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  • br Fig CLSM images of MCF cells treated with


    Fig. 10. CLSM images of MCF-7 2-Guanidinoethylmercaptosuccinic Acid treated with DHHC-GNRAH + NIR and DOX-DHHC-GNRAH + NIR. The lower left half of each well of culture plates was exposed to NIR irradia-tion, and the upper right half was kept in the dark. Viable cells were stained green with calcein AM and dead cells were stained red with EthD-1. (For interpretation of the refer-ences to colour in this figure legend, the reader is referred to the web version of this article).
    ensure equivalent therapeutic effect. Up to now, it is still an unfulfilled difficult problem how to keep the balance between effective anticancer dose and minimizing toxic side effects for tumor patients (Zhou et al., 2017). Our DOX-DHHC-GNRAH might have great potential for addres-sing the above-mentioned problem. Flow cytometry was also used to assess the apoptosis of MCF-7 cells treated with different formulations at a GNR concentration of 20 μg/mL. Annexin V-EGFP/PI apoptosis detection kit was used to distinguish viable cells from apoptotic or dead cells. As shown in Fig. 9D, the cell viability (39.6%) in DOX-DHHC-GNRAH + NIR group was obviously lower than those in other groups, which is in accordance with the results of MTT assay, further con-firming the effect of combined chemotherapy and photothermal therapy.
    To visualize the therapeutic effect of combined photothermal che-motherapy in vitro, MCF-7 cells were incubated with DHHC-GNRAH and DOX-DHHC-GNRAH for 24 h and then treated with NIR irradiation (2.0 W/cm2) for 10 min. Calcein AM and EthD-1 were used to stain viable and dead cells, respectively. As shown in Fig. 10, a large number of cells were destroyed in both DHHC-GNRAH and DOX-DHHC-GNRAH groups after exposure to NIR irradiation, demonstrating the effective GNR-based photothermal ablation. Moreover, compared with DHHC-GNRAH group, DOX-DHHC-GNRAH group showed a higher cytotoxicity in the NIR irradiation area and a circular boundary between living and dead cells was clearly observed. These results suggest that DOX-DHHC-GNRAH could more effectively destroy MCF-7 cells via the combination of DOX-mediated chemotherapy and GNR-based photothermal therapy. In a word, the in vitro studies consistently demonstrate that our DOX-DHHC-GNRAH has great potential as an effective and synergistic an-ticancer strategy.
    4. Conclusions
    A novel GNR-based conjugate DOX-DHHC-GNRAH was successfully developed for targeted delivery of DOX and combined photothermal-chemotherapy of breast cancer. DOX was loaded onto the conjugate via an acid-labile hydrazone linkage, and the DOX-loaded conjugate was decorated with AHA through Schiff base chemistry and electrostatic interaction. As we expected, the conjugate exhibited pH-responsive DOX release and surface charge reversal behaviors, desirable stability, and excellent photothermal conversion efficiency under physiological conditions. The results of in vitro studies showed that DHHC-GNRAH exhibited negligible cytotoxicity and could efficiently deliver DOX into breast cancer MCF-7 cells via active targeting. Under NIR light irra-diation, DOX-DHHC-GNRAH had excellent synergistic therapeutic effect of combined photothermal-chemotherapy in MCF-7 cells. However, 
    there are still several limitations for our nanoconjugate to be overcome, including relatively low DOX loading content and suboptimal targeting ability.
    This work was supported by Key Science and Technology Program of Shaanxi Province, China [grant numbers: 2017SF-179 and 2017SF-190] and The Foundation of First Affiliated Hospital of Xi’an Jiaotong University [grant number: 2016MS-04]. We thank Mr Chao Li at Instrument Analysis Center of Xi'an Jiaotong University for their as-sistance with HRTEM analysis.
    Appendix A. Supplementary data
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