Microbulles lyophilisées microfluidiques comme agent de contraste

The advantages of using ultrasound contrast agents (UCA) with monodisperse size distribution have been highlighted. Characterized by a coefficient of variation (CV) lower than 5%, monodisperse microbubbles (MMB) have the potential to improve the quality of ultrasound images. It also facilitates microbubble resonance frequency monitoring, opening possibilities in the areas of molecular imaging, non-invasive pressure measurements, and drug and gene delivery.But, contrarily to polydisperse microbubbles bubbles (PMB), freeze-drying monodisperse populations of fresh microbubbles, without deteriorating their monodispersity, remains a challenge. Thus, MMB can neither be stored nor transported. This represents a bottleneck for their use in clinical applications. Attempts made to solve the problem have used toxic solvents, raising regulatory issuesThe present work presents a new freeze-drying technique for MMB that did not degrade their size distribution, or their acoustic properties, without the use of toxic solvents. As the first step of the project, flow-focusing microfluidic devices were fabricated to produce microbubbles with highly monodisperse size distribution (CV<5%). During this step, the optimization of the microbubble formulation and cryoprotectant materials was performed. Geometric characterization of two microbubbles population with mean diameters of 40µm and 5µm was performed. Images of all stages of the freeze-drying process of the microbubbles were captured and analyzed, aiming the control of the size distribution and production rate. The steps of the freeze-drying process consisted of production, collection, freezing, lyophilization, and resuspension. The development of a new retrieval technique, where the microbubbles were stored in monolayers, resulted in a drastic reduction of the interaction between the bubbles during lyophilization. In this way, it was possible to preserve the monodispersity, resulting in a CV <6% for the resuspended microbubbles.In the second stage of the project, a characterization of the backscatter acoustic response (BSAR) of the freeze-dried monodisperse PVA-shelled microbubbles (FDMD), in comparison with fresh MMB, and PMB was conducted. Firstly, the BSAR of the microbubbles was evaluated in two different setups: the centimetric cell (large container), and the ‘milli-channel’ (confined system in which the liquid is at rest). Using a focused acoustic transducer with a frequency of 2.25MHz, the backscattering acoustic responses of the microbubbles, before and after the freeze-drying process was compared for the bubble population of 5µm diameter. The variation of amplitude and fundamental resonance frequency of the bubbles were within the experimental uncertainty range, suggesting that their acoustic properties were preserved. We also observed, in agreement with the literature, that there is a linear dependence between the concentration of the microbubble population and the amplitude of the backscatter coefficient. Also, in agreement with the literature, we observed an amplitude in the response signal of the monodispersed bubbles of 8 to 10 times higher than that for the polydispersed ones, for the same in vitro concentration. It was observed a lower uncertainty in monitoring the fundamental resonance peak of the bubbles and a smaller bandwidth for the FDMD. Finally, using the ultrasound matrix imaging approach, developed at Institut Langevin, the BSAR of the FDMD and PMB was evaluated in a phantom mimicking tissue. The results reinforced the findings obtained the centimetric cell and the milli-channel.We successfully presented a new technique developed to freeze-dry monodisperse microbubbles without degrading their geometrical and acoustic properties. Thus, we proposed a new generation of ultrasound contrast agents in the form of a stable freeze-dried powder that can be transported and stored for months and resuspended for use in clinical applications.Les avantages de l'utilisation d'agents de contraste ultrasonores (ACU) à distribution de taille monodisperse ont été mis en évidence. Caractérisées par un coefficient de variation (CV) de leur diamètre inférieur à 5%, les microbulles monodisperses (MBM) ont le potentiel d'améliorer la qualité des images ultrasonores (amélioration du rapport signal/bruit et réduction des effets d'ombre). Elles facilitent également le contrôle de la fréquence de résonance fondamentale (FRF) des microbulles, ouvrant des possibilités dans les domaines de l'imagerie moléculaire et des mesures de pression non invasives. En outre, les MBM peuvent optimiser l'administration de médicaments, et de gènesCependant, contrairement aux bulles polydisperses (MBP), la lyophilisation de MBM fraîches, sans détérioration de leur monodispersité, reste un défi. Ainsi, les MBM ne peuvent être ni stockées ni transportées. Cela représente un goulot d'étranglement pour leur utilisation dans des applications cliniques. Les tentatives faites pour résoudre ce problème ont utilisé des solvants toxiques, ce qui soulève des problèmes de réglementation.L'objectif était de développer une nouvelle technique de lyophilisation des MBM qui ne dégrade pas leur distribution de taille, ni leurs propriétés acoustiques, et ce sans utiliser de solvants toxiques. La première étape a consisté à fabriquer des dispositifs microfluidiques de focalisation du flux pour produire des microbulles avec une distribution de taille monodisperse (CV<5%). L'optimisation de la formulation des microbulles et des matériaux cryoprotecteurs a été réalisée. La caractérisation géométrique de deux microbulles avec des diamètres moyens de 40µm et 5µm a été menée. Des images de toutes les étapes du processus de lyophilisation des microbulles ont été capturées et analysées, pour contrôler la distribution de taille et le taux de production. Le développement d'une nouvelle technique de récupération a permis de réduire considérablement l'interaction entre les bulles pendant la lyophilisation. De cette manière, il a été possible de préserver la monodispersité pendant le processus de lyophilisation (CV