br vesicle in the control group
vesicle in the control group. In contrast, different RAP formu-lations used in single or in combination induced a large number of green spots of autophagic vesicles. Only a small amount of autophagic vesicles was induced by free PTX alone. This may be due to the fact that the free drug is highly lipophilic and easily diffuses into MCF-7 Imipenem through the cell membrane, leading to strong cytotoxicity, which resulted in adaptive autophagy for damage caused by chemotherapeutic drug.
In a word, these observations obtained from different methods consistently proved that 7pep-M-RAP could induce significantly more accumulation of autophagic vesicles than M-RAP as well as different PTX formulations. What’s more, combination with PTX did not hinder the autophagy induction of 7pep-M-RAP.
3.3.2. Satisfactory synergistic chemotherapy with PTX
After verified the autophagy initiation effect of 7pep-M-RAP, we then investigated the synergy effect with chemotherapy. As exhibited in Supporting Information Figs. S13 and S14, both RAP and PTX preparations inhibited cell proliferation in a dose-dependent manner. Compared with non-modified nanocarriers, 7pep-modified micelles exhibited significantly higher inhibition and much lower IC50 value (Table 2). These findings suggested that 7pep modification enhanced the anti-tumor activity in vitro, which was in accordance with cellular uptake studies.
It is worth mentioning that, when combined free drugs were administrated simultaneously or free RAP was administered after PTX, no synergistic anti-tumor effect was observed (data not shown), while the scheme that free RAP was administered 12 h prior to PTX showed synergistic effect. This observation indicated that the suitable administration schedule and sequence are very crucial for the therapeutic outcome of combination therapy. Therefore, in conjunction with the results of cell uptake studies, the follow-up studies were conducted with regimen that RAP formulations were applied prior to the PTX. Besides, the in vitro cytotoxicity assay revealed that cell viability rates in the combi-nation groups with molar ratio of 1:10 for PTX and RAP was lower than those in groups with concentration ratios of 1:1 or 1:5 (data not shown). The growth inhibition experiment of RAP monotherapy found that MCF-7 cells are less sensitive to low-dose RAP but more sensitive to high-dose RAP treatments. Hence, the concentration ratio of RAP formulations to PTX formulations was set at 10:1 (mol/mol) to achieve better cytotoxicity. The relative doseeeffect curve is presented in Fig. 4A. Sequential adminis-tration of 7pep-M-PTX after 7pep-M-RAP led to greater prolif-eration suppression of tumor cells than 7pep-M-PTX used in single. The combination therapy of 7pep-M-RAP and 7pep-M-PTX achieved better inhibitory effect than combined use of M-RAP and M-PTX (Supporting Information Fig. S15). Additionally, the relevant IC50 and CI50 values of two drugs combined are summarized in Table 2 and Fig. 4B. The IC50 of each nano-medicine in combination group exhibited lower values compared with that of single drug group, implying that the co-administration strategy improved the cytotoxic sensitivity towards MCF-7 cells. The CI50 values of the three combination groups (CI50 Z 0.362, 0.511 and 0.546 for Free Combi, M-Combi, and 7pep-M-Combi, respectively) were all below 0.90, further demonstrating the syn-ergy effect of RAP and PTX in MCF-7 cells.
Annexin V-FITC/PI apoptosis detections were utilized to further verify the synergy effects of combinational treatments on MCF-7 cells. Qualitative and quantitative results are displayed in Fig. 4C and Supporting Information Fig. S16, respectively. Compared with control group, the blank 7pep-modified micelles