Nanoparticles are small enough to pass into a tumour via its leaky vasculature. It was previously assumed that GNPs must localize within the cell nucleus to kill cancer cells, by destroying nuclear DNA. But here again, experiments undermined this thinking, as many cells take up GNPs in the cytoplasm and still exhibit significant sensitization. The question arises, therefore, as to which other cellular targets may drive radiosensitivity.

One possible extra-nuclear target is the mitochondria, which are small and distributed throughout the cytoplasm. GNPs have been observed to accumulate on the mitochondria surface, making mitochondrial DNA likely to be close to a GNP and thus exposed to a high dose. Researchers from Queen's University Belfast (QUB) and Massachusetts General Hospital/Harvard Medical School are investigating this premise. And in two recently published papers, they demonstrate that mitochondrial DNA may indeed provide a significant target for GNP radiosensitization.

"These papers underpin the changing paradigm of radiation modes of action from purely direct damage to nuclear DNA, to a more cell-based response," explains Kevin Prise from QUB's Centre for Cancer Research & Cell Biology. "This has important implications for future nanoparticle approaches and other molecularly targeted agents that may modulate DNA damage or metabolic signalling pathways."

Monte Carlo simulations

In the first study, the researchers used Geant4 Monte Carlo simulations to calculate the energy deposited by X-ray irradiation in cells with and without cytoplasmic GNPs. They used a simple model comprising an 11.5 µm diameter cell containing an 8.5 µm diameter nucleus and 30 mitochondria (1.8 × 1 × 0.6 µm) distributed throughout the cytoplasm. Fifty GNPs were distributed at the surface of each mitochondrion (J. Phys.: Conf. Ser. 777 012008).

The cells were exposed to 50 or 100 keV X-rays and the energies deposited within the mitochondria and nucleus were scored. To sample ionization cluster sizes, the researchers placed virtual DNA segments (10 x 5 nm cylinders) within the irradiated volume and used the density of ionizations in these volumes to estimate the degree of clustering, and thus DNA damage.

Simulations showed that the presence of GNPs within the cytoplasm significantly increased (three- to four-fold) the number of ionization clusters induced in mitochondria. As such clusters are strongly associated with DNA damage, this may lead to a sizeable increase in mitochondrial damage and significantly impact the cell's ability to survive irradiation.

The results indicate that, in cases where GNPs cannot penetrate the cell nucleus, mitochondria may be an important target for radiosensitization.

Targeted microbeams

In the second paper, Prise and co-authors used a soft X-ray microbeam to quantify DNA damage and repair after nuclear or cytoplasmic irradiation, with or without GNPs (1.9 nm Aurovist particles). They used a 5 µm-diameter microbeam to irradiate MDA-MB-231 breast cancer and AG01522 fibroblast cells (Scientific Reports 7 44752).

At the beam energy of 278 eV, secondary electrons (produced when GNPs interact with ionizing radiation) have ranges of less than 10 nm. This ensures that particles produced by cytoplasmic interactions cannot cause damage within the nucleus, enabling the researchers to separate physical and biological effects. At this low energy, no physical dose enhancement is expected from the GNPs. Despite this, differences in response were observed when GNPs were added to cells, suggesting a biological or chemical mechanism of action.

The researchers found that GNPs did not significantly impact the level of DNA damage following nuclear irradiation in either cell line. In contrast, cytoplasmic irradiation induced significantly different levels of DNA damage in GNP-treated cells in both cell lines. This finding further highlights the departure from the paradigm that nuclear targeting is most important in cellular response.

Mitochondrial mechanism

When mitochondria undergo stress – such as during exposure to GNPs – they lose their membrane potential. The researchers used TMRE dye to quantify this mitochondrial depolarization and subsequent recovery. When mitochondria are fully functional, TMRE is taken up by the polarized, negatively charged mitochondria and fluoresces red. Depolarized mitochondria no longer retain the dye and lose fluorescence.

The researchers quantified mitochondrial polarization recovery after removal of GNPs from the culture medium. With GNPs present, breast cancer cells showed a more dramatic depolarization than fibroblasts. After GNP removal, TMRE florescence increased across both cell lines, indicating recovery of polarization to the level of non-GNP-treated control cells. Fibroblast cells were fully recovered at 6 h, whereas breast cancer cells recovered only after 24 h. The finding that GNPs don't cause permanent alterations to cell function suggests that long-term side effects should be avoidable.

The researchers then irradiated the cytoplasm of TMRE-positive (functional mitochondria) and TMRE-negative (depolarized mitochondria) cells. Irradiating cells in the depolarized state led to significantly lower cell survival compared with non-GNP treated cells. These findings provide further evidence that GNP radiosensitization is mediated by mitochondrial function.

"These results have shown that mitochondria can be primed into a stressed state by the addition of GNPs alone, and that this can synergise with radiation exposure to cause greater cell killing than predicted from physical dose alone," Prise explains. He notes that these temporal effects may be important for clinical scheduling of these agents – ideally, radiotherapy should be delivered at the point of maximum sensitization, taking into account both the uptake of GNPs and the time required for them to drive biological effects.

Prise notes that the biological effects of GNPs are highly dependent on particle preparation and coating. "We have ongoing research into optimizing the preparation of GNPs to improve uptake and to better understand what drives the underlying biology, alongside further modelling studies to better understand how GNPs affect the dose distribution in all areas of the cell," he told medicalphysicsweb.

Related articles in PMB
Dose enhancement effects to the nucleus and mitochondria from gold nanoparticles in the cytosol
A L McNamara et al Phys. Med. Biol. 61 5993
Biological modeling of gold nanoparticle enhanced radiotherapy for proton therapy
Yuting Lin et al Phys. Med. Biol. 60 4149
The cytoplasm as a radiation target: an in silico study of microbeam cell irradiation
H L Byrne et al Phys. Med. Biol. 60 2325

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