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  • br Corresponding author at Laboratory of Biochemistry and


    Corresponding author at: Laboratory of Biochemistry and Biomedical Materials, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, PR China.
    E-mail address: [email protected] (B. Han).
    angiogenesis drugs may contribute to the treatment and prognosis of gastric cancer.
    Several kinds of polysaccharides have been applied in clinical ther-apy as injections and achieved excellent therapeutic effects, such as gin-seng polysaccharides and lentinan [9,10]. Carboxymethyl chitosan (CMCS) is one of the water-soluble polysaccharides derived from chito-san, exhibiting numerous outstanding physicochemical and biological properties [11]. Based on these favorable properties, CMCS has wide ap-plications in wound healing, gene therapy, drug delivery, tissue engi-neering and food technology [12–16]. CMCS has become a promising candidate for the delivery of numerous drugs due to the improved sol-ubility and structural advantages [17]. The enhanced solubility in water and other relevant solvents makes CMCS a suitable choice for de-livery of hydrophobic drugs. Besides, the presences of hydroxyl, car-boxyl and amino groups make it possible for combining with many drugs via amine or ester bonds [18]. In recent years, CMCS-based drug delivery systems have been designed and employed to control drug re-lease, reduce systematic toxicity and increase bioavailability, such as nanoparticles [19,20] and polymer-drug conjugates [21]. Particularly, polymer-drug conjugates based on CMCS have been developed for de-livery of methotrexate (MTX), 6-mercaptopurine (6-MP) and docetaxel (DTX) [22–24] and so on.
    Norcantharidin (NCTD) is a small-molecule anticancer drug derived from Chinese traditional medicine blister beetle (Mylabris) [25]. Numer-ous studies revealed that NCTD possessed potent inhibitory effects
    against hepatic carcinoma, leukemia, oral cancer and gallbladder carci-noma [26–29]. Besides, NCTD performed favorable antitumor effects in clinical treatments of gastrointestinal cancer such as gastric cancer and colorectal cancer [30]. Previous studies indicated that NCTD could inhibit the metastasis and induce apoptosis of multiple human cancer Cefepime [31]. Compared with other anticancer drugs, the distinguishing fea-tures of NCTD include the low degree of myelosuppression and the in-duction of leukocytosis [32]. However, the applications of NCTD in clinical therapy are limited by its poor intestinal absorption, short half-life and severe nephrotoxicity. In addition, NCTD is usually admin-istered by high-dose injection, which causes intense stimulation at the injection site [33]. Therefore, it is essential to explore new formulations to overcome these limitations in NCTD application.
    In the present study, NCTD was conjugated with CMCS via amide bonds and the antitumor efficacy of the conjugates was evaluated in vitro and in vivo. The in vitro studies were conducted to confirm the anti-proliferation and anti-angiogenesis effects after incubation with CNC. The in vivo antitumor efficacy and histological analysis were conducted and evaluated in BALB/c nude tumor bearing mice. Fur-thermore, the expressions of related proteins were determined to expli-cate the underlying mechanisms of the antitumor effects. Our results indicated that CNC was a promising and effective polymer derivative against gastric cancer in clinical treatment.
    2. Materials and methods
    2.1. Materials and reagents
    CMCS was synthesized and purified according to the previous pub-lished method [34]. NCTD (purity 99.00%) was purchased from Shan-dong Renhe Tang Pharmaceutical Co., Ltd. (Linyi, China). Commercially available sterile cisplatin (DDP) for injection (freeze-drier model) was kindly provided by Qilu Pharmaceutical Co., Ltd. (Jinan, China) (certifi-cate No. H20023461). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tet-razolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 96 well plates, 24 well plates and transwell plates were obtained from Corning INC. (Corning, NY, USA). Roswell Park Me-morial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were purchased from Gibco®, Life Technologies (Grand lsland, NY, USA). Hoechst 33258 staining kit, Annexin V-FITC/PI apoptosis detec-tion kit, radio immunoprecipitation assay (RIPA) lysis buffer, phenylmethanesulfonyl fluoride (PMSF), sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) gel preparation kit and BCA protein assay kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). β-actin mouse monoclonal antibody and Bcl-2/Bax rabbit polyclonal antibody were purchased from Abcam Inc. (Cambridge, MA, USA). CD34/Caspase-3 rabbit polyclonal antibody and MMP-2/MMP-9 rabbit polyclonal antibody, horse radish peroxidase (HRP)-labeled goat anti-rabbit/mouse IgG secondary antibody, 3, 3-diaminobenzidine (DAB), ECL luminescence reagent, mouse tumor ne-crosis factor-α (TNF-α) enzyme linked immunosorbent assay (ELISA) kit, mouse matrix vascular endothelial growth factor (VEGF) ELISA kit were purchased from Boster Biological Engineering Co., Ltd. (Wuhan, China). Polyvinylidene difluoride (PVDF) Cefepime membranes and bovine serum albumin (BSA) were obtained from Solarbio Science & Technol-ogy Co., Ltd. (Beijing, China). All other chemical reagents were of analyt-ical grade and used without further purification.