br The thermal properties of native BC
The thermal properties of native BC and BC/LMN were detected by thermogravimetric analysis (TGA) (Fig. 2F). The unbound water con-tent was observed in the temperature range between 30 °C and 80 °C. The percentage weight loss was approximately 2.72% for BC and 11.45% for BC/LMNs at 520 °C. The results show higher water content and stability compared to the BC membrane, since the LMNs have high hydrophilicity. The increased hydrophilicity and those of stability in the BC membrane could facilitate maximize drug loading in water-soluble molecules such as doxorubicin.
Finally, the SEM and AFM results indicate an inhomogeneous structure, which was attributed to a dense crossover network with a porosity that could adsorb water-soluble substances. Fibrils increased the random structure of BC membranes, which might reduce the strength and stability of the BC membrane (Fig. 3). In addition, the surfaces of BC and BC/LMNs with diﬀerent characteristics, the LMN particles were spherical shaped in morphology and homogeneous in distribution. The BC surface showed a typical network composed of entangled cellulose nanofibrils. And, the BC/LMNs was homogenous with a strong cooperative network. These results show that the reduc-tion of crystallinity can be attributed to the interconnection between
3.2. In vitro combination of chemotherapy and PDT for breast cancer
We investigated whether LMNs could exert tumor suppressor eﬀects in vitro. Results from the CCK-8 test showed that the growth of MCF7 Ac-DEVD-CHO transfected with M only, L only, and M + L for 24 h significantly decreased (p < 0.05) when compared with non-treated control group (Fig. S3A1-3). These results have shown a great targeting ability of LMNs to breast cancer cells. The results showed that the IC50 value was sig-nificantly (p < 0.05) delayed at 24 h, inhibiting the growth of these cells to 50% at 8 μg/100 μL in comparison with the non-treated control group (Fig. S3B). The tolerance study with MHNPs and LMNs were performed in vitro and showed no alterations in the inhibited the growth of human skin fibroblasts (HSF) cells, as shown in Fig. S3C and D. Results from the Flow Cytometry and FDA/PI staining showed that the growth of MCF7 cells treated with the LMNs + M, LMNs + L, LMNs + M + L and the BC/LMNs + M + L were significantly de-creased (p < 0.05) in comparison with non-treated control cells (Fig. 4A, B and C, Fig. S3E, F, G and H). Meanwhile, in order to evaluate the contribution of LMNs in cell migration and invasion, we performed scratch/wound-healing. The monolayer restoration was significantly delayed by 82% in MCF7 cells when compared with control cells at 24 h (Fig. 4D).
We also analyzed the activities of p53, Bcl-2, and Bax in response to treatments with saline only (NS), saline + M + L, LMNs only, LMNs + M + L, and BC/LMNs + M + L (n = 3 for each group) (Fig. 4E). The expression of p53, Bcl-2, and Bax at 53 and 26 kDa ex-hibited more significant down-regulation in the LMNs + M + L and BC/LMNs + M + L at 24 h than the M + L or LMNs only treatments with a similar period. In addition, Bax expression at 21 kDa was sig-nificantly up-regulation. The p53 and Bcl-2 data suggest that the p53 in the outer membrane of the MCF7 cells induced by the LMNs might be up-regulated significantly.
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3.3. In vivo combination of chemotherapy and PDT for breast cancer
In vivo chemotherapy and PDT were measured out on Balb/c mice bearing MCF7 cells with diﬀerent treatments. When the tumor volumes reached to 100 mm3, the mice were randomly divided into five groups randomly: saline only (NS), saline + M + L, LMNs only, LMNs + M + L, and BC/LMNs + M + L (n = 8 for each group). Based on the results, 633 nm laser irradiation was carried out 24 h after an i.v. injection of the saline and nanoparticle duet to achieve a high nano-particle accumulation.
As illustrated in Fig. 5A, no obvious changes can be observed in the tumor region of the saline + laser group after 10 min irradiation. In contrast, the tumor morphological changes in LMNs, LMNs + M + L, and BC/LMNs + M + L are obviously indicating the inhibition of the growth of tumor tissues. The higher inhibition in the LMN + M + L group was ascribed to the laser targeting ability of BC/LMNs + M + L to breast cancer cells, as well as the magnetic targeting ability. The eﬃcacy of in vivo chemotherapy and PDT was evaluated by measuring tumor volume every three days after treatment (Fig. 5B and C). In mice treated with saline only (NS), saline + M + L, LMNs only, LMNs + M + L, and BC/LMNs + M + L, the tumor volumes increased from about 102 ± 5.3 mm3 to approximately 208 ± 9.6 mm3 over 14 days.