HepG2 cells were obtained from the cell bank of the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cell culture reagents were purchased from Gibco, Carlsbad, CA, USA. Chlorauric acid (HAuCl₄·3H₂O), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), folic acid (FA) and (3-aminopropyl)triethoxysilane (APTS) were purchased from Sigma (St. Louis, MO, USA); cetyltrimethylammonium bromide (CTAB), silver nitrate (AgNO₃) and sodium borohydride (NaBH₄) were from Aladdin (Shanghai, China); tetraethoxysilane (TEOS) and ascorbic acid were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2,5-Diphenyltetrazolium bromide (MTT) and cell apoptosis reagents were purchased from BestBio (Shanghai, China). Polymerase chain reaction (PCR)-related reagents and cell incubators were purchased from Thermo Fisher Scientific, Inc. (Rockford, IL, USA). Anti-Bax (ab77566), anti-Bcl-2 (15071s) and anti-Ki-67 (PB0065) antibodies were purchased from Abcam (Cambridge, UK), Cell Signaling Technology (Danvers, MA, USA) and Boster Biological Technology, Ltd. (Wuhan, China), respectively.

The gold nanorods (GNRs) were synthesized as previously described (12). Briefly, 0.6 ml ice-cold 0.01 M NaBH₄ was mixed with 10 ml of aqueous solution containing 7.5 ml 0.2 M CTAB and 2.5 ml 0.001 M HAuCl₄ under vigorous stirring. It was then kept at 25°C for at least 2 h prior to use as a seed solution. The growth solution contained 50 ml 0.2 M CTAB, 50 ml 0.001 M HAuCl₄, 1 ml 0.004 M AgNO₃ and 0.7 ml 0.0778 M ascorbic acid. After gently mixing the growth solution, 80 µl of seed solution were added and kept at 30°C for 24 h to obtain the GNRs.

Next, the spherical core-shell silica-coated GNRs (GNRs@ SiO₂) were successfully prepared using the sol-gel method. First, the obtained GNRs were washed with deionized water twice to remove the excess CTAB and redispersed in 40 ml of water. Subsequently, aqueous ammonia solution was added to obtain a pH of approximately 10, and 8 ml of 10 mM TEOS/ethanol solution were added to the solution. The reaction mixture was allowed to react for 24 h under vigorous stirring. The resulting mesoporous silica-coated GNRs (GNRs@SiO₂) were redispersed in absolute ethyl alcohol. Finally, the GNRs@SiO₂ were conjugated with folic acid (GNRs@SiO₂-FA). EDC and NHS were used to activate the carboxyl groups of folic acid (Fig. 1).

The human hepatocellular carcinoma cell line, HepG2, was used in the experiments. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco) at 37°C in a 5% CO₂ humidified incubator (Thermo Fisher Scientific, Inc.).

The HepG2 cells were cultured in a 96-well plate at a density of 5×10³ cells/ml for 24 h, and the culture medium was then replaced with solutions containing either GNRs, GNRs@SiO₂, or GNRs@SiO₂-FA, which were pre-mixed with fresh DMEM supplemented with 10% FBS [gold (Au) concentrations were 40, 20, 10, 5 and 2.5 ppm]. Untreated cells served as the controls. The cells were cultured for a further 48 h, and the culture medium was then replaced with 0.2 ml fresh culture medium containing MTT assay reagent (4 mg/ml), followed by incubation for a further 4 h. DMSO (100 µl) was added to each well to dissolve the colored crystals. The optical density of the colored product was measured at 490 nm using a microplate reader (Bio-Tek ELx800; Bio-Tek Instruments Inc., Winooski, VT, USA).

The HepG2 cells were cultured in DMEM medium supplemented with 10% FBS, under standard cell culture conditions (5% CO₂, 37°C) for 24 h. The cells were washed 3 times with phosphate-buffered saline (PBS). The experimental group was treated with 9 ml of DMEM medium and 1 ml of 40 µg/ml GNRs@SiO₂-FA solution, and the control group (untreated) was treated with 10 ml DMEM medium. The cells in the experimental group were then fixed with 2.5% glutaraldehyde, followed by ethanol-acetone gradient dehydration and embedding in epoxy resin. The cells were then visualized under a transmission electron microscope (JEM-2100; JEOL, Tokyo, Japan).

The HepG2 cells were cultured in a 6-well plate at a density of 5×10⁴ cells/ml for 24 h. The cells were divided into 2 groups: in one group, 100 µl of GNRs@SiO₂ were added to each well, and in the other group, 100 µl of GNRs@SiO₂-FA were added to each well. The cells were then incubated in humidified air at 37°C with 5% CO₂. The cultivation of cells was terminated after 1, 4, 8, 16 and 24 h, and this was followed by the collection of the cells, the detection of Au elements by inductively coupled plasma mass spectrometry (ICP-MS), and the calculation of the cell solid Au element content per kilogram.

First, a total of 10 (2 ml) centrifuge tubes (mg) were measured for electronic balance. Before the cells were harvested, they were washed with PBS 3 times to remove the free GNRs@ SiO₂-FA and GNRs@SiO₂. The cells were then digested with 300 µl 0.25% trypsin. This was followed by the addition of 500 µl DMEM and 1 ml PBS, and the cells were then collected and placed within weighed 2 ml centrifuge tubes. Next, using a thermostat (60 degrees), the centrifuge tubes containing the cells were dried. They were then weighed for electronic balance, and the quality of the cell solid material was obtained by subtracting the previous record of quality. This was followed by the additino of 500 µl aqua regia to each tube, and the solid material was dissolved by ultrasound, with the addition of triple-distilled water to a final volume of 10 ml. Finally, the Au element was detected by ICP-MS and the Au element content was caclulated per kg cell solids (mg/kg).

HepG2 cells, cultured in vitro, were divided into the following 4 groups: i) the control group (untreated), ii) the GNRs@SiO₂-FA group (40 µg/ml), iii) the iodine 125 seeds group (9 grains, 0.8 mCi), and iv) the combination group (with GNRs@SiO₂-FA and iodine 125 seeds). The cells in the 4 groups were collected and washed twice with cold PBS. The cells were centrifuged at 2,000 rpm for 5 min and were then resuspended in 100 µl of Annexin V binding buffer at a density of 1×10⁶ cells/ml. The cells were incubated with 5 µl of Alexa Fluor 488 Annexin V conjugate and 5 µl of propidium iodide (PI) (both from BestBio) for 15 min at room temperature in the dark; 400 µl of 1X binding buffer was added to each sample tube, and the samples were immediately analyzed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Histograms and statistics were processed using FlowJo software version 7.6.1.

Bax and Bcl-2 mRNA expression levels were measured by RT-PCR. The HepG2 cells (1×10⁶), which were treated with GNRs in combination with iodine 125 seeds, were collected and counted. The analysis was performed using the two-step RT-PCR kit (Takara Co., Dalian, China). The purity of the total RNA was determined using a UV spectrophotometer (UV-2450; Shimadzu Corp., Kyoto, Japan) at OD260/OD280 (>1.8). The amplification method was as follows: Bax, 30 cycles; Bcl-2, 32 cycles; GAPDH, 32 cycles (94°C, 30 sec; 56°C, 30 sec; 72°C, 1 min). The amplified product was analyzed by gel electrophoresis on a 2% agarose gel and EB staining (ImageJ software). The primer sequences and PCR product sizes are listed in Table I.

For protein extraction, protein extraction solution with protease inhibitor (1% Triton-X100, 0.1% SDS, 150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% deoxycholic acid, 2 mM EDTA pH 8.0) was used, and the protein was quantified using a BCA Protein assay kit (Thermo Fisher Scientific, Inc.) to plot a standard curve. The protein samples were boiled for 5 min, loaded and subjected to SDS-polyacrylamide gel electrophoresis (PAGE), and were subsequently transferred onto a PVDF membrane. The membrane was blocked with 5% BSA for 1 h at room temperature and probed with rabbit antibodies against Bcl-2, Bax and caspase-3 (anti-caspase-3 antibody from Cell Signaling Technology) (all 1:1,000 dilution) overnight at 4°C. The membrane was washed with Tris-HCl buffer containing Tween-20 (TBST 20 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.05% Tween-20) and incubated in horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit antibody (Cat. no. 49620; Cell Signaling Technology) (1:4,000 dilution) for 1 h at room temperature. The blots were developed by adding electrochemiluminescence (ECL) detection reagents.

Both the treated and untreated HepG2 cells were fixed with 4% paraformaldehyde for 15 min. The cells were permeabilized for 10 min at room temperature in 0.4% Triton X-100 diluted in PBS. The fixed cells were incubated overnight at 4°C with each of the primary antibodies: anti-Bax (1:100), anti-Ki-67 (1:100) and anti-Bcl-2 (1:50). On the following day, after 3 washes with PBS, the secondary biotin-labeled antibody (SP9001; ZSGB-BIO, Beijing, China) was used at 1:200. For color development, streptavidin was labeled with HRP at 1:200, using the streptavidin-biotin-peroxidase complex (SABC) method.

Bax-positive staining (tan) was observed in the cytoplasm, and Bcl-2-positive staining (tan) sd was observed in the cytoplasm and cell membrane. To quantify protein expression in the various samples, a scoring method was applied. A mean percentage of positive cancer cells was determined from at least 5 areas at ×400 magnification and assigned to 1 of the following 5 categories, as previously described (13): 0 point, <5%; 1 point, 5–25%; 2 point, 26–50%; 3 point, 51–75%; and 4 point, >75%. Points for staining and percentages were multiplied for a 10-point scale as follows: 0 point, negative (−); 1–3 points, weakly positive (+); 4–6 points, positive (++); and 7–9 points, strongly positive (+++). For cells that showed heterogeneous staining, the predominant pattern was taken into account for scoring. The percentage of positive cancer cells and the staining intensity were multiplied to produce a weighted score for each case. Cases with a weighted surviving score <1 were considered to be negative.

Positive Ki-67 staining was indicated by the presence of fine tan particles in the nucleus. Each slice was observed at >10 typical views (at a high magnification, ×400), counting at least 200 cells. The percentage of positive cells indicated the Ki-67 cell proliferation index, which was termed KI.

Statistical analysis was performed using SPSS 16.0 software for Windows. The cell survival rate was determined by MTT assay and the differences in the apoptotic rate in the 4 groups was analyzed by one-way ANOVA. P-values <0.05 were considered to indicate statistically significant differences.

GNRs@SiO₂-FA distribution in the cells was determined by TEM. The prepared GNRs@SiO₂-FA had an ellipsoid shape. The GNRs were composed of a central rod, approximately 40 nm long and 10 nm wide, with a coating of silica on the surface. We demonstrated that GNRs@SiO₂-FA entered the cells in the form of nanoparticles. We also found that following entry into the cell, the ellipsoid shape of the GNRs@SiO₂-FA was retained (Fig. 2).

The results of MTT assay revealed that cell viability decreased as the GNR concentration increased in the culture medium. The cell survival rates in each group were as follows: GNRs group were 59.1, 71.8, 80.5, 85.3 and 91.1%; the GNRs@SiO₂ group were 80.3, 85.7, 88.8, 95.8 and 97.1%; and the GNRs@SiO₂-FA group were 92.3, 93.6, 95.4, 98.7 and 99.4%, respectively compared to treatment with 2.5, 5, 10, 20 and 40 ppm of the GNRs/GNRs@SiO₂/GNRs@SiO₂-FA. When the concentration of the Au element was ≥5 ppm, all 3 groups (GNRs, GNRs@SiO₂ and GNRs@SiO₂-FA) exhibited statistically significant differences compared to the untreated controls (P<0.05). The cell growth inhibitory effects of GNRs@SiO₂-FA were not as prominent as those of the GNRs and GNRs@SiO₂. The GNRs alone had the most prominent inhibitory effect on cell growth (Fig. 3).

As shown in Fig. 4, the cellular uptake rate of GNRs@SiO₂-FA was 2.65-fold higher than that of GNRs@SiO₂ at 4 h, and the cellular uptake rate in both groups reached a plateau at subsequent time points (16 and 24 h. This indicated that we had successfully prepared GNRs@SiO₂-FA with a high targeting ability, and with the ability to bind with folate receptors within a short period of time. This experiment also revealed that nanoparticles can enter cells by endocytosis; however, entry by endocytosis is less efficient compared with the folate receptor-mediated entry of nanoparticles into cells.

The apoptotic rates of the HepG2 cells were higher in the GNRs@SiO₂-FA group and the iodine 125 seeds group than in the control group (P<0.05). The apoptotic rate was also significantly higher in the combination group than in either the GNRs@SiO₂-FA group or iodine 125 seed group (P<0.05) (Fig. 5 and Table II).

RT-PCR revealed that the mRNA expression of Bax significantly increased, and the mRNA expression of Bcl-2 significantly decreased in the combination group to a greater extent compared with either the GNRs@SiO₂-FA group or the iodine 125 seed group (Fig. 6).

Western blot analysis revealed that the expression of the apoptosis-related proteins, Bax and caspase-3, was significantly upregulated in the combination group to a greater extent compared with either the GNRs@SiO₂-FA group or the iodine 125 seed group. The expression of Bcl-2 was, however, downregulated (Fig. 7).

In the 4 groups of HepG2 cells, the percentage of Bax-positive cells was found to be 0.95±0.18, 1.51±0.44, 4.29±0.86 and 7.55±1.40 in the control, GNRs@SiO₂-FA, iodine 125 seed and the combination group, respectively. Compared with the GNRs@SiO₂-FA and iodine 125 seeds groups, the protein expression level of Bax was significantly increased in the combination group (P<0.05) (Fig. 8 and Table III).

In the 4 groups of HepG2 cells, the percentage of Bcl-2-positive cells was 9.01±1.02, 8.39±0.47, 3.81±1.28 and 1.70±0.83 in the control, GNRs@SiO₂-FA, iodine 125 seed and the combination group, respectively. The protein expression level of Bcl-2 was much lower in the combination group than in the GNRs@SiO₂-FA and iodine 125 seeds groups (P<0.05) (Fig. 8 and Table III).

The cells in the control group were plump in shape. When the nuclei were hyper-chromatic, the results were positive. The protein expression level of Ki-67 was significantly decreased in the combination group compared with the GNRs@SiO₂-FA and iodine 125 seeds groups (P<0.05), and the cell nuclei were stained lightly. In the 4 groups of cells, the Ki-67 proliferation index (KI) was 75.59±6.29, 67.09±5.46, 46.84±7.90 and 27.90±5.83% in the control, GNRs@SiO₂-FA, iodine 125 seed and the combination group, respectively, which was significantly different between the 4 groups (P<0.05) (Fig. 8).