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  • Eight week old male Balb c mice obtained from Southern

    2020-08-18

    Eight-week-old male Balb/c mice obtained from Southern Medical University. 106 1380723-44-3 were injected below the breast into the abdomen of the mice for subcutaneous tumor xenografts. When the tissues grew to 90–120 mm3 (8–10 days after injecting the cells), the mice were grouped randomly into five groups: (1) the control group, which was the saline group, received no treatment; (2) the saline + M + L group, in which the mice subsequent irradiation with a short-period laser (100 mW·cm−2) for 10 min and a static magnetic field (100 mT·cm−3); (3) the LMNs (0.2 μg·20 g−1) group, LMNs were directly inserted into the tumors of mice; (4) the LMNs + M + L (0.2 μg·20 g−1) group, in which LMNs were inserted into the tumors of mice from the skin surface, ac-companied by irradiation using a procedure similar to that employed for the M + L group; and (5) the BC/LMNs + M + L (0.2 μg·20 g−1) group, in which the mice received transdermal LMNs into the tumor from the skin surface, accompanied with irradiation using a procedure similar to that employed for the M + L group.
    And then, the BC/LMNs was immobilized on a medical adhesive tape pasted to the surface of tumor tissue. Mouse body weights and tumor volumes (1/2 × length × width2) were collected every two days. Tumor sizes were evaluated by using caliper measurements. Immunohistochemistry, prussian blue staining, inductively coupled plasma-emission spectra (ICP), hematoxylin eosin (HE) staining, and serological detection were utilized as we previously described [36]. The results are shown in Figs. 5 and 6, respectively.
    Fig. 2. Characterization of LMNs, BC membrane, and BC/LMNs. Measured grafting dose of FA (A), encapsulation and loading efficiency of DOX and HMME (B), size distribution in PBS and cell medium (C), (D) cumulative release of DOX and HMME data at various time and temperatures, as measured by UV–visible spectro-photometer and High Performance Liquid Chromatography (HPLC), (mean ± SD, n = 3). FTIR spectra (E), TGA (F), and XRD (G) of LMNs and BC/LMNs.
    2.10. Statistical analysis
    Statistical results were analyzed using the statistical software SPSS17.0. One-way analysis of variance (ANOVA) was used to analyze statistical differences between groups under different conditions, and the Student’s t-test was performed. p < 0.05 was considered statisti-cally significant (in vitro, mean ± SD, n = 3; in vivo, mean ± SD, n = 8).
    3. Results
    3.1. Characterization of BC/LMNs nanocomposites
    Firstly, as shown in Fig. 2A, the FA grafting ratios were 68.58%. The evaluated encapsulation efficiency for DOX is 82.23% and the loading efficiency for NIPAm-AA is 24.67%, as shown in Fig. 2B. Moreover, the evaluated encapsulation efficiency for HMME is 31.75% and the loading efficiency for NIPAm-AA is 63.4%. We determined the size distribution of the LMNs. As shown in Fig. 2C and Table S1, the particle size of LMN in the PBS was 106.36 ± 15 nm, means that the DOX and HMME were effectively loaded. Similarly, the particle size of LMN in the cell culture medium was around 105.71 nm, show that the LMNs with a good stability. BC membrane has a nano-sized nanofiber with diameters of 30–100 nm [14–16]. In the present study, more than 50% 
    Next, the possible interactions between BC and LMNs were analyzed using Fourier Transform Infrared Spectroscopy (FTIR) (Fig. 2E). The BC had peaks at 3444 cm−1 and 2919 cm−1, which were assigned to –OH and –CH stretching, respectively. In addition, peaks at 1375 cm−1 and 1033 cm−1 were observed assigning to –CH bending and characteristic sugar-ring vibration. The results suggest that the water bound to the BC membrane might be at least partially replaced by the highly hydrophilic LMNs in the BC membrane. We attributed the observed shifts in band in the BC/LMNs spectrum to the inter hydrogen bonds based on the functional groups of LMNs and BC membrane, which molecular are rich in carboxylate and hydroxyl groups, respectively. These results in-dicated that LMNs play a decisive role in the biophysical properties of the BC membrane. The presence of LMNs in the BC network exposes an increased number of hydrophilic groups, which could further increase the membrane hydrophilicity. Increased hydrophilicity of BC
    Fig. 3. Microscopic surface and functional group analysis. AFM (A) and SEM (B) images of LMNs and BC/LMNs. The green bar stands for 200 nm.
    membrane is great since it can be related to increasing doxorubicin-loading. Meanwhile, the absorption of BC, MHNP, DOX, HMME, LMNs, BC/LMNs in PBS in 400–770 nm were detected and the results shown in Fig. S1.
    The structural properties of BC loading LMNs were examined using XRD (Fig. 2G). The characteristic Bragg’s angles of BC and BC/LMNs for both matrices at 2θ = 14.6° and 22.8°, respectively. The XRD spectra of BC and BC/LMNs exhibited profiles similar to the peak positions and distributions. The results indicate that the addition of LMNs does not alter the crystalline structure of the BC. Changes displayed in the BC/ LMNs could be attributed to interactions between the highly hydro-xylated LMNs biopolymers through hydrogen bridges. Moreover, the porosity of BC/LMNs were lower than BC membrane (Fig. S2). The atomic percent of N from LMNs in the BC-LMNs could be observed (Table S2), and demonstrated the success of adsorption.