SNs, with a particle size of ~60 nm, were provided by the College of Chemistry at Shandong University. Briefly, 2.5 ml tetraethylorthosilicate was added to a premixed ethanol solution (50 ml) containing 2 ml of ammonia and 1 ml of water. The mixture was stirred (150 RPM) at 40°C for 12 h and was then centrifuged at 11,294 x g and 37°C for 15 min to isolate the particles. After washing with deionized water three times, the particles were dispersed in 50 ml of deionized water and sterilized by autoclaving (0.1 MPa, 120°C, 20 min) as a concentrated suspension for further experiments. SNs were dispersed in normal saline, which provided osmotic pressure comparable to that of cell fluid, sonicated with 20 KHz at 37°C for 10 min, and then, after standing for 24 h, the morphology, particle size and dispersibility of the particles were observed via transmission electron microscopy (magnification, x100,000; JEM‑2010; JEOL, Ltd.). Briefly, samples were prepared for TEM observation by dropping a 10-l ethanol solution of ultrasonic dispersed SNs onto a 400-mesh carbon-coated copper grid. Image-Pro Plus software 6.0 (Media Cybernetics, Inc.) was used to analyze the data and calculate the mean particle size. SNs were diluted (3 mg/ml) with saline following exposure to high pressure, and the particle size distribution and polymerization state were monitored by dynamic light scattering using a particle size analyzer after standing at room temperature for 24 h. The zeta potential and hydrodynamic size of the different concentrations of SNs in the dispersion medium (RPMI-1640; Gibco; Thermo Fisher Scientific, Inc.) were examined using a zeta-potential granulometer (Zeta Sizer 3000HS; Malvern Panalytical Ltd.).

Human normal hepatocyte HL-7702 cells were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences). HL-7702 cells were maintained in RPMI-1640 medium, containing 10% fetal bovine serum (Biological Industries) with 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated at 37°C in a 5% CO₂ humidified atmosphere and continuously passaged every 2-3 days. Cells were seeded in culture plates at a density of 1x10⁵ cells/ml for experiments. Following incubation for 24 h, cells were treated with SNs dispersed in serum-free RPMI-1640 at concentrations of 0, 25, 50, 100 or 200 mg/ml at 37°C for 24 h. Cells maintained in RPMI-1640 without SNs were used as the control group.

The effects of SNs on cell viability were determined using an MTT assay according to the manufacturer's protocols. following exposure to various SN concentrations in serum-free RPMI-1640 (0, 25, 50, 100, and 200 µg/ml) at a density of 5,000 cells/sample in a 96-well plate for 12 and 24 h, MTT was used to detect cell viability. Each group was set up in triplicate, and then 10 µl MTT solution (5 g/l) was added to each well and incubated for an additional 4 h at 37°C. The optical density (OD) was detected at 490 nm using a microplate reader (Thermo Fisher Scientific, Inc.). The following formula was used to determine cell viability: [(Ae-Ab)/(Ac-Ab)] x100%, where Ae is the experimental OD, Ac is the control OD, and Ab is the blank OD.

H&E staining was conducted to observe cell morphology. The cells, after grouping, were seeded into six-well plates at a density of 1x10⁵ cells/sample. After exposure to SNs for 24 h, cells were washed 3 times with PBS. Following fixation with 95% ethanol at room temperature for 20 min, hematoxylin was used to stain the nuclei at room temperature for 3 min. After washing, the cytoplasm was stained with eosin at room temperature for 1 min. Cells were then washed, naturally dried and sealed with neutral balsam, and morphological changes were observed under an optical microscope (magnification, x200; DM4000M; Leica Microsystems GmbH).

Briefly, 1x10⁴ cells were inoculated onto clean and dry slides and exposed to SNs for 24 h. The cells were fixed in formaldehyde at room temperature for 15 min and then stained with Giemsa (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 10 min, according to the manufacturer's protocols. After staining, the cells were observed under an optical microscope (magnification, x400). In general, apoptotic cells stained blue and were darker in color than normal or necrotic cells.

Cells (1x10⁴ cells/sample) were stained by 5 µl Annexin V-FITC for 10 min and 5 µl PI for 5 min, respectively, at room temperature in the dark, according to the protocols provided by the manufacturer of an Annexin V-FITC/PI Apoptosis Assay kit (KGI Biosciences). The quantification of apoptosis induced by SNs in HL‑7702 cells was conducted via flow cytometry (FCM; BD Biosciences); cells were collected and evaluated using CellQuest software (version 5.0; BD Biosciences), and apoptosis was analyzed by ModFit software (version 3.2; Verity Software House). The results are expressed as the rate of apoptosis (the percentage of early + late apoptotic cells).

Rhodamine 123 is a cationic fluorescent dye that penetrates cell membranes and is an indicator of the MMP. Cells (1x10⁴ cells/sample) were trypsinized with 0.25% trypsin for 2 min, then mixed with 20 µg/ml Rhodamine 123 and incubated at 37°C in the dark for 30 min. The MMP was detected via FCM (Beckman Coulter, Inc.), with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Cells were evaluated using CellQuest software (version 5.0).

Damage of DNA in HL-7702 cells following treatment with SNs was analyzed using a Comet Assay kit (Trevigen; Bio-Techne). Cultured cells (1x10⁵ cells/sample) were treated with SNs and harvested, and live cells were embedded within agarose on a glass slide. Cells were then lysed, and DNA was unwound under alkaline conditions, followed by electrophoresis (1%) and 20 µg/ml ethidium bromide staining. Damaged DNA migrates towards the anode, leading to the appearance of comets; DNA damage was quantified by calculating the ratio of the tail length to the head diameter. Single-cell gel electrophoresis (SCGE) analysis was used to determine whether DNA damage had occurred in HL-7702 treated with SNs.

Cultured cells (1x10⁵ cells/sample) were trypsinized with 0.25% trypsin for 2 min, and centrifuged at 1,000 x g for 5 min at room temperature, then pre-cooled 70% ethanol was added to the cell pellet and stored overnight at 4°C. The cells were collected via centrifugation (1,000 x g) for 5 min at room temperature, following which 500 µl PI (Tianjin Sungene Biotech Co., Ltd.) was added, followed by FCM analysis. The results were analyzed using MultiCycle 6.0 (Phoenix Flow Systems, Inc.).

After fixing with 4% paraformaldehyde at room temperature for 15 min, cells were permeabilized with 0.5% Triton-X-100 for 20 min and blocked with 3% hydrogen peroxide for 30 min at room temperature to block endogenous peroxidase. Cells were blocked with 5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 30 min, followed by staining with primary and secondary antibodies, and then incubation for 10 min with DAB and 3 min with hematoxylin, both at room temperature. Immunocytochemistry was performed to detect the protein expression of Bax (1:500; cat. no. ab53154; Abcam), Bcl-2 (1:200; cat. no. ab59348; Abcam), Cyt C (1:1,000; cat. no. ab90529; Abcam) and caspase-3 (1:500; cat. no. ab13847; Abcam) using primary antibodies overnight at 4°C. The secondary antibody used was horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:1,000; cat. no. ab214880; Abcam) for 1 h at room temperature. After staining, the cells were observed under an optical microscope. Brown or yellow-stained cells were quantified for positive staining for each high‑power field (x400 magnification). Results are expressed as the total positive numbers of 50 random and continuous fields from each section using the software ImageJ (version 1.52; National Institutes of Health). All analyses were performed in a blinded manner without prior knowledge of the experimental groups.

Western blotting was conducted to detect the expression of apoptosis-associated proteins. Following exposure to 0, 25, 50, 100 or 200 µg/ml SNs as aforementioned, cells were cultured at 37°C in a 5% CO₂ incubator and cultured for 24 h. Cells were washed with cold PBS 2-3 times and lysed in RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) for 30 min on ice. Cells were then centrifuged for 10 min (8,430 x g at 4°C), and the supernatants were collected for protein determination using the BCA method (Beyotime Institute of Biotechnology). Protein samples (50 µg) were separated via SDS-PAGE (10% resolving gels, 5% stacking gels) and transferred to PVDF membranes (300 mA, 1.5 h). The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature, followed by incubation with the aforementioned primary antibodies (at a dilution of 1:1,000 for Bax, 1:1,000 for Bcl-2, 1:2,000 for caspase-3 and 1:1,000 for Cyt C) and anti-GAPDH antibody (1:20,000; cat. no. 10494-1-AP, ProteinTech Group. Inc.) overnight at 4°C. After washing with TBS-0.05% Tween 20 (TBST), the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibodies (cat. no. ab6721; Abcam) at 1:10,000 dilutions for 1.5 h at room temperature, followed by a further wash with TBST, prior to exposure to an enhanced chemiluminescence reagent (ECL detection kit; Pierce; Thermo Fisher Scientific, Inc.). Finally, the membranes were exposed to an Odyssey CLx near-infrared fluorescence imaging system (LI‑COR Biosciences). Results were quantified using AlphaEaseFC software (version 4.0; ProteinSimple). All experiments were conducted in triplicate.

All statistical analysis was performed using Prism 5 (GraphPad Software, Inc.). Data are presented as the mean ± SEM. One-way ANOVA with Dunnett's post hoc test was used to compare across multiple treatments. Experiments were repeated three times with at least triplicate wells per condition. P<0.05 was considered to indicate a statistically significant difference.

As presented in Fig. 1 and Table I, SNs were spherical, with an average particle size of 60.80±4.36 nm, and good dry dispersion characteristics in the absence of aggregation. Zeta potential analysis revealed that different concentrations of SNs in serum-free RPMI-1640 medium were negatively charged, and the absolute value of the zeta potential was ~−20 eV, with good dispersion (Table II).

A decrease in viability was observed for HL-7702 cells that were exposed to SNs for 12 h, with a significant difference emerging at 200 µg/ml compared with the control (P<0.05; Fig. 2). Cell viability decreased further with increasing exposure time (24 h), specifically at the concentrations of 50-200 µg/ml (P<0.05), with decreases in cell viability at 100 and 200 µg/ml of 24.6±4.3 and 44.5±0.09%, respectively. These results indicated that SNs reduced the viability of HL-7702 cells.

A number of morphological changes were observed following incubation of HL-7702 cells with SNs for 24 h. The control group resembled polygons with large and round nuclei, located in a central nucleus with well‑defined nucleoli; conversely, following treatment with 50 µg/ml SNs, cell swelling was evident and nuclei were mildly vacuolated (Fig. S1). With an increase in dose, the cell volume decreased, changes to the cytoplasm and nuclear vacuoles were notable, the chromatophore basophilic edge set was enhanced, the cytoplasm was dense, the eosinophilicity was enhanced and apoptotic bodies formed. At the highest concentration (200 µg/ml), nuclear membrane thickening, cell body shrinkage, nuclear pyknosis and disintegration were evident, indicating the occurrence of apoptosis (Fig. S1E). In summary, cells treated with SNs exhibited morphological changes associated with apoptosis.

Giemsa staining was employed to detect apoptosis in HL-7702 cells treated with SNs. In cells stained with Giemsa, nuclei are stained blue or purple-blue, and the cytoplasm is stained pink under a light microscope (34). In the present study, normal nuclei stained purple-blue and exhibited uniform color following Giemsa staining (Fig. S2). Conversely, there was nuclear hyperchromatism, shrinkage, edge aggregation and the formation of densely stained apoptotic bodies in the SN treatment groups. As presented in Fig. S2, with increasing concentrations of SNs, the number of cells was reduced, cells shrunk, the intercellular space increased, the cell volume decreased, the cytoplasm permeability disappeared, the cell surface became prominent and apoptotic body formation was evident.

Annexin V-FITC/PI double staining and a flow cytometer were used to detect apoptosis. A significant increase in apoptosis was observed at concentrations of SNs of 50-200 µg/ml, varying between 25-40% apoptosis by 24 h (P<0.05; Fig. 3F).

As a dose-dependent increase in apoptosis was observed following exposure to SNs, Rhodamine 123 was used to monitor the MMP in treated cells. Exposure of HL‑7702 cells to SNs for 24 h induced a significant decrease in MMP at 25 µg/ml, which decreased further with increasing concentrations of SNs (P<0.05; Fig. 4).

SCGE and an alkaline comet assay were used to determine the levels of DSBs in cultured HL-7702 cells treated with various concentrations of SNs. Whereas control cells appeared to be largely spherical, cells treated with different concentrations of SNs exhibited cell trailing or tail formation, indicating the formation of DSBs, which increased with the concentration of SNs (Fig. 5).

FCM analysis revealed that HL-7702 cells treated with SNs exhibited a significantly decreased percentage of cells in G₀/G₁ phase after 24 h, whereas the S and G2/M phase proportions significantly increased (Fig. 6). These results indicated that, following exposure to SNs, cells were impeded in their cycle progression and accumulated in the G2/M phase (Fig. 6F).

It was demonstrated via immunocytochemistry that exposure of HL-7720 cells to increasing concentrations of SNs induced a significant increase in the number of Bax‑positive, Bcl-2-positive, Cyt C-positive and caspase-3-positive cells (P<0.05; Figs. 7A and S3-S6). Furthermore, western blotting revealed that SN treatment significantly increased Bax expression and decreased the Bcl-2/Bax ratio (P<0.05; Fig. 7B-D). The expression levels of Cyt C and caspase-3 were also significantly upregulated following treatment with increasing concentrations of SNs, compared with control group (P<0.05; Fig. 7C and D).