Les nanoparticules (NPs) hybrides peuvent combiner les propriétés physiques uniques des éléments inorganiques pour des applications en imagerie et en thérapeutique avec la biocompatibilité des structures organiques. Cependant, leur utilisation en médecine est encore limitée par des risques potentiels de toxicité à long terme. Dans ce contexte, des NPs hybrides ultrafines pouvant être éliminées rapidement par la voie rénale apparaissent comme de bonnes candidates pour la nanomédicine. La NP à base de silice contenant des chélates du gadolinium appelée AGuIX (Activation et Guidage de l’Irradiation par rayon-X) a été développée avec un diamètre hydrodynamique de moins de 5 nm qui lui permet d’être éliminée rapidement via l’urine après injection intraveineuse. Cette NP s’est révélée être une sonde efficace en imagerie multimodale et un amplificateur local en radiothérapie pour le diagnostic et le traitement du cancer. Elle est en train d’être évaluée dans un essai clinique de phase I par radiothérapie des métastases cérébrales (NANO-RAD, NCT02820454). Néanmoins, la synthèse d’AGuIX est un procédé multi-étapes qui est difficilement modulable.Ce manuscrit rapporte, pour la première fois, le développement d’un protocole « one-pot » direct pour des nanoparticules de silice ultrafines (USNP) contenant des chélateurs complexés ou non à partir des précurseurs silanes chélatants moléculaires. Dans ce nouveau protocole, la taille des particules et les types des métaux chélatés peuvent être contrôlés facilement. Certaines des propriétés chimiques des USNP ont été clarifiées davantage pendant ce travail exploratoire. Les particules élaborées ont été caractérisées par différentes techniques analytiques complémentaires. Ces nouvelles nanoparticules USNPs présentent des caractéristiques similaires aux AGuIX en terms de propriétes biologiques et de biodistribution.Dans un second temps, un nouveau protocole de fonctionnalisation d’USNP par des précurseurs silanes chélatants a été développé. Ces chélatants libres fonctionnalisés sur la particule peuvent être alors utilisés afin de complexer des radiométaux pour l’imagerie bimodale. Enfin, d’autres stratégies de fonctionnalisation sont aussi décrites. La nouvelle sonde (17VTh031) combinant un petit chélateur cyclique (NODA) et un fluorophore proche-infrarouge tumeur ciblant (IR783) ainsi que le pyridinium quaternaire ont été greffés sur l’AGuIX pour créer une nouvelle sonde en imagerie multimodale et cibler des tumeurs chondrosarcomes respectivement / Hybrid nanoparticles (NPs) can combine unique physical properties for imaging and therapeutic applications of inorganic elements in bio-friendly organic structures. However, their uses in medicine are limited by the potential risks of long-term toxicities. In this context, ultrasmall renal clearable NPs appear as novel solutions. Silica based NP displaying gadolinium chelates named AGuIX (Activation and Guidance for Irradiation by X-ray) has been developed to have hydrodynamic diameter less than 5 nm which allows rapid elimination through urine after intravenous injection. This NP has been demonstrated as an efficient multimodal imaging probe and a local enhancer for radiotherapy for cancer diagnostics and treatment. It is now being evaluated in a phase I clinical trial by radiotherapy of cerebral metastases (NANO-RAD NCT02820454). Nevertheless, the synthesis of AGuIX implies a multisteps process that can be further improved.This manuscript shows, for the first time, the development of a straightforward one-pot protocol for ultrasmall silica nanoparticles (USNP) containing complexed or non-complexed chelators from molecular chelating silane precursors. In this new protocol, the size of particle and types of metals can be easily tuned. The chemical properties of USNP have been further clarified during this exploratory work. The produced particles have been characterized by different complimentary analytical techniques. These new nanoparticles USNPs show similar characteristics to AGuIX in terms of biological properties and biodistribution.Secondly, a new protocol of functionalization for USNP by chelating silane precursors has been developed. These functionalized free chelators on the particle can be used then to complex radiometals for bimodal imaging applications. Finally, other functionalization strategies have also been described. New probe (17VTh031) combining small cyclic chelator (NODA) and tumor targeting near-infrared fluorophore (IR783) as well as quaternary pyridinium have been grafted on AGuIX for creating new multimodal imaging probe and targeting chondrosarcoma tumors respectively

Two classes of C5 azido-modified pyrimidine nucleosides were synthesized and explored as radiosensitizers. The 5-azidomethyl-2'-deoxyuridine (AmdU) was prepared from thymidine and converted to its cytosine counterpart (AmdC). The 5-(1-azidovinyl) modified 2'-deoxyuridine (AvdU) and 2'-deoxycytidine (AvdC) were prepared employing regioselective Ag-catalyzed hydroazidation of 5-ethynyl pyrimidine substrates with TMSN3. AmdU and AmdC were converted to 5'-triphosphates AmdUTP and AmdCTP, and incorporated into DNA-fragments via polymerase-catalyzed reaction during DNA replication and base excision repair. Radiation-mediated prehydrated electrons formed in homogeneous aqueous glassy (7.5 M LiCl) systems in the absence of oxygen at 77 K led to site-specific formation of π-type aminyl radicals (RNH•) from AmdU, AmdC, AvdU, and AvdC. The ESR spectral studies and DFT calculations showed RNH• undergo facile conversion to thermodynamically more stable σ-type iminyl radicals, R=N•. For AmdU, conversion of RNH• to R=N• was bimolecular involving α-azidoalkyl radical as intermediate; however, for AvdU, RNH• tautomerized to R=N•. Our work provides the first evidence for the formation of RNH• attached to C5 position of azidopyrimidine nucleoside and its facile conversion to R=N• under reductive environment. These aminyl and iminyl radicals can generate DNA damage via oxidative pathways. The azido-nucleosides were successfully applied as radiosensitizers in EMT6 cancer cells in both hypoxic and normoxic conditions. To explore the generation and reactivity of 2'‑deoxyguanosin-N2-yl radical (dG(N2-H)•) postulated to generate from guanine moiety towards •OH, 2-azido-2'-deoxyinosine (2-N3dI) was prepared by conversion of 2-amino group in protected dG into 2-azido via diazotization with tert-butyl nitrite followed by displacement with azide and deprotection. The investigation of dG(N2-H)• generated from 2-N3dI and its subsequent reactions using ESR will be discussed. Cycloaddition between 5-ethynylpyrimidine or 8-ethynylpurine nucleosides and TMSN3 in the presence of Ag2CO3, CuI, or CuSO4/sodium ascorbate provided N-unsubstituted 1,2,3-triazol-4-yl analogues of the parental DNA bases (i.e. 5-TrzdU, 5‑TrzdC, 8-TrzdA, and 8-TrzdG). These novel triazolyl nucleosides showed excellent fluorescent properties: 8-TrzdA exhibits the highest quantum yield (ΦF) of 44% while 8‑TrzdG had ΦF of 9%. The 5-TrzdU and 5-TrzdC showed a large Stokes shift of ~110 nm. The application of these fluorescent nucleosides to cell imaging and DNA modifications will also be discussed.

The overexpression of the human epidermal growth factor receptor-2 (HER-2) in 20-25% of human breast cancers was investigated as a target for development of a gold nanoparticle (AuNP) based radiosensitizer for improving the efficacy of neoadjuvant X-radiation therapy of the disease. HER-2 targeted AuNPs were developed by covalently conjugating trastuzumab, a Health Canada approved monoclonal antibody for the treatment of HER-2-overexpressing breast cancer, to 30 nm AuNPs. Trastuzumab conjugated AuNPs were efficiently internalized by HER-2-overexpressing breast cancer cells (as assessed by darkfield microscopy and transmission electron microscopy) and increased DNA damage from X-radiation in these cells by more than 5-fold. To optimize delivery of AuNPs to HER-2-overexpressing tumors, high resolution microSPECT/CT imaging was used to track the in vivo fate of 111In-labelled non-targeted and HER-2 targeted AuNPs following intravenous (i.v.) or intratumoral (i.t.) injection. For i.v. injection, the effects of GdCl3 (for deactivation of macrophages) and non-specific (anti-CD20) antibody rituximab (for blocking of Fc mediated liver and spleen uptake) were studied. It was found that HER-2 targeting via attachment of trastuzumab paradoxically decreased tumor uptake as a result of faster elimination of the targeted AuNPs from the blood while improving internalization in HER-2-positive tumor cells as compared to non-targeted AuNPs. This phenomenon could be attributed to Fc-mediated recognition and subsequent sequestration of trastuzumab conjugated AuNP by the reticuloendothelial system (RES). Blocking of the RES did not increase tumor uptake of either HER-2 targeted or non-targeted AuNPs. Following i.t. injection, our results suggest that Au-NTs redistribute over time and traffick to the liver via the ipsilateral axillary lymph node leading to comparable exposure as seen with i.v. administration. In contrast, targeted AuNPs are bound and internalized by HER-2-overexpressing tumor cells following i.t. injection, with a lower proportion of AuNPs redistributing to normal tissues. In vivo, the combination of HER-2 targeted AuNPs injected i.t. and X-radiation (11 Gy) yielded a 46% decrease in tumor size over a 4 month period in contrast to an 11.5% increase in tumor size for X-radiation treatment alone. Toxicology studies (evaluated through complete blood cell counts, by serum transaminase and creatinine measurements and by monitoring the body weight) demonstrated no apparent normal organ toxicity from the combination of HER-2 targeted AuNPs and X-radiation. These results are promising for the clinical translation of HER-2-targeted AuNPs for radiosensitization of tumors to X-radiation.

The overexpression of the human epidermal growth factor receptor-2 (HER-2) in 20-25% of human breast cancers was investigated as a target for development of a gold nanoparticle (AuNP) based radiosensitizer for improving the efficacy of neoadjuvant X-radiation therapy of the disease. HER-2 targeted AuNPs were developed by covalently conjugating trastuzumab, a Health Canada approved monoclonal antibody for the treatment of HER-2-overexpressing breast cancer, to 30 nm AuNPs. Trastuzumab conjugated AuNPs were efficiently internalized by HER-2-overexpressing breast cancer cells (as assessed by darkfield microscopy and transmission electron microscopy) and increased DNA damage from X-radiation in these cells by more than 5-fold. To optimize delivery of AuNPs to HER-2-overexpressing tumors, high resolution microSPECT/CT imaging was used to track the in vivo fate of 111In-labelled non-targeted and HER-2 targeted AuNPs following intravenous (i.v.) or intratumoral (i.t.) injection. For i.v. injection, the effects of GdCl3 (for deactivation of macrophages) and non-specific (anti-CD20) antibody rituximab (for blocking of Fc mediated liver and spleen uptake) were studied. It was found that HER-2 targeting via attachment of trastuzumab paradoxically decreased tumor uptake as a result of faster elimination of the targeted AuNPs from the blood while improving internalization in HER-2-positive tumor cells as compared to non-targeted AuNPs. This phenomenon could be attributed to Fc-mediated recognition and subsequent sequestration of trastuzumab conjugated AuNP by the reticuloendothelial system (RES). Blocking of the RES did not increase tumor uptake of either HER-2 targeted or non-targeted AuNPs. Following i.t. injection, our results suggest that Au-NTs redistribute over time and traffick to the liver via the ipsilateral axillary lymph node leading to comparable exposure as seen with i.v. administration. In contrast, targeted AuNPs are bound and internalized by HER-2-overexpressing tumor cells following i.t. injection, with a lower proportion of AuNPs redistributing to normal tissues. In vivo, the combination of HER-2 targeted AuNPs injected i.t. and X-radiation (11 Gy) yielded a 46% decrease in tumor size over a 4 month period in contrast to an 11.5% increase in tumor size for X-radiation treatment alone. Toxicology studies (evaluated through complete blood cell counts, by serum transaminase and creatinine measurements and by monitoring the body weight) demonstrated no apparent normal organ toxicity from the combination of HER-2 targeted AuNPs and X-radiation. These results are promising for the clinical translation of HER-2-targeted AuNPs for radiosensitization of tumors to X-radiation.

La radiothérapie est souvent utilisée pour contrôler la progression d'un cancer. Cependant, la mauvaise spécificité de ciblage de la plupart des techniques de radiothérapie peut entraîner une réponse clinique ambiguë. Une stratégie alternative - et complémentaire - est d'utiliser des matériaux possédant un numéro atomique élevé et qui peuvent ainsi agir en synergie avec les rayonnements ionisants pour améliorer le ratio thérapeutique de la radiothérapie. Dans ce contexte, une nanoparticule (NP) théranostique à base de gadolinium (Gd) est particulièrement adaptée pour fournir simultanément une plus grande précision en Imagerie par Résonance Magnétique (IRM) et une meilleure efficacité en radiothérapie clinique. Au cours de cette thèse, nous avons étudié d'un point de vue préclinique la pharmacocinétique et le métabolisme de ces NP chez des rongeurs et des primates non humains afin d'élucider leurs voies d'élimination et de calculer la dose sans effet nocif observé (NOAEL). De plus, nous avons démontré la capacité d'imagerie et de thérapie de ces particules sur un modèle de souris porteuses de mélanome cérébral, ceci afin d'appuyer le potentiel des NP pour la radiothérapie guidée par IRM en clinique. Ces travaux de thèse - ainsi que des résultats précédents - ont contribué au début d'un essai clinique actuellement en cours / Combinations of chemotherapy and radiotherapy are often used to control cancer progression. However, the poor targeting specificity of most chemotherapies and radiotherapies can cause toxicity and ambiguous clinical response. In particular, dose escalation in radiotherapy inevitably increases radiation exposure for some surroundings normal tissues and organs, putting them at risk for debilitating damage. An alternative – and complementary – strategy is the use of materials with high atomic numbers (Z) that strongly interact with low energy photons to produce photoelectrons and Auger electrons In this context, a new efficient type of gadolinium (Gd)-based theranostic agent (AGuIX®) has recently been developed by the team of Prof. Tillement for MRI-guided radiotherapy. AGuIX® is a 3-nm size nanoparticles of 9 kDa, consist of a polysiloxane network surrounded by Gd chelates. In this thesis, we investigated the elimination kinetics of AGuIX nanoparticle’s (NPs) from sub-cellular to whole organ scale using original and complementary techniques. This combination of techniques allows the exact mechanism of AGuIX NPs elimination to be elucidated. We reported the preclinical pharmacokinetics and toxicology studies of intravenous AGuIX NPs administration in healthy and atherosclerosis non-human primates (NHP), the goal of which is to demonstrate the safety of AGuIX NPs, in particular, for pre-clinical evaluation. Subsequently, we performed experimental and theoretical studies to investigate the radiosensitization of AGuIX NPs, in particular with B16F10 mouse melanoma as a model for brain metastases. After, we implemented experimental and theoretical studies to precisely understand the mechanism of this radiosensitization, we suggest additional mechanism, potentially caused by chemical and biological effects induced by the combination of Gd and radiation (i.e. high yield of radicals formation and combination, and bystander effect)

Au moins 60% des patients atteints de cancer vont recevoir de la radiothérapie (RT). L’efficacité de la radiothérapie dans le traitement du cancer est limitée par le phénomène de radiorésistance des cellules cancéreuses et par la toxicité des radiations sur les tissus sains. La découverte de nouveaux agents radiosensibilisants et radioprotecteurs permettrait de surmonter ces difficultés. Les modèles cellulaires 3D tels que les sphéroïdes, émergent motivés par le besoin de modèles précliniques plus proches des tumeurs in vivo. L’objectif du projet est d’optimiser un système microfluidique pour tester facilement et à faible coût des agents thérapeutiques avec la RT sur des sphéroïdes dans un contexte de repositionnement. Le système microfluidique développé permet la formation de 336 sphéroïdes homogènes en deux jours avec intervention minimale de l’utilisateur. Les sphéroïdes sont répartis dans 16 chambres de culture séparées par un système de valve magnétique pour éviter des effets bystander entre sphéroïdes irradiés et non-irradiés. Une nouvelle technique d’irradiation a été développée permettant d’exposer un système à 4 doses de radiation différentes. En tout, 4 doses de radiation et 4 concentrations d’agents thérapeutiques peuvent être testées par système. En utilisant cette approche, l’efficacité de trois agents avec la RT a été évaluée avec des tests de survie clonogénique. Nous avons démontré que le Talazoparib, un inhibiteur de PARP, radiosensibilise les cellules de sarcome de tissus mous (STS) cultivés en sphéroïdes à 2 Gy. Le système développé permet d’évaluer le potentiel d’agents thérapeutiques avec la RT et contribue à l’adoption des sphéroïdes comme modèle préclinique. / At least 60% of cancer patients will receive radiotherapy (RT) as part of their treatment. The efficacy of radiotherapy in the treatment of cancer is limited by the phenomenon of radioresistance of cancer cells and by the toxicity of radiation on healthy tissues. The discovery of new radiosensitizers and radioprotectors is essential to overcome these challenges. 3D cellular models such as spheroids emerge motivated by the need for better preclinical models. The objective of the project was to optimize a microfluidic system for easy, fast and low-cost testing of therapeutic agents with RT on spheroids. The developed microfluidic system allows the formation of 336 homogeneous spheroids in two days with minimal user intervention. The spheroids are distributed in 16 culture chambers separated by a magnetic valve system to avoid bystander effects between irradiated and unirradiated spheroids. A new irradiation technique has been developed to expose a system with 4 different radiation doses. In total, 4 radiation doses and 4 concentrations of therapeutic agents can be tested per system. Using this approach, the efficacy of three agents with RT was evaluated with clonogenic assays. Radiosensitizing properties of Talazoparib, a PARP inhibitor, on soft tissue sarcoma (STS) cells cultured as spheroids at 2 Gy were demonstrated. The developed system enables the evaluation of therapeutic agents with RT and contributes to the wide adoption of spheroids as a preclinical model.