Effect of gadolinium-based nanoparticles on nuclear DNA damage and repair in glioblastoma tumor cells

Abstract

Background: Tumor targeting of radiotherapy represents a great challenge. The addition of multimodal nanoparticles, such as 3 nm gadolinium-based nanoparticles (GdBNs), has been proposed as a promising strategy to amplify the effects of radiation in tumors and improve diagnostics using the same agents. This singular property named theranostic is a unique advantage of GdBNs. It has been established that the amplification of radiation effects by GdBNs appears due to fast electronic processes. However, the influence of these nanoparticles on cells is not yet understood. In particular, it remains dubious how nanoparticles activated by ionizing radiation interact with cells and their constituents. A crucial question remains open of whether damage to the nucleus is necessary for the radiosensitization exerted by GdBNs (and other nanoparticles).

Methods: We studied the effect of GdBNs on the induction and repair of DNA double-strand breaks (DSBs) in the nuclear DNA of U87 tumor cells irradiated with γ-rays. For this purpose, we used currently the most sensitive method of DSBs detection based on high-resolution confocal fluorescence microscopy coupled with immunodetection of two independent DSBs markers.

Results: We show that, in the conditions where GdBNs amplify radiation effects, they remain localized in the cytoplasm, i.e. do not penetrate into the nucleus. In addition, the presence of GdBNs in the cytoplasm neither increases induction of DSBs by γ-rays in the nuclear DNA nor affects their consequent repair.

Conclusions: Our results suggest that the radiosensitization mediated by GdBNs is a cytoplasmic event that is independent of the nuclear DNA breakage, a phenomenon commonly accepted as the explanation of biological radiation effects. Considering our earlier recognized colocalization of GdBNs with the lysosomes and endosomes, we revolutionary hypothesize here about these organelles as potential targets for (some) nanoparticles. If confirmed, this finding of cytoplasmically determined radiosensitization opens new perspectives of using nano-radioenhancers to improve radiotherapy without escalating the risk of pathologies related to genetic damage.

Keywords: DNA double-strand breaks; DNA repair; Gadolinium; Nanomedicine; Nanoparticles; Radiosensitization; Radiotherapy; Theranostic.

Fig. 1

Localization of GdBNs-Cy5.5 nanoparticles in U87 cell. a Correlative fluorescence confocal image and transmission light image of U87 cell with internalized GdBNs-Cy5.5 (red) at the end of 16 h-long observation. The scale bar equals to 10 µm. The circles represent the regions of fluorescence spectroscopy measurements, cytoplasm (blue), nucleus (red), and plain medium (green). b Fluorescence emission spectra of the three regions

Fig. 2

Localization of GdBNs-Cy5.5 nanoparticles in U87 cells as a function of the incubation time. Correlative fluorescence confocal images and transmission light images of U87 cells incubated with 1 mM GdBNs-Cy5.5 (red) for three different incubation times: a—1 h, b—6 h and c—16 h. Scale bars equal to 10 µm

Fig. 3

Effect of GdBNs on DSBs formation in non-irradiated U87 cells. Distribution of DSBs foci numbers are compared for non-irradiated U87 cells never incubated with GdBNs (black) and incubated with 1 mM GdBNs for 1 h (purple) and 6 h (blue). The respective cell nuclei are shown as the maximum images (composed of 40 confocal slices 0.2 μm-thick) with 3D projections; γH2AX—green, 53BP1—red, chromatin—artificially blue

Fig. 4

Effect of GdBNs on DSBs formation and repair in irradiated (1 Gy) U87 cells. Distribution of DSBs foci numbers are compared for irradiated U87 cells a never incubated with GdBNs and b incubated with 1 mM GdBNs for 1 h. Non-irradiated controls are indicated as NI. The respective maximum images of representative nuclei for each period of time PI are shown above: γH2AX—green, 53BP1—red, chromatin—artificially blue

Fig. 5

Effect of GdBNs on DSBs formation and repair in irradiated (4 Gy) U87 cells. Distribution of DSBs foci in U87 cells never incubated with GdBNs (a) and incubated with 1 mM GdBNs for 1 h (b). Non-irradiated controls (NI) are also compared. The respective maximum images of representative nuclei for each period of time PI are shown above: γH2AX—green, 53BP1—red, chromatin—artificially blue

Fig. 6

Effect of incubation times with GdBNs on DSBs formation and repair in irradiated U87 cells. Distributions of DSBs foci numbers are compared for U87 cells irradiated with 1 Gy of γ-rays and never incubated with GdBNs (a) or incubated with 1 mM GdBNs for 1 h (b), 6 h (c) and 24 h (d). Non-irradiated controls (NI) are also compared. The respective maximum images of representative nuclei are shown above: γH2AX—green, 53BP1—red, chromatin—blue

Fig. 7

Effect of GdBNs on clonogenic survival fraction of U87 cells. Surviving fractions of U87 cells never incubated with GdBNs (grey) or in the U87 incubated with 1 mM GdBNs for 1 h (purple) and irradiated by 1 and 4 Gy of γ-rays (⁶⁰Co), respectively. Non-irradiated controls (dose 0) were normalized to 1