Characterization of surface-modified GNPs
The immobilization of herceptin and Cy5 dye on surface-modified GNPs was recorded by UV–vis absorption spectrum from aqueous solution at room temperature using a Hitachi U-3000 spectrophotometer. The core diameter and the surface morphology of GNPs were observed by TEM (Philips CM-200, 120 kV).
The samples for TEM observations were diluted with distilled water and deposited on Formvar-coated 400-mesh copper grids.
After drying the GNP–fluid thin film on the copper grid, a thin carbon film, approximately 10–30 nm in thickness, was deposited on the NP film. The size and hydrodynamic diameter distribution of the GNPs were determined by DLS using a light-scattering spectrometer BI90 (Brookhaven, Long Island, NY, USA).
SK-BR3 (breast cancer cells) was used as the target cell, and FB (human skin cells) was used as the control cell line. In a humidified atmosphere containing 5% CO2, the cells were cultured routinely in a polystyrene dish at 37°C using 10 mL of McCoy medium or DMEM, supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin G sodium. The medium was changed every 3 days.
For subculture, the cells were washed twice with PBS and incubated with a trypsin–EDTA solution (0.25% trypsin, 1 mM EDTA) for 10 minutes at 37°C to detach the cells. Complete media were then added to the polystyrene dish at room temperature to prevent the effects of trypsin.
The cells were washed thrice by centrifugation method and resuspended in a new culture dish. To observe the morphology of cells, the cells were seeded in a 10 mL Petri dish at a concentration of 1×105 cells/mL and incubated for 3 days with GNP and GNP–Her.
The surface morphology of adhered cells was observed by Nikon Eclipse TS100 optical microscopy.
To study the cytotoxic effects of GNP–Her, after 3 days of culture, the SK-BR3 and FB cells were suspended in PBS at a density of 1×105 cells/mL.
Subsequently, the cell suspension was mixed with 100 μL of the assay solution (10 μL propidium iodide–calcein AM) and incubated for 15 minutes at 37°C. The cells were then observed by fluorescence microscopy (Axioplan 2; Carl Zeiss Meditec AG, Jena, Germany) for the simultaneous observation of live and dead cells.
Intracellular uptake of GNPs
For the observation of effective cellular uptake of NPs via CLSM and fluorescence microscopy, the cells were seeded at a concentration of 1×105 cells/mL in a 10 mL culture dish and incubated for 24 hours.
After 1 day, the medium was swapped with GNP and GNP–Her, containing a particle concentration of 50 μg/mL, and incubated for a certain period (2–7 hours) for the specific internalization of the NPs into the cells.
The cells were then washed three times with PBS, and images were taken using a confocal laser microscope. A Carl Zeiss LSM 410 (Oberkochen, Germany) confocal laser scanning microscope was used to obtain the confocal images. The location and integrity of the internalized GNP–Her conjugates were estimated by CLSM using 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, blue) as a marker, which stains the nuclei of the cells.
The cell nuclei were stained by the addition of DAPI solution (10 μL) with proper mixing and incubated for 10 minutes. To track the GNP–Her NPs, GNP–Her and DAPI (488 nm) were added to the cells.
The stained cells were washed at least three times with 1 mL of fresh DMEM and images were then taken by CLSM. To measure the intracellular uptake of the NPs, cells were grown in 24-well culture plates with roughly 105 cells in 1 mL of the medium. After 20 hours’ incubation at 37°C, the cells were reseeded with culture medium containing GNP–Her at a concentration of 2×10−4 cells/mL. The intracellular gold concentration was quantified using ICP–MS.
Cells were washed with PBS, separated, resuspended, counted, and centrifuged, and the cell pellets were dissolved in 37% HCl aqueous solution at 60°C–85°C for 40 minutes. The experiment was repeated three times, and the results were averaged with standard deviation.
The cell multiplying experiment was conducted in triplicate and the results are presented as means ± standard deviations. The Student’s t-test was used to estimate the statistically significant difference between the results. Difference was considered statistically significant at P<0.05.