br In vitro combination of chemotherapy and PDT for breast
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 MCC950 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.
40.8 ± 5.4 mm3 in the BC/LMNs + M + L group (TGI: 80.38%). Due to the FA receptor/magnetic/laser multi-targeting capability of LMNs, the tumor growth was eﬀectively inhibited. Therefore, LMNs and BC/ LMNs have a great potential to be used as powerful agents for in vivo chemotherapy and PDT in breast cancer treatment.
We evaluated the in vivo biocompatibility of LMNs by body weight changes (Fig. 5C middle) and living rate. No significant weight loss can be found, and the body weight increased in the control at approxi-mately the same rate as the LMNs injected group. The survival results of rat correlated to the therapy response were observed in Fig. 5C (right). Compared to other groups, the LMNs + M + L group showed improved survival significantly in 14 days. The immunohistochemical analysis (Fig. 5D) revealed reduced light brown staining in LMN group when compared to the control group, indicating that the expression of Ki67 and Bcl-2 decreased after LMN administration, which further supported the results of the in vitro experiments.