5°C, which was conducted in triplicate. The amount of released drug was measured at 593 nm by fluorescence spectrometry. These results are shown as average ± standard deviation (n = 3).
In addition, the drug loading efficiency (7.2 wt.%) was measured in the same manner. Briefly, NChitosan-DMNPs’ weight was measured after lyophilization and then dissolved in 1 mL of DMSO. The loaded amount of drug was measured by fluorescence spectrometry, using the following formula: Cellular internalization of NChitosan-DMNPs MR imaging and fluorescence microscopy confirmed cellular internalization of NChitosan-DMNPs. NIH3T6.7 cells were obtained from American Type Culture Collection. First, these cells were seeded at a density of 1.0 × 106 cells/well in six wells for growth BAY 57-1293 overnight at 37°C and then further incubated with NChitosan-DMNPs in 5% CO2 for 24 h at 37°C. The cells were washed three times with
PBS and stained by Hoechst (Molecular Probes TM, OR, USA) to show nucleus location. Fluorescence microscopic images were obtained using a laser scanning confocal microscope (LSM700, Carl Zeiss, Jena, Germany). Under the same conditions, NIH3T6.7 cells Z-IETD-FMK molecular weight treated with NChitosan-DMNPs were washed twice, collected, and then re-suspended in 0.2 mL of 4% paraformaldehyde for MR imaging analysis. All experiments C59 wnt ic50 were conducted in triplicate. Determination of cell viability using MTT assay The cell viability of NChitosan-DMNPs was evaluated by measuring cell growth inhibition using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche Molecular Biochemicals, Mannheim, Germany) compared to DOX as a control. NIH3T6.7 cells (1.0 × 104 cells/well) were implanted in a 96-microwell plate with temperature at 37°C overnight and treated with various concentrations of NChitosan-DMNPs. After 24 h, the cells were washed and incubated for an additional 48 h. The yellow tetrazolium salt of MTT solution was
tuclazepam reduced to purple formazan crystals in metabolically active cells. The cell viability was determined from the ratio of treated cells to non-treated control cells. The results are shown as average ± standard deviation (n = 4). Animal experiments All animal experiments were conducted with approval from the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Tumor-bearing mice were developed, and NIH3T6.7 cells (5 × 106 cells suspended in 50 μL saline per animal) were implanted into the proximal thighs of female BALB/nude mice (4 to 5 weeks of age) to investigate NChitosan-DMNPs’ distribution and tumor growth rate. After tumor volume reached approximately 40 mm3 at 3 days post-implantation (0 days), in vivo magnetic resonance imaging (MRI) experiments were performed using NChitosan-DMNPs (five mice).
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