 Western blotting analysis for P53, Bax, Bcl-2, caspase-3, and PARP. Different expression of apoptotic biomarkers (P53, Bax, Bcl-2, caspase-3, and PARP) under treatment with VPA-MSNs, VPA, and/or MSNs when exposed to X-ray (A and B). Quantitative expression changes of proteins P53, Bax, caspase 3, and Bcl-2 are shown in histograms (C and D) (*P<.05, **P<.01). Translational Oncology 2017 10, 229-240DOI: (10.1016/j.tranon.2016.12.011) Copyright © 2017 The Authors Terms and Conditions