Les thérapies par irradiation sont utilisées pour traiter la plupart des cas de cancer. Une limitation majeure est l’induction de dommages dans les tissus sains. Par conséquent, l’amélioration du ciblage tumoral est un défi majeur. L'addition de nanoparticules (NPs) est proposée comme une nouvelle stratégie pour amplifier les effets des radiations dans les tumeurs (radiosensibilisation ). Les nanoparticules de Z élevé (platine, or, gadolinium) se révèlent être de bons candidats. Afin de développer de nouveaux nanoagents et d’améliorer les plans de traitement, il est nécessaire de mieux comprendre les mécanismes fondamentaux impliqués. Il a été observé que les radiosensibilisateurs augmentent l'effet létal des radiations (ions rapides ou rayons gamma). Ceci est attribué à une cascade d'événements multi-échelle qui comprend l'activation des NPs, leur relaxation, suivi de la production de radicaux responsables de la mort cellulaire (dans les eucaryotes). Il n'est pas encore clair laquelle des étapes, entre l’excitation/relaxation électronique des NPs ou la réponse biologique joue le rôle prédominant. Par conséquent, le défi de ma thèse était de tester les effets de radiosensibilisateurs (NPs d'or, de platine ou à base de gadolinium) sur des cellules autres que des cellules eucaryotes. Pour la première fois, l’effet des NPs a été testé sur la bactérie la plus radiorésistante jamais rapportée, D. radiodurans. Les NPs ont également été testées sur E. coli. Des études à l'échelle moléculaire ont été utilisées pour comprendre les mécanismes élémentaires. En résumé, ce travail montre que les NPs radiosensibilisantes amplifient les effets des rayons γ dans les bactéries radiosensibles et radiorésistantes. Ceci est attribué à la production de grappes de radicaux et à l’induction de dommages nanométriques dans l'ADN mais également dans les protéines de réparation. Finalement la radiosensibilisation est un phénomène «universel» qui peut être induite dans tout organisme vivant. En d'autres termes, les mécanismes élémentaires liés à l’excitation/relaxation de la NP jouent un rôle majeur par rapport à la réponse biologique de la cellule. Enfin, un ensemble de méthodes ont été optimisées pour évaluer la toxicité et observer l’internalisation des NPs dans les bactéries. / Radiation therapies are used to treat most of the cancer cases. One major limitation is the damage induced in the healthy tissues and tumor targeting is a major challenge. The addition of nanoparticles (NPs) is proposed as a novel strategy to amplify the radiations effects in the tumors (radiosensitization). The high-Z nanoparticles (platinum, gold, gadolinium) are found to be good candidates. To develop new nanoagents and improve treatment planning, a deeper knowledge of the fundamental mechanisms is required. It was found that radiosensitizers enhance the lethal effect of radiations (fast ions and gamma rays). This is attributed to a multiscale cascade of events, which includes the NPs activation and relaxation, the production of water radicals up to the biological impact in mammalian cells. It is not clear yet what from the early stage processes or from the (eukaryotic) cell response is the key stage of the radiosensitization. Hence, the challenge of my thesis was to probe the effects of radiosensitizers (gold, platinum and gadolinium based nanoparticles) on cells other than eukaryotic cells. For the first time, their effect was tested on the most radioresistant bacterium ever reported Deinoccocus radiodurans (D. radiodurans). For comparison, the nanoparticles were tested on the radiosensitive bacterium E.coli. Additional studies at molecular scale were used to understand the elementary mechanisms. In summary, this work demonstrates that the radiosensitizing nanoparticles amplify the effects of -rays in radiosensitive and also radioresistant bacteria. This is attributed to the production of radical clusters and to the inducetion of nano-size biodamages in DNA but also in repair proteins. Finally, this work proves that the radiosensitization is a “universal” phenomenon that can take place in all living organisms. In other words, it tells that elementary mechanisms play a major role compared to the biological response of the cell. A set of standardized methods for evaluating the cellular uptake and the toxicity of the potential nanodrug was established throughout this study.

Radiosensitization is a novel targeted therapy strategy where chemical compounds are being explored to enhance the sensitivity of the tissue to the effects of ionizing radiation. Among the different radiosensitizers alternatives, nanomaterials have shown promising results by enhancing tumor injury through the production of free radicals and reactive oxygen species (ROS). In this work, Gold-supported titania (Au@TiO2) nanocomposites were synthesized through an innovative strategy using X-ray irradiation, and their potential as radiosensitizers was investigated. Radiosensitization of Au@TiO2 nanocomposites was assessed by monitoring the decomposition of Methylene Blue (MB) under X-ray irradiation in the presence of the nanomaterial. Radiosensitization of Au@TiO2 was thoroughly investigated as a function of parameters such as Au loading, TiO2 particle size, nanomaterial concentration, different irradiation voltages, and dose rates. Results showed that the presence of Au@TiO2 increases significantly the absorbed dose, thus enhancing MB decomposition. The mechanism behind Au@TiO2 radiosensitization relies on their interaction with X-rays. TiO2 produces reactive ROS whereas Au leads to the generation of photoelectrons and Auger electrons upon exposure to X-rays. These species lead to an enhanced degradation rate of the dye, a feature that could translate to cancerous cells damage with minimal side effects. The radiosensitization effect of Au@TiO2 nanocomposites was also tested in biological settings using Microcystis Aeruginosa cells. The results showed an increase in cell damage when irradiated in the presence of Au@TiO2 nanocomposites. Au@TiO2 nanocomposites were fabricated using X-ray radiolytic synthesis, a method that diverges from conventional fabrication processes and leads to negligible by-product formation, an important feature for medical and catalytic applications. In this work, Au nanoparticles are supported on TiO2 with a mean particle size of either 6.5 nm or 21.6 nm, using different ligands such as NaOH or urea, and under different absorbed doses to determine the effects of these parameters on the nanomaterials’ characteristics. Overall, Au@TiO2 synthesized by X-rays showed remarkable promise as radiosensitizers, a concept relevant to a number of medical, biological and environmental applications.

Fresh produce is increasingly implicated in outbreaks of foodborne illness. Internalization of bacterial pathogens into produce is of particular concern as internalized pathogens are unlikely to be removed by surface sanitizers. It is therefore necessary to develop treatments that will reduce their prevalence and numbers on fresh produce. Irradiation is a penetrating nonthermal treatment that effectively eliminates bacteria. Irradiated baby spinach leaves up to 1.0 kGy showed negligible (P>0.05) changes in color, texture, vitamin C, total carotenoids, and chlorophyll content compared to non-irradiated controls throughout storage (15 days at 4oC). This research also shows that irradiation effectively reduces viable Escherichia coli cells internalized in lettuce, and that decontamination is not influenced (P>0.05) by lettuce variety. Irradiation effectively reduced the population of internalized pathogens in a dose-dependent manner (3-4 log reduction at 1.0 kGy). Microscopy images suggest that the contamination sites of pathogens in leafy vegetables are mainly localized on crevices and in the stomata. A careful design of the treatment (understanding dose distribution) will effectively eliminate pathogens while maintaining produce quality. The use of modified atmosphere packaging increased (P<0.05) the sensitivity of pathogens (Salmonella spp. and Listeria spp.) to irradiation in baby spinach leaves (up to 25%). Increasing concentration of oxygen increased (P<0.05) sensitivity of both microorganisms. Radiosensitization could be affected (P<0.05) by production of ozone, which increases with increasing dose-rate and oxygen concentration, and reducing temperatures. Antimicrobial effectiveness of various active compounds was determined against Salmonella spp. and Listeria spp. Inclusion complexes were prepared with antimicrobial compounds and -cyclodextrin. The effectiveness of the microencapsulated compounds was tested by spraying them on the surface of baby spinach leaves inoculated with Salmonella spp. The increase in radiosensitivity (up to 40%) varied with the antimicrobial compound. Spherical poly (DL-lactide-co-glycolide) (PLGA) nanoparticles with entrapped eugenol and trans-cinnamaldehyde were synthesized for future antimicrobial delivery applications. All loaded nanoparticles proved to be efficient in inhibiting growth of Salmonella spp. and Listeria spp. The entrapment efficiency for eugenol and trans-cinnamaldehyde was 98% and 92%, respectively. Controlled release experiments (in vitro at 37oC for 72 hrs) showed an initial burst followed by a slower release rate of the antimicrobial entrapped inside the PLGA matrix.

The goal of radiotherapy is to operate within the therapeutic window - delivering doses of ionizing radiation to achieve locoregional tumour control, while minimizing normal tissue toxicity. A greater therapeutic ratio can be achieved by utilizing radiosensitizing agents designed to enhance the effects of radiation at the tumour. Gold nanoparticles (AuNP) represent a novel radiosensitizer with unique and attractive properties. AuNPs enhance local photon interactions, thereby converting photons into localized damaging electrons. Experimental reports of AuNP radiosensitization reveal this enhancement effect to be highly sensitive to irradiation source energy, cell line, and AuNP size, concentration and intracellular localization. This thesis explored the physics and some of the underlying mechanisms behind AuNP radiosensitization. A Monte Carlo simulation approach was developed to investigate the enhanced photoelectric absorption within AuNPs, and to characterize the escaping energy and range of the photoelectric products. Simulations revealed a 10^3 fold increase in the rate of photoelectric absorption using low-energy brachytherapy sources compared to megavolt sources. For low-energy sources, AuNPs released electrons with ranges of only a few microns in the surrounding tissue. For higher energy sources, longer ranged photoelectric products travelled orders of magnitude farther. A novel radiobiological model called the AuNP radiosensitization predictive (ARP) model was developed based on the unique nanoscale energy deposition pattern around AuNPs. The ARP model incorporated detailed Monte Carlo simulations with experimentally determined parameters to predict AuNP radiosensitization. This model compared well to in vitro experiments involving two cancer cell lines (PC-3 and SK-BR-3), two AuNP sizes (5 and 30 nm) and two source energies (100 and 300 kVp). The ARP model was then used to explore the effects of AuNP intracellular localization using 1.9 and 100 nm AuNPs, and 100 and 300 kVp source energies. The impact of AuNP localization was most significant for low-energy sources. At equal mass concentrations, AuNP size did not impact radiosensitization unless the AuNPs were localized in the nucleus. This novel predictive model of AuNP radiosensitization could help define the optimal use of AuNPs in potential clinical strategies by determining therapeutic AuNP concentrations, and recommending when active approaches to cellular accumulation are most beneficial.

The goal of radiotherapy is to operate within the therapeutic window - delivering doses of ionizing radiation to achieve locoregional tumour control, while minimizing normal tissue toxicity. A greater therapeutic ratio can be achieved by utilizing radiosensitizing agents designed to enhance the effects of radiation at the tumour. Gold nanoparticles (AuNP) represent a novel radiosensitizer with unique and attractive properties. AuNPs enhance local photon interactions, thereby converting photons into localized damaging electrons. Experimental reports of AuNP radiosensitization reveal this enhancement effect to be highly sensitive to irradiation source energy, cell line, and AuNP size, concentration and intracellular localization. This thesis explored the physics and some of the underlying mechanisms behind AuNP radiosensitization. A Monte Carlo simulation approach was developed to investigate the enhanced photoelectric absorption within AuNPs, and to characterize the escaping energy and range of the photoelectric products. Simulations revealed a 10^3 fold increase in the rate of photoelectric absorption using low-energy brachytherapy sources compared to megavolt sources. For low-energy sources, AuNPs released electrons with ranges of only a few microns in the surrounding tissue. For higher energy sources, longer ranged photoelectric products travelled orders of magnitude farther. A novel radiobiological model called the AuNP radiosensitization predictive (ARP) model was developed based on the unique nanoscale energy deposition pattern around AuNPs. The ARP model incorporated detailed Monte Carlo simulations with experimentally determined parameters to predict AuNP radiosensitization. This model compared well to in vitro experiments involving two cancer cell lines (PC-3 and SK-BR-3), two AuNP sizes (5 and 30 nm) and two source energies (100 and 300 kVp). The ARP model was then used to explore the effects of AuNP intracellular localization using 1.9 and 100 nm AuNPs, and 100 and 300 kVp source energies. The impact of AuNP localization was most significant for low-energy sources. At equal mass concentrations, AuNP size did not impact radiosensitization unless the AuNPs were localized in the nucleus. This novel predictive model of AuNP radiosensitization could help define the optimal use of AuNPs in potential clinical strategies by determining therapeutic AuNP concentrations, and recommending when active approaches to cellular accumulation are most beneficial.

This thesis focuses on the evaluation of biomarkers for radio-immunodiagnostics and radio-immunotherapy and on radiosensitization strategies after HSP90 inhibition, as a step towards more personalized cancer medicine. There is a need to develop new tracers that target cancer-specific biomarkers to improve diagnostic imaging, as well as to combine treatment strategies to potentiate synergistic effects. Special focus has been on the cell surface molecule CD44 and its oncogenic variants, which were found to exhibit unique expression patterns in head and neck squamous cell carcinoma (HNSCC). The variant CD44v6 seems to be a promising target, because it is overexpressed in this cancer type and is associated with radioresistance. Two new radioconjugates that target CD44v6, namely, the Fab fragment AbD15179 and the bivalent fragment AbD19384, were investigated with regard to specificity, biodistribution and imaging performance. Both conjugates were able to efficiently target CD44v6-positive tumors in vitro and in vivo. PET imaging of CD44v6 with 124I-AbD19384 revealed many advantages compared with the clinical standard 18F-FDG. Furthermore, the efficacy of the novel HSP90 inhibitor AT13387 and its potential use in combination with radiation treatment were evaluated. AT13387 proved to be a potent new cancer drug with favorable pharmacokinetics. Synergistic combination effects at clinically relevant drug and radiation doses are promising for both radiation dose reduction and minimization of side effects, or for an improved therapeutic response. The AT13387 investigation indicated that CD44v6 is not dependent on the molecular chaperone HSP90, and therefore, radio-immunotargeting of CD44v6 in combination with the HSP90 inhibitor AT13387 might potentiate treatment outcomes. However, EGFR expression levels did correlate with HSP90 inhibition, and therefore, molecular imaging of EGFR-positive tumors may be used to assess the treatment response to HSP90 inhibitors. In conclusion, these results demonstrate how tumor targeting with radiolabeled vectors and chemotherapeutic compounds can provide more specific and sensitive diagnostic tools and treatment options, which can lead to customized treatment decisions and a functional diagnosis that provides more precise and safer drug prescribing, as well as a more effective treatment for each patient.

Le glioblastome est la forme la plus commune et la plus mortelle de tumeur cérébrale chez l’adulte. La prise en charge thérapeutique conventionnelle de ce cancer consiste en une exérèse chirurgicale de la tumeur suivie d’une radiothérapie et d’une chimiothérapie par témozolomide (Temodal®). En dépit de ces traitements pourtant agressifs, la plupart des patients rechutent et leur survie n’excède généralement pas 15 mois. Plusieurs études ont été menées afin de comprendre les mécanismes qui conduisent à une résistance de la tumeur vis-à-vis de ces traitements et récemment, un contingent cellulaire appelé cellule souche de gliome(CSG) a été mis en évidence. L’objectif de cette thèse a été de développer un nanovecteur capable de cibler ces CSGs afin de concentrer l’efficacité de la radiothérapie au niveau des cellules radiorésistantes et notamment des cellules CXCR4-positives impliquées dans la prolifération, la migration cellulaire et la résistance à l’apoptose. Pour cela, nous avons développé des nanocapsules lipidiques (LNC) contenant du rhénium-188 (188Re), un émetteur bêta -, et fonctionnalisées au moyen d’un anticorps bloquant(12G5) dirigé contre le récepteur à chimiokine CXCR4. L’efficacité de cet objet a été testée dans un modèle orthotopique de glioblastome humain chez la souris et nous avons montré que les souris traitées avec cesLNC-188Re couplées au 12G5 présentent les meilleures médianes de survie. En parallèle de ce travail, nous avons conçu un autre nanovecteur contenant de la rapamycine, un inhibiteur de la voie PI3K/Akt/mTOR impliquée dans la radiorésistance et seulement soluble dans des solvants organiques. L’efficacité de ce vecteur à rendre la rapamycine biodisponible au niveau cellulaire et à bloquer la voie mTOR a été validée in vitro. Son activité antitumorale propre et son rôle en tant que radiosensibilisant ont de plus été caractérisés en amont d’investigations précliniques. En conclusion, cette thèse a permis de développer un outil de radiothérapie interne dans le cadre d’une thérapie ciblée dans le glioblastome. Nous avons pour la première fois montré que des LNC188Re couplées à un anticorps présentent un intérêt dans le traitement du glioblastome. / Glioblastoma is the most common and deadly primarily brain tumor in adult. Conventional therapy consists on a surgical resection of the tumor followed by radiotherapy and radiotherapy. Despite this treatments, most patients rescue. These recurrences have recently been assign to radio-chemotherapeutic resistant cell contingents called glioblastoma stem-cells (GSCs). The aim of this thesis was to develop nanovector targeting these GSCsCXCR4 positives cells implicated in proliferation, cell migration and apoptose resistance. Then, we have developed lipid nanocapsules(LNC) loaded with rhenium-188 (188Re), a beta-emitter, and functionalized with a blocking antibody (12G5) directed to CXCR4. Nanovector efficiency was evaluated in an orthotopic human glioblastoma mice model and we showed that 12G5-LNC188Re treated mice had the best median survival. Concurrently of this study, we have developed another nanovector loaded with rapamycin, an inhibitor of PI3K/Akt/mTOR signaling pathway implicated in radioresistance and only soluble in organics solvants. Efficiency of this new nanovector to improve rapamycine bioavaibility and to block mTOR phosphorylation was assessed in vitro. Its own antitumor activity and its role as radiosensitizer have been evaluated in up-stream of preclinical studies.To conclude, this thesis allowed the development of a new tool forvectorized internal radiotherapy in glioblastoma. We have shown for the first time that LNC-188Re functionalized with a blockin antibody present an interest in glioblastoma therapy.

Les SRPs (Small Rigid Platforms) sont des nanoparticules de taille inférieure à 5 nm, constituées d'une matrice de polysiloxane, à la surface de laquelle sont greffés de manière covalente des complexes de gadolinium. Les SRPs ont été développées pour des applications en imagerie multimodale, notamment en imagerie par résonance magnétique, en scintigraphie et en imagerie par fluorescence, ainsi que pour des applications en théranostic, et plus particulièrement, en radiosensibilisation. L'objectif de la thèse consiste à fonctionnaliser les SRPs par des entités de ciblage, capables d'identifier et de se lier à des récepteurs surexprimés par les tumeurs ou par l'endothélium qui leur est associé. Plus précisément, trois molécules ciblantes ont été retenues : (i) le c(RGDfK) – un pentapeptide cyclique contenant le motif Arg-Gly-Asp ciblant spécifiquement l'intégrine αuß3, récepteur de cellule endothéliale pour les protéines de la matrice extracellulaire et surexprimée par de nombreuses cellules cancéreuses, (ii) une molécule dérivée de la quinoxaline – ciblant la mélanine, sur-exprimée par de nombreux types de mélanomes (cancers de la peau), et (iii) un pyridinium – ciblant les protéoglycanes, sur-exprimés par le chondrosarcome (cancer du cartilage). Les différentes molécules ciblantes ont alors été greffées en surface des SRPs afin d'optimiser leur accumulation et leur rétention au sein des tumeurs, et par conséquent, d'augmenter le potentiel de ces SRPs en tant qu'agents théranostiques. Le couplage covalent des différents vecteurs aux SRPs et leur quantification ont été mis en évidence par diverses techniques de caractérisation physicochimiques et complémentaires. Enfin, le ciblage des nanoparticules vectorisées a été démontré par plusieurs tests in vitro et/ou in vivo lors de nombreuses collaborations pour les trois différents vecteurs / SRPs (Small Rigid Platforms) are sub-5 nanometre nanoparticles, composed of a polysiloxane network surrounded by gadolinium chelates. SRPs have previously demonstrated their efficiency for multimodal imaging and theranostic applications, especially magnetic resonance imaging, scintigraphy, fluorescence imaging, and radiosensitization. The purpose of this thesis deals with the functionalization of these SRPs by targeting entities able to recognize and to bind to receptors that are over-expressed by tumours or by the matching endothelium. More specifically, three targeting molecules drew our attention: (i) c(RGDfK) – a cyclic pentapeptide containing the Arg-Gly-Asp motif, able to target the αuß3 integrin, an endothelial cell receptor for the proteins of the extracellular matrix and frequently expressed by tumour cells, (ii) a quinoxaline derivative – targeting melanin, which is over-expressed by melanomas (skin cancers) and (iii) a pyridinium moiety – targeting proteoglycans, which are over-expressed in chondrosarcoma (cartilage cancer). The different targeting molecules were then grafted to the SRPs in order to maximize their accumulation and retention in tumours with the aim to enhance the SRP efficiency as theranostic agents. The covalent coupling of the different targeting molecules to the SRPs and their quantification were performed by various physicochemical characterization techniques. Finally, the targeting of the functionalized SRPs was highlighted by several in vitro and/or in vivo assays thanks to numerous successful collaborations

La radiothérapie basée sur l'utilisation des photons de haute énergie (rayons X) est l'approche la plus courante dans le traitement du cancer. Toutefois, elle est limitée par la tolérance des tissus sains. Par conséquent, il est d'un intérêt majeur de développer de nouvelles techniques et protocoles pour améliorer le ciblage dans les tumeurs. Dans cette perspective, la hadronthérapie (irradiation de la tumeur par des protons ou des ions carbone) est considérée comme l'une des techniques les plus prometteuses car le dépôt d'énergie est maximum en fin de parcours, ce qui permet de cibler la tumeur. Pourtant, l’utilisation de cette modalité reste limitée du fait de la dose reçue par les tissus sains situés à l'entrée du faisceau.Pour améliorer les performances des thérapies par radiation, une nouvelle stratégie basée sur la combinaison de nanoparticules métalliques (nano-médecine) avec des rayonnements ionisants a été développée par le groupe. En effet, les nanoparticules ont une chimie de surface remarquable qui permet de les fonctionnaliser avec des ligands qui les rendent plus futiles et moins reconnus des macrophages afin de les concentrer dans les tumeurs.Le but de mon travail a été de développer des nanoparticules à base de platine (NPs de platine pelylée et des nanoparticules bimétalliques) visant à améliorer l’effet des rayonnements ionisants (photons et ions carbone) dans les cellules.Une méthode originale de synthèse en une seule étape combinant la radiolyse et la PEGylation in situ a été optimisée. Cette méthode a permis d’obtenir des NPs stables, de taille homogène (cœur métallique proche de 3 nm).L'impact biologique de ces nouvelles NPs a été évalué sur deux lignées de cellules cancéreuses humaines. Il a été observé que les NPs, non-toxiques, ont un mode d’internalisation qui dépend de la lignée cellulaire. Celles-ci sont, dans tous les cas, localisées exclusivement dans le cytoplasme. Les NPs de platine développées dans ce travail permettent d’amplifier significativement la destruction des cellules cancéreuses, en particulier lorsqu’un faisceau médical d’ions carbone est utilisé comme rayonnement. Les mécanismes moléculaires à l’origine de cet effet ont été étudiés grâce à l’utilisation d’une nanosonde biologique. Ces expériences ont montré que les NPs sont responsables de l’augmentation de dommages nanométriques, qui peuvent être létaux pour les cellules. Cet effet est attribué à des processus électroniques d’activation et de reneutralisation de la NP qui engendre une forte perturbation dans le volume nanométrique qui l’entoure tel que la production groupée de radicaux fortement réactifs et toxiques.En conclusion, ce travail à l’interface de la physique, chimie et biologie montre les capacités des NPs à base de platine à améliorer l’éradication par radiation des cellules cancéreuses. / Radiotherapy based on the use of high energy photons (X-rays) is the most common approach in cancer treatment. However, its implementation is limited by the tolerance of healthy tissue. Therefore, it is of major interest the development of new techniques and protocols to improve the selectivity of radiation effects within the tumor. In this perspective, the hadrontherapy (tumor irradiation by protons or carbon ions) is considered as one of the most promising techniques due to the energy deposition of ions in depth which is maximum at the end of the track. However, the use of this modality remains restricted by the lower but significant damage induced to the normal tissue located at the entrance of the ion beam.To improve the performance of radiation therapies, a new strategy based on the combination of metallic nanoparticles (nanomedicine) with ionizing radiations was studied. These treatments have been developed by the group. Indeed, the nanoparticles present a remarkable surface chemistry that allows their functionalization with ligands which make them less recognized by macrophages allowing an important accumulation of these nano-agents selectively into the tumors.The goal of my work was thus to develop platinum based nanoparticles (mono- and bimetallic Pt NPs) to enhance the effect of radiations (photons and carbon ions) into the cells.A novel one-step method of synthesis combining radiolysis and in situ PEGylation has been optimized. This method enabled to obtain stable NPs with a uniform size (metallic core diameter close to 3 nm) and shape. The biological impact of these new Pt NPs was evaluated in two human cancer cell lines.It has been observed that non-toxic Pt NPs have an internalization pathway that strongly depends on the cell line. These are, in all cases, exclusively localized in the cytoplasm. The Pt NPs developed in this work significantly enhanced cancer cell killing, particularly when medical carbon ions are used to irradiate.The molecular mechanisms underlying this effect were investigated through the use of a bio-nanoprobe. These experiments showed that NPs are responsible for the increase of nanometric damage, lesions that can be lethal to cells. This effect is attributed to an electronic activation processes and to the reneutralisation of NPs, which generates a strong perturbation in the surrounding nanometer volume producing highly reactive and toxic free radical clusters.In conclusion, this work at the interface of physics, chemistry and biology shows the potential of platinum NPs to improve the eradication of cancer cells by radiation.

Les agents ciblant les microtubules sont des médicaments anticancéreux efficaces. Leur utilisation dans le cadre d’un traitement combiné avec des rayonnements ionisants est également une stratégie prometteuse. Cependant, l’apparition de résistances aux produits chimiques et aux radiations nécessite de rechercher d'autres types de traitements. Nos laboratoires ont récemment décrit deux médicaments qui ciblent directement ou indirectement les microtubules. Premièrement, un analogue du 2-méthoxyestradiol, un poison de fuseau se liant à des microtubules et provoquant la formation de fuseaux mitotiques anormaux. Il s'agit du 2-éthyl-3-O-sulphamoyle-estra-1,3,5 (10) 16-tétraène (ESE-16). Deuxièmement, le 9-benzoyloxy-5,11-diméthyl-2H, 6H-pyrido [4,3-b] carbazol-1-one (LimPyr1), un nouvel inhibiteur des LIM kinases induisant indirectement la stabilisation des microtubules. Il a été démontré récemment que LimPyr1 est actif sur les modèles de cancer du sein résistants au taxol. En tant que médicaments ciblant les microtubules, les deux agents, ESE-16 et LimPyr1, induisent des défauts mitotiques. Nous émettons donc l’hypothèse qu’ils pourraient sensibiliser les cellules aux radiations. Le but de ce projet de thèse était de vérifier cette hypothèse et, plus précisément, de déterminer si de faibles doses de ESE-16 et de LimPyr1 pourraient augmenter l'apoptose et retarder la réparation nucléaire induite par le rayonnement dans les cellules du cancer du sein in vitro.Différentes lignées cellulaires cancéreuses, les cellules MCF-7, MDA-MB-231 et BT-20, ont été exposées à ESE-16 et à LimPyr1 pendant 24 heures avant un rayonnement de 8 Gy. Les effets de ces combinaisons thérapeutiques ont été comparés à ceux obtenus à partir de cellules exposées aux composés seuls ou aux seules radiations. L'activation des voies de survie et des voies apoptotiques intrinsèques a été étudiée. Les résultats ont révélé une augmentation de la signalisation de la survie et de la mort dans les cellules exposées aux traitements individuels. Les traitements combinés ont diminué la survie des cellules alors que la signalisation apoptotique augmentait, entraînant une augmentation de l'apoptose. En outre, les traitements combinés ont augmenté de manière significative la présence de micronoyaux dans les cellules BT-20, indiquant une augmentation des dommages à l'ADN. Les cellules MCF-7 et MDA-MB-231 présentent une formation de micronoyaux similaire lorsqu'elles sont exposées à la combinaison de traitements ou au rayonnement uniquement. La phosphorylation de H2AX (γH2AX) (normalement augmentée lors de dommages à l'ADN) et l'expression de Ku70 (nécessaire pour la réparation de l'ADN) étaient diminuées dans les cellules de cancer du sein prétraitées 2 heures après l'irradiation par rapport aux cellules exposées à l'irradiation uniquement. L'expression de H2AX et Ku70 est cependant significativement accrue 24 heures après irradiation des cellules prétraitées par rapport aux cellules exposées aux traitements individuels. Des expériences portant sur la réponse adaptative ont révélé que LimPyr1 diminuait le développement de la résistance aux radiations en augmentant la perméabilité transmembranaire mitochondriale et en générant des ROS, un mécanisme qui n'est pas observé dans cellules traitées par ESE-16. Nous avons également observé une communication intercellulaire entre les cellules exposées au rayonnement et les cellules non exposées via l'effet induit par le rayonnement.En conclusion, le blocage mitotique partiel induit par ESE-16 et LimPyr1 rend les chromosomes plus exposés aux dommages dus aux radiations, comme l'indique l'augmentation de la présence de micronoyaux. De plus, les deux composés diminuent la signalisation et le trafic des protéines de protection et de dommages à l'ADN. En outre, LimPyr1 empêche le développement de résistances aux radiations dans les cellules exposées aux radiations. / Microtubule targeting agents are effective anti-cancer drugs. Their use as part of a combined treatment modality with ionising radiation is also a promising strategy. However, the emergence of resistance to chemical and radiation requires searching for alternative treatments. Our laboratories have recently described two drugs that directly or indirectly target the microtubules. Firstly, an analogue of 2-methoxyestradiol, a spindle poison binding to microtubules and causing the formation of abnormal mitotic spindles. This is 2-ethyl-3-O-sulphamoyl-estra-1,3,5 (10) 16-tetraene (ESE-16). Secondly, 9-benzoyloxy-5,11-dimethyl-2H, 6H-pyrido [4,3-b] carbazol-1-one (LimPyr1), a novel inhibitor of LIM kinases indirectly inducing microtubule stabilization. It has been recently shown that LimPyr1 is active on taxol-resistant breast cancer models. As microtubule-targeting drugs, both agents, ESE-16 and LimPyr1, induce mitotic defects. We thus hypothesize that they could sensitize cells to radiation. The aim of this PhD project was to test that hypothesis and, more specifically, to investigate whether low-dose ESE-16 and LimPyr1 could increase apoptosis and delay nuclear repair induced by radiation in breast cancer cells in vitro.Various cancer cell lines, MCF-7-, MDA-MB-231- and BT-20 cells, were exposed to ESE-16 and LimPyr1 for 24-hours prior to 8 Gy radiation. The effects of these combination therapies were compared to those obtained from cells exposed to the compounds alone or only to radiation. The activation of the survival and intrinsic apoptotic pathways were investigated. Results revealed an increase in survival and -death signaling in cells exposed to the individual treatments. The combination treatments decreased the cell survival while apoptotic signaling was increased, resulting in increased apoptosis. Furthermore, the combination treatments significantly increased the presence of micronuclei in BT-20 cells, indicating an increase in DNA damage. MCF-7- and MDA-MB-231 cells displayed similar micronuclei formation when exposed to the combination treatments or radiation only. Phosphorylation of H2AX (γH2AX) (normally increased upon DNA damage) and Ku70 expression (required for DNA repair) were decreased in pretreated breast cancer cells 2 hours after irradiation compared to cells exposed to irradiation only. The expression of H2AX and Ku70, however, is significantly increased 24 hours after irradiation of the pretreated cells relative to the cells exposed to the individual treatmentsExperiments investigating the adaptive response revealed that LimPyr1 decreased radiation resistance development by increasing the permeability of the mitochondrial transmembrane (flow cytometry measuring Mitocapture™) and the generation of ROS (flow cytometry employing hydroethidine), a mechanism not observed in ESE-16 pre-treated cells. We also observed an intercellular communication between cells exposed to radiation and non-exposed cells via the radiation induced bystander effect.In conclusion, the anti-mitotic effect of ESE-16 and LimPyr1 renders the chromosomes more exposed to radiation damage, as assessed by the increased occurrence of micronuclei. Moreover, both compounds decrease the signaling and trafficking of DNA damage and repair proteins. Additionally, LimPyr1 prevented the development of radiation resistance in cells exposed to radiation.