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  • br Fig A Cytotoxicity of GNR and DHHC GNRAH


    Fig. 9. (A) Cytotoxicity of GNR and DHHC-GNRAH after 24 h of incubation in the absence of DOX and NIR irradiation. (B) Viability of MCF-7 Bradykinin (acetate) incubated with different concentrations of free DOX, DHHC-GNRAH, [email protected] and DOX-DHHC-GNRAH for 24 h in the presence and absence of NIR irradiation (2.0 W/cm2, 10 min). (C) Comparison of synergistic efficacy of photothermal chemotherapy (DOX-DHHC-GNRAH + NIR irradiation) and additive efficacy of in-dependent PTT (DHHC-GNRAH + NIR irradiation) and chemotherapy (DOX-DHHC-GNRAH). (D)Apoptotic effects of MCF-7 cells treated with PBS, free DOX, DHHC-GNRAH, DOX- DHHC-GNRAH, [email protected] + NIR irradiation, and DOX-DHHC-GNRAH + NIR irradiation. Statistically significant differences (p < 0.05) are marked with asterisks.
    amount of Au in MCF-7 cells. The number of GNRs per cell could be calculated according to a reported method (Qiu et al., 2010), and the results are shown in Fig. 8D. As indicated in Fig. 8D, the GNR number in MCF-7 cells in both DOX-DHHC-GNRAH and [email protected] groups increased obviously with incubation time. After incubation for 24 h, the GNR number in DOX-DHHC-GNRAH group was about 2.4 × 105 per cell that was 1.5 times higher than that (1.6 × 105 per cell) in [email protected] group. All the results of CLSM, flow cytometry and ICP-MS analysis demonstrated that DOX-DHHC-GNRAH could be efficiently internalized by MCF-7 cells via HA-mediated active targeting. This is of great importance for combined photothermal che-motherapy of breast cancer.
    3.7. In vitro biocompatibility and photothermal chemotherapy
    The cytotoxicities of both pristine GNR and empty DHHC-GNRAH conjugate were evaluated using the MTT assay, and the results are shown in Fig. 9A. As displayed in Fig. 9A, the cell viability in GNR group obviously decreased when GNR concentration increased up to 10 μg/mL. The noticeable toxicity was attributed to the presence of toxic CTAB molecules on the GNR surface (Qiu et al., 2010). In contrast, empty DHHC-GNRAH conjugate showed a negligible toxicity to MCF-7 cells within the experimental concentration range. The cells still re-tained high viability of about 80% even if its concentration was up to 80 μg/mL. This confirmed the excellent biocompatibility of DHHC-GNRAH conjugate.
    To verify the therapeutic effect of combined photothermal che-motherapy based on DOX-DHHC-GNRAH, MCF-7 cells were incubated with different formulations with or without NIR irradiation. Cell via-bility was evaluated by the MTT assay and the results are shown in Fig. 9B. It can be seen from Fig. 9B that the viability of MCF-7 cells was concentration-dependent. The cell viability in free DOX + NIR group was approximately equal to that in free DOX group, indicating that NIR 
    laser did not cause obvious cytotoxicity on MCF-7 cells. The DHHC-GNRAH + NIR group showed obvious cytotoxicity and the cell viability decreased to 34.6% when the GNR concentration was 80 μg/mL, in-dicating the excellent PTT effect of DHHC-GNRAH. Compared with free DOX group, DOX-loaded conjugate groups (DOX-DHHC-GNRAH and [email protected]) displayed lower cytotoxicity, which is due to the gradual release of DOX from the conjugate (Zhang et al., 2014). Furthermore, DOX-DHHC-GNRAH group showed a lower cell viability than [email protected] group at the equivalent concentration of DOX. The lower cell viability was the result of higher cellular uptake (Zhou et al., 2017). This suggests that the HA moiety on DOX-DHHC-GNRAH facilitated CD44 receptor-mediated endocytosis, resulting in higher cytotoxicity. When the GNR concentration was 20 μg/mL, DOX-DHHC-GNRAH + NIR group could effectively inhibit proliferation of MCF-7 cells, and cell viability greatly decreased to 43%. In contrast, the cell viabilities in DOX-DHHC-GNRAH and DHHC-GNRAH + NIR groups were 71% and 72%, respectively. Compared with DOX-DHHC-GNRAH and DHHC-GNRAH + NIR groups, DOX-DHHC-GNRAH + NIR group exhibited a much lower cell viability at the same GNR concentration. The enhanced therapeutic effect is attributed to combined photo-thermal chemotherapy. In addition, the synergistic and additive ther-apeutic efficacies of combined photothermal chemotherapy were cal-culated according to reported methods (2010b, 2017b, Li, Chen et al., 2010; Wang, Liu et al., 2017), and the results are shown in Fig. 9C. It was found that the therapeutic efficacy of DOX-DHHC-GNRAH + NIR was always higher than the additive therapeutic efficacy of che-motherapy (DOX-DHHC-GNRAH group) and photothermal therapy (DHHC-GNRAH +NIR group) at the same GNR concentration. This re-sult indicates that DOX-DHHC-GNRAH + NIR exhibited synergetic ef-fect rather than just the additive effect. The mechanism of synergistic effect is the result of heating-accelerated DOX diffusion and heating-induced sensitization of cancer cells to DOX. The synergistic effect implies that the maximum DOX dosage can be significantly reduced but