Cell culture
The HepG2 cells were cultured in DMEM (GIBCO, Grand Island, USA) supplemented with 10 % heat-inactivated fetal calf serum (FCS, GIBCO, Grand Island, USA) and 1 % penicillin–streptomycin (GIBCO, Grand Island, USA) at 37 °C under a humidified atmosphere containing 5 % CO2 and maintained in an exponential growth state. Cells were passaged once every 3 days by using 0.25 % trypsin.
Preparation of GAL-PEG-GNPs
Synthesis of GNPs in 20 nm diameter was done with sodium citrate reduction method. The preparation method can be found elsewhere [21]. To graft amine groups onto bare GNPs surfaces, 0.1 mL of GNPs at 0.1 mg/L (pH 7.5) were well mixed with excessive amount of thiol-PEG-amino (5000 Da, Seebio, ShangHai, China) and incubated at 4 °C overnight. The following thiol-PEG (5000 Da, Seebio, ShangHai, China) surface modification was performed to thoroughly replace the residual citrate groups. The products were purified and buffer exchanged into 1× PBS (pH 7.5) using ultrafiltration. 100 μL of N-hydroxysulfosuccinimide (sulfo-NHS) and carbodiimide (EDC) (5:1, pH 5.0) were incubated with 20 μg of GAL for 6 h at 37 °C to active carboxyl groups of GAL. At pH 7.2, 20 μL of PEGylated GNPs at 0.1 mg/L were added and incubated with the mixture at 37 °C for 2 h to finally produce GAL-PEG-GNPs (Fig. 1). The products were concentrated, filtered and stored at 4 °C.
Characterization of GAL-PEG-GNPs
Dynamic light scattering (DLS) was used to determine the size of GNPs. The DLS and Z-potential experiments were performed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Southborough, Massachusetts). Samples filtered followed by equilibration (typically 5 min) in a quartz cuvette to 37 °C. The software was arranged with the specific parameters of refractive index and absorption coefficient of the material and the viscosity of the solvent. DLS allows determination of hydrodynamic diameter of colloidal particles and conjugates, that is the diameter of sphere with the same Brownian motion as the analyzed particle. The concentration of GNPs and GAL-PEG-GNPs were determined by using inductively couple plasma mass spectrometry (ICP-MS). UV–visible adsorption spectrum of GNPs or GAL-PEG-GNPs was acquired over the wavelength range from 250 to 800 nm with UV-2450 spectrophotometer (Tianjin Gangdong Sci. & Tech. Development CO. LTD, China) using quartz cuvettes with an optical path length of 0.5 cm at room temperature (RT). The size and morphology of GAL-PEG-GNPs are analyzed by FEI Tecnai Spirit transmission electron microscopy (TEM) (JEM-100CX II, Japan) at 200 kV using AnalySIS software (Soft Imaging Systems).
CCK8 assay
The CCK8 assays were performed as instructed by the manufacturer to assess cell viability. Briefly, the cells were seeded in 96-well plates (approximately 3000 cells/well). GNPs and GAL-PEG-GNPs were diluted to various concentrations in 1× PBS (pH 7.4), then added into the wells. After 24 h incubation, 20 μL CCK8 (keygentec company, Nanjing, Jiangsu, China) was added to each well for 4 h. Optical density (OD) was measured at 490 nm using a Microplate reader (BioRad, DG3022, USA) and the proliferation index was calculated as experimental OD value/control OD value. Estimate value of 50 % inhibition concentration (IC50).
ICP-MS assay
The assay was performed in triplicate. A total of 1 × 106 HepG2 cells were seeded onto a culture dish (dia. 15 mm) and cultured for 24 h. When the cells reached a 70 % confluence, cells were exposed to GNPs (1/5 IC50 = 1.0 μg/mL) and GAL-PEG-GNPs (1/5 IC50 = 1.0 μg/mL) at 37 °C for 1, 2, 4, 8, 12, 24, 48, 72, 96 h, respectively. Cells were washed with 1× PBS twice, detached with 0.25 % trypsin, and suspend in 5 mL of 1× PBS. 5 mL of Aqua Regia (HNO3:HCL = 1:3) solution was added to cell suspension to fully lyse cells at RT for 48 h. Concentration of Au in lysates was determined by ICP-MS, the total number of nanoparticles and cells were recorded as n and N, respectively. Thus, the number of nanoparticles contained in each cell calculated as n/N.
Transmission electron microscopy
A total of 1 × 106 HepG2 cells were seed onto a culture dish (dia. 15 mm) and cultured for 24 h. After another 24 h of incubation with GNPs or GAL-PEG-GNPs, cells were fixed by 4 % paraformaldehyde/2.5 % glutaraldehyde in PBS (0.7 mL) for 3 h. The cells were next rinsed with PBS and post-fixed using 1 % aqueous solution of OsO4 (0.5 mL) for 1 h. Subsequently, the cells were washed with DI water, 30 % ethanol solution and stained with 0.5 % uranyl acetate (0.5 mL, in 30 % ethanol) for 1 h. Cells were then gradually dehydrated using a series of ethanol solutions (30, 60, 70, 80, and 100 %) and embedded in epoxy resin. The resin was polymerized at 60 °C for 48 h. Ultra-thin sections (70–100 nm) were cut using a diamond knife on a Leica Ultramicrotome and mounted on Formvarcoated copper grids. The sections were then post-stained with 5 % uranyl acetate in 50 % ethanol and 2 % aqueous lead citrate solution and imaged with TEM at 200 kV.
Cells cycle assay
The cells exposed to 0.25 Gy X-ray (6 MeV, Medical linear accelerator, Germany Siemens Primus Company) after incubating with GNPs (1.0 μg/mL) or GAL-PEG-GNPs (1.0 μg/mL) for 24 h and then were harvested using 0.25 % trypsin with 1 mM EDTA solution and fixed for 12 h in 70 % ethanol at 4 °C. The fixed cells were then centrifuged at 3000 rpm for 15 min to remove the ethanol thoroughly. The cells were then washed twice with 3 mL of PBS, resuspended in 1 mL of PI (Sigema, USA) staining solution, and incubated for 15 min at RT. The staining solution consisted of 20 mg/mL PI and 0.2 mg/mL RNase in PBS. The samples were subsequently analyzed using a BD FACS CantoII instrument (BD Biosciences, USA). Twenty thousand events were collected from each sample. The percentages of cells in the G0/G1, S, and G2/M phases of the cell cycle were determined using the ModFit software (BD, USA).
Clonogenic assay
The radiosensitization of GAL-PEG-GNPs or GNPs to HepG2 cells were assessed by the clonogenic assay. Different number of HepG2 cells (100, 300, 1000, 5000, 10,000) were plated in 6-well and incubated with GNPs (1.0 μg/mL) or GAL-PEG-GNP (1.0 μg/mL) for 24 h, then irradiated with 0, 1, 2, 4, 6, 8 Gy X-ray and incubated for 9–14 days. The colonies were fixed with methanol and stained with 0.4 % crystal violet. Finally, the plates were inspected by microscopy and the number of the colonies was counted. Each assay was made in triplicate and only colonies containing at least 50 cells were counted. The sensitizer enhancement ratio (SER) was calculated as the radiation dose needed for radiation alone divided by the dose needed for various concentrations of nanoparticles plus radiation at a survival fraction of 37 % (D0 in radiobiology).
DNA damage immunofluorescence microscopy
The ability of GAL-PEG-GNPs or GNPs to enhance DNA double-strand breaks (DSBs) in HepG2 cells exposed to 0.5 Gy X-ray was evaluated using the γ-H2AX assay. This assay detects the phosphorylation of histone-H2AX at serine-139 (γ-H2AX), which is visualized as discrete nuclear foci by laser confocal microscopy using γ-H2AX-specific antibodies.
Cells were cultured (6 × 104 cells/well) on 24-well plates overnight at 37 °C in medium. After 24 h, the culture medium was replaced with 300 mL of fresh medium or medium containing GAL-PEG-GNPs or GNPs, respectively, and incubated overnight at 37 °C. The treated cells were then exposed to 0.5 Gy of X-radiation using an X-ray source, operating at 6 MeV. Cells were subsequently fixed using 4 % paraformaldehyde (Sigma-Aldrich, USA), permeabilized with 0.5 % Nonidet P40 (Sigma-Aldrich, USA) in PBS for 15 min, and blocked in 2 % bovine serum albumin (BSA, Sigma-Aldrich, USA) for 1 h at RT. Cells were then incubated with anti-γ-H2AX mouse monoclonal IgG1 (Upstate Biotechnology, Billerica, MA, USA) at a 1:800 dilution in 3 % BSA-PBS overnight at 48 °C and then with anti-mouse IgG (H + L) (Invitrogen Molecular Probes, Carlsbad, CA, USA) at 1:500 dilution for 45 min at RT. Cover glasses were mounted on microscope slides (25 × 75 × 1 mm, Fisherbrand, Fisher Scientific Co.) The edges of cover glasses were sealed with clear nail polish. From the step using the secondary antibody onwards, all procedures were performed in the dark. Cover glasses were wrapped in aluminum foil and stored at 4 °C for later image acquisition. Images of γ-H2AX foci and nuclei were acquired with a Confocal Microscope Images were taken with an inverted laser confocal microscope (Zeiss510, Germany). Excitation was at 364 or 488 nm for visualization of DAPI or AlexaFluor-488 with emission filters of 385–470 nm or 505–550 nm, respectively. The number of γ-H2AX foci present in each cell was quantified with Image J software (version 1.36b, National Institutes of Health) using customized macros recently developed by our group.
Western blot analysis
Cellular proteins were extracted, quantified, and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE, keygentec company, KGP113). Proteins samples were then blotted onto a nitrocellulose membrane. After incubation with a blocking buffer, the membranes were incubated for 2 h at RT with each primary antibody at the appropriate dilution, as recommended by the supplier. Antibodies included Bax, Bcl-2, Caspase-3, Caspase-9, and Cytochrome C. After washing, the membranes were subsequently incubated for 1 h at RT in Goat Anti-mouse Immunoglobulin G (IgG, keygentec company, KGAA36) followed by the use of enhanced chemiluminescence kits. β-actin was used as an internal control.
Expression of CAT, SOD, and T-GSH in HepG2 cells14
Catalase (CAT) activity was estimated by the method of Aebi. Activity of the enzyme superoxide dismutase (SOD) was measured by nitro blue tetrazolium reduction method of McCord and Fridovich. The level of glutathione (GSH) was assayed by the method of Moron et al. based on the reaction with dithiobis(2-nitrobenzoic acid). The method measured the rate of decomposition of hydrogen peroxide (H2O2) at 240 nm.
Statistical analysis
In the statistical analysis, differences between the treated and control groups were compared using Student’s t tests, with the differences at the P < 0.05 level considered to be statistically significant.
