• 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • br Statistical analysis br All data were


    2.10. Statistical analysis
    All data were presented as means ± standard deviation (SD). Statistical analysis was conducted by Student’s t-test. p < 0.05 was considered statistically significant.
    3. Results and discussion
    3.1. Preparation and characterization of DOX-DHHC-GNRAH conjugate
    To obtain a GNR/DOX-based combination anticancer strategy in breast cancer treatment, a novel conjugate DOX-DHHC-GNRAH was designed and synthesized, and the corresponding schematic diagram is shown in Fig. 1. DHHC was first synthesized and its chemical structure is displayed in Fig. 1A. Subsequently, DHHC was chemically conjugated onto GNR surface via Au-catechol bonds. DOX was loaded onto the conjugate through a hydrazone linkage between the carbonyl group of DOX and the acylhydrazine group of DHHC. AHA was decorated on the DOX-loaded DHHC-GNR conjugate via Schiff Stearamide chemistry and
    Fig. 1. Schematic diagram of the preparation process of DOX-DHHC-GNRAH and its application for combined photothermal-chemotherapy of breast cancer. (A) Chemical structure of DHHC. (B) Preparation process of DOX-DHHC-GNRAH. (C) Combined chemotherapy and PTT in breast cancer MCF-7 cells.
    electrostatic interaction. The AHA molecules bounded on the conjugate acted as active targeting moiety to interact with CD44 receptors that are overexpressed in breast cancer (Zhong et al., 2016). The final DOX-DHHC-GNRAH was expected to exhibit pH-sensitive surface charge-re-versal behavior due to the dissociation of Schiff base and the amino protonation in acidic environment (Fig. 1B). The conjugate with posi-tively charged surface can not only significantly improve the cellular uptake of the conjugate but also facilitate its endosomal/lysosomal escape because of favorable electrostatic interaction between plasma membranes and the conjugate. Once the conjugate enters tumor cells, DOX will be released due to acid-induced breakage of hydrazone linkage. Under NIR irradiation, GNR can absorb NIR light and convert into heat that may not only kill cancer cells but also accelerate the diffusion of DOX. Obviously, breast cancer MCF-7 cells can be effec-tively killed by the combination of chemotherapy and PTT.
    According to our previous studies, AHA and HECS were synthesized and characterized by 1H-NMR, and the results are shown in Fig. S1 and 2A, respectively. The oxidation degree of AHA was calculated to be about 16.58%, and the degree of substitution of hydroxyethyl groups of HECS was approximately 72.58%. HECS was subsequently modified with DA to obtain HECS-DA and its 1H NMR spectrum is presented in Fig. 2B. Compared with the 1H NMR spectrum of HECS, some new chemical shifts at 2.84 and 6.59–6.88 ppm attributed to the protons of methylene and phenyl group of DA appeared, suggesting the successful conjugation of DA onto HECS (Yang et al., 2014). The grafting degree of DA onto HECS was about 5.7%. The 1H NMR spectrum of DHHC is displayed in Fig. 2C. The chemical shifts at 1.19, 1.50, 3.17 and 3.79 ppm were ascribed to the protons of methylene and methine of MA, and the modification degree of MA onto DHHC was about 15.1%. The signals at 1.99–2.41 ppm were due to the methylene protons of MPH, which indicated that MPH was successfully grafted onto HECS via bridging MA molecule. These results confirm that DHHC was success-fully synthesized and could act as a bridging macromolecule to con-jugate GNR, DOX and AHA.
    FT-IR spectroscopy was used to characterize the chemical structures of AHA and DHHC and confirm their successful decoration on the GNR surface. As shown in Fig. 3A, the FT-IR spectrum of GNR showed two strong absorption peaks at 2918 and 2848 cm−1, indicating the 
    existence of a large number of CTAB molecules (Tang et al., 2012). In the FT-IR spectrum of DHHC-GNR, the two bands at 2922-2857 and 1108-1028 cm−1 were attributed to the stretching vibrations of me-thylene and OeCeO groups of DHHC, respectively (An, Thien, Dong, & Dung, 2009). These results suggest that CTAB molecules on the GNR surface were successfully replaced by DHHC. In the FT-IR spectrum of DHHC-GNRAH, a new peak at 1047 cm−1 due to the CeOH stretching vibrations of AHA molecules was observed (Gilli, Kacuráková, Mathlouthi, Navarini, & Paoletti, 1994), confirming the successful