Les microsphères d'embolisation, apparues dans les années 2000, sont des dispositifs médicaux dirigées contre les tumeurs hépatiques non opérables. Elles sont calibrés et peuvent être chargées en anticancéreux. Ces avancées majeures permettent de contrôler le niveau d'occlusion et la concentration en principe actif à injecter dans la tumeur. Cependant, le type, la taille des microsphères ou encore la concentration en anticancéreux varient d'un centre à un autre et d'un pays à un autre. Notre travail vise à comparer, sur des modèles de tumeurs hépatiques, les propriétés d'élution et l'efficacité antitumorale de différentes préparations de microsphères. La microspectroscopie infrarouge est utilisée pour mesurer la quantité de doxorubicine présente dans les microsphères à différents délais alors que la microspectrofluorimétrie permet d'évaluer la concentration et la distribution de la doxorubicine autour des billes. L'évaluation de l'activité antitumorale du traitement est mesurée sur les images spectrales infrarouge grâce à un modèle de prédiction et confirmée par un examen histopathologique. Les résultats ont montré que la vitesse d'élution dépend des propriétés physicochimiques de la microsphère, de sa taille et de la concentration de chargement. Les concentrations tissulaires de doxorubicine mesurées induisent une réduction significative de la viabilité tumorale. Le model de prédiction est un outil robuste et précis pour évaluer les modifications tissulaires. Ces résultats permettent de formuler des hypothèses mécanistiques sur l'activité antitumorale de différentes préparations de microsphères afin d'optimiser leur utilisation dans une stratégie thérapeutique clinique. / Transarterial chemoembolization is the most common treatment for patients with unresectable liver tumors. Calibrated drug eluting beads offer the advantages of controlling the level of occlusion, the amount of drug delivered, and the duration of drug delivery to the tumor. However, optimal procedure still remains unanswered and treatments differ through the use of various beads sizes or dose of loading. Our work is to compare, on experimental liver tumor model, the release properties and antitumor effects for different preparations of doxorubicin eluting beads. The amount of drug retained inside the beads, at different time point, is assessed by infrared microspectroscopy. Doxorubicin concentration and distribution in the tissue are determined by microspectrofluorimetry. Tissue modifications are quantified by a prediction model on infrared images and compared with the conventional pathological examination of stained tissue sections. Results show that elution rate of doxorubicin depend on the beads composition, the size and the loaded concentration. The doxorubicin tissue concentration induces a significant decrease of tumor viability. The prediction model established by infrared microspectroscopy is an accurate and robust tool to quantify tissue modifications. These results allow the formulation of mechanistic hypotheses on antitumor activity of different preparations of beads to optimize their use in a clinical therapeutic strategy.

Tissue engineering (TE) uses a combination of biomaterial scaffolds, cells, and drug delivery systems (DDS) to create tissues that resemble the human physiology. Such engineered tissues could be used to treat, repair, replace, and augment damaged tissues or organs, for disease modeling, and drug screening purposes. This work describes the development and use of novel strategies for engineering neural tissue using a combination of drug delivery systems (DDS), human induced pluripotent stem cells (hiPSCs), and bioprinting technologies for the generation of a drug screening tool to be used in the process of drug discovery and development. The DDS consisted of purmorphamine (puro) loaded microspheres that were fabricated using an oil-in-water single emulsion with 84% encapsulation efficiency and showed the slow release of puro for up to 46 days in vitro. Puro and retinoic acid (RA)-loaded microspheres were combined with hiPSCs-derived neural aggregates (NAs) that differentiated into neural tissues expressing βT-III and showed increased neural extension. hiPCS-derived neural progenitor cells (NPCs) were bioprinted on a layer-by-layer using a fibrin based-bioink and extrusion based- bioprinting. The bioprinted structures showed >81% cellular viability after 7 days of culture in vitro and the expression of the mature motor neuron (MN) markers HB9 and CHAT. Lastly, hiPCS-derived NPCs were bioprinted in combination with puro and RA-loaded microspheres and cultured for 45 days in vitro. The microspheres slowly released the drug and after 30 and 45 days the tissues contained mature neurons, astrocytes and oligodendrocytes expressing CHAT, GFAP, and O4, respectively. Changes in membrane potential indicated tissue responsiveness to different types of treatments such as acetylcholine and gamma-aminobutyric acid (GABA). In the future the bioprinted tissues could contain localized regions of varied drug releasing microspheres using a concentration gradient to promote differentiation into specific cell types in order to create more complex tissues. Moreover, these tissues will benefit from the presence of a neurovascular unit (NVU). Upon validation, the engineered tissues could be used as preclinical tools to test potential drugs and be used for personalized medicine by using patient specific hiPSCs. / Graduate / 2020-11-19

The retina is a highly metabolically active tissue with large demands on the supply of nutrients. Disorders affecting the retina often include some vasculopathy with an impact on retinal circulation. Studies of retinal haemodynamics could thus help to detect, differentiate and diagnose diseases, to monitor changes in disease as well as progression and efficiency of the therapy. The present studies were an attempt to validate and determine the clinical usefulness of a newly developed technique for studying the retinal circulation in human eyes. We used different techniques to evaluate different parameters of retinal circulation. We examined how leukocyte velocity determined with Blue Field Simulation and transit times, mean transite time (MTT) and arterio-venous passage (AVP), and vessel diameter, determined from fluorescein angiograms, together reflects the retinal circulation. MTT was determined with a method based on an Impulse-Response technique, MTTIR. In a study on monkeys we compared our method, together with two conventional methods, with an absolute measurement of retinal blood flow (RBF) determined with labelled microspheres. There was a weak, but not statistically significant, correlation between retinal blood flow and MTTIR (r2 = -0.60, p = 0.06), but no useful correlation between retinal blood flow and either of the other two measures of transit times. In a study on healthy eyes we determined the effect of a physiological provocation, changes in arterial blood gases, on retinal circulation. Breathing pure oxygen or increased level of carbon dioxide in inspired air had no effect on MTT, but oxygen reduced leukocyte velocity and vessel diameter and carbon dioxide increased leukocyte velocity significantly. We concluded that unchanged transit time trough the retinal tissue was not due to a lack of effect of the gas provocation but a result due to concomitant changes in volume and flow. In a study on eyes of patients with diabetic retinopathy we investigated the relation between the extent of retinal circulation changes and the severity of the diabetes retinopathy (DRP). Transit times were relatively unaffected until proliferative DRP (PDRP) developed. In eyes with PDRP both MTTIR and AVP were increased. After panretinal photocoagulation treatment MTTIR returned to normal levels and vessel diameters tended to decrease while leukocyte velocity and AVP remained unchanged. We concluded that the increase in MTTIR in eyes with PDRP is at least partly explained by vessel dilation, causing an increased volume of the retinal vascular bed.

The thesis concerns polyelectrolyte gels in contact with oppositely charged proteins and surfactant micelles, and includes of four papers (I-IV). In paper I confocal Raman spectroscopy was introduced as a method to trace micelles and investigate the structure of gel-surfactant complexes, in phase separated gel spheres. In paper II, the binding of surfactants to microspheres (~50-100 µm) was investigated by means of a micromanipulator-assisted microscopy method. The two surfactants were found to display qualitative difference respect to degree of swelling, surfactant distribution in the gels, and the difference is discussed in terms of absence/presence of hydrophobic attraction to the polyelectrolyte gel network. Kinetics of volume change in gels were analyzed. Aggregation numbers of micelles in polystyrenesulfonate (PSS) solutions, obtained from fluorescence quenching measurements, are presented. In paper III, phase behaviour, protein assembly and diffusion, was studied in PSS gel microspheres. Interpretation of results was aided by measurements of osmotic swelling of individual gel networks, and by combining the results with studies of protein diffusion in macroscopic (cm-sized) gel spheres. Complexes formed were further analyzed with small angle x-ray spectroscopy. In paper IV phase behaviour of mixed ionic/nonionic surfactant micelles is investigated in cm-sized gel spheres. The coexistence of three phases, the formation of dense shells in the bulk of the gels and other phenomena are described for the first time, and the results are presented along with discussion on the charge-density of spherical micelles and of  network induced hysteresis effects in gels. The composition and microstructure of phases are investigated by confocal Raman spectroscopy and small-angle x-ray scattering respectively. The results are interpreted with aid of highly detailed theoretical model calculations.

Ce travail de thèse a permis de montrer que la résolution latérale de gravure de lasers proches infrarouges nanosecondes pouvait être réduite en faisant passer le faisceau à travers des microbilles de verre ou des fibres de silice à embouts façonnés de telle manière à générer des jets photoniques. Sur du silicium la taille de gravure peut être réduite d’un facteur 44 comparée à celle d’une gravure directe sans jet photonique. Les densités de puissances atteintes permettent même d’envisager de graver le verre avec ce type de laser malgré sa très faible absorption à ces longueurs d’onde. Pour la première fois nous avons montré la possibilité d’obtenir des jets photoniques en sortie de fibre optique à embout façonné. Nous avons montré leur capacité à graver le silicium à l’échelle du micron et ce avec un laser proche infrarouge nanoseconde. La possible utilisation de fibres optiques est une condition clairement décisive à la réalisation d’un réel procédé laser capable de graver des motifs complexes et de façon répétée. / This work has shown that the lateral resolution etching using near infrared lasers nanoseconds could be reduced by passing the beam through a glass microsphere or a silica fibers with spheroid shape in such a way to generate photonic jets. Etching on silicon size with a glass microsphere can be reduced by a factor of 44 compared to that of a direct etching without photonic jet. Powers reached densities allow even consider burning the glass with this type of laser, despite its very low absorption at these wavelengths. For the first time we have shown the possibility of obtaining photonic jets coming-out of a spheroid-tip of fiber optic. We also have shown the ability to etch silicon at the micron scale using the near infrared nanosecond laser. The possible use of optical fibers is clearly a decisive condition for the realization of a laser capable of etching process real intricate designs and repeatedly.

No description available.