Les nanomédecines injectées par voie intraveineuse interagissent avec les éléments biologiques qui les entourent dans le compartiment sanguin. Parmi ces interactions, celles avec les protéines sanguines se révèlent être très importantes dans le devenir de ces nanovecteurs, leur conférant une identité biologique influençant leur chemin jusqu’au tissu et aux cellules cibles. La compréhension et le contrôle de ces phénomènes reste un enjeu crucial dans le développement des nanomédecines. Des méthodes permettant une étude facilitée de ces interactions sont nécessaires à cet égard. Les travaux de cette thèse ont eu pour but de développer des méthodes, utilisables en routine, permettant une caractérisation fine des nanomédecines et de leurs interactions avec les protéines plasmatiques, applicables dans un contexte clinique. Ils s’inscrivent dans un projet intitulé « Nano Innovation for CancEr » (NICE, BPI France) regroupant un consortium de partenaires industriels en développement clinique de nanomédecines.Dans un premier temps, un travail bibliographique sur les méthodes actuellement mises en œuvre pour une telle caractérisation ont pu mettre en avant deux limitations majeures. (i) D’une part, la complexité des méthodes actuelles disponibles pour lesquelles la spécificité des équipements et l’expertise requise limitent une utilisation à large échelle. (ii) D’autre part, les propriétés aujourd’hui caractérisées en routine (taille, morphologie globale, charge) ne sont que grossières comparées à la finesse des processus biologiques qui interagissent et « analysent » les nanovecteurs une fois introduits dans le milieu biologique. Ces deux aspects limitent aujourd’hui un développement plus sûr des nanomédecines pour une bonne reproductibilité en clinique et garantir des essais de contrôle qualité fiables.Au cours de nos travaux, nous avons développé des méthodes permettant de répondre en partie à la problématique posée par la caractérisation des nanomédecines. Une méthode d’analyse à haut débit de l’activation du système du complément par immunoélectrophorèse en deux dimensions a été développée et validée. Elle permet l’analyse reproductible de l’activation de la protéine C3. Elle est applicable à l’étude de l’effet de la présence de nanoparticules dans le sérum humain et leur degré d’action sur la cascade du complément. Cette méthode a été utilisée pour mener une étude plus fondamentale du mécanisme de l’activation du système du complément en regard de l’architecture de la surface de nanoparticules.Une deuxième méthode d’étude de l’activation du complément produit par des nanomédecine a été proposée sur la base de la résonnance plasmonique de surface (SPR). Une puce permettant un screening automatisé de l’activation du complément a été développée. L’application de cette méthode comparée à d’autres méthodes d’études de l’activation du système du complément (Immunoélectrophorèse 2D, ELISA) a permis d’identifier des biais lors de leur application à l’évaluation des nanomédecines.Enfin, une approche originale de caractérisation de la surface de nanoparticules a été proposée utilisant des protéines pour sonder la capacité de la surface des nanoparticules à adsorber ou repousser ces dernières. Dans cette méthode, l’électrophorèse capillaire est utilisée comme outil analytique permettant une analyse directe de l’échantillon sans séparation préalable des nanomédecines.Les méthodes développées au cours de ces travaux peuvent être appliquées à la caractérisation de nanomédecines et proposées comme des méthodes de contrôle en routine de façon plus générale. Un développement de la caractérisation dans ce sens constitue l’un des leviers pour une translation plus fructueuse des nanomédecines entrant en phase clinique. / Nanomedicines injected intravenously interact with surrounding biological elements in the bloodstream. Among these interactions, those with blood proteins turn out to be very important regarding the becoming of the nanovectors. They acquire a biological identity upon interaction with proteins which influence their path to target tissue and cells. The understanding and mastering of these phenomena remains a crucial issue in nanomedicine development. Methods allowing an easier study of these interactions are needed. The aim of these PhD thesis was to develop such methods, usable on a routine basis in a clinical context, allowing a fine characterization of nanomedicines and their interactions with plasmatic proteins. This PhD is part of the project “Nano Innovation for CancEr” (NICE, BPI France), gathering a consortium of industrials partners developing clinical nanomedicines.In a first time, a bibliographic study about current methods used for such a characterization could identify two major limitations. (i) On one hand, the complexity of current available methods for which the equipment specificity and required expertise prevent their use at a large scale. (ii) On the other hand, properties today characterized on a daily basis (size, morphology, charge) are too rough compared to the sharpness of biological processes who interact and “analyze” the nanovectors introduced in biological media. These two aspects are limiting a safer development of nanomedicines as well as a good reproducibility of their action in clinics.During this thesis, we developed methods allowing a beginning of answer to the wide problematic of nanomedicine characterization. A method for a high throughput analysis of complement activation by nanomedicines via 2D immunoelectrophoresis was developed and validated. It allows the reproducible analysis of protein C3 fragmentation. This method is applicable to the study of the impact of nanoparticles in human serum and their degree of action on the complement cascade. This method has been used for a more fundamental study on complement activation pathways activated according to the architecture of nanoparticles surface.A second method for the study of complement activation produced by nanoparticles has been proposed using surface plasmon resonance (SPR). A chip allowing an automated screening of complement activation has been developed. This method was compared to other methods for complement activation study (2D immunoelectrophoresis, ELISA) and allowed the identification of bias during nanomedicine evaluation.Finally, an original approach for the characterization of nanomedicine’s surface architecture using proteins as molecular probes has been proposed. In this method, capillary electrophoresis has been used as analytical tool to allow a direct analysis of sample without preliminary nanoparticle removal step.Methods developed during this work can be applied to the characterization of nanomedicines and proposed as routine methods for quality control. A development of nanomedicines characterization in this direction constitute one of the lever for a more fruitful translation of nanomedicines entering in clinical phase.

<p>Approximately 50% of all cancer patients undergo radiotherapy (RT) as part of their treatment regimen. However, the development of genetic mutations can severely impair cell death arising from radiation-induced DNA damage, leading to cancer recurrence and poor disease prognosis after treatment. Photodynamic therapy (PDT) offers an alternative approach to induce localized cancer cell death by damaging the cell and organelle membranes instead of relying on DNA damage. Yet, its clinical application is typically limited to surface-level lesions due to the poor tissue-penetration properties of visible light photons, which are required as an activation source.</p><p>Herein, we report the usage of calcium tungstate nanoparticles (CWO NPs) as energy transducers for potentiating PDT using X-ray photons from RT as the activation source. CWO NPs undergo “radioluminescence” wherein they can absorb incident high energy X-ray photons and emit lower energy UV-A and blue photons. Therefore, by intratumorally administering NPs, visible light photons can be generated <i>in situ</i> during RT. Since X-ray photons can penetrate tissue more efficiently than visible light photons, this strategy addresses the limitations of both RT and PDT.</p><p>Firstly, we demonstrate the compatibility of CWO NPs as energy transducers for activating two different photosensitizers: bilirubin (BR) and protoporphyrin IX (PPIX). In the case of bilirubin, we conjugated it with poly(ethylene glycol) (PEG) to form amphiphilic chains that self-assembled to encapsulate CWO NPs. For PPIX, CWO NPs were formulated by encapsulating them with poly(ethylene glycol-b-D,L-lactic acid) block copolymer (PEG-PLA/CWO NPs), while systemically delivering PPIX through its hydrophilic prodrug 5-aminolevulinic acid (ALA). In both scenarios, mechanistic studies revealed that X-ray irradiated CWO NPs generated sufficient blue light photons <i>in situ </i>to activate photosensitizers. This yielded significant improvement in cell-killing effects compared to RT alone, as demonstrated by clonogenic assays conducted in radio-resistant 4T1 and HN31 cell lines. The inherent non-toxicity of both formulations was also demonstrated through MTT assays. <i>In vivo</i> efficacy studies using intratumorally administered NPs demonstrated a significant improvement in tumor growth control and mouse survival compared to conventional RT treatments.</p><p>These studies highlight the potential of RT-PDT in achieving enhanced local tumor control. However, a notable limitation of this approach is its inability to effectively treat metastatic lesions. To address this challenge, recent research has explored the combination of RT-PDT with immune checkpoint inhibition, particularly targeting indoleamine-2,3-dioxygenase (IDO) to induce systemic abscopal responses. To investigate this idea, we conducted efficacy studies in mice upon simultaneous treatment with Epacadostat, a small molecule IDO inhibitor. Although some improvement in tumor control and survival was observed across two separate studies, these results did not reach statistical significance. Consequently, further optimization of treatment schedules and immune checkpoint inhibitor delivery is necessary to obtain a more conclusive understanding of the compatibility of these treatment modalities.</p><p>Next, computed tomography (CT) imaging studies revealed that the current formulation of PEG-PLA/CWO NPs exhibits limited spreading in collagen-dense tumors like 4T1 when administered intratumorally. To overcome this, a modified formulation was developed by surface-functionalization with collagenase (Col-PEG-PLA/CWO NPs) to degrade collagen within tumors. The results suggest approximately 2.4× improvement in intratumoral spreading volume relative to non-functionalized NPs. In the context of RT-PDT, this could imply significantly improved illumination of the tumor volume.</p><p>Lastly, one limitation of the current platform design is the requirement of intratumoral administration to deliver NPs. When administered systemically, less than 1% of NPs passively accumulate in the tumor. To address this, NPs were loaded into chimeric antigen receptor-functionalized neutrophils (CAR-neutrophils) differentiated from human pluripotent stem cells. Specifically, the receptors were modified with chlorotoxin peptide which is capable of selectively targeting glioblastomas. The results presented in this study demonstrate the optimal conditions for uptake of NPs by CAR-neutrophils. Furthermore, purification steps to separate NP-loaded CAR-neutrophils from unloaded NPs are described.</p><p>In summary, these studies describe the development and biological evaluation of two distinct NP platforms for RT-PDT. However, a few key limitations currently hinder the clinical translation of these technologies, including the inability to treat metastases, poor intratumoral spreading, and the need for intratumoral injections. Preliminary solutions have been identified for each of these challenges, providing a foundation for future investigations.</p>

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In recent years, the field of nanomedicine has provided promising alternatives to traditional ways of gene and drug delivery, such as nanobubbles instead of viral vectors. Manufacturing these nanomaterials is cost-effective and tends to be safer than previous forms of treatment for clinical use. In the realm of osteoporosis, a disease based around a higher rate of bone resorption to bone formation, leads to weak and brittle bones, leaving sufferers at risk for fractures, which are sudden and unexpected. These fractures are estimated to cost USD 22 Billion annually in the US alone. Reports show a significant effect on the quality of life after these fractures. Osteoclast-inhibiting oral medicines have been used to slow down bone resorption, but they have had severe side effects. This project aimed to study the impact of nanobubbles synthesized to target bone and filled with CTSK-siRNA to silence the Cathepsin K gene in an ovariectomized mice model. Cathepsin K is pivotal in osteoporosis as it is responsible for collagen degradation in bone resorption. Low-intensity pulsed ultrasound (LIPUS) for nanobubble destruction to facilitate targeted CTSK-siRNA release. LIPUS can also promote bone formation. We studied the effects of the treatment through histological analysis. Microarchitecture in the mice femur was highlighted through different types of staining such as Masson’s Trichrome, Hematoxylin & Eosin, and tartrate-resistant acid phosphatase staining. ImageJ was used for all measurements taken to interpret bone resorption in experimental groups. The major organs were studied to ensure the biocompatibility of nanobubble treatment. Our results show an increase in the average cortical thickness and trabecular spacing, but there was no significant increase in trabecular thickness in the group treated with CTSK-siRNA nanobubbles.

<p dir="ltr">Nucleic acids are promising drug candidates as they can address diseases with few “druggable” targets. Nevertheless, nucleic acids are challenging to deliver because of their large molecular weights, dense negative charges, proinflammatory activities, and short half-lives in biological fluids. Synthetic gene carriers based on cationic polymers or lipids have been used to overcome these challenges; however, their cationic nature results in dose-limiting toxicities and accelerated removal by the filtering MPS organs after systemic administration. In the past six years, several nucleic acid-based therapeutics have been approved by the FDA, formulated as lipid nanoparticles (LNPs). Nonetheless, LNPs show extensive liver accumulation after intravenous administration and, hence, are only indicated for hepatic or local vaccine delivery applications. Therefore, there is a critical unmet need for a nanocarrier that delivers nucleic acids to the extrahepatic organs without significant toxicities. To address this need, we developed Nanosac, a deformable and non-cationic nanocarrier, to deliver siRNA to solid tumors. Deformability can improve multiple aspects of the nanoparticle biotransport, ranging from circulation time and protein corona composition to biodistribution and interactions with the target cells. Meanwhile, a non-cationic carrier avoids proinflammatory complications and rapid clearance of cationic nanoparticles. For this application, we used siRNAs targeting CD47/SIRPa and PD-l/PD-L1 immune checkpoints due to their critical roles as “don't-eat-me” and “don't-find-me” signals to immune cells, respectively, which interfere with the development of innate and adaptive antitumor immune responses.</p><p dir="ltr">In the same context of enhancing the tumor delivery of nanomedicine, we developed two formulations for the small-molecule chemo drug, carfilzomib (CFZ). A nanocrystal formulation with optimized particle size had high CFZ loading, adequate colloidal stability in circulation and better antitumor activity in mice than the FDA-approved CFZ formulation. Despite its improved efficacy, the stiff nanocrystals aggravated CFZ immunotoxicity due to its excessive accumulation in mice spleens. To address this issue, we employed Nanosac technology for CFZ delivery, exploiting its deformability to reduce the non-specific spleen distribution and enhance CFZ tolerability.</p><p dir="ltr">Our results showed that Nanosac delivered siRNA to tumor cells and silenced the target protein expression better than LNPs. <i>In vivo</i>, Nanosac reduced siRNA accumulation in the MPS organs and achieved greater siRNA-mediated tumor suppression than LNPs in two murine tumor models. Moreover, Nanosac achieved greater checkpoint protein silencing in tumors, but less silencing in the MPS organs than LNPs, highlighting their differential biodistribution. The superior Nanosac performance relative to LNPs after systemic delivery is likely due to the difference in their protein coronas and cellular delivery capabilities. In addition, CFZ loading in Nanosac ameliorated CFZ immune cell toxicity <i>in vitro</i> and improved its tolerability in mice while maintaining similar therapeutic efficacy compared to the stiff nanocrystal formulation. Collectively, these findings highlight nanocarrier deformability and corona composition as viable strategies to improve the extrahepatic delivery of nucleic acids as well as to minimize toxicities related to extensive NP distribution to the off-target MPS organs.</p>

Iron oxide nanoparticles have received sustained interest for biomedical applications as synthetic approaches are continually developed for precise control of nanoparticle properties. This thesis presents an investigation of parameters in the benzyl alcohol synthesis of iron oxide nanoparticles. A modified seed growth method was designed for obtaining optimal nanoparticle properties for magnetic fluid hyperthermia. With a one or two addition process, iron oxide nanoparticles were produced with crystallite sizes ranging from 5-20 nm using only benzyl alcohol and iron precursor. The effects of reaction environment, temperature, concentration, and modified seed growth parameters were investigated to obtain precise control over properties affecting radiofrequency heat generation. The reaction A2-24(205)_B2-24(205) produced monodispersed (PDI=0.265) nanoparticles with a crystallite size of 19.5±1.06 nm and the highest radiofrequency heating rate of 4.48 (°C/min)/mg (SAR=1,175.56 W/g, ILP=3.1127 nHm2/kg) for the reactions investigated. The benzyl alcohol modified seed growth method offers great potential for synthesizing iron oxide nanoparticles for radiofrequency hyperthermia.

La thérapie génique est une forme de médecine moléculaire qui a des potentialités majeures dans le traitement d'un grand nombre de maladies héréditaires ou de cancers. Les acides nucléiques doivent pénétrer à l'intérieur des cellules et interagir avec la machinerie génétique présente. Une des plus grandes limitations dans l'application de ce type de traitement est le développement d'un vecteur sûr et efficace pour transporter ces molécules dans les cellules. Les vecteurs sont classifiés traditionnellement en deux grandes catégories : viral et non-viral. Les vecteurs de type viral sont les plus efficaces et utilisés dans la majorité des tests cliniques, cependant ils peuvent provoquer une forte réponse immunitaire et ils ont des coûts importants de production. Les vecteurs de type non viral peuvent être fabriqués facilement en grande quantité, ne sont pas immunogéniques et possèdent l'avantage de pouvoir être modulés en fonction des applications. Les lipides et les polymères cationiques sont les plus étudiés à cause de leurs propriétés de complexation avec les acides nucléiques. Cependant ces complexes chargés positivement peuvent être toxiques à cause de leur interaction non spécifique avec les membranes cellulaires, ce qui limite leur utilisation in vivo. Cela a conduit à la recherche de nouveaux vecteurs, neutres ou anioniques, pour diminuer les problèmes de cytotoxicité. Les nucléolipides (NL) sont des molécules hybrides bioinspirées amphiphiles formées d’une partie hydrophile nucléotidique et d’une partie hydrophobe lipidique. Ces molécules sont donc capables de s'autoassembler et de former des structures supramoléculaires avec des propriétés physico-chimiques principalement liées à la nature chimique de la tête polaire nucléotidique. L'interaction avec des acides nucléiques est alors possible grâce à des interactions de type Watson-Crick, cependant elles ne sont pas suffisamment élevées pour former un complexe stable pour être utilisées dans les conditions biologiques. La synthèse d’une nouvelle famille de molécules hybrides de type amino-nucléo-lipides (ANL) a été développée dans l'objectif d'améliorer les interactions et donc la complexation grâce à la présence d'un acide aminé sur la partie polaire de l’amphiphile. La synthèse chimique des ANLs avec différents acides aminés (glycine, phénylalanine) en position 5' du sucre et différentes lipides (dimiristoyl, dioleoyl) estérifié au phosphate présent en position 3' a été réalisée. Les études physico-chimiques réalisées par diffusion dynamique de la lumière (DLS), cryo-microscopie électronique (cryo-TEM) et diffusion des rayons X aux petits angles (SAXS) ont permis d'étudier la morphologie et l'organisation structurale des objets supramoléculaires formés par ces molécules. Ces études ont en particulier permis de mettre en évidence la relation entre la structure chimique et les propriétés physico-chimiques. Les NLs forment des vésicules unilamellaires et les ANLs, grâce à la présence de l'acide aminé sur la tête polaire, forment des vésicules de type multilamellaire. L'étude des interactions, réalisée par calorimétrie à titration isotherme (ITC), entre les vésicules et un acide nucléique modèle (poly A) montre qu'il existe une interaction entre les deux partenaires, mais que cette interaction n'est pas suffisamment forte pour envisager des applications biologiques. La stratégie d'utiliser un cation divalent, comme le calcium, pour ponter les charges négatives des nucléolipides et les charges négatives des acides nucléiques a été envisagé et les complexes ont été étudiés par SAXS. Enfin, des transitions de morphologies observées en fonction de la concentration ont été étudiées par DLS et cryo-TEM. Ces transitions de morphologies ont pu être corrélées à des effets de force ionique sur les propriétés de la tête polaire. / Gene therapy is a molecular medicine and a very powerful tool for the treatment of several diseases such as inherited disorder and cancer. Nucleic acids must penetrate into cells in order to interact with their genetic material. Currently the main limitation to the application of this treatment towards clinics is the lack of robust, safe and efficient gene delivery vectors. The two major classes of vectors are those based on recombinant viruses and those based on non-viral systems. Viral vectors are the most efficient and used in several clinical trials, however they can elicit a strong immune reaction and they possess high cost of production. Non-viral vectors are less immunogenic and can be easily produced on a large scale. A large variety of both cationic lipids and polymers have been developed due to their ability to interact spontaneously with negatively charged nucleic acids to form complexes. However these positively charged complexes can present some toxicity due their non-specific interaction with cell membranes and seric proteins. This is the main limitation for their clinical use. For this purpose, new vectors, neutral or anionic, have to be developed in order to diminish the cytotoxicity and increase the circulation time. Nucleotide-lipids (NLs) are bio-inspired amphiphilic hybrid molecules composed of a hydrophilic nucleotidic moiety and a hydrophobic lipophilic moiety. These molecules are able to self-assembly to form supramolecular structures which possess particular physico-chemical properties due to the chemistry of their polar head. These molecules can interact with a nucleic acid by Watson-Crick base pair interactions, however they are not sufficiently strong to form a stable complex that can be used for a biological application. A new chemical family of hybrid amphiphile, amino acid-nucleotide-lipids (ANLs), has been developed in order to increase the interactions and the stability of the complex thank to the presence of the amino acid on the polar head. Herein, we have synthesized novel amino acid-nucleotide-lipids, presenting phenylalanine (or glycine) and thymidine residues and saturated (dimiristoyl) or unsaturated (dioleoyl) diacyl glycerol lipid. The morphology and the structural organization of the supramolecular objects formed by these molecules was studied by dynamic light scattering (DLS), cryo-electron microscopy (cryo-TEM) and small angle X-ray scattering (SAXS). These studies allowed investigating the relation between the chemical structure and the physic-chemical properties. The amino acids, inserted at the 5′ position of the nucleotide-lipids, stabilize multilamellar systems, whereas unilamellar vesicles are formed preferentially in the case of nucleotide-lipids. Both NLs and ANLs exhibit weak interactions with complementary polyA RNA as revealed by isothermal titration calorimetry (ITC) investigations, however they are not sufficiently strong to form a stable complex that can be used for a biological application. The use of multivalent cations, such as Ca2+, which bridge the phosphate groups on the lipid polar heads with those of the backbone of nucleic acids, to form ternary complexes, has been investigated by SAXS. Finally, a structural study, by DLS and cryo-TEM of NLs aggregates in aqueous solutions as a function of ionic strength and surfactant concentration, has been conducted in order to investigate the different morphologies of the systems.

Une nouvelle plateforme hydrogel uniquement formée par l’association de nanocapsules lipidiques (NCLs) a été développée en s’inspirant de précédents travaux utilisant une gemcitabine modifiée. Afin de limiter la toxicité de l’hydrogel, la gemcitabine a été remplacée par la cytidine, rendue amphiphile par une chaîne aliphatique (Cyt-C16). Placée à l’interface huile/eau des NCLs, la Cyt-C16 permet la formation d’un réseau tridimensionnel de NCLs à l’origine de la gélification. Un plan de mélange a permis d’optimiser les procédés de formulation de 4 tailles de NCLs modèles. Les propriétés viscoélastiques des hydrogels sont modulables. Plus les concentrations en NCLs et Cyt-C16 sont élevées, plus le gel est « rigide », indépendamment de la taille des NCLs qui doit être supérieure à 50 nm pour permettre la gélification. Les hydrogels sont injectables et permettent une libération prolongée de NCLs (de taille mono-disperse), sans toxicité supplémentaire in vitro, du fait de la présence de la Cyt-C16. De plus, uniquement solubilisée dans l’huile,la Cyt-C16 permet d’obtenir un organogel, dont les propriétés viscoélastiques sont renforcées en augmentant sa concentration. L’injection sous-cutanée (SC) in vivo des deux gels est bien tolérée et entraine une réaction inflammatoire locale comparable à celle provoquée par un excipient pharmaceutiquement acceptable. Ces deux formes pourront être utilisées pour libérer de façon prolongée différents actifs. Deux applications précliniques des hydrogels ont été explorées, l’une utilisant la voie SC pour cibler les ganglions lymphatiques, la seconde permettant un traitement local des suites opératoires d’une résection de glioblastome. / An innovative hydrogel platform obtained by the association of lipid nanocapsules (LNCs) was based on the previous work on modified gemcitabine. To limit the inherent toxicity of the hydrogel, gemcitabine was replaced by cytidine, then modified by an aliphatic chain (Cyt-C16). The hydrogel network was allowed by H-bond interactions between cytidine moieties exposed at the oil/water interfaces of LNCs. An experimental plan provided the formulation processes for 4 optimized sizes of model LNCs. The gelation was only possible for LNC sizes higher than 50 nm, and the hydrogel viscoelastic properties are versatile. The hydrogel is more “rigid” when LNC and Cyt-C16 concentrations increase, independently of the LNC size. The hydrogels are injectable and allow a sustained release of LNCs (withmonodisperse size), without additional in vitrocytotoxicity due to Cyt-C16. Moreover, when solubilized in oil, Cyt-C16 alone produced an organogel platform, whose viscoelastic properties are strengthened increasing its concentration. Both types of gels showed a good biocompatibility after an in vivo subcutaneous (SC) injection, with a local inflammatory response similar to that of induced by an approved excipient. These two forms could be used to sustain the release of various drugs, and two preclinical applications of hydrogels have been explored : one using the SC route to target lymph nodes, and the second for local treatment after glioblastoma resection.