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 buy JNJ-26481585 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 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 this website 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,LY2603618 supplier 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
Phenylethanolamine N-methyltransferase 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).