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  • br Statistical analysis br All statistical analyses


    2.11. Statistical analysis
    All statistical analyses were performed using SPSS software (version 18.0, SPSS Inc., Chicago, IL). Student’s t-test for unpaired data was used to evaluate statistical differences between mean val-ues. All data are presented as the mean ± standard deviation (SD), and data were considered statistically significant at P < 0.05.
    3. Results
    3.1. Synthesis and characterization of [email protected] nanoparticles
    HA-coated PAMAM-Pt-Dox was prepared as presented in Scheme 1. First, Pt and Dox were covalently conjugated with fourth-generation PAMAM (G4.0 NH2) through an esterification reaction (PAMAM-Pt-Dox). Then, PAMAM-Pt-Dox was conjugated with HA through electrostatic interactions to generate the target-ing nanocarrier [email protected] The prepared [email protected] was purified through the dialysis method [35] and then characterized by dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM), and zeta potential was calculated. TEM results demonstrated that [email protected] possessed a typical struc-ture with a spherical shape (Fig. 1, an enlarged Fig. 1 is presented in
    Fig. 1. Transmission electron microscopy images of different types of nanoparticles including PAMAM, [email protected], PAMAM-Pt, PAMAM-Pt-Dox, and [email protected]
    Table 1
    Characteristics of different types of nanoparticles.
    Nanoparticle Size (nm) Zeta potential (mV) PDI
    Fig S1). DLS results demonstrated that [email protected] had uniform size distribution and adequate polydispersity (Fig S1 and Table 1). As shown in Table 1, when conjugated with HA, the size of PAMAM-Pt-Dox was increased by approximately 10 nm, thereby indicating successful preparation of [email protected] The zeta potentials were maintained at 28.4 mV for the PAMAM-Pt and 26.6 mV for the PAMAM-Pt-Dox, respectively. In addition, after mixing with HA, the presence of the negatively charged HA resulted in a shift of the zeta potential to 6.8 mV. In addition, the [email protected] was characterized by FTIR (Fig. 2a and Fig S2), UV–Vis Oxidopamine spectroscopy, and fluorescence spec-troscopy (Fig. 2b, c). It was observed that the CH3 group (2800– 3000 cm 1) belonging to Pt and Dox could be found in FTIR results, thus suggesting that Pt and Dox have been conjugated with PAMAM. The reason is as follows: there is no CH3 group on PAMAM, whereas both Pt and Dox possess the CH3 group on their molecules, thus indicating that the existence of CH3 infers to the conjugation of Pt and Dox with PAMAM. Moreover, it could be observed from 1HNMR, which showed that the signal (6.9 ppm) was assigned to the protons of Dox (Fig. S3).
    The composition of PAMAM-Pt was characterized by X-ray pho-toelectron spectroscopy (XPS), which showed that PAMAM was mainly composed of C, O, and N elements. In addition, the binding energy of Pt4f7/2 and Pt4f5/2 was observed at 74.0 eV and 77.6 eV (Fig. S4), thus suggesting that Pt has been successfully conjugated with PAMAM. Based on the results of XPS analysis, it could be cal-culated that PAMAM-Pt contained 1.4 wt% of Pt. Moreover, as shown in Fig. 2a, the peak that was observed at 1400–1600 cm 1 
    could be ascribed to the stretching vibration of the benzene ring, whereas the peak at 2876 cm 1 was characteristic of C–H (CH3) stretching. As shown in Fig. 2b, the UV–Visible spectrum of PAMAM-Pt-Dox displayed a characteristic absorption peak of Dox at 480 nm. These results suggested that Dox was successfully con-jugated on PAMAM-Pt. Next, the loading efficiency of Pt and Dox on the PAMAM-Pt-Dox was evaluated by UV–Visible spectroscopy and inductively coupled plasma mass spectrometry, which showed that the drug-loading efficiency of Pt and Dox was 13.6% and 18.3%, respectively. In our study, we also investigated the stability of PAMAM-Pt-Dox and [email protected] in PBS (pH 7.4). As shown in Fig. S5, both PAMAM-Pt-Dox and [email protected] had adequate stability with a minimal change in size in 7 days.
    Next, the release study of Pt-Dox was studied in an acidic envi-ronment (pH 5.5) and under simulated physiological conditions (pH 7.4). As shown in Fig. 2d and S6, almost no release of Pt/Dox from PAMAM-Pt-Dox was observed within 2 h and 24 h at pH 5.5 and 7.4, respectively, thereby indicating that PAMAM-Pt-Dox was stable enough to prevent clearance from the blood before reaching the target organs.
    3.2. Cellular uptake of [email protected]
    The cellular uptake of [email protected] in MCF-7 and MDA-MB-231 breast cancer cells was investigated by confocal laser scanning microscopy. Considering the cytotoxicity of Dox on MDA-MB-231 cells, Cy7.5-labeled [email protected] was used for tracking the cellular uptake of [email protected], although Dox possessed red fluorescence. First, Cy7.5-labeled [email protected] was synthesized and used for intracellular tracking. As shown in Fig. 3, Cy7.5 fluorescence was observed in the red channel, whereas the lysosome was stained with LysoTracker in green. After 30 min of pretreatment with [email protected], red fluorescence was observed in MDA-MB-231 cells. The red fluorescent intensity was further increased after 6 and 12 h of incubation. These results indicated that the cellular uptake of [email protected] occurred in a time-dependent manner. The yellow staining in the merged image that was generated by superposition