Plusieurs types cancéreux prolifèrent par leur capacité à exprimer les marqueurs de régulation négative du système immunitaire, tels que les récepteurs PD-L1 et CD80/86 qui inhibent l’activation et la prolifération des lymphocytes T. L’inhibition de ces voies par des anticorps peut ainsi réactiver la réponse immunitaire chez certains patients. D’autres voies de signalisations sont aujourd’hui explorées, incluant la signalisation purinergique (ATP/adénosine) dans la modulation du microenvironnement tumoral. L’adénosine triphosphate extracellulaire (ATPe) est classifiée parmi les molécules de danger extracellulaire et joue un rôle crucial dans l’activation de l’inflammasome NLRP3, un médiateur important de l’activation des réactions pro-inflammatoires. Les ultrasons sont des ondes mécaniques de haute pression capable d’engendrer la cavitation inertielle des microbulles. Il a été démontré que les microbulles (MB) stimulées par ultrasons (US) libèrent de l’ATP dans le muscle squelettique et dans le muscle cardiaque. Nous posons l’hypothèse selon laquelle le traitement US+MB appliqué sur une tumeur de cancer du sein murin (4T1) in vivo peut libérer de l’ATPe localement dans le but d’activer des réactions pro-inflammatoires pour l’immunothérapie du cancer. Dans ce mémoire, nous présentons la quantification du signal d’ATPe d’une culture de cellules 4T1, puis in vivo dans le muscle et dans une tumeur solide sous-cutanée chez la souris à la suite d’une stimulation par US+MB. Nos études démontrent que la thérapie US+MB libère de l’ATP in vitro et in vivo. En comparant le signal découlant de l’injection IM d’ATP avec celui du muscle et des tumeurs post-US+MB, nous pouvons conclure que le traitement US+MB libère une quantité d’ATPe supérieure à 250 µM, ce qui est supérieur à la quantité d’ATPe dans un microenvironnement tumoral et qui persiste pour une durée d’au moins 60 min dans le muscle et 45 min dans la tumeur. La transfection stable de cellules MC38 (carcinome colorectal) à travers le gène PLenti-PmeLUC, codant la synthèse de luciférase sur la face externe de la membrane cellulaire, est explorée afin d’augmenter le rapport signal sur bruit en bioluminescence (annexe A). L’utilisation de POM-1 (inhibiteur pharmacologique de CD39) et l’utilisation de souris knockout du gène CD39 sont discutées pour la suite du projet afin d’inhiber la dégradation de l’ATP extracellulaire (Annexe B). / Several cancer types proliferate due to their ability to express the negative regulatory markers of the immune system (PD-L1 and CD80/86) which inhibit the activation and proliferation of T cells. Inhibition of these pathways by antibodies (anti-PDL-1, anti-PD-1, anti-CTLA-4) can thus reactivate the immune system in some patients. Other signaling pathways are currently being explored, including purinergic signaling (ATP/adenosine) in the modulation of the tumor microenvironment. Extracellular Adenosine triphosphate (eATP) is classified as danger signal plays a critical role in the activation of the NLRP3 inflammasome, an important mediator of the innate immune response. Ultrasound (US) and microbubbles (MB) have been shown to release ATP in skeletal and cardiac muscle. Thus, we hypothesized that US+MB treatment in 4T1 breast cancer cells could locally activate pro-inflammatory responses by releasing an eATP in tumors for cancer immunotherapy. In this thesis, I present the quantification of the eATP signal after US+MB stimulation in vitro (4T1 cell culture), then in muscle and subcutaneous solid tumors in the mouse. Our studies demonstrate that US+MB treatment releases ATP both in vitro and in vivo. In comparison with the IM injection of ATP, we can conclude that US+MB released a large amount of ATP (>250 µM), which is more than the eATP concentration in the untreated tumor microenvironment, and which persisted for at least 60 min in muscle and 45 min in tumor. The stable transfection of MC38 cells (colorectal carcinoma) through the Plenti-PmeLUC gene, encoding the synthesis of luciferase on the external surface of cell membrane is explored to increase the signal to noise ratio in bioluminescence (see appendix A). The use of POM-1 (pharmacological inhibitor of CD39) and CD39 gene knockout mice to inhibit the degradation of eATP signal are discussed for the continuation of the project.

Dans l'imagerie moléculaire par ultrasons, des microbulles sont fonctionnalisées avec des ligands. Après injection intraveineuse, ces microbulles ciblées s'accrochent aux marqueurs présents sur l'endothélium tumoral et permettent une détection non-invasive. Dans cette thèse, l'imagerie moléculaire par ultrasons a été développée pour la détection de la nétrine-1, qui est surexprimée dans 70% des cancers du sein et promeut la survie cellulaire. Une nouvelle thérapie moléculaire interférant avec la nétrine-1 a été développée et nécessite l'identification des patientes qui pourront bénéficier de ce traitement. Avec l'imagerie moléculaire de la nétrine-1, il a été possible de discriminer les tumeurs positives pour la nétrine-1 des tumeurs négatives. Par sa capacité à détecter de manière spécifique la nétrine-1 présentée sur l'endothélium des tumeurs, cette technique d'imagerie pourrait donc devenir un test d'accompagnement pour la thérapie d'interférence de la nétrine-1 chez les patientes atteintes de cancer du sein.La destruction ciblée des microbulles par ultrasons induit la cavitation et la sonoporation qui perméabilisent le tissu et facilite la délivrance locale de médicaments. De plus, cette destruction ciblée peut induire l'infiltration de cellules immunitaires et la libération d'antigènes tumoraux, déclenchant une réponse immunitaire anti-tumorale. Dans cette thèse, nous avons quantifié l'activation de la réponse immunitaire dans le carcinome hépatocellulaire, suivant la délivrance de nanoparticules chargés en microARN-122 et anti-microARN-21. Dans les nœuds lymphocytaires tumoraux, une baisse d'expression des cytokines pro-tumorales et une augmentation d'expression des cytokines anti-tumorales ont été observées, suggérant une réponse thérapeutique positive. L'approche thérapeutique de destruction ciblée des microbulles par ultrasons pour la délivrance de micro-ARN s'avère donc être un outil immuno-modulatoire puissant / Ultrasound molecular imaging uses microbubbles as ultrasound contrast agents which are functionalized with targeting ligands. Upon intravenous injection, targeted microbubbles bind to molecular markers presented on the tumor endothelium and enable the non-invasive assessment cancer-related biomarkers. In the present thesis, ultrasound molecular imaging was developed for detection of netrin-1, which is upregulated in 70% of metastatic breast cancer and promotes cell survival. A newly developed netrin-1 interference therapy requires the identification of patients who overexpress the target protein and, could benefit from anti-netrin-1 therapy. In vivo imaging of netrin-1 showed a significantly increased imaging signal in netrin-1-positive breast tumors compared to netrin-1-negative breast tumors and normal mammary glands. The results suggest that ultrasound molecular imaging allows accurate detection of netrin-1 on the endothelium of netrin-1-positive tumors and has the potential to become a companion diagnostic for netrin-1 interference therapy in breast cancer patients.Ultrasound-targeted microbubble destruction triggers cavitation and sonoporation thereby permeabilizing the tissue and facilitating local drug delivery. Further, immune cell infiltration and tumor antigen release are induced and trigger anti-tumor immune responses. In the present thesis, ultrasound-targeted microbubble destruction-mediated delivery of anti-cancer microRNA-122 and anti-microRNA-21 is studied for immune response activation in hepatocellular carcinoma, in which the immune microenvironment is deregulated. Tumor lymph nodes showed pro-tumor cytokine downregulation and anti-tumor cytokine upregulation, suggesting an overall positive therapy response with regard to the tumor immunology. The results identified ultrasound-targeted microbubble destruction-mediated miRNA delivery as a potent immuno-modulatory therapeutic approach

This dissertation offers demonstrations of autonomous catalytic microtubes (microjet engines) with tunable diameters ranging from micro- to nanoscale and lengths from 50 μm to 1 mm. These results open the door to effective microengines and represent the entry in the Guinness Book of World Records for “the smallest man-made jet engine.” Several attractive methodologies of machine-based functions at the micro- and nanoscale are shown. For instance, catalytic Ti/Cr/Pt microjets, which are integrated on a planar substrate, can operate as “on chip” chemical micropumps by decomposition of hydrogen peroxide fuel into oxygen bubbles and water. When released from a substrate, microjets self-propel autonomously in solution. The incorporation of ferromagnetic layer (Fe) into the rolled-up geometry enables their remote control using external magnetic field. Such microjets were used to load, transport, deliver and assemble multiple cargo particles, including biological cells in bulk solutions and microfluidic channels. Furthermore, it is demonstrated that for microjets that are fixed to or self-propelled above a platinum patterned surface, the microengine power/speed can be controlled using a white lightsource. A change in intensity of the white light leads to a controllable switching “off” and “on” of the microengine power on demand. Light degrades a local concentration of the hydrogen peroxide fuel and surface tension and subsequently suppresses the generation of oxygen microbubbles. In the next step, the diameter of the microjets was rigorously reduced to 250 nm by using hybrid heteroepitaxial/catalytic InGaAs/GaAs/Cr/Pt nanotubes. Due to asymmetry of the rolled-up layers, these nanojets move in corkscrew-like motions and act as “self-propelled nanotools,” which were used in the next step to transport yeast cells and drill into fixed cancer Hela cells. Although it is well-known that hydrogen peroxide cannot be used to sustain viable cellular function, it is however conceivable that alternative fuels, such as glucose, might enable operation of such nanotools under biologically compatible conditions. As a first step to achieve this goal, demonstrations were made using metal-enzyme biocatalytic Ti/Au/SAM/Catalase microengines. Synthetic components with competing interactions are well-suited to study the emergence of their collective behavior, such as swarms of large numbers of individuals. Microengines’ self-organization in bistable swarms is shown at the air-liquid interface of the mixture of propylene carbonate and hydrogen peroxide. Microengines act as “water striders.” Buoyed by oxygen bubbles, they self-propel via the microbubble recoiling mechanism and, depending on the bubbles’ sizes, self-organize into swarms due to the meniscus climbing effect. These reversible swarms depend on the microengine power, which competes against attracting surface tension force. The demonstrated microjet engines show great promise for emerging applications, including biomedical, on-chip, environmental, and robotic micromachines. Furthermore, a key method discovered, entitled “rolled-up nanotechnology on polymers,” allowed for the fabrication of highly parallel arrays of microtubes with multiple functionalities and aimed for different purposes.

Les fluorocarbures ont un fort potentiel en médecine. Cependant, et en dépit du fait que certaines formulations employant des fluorocarbures sont utilisées en clinique, il n’existe que relativement peu d’études visant à déterminer les interactions entre un fluorocarbure et une membrane de phospholipides. Notre étude concentre à l’interface fluorocarbure/phospholipide, qui représente d’une part un modèle simplifié du surfactant pulmonaire natif dont le composant majoritaire est la dipalmitoylphosphatiylcholine (DPPC), et d’autre part la paroi de microbulles développées comme nouveaux agents théranostiques.Tout d’abord, nous montrons que les fluorocarbures abaissent considérablement la tension interfaciale d’équilibre d’une série de phospholipides et accélèrent fortement leur adsorption. Nous montrons que des oscillations périodiques appliquées à la bulle induisent une transition du film de DPPC vers un état d’organisation plus dense. L’application d’oscillations périodiques permet aussi à la DPPC d’expulser du film interfacial une protéine, l’albumine, dont la présence est souvent liée aux troubles dus au mauvais fonctionnement du surfactant pulmonaire. L’effet des fluorocarbures, qui accélère considérablement l’expulsion de l’albumine par la DPPC, est également étudié. D’autre part, nous avons obtenu des microbulles exceptionnellement stables grâce à une série homologue de phosphates perfluoroalkylés. Nous avons également réussi à former des microbulles couvertes par des nanoparticules magnétiques, tout en gardant les propriétés échogènes des bulles. De telles microbulles offrent un potentiel comme des agents de contraste bimodaux pour l’IRM et l’échosonographie. / Fluorocarbons have a great potential in medicine. However, and despite the fact that some formulations using fluorocarbons are used clinically, only a few studies are reported that aim to determining the interactions between a fluorocarbon and a membrane of phospholipids. Our work concentrated on the fluorocarbon/phospholipid interface, which represents, on one hand, a simplified model of the lung surfactant, the major component of which is dipalmitoylphosphatiylcholine (DPPC), and on the other hand, the shell of microbubbles developed as new theranostic agents. In a first part, we show that fluorocarbons significantly reduce the equilibrium interfacial tension of a series of phospholipids and greatly accelerate their adsorption rate. We also show that periodical oscillations applied to the bubble induce a transition of DPPC film to state with a denser organization. The application of periodical oscillations also allows DPPC to expel from the interfacial film a protein, albumin, whose presence is often associated with disorders caused by dysfunction of the lung surfactant. The impact of fluorocarbons, which considerably accelerate the expulsion from the interfacial film of albumin, is also studied. In a second part, we have obtained exceptionally stable microbubbles with a homologous series of perfluoroalkylated phosphates. We were also able to form microbubbles covered by magnetic nanoparticles, while preserving the echogenicity of the bubbles. Such microbubbles offer a potential as bimodal contrast agents for MRI and echography.

This dissertation offers demonstrations of autonomous catalytic microtubes (microjet engines) with tunable diameters ranging from micro- to nanoscale and lengths from 50 μm to 1 mm. These results open the door to effective microengines and represent the entry in the Guinness Book of World Records for “the smallest man-made jet engine.” Several attractive methodologies of machine-based functions at the micro- and nanoscale are shown. For instance, catalytic Ti/Cr/Pt microjets, which are integrated on a planar substrate, can operate as “on chip” chemical micropumps by decomposition of hydrogen peroxide fuel into oxygen bubbles and water. When released from a substrate, microjets self-propel autonomously in solution. The incorporation of ferromagnetic layer (Fe) into the rolled-up geometry enables their remote control using external magnetic field. Such microjets were used to load, transport, deliver and assemble multiple cargo particles, including biological cells in bulk solutions and microfluidic channels. Furthermore, it is demonstrated that for microjets that are fixed to or self-propelled above a platinum patterned surface, the microengine power/speed can be controlled using a white lightsource. A change in intensity of the white light leads to a controllable switching “off” and “on” of the microengine power on demand. Light degrades a local concentration of the hydrogen peroxide fuel and surface tension and subsequently suppresses the generation of oxygen microbubbles. In the next step, the diameter of the microjets was rigorously reduced to 250 nm by using hybrid heteroepitaxial/catalytic InGaAs/GaAs/Cr/Pt nanotubes. Due to asymmetry of the rolled-up layers, these nanojets move in corkscrew-like motions and act as “self-propelled nanotools,” which were used in the next step to transport yeast cells and drill into fixed cancer Hela cells. Although it is well-known that hydrogen peroxide cannot be used to sustain viable cellular function, it is however conceivable that alternative fuels, such as glucose, might enable operation of such nanotools under biologically compatible conditions. As a first step to achieve this goal, demonstrations were made using metal-enzyme biocatalytic Ti/Au/SAM/Catalase microengines. Synthetic components with competing interactions are well-suited to study the emergence of their collective behavior, such as swarms of large numbers of individuals. Microengines’ self-organization in bistable swarms is shown at the air-liquid interface of the mixture of propylene carbonate and hydrogen peroxide. Microengines act as “water striders.” Buoyed by oxygen bubbles, they self-propel via the microbubble recoiling mechanism and, depending on the bubbles’ sizes, self-organize into swarms due to the meniscus climbing effect. These reversible swarms depend on the microengine power, which competes against attracting surface tension force. The demonstrated microjet engines show great promise for emerging applications, including biomedical, on-chip, environmental, and robotic micromachines. Furthermore, a key method discovered, entitled “rolled-up nanotechnology on polymers,” allowed for the fabrication of highly parallel arrays of microtubes with multiple functionalities and aimed for different purposes.