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  • Liproxstatin-1 br iv provide highly effective treatment of t


    iv) provide highly effective treatment of tumors while ameliorating the off-target side effects common with chemotherapy. The materials de-veloped in our work offer a new chemical engineering approach to prepare multifunctional nanoparticles. The platform approach reported here has broad applicability in the treatment of a range of diseases and conditions.
    2. Materials and methods
    CS was purchased from the SinoPharm Chemical Reagent Co., Ltd (Shanghai, China), with a deacetylation degree of 95% and average molecular weight (MW) of 100 kDa. 4-(dimethyl-amino) pyridine (DMAP), pyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), 3-(4,5-di-methyl-thiazol-yl)-2, 5-diphenyltetrazolium bromide (MTT), calcein-AM, propidium iodide (PI), OA and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). FA (MW = 441 Da) was supplied by Wako Pure Chemical Industries, Ltd. (Osaka, Japan). DOX (C27H29NO11HCl, 98%) and dialysis tubing were purchased from Shanghai Yuanye Biotech Co., Ltd. (Shanghai, China). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and trypsin-EDTA were obtained from Gibco (Carlsbad, CA, USA). 4′,6-diamidino-2-phenylindole (DAPI) was pur-chased from the Dingguochangsheng Biotechnology Co. Ltd. (Beijing, China). DiR (1,10-dioctadecyl-3,3,30,30-tetramethylindotricarbo cya-nine iodide) was procured from Biotium (Hayward, CA, USA). Tween-
    20, TUNEL and Ki67 commercial detection kits were obtained from Beyotime (Beijing, China). P-gp, MRP1, PARP, PTEN, p53, MMP-2, TGF-β, Collagen I and β-actin Liproxstatin-1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Deionized water, used throughout the research work, was produced using a Milli-Q Gradient A10 System (Merck Millipore, Burlington, MA, USA). All chemicals were used as obtained without any further purification.
    2.2. Cell lines and culture procedures
    The human breast adenocarcinoma cell line (MDA-MB-231) and the control HUVEC cell line were kindly provided by the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM sup-plemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37 °C.
    2.3. Animals and the murine xenograft MDA-MB-231 tumor model
    All animal care and handling was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US
    National Institutes of Health (NIH Publication No. 8523, revised 1985). All experimental protocols were approved by the Animal Care and Use Committee of Kunming Medical University (reference: KMMU 2015002). Adult male Sprague-Dawley (SD) rats (180–200 g, 3–4 weeks old) were obtained from the Kunming Medical University (Kunming, China), and raised with free access to food and water under specific pathogen-free conditions in a room maintained under a 12 h light/dark cycle. For tumor models, female nude mice (16–20 g, 5–6 weeks old) were sup-plied by the Animal Center of Kunming Medical University (Kunming, China) and housed under specific pathogen-free conditions with free access to standard food and water. The temperature was maintained at 20–22 °C. Tumor-bearing xenografts were established by subcutaneous inoculation of 5 × 106 MDA-MB-231 cells (150 μL) into the right flank of each mouse. The tumor sizes of all mice were monitored with a di-gital caliper and tumor volume calculated using the formula
    Vtumor = LW2/2 (L: tumor length, W: tumor width). When the tumor reached a designated volume (100 mm3), the mice were randomly
    grouped for experiments.
    Scheme 1. Schematic illustration of the preparation, microstructure, and application of the [email protected] NPs.