Afin d’améliorer les traitements par radiothérapie, des radiosensibilisateurs tels que les nanoparticules d’or (NPo) sont étudiés. Mais leur translation en clinique nécessite une bonne compréhension des phénomènes en jeu. Si l’effet radiosensibilisateur a bien été confirmé sur des cibles biologiques (ADN, cellules et in vivo) et si les radicaux hydroxyle ont souvent été proposés comme intermédiaires, aucune preuve claire n’a encore été apportée. Ce travail avait pour premier objectif d’élaborer un protocole de « référence » afin de quantifier les radicaux hydroxyle et les électrons produits par les NPo en interaction avec un rayonnement ionisant. Cette étude a mis en évidence des productions massives de ces deux espèces pour des NPo non-fonctionnalisées. De plus, l'étude de différents paramètres, tels que la quantité de dioxygène en solution ou le rayonnement incident, nous a conduits à proposer un nouveau mécanisme permettant de rendre compte de nos résultats. Néanmoins, l'application biologique des NPo ne peut être envisagée que si ces nano-objets sont fonctionnalisés afin de les rendre furtifs et de les adresser spécifiquement à la tumeur. Après synthèse et caractérisation poussée de différents types de NPo fonctionnalisées, nous avons comparé la production de radicaux hydroxyle avec la dégradation d’une cible biologique, l'ADN, et mis en évidence l'impact significatif de la fonctionnalisation sur l'effet radiosensibilisateur. Ainsi, cette étude apporte des informations essentielles en vue de l’optimisation de la conception des NPo les plus efficaces pour la radiosensibilisation, une première étape vers leur application radiothérapeutique. / To improve radiotherapy efficiency, radiosensitizers such as gold nanoparticles (GNP) are developed. But to translate them to clinics, a good knowledge of the processes at stage is needed. GNP radiosensitizing effect was well-confirmed on biological targets (DNA, cells and in vivo) and hydroxyl radicals are often proposed to be key intermediates, but no clear evidence has been given yet. In this work, we first developed a ‘reference’ protocol to quantify hydroxyl radicals and electrons produced by GNP in their interaction with ionizing radiation. These investigations reveal a massive production of both species for non-functionalized GNP. Moreover the study of various parameters such as the concentration of dissolved dioxygen or the energy of the incident radiation leads us to propose a new mechanism on the origin of the radiosensitizing effect. Nevertheless, biological applications of GNP can only be considered if the nano-objects are functionalized to make them furtive, address them or deliver medicines to the tumor. After synthesis and characterization of different functionalized GNP, we compared hydroxyl radicals production with the damages induced on DNA and highlighted a significant impact of functionalization on the radiosensitizing effect. Finally, this work gives valuable information for the design of the most efficient GNP for radiotherapy which is a first step towards their medical application.

Nesse trabalho foram produzidas nanopartículas (NPs) de Gd2O3 e Er2O3 para aplicação como radiossensibilizadores em feixes de radioterapia. Elas foram sintetizadas no Laboratório de Interações Hiperfinas do IPEN pelo método da decomposição térmica e caracterizadas utilizando difração de raios-X, para verificar a estrutura cristalina, microscopia eletrônica de transmissão, para obter informações sobre forma, tamanho e distribuição de tamanho, análise por ativação neutrônica, por meio da qual foi possível determinar a pureza das amostras e calcular a concentração de gadolínio e érbio. Medições de magnetização e de espectroscopia de correlação angular γ-γ perturbada (PAC) foram realizadas a fim de estudar o comportamento magnético e a interação quadrupolar das partículas, respectivamente. Os resultados da caracterização mostram a formação de uma estrutura cristalina do tipo bixbyite, com aproximadamente 5 nm de diâmetro e estreita distribuição de tamanho, para as amostras pós-síntese. A determinação da massa de terra-rara em cada amostra foi importante para realizar a normalização nas medições de susceptibilidade magnética, tornando possível a visualização de um grande aumento na magnetização abaixo de 30 K, nas amostras pós-síntese, o que não é observado em partículas maiores, além de um aumento no momento magnético efetivo das NPs em relação aos respectivos bulks e uma mudança na temperatura de ordenamento antiferromagnético para o Er2O3. Os resultados da espectroscopia PAC evidenciam possíveis efeitos de superfície. A falta de uma frequência bem definida nas amostras de 5 nm indicam que a quantidade de 111In(111Cd) na superfície da partícula é maior do que no interior da mesma, fazendo com que a interação hiperfina do núcleo de prova com o host não seja evidente. Já a união da técnica de difração de raios-X com a espectroscopia PAC foi fundamental para o entendimento do dano causado às partículas pela irradiação com 60Co. Quanto às medições de radiossensibilização a dosimetria Fricke gel foi fundamental para a verificação de um fator de aumento de dose (DEF) de até 1,67 e 1,09 para NPs de Gd2O3 irradiadas com 60Co e 6MV, respectivamente. Nas mesmas condições, para as amostras de Er2O3, foram encontrados valores de DEF de até 1,37 e 1,06. Isso comprova os efeitos radiossensibilizadores dessas NPs. Os resultados alcançados nesse trabalho não apenas fornecem dados importantes para o estudo de NPs de terra-rara na área de física da matéria condensada como também uma base sólida para a aplicação desses elementos como radiossensibilizadores em feixes de radioterapia, possibilitando a utilização da imagem por ressonância magnética para localizar e obter a concentração dessas NPs dentro do paciente, aumentando assim a eficiência do tratamento do câncer. / In this study Gd2O3 and Er2O3 nanoparticles were produced for application as radiosensitizers in radiotherapy beams. They were synthesized at the Hyperfine Interactions Laboratory, IPEN, using thermal decomposition method and characterized by X-ray diffraction, to verify crystalline structure, transmission electron microscopy, to obtain information about shape, size and size distribution, neutron activation analysis, whereby it was possible to determine samples purity and gadolinium and erbium concentration. Magnetization and perturbed γ-γ angular correlation (PAC) measurements were performed in order to study particles magnetic behavior and quadrupole interactions, respectively. Characterization results showed a bixbyite structure, 5 nm diameter post-synthesis particles with narrow size distribution. Rare-earth mass determination in each sample was important to perform normalization in magnetic susceptibility measurements, making possible the view of a high magnetization under 30 K for post-synthesis samples, what was not observed in larger particles, together with an effective magnetic moment enhancement for nanoparticles, not seen in bulk samples, and a change in the antiferromagnetic ordering temperature for Er2O3. PAC spectroscopy results show possible surface effects. The absence of a well-defined frequency in 5 nm samples indicates the amount of 111In(111Cd) at particle surface is bigger than in the core, resulting in a non-evident hyperfine interaction between the probe nuclei and the host. The X-ray diffraction and PAC spectroscopy joint was vital to understand the particles structural damage caused by 60Co irradiation. About radiosensitizer measurements a dose enhancement factor (DEF) of up to 1,67 and 1,09 for Gd2O3 nanoparticles under 60Co and 6MV irradiation, respectively, were observed. Under same conditions DEF values of up to 1,37 and 1,06 were found for Er2O3 samples. Results reached in this study provide not only important data for rare-earth oxides study in condensed matter physics but also a solid ground for the application of these elements as radiosensitizers in radiotherapy beams, allowing the use of magnetic resonance imaging to locate and obtain the concentration of these particles inside patient body, increasing cancer treatment efficiency.

Nesse trabalho foram produzidas nanopartículas (NPs) de Gd2O3 e Er2O3 para aplicação como radiossensibilizadores em feixes de radioterapia. Elas foram sintetizadas no Laboratório de Interações Hiperfinas do IPEN pelo método da decomposição térmica e caracterizadas utilizando difração de raios-X, para verificar a estrutura cristalina, microscopia eletrônica de transmissão, para obter informações sobre forma, tamanho e distribuição de tamanho, análise por ativação neutrônica, por meio da qual foi possível determinar a pureza das amostras e calcular a concentração de gadolínio e érbio. Medições de magnetização e de espectroscopia de correlação angular γ-γ perturbada (PAC) foram realizadas a fim de estudar o comportamento magnético e a interação quadrupolar das partículas, respectivamente. Os resultados da caracterização mostram a formação de uma estrutura cristalina do tipo bixbyite, com aproximadamente 5 nm de diâmetro e estreita distribuição de tamanho, para as amostras pós-síntese. A determinação da massa de terra-rara em cada amostra foi importante para realizar a normalização nas medições de susceptibilidade magnética, tornando possível a visualização de um grande aumento na magnetização abaixo de 30 K, nas amostras pós-síntese, o que não é observado em partículas maiores, além de um aumento no momento magnético efetivo das NPs em relação aos respectivos bulks e uma mudança na temperatura de ordenamento antiferromagnético para o Er2O3. Os resultados da espectroscopia PAC evidenciam possíveis efeitos de superfície. A falta de uma frequência bem definida nas amostras de 5 nm indicam que a quantidade de 111In(111Cd) na superfície da partícula é maior do que no interior da mesma, fazendo com que a interação hiperfina do núcleo de prova com o host não seja evidente. Já a união da técnica de difração de raios-X com a espectroscopia PAC foi fundamental para o entendimento do dano causado às partículas pela irradiação com 60Co. Quanto às medições de radiossensibilização a dosimetria Fricke gel foi fundamental para a verificação de um fator de aumento de dose (DEF) de até 1,67 e 1,09 para NPs de Gd2O3 irradiadas com 60Co e 6MV, respectivamente. Nas mesmas condições, para as amostras de Er2O3, foram encontrados valores de DEF de até 1,37 e 1,06. Isso comprova os efeitos radiossensibilizadores dessas NPs. Os resultados alcançados nesse trabalho não apenas fornecem dados importantes para o estudo de NPs de terra-rara na área de física da matéria condensada como também uma base sólida para a aplicação desses elementos como radiossensibilizadores em feixes de radioterapia, possibilitando a utilização da imagem por ressonância magnética para localizar e obter a concentração dessas NPs dentro do paciente, aumentando assim a eficiência do tratamento do câncer. / In this study Gd2O3 and Er2O3 nanoparticles were produced for application as radiosensitizers in radiotherapy beams. They were synthesized at the Hyperfine Interactions Laboratory, IPEN, using thermal decomposition method and characterized by X-ray diffraction, to verify crystalline structure, transmission electron microscopy, to obtain information about shape, size and size distribution, neutron activation analysis, whereby it was possible to determine samples purity and gadolinium and erbium concentration. Magnetization and perturbed γ-γ angular correlation (PAC) measurements were performed in order to study particles magnetic behavior and quadrupole interactions, respectively. Characterization results showed a bixbyite structure, 5 nm diameter post-synthesis particles with narrow size distribution. Rare-earth mass determination in each sample was important to perform normalization in magnetic susceptibility measurements, making possible the view of a high magnetization under 30 K for post-synthesis samples, what was not observed in larger particles, together with an effective magnetic moment enhancement for nanoparticles, not seen in bulk samples, and a change in the antiferromagnetic ordering temperature for Er2O3. PAC spectroscopy results show possible surface effects. The absence of a well-defined frequency in 5 nm samples indicates the amount of 111In(111Cd) at particle surface is bigger than in the core, resulting in a non-evident hyperfine interaction between the probe nuclei and the host. The X-ray diffraction and PAC spectroscopy joint was vital to understand the particles structural damage caused by 60Co irradiation. About radiosensitizer measurements a dose enhancement factor (DEF) of up to 1,67 and 1,09 for Gd2O3 nanoparticles under 60Co and 6MV irradiation, respectively, were observed. Under same conditions DEF values of up to 1,37 and 1,06 were found for Er2O3 samples. Results reached in this study provide not only important data for rare-earth oxides study in condensed matter physics but also a solid ground for the application of these elements as radiosensitizers in radiotherapy beams, allowing the use of magnetic resonance imaging to locate and obtain the concentration of these particles inside patient body, increasing cancer treatment efficiency.

In the past decades, tumors have progressively been perceived as highly integrated systems in which the genetically unstable tumor cells and the genetically stable host cells cooperate to promote tumor growth. This view suggests that, beside tumor cells (that are targeted by conventional anticancer treatments such as radio- and chemotherapy), host cells within the tumor microenvironment can be targeted by antitumor therapy. Such alternative strategies are strongly supported by the need to overcome several limitations of the conventional therapies targeting tumor cells, such as collateral toxicity due to lack of tumor selectivity, limited tumor accessibility, and the selection of treatment-resistant variants. By contrast to tumor cells, the genetically stable host cells should not develop resistance to treatments. In this context, the observation that tumor growth is fundamentally dependent on the onset of a private tumor neovasculature (tumor angiogenesis) has revolutionized the field of cancer research. Several treatments have been developed aimed to prevent tumor angiogenesis (anti-angiogenic strategies) or to erase the existent tumor vasculature (anti-vascular approaches) supporting the survival and growth of thousands of tumor cells. However, although such therapies achieved cancer cure in animal models, they turned out to be rather inefficient when tested in patients. This can be attributed to differences in the angiogenic status between fast-growing animal tumors and slow-growing human tumors at the time of clinical detection. Another reading of the above-mentioned observations is that anticancer treatments could benefit from interventions aimed at increasing their efficiency. For instance, radiotherapy could benefit from tumor reoxygenation while a decrease in tumor interstitial pressure could facilitate tumor accessibility to circulating agents. In this context, the mature vasculature is an attractive target since it controls tumor blood supply and is highly accessible for therapy. Therefore, strategies aimed at exploiting its functional reactivity by inducing vasorelaxation have the potential to improve tumor perfusion/drug delivery and oxygenation/radiosensitivity. To be exploited in the clinics, such pro-vascular approaches have to fulfill essential requirements. First, they need to achieve high selectivity for tumor vessels. It should prevent systemic toxicity as well as the stealing of the blood flow towards the peripheral vasculature. Second, vasodilation has to be transient, so that the tumor should not take advantage of an increased energetic supply to grow faster. Third, the therapeutic effects have to be achieved in several tumor types and in different host strains to gain a wide therapeutic range of applicability. Finally, vasomodulation has to be achieved with interventions relevant to the clinical situation, ensuring direct therapeutic significance. However, the therapeutic exploitation of agents modulating tumor perfusion was generally hampered by confounding effects on the systemic blood pressure. In our studies, we have documented that this lack of tumor selectivity can be overcome by identifying vasomodulatory pathways that are selectively altered within the tumor microenvironment, allowing selective vasomodulatory interventions. According to the criteria detailed above, to identify a differential tumor vascular reactivity, we had to work with mice models of mature tumor vascularization. We reasoned that preexisting host arterioles in mice, if coopted, should retain architectural characteristics (such as a muscular coat) necessary for functional reactivity but also be influenced by the tumor microenvironment at both molecular and functional levels. To gain in reproducibility, this model was developed by injecting syngeneic tumor cells in the vicinity of the saphenous arteriole (i.e., a collateral branch of the femoral artery) in the rear leg of mice. With tumor growth, this arteriole was progressively included in the tumor cortex (coopted), with side branches running deeply into tumors. This model was developed using several tumors and mice strains. It provides the unique advantage to allow the easy identification and isolation of mature tumor vessels from fast-growing animal tumors. To evaluate differential vasoreactivity in those tumor-coopted vessels, we adapted pressure myography, a device initially dedicated to the study of the reactivity of coronary arterioles (see annex 1). In our hands, the unprecedented application of pressure myography to the study of small tumor vessels proved to be very efficient. Indeed, this technique not only served us to confirm that arterioles remain sensitive to vasomodulation under tumor cooption, but also allowed us to evidence two major adaptations of host vessels to the tumor microenvironment: the acquisition of an ET-1-mediated basal constrictive tone and a defect in the vasodilatory NO pathway. Furthermore, we used pressure myography to identify and characterize vasomodulatory strategies exploiting these differential reactivities. More particularly, we showed that both BQ123 (an ETA inhibitor) and ionizing radiations (that restored a functional NO pathway) promoted the vasodilation of the tumor-coopted vessels. In vivo, we verified that these strategies fulfilled the essential requirements of pro-vascular approaches: tumor selectivity, transient effects, broad range of applicability, and therapeutic significance in clinically relevant regimens. This latter study led us to further explore the effects of radiotherapy on the status of the tumor vasculature. Hence, we showed that fractionated radiotherapy induced tumor angiogenesis, thereby providing a rationale to combine radiotherapy to anti-angiogenic therapies.

xHypoxia and repopulation of tumour clonogens are two important determinants of treatment outcome in radiotherapy. In general clinical evidence indicates that loco-regional control may be reduced with long overall treatment times and for tumours with low pre-treatment levels of oxygen. Experimental studies with normobaric carbogen and oxygen showed a two-fold enhancement of the efficacy of radiation in a mouse tumour model when combining oxygen with treatment acceleration. It was then demonstrated that substituting carbogen for oxygen and adding high-dose nicotinamide (NAM) further increased the effect. These findings became the basis for a multi-factorial approach designed to overcome the radioprotective effect of tumour repopulation and that of perfusion–limited and diffusion–limited hypoxia. The strategy, named ARCON, combines Accelerated Radiotherapy with CarbOgen and Nicotinamide. Experimental evaluation of ARCON The therapeutic potential of carbogen combined with NAM (CON) focusing on treatment schedules that use clinically relevant radiation and drug dose levels was evaluated in tumour and normal tissue animal models. Some of the conditions under which ARCON gives the largest degree of tumour radiosensitization and therapeutic benefit were identified. Specifically, NAM-dose level, pharmacokinetics and scheduling, and the effect of NAM on repair processes in vivo were also investigated. The results showed that in conventional and accelerated radiotherapy, carbogen and CON are effective and relatively non-toxic tumour sensitizers. They also demonstrated that tumour sensitization with CON was independent of time of NAM administration but that it was drug dose dependent. Some degree of normal tissue sensitization was observed but even relative to mouse skin a significant therapeutic gain was achieved. The mechanism of action for NAM sensitization originally proposed was that of repair inhibition. In the in vivo mouse models tested, namely skin and kidney, NAM did not alter the rate nor the magnitude of repair of radiation induced damage. Clinical evaluation of ARCON In the early 90s, various centres, particularly in the UK, Sweden, Holland and Switzerland, undertook clinical trials of ARCON. The protocols were designed based on detailed considerations of the rodent and human radiation and pharmacokinetic studies. This document also discusses the findings of a phase II non-randomized trial in advanced bladder cancer of accelerated radiotherapy combined with carbogen alone and ARCON. The aim of the study was to establish the feasibility of administering carbogen and NAM to patients and to determine the extent of early and late normal tissue damage. Historical comparisons suggested no overt increase in normal tissue radiosensitivity and the data indicate that ARCON could achieve a therapeutic gain in advanced bladder cancer.

Le traitement des gliomes de haut grade constitue aujourd'hui encore un réel enjeu médical. Les techniques actuellement disponibles sont principalement palliatives et permettent d'augmenter la survie des patients de quelques mois seulement. Une technique innovante de radiothérapie consiste à renforcer la dose déposée dans la tumeur grâce à l'injection d'atomes lourds de manière spécifique dans celle-ci au préalable d'une irradiation de photons de basse énergie (50-100 keV). Cette technique a fait l'objet d'essais précliniques et maintenant d'essais cliniques de phases I et II sur la ligne médicale du synchrotron Européen dont le rayonnement monochromatique et intense est particulièrement adapté pour l'application. L'utilisation d'un agent de contraste (AC) iodé (Z=53) injecté par voie veineuse permet d'améliorer le bénéfice de la radiothérapie mais n'est cependant pas suffisante pour l'élimination complète de la tumeur. En effet, l'accumulation passive d'atomes lourds dans la tumeur n'est pas assez importante et le caractère extracellulaire d'un AC ne maximise pas l'efficacité biologique de l'irradiation. Les nanoparticules (NPs) métalliques apparaissent comme un moyen efficace pour repousser ces limites. Dans le cadre de cette thèse, des études ont été conduites sur la lignée cellulaire de gliome F98 afin de caractériser la toxicité et l'internalisation de trois types de nanoparticules différents : nanoparticules de gadolinium (GdNPs 3 nm), d'or (AuNPs 13 nm) et de platine (PtNPs 6 nm). La survie cellulaire a également été évaluée après différentes conditions d'irradiation de photons monochromatiques en présence de ces nano-objets. La dépendance de la réponse cellulaire à l'énergie du rayonnement incident ainsi qu'à la distribution subcellulaire des NPs a permis de mettre en évidence plusieurs mécanismes mis en jeu dans ce traitement. A concentration identique, les NPs diminuent la survie cellulaire de manière plus importante qu'un AC, validant ainsi l'intérêt microdosimétrique des NPs. L'effet est préférentiel à basse énergie (keV) indiquant que la photoactivation des atomes lourds est en partie responsable la réponse cellulaire. Par ailleurs, les GdNPs et les PtNPs se sont aussi montrées efficaces pour diminuer la survie cellulaire en combinaison à une irradiation à haute énergie (1.25 MeV) indiquant qu'un mécanisme de radiosensibilisation différent de la photoactivation intervient également. Les études précliniques, ont montré que le recouvrement complet de la tumeur par les NPs constitue un point clé pour garantir le bénéfice thérapeutique du traitement. Dans cette optique, une méthode de tomographie à deux énergies développée au synchrotron, a été caractérisée dans le cadre de ce travail. L'étude a permis d'imager de manière quantitative et simultanée la tumeur (mise en évidence par un AC iodé) et son recouvrement par des GdNPs (injectées par voie directe) chez le rongeur porteur d'un gliome. La correspondance entre la distribution de l'AC et la tumeur a également été étudiée à l'aide de techniques d'imagerie à haute résolution (IRM, tomographie X par contraste de phase et histologie). / Gliomas treatment is still a serious challenge in medicine. Available treatments are mainly palliative and patients' survival is increased by a few months only. An original radiotherapy technique consists in increasing the dose delivered to the tumor by loading it with high Z atoms before an irradiation with low energy X-rays (50-100 keV). Preclinical studies have been conducted using iodine contrast agent (CA) (Z=53) and 50 keV X-rays. The increase of the animals' survival leads today to the beginning of clinical trials (phases I and II) at the medical beamline of the European synchrotron, where the available monochromatic and intense photons beam is well suited for this treatment. The use of intravenously injected CA is however insufficient for curing rat's bearing glioma. Indeed, the contrast agent's accumulation is limited by the presence of the BBB and it remains extracellular. Metallic nanoparticles (NPs) appear interesting for improving the treatment efficacy. During this work, three different types of NPs have been studied: GdNPs (3 nm), AuNPs (13 nm) and PtNPs (6 nm). Their toxicity and internalization have been evaluated in vitro on F98 rodent glioma cells. Cells' survival has also been measured after different irradiation conditions in presence of these NPs and with monochromatic photons beams. Several mechanisms implicated in the treatment have been highlighted by the study of the cells' response dependence to the incident particles energy and to the sub cellular NPs distribution during irradiation. For identical concentrations, NPs were more efficient in cells killing than CA, illustrating their microdosimetric potential. The effect was also preferential for low energy X-rays, indicating that photoactivation of heavy atoms plays a role in the cells' death. GdNPs and PtNPs have also lead to an effect in combination to high energy photons (1.25 MeV), indicating that another mechanism may also increase the cell sensitivity to radiations with such NPs. Preclinical trials, performed on rats bearing F98 glioma, have shown that the complete tumor's overlap with NPs is a key point for the success of this treatment. Dual energy computed tomography (CT) has been developed at the synchrotron medical beamline and evaluated during this PhD thesis. The study has allowed quantitatively and simultaneously imaging the tumor (highlighted by iodinated CA) and the GdNPs distribution injected intracerebrally in rodents bearing glioma. The comparison between the CA distribution and the tumor's volume has also been performed using high spatial resolutions imaging methods (MRI, X-rays phase contrast tomography and histology).

Le médulloblastome est la tumeur du système nerveux central la plus répandue et la plus meurtrière chez l’enfant. Malgré de bons taux de survie globale, le traitement du médulloblastome est confronté à deux problèmes cliniques majeurs: les survivants présentent souvent des séquelles graves et irréversibles causées par les traitements, et certains sous-groupes de la maladie répondent mal au traitement. Dans ce travail, nous avons cherché à traiter les deux problèmes en améliorant l'efficacité/tolérance de la radiothérapie dans des modèles précliniques de médulloblastome grâce à l’utilisation d’AsiDNA, un inhibiteur de la réparation de l'ADN déjà testé dans des essais cliniques. Nous avons caractérisé l'effet des molécules AsiDNA sur la réponse à l’irradiation des cellules tumorales, in vitro et in vivo. Nous avons observé qu'AsiDNA peut pénétrer dans les jeunes cerveaux et des tumeurs cérébrales après administration systémique. Aucune preuve d’augmentation de la toxicité liée à l‘irradiation n’a été observée. De plus, l’addition d'AsiDNA aux doses létales de radiations délivrées dans le cerveau murin en développement semble protéger la toxicité radio-induite. Nos résultats démontrent que la combinaison d'AsiDNA avec l’irradiation augmente la survie des animaux greffés avec un modèle de médulloblastome sur le flanc ou dans le cervelet. Les analyses de transcriptome indiquent qu’AsiDNA amplifie les modifications de la transcription induites par les radiations d’une manière similaire à une augmentation de dose. Ses effets sont indépendants de la cellule d'origine ou du statut de TP53. Les données obtenues suggèrent qu’AsiDNA est une drogue qui pourrait potentiellement améliorer la prise en charge des enfants ayant des tumeurs cérébrales et leur qualité de vie. / Medulloblastoma is the most common central nervous system malignancy in pediatric oncology. Despite good overall survival rates, medulloblastoma treatment plans face two major clinical problems: survivors of medulloblastoma often present severe and irreversible sequela caused by the treatments and, certain subgroups of the disease will respond poorly to the treatments. In this work we aimed at tackling both clinical issues by improving radiotherapy efficacy in medulloblastoma preclinical models using a DNA repair inhibitor in clinical trials, AsiDNA. We have characterized the effect of AsiDNA in combination with radiation in vitro and in vivo studies. We have observed that AsiDNA can penetrate young brains and brain tumors through systemic delivery. No evidences of additional radiation-associated brain toxicity were observed. In addition, combination of AsiDNA treatments to lethal doses of radiation delivered in developing murine brain seem to protect from the radiation-induced toxicity. Our results show that the combination of AsiDNA to radiation improves survival of subcutaneous and orthotopic models of medulloblastoma. Transcriptome analysis on medulloblastoma cell lines indicate that AsiDNA intensifies the transcriptional response to radiation resembling the increase in radiation doses. This effect is independent of the cell of origin or TP53 status of the cells. The obtained results indicate that AsiDNA may be a good candidate to improve the efficacy of treatment protocols and quality of life of medulloblastoma patients.

Parmi tous les nanomatériaux carbonés, les nanodiamants de détonation (NDs) possèdent des propriétés physico-chimiques exceptionnelles faisant d’eux un matériau idéal pour les applications en biologie. Aujourd’hui, la production industrielle permet de synthétiser des NDs ayant une taille de 5 nm comportant un cœur diamant et une enveloppe de surface possédant différentes terminaisons. La chimie de surface des NDs peut être modifiée par recuit ou par plasma donnant des NDs négativement ou positivement chargés en suspension dans l’eau. Notre équipe a récemment démontré des propriétés radiosensibilisantes des NDs hydrogénés par plasma (H-NDs) sur des lignées cellulaires cancéreuses radiorésistantes. Ces résultats prouvent leur aptitude thérapeutique comme agents radiosensibilisants. Cependant, les mécanismes impliqués dans cet effet ne sont pas bien compris. L’objectif principal de ce travail de thèse est d’étudier le comportement des NDs en suspension dans l’eau sous irradiation (rayons X et gamma) et de mesurer la production d’espèces réactives de l’oxygène (ROS) en particulier les radicaux hydroxyles HO. Des expériences complémentaires ont permis de détecter la production d’électrons solvatés (eaq). La détection des radicaux HO et des électrons solvatés (eaq) a été réalisée en utilisant une sonde fluorescence, la 7 OH-coumarine, dans des atmosphères différentes (air and N2O/O2). Différentes chimies de surface ont été comparées (oxydée, hydrogénée, graphitisée en surface) préparées à partir de la même source de NDs. En parallèle, les propriétés colloïdales et la stabilité de ces NDs dans l’eau ont été étudiées à court et à long terme en fonction de leur chimie de surface. Une surproduction de radicaux HO a été mesurée pour les H-NDs hydrogénés par les deux méthodes et pour les NDs recuites sous vide à 750°C. De plus, une surproduction d’électrons solvatés a été mise en évidence pour les H-NDs. Ces résultats sont discutés en fonction de la chimie de surface, la stabilité colloïdale et les interactions spécifiques des molécules d’eau avec les NDs. / Among all nanocarbons, detonation nanodiamonds (NDs) possess outstanding chemical and physical properties suitable for bio-applications. Well-controlled mass production provides NDs with a primary size of 5 nm made of a diamond-core and a shell-coating containing various surface terminations. Surface chemistry of NDs can be tuned via thermal or plasma treatments providing either positively or negatively charged NDs in water suspension. Our group recently showed that plasma hydrogenated NDs (H-NDs) behave a radiosensitizing effect on radioresistant cancer cell lines providing potential therapeutic abilities as radiosensitizing agents. Nevertheless, the mechanisms involved behind this effect are not currently well understood. The main goal of this PhD is to study the behaviour of NDs suspended in water under ionizing radiations (X-ray and Gamma) and to investigate the production of reactive oxygen species (ROS), in particular hydroxyl radicals (HO). Additional experiments allow to detect also produced solvated electrons (eaq). The detection of HO radicals and solvated electrons was realized in the presence of a fluorescence probe, the 7 OH-coumarin, under various atmospheres (air and N2O/O2). Starting from the same source of NDs, different surface chemistries were compared (oxidized, hydrogenated and surface graphitized). In parallel, colloidal properties and stability of these modified NDs in water with respect to their surface chemistry were investigated at short and long term. An overproduction of HO was observed for H-NDs for both hydrogenation methods and vacuum annealed NDs at 750°C. In addition, the production of solvated electrons was confirmed for H-NDs. These results were discussed taking into account the surface chemistry, the colloidal stability and specific interactions of water molecules with NDs.

Une approche thérapeutique innovante utilisant l'adjonction d'éléments de numéro atomique élevé à une radiothérapie de basse énergie semble offrir une voie prometteuse pour le traitement des tumeurs cérébrales résistantes. Une telle technique est notamment développée sur la ligne médicale de l'ESRF (European Synchrotron Radiation Facility) utilisant un rayonnement monochromatique allant de 25 à 90 keV [Adam 2003, Adam 2006]. Des résultats encourageants ont été obtenus en traitant des souris cancéreuses après injection de nanoparticules d'or (AuNP) [Hainfeld 2004]. Cependant, les processus physiques et l'impact biologique issus de la photoactivation de nanoparticules sont encore aujourd'hui mal compris et ne peuvent être expliqués par des calculs de doses macroscopiques [Cho 2005, Zhang 2009]. Le but de ce travail est d'évaluer par simulation Monte Carlo l'augmentation locale de dose en présence de nanoparticules ainsi que les caractéristiques des électrons secondaires produits. Dans un premier temps, des simulations ont été réalisées en utilisant une géométrie cellulaire, de manière à comparer les données simulées aux expérimentations menées à l'ESRF. Des tests de clonogénicité ont été réalisés pour mesurer le taux de radiosensibilité des cellules pour une irradiation de 4 Gy (SER4Gy) en présence de gadolinium, pour différentes énergies d'irradiation (25 keV à 1250 keV). Ces études, expérimentales et numériques, ont permis l'évaluation de l'influence de la localisation du gadolinium au sein de la cellule et la forme de ce dernier (nanoparticules ou agent de contraste). D'autre part, une étude comparative a été menée pour caractériser le comportement d'une nanoparticule sous irradiation à une échelle nanométrique, en fonction de l'énergie de faisceau, du rayon de la nanoparticule et de l'élément lourd (or et gadolinium). / An innovative approach using X-ray interactions with heavy elements seems to open a promising way of treatment for resistant cancers, such as high-grade gliomas. Such a technique is developed at the medical beam line of ESRF using monochromatic X-rays in the 25-90 keV range for the treatment of brain tumors [Adam 2003, Adam 2006]. The use of gold nanoparticles (AuNP) to treat mice bearing subcutaneous tumors led to encouraging results [Hainfeld 2004]. However, the physical processes and biological impact of the photon activation of nanoparticles are not yet well understood. The experimental results cannot be explained by macroscopic dose calculations [Cho 2005, Zhang 2009]. The aim of this work was to evaluate, at the sub-cellular level, the dose enhancement in presence of nanoparticles and the properties of the secondary electrons production using Monte Carlo simulations. In a first step, simulations were performed using cell geometry, in order to compare the simulated data to the experiments realized on the ID17 beamline of ESRF. Clonogenic assays have been performed on F98 cells to measure the “Sensitizer Enhancement Ratio” for an irradiation of 4 Gy (SER4Gy) in the presence of gadolinium, for several beam energies (25 to 80 keV). These experimental and numerical studies were done to evaluate the influence of the gadolinium location within the cell and its shape (nanoparticles or contrast agent). On the other hand, a comparative study has been performed to evaluate the behavior of a nanoparticle under irradiation at a nanometer scale. Electron spectra have been studied for two heavy elements - gold and gadolinium - and several beam energies from 25 keV to 2 MeV. Experiments have shown that gadolinium nanoparticles (GdNP) incubated during 5 h with the cells were strongly effective compared to non-incubated nanoparticles and contrast agent, for the same concentration of gadolinium. A part of radiosensitivity could possibly be explained by a biological action of GdNP on the cell cycle. Another part could be attributed to the important dose enhancement factor (DEF) calculated in the vicinity of GdNP, highlighted from two-dimension DEF maps. The DEF can reach two orders of magnitude within a few nanometers of the GdNP surface and is mainly due to high-linear energy transfer electrons (< 5 keV). By modeling the case of nanoparticles randomly distributed on the cell membrane (closest to the experimental case), we showed that a good correlation exists between the SER4Gy and the membrane DEF. On the other hand, the comparison of the two elements showed that GdNP could produce more electrons (of lower energy) than AuNP (with same mass), but that the local DEF due to AuNP was more important. Interesting results were obtained by comparing the local DEF with experimental results on plasmid DNA. However, it seems important to carry on these studies by taking into account the post-irradiation chemical processes in modeling.

Résumé : Il est généralement admis que les cassures double-brin (CDB) de l’ADN sont parmi les lésions les plus toxiques induites par les radiations ionisantes (RI). Les CDBs non ou mal réparées peuvent conduire à une instabilité génomique et à la mort cellulaire. La chimioradiothérapie concomitante est l’une des modalités la plus efficace pour le traitement de certains cancers surtout en stade avancé. Le rendement des CDBs a augmenté quand l’ADN a été irradié en présence de cisplatine avec des électrons de basse énergie (EBEs). Notre étude a pour objectif de réévaluer la contribution des CDBs et d’autres lésions induites par les RI dans la létalité cellulaire. L'effet des RI sur la fonctionnalité de l’ADN plasmidique modifié ou non de façon covalente par le cisplatine a été étudié par mesure de l'efficacité de transformation du plasmide dans E. coli. Les complexes cisplatine-ADN ont été préparés de telle sorte qu’il y avait en moyenne deux adduits de cisplatine par plasmide tel que mesuré par ICP-MS. Nos échantillons ont été irradiés en solution avec des doses croissantes de rayonnements gamma (137Cs). La présence de cisplatine a augmenté la formation des CDBs par un facteur de 2.6 par comparaison avec l'ADN non modifié. Malgré cette augmentation, le rendement des CDBs reste très faible et ne peut pas expliquer la perte de fonctionnalité observée. Alors que, les dommages multiples localisés (LMDS) (non-DSB cluster damage) donnant naissance à des CDBs sous l’action des enzymes de réparation la formamidopyrimidine [fapy]-DNA glycosylase (Fpg) et l’endonuclease III (Nth) où leur rendement a été augmenté d’un facteur de 2.1 lorsque l’ADN a été irradié en présence de cisplatine, ont pu expliquer la perte de fonctionnalité observée. Ces résultats suggèrent que le cisplatine peut agir, non seulement comme un agent chimiothérapeutique, mais aussi comme un radiosensibilisateur efficace par addition d’autres lésions à l’ADN. Aussi, pour la première fois nous avons pu évaluer l’effet des EBEs sur la létalité cellulaire. Des films d'ADN ont été préparés en utilisant la méthode d’adsorption douce sur un substrat de graphite pyrolytique, en présence de 1,3- diaminopropane (Dap[indice supérieur]2+) et ont été irradiées avec des EBEs 10 eV. Nous avons pu conclure, qu’en plus des CSBs, CDBs et des dommages de base, les EBEs sont capables aussi d’induire des LMDS (non-DSB cluster damage) et induire la perte de fonctionnalité de l’ADN. Le rendement des CDBs est très faible d’où ils n’ont pas pu expliquer la perte de fonctionnalité de plasmide observée, après irradiation avec les EBEs. Le rendement très faible des LMDS (non-DSB cluster damage) ne peut pas expliquer la perte de fonctionnalité de l’ADN. Il semble que les EBEs sont capables d’induire des dommages très proches les uns des autres et qui ne peuvent pas être révélés par les enzymes de réparation Fpg et Nth. Plus les dommages sont proches les uns des autres, plus leur réparation est difficile, car une de ces lésions peut inhiber la réparation de l’autre la plus proche. // Abstract : It is generally accepted that DNA double-strand breaks (DSB) are among the most toxic lesions induced by ionizing radiation (IR). Unrepaired or misrepaired DSB can lead to genomic instability and cell death. It is known that concomitant chemoradiation therapy is one of the most preferred methods for the treatment of certain cancers especially in advanced stage. The yield of DSBs was increased when DNA was irradiated with low energy electron (LEEs). The aims of our study was to reassess the contribution of DSBs and other lesions induced by indirect and direct effect of IR in cell lethality. The effect of IR on the DNA functionality of the plasmid modified covalently with cisplatin was studied by measuring the transformation efficiency of the plasmid in E. coli. Cisplatin-DNA complexes were prepared such that there was an average of two cisplatin adducts per plasmid as measured by ICP-MS. Aqueous solutions of the samples were irradiated with 137Cs [gamma]-rays at various doses. Gel electrophoresis analysis shows that cisplatin enhances, by a factor of 2.6, the formation of DSB by [gamma]-rays relative to those in unmodified DNA. Despite this increase, the yield of DSBs is very low and cannot explain the loss of functionality observed after transformation with plasmids modified with cisplatin. While locally multiple damaged sites (LMDS) revealed by repair enzymes Fpg (Formamidopyrimidine [fapy]-DNA glycosylase) and Nth (Endonuclease III) as DSB (nonDSB cluster damage), where their yield was increased by a factor of 2.1 when DNA was irradiated in the presence of cisplatin were able to explain the observed loss of DNA functionality. These results suggest that cisplatin may act not only as a chemotherapeutic agent, but also as an effective radiosensitizer by addition of other DNA lesions. For the first time, we could also evaluate the effect of low energy electrons (LEEs) on DNA functionality. Highly ordered DNA films were prepared on pyrolytic graphite by molecular self-assembly using 1,3-diaminopropane ions (Dap[superscript]2+) to bind together the plasmids and irradiated with LEE (10 eV). We concluded that in addition to CSBs, DSBs and base damage, LEEs induced the formation of non-DSB cluster damage and also induced the loss of DNA functionality under LEE irradiation. The yields of DSBs and of non-DSB cluster damage are too low and so one unable to explain the loss of DNA functionality. It seems that LEEs are able to induce a high complex damage that cannot be revealed by repair enzymes Fpg and Nth. The high complex damage is difficult to repair possibly because the repair of one lesion, may inhibit the repair of another.