Sodium borohydride (≥98.0%), gold(III) chloride trihydrate (≥99.9%, HAuCl4 · 3H2O), trisodium citrate tribasic dihydrate (≥99.0%), and N-(2-mercaptopropionyl)glycine (Tiopronin, C5H9O3NS) were purchased from Sigma-Aldrich (St Louis, USA). L(+)-Ascorbic acid (99%) was supplied by Acros (USA). Nitric acid and hydrogen peroxide of MOS grade were bought from the Beijing Chemical Reagents Institute (China). Standard stock solution of Au (1000 μg/mL) was obtained from the National Analysis Center for Iron and Steel, China. All chemicals were used without further purification, and Milli-Q water was used throughout this study.
Synthesis of 2 and 6 nm Au@tiopronin NPs
The procedure for gold nanoparticle synthesis was as reported.26
Au@tiopronin NPs of 2 nm were prepared by dissolving gold(III) chloride trihydrate (0.15 g, 0.4 mmol) and N
-(2-mercaptopropionyl)glycine (tiopronin, 0.19 g,1.2 mmol) in 20 mL of methanol/acetic acid (6:1), producing a ruby red solution. Sodium borohydrate (0.30 g, 8.0 mmol) in 7.5 mL of H2
O was added dropwise with rapid stirring. After continuous stirring for 2 h, a black suspension was formed, and the solvent was then removed under vacuum at 40 LC. The residues were dissolved in 20 mL of H2
O, the pH was adjusted to 1 by dropwise addition of 1 M HCl, and then the solution was dialyzed (dialysis membrane, Solarbio, MWCO = 8000–14 000) for 72 h against Milli-Q water, which was changed every 8 h. The resulting Au@tiopronin NPs were lyophilized and dried completely before further use. The method to prepare 6 nm Au@tiopronin was the same except that the amount of tiopronin was decreased to 0.021 g (0.13 mmol).
Synthesis of 15 nm Au@tiopronin NPs
A surface molecule exchange reaction was adopted to obtain 15 nm Au@tiopronin NPs. Au@citrate NPs of 15 nm were first prepared by the standard citrate reduction method.48
Briefly, 0.5 mL of HAuCl4
O solution (1%, w/v) in 50 mL of Milli-Q water was heated to boiling; then 1.5 mL of trisodium citrate tribasic dihydrate solution (1%, m/v) was added quickly with vigorous stirring. After the color change finished in 5 min, the solution was kept boiling for another 15–30 min and then allowed to cool to room temperature while stirring. Subsequently, the tiopronin replacement was performed while stirring at 40 °C for more than 48 h by mixing Au@citrate solution, prepared as described above, with an aqueous solution containing a large excess of tiopronin. The molar ratio of tiopronin was 20 times more than that of HAuCl4
to ensure the complete replacement of citrate by tiopronin in AuNPs, as the binding affinity of tiopronin to AuNPs with the formation of a Au–S bond is stronger than the electrostatic interaction between AuNPs and citrate. After the reaction, 15 nm Au@tiopronin NPs were purified by centrifugation several times at 9000 rpm for 30 min to remove the unbound tiopronin and the citrate from the AuNPs.
Characterization of AuNPs
The morphology of the Au@tiopronin NPs was determined using a Tecnai G2 20 STWIN transmission electron microscope (TEM, Philips, Netherlands) with 200 kV acceleration voltage. Optical absorption spectra were measured with a Lambda 950 UV/vis/NIR spectrophotometer (Perkin-Elmer, USA). X-ray diffraction spectra and X-ray photo-electron spectra were tested separately with an X’Pert PRO MPD X-ray diffractometer (PANalytical B.V., The Netherlands) and an ESCALAB250Xi X-ray photoelectron spectrometer (Thermo Fisher Scientific, UK). Zeta-potential distribution of AuNPs was determined by a Zetasizer Nano ZS (Malvern, England), and the measurements were recorded at 25 °C suspended in Milli-Q water. The concentration of gold was determined via an Optima 5300DV inductively coupled plasma optical emission spectrometer or an ELAN DRC-e inductively coupled plasma mass spectrometer (Perkin-Elmer, USA).
Uptake of AuNPs by Monolayer Cells
The human breast cancer cell line MCF-7 was maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37 °C. MCF-7 cells were cultured in six-well plates at about 60% confluence. After 24 h incubation, the medium was removed and 1 mL of fresh medium containing 1 nM 2, 6, and 15 nm AuNPs was added to the wells. After 24 h, cells were washed gently with PBS, digested with 0.25% trypsin, centrifuged for 3 min at 1000 rpm, and then collected and counted by Vi-CELL (Beckman Coulter, USA).
Isolation of Cell Nuclei and Qualitative Determination of Au Content
MCF-7 cells were grown at about 60% confluence in six-well plates for 24 h. Afterward, 1 nM gold nanoparticles of 2 and 6 nm were added to the wells. After 24 h incubation, the cells were trypsinized, washed with PBS, and centrifuged for 3 min at 1000 rpm. The treated cells were then divided into two equal parts, one part for direct analysis by ICP-MS and the other for isolation of nuclei using a nuclear extraction kit (Solarbio, Shanghai, China). The percentage of Au nanoparticles in the nucleus was calculated according to the Au content in the isolated nuclei compared to the whole cells.
Cell Viability Assay
Cell viability was determined using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan). First, cells were seeded into 96-well plates (Costar, Corning, NY, USA). After incubation for 24 h, the culture medium was removed and replaced with complete medium containing 2 nm AuNPs at a dose from 4 to 411 nM, 6 nm AuNPs from 0.38 to 38 nM, and 14.8 nm AuNPs from 0.625 to 10 nM. Cells unexposed to Au nanoparticles were regarded as controls. Cell viability was calculated as the ratio of the absorbance of treated and control wells. The absorbance was measured at 450 nm with a reference wavelength of 650 nm using an Infinite M200 microplate reader (Tecan, Durham, NC, USA).
MCF-7 Multicelluar Spheroid Culture
MCF-7 spheroids were produced by the liquid overlay method as previously described.49
Briefly, cells were detached from monolayers, and single cell suspensions (200 μ
L per well containing 600 cells) were transferred into flat-bottomed 96-well plates precoated with 1% agarose. Cells were incubated for about 7 days as described above for monolayer cells, except that the culture medium was partially (100 μ
L) replaced by fresh medium every other day.
SEM Observation of MCF-7 Tumor Spheroids
Environmental scanning electron microscopy was used to examine the outer morphology of the spheroid. After incubation for one week, the spheroids were removed from the wells, washed with PBS (150 mM, pH 7.4), and fixed overnight at room temperature in 5% glutaraldehyde solution. The spheroids were then progressively dehydrated in an ethanol series from 70% to 100%, followed by critical point drying with isoamyl acetate. Finally, the spheroids (without gold coating) were observed by SEM (FEI Quanta 200). For monolayer cells, cells were cultured on cover-slips for 24 h; then the medium was removed and the cells were treated in the same way as the spheroids.
Penetration of AuNPs into Spheroids
For 7-day-old spheroids seeded at a concentration of 600 cells per well, 100 μL of medium was replaced with 2 nM nanoparticles at a final concentration of 1 nM. After 24 h incubation, the spheroids were removed by pipet and gently washed with PBS. The distribution of AuNPs in the spheroid was qualitatively observed using bright field and dark field microscopy. The ultramicrolocalization of gold nanoparticles in the outer and inner cells of the spheroid was evaluated by TEM. In addition, the Au content in every spheroid was quantified by ICP-MS.
Pharmacokinetics and Biodistribution of AuNPs in Vivo
Female Balb/c nude mice (18–20 g) were purchased from Beijing Vital River Laboratories. All care and handling of animals were performed with the approval of the Animal Ethics Committee of the Medical School, Peking University. To establish tumor-bearing mice, animals were subcutaneously inoculated in the right flank with 4 × 106 MCF-7S cells. When the average volume of the tumors reached about 100 mm3, the mice were divided randomly into groups for the pharmacokinetics and distribution studies.
For the pharmacokinetics study, tumor-bearing mice were intravenously injected with AuNPs of 2, 6, and 15 nm at a dose of 5 mg Au/kg. About 30–100 μL blood was taken from the tail vein with a quantitative capillary at 10 min, 1 h, 3 h, 8 h, and 24 h after administration. The nanoparticles were injected at the lower part of one tail vein, and the blood sample was taken at the other side of the tail vein, thereby protecting the sample from contamination. The blood volume was calculated as 0.0778 mL/g body weight. For the tissue distribution evaluation, tissues including tumor, heart, liver, spleen, lung, and kidney were removed 24 h after injection. The tissues were used for ICP-MS measurement, TEM observation, and histological evaluation. The blood and part of the tissue samples were used to quantitate the Au content by ICP-MS.
TEM Observation of Spheroids and Tumors
SEM observation was used to evaluate the morphology of the outer cells in the spheroid. In order to evaluate the inner cells of the spheroid, TEM was carried out. Briefly, 7-day-cultured spheroids were removed, washed with PBS, and fixed overnight at room temperature using 3% glutaraldehyde solution. This was followed by secondary fixation with 1% osmium tetraoxide, then serial dehydration in a graded ethanol series. Each spheroid was embedded in Epon resin and polymerized for 3 days at 60 °C. Embedded samples were sectioned, stained with uranyl acetate, and examined under an electron microscope (JEM-1400, JEOL). For tumor tissues, small pieces of the tumor were collected and processed as described for spheroids, except that the sections were not stained with uranyl acetate.
After the biodistribution study, tissue samples including tumor, heart, liver, spleen, lung, and kidney were collected and fixed in 4% formalin for at least one day. The samples were then dehydrated in an ethanol series, embedded in paraffin, and sectioned. Subsequently, sections were stained with hematoxylin and eosin (HE) according to the standard protocol.
For the quantitative determination of Au content, cell samples, blood samples, and tissue samples were digested in aqua fortis composed of nitric acid and hydrochloric acid (3:1, v/v), diluted with 2% nitric acid and 1% hydrochloric acid, and then subjected to ICP-MS.