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  • br Confocal microscope images Fig B further verified the

    2020-03-24


    Confocal microscope images (Fig. 2B) further verified the re-sults from flow cytometry. The intracellular fluorescence intensity in the 7pep-M-C6 group was greater than that of M-C6, and highly reduced by saturation of TfR. Intracellular distribution study was performed to validate the targeting efficiency of 7pep-M-C6. Fig. 2C and D manifested that 7pep-M-C6 colocalized more with lysosomes than M-C6 after being incubated with MCF-7 Conessine for 2 h. The different lysosomal distribution characteristics
    Figure 2 In vitro 7pep targeting efficiency in MCF-7 cells. (A) The competitive cellular uptake of various C6 formulations by flow cytometry. MCF-7 cells were incubated with M-C6 or 7pep-M-C6 at a final concentration of 100 ng/mL for 2 h at 37 C. For receptor competitive experiment, excess anti-TfR antibody was added 0.5 h prior to the 7pep-M-C6. (B) Confocal microscopy images of MCF-7 cells after incubation with M-C6 or 7pep-M-C6 for 2 h at 37 C. Green and blue represent the fluorescence of C6 and Hoechst 33258, respectively. The white scale bars represent 25 mm. (C) CLSM Images of colocalization of lysosomes (red) with C6-loaded micelles (green). The white bar scales represent 10 mm. (D) Quantitative colocalization analysis of micelles with lysosomes (mean SD, n Z 6). *P < 0.05 vs M-C6.
    Please cite this article as: Mei D et al., Actively priming autophagic cell death with novel transferrin receptor-targeted nanomedicine for synergistic chemotherapy against breast cancer, Acta Pharmaceutica Sinica B, https://doi.org/10.1016/j.apsb.2019.03.006
    + MODEL
    Priming autophagic cell death with transferrin receptor-targeted nanomedicine 7
    between 7pep-M-C6 and M-C6 revealed that the 7pep modifica-tion facilitated the faster and more internalization of micelles into cells34. It is worth mentioning that, because of this phenomenon of receptor saturation, the two active targeting nanomedicines which separately encapsulated RAP and PTX might have a competitive inhibitory effect on cellular uptake when administered simulta-neously. Therefore, we used HPLC method to investigate the cellular uptake of PTX micelles and RAP-loaded micelles added at the same time point or at certain intervals, thus determining the optimal dosing regimen for the combined administration. The results displayed in Supporting Information Fig. S11 verified our inferences. Based on this, we suggested that RAP and PTX for-mulations could be administered at certain intervals in following experiments, thus avoiding mutual inhibition of endocytosis.
    Totally, the above quantitative and qualitative results consis-tently demonstrated that the 7pep-modified nanocarriers could increase the delivery to TfR-overexpressing cells via a receptor-mediated endocytosis mechanism.
    3.3. Superior autophagy induction and synergistic chemotherapy of 7pep-M-RAP
    3.3.1. Active autophagy modulation of 7pep-M-RAP
    After verified the active targeting efficacy of 7pep-M, we then studied the autophagy inducing ability of 7pep-M-RAP alone or combination with 7pep-M-PTX. The autophagic vesicular accu-mulation in MCF-7 cells was characterized by the detection of autophagy marker protein LC3B maturation/aggregation, the ratios of LC3-II to LC3-I expression, MDC, as well as Cyto-ID autophagy detection kit. First, we investigated the effect of different treatments on the maturation/aggregation of LC3B protein by ELISA, which is produced during autophagosome formation and indicates the initiation of autophagy. As demon-strated in Fig. 3A, compared with control and PTX formulation groups, the quantity of LC3B obviously increased in MCF-7 cells treated with RAP formulations in single or combination use. During the formation of autophagosomes, the cytosolic LC3-I protein is activated by Atg7, and then transferred to Atg3, becoming a membrane-bound form LC3- II protein which is located on the autophagosome membranes. Thus, we further measured the conversion of LC3-I to LC3-II by immunoblot (Fig. 3B). Compared with control, the ratios of LC3-II to LC3-I in both 7pep-M-RAP group and combination group increased obviously (P < 0.05), but there was no significant difference between these two groups (P > 0.05). Then, the colocalization of the LC3B-labeled autophagic vesicles and lysosomes was observed by CLSM to monitor the formation of autolysosomes which are produced during the last stage of autophagy. The fluorescent images and semi-quantitative results in Fig. 3C revealed that both 7pep-M-RAP used alone and in combination caused aggregation of green labeled LC3B protein and higher colocalization rate than control and 7pep-M-PTX monotherapy group. Furthermore, MDC is a tracer for autophagic vacuoles, and the incorporation of MDC into MCF-7 cells was observed with fluorescence microscopy. As shown in Supporting Information Fig. S12, MCF-7 cells treated with 7pep-M-RAP alone or in combination with 7pep-M-PTX demonstrated a punctate pattern of MDC-labeled fluorescence. By contrast, cells in other groups exhibited a diffused distribution of MDC-labeled fluorescence. Cyto-ID is a novel dye that selectively labels autophagic vacuoles and monitors autophagic flux in live Conessine cells. As can be seen from Fig. 3D, there is almost no autophagic