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

(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 Co-60 gamma-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 gamma H2AX, 53BP1 and H3K9me3. The extent of DSB induction, multi-parametric micro- and nano-morphology of gamma H2AX and 53BP1 repair foci, DSB repair kinetics, persistence of unrepaired DSBs, nanoscale clustering of gamma H2AX 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 gamma H2AX/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 gamma H2AX/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 gamma H2AX/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 gamma H2AX/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)

Challenges and Contradictions of Metal Nano-Particle Applications for Radio-Sensitivity Enhancement in Cancer Therapy

From the very beginnings of radiotherapy, a crucial question persists with how to target the radiation effectiveness into the tumor while preserving surrounding tissues as undamaged as possible. One promising approach is to selectively pre-sensitize tumor cells by metallic nanoparticles. However, though the “physics” behind nanoparticle-mediated radio-interaction has been well elaborated, practical applications in medicine remain challenging and often disappointing because of limited knowledge on biological mechanisms leading to cell damage enhancement and eventually cell death. In the present study, we analyzed the influence of different nanoparticle materials (platinum (Pt), and gold (Au)), cancer cell types (HeLa, U87, and SKBr3), and doses (up to 4 Gy) of low-Linear Energy Transfer (LET) ionizing radiation (- and X-rays) on the extent, complexity and reparability of radiation-induced H2AX + 53BP1 foci, the markers of double stand breaks (DSBs). Firstly, we sensitively compared the focus presence in nuclei during a long period of time post-irradiation (24 h) in spatially (three-dimensionally, 3D) fixed cells incubated and non-incubated with Pt nanoparticles by means of high-resolution immunofluorescence confocal microscopy. The data were compared with our preliminary results obtained for Au nanoparticles and recently published results for gadolinium (Gd) nanoparticles of approximately the same size (2–3 nm). Next, we introduced a novel super-resolution approach—single molecule localization microscopy (SMLM)—to study the internal structure of the repair foci. In these experiments, 10 nm Au nanoparticles were used that could be also visualized by SMLM. Altogether, the data show that different nanoparticles may or may not enhance radiation damage to DNA, so multi-parameter effects have to be considered to better interpret the radiosensitization. Based on these findings, we discussed on conclusions and contradictions related to the effectiveness and presumptive mechanisms of the cell radiosensitization by nanoparticles. We also demonstrate that SMLM offers new perspectives to study internal structures of repair foci with the goal to better evaluate potential differences in DNA damage patterns

Geocaching as a tool for development of tourism

The aim of this thesis is to examine the impact of geocaching on tourism. The theoretical part explains definition of geocaching, its history, rules and principles. After that the theoretical part analyzes geocaching from the tourism perspective. In this part, there are also described the different projects where geocaching was used for tourism development in the regions of the Czech Republic. In the practical part, a survey is performed to examine the behavior of geocaching participants and their impact on tourism. In the final part, geocaching application for the development of tourism in the region is designed. The outcome of this work is the evaluation of potential visitors-geocaching players as a target group for the development of local tourism

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

International audienceBackground: 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