Les systèmes théranostiques associant des propriétés thérapeutiques et des propriétés d'imagerie sont développés pour permettre le suivi de traitement in vivo. La stratégie que nous proposons dans cette thèse est de formuler des liposomes magnétiques thermosensibles chargés en principe actif pour traiter des tumeurs murines superficielles du côlon CT26. Ces nanovecteurs peuvent être accumulés dans les tumeurs par ciblage magnétique et le relargage du principe actif peut être déclenché par HIFU (Ultrasons Focalisés de Haute Intensité). Ces liposomes sont développés par co-encapsulation de nanoparticules de maghémite (?-Fe2O3) et de Combrétastatine A4 Phosphate (CA4P) dans des liposomes thermosensibles. La forte encapsulation des nanoparticules magnétiques dans les Liposomes Ultra-Magnétiques (LUM) permet à la fois le ciblage magnétique et leur suivi in vivo par IRM. Le chauffage par HIFU in vitro à la température de transition des membranes des LUM a permis une amélioration du relargage de la CA4P. La biodistribution des LUM in vivo a été évaluée par IRM dynamique de contraste de susceptibilité avec une résolution temporelle adaptée à la cinétique de capture des nanovecteurs, notamment par le foie. L'efficacité du ciblage magnétique a été démontrée grâce à une nouvelle méthode de traitement de l'histogramme des intensités IRM. Enfin, l'ajustement in vivo des séquences d'HIFU permet le relargage du principe actif. L'efficacité du traitement est ensuite suivie par IRM multiparamétrique anatomique, pondérée T2* de diffusion et de perfusion pour évaluer l'impact sur la fonctionnalité vasculaire et l'évolution tumorale, ainsi que par histologie. / Theranostic systems with imaging and therapeutic properties are developed to monitor treatments in vivo. The strategy we propose here is to design thermosensitive drug-loaded magnetic liposomes to treat superficial colon tumors CT26 on mice. These nanocarriers can be accumulated in the tumor by using a magnetic field gradient and the drug release can be triggered by a local heating induced by HIFU (High Intensity Focused Ultrasounds). They have been developed by co-encapsulation of magnetic nanoparticles and the antitumoral drug CA4P (Combretastatin A-4 Phosphate) in thermosensitive liposomes. The high loading of maghemite (γ-Fe2O3) magnetic nanoparticles enables both magnetic targeting and in vivo monitoring by MRI of this Ultra-Magnetic Liposomes (UML). In vitro HIFU heating at the UML membrane transition temperature improved the drug release. In vivo UML biodistribution was evaluated with dynamic susceptibility contrast imaging adjusted in time acquisition in MRI and the magnetic targeting efficiency was shown with a new MRI imaging processing. Adjustments of in vivo HIFU sequences to locally heat the tumor at the UML transition temperature allowed the triggering of drug release. Treatment efficiency was monitored by multiparametric diffusion, T2* weighted, anatomical and perfusion MRI and histology.

Cette thèse traite de deux projets en rapport avec la microfluidique. Le premier est consacré à la réalisation d'actionneurs à base d'hydrogel thermosensible. Avec une approche technologique basée sur le greffage et la structuration d'un hydrogel thermosensible sur des surfaces en verre, nous montrons comment un matériau capable de subir de fortes variations de volume peut être utilisé comme actionneur microfluidique dans les milieux aqueux. D'abord, en tant que vanne, nous démontrons que la stratégie de greffage des structures permet d'obtenir des actionneurs robustes et durables capables de réguler la circulation de fluides en micro-canaux avec une réponse rapide en miniaturisant la stimulation thermique. Ensuite, une autre configuration, dite de compartimentation libre, est mise en oeuvre et appliquée en biologies moléculaire et cellulaire. Le second est quant à lui dédié à l'étude du glissement par nanovélocimétrie en utilisant la technique de microscopie de fluorescence par réflexion totale interne. Cette technique permet de suivre les écoulement de nanoparticules à proximité immédiate de la paroi d'un micro-canal (dans les premiers 800nm de fluide). En utilisant les modèles de Langevin, nous avons été en mesure de prendre en compte différents artéfacts expérimentaux et les corriger. Nous confirmons l'absence de glissement de l'eau pure sur surface hydrophile et l'existence du glissement sur surface hydrophobe. Cette partie est traitée sous la forme d'une brève introduction, suivie d'un article publié dans Journal of Fluid Mechanics. / In this work, we have addressed two projects related to microfluidics.The first one is dedicated to the integration of hydrogel-based actuators in microsystems. Thanks toa technological approach based on the grafting and patterning of a thermosensitive hydrogel on glasssubstrates, we show how a material that can undergo large volume changes can be embedded as anactuator for aqueous solutions. First as a valve, we prove that the strategy consisting in grafting patternsof hydrogel enables us to obtain robust actuators able to regulate flows in micro-channels, and with afast response by miniaturising the thermal stimulation. Then we report another configuration called freecompartmentalization, applied to molecular and cellular biology. The second part of this manuscript deals with the study of slippage by nanovelocimetry using the totalinternal reflection fluorescence microscopy technique. This method enables us to follow fluorescent nanoparticlesflowing near the wall of a microchannel (within the first 800nm ). Using Langevin simulations,we have been able to take several experimental biases into account and correct them. We can confirmthe no-slip condition of water on hydrophilic surfaces and the actual slippage over hydrophobic surfaces.This part is made of a brief introduction followed by an publication in Journal of Fluid Mechanics.

Presented diploma thesis deals with drug encapsulation of Sulfathiazole (STA) in thermosensitive hydrogel composed of amphiphilic fibrous molecules of copolymer poly(D,L-lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(D,L-lactide-co-glycolide) (PLGA-PEG-PLGA). In the experimental part, the structure of the micelles which is dependent on the concentration of the default copolymer, was thoroughly described. The method of dynamic light scattering (DLS) was used to dimensionally characterize the micellar size in aqueous solution. At the same time, the Cryo-Transmission Electron Microscopy (Cryo-TEM) imaging technique confirmed the transition from spherical micelles to fibrous branched structures with a number of topological interactions. The change in viscoelastic properties of micellar hydrogel after addition of STA was studied by rheological analysis. The studied triblock copolymer appears to be a suitable tissue engineering material as a carrier in applications for targeted drug delivery and tissue regeneration.

No description available.

<p>N-chloramide containing and primary amine-containing microgels were prepared by post-modification of thermosensitive microgels in alkaline bleach. The objective of this project was to develop simple strategies for preparation of functionalized microgels.</p> <p>N-chlorination of linear poly(N-isopropylacrylamide) (PNIPAM) in bleach at high pH resulted in a novel N-chloramide containing copolymer: poly(NIPAM-co-NIPAMCl). The chlorinated PNIPAM showed controlled phase transition temperature and oxidative ability. The N-chlorination of linear PNIPAM inspired the preparation of N-chloramide containing PNIPAM microgels in a similar way. The phase transition temperature of the resulted chlorinated microgels, which corresponds to the extent of N-chlorination, was affected by the reaction temperature and salt concentration. The reaction between the chlorinated microgels and glutathione is proposed as diffusion controlled.</p> <p>The N-chlorination of poly(N-isopropylmethacrylamide) (PNIPMAM) microgels in bleach was restricted, in comparison with PNIPAM microgels. The active chlorine content of chlorinated PNIPMAM microgels was about one-tenth of that of chlorinated PNIPAM microgels under the same N-chlorination condition. It is proposed that the high stability of PNIPMAM in bleach is a result of the electron-donating effect of methyl groups on PNIPMAM backbone. Hence, core-shell microgels with PNIPAM cores and poly(NIPAM-co-NIPMAM) shells showed improved colloidal stability after N-chlorination because the shell was less chlorinated and served as a steric stabilizer.</p> <p>Finally, primary amine-containing microgels were prepared via Hofmann rearrangement of copolymers of methacrylamide, which decomposed to give amines, and NIPMAM, which did not react. The method was further extended to give amphoteric microgels by including acrylic acid in the starting microgels. Although other approaches to aminated and amphoteric microgels have been developed, this approach is particularly attractive because of the ease of the reaction and the ability to control the microgel isoelectric points.</p> / Doctor of Philosophy (PhD)

This study seeks to develop smart hydrogel formulations for injectable controlled drug delivery from Pluronics to enhance patients compliance, decrease side effects, reduce dose and frequency. A biocompatible copolymer, Pluronic F127 was probed as the main ingredient for the injectable systems owing its low gelation concentration and ease of modification the system properties through excipients addition. The matrix properties were studied through a series of thermal, rheological and structural (SAXS/SANS) experiments as a function of concentration and shear rate, covering both static and dynamic environments. It has shown that gelled viscosity (and structure) can be critically controlled by shear rate and the structures recorded do not match those predicted for sheared colloids. Two further Pluronics F68 and F108, were studied showing similar but shifted gelation properties to F127. Effects of additives were studied by introducing different Mw PEGs and a model hydrophobic drug ‘ibuprofen’ to a F127 20% formulation. PEGs addition effects on the system properties and gelation transition were largely dependent on the Mw used in the blend, which became more prominent with increasing chain length. Ibuprofen’s addition has resulted in reduced gelation temperature and smaller hard spheres without having a great effect on the system rheological properties compared to neat gels. Blends containing both additives PEG and ibuprofen exhibited a synergistic effect, where comparisons show that Ibuprofen had the largest effect on the blends lowering gelation boundaries and slightly increasing the size of the hard spheres indicating the necessity of full characterisation of the formulation with any API.

Nous avons optimisé des liposomes thermosensibles, encapsulant un principe actif anticancéreux, le 5-Fluorouracile (5-FU), afin de déclencher sa libération par une hyperthermie locale modérée (39-42°C) induite par des ultrasons focalisés. L'hyperthermie sera appliquée au niveau de la tumeur, afin d'améliorer l’efficacité thérapeutique et de réduire la toxicité systémique. Les liposomes ont été formulés par hydratation du film lipidique en mélangeant la 1,2-dipalmitoyl-sn-glycéro-3-phosphocholine (DPPC) pour sa thermosensibilité à 41,5 ± 0,5°C, le cholestérol (CHOL) pour favoriser la stabilité des liposomes vis-à-vis des composants du sang, et le 1,2-distéaroyl-sn-glycéro-3-phosphoéthanolamine-N-[méthoxy(polyéthylène glycol)-2000] (DSPE-PEG) pour assurer la furtivité de la formulation. Les expériences ont confirmé que les liposomes formulés à base de DPPC/CHOL/DSPE-PEG dans un ratio molaire 90 : 5 : 5 mol% sont thermosensibles. Des liposomes composés du même mélange lipidique dans un rapport 65 : 30 : 5 mol% ont été considérés comme contrôle négatif non thermosensible. L’optimisation de l’encapsulation passive du 5-FU a permis d’obtenir une efficacité d’encapsulation (5-FU encapsulé/5-FU total) de 13%, mais le 5-FU est très faiblement retenu (12%) dans la cavité aqueuse des liposomes du fait du gradient osmotique à la dilution. La rétention du 5-FU a été optimisée (93%) par la technique d’encapsulation active basée sur la complexation intraliposomale du 5-FU avec le complexe cuivre-polyéthylèneimine préalablement encapsulé dans les liposomes. Cette technique a également permis d’améliorer l’efficacité d’encapsulation d’un facteur trois environ (37%), avec un taux de charge (ratio final 5-FU/lipides, mole/mole) de 50% environ. Nous avons alors obtenu des liposomes thermosensibles d'un diamètre hydrodynamique de 65 nm et de charge de surface de -10 mV. Les liposomes non thermosensibles, ont été caractérisés par un diamètre hydrodynamique de 105 nm et une charge de surface de -4,9 mV. La libération du 5-FU déclenchée par une hyperthermie induite par des ultrasons focalisés a été mesurée in vitro. En réponse à une hyperthermie de 42°C, les liposomes thermosensibles libèrent 68% de leur contenu, au bout de 10 min, alors que les liposomes non thermosensibles en libèrent moins de 20%. En outre, la cytotoxicité des liposomes encapsulant le complexe 5-FU-cuivre-polyéthylèneimine a été évaluée vis-à-vis de la lignée cellulaire HT-29 du carcinome colorectal humain. Les résultats ont révélé que les lipides à une concentration de 800 µM ne sont pas cytotoxiques (80% de viabilité). De plus, la complexation du 5-FU n’influence pas sa cytotoxicité ce qui prouve que la toxicité provient du 5-FU et non des excipients. En revanche, l’encapsulation du complexe 5-FU-cuivre-polyéthylèneimine dans les liposomes induit une diminution de la concentration inhibitrice médiane de 115 (solution du complexe) à 49 µM environ, corrélée à leur internalisation cellulaire. La pharmacocinétique chez des souris porteuses d’un modèle de tumeur colorectale HT-29 xénogreffée a montré que les liposomes permettent de prolonger d’un facteur 1,4 la demi-vie plasmatique de distribution du 5-FU. De plus, les aires sous la courbe des concentrations plasmatiques sur 24 h sont 1,9 et 2,9 fois plus élevées lorsque le 5-FU est administré sous forme de liposomes thermosensibles et non thermosensibles, respectivement, par rapport à la solution de 5-FU. Enfin, les liposomes non thermosensibles augmentent significativement d'un facteur 2 l'accumulation du 5-FU dans la tumeur par rapport à la solution de 5-FU. En conclusion, les liposomes thermosensibles développés présentent un fort intérêt pour une application thérapeutique en combinaison avec des ultrasons focalisés. / We optimized thermosensitive liposomes encapsulating an anticancer drug, 5-Fluorouracil (5-FU), in order to trigger the release upon focused ultrasound-mediated mild hyperthermia at the tumor. This approach would improve drug efficacy and would lower side effects. Liposomes were prepared by the lipid hydration method by mixing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) for its temperature sensitivity at 41.5 ± 0.5°C, cholesterol (CHOL) to promote liposome stability towards blood components, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) to confer stealthiness to the formulation. The experiments confirmed that the liposomes formulated with DPPC/CHOL/DSPE-PEG in a molar ratio 90:5:5 mol% are thermosensitive, while liposomes composed of the same lipid mixture in a ratio 65:30:5 mol% were considered non thermosensitive negative control. The optimization of passive encapsulation of 5-FU yielded an encapsulation efficacy (encapsulated 5-FU/total 5-FU) of 13%. 5-FU was, however, very weakly retained (12%) in the aqueous core of liposomes following dilution due to the generation of an osmotic gradient. The retention of 5-FU has been optimized (93%) by the active encapsulation technique based on the intraliposomal complexation of 5FU with copper-polyethylenimine complex encapsulated beforehand into liposomes. This technique also improved 5-FU encapsulation efficacy by 3-fold (37%), yielding a loading efficiency (final drug/lipid ratio, mol/mol) of approximately 50%. The resulting thermosensitive liposomes and non thermosensitive liposomes have a hydrodynamic diameter and a surface charge around 65 nm and -10 mV, and 105 nm and -4.9 mV, respectively. Heat-triggered drug delivery was evaluated using focused ultrasound, and showed a release of 68% of the encapsulated 5-FU from thermosensitive liposomes, within 10 min, whereas release remained below 20% for the non thermosensitive formulation. Furthermore, the cytotoxicity of 5-FU-copper-polyethylenimine complex-loaded liposomes towards HT-29 human colorectal carcinoma cell line was evaluated. Results revealed that lipids at a concentration of 800 µM are not cytotoxic (80% viability). Moreover, 5-FU complexation has no impact on its cytotoxic activity, disclosing that liposomes toxicity arose from 5-FU and not from the excipients. Nevertheless, 5-FU-copper-polyethylenimine complex-loaded liposomes exhibited a lower half maximal inhibitory concentration of 49 µM compared to 115 µM for complex solution. This enhancement of cytotoxicity was attributed to the cellular internalization of liposomes. Pharmacokinetics in mice bearing HT-29 xenograft tumor showed that liposomes can extend the plasma distribution half-life of 5-FU by a factor 1.4. Furthermore, areas under the concentration-time curve over 24 h were higher by 1.9- and 2.9-fold when the drug was encapsulated into thermosensitive and non thermosensitive liposomes, respectively, compared to free 5-Fluorouracil. Finally, non thermosensitive liposomes significantly increased 5-FU accumulation in tumor by 2-fold, compared to 5-FU solution. In conclusion, these 5-FU-loaded thermosensitive liposomes represent valuable carriers to investigate the therapeutic efficacy following focused ultrasound-mediated heat application.

Développer de nouveaux nanomatériaux, interrupteurs et machines nanométriques sensibles à de petites variations de température spécifiques devrait être de grande utilité pour une multitude de domaines œuvrant dans la nanotechnologie. De plus, l’objectif est de convaincre le lecteur que les nanotechnologies à base d’ADN offrent d’énormes possibilités pour la surveillance de température en temps réel à l’échelle nanométrique. Dans la section Résultats, nous exploitons les propriétés de l’ADN pour créer des thermomètres versatiles, robustes et faciles à employer. En utilisant une série de nouvelles stratégies inspirées par la nature, nous sommes en mesure de créer des nanothermomètres d’ADN capables de mesurer des températures de 25 à 95°C avec une précision de <0.1°C. En créant de nouveaux complexes d’ADN multimériques, nous arrivons à développer des thermomètres ultrasensibles pouvant augmenter leur fluorescence 20 fois sur un intervalle de 7°C. En combinant plusieurs brins d’ADN avec des plages dynamiques différentes, nous pouvons former des thermomètres montrant une transition de phase linéaire sur 50°C. Finalement, la vitesse de réponse et la précision des thermomètres développés et leur réversibilité sont illustrées à l’aide d’une expérience de surveillance de température à l’intérieur d’un unique puits d’un appareil de qPCR. En conclusion, les applications potentielles de tels nanothermomètres en biologie synthétique, imagerie thermique cellulaire, nanomachines d’ADN et livraison contrôlée seront considérées. / Developing nanomaterials, probes, switches or nanomachines that are able to respond to specific temperature changes should prove of utility for several applications in the fields of in vivo imaging, clinical diagnostics, and drug-delivery. Here, we describe various bio- inspired strategies to engineer DNA thermoswitches with programmable linear response ranges for precise temperature sensing between 25 to 95°C with thermal precision <0.1°C. Using multimeric switch architectures, we are able to create ultrasensitive thermometers that display large 20-fold, quantitative signal changes within only 7°C. Lastly, by combining thermoswitches of different stabilities, or a mix of stabilizers of various strengths, we can create extended thermometers that respond linearly in a 50°C temperature window. Using these programmable DNA thermometers we measured, for the first time, the temperature equilibration time inside PCR wells using a fluorescent readout. Their potential applications in in vivo imaging, DNA nanomachines, drug delivery systems and synthetic biology are further discussed.

The clinical efficacy of chemotherapy in solid tumours is limited by systemic toxicity and the inability to deliver a cytotoxic concentration of anticancer drugs to all tumour cells. Temperature sensitive drug carriers provide a mechanism for triggering the rapid release of chemotherapeutic agents in a targeted region. Thermally mediated drug release also leverages the ability of hyperthermia to increase tumour blood flow, vessel permeability, and drug cytotoxicity. Drug release from thermosensitive liposome drug carriers in the tumour vasculature serves as a continuous intravascular infusion of free drug originating at the tumour site. However, localized drug release requires precise heating to improve drug delivery and efficacy in tumours while minimizing drug exposure in normal tissue. Focused ultrasound can noninvasively heat millimeter-sized regions deep within the body, and can be combined with MR thermometry for precise temperature control. This thesis describes the development of strategies to achieve localized hyperthermia using MRI-controlled focused ultrasound, for the purpose of image-guided heat-triggered drug release from thermosensitive drug carriers. First, a preclinical MRI-controlled focused ultrasound system was developed as a platform for studies of controlled hyperthermia and drug delivery in rabbits. The feasibility of using ultrasound hyperthermia to achieve localized doxorubicin release from thermosensitive liposomes was demonstrated in normal rabbit muscle. Second, strategies were described for using MR thermometry to control ultrasound heating at a muscle-bone interface based on MR temperature measurements in adjacent soft tissue, demonstrating localized drug delivery in adjacent muscle and bone marrow. Third, fluorescence microscopy was employed to demonstrate that increased overall drug accumulation in rabbit VX2 tumours corresponds to high levels of bioavailable drug reaching their active site in the nuclei of tumour cells. The results of this thesis demonstrate that image-guided drug delivery using thermosensitive liposomes and MRI-controlled focused ultrasound hyperthermia can be used to noninvasively achieve precisely localized drug deposition in soft tissue, at bone interfaces, and in solid tumours. Clinical application of this work could provide a noninvasive means of enhancing chemotherapy in a variety of solid tumours.

The clinical efficacy of chemotherapy in solid tumours is limited by systemic toxicity and the inability to deliver a cytotoxic concentration of anticancer drugs to all tumour cells. Temperature sensitive drug carriers provide a mechanism for triggering the rapid release of chemotherapeutic agents in a targeted region. Thermally mediated drug release also leverages the ability of hyperthermia to increase tumour blood flow, vessel permeability, and drug cytotoxicity. Drug release from thermosensitive liposome drug carriers in the tumour vasculature serves as a continuous intravascular infusion of free drug originating at the tumour site. However, localized drug release requires precise heating to improve drug delivery and efficacy in tumours while minimizing drug exposure in normal tissue. Focused ultrasound can noninvasively heat millimeter-sized regions deep within the body, and can be combined with MR thermometry for precise temperature control. This thesis describes the development of strategies to achieve localized hyperthermia using MRI-controlled focused ultrasound, for the purpose of image-guided heat-triggered drug release from thermosensitive drug carriers. First, a preclinical MRI-controlled focused ultrasound system was developed as a platform for studies of controlled hyperthermia and drug delivery in rabbits. The feasibility of using ultrasound hyperthermia to achieve localized doxorubicin release from thermosensitive liposomes was demonstrated in normal rabbit muscle. Second, strategies were described for using MR thermometry to control ultrasound heating at a muscle-bone interface based on MR temperature measurements in adjacent soft tissue, demonstrating localized drug delivery in adjacent muscle and bone marrow. Third, fluorescence microscopy was employed to demonstrate that increased overall drug accumulation in rabbit VX2 tumours corresponds to high levels of bioavailable drug reaching their active site in the nuclei of tumour cells. The results of this thesis demonstrate that image-guided drug delivery using thermosensitive liposomes and MRI-controlled focused ultrasound hyperthermia can be used to noninvasively achieve precisely localized drug deposition in soft tissue, at bone interfaces, and in solid tumours. Clinical application of this work could provide a noninvasive means of enhancing chemotherapy in a variety of solid tumours.