The Triple Negative “Basal-like” breast cancer (TNBL) tumours have a high proliferative capacity and develop a resistance phenotype associated with metastases. However, the management of TNBL carcinomas is still not standardized. Among the promising trails, gold nanoparticles could be a relevant tool for the development of a targeted treatment for this breast cancer subtype in monotherapy, associated and/or conjugated with other drugs. In this work, we report the cytotoxicity impact of gold nanoparticles wrapped in Poly-Ethylene Glycol (PEG) on the TNBL HCC-1937 breast cancer cell line. PEG-coated gold nanoparticles (PEG-Au NPs) were synthesized by a two-step method using a reduction process followed by a post - functionalization called PEGylation. PEG-Au NPs were characterized using transmission electron microscopy and X-ray diffraction. The gold content of the samples was determined using atomic absorption spectrometer. The cytotoxicity tests were performed using Sulforhodamine B survival test and resazurin viability test. PEG-Au NPs impact analysis on HCC1937 TNBL cell line showed a clear toxic action of type dose dependent and at long term. These PEGylated gold nanoparticles present a promising tool for the development of tumor-specific radiosensitizing vectors, with or without the association of other treatment strategies.
Gold nanoparticles (GNPs) are promising candidates in the field of medicine for the treatment of illnesses such as Alzheimer’s [
Before the experiment, all the glassware was carefully cleaned using aqua regia (mixture of concentrated nitric and hydrochloric acid in 1:3 volume ratio) and rinsed with ultra-pure water (18 MΩ.cm), blown dried with nitrogen and stored at 110˚C. The starting solution is obtained by dissolving the designated amount of hydrogen tetrachloroaurate (III) hydrate (HAuCl4, xH2O) purchased from Sigma Aldrich to reach suitable gold concentration. Gold nanoparticles in suspension were synthesized using a two step method. First, reduction of hydrogen tetrachloroaurate (III) precursor species was carried out using sodium borohydride under vigorous stirring for 15 minutes at room temperature; gold metal nano-cores were obtained. Secondly, gold nanoparticles surface were post functionalized using thiol-terminated poly(ethylene) glycol (HS-PEG, of MW 5000 from Sigma Aldrich). After this so called PEGylation step, red ruby, stable gold nanoparticles suspensions are obtained.
A Hitachi transmission electron microscope (H7650) was used for morphological characterization.
The crystalline structure of the gold nanoparticles was determined using a Bruker D2 Phaser diffractometer. The operating parameters are listed in
The gold content of the samples was determined using an AA 7000 atomic absorption spectrometer (Shimadzu) with gold hollow-cathode lamp. All analyses were performed at a wavelength of 242.8 nm using 0.7 nm slit width. Before the experiments, the machine was calibrated using a gold standard for AAS (Sigma Aldrich) at 1000 ppm in 5% HCl. Calibration line was performed using thirteen diluted standard solutions prepared in 5 % HCl suitable for trace analysis and high-purity water, 18.2 MΩ, ranging from 0.01 to 20 ppm, respectively.
The HCC1937 TNBL breast cancer cell line (ATCC®) was maintained in RPMI 1640 medium (Gibco®), supplemented with 10% decomplemented fetal calf serum and 20 mg/mL gentamycin, in 75 cm2 culture dishes (Falcon®) at 37˚C under 5% CO2, according to supplier’s instructions [
radiation | tube voltage | tube current | scanning range (2θ) scanning speed | time constant | spinning speed |
---|---|---|---|---|---|
CuKα1 λ = 1.5406 Å | 30 kV | 10 mA | 30˚ to 80˚ 0.24 ˚/min | 5 seconds | 15˚/min. |
Sulforhodamine B (SRB) is an anionic dye that binds electrostatically to cellular proteins, reflecting indirectly the number of living cells in the wells. Its absorbance peak is at 540 nm. After each treatment period (from Day 1 to Day 7) cells were fixed with 50 μL of a 50% (weight/volume) trichloroacetic acid (TCA) solution added to each well for 1 hour at 4˚C. Plates were then washed, dried, and 50 μL of a 0.4% (w/v) solution of SRB in 1% (v/v) acetic acid was added to each well. After 15 minutes of incubation at room temperature, plates were washed with 1% acetic acid solution and dried. 200 μL of a 10 mM tris-base buffer solution was finally added to the wells for dye solubilisation and plates were stirred for 30 min. The optical density (OD) of each well was determined at 540 nm using a microplate reader (VWR Multiskan FC®). Cell survival ratio was calculated by the following formula: cell survival (%) = (Mean (OD λ540 nm) of treated wells/ Mean (OD λ540 nm) of untreated control wells) × 100.
Resazurin test is a colorimetric metabolic assay based on the reduction of resazurin into resorufin by metabolically active cells. Resorufin is a fluorescent dye exhibiting a pink colouring. After a time of incubation with resazurin, the amount of resorufin formed is proportional to the number of living cells in the wells. For these experiments, after each treatment period (from Day 1 to Day 7), medium was removed from the plates and cells were incubated with 100 µL of 60 µM resazurin diluted in PBS*, for 4 hours. Optical density (OD) of resorufin and resazurin were measured at 570 and 620 nm, respectively with a microplate reader (VWR Multiskan FC®). The corrected OD of resorufin was calculated as follows: (OD reso corr) = ((OD) λ570 nm − (OD) λ62 0nm.) Then, the percentage of cell survival was obtained with the following formula: Cell survival (%) = (Mean (OD reso corr) of treated wells/Mean (OD reso corr) of untreated control wells) × 100.
The half maximal inhibitory concentration (IC50) was calculated with the following formula, IC50 = EXP (LN (concentration > 50% inhibition) − ((signal > 50% inhibition − 50)/(signal > 50% inhibition − signal < 50% inhibition)*LN (concentration >50% inhibition/concentration < 50% inhibition))) (1) [
Cytotoxicity results were expressed as means ± standard error of n independent experiments. All experiments were performed at least in triplicate and then statistically compared using a Student’s t-test. Tests were two-sided and the nominal level of significance was p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****) and p < 0.00001 (*****).
The TEM images of the synthesized gold nanoparticles in suspension are shown
in
(PEG-Au NPs) pictures exhibited a quasi-spherical shape, with some aggregates. Taking into account this spherical geometry, the average core diameters of PEG-coated gold nanoparticles are found to be 7 nm. PEG-coated gold nanoparticles are synthesized using sodium borohydride as a strong reducing reagent. The PEG-coating of the gold nanoparticles, also known as pegylation, is dedicated to improve the residence in body [
XRD pattern of the analyzed gold nanopowder is presented in
D = 0.9 * λ β cos θ (2)
where λ is the wavelength of the Cu K α1 line in Å, β is the full-width at half max (FWHM) of the peak. The size of the crystallites was found and is approximately
8 nm for gold nanopowder. This result is in good agreement with the average core diameter of 7 nm previously found.
The calibration line for gold assay is presented in
We measured the gold nanoparticles suspensions using the 5% HCl solution to dilute the suspension. The gold concentration in our samples is 19.5 ppm.
The action of increasing concentrations of PEG-Au nanoparticles (2 to 25 µM) was studied on the Triple-Negative Basal-Like breast cancer cell line HCC1937, during seven days. For this, i) SRB cell survival test coupled to cell microscopy analyses and ii) cell viability via resazurin test were performed.
The rate of HCC1937 TNBL cell viability via Sulforhodamine B quantitative survival test was performed in order to quantify PEG-Au NPs toxicity on this breast cancer cell line. For this, after each treatment period, the absorbance of the Sulforhodamine B dye fixed in each well was quantified and allowed the calculation of the percentage of cell survival compared to untreated cells presented in
Control cells treated with PEG alone showed no difference in cell survival compared to untreated cells (data not shown). After two days of treatment with 2 and 4 µM of nanoparticles, cell survival was respectively of 108% ± 2% and 109% ± 2% =. Then, after 5 days, it decreased to 82% ± 1% with 2 µM and to 61% ± 2% with 4 µM (p < 0.00001 and p < 0.00001 respectively), and remained stable until 7 days with 85% ± 1% with 2 µM, (p = 0.80) and 59% ± 3% with 4 µM (p = 0.25). Cell survival ratio of HCC1937 cell line treated with 7 µM nanoparticles was of 98% ± 2% after one day of treatment, and decreased significantly to 39% ± 1% (p < 0.00001) and 28% ± 2% (p < 0.00001) after 5 and 7 days of treatment, respectively. Then, with higher nanoparticles concentrations (14 and 25 µM), cell survival was of 96% ± 1% and 89% ± 1% after one day of treatment, and
dropped dramatically to 20% ± 0.7% (p < 0.00001) and 13% ± 0.4% (p < 0.00001) after 5 days of treatment. Then, it continued to decrease until 7 days of treatment, with survival rates of 10% ± 0.4% (p < 0.00001) and 6% ± 0.2% (p < 0.00001) for 14 and 25 µM of nanoparticles, respectively. All these results showed that PEG-Au NPs toxicity on HCC1937 cell line was both dose and time-dependent. In addition, microscopy analyses presented in
Then, the resazurin viability test involving the reduction of resazurin into resorufin by metabolically active cells was performed. The percentages of cell viability after treatment with increasing concentrations of PEG-Au NPs were calculated by the ratio of metabolic activity of untreated cells, after 1 to 7 days of treatment, see
performed, and toxicity was not noted compared to untreated cells (Data is not shown). Then, after a treatment with 2 µM of nanoparticles, cell viability was of 102% ± 1% at day 1, and remained stable until the end of the treatment with 99% ± 1% at day 7 (p = 0.28). A slight decrease in cell viability was detected after 3 days of treatment with 4 µM (92% ± 1%, p < 0.01), which remained stable until seven days (96% ± 1%, p = 0.79). After a treatment with 7 µM of nanoparticles, cell viability was of 96% ± 1% after one day, decreased significantly to 78% ± 2% (p < 0.00001) after 4 days, and remained stable until the seventh day (75% ± 3%, p = 0.54). In contrast, with a higher nanoparticles concentration of 14 µM, cell viability dropped from 91% ± 1% after one day to 48% ± 3% after 4 days (p < 0.00001), remained relatively stable until the sixth day (41% ± 3%, p = 0.09) and decreased at day 7 (27% ± 2%, p < 0.01). Finally, the highest nanoparticles concentration of 25 µM induced a dramatic decrease in cell viability from 84% ± 1% at day 1, to 35% ± 1% at day 4 (p < 0.00001), and to 19% ± 2% at day 7 (p < 0.00001). Similarly to SRB results, these results showed a clear dose and time-dependent toxicity of PEG-Au NPs nanoparticles on HCC1937 breast cancer cell line.
PEG-Au NPs impact analysis on HCC1937 TNBL cell line showed a clear toxic action of type dose dependent and at long term. Indeed, TNBL cells mortality increased by the time, with IC50 (SRB test) of 9 µM at day 4 and of 5 µM at day 7. Similarly, IC50 (Resazurin test) was of 12 µM at day 4 and decreased to 10 µM at day 7. The variability between SRB survival test and resazurin viability test can be explained by a difference in sensibility thresholds due to the inner mechanism of both tests. Nevertheless, these two assays described the same toxicity profile of PEG-Au NPs nanoparticles on HCC1937 cell line, probably correlated with nanoparticles accumulation in cells.
Similarly, in previous in vitro and in vivo studies, gold nanoparticles have been used as tool both to detect and to treat breast cancer [
PEG-Au NPs were synthesized using a two-step method using a reduction process followed by a post-functionalization called PEGylation. Stable gold nanoparticles in suspension, suitable for biological experiments, with a gold concentration of 19.5 ppm, are obtained. The two cytotoxicity tests described the same PEG-Au NPs nanoparticles toxicity profile of type dose and time dependent on HCC1937 cell line, probably correlated with nanoparticles accumulation in cells. These works open the way to further experiments aiming to determine the PEG Au NPs uptake and intracellular localisation in TNBL cell line models.
The authors would like to thank the Centre Imagerie Cellulaire Santé (CICS), Université Clermont Auvergne for the electronic microscopy characterizations.
Massard, C., Dubois, C., Raspal, V., Daumar, P., Sibaud, Y., Mounetou, E., Bamdad, M. and Awitor, O.K. (2018) Cytotoxicity Study of Gold Nanoparticles on the Basal-Like Triple-Negative HCC-1937 Breast Cancer Cell Line. Journal of Biomaterials and Nanobiotechnology, 9, 13-25. https://doi.org/10.4236/jbnb.2018.91002