Materials

Silver nitrate (AgNO3) and trisodium citrate dihydrate (Na3C6H5O7) were purchased from Sinopharm Chemical Reagent Co., Ltd., polyvinylpyrrolidone (PVP, Mw = 29.0 kg·mol−1) was purchased from Tianjin Kermel Chemical Reagent Co., Ltd., and sodium borohydride (NaBH4, purity 98%) was purchased from Alfa. Human OC SKOV3 cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Dojindo, Japan). The cell cycle, Annexin V-FITC/PI and ROS detection kits were all purchased from Beyotime. Antibodies for western blot (WB) were purchased from Cell Signaling Technology (CST) and Abcam. BALB/c-nu mice were purchased from the Jinan Pengyue Experimental Animal Breeding, Co., Ltd. (Shandong, China).

Synthesis and characterization of tAgNPs

Five types of AgNPs were synthesized using a chemical reduction method. AgNO3 was added to Na3C6H5O7, PVP, and H2O2 with stirring, and stirring was continued for approximately 5 min. An appropriate amount of NaBH4 was weighed and directly added to this solution; the solution was placed in a water bath at 27 °C, and stirring was continued for approximately 30 min until the colour of solution changed. The dynamic light scattering (DLS) experiment was carried out with Malvin Laser Particle Sizer (ZEN3690, Malvin company, UK). The synthesized AgNPs were characterized using a scanning electron microscope (SEM, Zeiss, Germany) to obtain SEM images. According to the SEM images, the particle size distribution was plotted using Origin software, and the biological reduction of silver ions at 420 nm was monitored using spectrophotometry.

Cell viability assay

Human OC SKOV3 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 5 × 103 cells/well and cultured overnight in an incubator (37 °C and 5% CO2). Solutions containing different concentrations of tAgNPs (500 ng/ml, 1000 ng/ml, 1500 ng/ml, 2000 ng/ml, 2500 ng/ml, 3000 ng/ml) were prepared with RPMI 1640 complete medium and added into wells at 100 μl/well. A control group and a blank group were also prepared. After 24 h of exposure, the old medium was removed and replaced with 100 μl of new medium containing CCK-8, and the culture was continued for 1–2 h. The optical density (OD) at 450 nm was measured with a microplate reader (BioTek). The cell survival rate was calculated using the following formula:

$$\mathrm(\mathrm) = (}_} - }_})/(}_} - }_}) \times 100\mathrm$$

Plate colony formation assay

Human OC SKOV3 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 1000 cells/well and cultured overnight in an incubator (37 °C, 5% CO2). Complete RPMI 1640 medium was used to prepare a solution of tAgNPs (d = 50 nm) at a concentration of 1000 ng/ml, and a control group was prepared. Cells were cultured in an incubator for 2 weeks (37 °C, 5% CO2), and the medium was changed every 3 days. After 2 weeks, the cells were fixed with 4% paraformaldehyde and stained with an appropriate amount of 0.5% crystal violet for 15 min. The excess staining solution was slowly removed with running water, and the cells were air-dried and counted under a microscope (Olympus). The colony formation rate was calculated using the following formula:

$$\mathrm=\mathrm/\mathrm\times 100\mathrm$$

Cell cycle experiment

Human OC SKOV3 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 2 × 105 cells/well and cultured overnight in an incubator (37 °C, 5% CO2). The culture medium in the 6-well plates was discarded. A solution of tAgNPs (d = 50 nm) at a concentration of 1000 ng/ml was prepared in RPMI 1640 complete medium, and a control group was prepared. Cells were cultured in an incubator for 24 h, 48 h, and 72 h (37 °C, 5% CO2). The procedure was performed according to the instructions of the cell cycle detection kit (Beyotime), and detection was performed using a flow cytometer (Beckman Coulter). The data are processed and analyzed by flow JO software (Version 10).

Cell apoptosis assay

Human OC SKOV3 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 2 × 105 cells/well and cultured overnight in an incubator (37 °C, 5% CO2). Complete RPMI 1640 medium was used to prepare a solution of tAgNPs (d = 50 nm) at a concentration of 1000 ng/ml, and a control group was prepared. Cells were cultured in an incubator for 24 h, 48 h, and 72 h (37 °C, 5% CO2). The procedure was performed according to the instructions of the Annexin V-FITC/PI apoptosis detection kit (Beyotime), and detection was performed using a flow cytometer (Beckman Coulter). The data are processed and analyzed by CytExpert for DxFLEX software.

ROS detection

Human SKOV3 OC cells in the logarithmic growth phase were seeded in 6-well plates at a density of 2 × 105 cells/well and cultured overnight in an incubator (37 °C, 5% CO2). After 24 h of culture, a solution of tAgNPs (d = 50 nm) at a concentration of 1000 ng/ml was added to the experimental group and cultured for 24 h. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) was diluted with serum-free medium at a ratio of 1:1000, and 1 ml of diluted DCFH-DA was added to each well, followed by an incubation in an incubator for 20 min (37 °C). DCFH-DA was discarded, and the serum-free medium was used to fully wash away the DCFH-DA that did not enter the cells. Cells were observed under an inverted fluorescence microscope (Leica).

WB

Human OC SKOV3 cells in the logarithmic growth phase that were untreated and treated with 1000 ng/ml tAgNPs were collected, fully lysed and resuspended in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. The OD of the extracted protein at 562 nm was determined using the bicinchoninic acid (BCA) protein assay kit (Beyotime), and the protein concentration was calculated. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes after separation on PAGE gels and then incubated with Tris-buffered saline supplemented with Tween (TBST) blocking solution containing 5% nonfat milk for 2 h at room temperature. The membranes were washed with TBST and incubated with antibodies against specific proteins at 4 °C overnight. Rabbit pAb: anti-caspase-3(diluted 1/5000), cyclinA2(diluted 1/2000) and cyclinD1(diluted 1/1000) were used. The membrane was washed three times with TBST and incubated with the secondary antibody (diluted 1/2000) for 1.5 h. TBST was used to remove the unbound secondary antibody, an appropriate amount of chromogenic solution was added to visualize the bands, and images were captured using a luminometer (Tanon).

In vivo tumorigenesis experiment

BALB/c female nude mice aged 4–5 weeks and weighing 16–18 g were housed in separate cages in a specific-pathogen-free (SPF) animal room, with five animals in each cage. Mice were fed with national standard solid mixed feed, with the free access to water. Human OC SKOV3 cells in the logarithmic growth phase were inoculated subcutaneously into the right thigh of nude mice at a concentration of 2 × 107 cells/ml in a volume of 100 μl per mouse. Mice were randomly divided into Groups A and B, with six mice in each group. Immediately after modelling, mice in Group A were intraperitoneally injected with a solution of tAgNPs (d = 50 nm) at a concentration of 1.513 × 104 ng/ml once every other day in a volume of 300 μl per mouse. In Group B, the injection was started when the tumour grew to approximately 100 mm3, and the concentration and dose of tAgNPs (d = 55.7 nm) were the same as those in Group A. The total intervention time was approximately 2 weeks. During the administration period, the mental state, diet and water consumption of the mice were observed daily. The long axis a and short axis b of the tumours in the tumour-bearing mice were measured every 2 days, and the changes in the body weight of the mice were measured. The tumour volume was calculated using the following formula, and the tumour growth and mouse body weight change curves were plotted:

Haematoxylin and eosin (HE) staining

After the nude mice were sacrificed, the heart, liver, spleen, lung, and kidney were removed, washed with normal saline to remove the blood, fixed with 4% paraformaldehyde, processed into paraffin sections for HE staining, and observed under a microscope (Olympus).

Statistical analysis

All experiments were repeated at least three times. The results are presented as the means ± standard deviations (SD). A T test or one-way analysis of variance (ANOVA) was used to compare all experimental data, and p < 0.05 was considered statistically significant.