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Injection grade hydroxypropyl-β-cyclodextrin (HP-βCD, Fw = 1541.54, purity ≥ 98%) was purchased from Zhiyuan Biotechnology Co., Ltd. (China). Anhydrous N,N-dimethylformamide (DMF), anhydrous dichloromethane (DCM) and N,N"-carbonyldiimidazole (CDI) were obtained from J&K (China). 4-(Hydroxymethyl)phenylboronic acid (PBA) and 4-acetamidophenol (APAP) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Rifampicin (RFP) was purchased from Tokyo Chemical Industry Co., Ltd. (Japan). Baicalein (BE) was purchased from MedChemExpress (purity > 98%). Cyanine5 NHS ester (Cy5) and Cyanine7.5 NHS ester (Cy7.5) were obtained from Lumiprobe (USA). William"s E Medium, Dulbecco"s Modified Eagle"s Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin and trypsin for cell culture were purchased from Gibco (USA). FITC Annexin V Apoptosis Detection Kits with 7-aminoactinomycin D (#640922) was purchased from Biolegend (USA), while MTT Cell Proliferation and Cytotoxicity Assay Kit (#C0009), Enhanced BCA Protein Assay Kit (#P0010S) and Reactive Oxygen Species Assay Kit based on DCFH-DA (#S0033) were supplied by Beyotime Biotechnology Co. Ltd. (China). N-(4-Diphenylphosphinophenyl)-N"-(3, 6, 9, 12-tetraoxatridecyl)perylene-3, 4, 9, 10-tetracarboxydiimide (LiperFluo™, #L248) was obtained by Dojindo Molecular Technologies (Japan). TMRE-Mitochondrial Membrane Potential Assay Kit (#ab113852) was obtained from Abcam (USA). Collagenase Ⅱ, DNAse I, Lymphoprep and D-Hank"s were purchased from Solarbio (China). APC-labeled anti-mouse Ly6G antibody (#127613) and FITC-labeled anti-mouse CD11b antibody (#101205) were purchased from Biolegend (USA). With the exception of myeloperoxidase (MPO) ELISA kits (#E4580) was obtained from BioVision (USA), glutathione (GSH), glutathion reductases (GR), malondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (China). QuantiCyto® Mouse ELISA kits for TNF-α and IL-1β were purchased from Neobioscience (China). Kits for H2O2 (#DIOX-250) were purchased from BioAssay Systems (USA). All the other reagents are commercially available and used as received.

To measure the drug loading content of nanoprodrug, 200 mg lyophilized cPBA-BE was complete hydrolyzed in acetonitrile with 1 mM H2O2 at pH 6.0. The concentration of BE in hydrolysate was quantified by LC-MS referring to the standard curve + is 271.06 m/z. The mobile phase was 75% acetonitrile and the flow rate was 1 mL/min. Similarly, cPBA-BE-Cy5 and cPBA-BE-Cy7.5 were hydrolyzed completely in acetonitrile with 1 mM H2O2 at pH 6.0, the concentrations of dyes were determined by UV-Vis spectrophotometry at 646 and 788 nm, respectively.

HepaRG cells were seeded in 12-well plates at a density of 4 × 105 cells/well in 1 mL of complete medium. After 24 h, the medium was replaced with 1 mL of fresh medium containing 10 μg/mL Cy5-loaded cPBA-BE (cPBA-BE-Cy5) and incubated for various periods of time (0.5, 1, 2, 4, 6, 12 and 24 h). Then, the cells were digested and fluorescence intensity was determined using a flow cytometer (BD Accuri C6). Through similar procedures, dose-dependent (1.25, 2.5, 5 and 10 μg/mL cPBA-BE-Cy5) internalization profiles were examined after 2 h of incubation.

Meanwhile, the drug release of cPBA-BE in cells was examined by a parallel test. 80 μg/mL cPBA-BE was incubated with HepaRG cells for 8 h. Then replaced with fresh medium, the cells were digested and menthol/acetonitrile (1:1) was added to extract the drug. The content of BE was analyzed by LC-MS with 75% acetonitrile as mobile phase and the flow rate was 1 mL/min.

The concentration-time relationships of BE in liver and blood were focused in the pharmacokinetic studies. AIH mice were injected with 100 μL 5.1 mg/mL BE or 20 mg/mL cPBA-BE (equal to 5.1 mg/mL BE) through tail vein. Blood samples and liver tissues were collected at predefined time points (0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 3, 4, 6, 8, 12, 16 and 24 h). According to previously reported methods with modification g for 2 min to obtain plasma. Then 800 μL of ice-cold methanol was added into 200 μL of plasma to precipitate protein. After centrifugation at 12,000 g for another 2 min, the supernatant was purged with nitrogen, reconstituted with methanol, and detected by LC-MS. The mobile phase was consisted of acetonitrile and water at 75:25 (v/v). To determine the concentration of BE in liver, the liver tissues were homogenized and centrifuged. BE in the supernatant was extracted with methanol/acetonitrile then quantified by LC-MS. Typical pharmacokinetic parameters such as the maximum concentration (Cmax) in plasma or liver, time to reach Cmax (Tmax), and the area under the plasma/liver BE concentration-time curve (AUC) were calculated.

Hepatic leukocytes were purified as described ,g for 10 min. Lymphocytes were purified by layering the cell suspension on Lymphoprep™ and centrifuged at 800 g for 20 min at room temperature. The emigrated neutrophils in livers were analyzed by flow cytometry. Specifically, the suspension concentration was adjusted to 1 × 106 cells/mL and labeled for 30 min with the following antibodies: APC-conjugated anti-mouse Ly6G antibody and FITC-conjugated anti-mouse CD11b antibody. The cells were collected in a flow cytometry (Accuri C6, BD) and analyzed using FlowJo 10 software.

On Friday, China Rongsheng Heavy Industries Group, China’s largest private shipbuilder, appealed for financial help from the government and big shareholders, after cutting its workforce and delaying payments to suppliers.