Incorporation of Low Concentrations of Gold Nanoparticles: Complex Effects on Radiation Response and Fate of Cancer Cells

Abstract

(1) Background: In oncology research, a long-standing discussion exists about pros and cons of metal nanoparticle-enhanced radiotherapy and real mechanisms behind the tumor cell response to irradiation (IR) in presence of gold nanoparticles (GNPs). A better understanding of this response is, however, necessary to develop more efficient and safety nanoparticle (NP) types designed to disturb specific processes in tumor cells. (2) Aims and Methods: We combined 3D confocal microscopy and super-resolution single molecule localization microscopy (SMLM) to analyze, at the multiscale, the early and late effects of 10 nm-GNPs on DNA double strand break (DSB) induction and repair in tumor cells exposed to different doses of photonic low-LET (linear energy transfer) radiation. The results were correlated to different aspects of short and long-term cell viability. SkBr3 breast cancer cells (selected for the highest incidence of this cancer type among all cancers in women, and because most breast tumors are treated with IR) were incubated with low concentrations of GNPs and irradiated with 60Co γ-rays or 6 MV X-rays. In numerous post-irradiation (PI) times, ranging from 0.5 to 24 h PI, the cells were spatially (3D) fixed and labeled with specific antibodies against γH2AX, 53BP1 and H3K9me3. The extent of DSB induction, multi-parametric micro- and nano-morphology of γH2AX and 53BP1 repair foci, DSB repair kinetics, persistence of unrepaired DSBs, nanoscale clustering of γH2AX and nanoscale (hetero)chromatin re-organization were measured by means of the mentioned microscopy techniques in dependence of radiation dose and GNP concentration. (3) Results: The number of γH2AX/53BP1 signals increased after IR and an additional increase was observed in GNP-treated (GNP(+)) cells compared to untreated controls. However, this phenomenon reflected slight expansion of the G2-phase cell subpopulation in irradiated GNP(+) specimens instead of enhanced DNA damage induction by GNPs. This statement is further supported by some micro- and nano-morphological parameters of γH2AX/53BP1 foci, which slightly differed for cells irradiated in absence or presence of GNPs. At the nanoscale, Ripley’s distance frequency analysis of SMLM signal coordinate matrices also revealed relaxation of heterochromatin (H3K9me3) clusters upon IR. These changes were more prominent in presence of GNPs. The slight expansion of radiosensitive G2 cells correlated with mostly insignificant but systematic decrease in post-irradiation survival of GNP(+) cells. Interestingly, low GNP concentrations accelerated DSB repair kinetics; however, the numbers of persistent γH2AX/53BP1 repair foci were slightly increased in GNP(+) cells. (4) Conclusions: Low concentrations of 10-nm GNPs enhanced the G2/M cell cycle arrest and the proportion of radiosensitive G2 cells, but not the extent of DNA damage induction. GNPs also accelerated DSB repair kinetics and slightly increased presence of unrepaired γH2AX/53BP1 foci at 24 h PI. GNP-mediated cell effects correlated with slight radiosensitization of GNP(+) specimens, significant only for the highest radiation dose tested (4 Gy).

Catalytic activity imperative for nanoparticle dose enhancement in photon and proton therapy

“…Interestingly, cytoplasmic processes leading to the disruption of organelles, such as mitochondria or lysosomes, may play a major role in nanoparticle-mediated radioenhancement 69 , 70 . Most recently, it was also shown that even very low concentration of 10 nm Au nanoparticles can have an effect on cell cycle phase, the proportion of radiosensitive G2 cells as well as the DSB repair kinetics 71 . Thus, the nanoparticle-mediated radiation response is complex with sensitization of cancer cells as well as dose enhancement both contributing to the overall response.…”

Advanced image-free analysis of the nano-organization of chromatin and other biomolecules by Single Molecule Localization Microscopy (SMLM)

“…Importantly, IRIFs contain proteins common to all repair mechanisms as well as proteins exclusively involved only in one of the repair pathways (non-homologous end joining, NHEJ, or homologous recombination, HR). The interaction between chromatin structure, type of ionizing radiation ( [50] , [82] , [83] ; reviewed in [84] ), cell cycle phase [85] and cell type [50] , [53] , [86] has been shown to influence the formation and internal organization of IRIFs at micro- and nanoscale resolution. Various radioprotectants and radiosensitizers (e.g., amifostine [87] and metal nanoparticles [88] , respectively) can also directly or indirectly affect DSB repair and the structure of IRIFs and chromatin [85] , [89] .…”

“…The interaction between chromatin structure, type of ionizing radiation ( [50] , [82] , [83] ; reviewed in [84] ), cell cycle phase [85] and cell type [50] , [53] , [86] has been shown to influence the formation and internal organization of IRIFs at micro- and nanoscale resolution. Various radioprotectants and radiosensitizers (e.g., amifostine [87] and metal nanoparticles [88] , respectively) can also directly or indirectly affect DSB repair and the structure of IRIFs and chromatin [85] , [89] . Importantly, at nanoscale resolution, IRIFs can be divided into (sub)clusters [80] , [90] .…”

“…In addition to questions concerning the basic principles of biological regulation, as in the case of the aforementioned hypothesis, the proposed procedures of geometric and topological analysis can offer answers to an unexpected number of specific questions related to radiobiology and cancer biology, as we show below. For example, since different types of ionizing radiation damage chromatin in different ways, knowledge of the geometry and topology of IRIF point clusters at different chromatin locations, and their changes over time after irradiation, appear to be promising indicators of cell fate after exposure to specific types of ionizing radiation in radiotherapy or space research [3] , [38] , [53] , [54] , [55] , [68] , [80] , [85] , [89] , [91] , [92] .…”

Cancer Treatment Using Different Shapes of Gold-Based Nanomaterials in Combination with Conventional Physical Techniques

“…The efficacy of radiotherapy in the presence of AuNPs can be measured by the dose enhancement factor (DEF). This factor related to AuNPs is expressed as the ratio of the radiation dose absorbed by the tumor cells to that which is absorbed without the presence of AuNPs [ 69 , 70 ]. The value depends, in turn, on certain factors such as the concentration of the NPs and their chemical and physical properties, as well as their intracellular localization.…”