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  • br that NCTD induced apoptosis via both extrinsic and intrinsic

    2022-05-22


    that NCTD induced apoptosis via both extrinsic and intrinsic pathways by activation of the CD95 receptor system and many Caspases as well as the regulation of Bcl-2 protein family [46]. In our study, CNC as a mac-romolecular derivative could change nuclear morphology of tumor Cyclophosphamide and induce a higher apoptosis rate compared with free NCTD at equiv-alent dose.
    3.6. In vivo antitumor efficacy
    To evaluate the antitumor efficacy of the conjugates in gastric cancer cell line, BALB/c nude mice were xenografted with SGC-7901 tumor cells. Fig. 4A showed the body weight variations during the treatments with normal saline, DDP, free NCTD and different concentrations of CNC. Fig. 4B depicted the tumors excised from mice after treatments with various formulations. The body weights of the DDP group exhib-ited notable weight losses due to the severe side effects. Compared with control group, the body weights of free NCTD group decreased in the initial procedure, indicating the toxicity of the antitumor drug. Moreover, the body weights of mice in conjugates treated group did not clearly differ from that of the control group, suggesting the reduced toxicity and excellent biocompatibility.
    In terms of tumor inhibitory rates, DDP possessed the best antitumor efficiency as a clinical chemotherapeutic drug. Compared with control group, the inhibitory rates of DDP, free NCTD, high and low dose of CNC were 76.32%, 40.46%, 59.57% and 50.64%, respectively (Table. 2). El-evated antitumor capacity was observed with high dose of CNC treat-ment, which was higher than that of free NCTD at equal dose. Histological analysis by H&E staining showed phenomenon of cell apo-ptosis or tumor necrosis in the tumors of mice administered with differ-ent formulations. As exhibited in Fig. 5A, large, densely arranged hyperchromatic nuclei and obvious nucleolus cleavage could be ob-served in tumor tissues of control group. In addition, H&E sections disclosed that substantial amount of apoptotic tumor cells and evident necrosis areas occurred in tumor tissues of conjugates groups, along with apparent vacuoles degeneration of tumor cells, which revealed the advantage of conjugates in improving antitumor efficacy. As a mac-romolecular derivative, CNC could accumulate at the tumor sites through enhanced permeability and retention (EPR) effect and NCTD could continuously release from the conjugates under physiological conditions in the tumor microenvironment [47]. Therefore, CNC could be considered as a prodrug. According to the above evaluation, CNC showed enhanced antitumor activity and superior security compared with free NCTD, thus it might be a promising polymer-drug conjugates for cancer therapy.
    3.7. Immunohistochemical analysis
    To further investigate the antitumor efficacy and mechanisms medi-ated by CNC, the cell apoptosis and angiogenesis in tumor tissues were evaluated by immunochemistry staining of CD34, Bcl-2, Bax and Caspase-3, respectively. Angiogenesis is the development of new capil-lary blood vessels derived from preexisting vessels and is essential for the growth and progression of neoplasms [48]. CD34 is an antigen pres-ent in hematopoietic progenitor cells and endothelial cells, which has been studied as a marker for vascular tumors [49]. As shown in Fig. 5B, CD34 staining for microvessels revealed significant differences in microvessel number and morphology among different formulations. The staining of CD34 positive microvessels were relatively abundant in tumor tissues of control group. However, treatments with CNC re-sulted in small and irregularly shaped microvessels in the tumor tissues,
    Fig. 5. Histopathological and immunohistochemical detections of tumor tissues (original magnification, 100×). (A) H&E stained sections of tumor tissues. (B) Photographs of protein expressions in tumor tissues. (C) Microvessles density of tumor tissues in different groups. (D) Relative protein expressions in different groups. (a) mice treated with normal saline;