Au NS particles were prepared by a seed-mediated reduction method. The morphology and size distribution of the particles were analysed by a laser particle size analyser, a spectrophotometer and a transmission electron microscope (TEM). The uptake of Au NS by the endometrial cancer cell line (HEC-1A) at different temperatures was observed by spectrophotometry and using an inverted microscope. Cytotoxicity tests and apoptosis experiments were then carried out by MTT assays and flow cytometry, respectively, to analyse the effects of Au NS, cordycepin and their combination on HEC-1A cells.
Preparation of Au NS
Au NS was prepared by seed-mediated reduction . A tetrachloroauric acid solution with a concentration of 1 mmol was prepared using 0.08494 g of tetrachloroauric acid (HAuCl4) and 250 ml of deionised water. The solution was heated for 40 minutes. Then, 4.5 ml of freshly prepared 1% sodium citrate solution was added to 30 ml of the tetrachloroauric acid solution, followed by another 15 minutes of heating and vigorous stirring. The solution was allowed to cool and filtered through a 0.22 μm filter membrane, which was used as the seed. The hydrodynamic size and polydispersity of the particles in the seed solution were measured using a laser particle size analyser (ZETASIZER Nano series, Malvern).
Stock solutions of 1 mmol hydrochloric acid solution, 4 mmol silver nitrate solution, 100 mmol ascorbic acid solution and 0.25 mmol tetrachloroauric acid solution were prepared. Next, 10 μl of 1 mmol hydrochloric acid solution was mixed with 10 μl of 1 mmol hydrochloric acid solution at 700 rpm. After 30 seconds, 100 μl of the seed solutions was added to the mixture. After another 30 seconds, 100 μl of a 4 mmol silver nitrate solution and 50 μl of a 100 mmol ascorbic acid solution were also added. After centrifugation at 5000 rpm for 15 minutes, the precipitate was removed and resuspended in 1 ml of deionised water. Finally, the concentration of the Au NS solution was determined by the extinction coefficient and the Beer-Lambert law.
Morphology and size
The morphology of the Au-NS particles was observed by TEM (FEI Tale, 200keV) and the localised surface plasmon resonance (LSPR) was measured by UV-visible spectrophotometry (SPECTROstar Nano). The highest absorption peak of LSPR in this study was 800-850 nm . The laser particle size analyser (ZETASIZER Nano series) were used to measure the particle sizes, aiming for a diameter between 50 and 80 nm ; the gold nanostar morphology was observed by TEM.
Photothermal effect of Au NS and its conjugates
Different concentrations of Au NS alone and Au NS-cordycepin solutions were cultured with Hec-1A cells for 24 hours. The supernatant was removed and the cells were washed three times with phosphate buffered saline (PBS). The temperature change induced by each concentration (Fig. 2) was detected after 10 minutes of 808 nm light exposure using an infrared temperature detector (Fluke Ti400). The experiments were repeated three times and the temperature changes of the cell culture medium was used as the control.
Detection of Au NS uptake by ultraviolet-visible spectrophotometer
Different concentrations of Au NS alone and Au NS-cordycepin solutions were cultured with HEC-1A cells for 24 hours. Two hundred grams of the cell suspension was centrifuged for 3 minutes and the cells were washed three times. The supernatant was collected for absorbance measurements by spectrophotometry at 808 nm. The experiments were repeated three times and the absorbance of the cell culture medium was used as the control. The optical density (OD) was calculated using the following formula: OD = (Mean absorbance of test group – Mean absorbance of medium control group) / (Mean absorbance of untreated group – Mean absorbance of medium control group).
The HEC-1A cell line (ATCC, Manassas, VA, USA) was cultured in RPMI-1640 (Gibco, 11875, Waltham, MA, USA) supplemented with 10% foetal bovine serum (Gibco, 16000044, Waltham, MA, USA). Cells were incubated at 37°C in a humidified 5% CO2 atmosphere.
MTT (tetrazolium) assays were used to quantitatively evaluate the effect of Au NS, cordycepin and their combination on HEC-1A cells. The concentrations of Au NS were 3x1010 particles/ml, 6x1010 particles/ml and 1.2x1011 particles/ml; the concentrations of cordycepin were 0.05 mg/ml, 0.1 mg/ml, 0.2 mg/ml and 0.4 mg/ml. The concentration of the Au NS-cordycepin mixtures were 6x1010 particles/ml (AU Ns) + 0.05 mg/ml (cordycepin) and 3x1010 particles/ml (AU Ns) + 0.1 mg/ml (cordycepin). The HEC-1A cells were cultured in a 96-well plate and the exposure time for all samples was 24 hours. PBS was used to wash the cells and 120μl of MTT solution was added. The mixtures were incubated at 37°C and in a humidified 5% CO2 atmosphere. After another 4 hours of incubation, 150 μl of DMSO (Sigma-Aldrich, D4540, Burlington, MA, USA) was added to each well to dissolve the formazan crystals. The plates were gently shaken for 15 minutes at room temperature. The optical density (OD) of the solution was then measured using a microplate reader (SPECTROstar Nano, Ortenberg, Allmendgrün, Germany) at 550 nm. The inhibition percentage was calculated as follows :
Percentage viability = 100% × mean of test OD / mean of control OD
Percentage inhibition = 100 − percentage viability
Clonogenic survival assay
Clonogenic assays (CA) were performed to determine the division ability of HEC-1A cells, and hence, to determine the ability of Au NS and cordycepin to inhibit the growth of HEC-1A cells. Two hundred Hec-1A cells were seeded in a 48-well plate for 24 hours in a humidified 5% CO2 atmosphere at 37°C. Cordycepin, Au NS and the combinations were added to the cells and left for 24 hours. The culture medium was then substituted with complete medium and cells were incubated for 7 days. Wright-Giemsa staining (Sigma, WG16) was used to stain the cell colonies, and those with 50 cells or more were counted. The plating efficiency (PE) and survival fraction (SF) were used to determine the ability of the HEC-1A cells to grow into colonies. This was calculated by the following equations :
Plating efficiency (PE) = (No. of colonies formed / No. of cells seeded) x 100%
Survival fraction (SF) = No. of colonies form / (No. of cells seeded x PE)
Annexin V-FITC/PI staining was performed to detect apoptosis. Two millilitres of 1.0 × 105 HEC-1A cells/ml were inoculated in 35-mm culture dishes and incubated for 24 hours at 37°C, with 5% CO2 and 95% humidity. The cell supernatants were then collected and 1 ml of each experimental solution was added to the dishes and incubated at 37°C, with 5% CO2 and 95% humidity. After 24 hours, the cells were harvested and resuspended using 100 μl of the binding buffer provided with the apoptosis kit (BD Biosciences, 556547, San Diego, CA, USA). The suspension was stained by adding 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI) (Sigma-Aldrich, P4170, Burlington, MA, USA) for 15 minutes in the dark, followed by adding 400 μl of binding buffer for the analysis of apoptosis using an Applied Biosystems Attune flow cytometer (Waltham, MA, USA) and the manufacturer’s software.
Microsoft Office Excel 2013 and Statistical Product and Service Solutions (SPSS) software, version 24.0 were used to analyse all the results. Data are presented as the mean ± the standard deviation (SD). A two-tailed t-test was used to calculate the P values of the differences between two groups of the cytotoxic assay and apoptosis assay while one-way analysis of variance multiple comparisons (one-way ANOVA) was used for the differences between three or more groups. If the p-value was smaller than 0.01, it was considered to be statistically significant.