Green One-Step Synthesis of Medical Nanoagents for Advanced Radiation Therapy.

PURPOSE: Metal-based nanoparticles (M-NPs) have attracted great attention in nanomedicine due to their capacity to amplify and improve the tumor targeting of medical beams. However, their simple, efficient, high-yield and reproducible production remains a challenge. Currently, M-NPs are mainly synthesized by chemical methods or radiolysis using toxic reactants. The waste of time, loss of material and potential environmental hazards are major limitations. MATERIALS AND METHODS: This work proposes a simple, fast and green strategy to synthesize small, non-toxic and stable NPs in water with a 100% production rate. Ionizing radiation is used to simultaneously synthesize and sterilize the containing NPs solutions. The synthesis of platinum nanoparticles (Pt NPs) coated with biocompatible poly(ethylene glycol) ligands (PEG) is presented as proof of concept. The physicochemical properties of NPs were studied by complementary specialized techniques. Their toxicity and radio-enhancing properties were evaluated in a cancerous in vitro model. Using plasmid nanoprobes, we investigated the elementary mechanisms underpinning radio-enhancement. RESULTS AND DISCUSSION: Pt NPs showed nearly spherical-like shapes and an average hydrodynamic diameter of 9 nm. NPs are zero-valent platinum successfully coated with PEG. They were found non-toxic and have the singular property of amplifying cell killing induced by γ-rays (14%) and even more, the effects of carbon ions (44%) used in particle therapy. They induce nanosized-molecular damage, which is a major finding to potentially implement this protocol in treatment planning simulations. CONCLUSION: This new eco-friendly, fast and simple proposed method opens a new era of engineering water-soluble biocompatible NPs and boosts the development of NP-aided radiation therapies.

Uptake and excretion dynamics of gold nanoparticles in cancer cells and fibroblasts.

Radiotherapy is one of the main treatments used to fight cancer. A major limitation of this modality is the lack of selectivity between cancerous and healthy tissues. One of the most promising strategies proposed in this last decade is the addition of nanoparticles with high-atomic number to enhance radiation effects in tumors. Gold nanoparticles (AuNPs) are considered as one of the best candidates because of their high radioenhancing property, simple synthesis and low toxicity. Ultra small AuNPs (core size of 2.4 nm and hydrodynamic diameter of 4.5 nm) covered with dithiolated diethylenetriaminepentaacetic acid (Au@DTDTPA) are of high interest because of their properties to bind MRI active or PET active compounds at their surface, to concentrate in some tumors and be eliminated via renal clearance thanks to their small size. These key figures make Au@DTDTPA the best candidate to develop image-guided radiotherapy. Surprisingly the capacity of the nanoparticles to penetrate cells, an important issue to predict radioenhancement, has not been established yet. Here, we report the uptake dynamics, internalization routes and excretion dynamics of Au@DTDTPA nanoparticles in various cancer cell lines including glioblastoma (U87-MG), chordoma (UM-Chor1), cervix (HeLa), prostate (PC3), and pancreatic (BxPC-3) cell lines as well as fibroblasts (Dermal fibroblasts). This study demonstrates a strong cell line dependence of the nanoparticle uptake and excretion dynamics. Different pathways of cell internalization evidenced here explain this dependence. As a major finding, the retention of Au@DTDTPA nanoparticles was found to be higher in cancer cells than in fibroblasts. This result strengthens the strategy of using nanoagents to improve tumor selectivity of radiation treatments. In particular Au@DTDTPA nanoparticles are good candidates to improve the treatment of radioresitant gliobastoma, pancreatic and prostate cancer in particular. In conclusion, the variability of cell-to-nanoparticle interaction is a new parameter to consider in the choice of nanoagents in a combined treatment.

Fluorescent Radiosensitizing Gold Nanoparticles.

Ultrasmall polyaminocarboxylate-coated gold nanoparticles (NPs), Au@DTDTPA and Au@TADOTAGA, that have been recently developed exhibit a promising potential for image-guided radiotherapy. In order to render the radiosensitizing effect of these gold nanoparticles even more efficient, the study of their localization in cells is required to better understand the relation between the radiosensitizing properties of the agents and their localization in cells and in tumors. To achieve this goal, post-functionalization of Au@DTDTPA nanoparticles by near-infrared (NIF) organic dyes (aminated derivative of cyanine 5, Cy5-NH2) was performed. The immobilization of organic Cy5-NH2 dyes onto the gold nanoparticles confers to these radiosensitizers fluorescence properties which can be exploited for monitoring their internalization in cancerous cells, for determining their localization in cells by fluorescence microscopy (a common and powerful imaging tool in biology), and for following up on their accumulation in tumors after intravenous injection.

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 (γ- and X-rays) on the extent, complexity and reparability of radiation-induced γH2AX + 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.

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

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.

Improving proton therapy by metal-containing nanoparticles: nanoscale insights.

The use of nanoparticles to enhance the effect of radiation-based cancer treatments is a growing field of study and recently, even nanoparticle-induced improvement of proton therapy performance has been investigated. Aiming at a clinical implementation of this approach, it is essential to characterize the mechanisms underlying the synergistic effects of nanoparticles combined with proton irradiation. In this study, we investigated the effect of platinum- and gadolinium-based nanoparticles on the nanoscale damage induced by a proton beam of therapeutically relevant energy (150 MeV) using plasmid DNA molecular probe. Two conditions of irradiation (0.44 and 3.6 keV/µm) were considered to mimic the beam properties at the entrance and at the end of the proton track. We demonstrate that the two metal-containing nanoparticles amplify, in particular, the induction of nanosize damages (>2 nm) which are most lethal for cells. More importantly, this effect is even more pronounced at the end of the proton track. This work gives a new insight into the underlying mechanisms on the nanoscale and indicates that the addition of metal-based nanoparticles is a promising strategy not only to increase the cell killing action of fast protons, but also to improve tumor targeting.

Two New Faces of Amifostine: Protector from DNA Damage in Normal Cells and Inhibitor of DNA Repair in Cancer Cells.

Amifostine protects normal cells from DNA damage induction by ionizing radiation or chemotherapeutics, whereas cancer cells typically remain uninfluenced. While confirming this phenomenon, we have revealed by comet assay and currently the most sensitive method of DNA double strand break (DSB) quantification (based on γH2AX/53BP1 high-resolution immunofluorescence microscopy) that amifostine treatment supports DSB repair in γ-irradiated normal NHDF fibroblasts but alters it in MCF7 carcinoma cells. These effects follow from the significantly lower activity of alkaline phosphatase measured in MCF7 cells and their supernatants as compared with NHDF fibroblasts. Liquid chromatography-mass spectrometry confirmed that the amifostine conversion to WR-1065 was significantly more intensive in normal NHDF cells than in tumor MCF cells. In conclusion, due to common differences between normal and cancer cells in their abilities to convert amifostine to its active metabolite WR-1065, amifostine may not only protect in multiple ways normal cells from radiation-induced DNA damage but also make cancer cells suffer from DSB repair alteration.

Cell localisation of gadolinium-based nanoparticles and related radiosensitising efficacy in glioblastoma cells.

Recently, the addition of nanoparticles (NPs) has been proposed as a new strategy to enhance the effect of radiotherapy particularly in the treatment of aggressive tumors such as glioblastoma. The physical processes involved in radiosensitisation by nanoparticles have been well studied although further understanding of its biological impact is still lacking, and this includes the localisation of these NPs in the target cells. Most studies were performed with NPs tagged with fluorescent markers. However, the presence of these markers can influence the NPs uptake and localisation. In this study, a set of methods was used to unambiguously and fully characterise the uptake of label-free NPs, their co-localisation with cell organelles, and their radiosensitising efficacy. This set was applied to the case of gadolinium-based nanoparticles (GdBN) used to amplify the radiation killing of U87 glioblastoma cells extracted from highly aggressive human tumor. For the first time, Synchrotron Radiation Deep UV (SR-DUV) microscopy is proposed as a new tool to track label-free GdBN. It confirmed the localisation of the NPs in the cytoplasm of U87 cells and the absence of NPs in the nucleus. In a second step, Transmission Electron Microscopy (TEM) demonstrated that GdBN penetrate cells by endocytosis. Third, using confocal microscopy it was found that GdBN co-localise with lysosomes but not with mitochondria. Finally, clonogenic assay measurements proved that the presence of NPs in the lysosomes induces a neat amplification of the killing of glioblastoma cells irradiated by gamma rays. The set of combined experimental protocols-TEM, SR-DUV and confocal microscopy-demonstrates a new standard method to study the localisation of label-free NPs together with their radiosensitising properties. This will further the understanding of NP-induced radiosentisation and contribute to the development of nanoagents for radiotherapy.

Determining Omics spatiotemporal dimensions using exciting new nanoscopy techniques to assess complex cell responses to DNA damage: part A--radiomics.

Recent ground-breaking developments in Omics have generated new hope for overcoming the complexity and variability of biological systems while simultaneously shedding more light on fundamental radiobiological questions that have remained unanswered for decades. In the era of Omics, our knowledge of how genes and proteins interact in the frame of complex networks to preserve genome integrity has been rapidly expanding. Nevertheless, these functional networks must be observed with strong correspondence to the cell nucleus, which is the main target of ionizing radiation. Nuclear architecture and nuclear processes, including DNA damage responses, are precisely organized in space and time. Information regarding these intricate processes cannot be achieved using high-throughput Omics approaches alone, but requires sophisticated structural probing and imaging. Based on the results obtained from studying the relationship between higher-order chromatin structure, DNA double-strand break induction and repair, and the formation of chromosomal translocations, we show the development of Omics solutions especially for radiation research (radiomics) (discussed in this article) and how confocal microscopy as well as novel approaches of molecular localization nanoscopy fill the gaps to successfully place the Omics data in the context of space and time (discussed in our other article in this issue, "Determining Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part B--Structuromics"). Finally, we introduce a novel method of specific chromatin nanotargeting and speculate future perspectives, which may combine nanoprobing and structural nanoscopy to observe structure-function correlations in living cells in real time. Thus, the Omics networks obtained from function analyses may be enriched by real-time visualization of Structuromics.

Determining Omics spatiotemporal dimensions using exciting new nanoscopy techniques to assess complex cell responses to DNA damage: part B--structuromics.

Recent groundbreaking developments in Omics and bioinformatics have generated new hope for overcoming the complexity and variability of (radio)biological systems while simultaneously shedding more light on fundamental radiobiological questions that have remained unanswered for decades. In the era of Omics, our knowledge of how genes and dozens of proteins interact in the frame of complex signaling and repair pathways (or, rather, networks) to preserve the integrity of the genome has been rapidly expanding. Nevertheless, these functional networks must be observed with strong correspondence to the cell nucleus, which is the main target of ionizing radiation. Information regarding these intricate processes cannot be achieved using high-throughput Omics approaches alone; it requires sophisticated structural probing and imaging. In the first part of this review, the article "Giving Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part A--Radiomics," we showed the development of different Omics solutions and how they are contributing to a better understanding of cellular radiation response. In this Part B we show how high-resolution confocal microscopy as well as novel approaches of molecular localization nanoscopy fill the gaps to successfully place Omics data in the context of space and time. The dynamics of double-strand breaks during repair processes and chromosomal rearrangements at the microscale correlated to aberration induction are explained. For the first time we visualize pan-nuclear nucleosomal rearrangements and clustering at the nanoscale during repair processes. Finally, we introduce a novel method of specific chromatin nanotargeting based on a computer database search of uniquely binding oligonucleotide combinations (COMBO-FISH). With these challenging techniques on hand, we speculate future perspectives that may combine specific COMBO-FISH nanoprobing and structural nanoscopy to observe structure-function correlations in living cells in real-time. Thus, the Omics networks obtained from function analyses may be enriched by real-time visualization of Structuromics.

Heterochromatinization associated with cell differentiation as a model to study DNA double strand break induction and repair in the context of higher-order chromatin structure.

Cell differentiation is associated with extensive gene silencing, heterochromatinization and potentially decreasing need for repairing DNA double-strand breaks (DSBs). Differentiation stages of blood cells thus represent an excellent model to study DSB induction, repair and misrepair in the context of changing higher-order chromatin structure. We show that immature granulocytes form γH2AX and 53BP1 foci, contrary to the mature cells; however, these foci colocalize only rarely and DSB repair is inefficient. Moreover, specific chromatin structure of granulocytes probably influences DSB induction.

Function of chromatin structure and dynamics in DNA damage, repair and misrepair: γ-rays and protons in action.

According to their physical characteristics, protons and ion beams promise a revolution in cancer radiotherapy. Curing protocols however reflect rather the empirical knowledge than experimental data on DNA repair. This especially holds for the spatio-temporal organization of repair processes in the context of higher-order chromatin structure-the problematics addressed in this work. The consequences for the mechanism of chromosomal translocations are compared for gamma rays and proton beams.

Expression of human BRCA1Δ17-19 alternative splicing variant with a truncated BRCT domain in MCF-7 cells results in impaired assembly of DNA repair complexes and aberrant DNA damage response.

Alternative pre-mRNA splicing is a fundamental post-transcriptional regulatory mechanism. Cancer-specific misregulation of the splicing process may lead to formation of irregular alternative splicing variants (ASVs) with a potentially negative impact on cellular homeostasis. Alternative splicing of BRCA1 pre-mRNA can give rise to BRCA1 protein isoforms that possess dramatically altered biological activities compared with full-length wild-type BRCA1. During the screening of high-risk breast cancer (BC) families we ascertained numerous BRCA1 ASVs, however, their clinical significance for BC development is largely unknown. In this study, we examined the influence of the BRCA1Δ17-19 ASV, which lacks a portion of the BRCT domain, on DNA repair capacity using human MCF-7 BC cell clones with stably modified BRCA1 expression. Our results show that overexpression of BRCA1Δ17-19 impairs homologous recombination repair (sensitizes cells to mitomycin C), delays repair of ionizing radiation-induced DNA damage and dynamics of the ionizing radiation-induced foci (IRIF) formation, and undermines also the non-homologous end joining repair (NHEJ) activity. Mechanistically, BRCA1Δ17-19 cannot interact with the partner proteins Abraxas and CtIP, thus preventing interactions known to be critical for processing of DNA lesions. We propose that the observed inability of BRCA1Δ17-19 to functionally replace wtBRCA1 in repair of DNA double-strand breaks (DDSB) reflects impaired capacity to form the BRCA1-A and -C repair complexes. Our findings indicate that expression of BRCA1Δ17-19 may negatively influence genome stability by reducing the DDSB repair velocity, thereby contributing to enhanced probability of cancer development in the affected families.

Molecular subsets of mantle cell lymphoma defined by the IGHV mutational status and SOX11 expression have distinct biologic and clinical features.

Mantle cell lymphoma (MCL) is a heterogeneous disease with most patients following an aggressive clinical course, whereas others having an indolent behavior. We conducted an integrative and multidisciplinary analysis of 177 MCL to determine whether the immunogenetic features of the clonotypic B-cell receptors (BcR) may identify different subsets of tumors. Truly unmutated (100% identity) IGHV genes were found in 24% cases, 40% were minimally/borderline mutated (99.9%-97%), 19% significantly mutated (96.9%-95%), and 17% hypermutated (<95%). Tumors with high or low mutational load used different IGHV genes, and their gene expression profiles were also different for several gene pathways. A gene set enrichment analysis showed that MCL with high and low IGHV mutations were enriched in memory and naive B-cell signatures, respectively. Furthermore, the highly mutated tumors had less genomic complexity, were preferentially SOX11-negative, and showed more frequent nonnodal disease. The best cut-off of germline identity of IGHV genes to predict survival was 97%. Patients with high and low mutational load had significant different outcome with 5-year overall survival (OS) of 59% and 40%, respectively (P = 0.004). Nodal presentation and SOX11 expression also predicted for poor OS. In a multivariate analysis, IGHV gene status and SOX11 expression were independent risk factors. In conclusion, these observations suggest the idea that MCL with mutated IGHV, SOX11-negativity, and nonnodal presentation correspond to a subtype of the disease with more indolent behavior.

The BRCA1 alternative splicing variant Δ14-15 with an in-frame deletion of part of the regulatory serine-containing domain (SCD) impairs the DNA repair capacity in MCF-7 cells.

The BRCA1 gene codes for a protein involved in the DNA double strand break (DDSB) repair. Alongside the dominant full-length splicing form of BRCA1, numerous endogenously expressed alternative splicing variants of unknown significance have been described in various tissues. Some of them retain the original BRCA1 reading frame but lack several critical BRCA1 structural domains, suggesting an altered function of the resulting protein in the BRCA1-regulated processes. To characterize the effect of the BRCA1Δ14-15 splicing variant (with an in-frame deletion affecting the regulatory serine-containing domain) on the DDSB repair, we constructed the MCF-7 clones stably expressing the analyzed variant with/without a shRNA-mediated downregulation of the endogenous full-length wild-type BRCA1 expression. Our results show that the expression of the BRCA1Δ14-15 variant delays the γ-radiation-induced DDSB repair, alters the kinetics of irradiation-induced foci formation/decomposition and reduces the non-homologous end-joining capacity in MCF-7 cells. Therefore, the BRCA1Δ14-15 is not able to functionally replace the full-length wt BRCA1 in the DDSB repair. Our findings indicate that the endogenously expressed BRCA1 alternative splicing variants may negatively influence genome stability and support the growing evidence of the pathological potential of the sequence variants generated by an altered or misregulated alternative splicing in the process of mammary malignant transformation.

Prognostic impact of p53 aberrations for R-CHOP-treated patients with diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoma in adults. There are specific alterations that appear repeatedly in DLBCL cases and play a role in lymphomagenesis or progression of the disease. Some aberrations were used as prognostic markers in the pre-rituximab era. Addition of rituximab to the classical anthracycline-based chemotherapy significantly increased the survival rate in DLBCL. Only few prognostic factors have been re-evaluated for patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone). We performed complex analysis of the p53 tumor suppressor in collection of 75 DLBCL cases. Fifty-four patients were de novo cases, twenty-one cases developed into DLBCL by transformation from less aggressive disease. We determined functional status by analysis of separated alleles in yeast (FASAY) and analyzed the p53 mutations by cDNA sequencing. We assessed the level of the p53 protein by immunoblot analysis. We used FISH to analyze loss of the p53 and ATM (ataxia telangiectasia mutated) gene deletions. We detected 16 p53 mutations (21.3%) including the mutation activating non-sense-mediated RNA decay pathway. Deletion of the p53 allele was more common in cases with p53 mutation. Mutations and/or deletions of p53 had statistically significant negative impact on progression-free survival and tended to decrease also overall survival in 46 de novo DLBCL patients treated with R-CHOP. p53 aberrations are negative predictors for survival of DLBCL patients treated with R-CHOP.

Loss of the p53 tumor suppressor activity is associated with negative prognosis of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is typified by translocation t(11;14)(q13;q32) causing upregulation of cyclin D1 and deregulation of cell cycle. The cyclin D1 activation plays a critical role in MCL pathogenesis but additional oncogenic events, such as aberrations of the ARF/MDM2/p53 pathway are also necessary for progression of the disease. We analyzed the p53 tumor suppressor in tumor tissue of 33 patients with MCL. The p53 status was determined by functional analyses in yeast (FASAY) and by cDNA sequencing. The level of the p53 protein was assessed by immunohistochemistry and immunoblotting. Loss of the p53-specific locus 17p13.3 was detected by FISH. Mutations in the p53 gene were detected in nine samples and they included eight missense mutations and one short deletion causing frame shift and premature stop codon formation in position 169. This mutation was associated with mRNA decay as revealed by sequencing of the p53 gDNA. All eight missense mutations were manifested by accumulation of the p53 protein in nuclei of tumor cells and three of them exhibited loss of the p53-specific locus 17p13.3. The p53 mutations were shown to be a negative prognostic marker in MCL.