Contrôle spatio-temporel de la formation de NO et imagerie tumorale par excitation biphotonique de nanodéclencheurs – TRIGNOSTUMOR

Tumor vasculature brings nutrients to the tumor and also constitutes the main entry path for chemotherapy. Tumor vasculature usually fails to meet the demands of the tumor cells for oxygen and nutrients and this failure results in tumor blood flow heterogeneity often inducing intermittent hypoxia, and acidic conditions within the tumor. This limits penetration and efficacy of chemotherapy. This project applies a new tool to control NO-dependent vasodilation in tumor blood vasculature in precise spatial and temporal way. The localized increase in blood flow will be achieved just before the treatment with chemotherapy. This 'adjuvant' strategy should enhance the efficiency of the chemotherapeutics at lower doses, with minimal risks of angiogenesis and metastasis. In mammals, nitric oxide (NO) is a free radical which has important functions in the cardiovascular, nervous and immune systems. NO is endogenously produced by the NO-synthases, NOS. The observed biological activities of NO can be parted along two different registers. On one hand, NOSs are involved in signalling processes which relies on the ability of NO to activate its main target, soluble guanylate cyclase. On the other hand, NOSs are involved in the generation of Reactive Oxygen and Nitrogen (RONS) species commonly associated with the development of several pathologies such as neurodegenerative diseases, diabetes or cancers. These roles for NO can be tuned at different levels involving redox environment, phosphorylation cascades, NOSs/proteins interactions, availability in substrate and cofactors. In this project, we take advantage of the vasodilation ability of NO produced by the endothelial NO-synthase (eNOS) of the tumor endothelium. NO formation by eNOS will be switch on with a laser pulse using novel tools. These molecular tools, called nanotriggers NT, initiate the enzymatic catalysis of the NO-synthase in a synchronous manner with a laser pulse by biphotonic absorption. The nanotrigger molecular tools combine a 'docking' subunit responsible for the recognition of NADPH sites within NOS and a 'chromophoric' subunit responsive to biphotonic excitation and able to transfer electrons to the flavin moieties. By designing nanotriggers with two-photon absorption cross-sections much larger than endogeneous chromophores (such as flavins, NADPH….), selective and synchronous initiation of the enzymatic processes is possible by using short pulsed biphotonic excitation. Turning on with light the endothelial NO-synthase will increase the blood flow (via NO signaling) within the tumor just before treatment with chemotherapy. Because switching off the light turns off NO production, vasodilation of the vessels will be controlled in a short time window and spatially confined to the tumor. Therefore, this new approach should avoid angiogenesis and tumor metastasis. Moreover, the presence of NT in the vessel endothelium can be monitored in situ by means of its fluorescence. Thus, the nanotrigger molecular tools will also be used for imaging by two-photon microscopy in cells and in tumor tissues. When combined with fluorescent markers of the vasculature, the NT tools provide a way to monitor the adjuvant pre-treatment and the chemotherapy therapeutics in the tumor vasculature. The nanotriggers would thus provide innovative tools for both selective and non-damaging activation and synchronization of ensemble of native eNOS proteins in tumors. The reactivity of the nanotriggers will be tuned and optimized by molecular modeling and chemical synthesis. Their activity/ selectivity tested in vitro using purified enzymes, at the cellular level and in tumors carried out by nude mice. The proposed Nanotrigger technology should be useful as an adjuvant strategy improving current chemotherapies.