Application of Gold Nanoparticles as Radiosensitizer for Metastatic Prostate Cancer Cell Lines

Abstract

More than 50% of all prostate cancer (PCa) patients are treated by radiotherapy (RT). Radioresistance and cancer recurrence are two consequences of the therapy and are related to dose heterogeneity and non-selectivity between normal and tumoral cells. Gold nanoparticles (AuNPs) could be used as potential radiosensitizers to overcome these therapeutic limitations of RT. This study assessed the biological interaction of different morphologies of AuNPs with ionizing radiation (IR) in PCa cells. To achieve that aim, three different amine-pegylated AuNPs were synthesized with distinct sizes and shapes (spherical, AuNP_sp-PEG, star, AuNP_st-PEG, and rods, AuNP_r-PEG) and viability, injury and colony assays were used to analyze their biological effect on PCa cells (PC3, DU145, and LNCaP) when submitted to the accumulative fraction of RT. The combinatory effect of AuNPs with IR decreased cell viability and increased apoptosis compared to cells treated only with IR or untreated cells. Additionally, our results showed an increase in the sensitization enhancement ratio by cells treated with AuNPs and IR, and this effect is cell line dependent. Our findings support that the design of AuNPs modulated their cellular behavior and suggested that AuNPs could improve the RT efficacy in PCa cells.

Keywords: gold nanoparticles; in vitro assay; prostate cancer; radiosensitizing effect; radiotherapy.

Figure 1

Characterization of AuNPs. (A–C) Visual appearance of samples; (D–F) UV-visible spectra of bare AuNP_sp, AuNP_st, AuNP_r (continuous line) and PEG-functionalized AuNPs, AuNP_sp-PEG, AuNP_st-PEG, and AuNP_r-PEG (dashed line); (G–I) TEM imaging shows the morphology of the AuNPs; (J–L) The DLS peaks of AuNP_sp-PEG, AuNP_st-PEG, and AuNP_r-PEG. AuNP_sp-PEG, PEGylated spherical gold nanoparticles; AuNP_st-PEG, PEGylated gold nanostars; AuNP_r-PEG, PEGylated gold nanorods.

Figure 2

(A) Photos of the image acquisition set-up using clinical Light Speed VCT CT imaging system (GE Medical Systems, Milwaukee, WI, USA). (B) Representative histogram measurements of CT contrast attenuation rate values in Hounsfield units of different conformations of AuNPs under four different concentrations ([Au] = 0.001 to 4 mM) with PBS as control. Iomeprol was used as a reference of clinical practice. AuNP_sp, spherical gold nanoparticles; AuNP_st, gold nanostars; AuNP_r, gold nanorods.

Figure 3

Fluorescence microscopy images of cellular uptake of AuNPs labeled with rhodamine B-labeled AuNPs in PC3 (A–D), DU145 (E–H) and LNCaP (I–L) cell lines. (A,E,I) were control groups (without treatment of AuNPs); (B,F,J) cells were treated with rhodamine-AuNP_sp; (C,G,K) were treated with rhodamine-AuNP_st; and (D,H,L) were treated with rhodamine-AuNP_r. AuNPs-Rhodamine (red) cell nuclei were stained with DAPI (blue). AuNP_sp-PEG, PEGylated spherical gold nanoparticles; AuNP_st-PEG, PEGylated gold nanostars; AuNP_r-PEG, PEGylated gold nanorods. Scale bars = 20 µm.

Figure 4

Effect of AuNPs and ionizing radiation (IR) on the viability of prostate cancer cell lines ((A–C), PC3; (D–F), DU145; (G–I) LNCaP). The cells were previously treated with different concentrations of AuNPs (0–1 mM), and then they were exposed to a cumulative dose of 7.5 Gy in three fractions of 6MV photon beam. An indication of cell viability was obtained by analyzing the overall metabolic activity of the cell population by PrestoBlue™ assay. Results were expressed as the mean ± SD, n = 6. AuNP_sp-PEG, PEGylated spherical gold nanoparticles; AuNP_st-PEG, PEGylated gold nanostars; AuNP_r-PEG, PEGylated gold nanorods. The results were considered to be statistically significant when * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. The treatment groups were compared to the control group of the respective day, represented by black lines.

Figure 5

Effect of AuNPs and ionizing radiation (IR) on the wound healing of prostate cancer cell lines ((A–D) PC3; (E–H) DU145; (I–L) LNCaP). The cells were treated with different concentrations (0–1 mM) for 24 h before being exposed to a cumulative dose of 7.5 Gy in three fractions of 6 MV photon beam. Relative migration (%) was measured 0, 6, 12 and 24 h after scratching. The results are expressed as the mean ± SD of 3 replicates. AuNP_sp-PEG, PEGylated spherical gold nanoparticles; AuNP_st-PEG, PEGylated gold nanostars; AuNP_r-PEG, PEGylated gold nanorods.

Figure 6

Survival curves of prostate cancer cells (PC3 (A), DU145 (B), and LNCaP (C)) plated immediately after respective RT treatments with fractions of 2.5 Gy until completion of a cumulative dose of 2.5, 5, and 7.5 Gy. The data presented are the mean ± SD of at least three independent experiments. The survival curves derived from clonogenic assay experiments are significantly different. AuNP_sp-PEG, PEGylated spherical gold nanoparticles; AuNP_st-PEG, PEGylated gold nanostars; AuNP_r-PEG, PEGylated gold nanorods. Significance of different treatments compared to the control of the respective day shown as * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

Cell apoptosis assay using annexin V–CF Blue/7-aminoactinomycin D (7-AAD). Graphs show the quantification of the percentage of apoptotic cells in PC3, DU145, and LNCaP cells. The results were obtained using flow cytometry assay after Annexin V/7-AAD staining and average percentage of apoptotic cells in (A) PC3, (B) DU145, and (C) LNCaP cells treated with different conformations of AuNPs—spherical (AuNP_sp-PEG), star (AuNP_st-PEG), and rod (AuNP_r-PEG) and irradiated with 3 fraction of 2.5 Gy of RT. The obtained results were compared to the respective control groups with and without IR and are presented as mean ± SD. AuNP_sp-PEG, PEGylated spherical gold nanoparticles; AuNP_st-PEG, PEGylated gold nanostars; AuNP_r-PEG, PEGylated gold nanorods. The significance of the different treatments compared to the control of the respective day are shown as * p < 0.05, *** p < 0.001.

Figure 8

Relative mitochondrial ROS levels in (A,D) PC3, (B,E) DU145, and (C,F) LNCaP cells before (A–C) and after 2.5 Gy of RT (D–F) without AuNPs and with AuNPs. The measurements were taken 24 h after treatment. H₂O₂ was used as positive control. Data are presented as mean values ± SD of five replicate samples. AuNP_sp-PEG, PEGylated spherical gold nanoparticles; AuNP_st-PEG, PEGylated gold nanostars; AuNP_r-PEG, PEGylated gold nanorods. Significance of different treatments compared to control of the respective day are shown as * p < 0.05, ** p < 0.01.

Figure 9

(A) Schematic diagram of cell irradiation setup with 6 MV photon beam; (B) representative dosimetric plan using the software XIO-Release version 4.70.02. The dose was prescribed to the isocenter using two treatment fields, and the treatment field was covered with different isodose lines, including 100% of the prescribed amount. Pink: 80% isodose line; Dark blue: 90% isodose line; Green: 95% isodose line; Orange: 100% isodose line; Red: 101% isodose line; Yellow: 110% isodose line; Cyan: 115% isodose line.