br Stability of DOX DHHC GNRAH br DOX DHHC
2.5. Stability of DOX-DHHC-GNRAH
DOX-DHHC-GNRAH was suspended in water and PBS solution
(10 mM, pH 7.4) at 37 °C, respectively, using pristine GNR as control. After 1 h, the UV–vis-NIR spectra and photographs of GNR and DOX-DHHC-GNRAH in water and PBS solution were recorded using UV–vis spectrometer and digital camera, respectively. Furthermore, the UV–vis-NIR spectra of DOX-DHHC-GNRAH in water and PBS solution over 5 days were monitored with the UV–vis spectrometer at preset time points.
2.6. Photothermal properties of DOX-DHHC-GNRAH
GNR, DHHC-GNR, DOX-DHHC-GNR and DOX-DHHC-GNRAH sus-pensions were irradiated with an 808 nm laser (2.0 W/cm2, 10 min), and the temperature was measured using a thermic camera-FLIR E60. To explore the eﬀects of suspension concentration and laser power on photothermal conversion, DOX-DHHC-GNRAH suspensions with dif-ferent concentrations (20, 30, 40 and 50 μg Au/mL) were irradiated at diﬀerent powers (1, 2 and 4 W/cm2). To clarify the photostablity, DOX-DHHC-GNRAH suspension (20 μg Au/mL) was exposed to NIR irradia-tion for three heating-cooling cycles and temperature changes were recorded.
Human breast cancer MCF-7 cells were used for in vitro biological experiments. MCF-7 cells were maintained in DMEM medium con-taining 10% FBS and 1% penicillin-streptomycin in an FLAG tag Peptide of 5% CO2 at 37 °C and routinely sub-cultured. Carbohydrate Polymers 212 (2019) 334–344
2.8. In vitro cellular uptake
The cellular uptake of DOX-DHHC-GNRAH was evaluated using flow cytometry and confocal laser scanning microscopy (CLSM). Briefly, MCF-7 cells were seeded in 6-well plates at a density of 5 × 105 cells/ well for 24 h. The cells were treated with sterilized suspensions of DOX-DHHC-GNRAH and [email protected] (DOX-DHHC-GNRAH + free HA). After 1, 4 and 24 h of incubation, the cells were washed with PBS and collected. Cellular uptake was quantitatively characterized using a FACS Canto II flow cytometer (BD Biosciences, USA). For CLSM studies, MCF-7 cells were seeded in confocal dishes at a density of 5 × 104 cells/well. After incubation for 1, 4 and 24 h, the cells were stained with DAPI and observed using a confocal laser scanning mi-croscope (Leica, TCS SP8 STED 3X).
The amount of Au internalized into MCF-7 cells was quantitatively analyzed by ICP-MS. Briefly, MCF-7 cells were seeded in 24-well plates at a density of 1 × 105 cells/well for 24 h. After that, cells were treated with diﬀerent formulations and then incubated for 1, 4 or 24 h. Subsequently, cells were washed, collected, quantified by Trypan blue cell counting assay, and digested in 1 mL of aqua regia. The con-centration of Au was measured by ICP-MS and then converted to GNR number that was calculated based on the estimated volume of GNR in TEM images.
2.9. In vitro photothermal chemotherapy
To assess the cytocompatibility and treatment eﬀect of DOX-DHHC-GNRAH, MTT, Live/Dead and apoptosis assays were performed. For the MTT assay, MCF-7 cells were seeded into 96-well plates at a density of 1 × 104 cells/well for 24 h. Subsequently, the cells were treated with diﬀerent concentrations of formulations and incubated for another 24 h. Each group was then subdivided into two subgroups: with or without NIR irradiation. Irradiation groups were irradiated with 808 nm laser for 10 min (2.0 W/cm2) and then cells were cultured for 4 h. Cell viability was determined using the MTT assay.
To reveal apoptosis and necrosis, MCF-7 cells were cultured for 24 h in 6-well plates at a density of 5 × 105 cells/well and then treated with preset formulations for another 24 h. Afterwards, the cells were irra-diated with NIR light (2.0 W/cm2) for 10 min and cultured for a further 4 h. The cells were washed, collected and stained with annexin V-EGFP/ PI. Flow cytometry analysis was performed immediately. For Live/Dead assay, MCF-7 cells were seeded in confocal dishes at a density of 2 × 105 cells/well for 24 h. The cells were treated in the same way as described above for flow cytometry analysis. Finally, the cells were stained with calcein-AM/EthD-1 according to the manufacturer’s in-structions and observed by CLSM.