Utilizing two-dimensional monolayer and three-dimensional spheroids to enhance radiotherapeutic potential by combining gold nanoparticles and docetaxel

Abstract

Background: Much in vitro research on the applicability of gold nanoparticles (GNPs) in cancer treatment has been focused on two-dimensional (2D) monolayer models. To improve this, we explored the effect of the combination of GNPs and docetaxel (DTX) with radiotherapy (RT) in a more complex three-dimensional (3D) spheroid that can better mimic a real tumour microenvironment.

Methods: Two cell lines, prostate cancer LNCaP and cervical cancer HeLa, were grown in monolayer and spheroids. Cells were dosed with GNPs at a concentration of 10 $\mu g/\mathrm{mL}$ and with DTX at a dose that inhibited growth-rate by 50%. Samples were irradiated 24 h after drug dosing with 2 Gy, 5 Gy, or 10 Gy using a 6 MV beam. Monolayer cells had the DNA double-strand breaks (DSBs) probed 24 h post-radiation, and cell proliferation observed over 7 days. Spheroid proliferation was monitored over 14 days along with spheroid volume measurements.

Results: In DTX and GNP-treated monolayer samples, there is decreased survival after irradiation with 5 and 10 Gy of 16-24% and an increase in DSBs of 91.6-109.9%, compared to DTX. In spheroids, GNPs decreased the surviving cells by 10.54-15.61% compared to control, while GNPs and DTX decreased survival by 20.9-31.04%. There is reduced spheroid volume 14 days after treatment with the triple combination.

Conclusions: Combining GNPs and DTX leads to a synergistic radiosensitization effect in spheroids, which can better mimic the tumour microenvironment. Testing treatment modalities with spheroids and RT may allow a quicker translation to the clinic.

Supplementary information: The online version contains supplementary material available at 10.1186/s12645-023-00231-5.

Keywords: Cancer; Docetaxel; Gold nanoparticles; Nanomedicine; Radiotherapy; Spheroids.

Fig. 1

Improved cell kill with combined cancer therapy. a Multicellular spheroid is a three-dimensional in vitro tissue structure that can better approximate the complex heterogeneous in vivo tumour microenvironment. This includes modelling the ECM, which has a proliferation zone on the outer edge of the spheroid, a quiescent zone in the middle of the spheroid, where the cancer does not grow effectively, and possible necrotic core, where the cells lack the nutrients to survive due to the concentration gradient and are either hypoxic or are dead. b With the addition of GNPs and DTX, the cancer cells become more sensitized to radiation, and we see an improved cell kill response. This occurs even in clinically relevant 6 MV radiation

Fig. 2

Characterization of gold nanoparticles. a Hyper spectral imaging of gold nanoparticles decorated with polyethylene glycol (PEG) and a peptide containing RGD. Scale bar is 20 $\mathrm{\mu }$m. Inset is hyper spectral spectrum of gold nanoparticles and background. b Secondary electron image from a transmission electron microscope of gold nanoparticles. Scale bar is 300 nm. c Size of gold nanoparticles measured by dynamic light scattering while bare, with PEG, and with PEG and RGD. d Surface $\mathrm{\zeta }$-potential of bare and decorated GNPs. e Size as measured by the UV–visible absorbance spectrum of bare and decorated GNPs

Fig. 3

Docetaxel characterization in HeLa. a, b Proliferation assays for a (a) two-dimensional monolayer and a (b) three-dimensional spheroid, for the cervical cancer cell line HeLa, treated with docetaxel. c, d Cell cycle analysis of a (c) monolayer and (d) spheroids of HeLa cells treated with the GR50 dose of docetaxel, calculated per modality

Fig. 4

Gold nanoparticle uptake in monolayer and spheroid. a, b Uptake of gold nanoparticles in a monolayer and b spheroids of HeLa and LNCaP with and without docetaxel. Error bars are the 95% confidence interval from three independent experiments. c, e Confocal images of a monolayer of HeLa with gold nanoparticles, c without and e with docetaxel. d, f Confocal images of spheroids of HeLa with gold nanoparticles, d without and f with docetaxel. Gold nanoparticles and nuclei are marked in red and blue, respectively. Scale bars are 25 $\mathrm{\mu }$m. ns indicates no significance, * indicates 0.01 < p < 0.05, ** indicates 0.001 < p < 0.01, *** indicates 0.0001 < p < 0.001

Fig. 5

Radiation response in monolayer. a, b Normalized PrestoBlue proliferation assay after 6 days following radiation for a HeLa and b LNCaP. Error bars are the 95% confidence interval from three independent experiments. Cells were irradiated with 0, 2, 5, and 10 Gy using a 6 MV linear accelerator. c, d Normalized 53BP1 foci per nuclear area 24 h following radiation in c HeLa and b LNCaP. Error bars are the 95% confidence interval from at least 50 nuclei from three experiments. ns indicates no significance, * indicates 0.01 < p < 0.05, ** indicates 0.001 < p < 0.01, *** indicates 0.0001 < p< 0.001, **** indicates p < 0.0001

Fig. 6

Radiation response in spheroids. a, b Normalized CellTiter-Glo 3D cell viability assay after 13 days following radiation for spheroids of a HeLa and b LNCaP. c, d Normalized increase of diameter relative to starting size of c HeLa spheroids and d LNCaP spheroids over 13 days following radiation. Error bars are the 95% confidence interval from three independent experiments with at least 5 repeats per condition per experiment. ns indicates no significance, * indicates 0.01 < p < 0.05, ** indicates 0.001 < p < 0.01, *** indicates 0.0001 < p < 0.001, **** indicates p < 0.0001

Fig. 7

Growth of HeLa spheroids following radiation. Brightfield images of HeLa spheroids that have been untreated, treated with docetaxel, treated with gold nanoparticles, and treated with docetaxel and gold nanoparticles, and then radiated with 2 Gy, 5 Gy, and 10 Gy. Scale bar is 100 $\mu \mathrm{m}$