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  • Preparation of DOX loaded HA NB SC nanomicelles br

    2019-11-05

    2.5. Preparation of DOX-loaded HA-NB-SC nanomicelles
    DOX loaded nanoparticles were prepared by a nanoprecipitation method with minor modification (Wei, Dong, & Liu, 2015; Zhang et al., 2016). Briefly, 5 mg DOX·HCl in the presence of 100 μL triethylamine (TEA) was dissolved in DMSO and stirred for 24 h at room temperature. HA-NB-SC was dissolved in DMSO. The two solutions were well mixed by magnetic stirring, which was added dropwise into 8 volumes of distilled water. Then the solution was dialyzed against distilled water in a dialysis membrane (MWCO 3500–5000 Da) to remove the unloaded drug, DMSO, TEA and trimethylamine chloride (TEA·HCl), and then lyophilized. The amount of DOX was determined using UV–vis spec-trophotometer (wavelength at 484 nm). For determination of drug
    Scheme 1. Schematic illustration of photo-controlled HA-NB-SC nanomicelles for CD44-mediated delivery and UV light-triggered intracellular release of DOX to HeLa cells.
    loading content (DLC), lyophilized DOX loaded HA-NB-SC nanomicelles were dissolved in formamide and analyzed with UV–vis spectro-photometer, wherein calibration curve was obtained with DOX/for-mamide solutions with different DOX concentrations. DLC and drug loading efficiency (DLE) were calculated according to the following formula:
    2.6. Light-triggered release study
    To study the photo-controlled release of host molecules in micelles, NR was encapsulated into HA-NB-SC nanomicelles. NR-loaded HA-NB-SC nanomicelles in water were first irradiated for the specific periods of time, and fluorescence emission spectra of NR with excitation wave-length at 550 nm were recorded after each irradiation. The concentra-tions of particle solutions were 1 mg/mL.
    2.7. UV–vis Cucurbitacin I analysis
    The UV–vis absorption spectra of HA-NB-SC nanomicelles irradiated for different time were recorded on Thermo Evolution 300 spectro-photometer in the range of 200–600 nm, respectively. 
    NR-loaded HA-NB-SC nanomicelles were used for the stability test, which was carried out at different temperature (4 °C and 37 °C) for 7 days. And NR-loaded HA-NB-SC nanomicelles in different pH conditions (pH 4, 5, 6, 7.4, 8) were also used for stability test. 2 mL nanomicelles were taken out every day for fluorescence analysis.
    HeLa and 293T cell lines were cultured in regular growth medium consisting of Roswell Park Memorial Institute medium (RPMI) 1640 and Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C under a 5% CO2 atmosphere. The cells were routinely harvested by treating with a trypsin solution (0.25%).
    2.10. In vitro cytotoxicity assays of blank nanomicelles
    HeLa or 293T cells were seeded at a density of approximate 1.8 × 104 or 2 × 104 cells/well in a 96-well plate, respectively, and were incubated for 4 h to allow cells to attach to the bottom of the wells. Then the cells were exposed to various concentrations of different samples. PBS was used as blank control. After incubated for 24 h or 48 h, media were discarded, and 100 μL fresh media containing 10 μL MTT stock solution (5 mg/mL) were added into each well and further incubated for 4 h. After that, media were discarded, and 200 μL DMSO was added to dissolve the formazan. After shaking for 10 min, the op-tical density of each well at 490 nm was measured by a microplate
    reader. Cell viability was evaluated in comparison to the control culture (taken as 100%).
    The cytotoxicity of blank nanomicelles irradiated by UV light, blank nanomicelles residues, were also measured. The blank nanomicelles aqueous solutions were irradiated for 0.5 h or 1 h, and the cell viability of blank nanomicelles residues was studied as described above.
    2.11. Cell cytotoxicity of DOX-loaded HA-NB-SC nanomicelles
    For cell cytotoxicity of DOX-loaded HA-NB-SC nanomicelles, HeLa cells were seeded at a density of 1.8 × 104 cells/well in a 96-well plate and incubated for 4 h to allow cells to attach to the bottom of the wells. The cells were then treated to DOX-loaded HA-NB-SC nanomicelles of miscellaneous concentrations around the IC50 value of DOX. PBS was used as blank control. After 2 h incubation, cells treated with DOX-loaded HA-NB-SC nanomicelles were thoroughly washed with HBSS buffer for three times and then irradiated with UV light for 0.5 h or 1 h. After another 24 h co-culture, the cell viability was measured using a modified MTT-based colorimetric method mentioned above.