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  • Chemical Engineering Journal br in the blood was then


     Chemical Engineering Journal 369 (2019) 134–149
    in the blood was then determined as reported in the literature [30].
    2.12. In vivo biodistribution
    In order to monitor release from the NPs, in vivo drug distribution in female nude mice bearing MDA-MB-231 tumors was studied using a Maestro™ in-vivo imaging system (CRi Inc., Woburn, MA, USA). The model fluorescent molecule DiR was loaded into FA-CS-g-OA NPs fol-lowing the same protocol as for DOX loading. When the size of tumors reached approximately 100 mm3, the mice were intravenously (i.v.)
    Fluorescent scans were then performed at various time points (1, 4, 8, 12, 24 and 48 h) post i.v. injection [31]. After in vivo imaging, the mice were sacrificed and the tumor, liver, kidney, lung, spleen and CFTRinh-172 were removed and collected for further ex vivo fluorescence imaging of DiR.
    2.13. In vivo antitumor efficacy
    150 µL of a MDA-MB-231 cell suspension (ca. 5 × 106 MDA-MB-231 cells) was subcutaneously injected into the right flank area of the nude mice. When the tumor volume reached approximately 100 mm3, the mice were randomly divided into 5 groups (8 mice per group) and treated with saline, DOX, OA, FA-CS-g-OA and [email protected] (7.5 mg/kg DOX equiv.) via i.v. injection every two days. The tumor sizes of the mice were monitored with a digital caliper and the volume
    calculated as Vtumor = L × W2/2 (L: tumor length, W: tumor width). After each injection, the weight of each mouse was determined. The
    survival time of each mouse was also recorded and evaluated by Kaplane-Meier analysis [32].
    2.14. Histological and immunohistochemical examinations
    At the end of the treatment (day 30), one mouse from each group was sacrificed. The tumor and major organs were excised, fixed with paraformaldehyde for 48 h, embedded in paraffin, and cut into 5 mm slices. To observe cell apoptosis in the tumor tissue, it was further sliced into thin sections and stained with a terminal deoxynucleotidyl trans-ferase dUTP nick end labeling (TUNEL) apoptosis detection kit. The tumor tissue sections were also subjected to Ki67 analysis for de-termining the inhibition rate of tumoral proliferation, using a com-mercial detection kit according to the manufacturer’s instructions. For histological examination, tumor and organ sections were stained with hematoxylin and eosin (H&E) and observed with an optical microscope.
    2.15. Cytokine detection
    All remaining mice were sacrificed on day 31 after fasting overnight [33]. Blood samples were collected via heart puncture, with the animals under terminal anesthesia using 0.01 mg/kg phenobarbital (Kefeng Chemical Reagent Co., Shanghai, China). These blood samples were then centrifuged (3500 rpm, 15 min, 4 °C) to obtain the serum. The concentrations of hepatic function indices (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)), and renal function in-dices (creatinine (Scr), urea nitrogen (BUN)) in the serum were mea-sured using an automated AU5400 biochemistry analyzer (Olympus, Tokyo, Japan). In addition, serum levels of the inflammatory cytokines interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), together with oxidative stress biomarkers (glutathione per-oxidase (GSH-Px), malondialdehyde (MDA), and superoxide dismutase (SOD)) were determined using commercial ELISA kits (Bioswamp, Shanghai, China). All procedures complied with the manufacturer’s instructions.
    2.16. Western blot analysis
    The tumors from the xenograft nude mice were excised and washed with physiological saline, then homogenized in modified RIPA buffer supplemented with 1:100 (v/v) of a proteinase/phosphatase inhibitor cocktail (Solarbio, Beijing, China). Insoluble material was removed by centrifugation at 12,000 g and 4 °C for 30 min. Protein content was then determined with a commercial BCA kit (Beyotime, Beijing, China). Subsequently, equal amounts of 40 μg total protein were loaded per lane on a 10% SDS–PAGE gel for separation. After SDS-PAGE, the proteins were transferred to polyvinylidene difluoride (PVDF) mem-branes, preincubated in blocking solution at room temperature for 2 h, and incubated with the primary antibodies for P-gp (1:500), MRP1 (1:500), poly(ADP-ribose polymerase) (PARP, 1:1000), phosphatase and tensin homolog (PTEN, 1:100), p53 (1:100), matrix metallopro-teinase-2 (MMP-2, 1:800), transforming growth factor (TGF-β, 1:800), collagen I (1:500) and β-actin (1:2000, Santa Cruz, Dallas, TX, USA) overnight at 4 °C. After being washed three times for 10 min with Tris-buffered saline containing 0.5% Tween-20, the membrane was in-cubated with the corresponding secondary antibody linked to horse-radish peroxidase (goat antimouse IgG, 1:10,000; Santa Cruz, Dallas, TX, USA) for 2 h at room temperature. Immunoreactive proteins were visualized using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Finally, membranes were treated with an enhanced chemiluminescent reagent (Merck Millipore, Burlington, MA, USA) and exposed to a Kodak X-Omat film (Kodak,