Hybri care

Cancer cell lines were purchased from ATCC except for Huh7 (JCRB), PC-9 (Riken BRC), BEL7404 (SUZHOU BEILE BIOTECH), SK-OV-3, A2780, RPMI-8226 (ECACC), HCC78 (DSMZ) and KM-12 (HUATUO). Cell culture medium included RPMI-1640 (#22400-089), McCoy’s 5a (#16600082), DMEM (#11995-065), F-12K (#21127-022) and IMDM (#12440053) was purchased from Gibco. EMEM (#30-2003) and Hybri-Care (#46-X) medium was purchased from ATCC. Leibovitz’s L-15 (#L1518) was purchased from SIGMA. All the medium was contained 10% or 20% FBS (#FND500, ExCell Bio). The 2D and 3D models used the same culture medium. Horse serum (#041241A) was bought from BI. Trypsin (#25200072) and antibiotic-antimycotic (#15240-062) was purchased from Gibco. Dulbecco’s PBS (#21-031-CVC) and matrigel (#354234) were purchased from Corning. DMSO (#D2650) was bought from Sigma. CellTiter-Glo Cell Viability Assay kit (#G7573) and CellTiter-Glo 3D Cell Viability Assay kit (#G9683) were purchased from Promega.

BT-474 and MDA-MB-231 breast cancer cells (ATCC) were cultured at 37 °C and 5% CO₂ in Hybri-Care (ATCC) and RPMI 1640 (Life Technologies), respectively supplemented with 10% FBS (Corning) and 1% (100 units/ml) Penicillin–Streptomycin (Gibco). Cells in the exponential growth phase were seeded in a 96-well plate at an initial density of 10,000 cells/well and incubated overnight. Different concentrations of PLL–Au–Fe₃O₄ NPs (0–500 μg/ml) were added to the cell culture media. After 2 h of incubation, the media was removed, and the cells were washed using PBS followed by re-incubation in cell culture media for additional 72 h. The viable and dead cells were measured using a live–dead (calcein AM; CAM and EthD–1) assay (Invitrogen) and calculated according to the manufacturer’s protocol. The cells without any NP treatments were used as the control. The percentage cell death was calculated by the following equation: $$\%\text{cell}\text{death}=\frac{F_{\mathit{NPs}}-F_{\mathit{blank}}}{F_{\mathit{control}}-F_{\mathit{blank}}}\times 100$$ where F_NPs is the Ethd-1 fluorescence of the cells treated with PLL–Au–Fe₃O₄ NPs, F_blank is the Ethd-1 fluorescence of the cell culture medium alone, and F_control is the Ethd-1 fluorescence of the cells without any NP treatments. The concentration at which cell death was less than 10% was identified as a non-toxic dose.