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  • br Please cite this article as Mei D et al


    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,
    + MODEL
    Priming autophagic cell death with transferrin receptor-targeted nanomedicine 5
    a dose of 100 mg/kg, respectively. For the study of the effect of co-administration on the bio-distribution of nanocarriers, mice were injected with 7pep-M-DiD, 7pep-M-DiR, 7pep-M-DiD plus 7pep-M-DiR, 7pep-M-DiD (pre-injected for 24 h) plus 7pep-M-DiR, respectively. At the predetermined time points, the mice were anaesthetized by isoflurane and imaged by an in vivo imaging system (Carestream Health, Fx Pro, USA). The fluorescent images were taken with an excitation at 748 nm and an emission at 780 nm for DiR, as well as an excitation at 630 nm and an emission at 700 nm for DiD. After imaging at the last time point, the mice were sacrificed, then tumors and major organs were excised to collect ex vivo fluorescent images.
    2.8. In vivo therapeutic efficacy and toxicity studies
    The MCF-7 tumor-bearing nude mice model described above was used to evaluate the therapeutic efficacy and toxicity in vivo. When the tumor volume reached 30e50 mm3, mice were injected with different RAP and PTX formulations in single or combina-tion after randomization, including PBS (control), free RAP, M-RAP, 7pep-M-RAP, free PTX, M-PTX, 7pep-M-PTX, Free Combi, M-Combi and 7pep-M-Combi. Both free RAP and free PTX are prepared with Cremophor EL and ethanol (1:1, v/v). The dosage of RAP or PTX was both 10 mg/kg. Tumor volumes and body weights were measured every one day. Tumor volume was calculated by the following formula: V Z 1/2
    (Length) (Width)2. Relative body weight was calculated as follow: Relative body weight Z Body weight/Primary body weight.
    At day 18, blood was collected from the orbital sinus, and white blood Galactose 1-phosphate  (WBC), neutrophils (GRN) and platelets (PLT) were counted to assess the myelosuppressive toxicity in each administration group. At day 20, all animals were sacrificed, and tumors and livers were then dissected. The excised tumors were weighed and pictured to assess the in vivo antitumor efficacy. Afterwards, frozen sections made by tumor tissues from each group were stained with TUNEL in situ cell death detection kit or treated with TfR antibody (PE labeled-mouse antihuman CD71 antibody), followed by imaged with CLSM. Meanwhile, the other tumor tissues from each group were fixed to evaluate the apoptosis and autophagy of tumor cells by TEM. Lastly, paraffin section made by excised livers were subjected to H&E staining, observed by light microscope to evaluate the organ toxicity.
    2.9. Statistical analysis
    All quantitative results are reported as mean standard deviation of the mean (SD) unless otherwise specified. Statistical signifi-cance was analyzed using two-tailed Student’s t-test or one-way
    analysis of variance (ANOVA) followed by a Tukey-Kramer multiple comparison test. A P-value less than 0.05 is considered statistically significant, while P less than 0.01 was considered highly significant.
    3. Results and discussion
    3.1. Preparation and characterization of transferrin receptor-targeted micelles
    3.1.1. Synthesis of 7peptide conjugated PEG-DSPE polymer 7pep was conjugated to the distal end of PEG through a nucleo-philic substitution reaction (Supporting Information Scheme S1). As shown in Supporting Information Fig. S1, the retention time for 7pep monitored by RP-HPLC with gradient elution was around 8.5 min. The peak of 7pep almost disappeared after 120 h reaction, indicating that 7pep had been successfully linked with NHS-PEG-DSPE. As displayed in Supporting Information Fig. S2, there was a strong absorption peak of 7pep at around 281 nm in the UV spectrum of 7pep-PEG-DSPE, while no such peak was observed for NHS-PEG-DSPE, suggesting that 7pep was successfully conjugated to NHS-PEG-DSPE. Moreover, the molecular weight (MW) of final product determined by MALDI-TOF MS was in accordance with theoretical MW (Supporting Information Fig. S3).