Biofunctional surfaces have been under extensive research due to the numerous applications they have in science and technology. In biofunctional surfaces, different biomolecules are immobilized on an interface in order to achieve a stable and selective biorecognition capability. A key characteristic of biofunctional interfaces that is sought after is prevention of non-specific adhesion, which will lead to an improved and selective interaction between the biological elements and the surface as well as reduction of noise in the system. In this work, we developed biofunctional surfaces which simultaneously have the capability to prevent non-specific binding. For the repellant characteristics, omniphobic liquid-infused coatings were implemented which were developed by producing self-assembled monolayers (SAMs) of fluorosilanes. The biofunctional characteristics were integrated with the interface by two means: (i) producing mixed SAMs of aminosilanes and fluorosilanes to act as a bridge to chemically bind biological recognition elements while simultaneously add omniphobic characteristics, and furthermore, promote controlled biofunctionality (ii) microcontact printing patterns of proteins and further on producing SAMs of fluorosilanes on the surface, therefore resulting in an omniphobic micropatterned biofunctional surface. In order to investigate the biofunctionality, cells specific to the immobilized biomolecules were incubated on the biofunctional lubricant-infused interfaces. Here, we report that by varying the mixed SAMs ratio, we were able to control the degree of cell adherence to the interface. Furthermore, in the case of the micropatterned surfaces, we demonstrated localized cell attachment and enhanced cell specific adhesion. / Thesis / Master of Applied Science (MASc) / Biofunctional surfaces, consist of different biomolecules which are immobilized on a desired surface by various means. These surfaces have countless applications in bioengineering, leading to interdisciplinary research, such as lab on chip devices, tissue engineering, diagnostic tools, and medical implants. Therefore, preserving the biofunctionality of the surface as well as preventing non-specific adhesion are required when considering an ideal biofunctional surface. In this work, we designed and developed biofunctional omniphobic lubricant-infused interfaces in order to investigate cell adhesion and non-specific adhesion, simultaneously. This was achieved by producing mixed self-assembled monolayers of organosilanes and also by combining microcontact printing of proteins and self-assembled monolayers of fluorosilanes.

La conception de biomatériaux actifs est actuellement encouragée par le manque de thérapies régénératives efficaces pour des tissus endommagés présentant une faible capacité d’autoréparation. Les progrès récents concernant les techniques de préparation de matériaux structurés (électrospinning, microfluidique) ainsi que la découverte du fort potentiel régénératif des cellules souches ont suscité un regain d’intérêt pour des projets collaboratifs à l’interface entre biologie et sciences des matériaux. Une approche prometteuse de régénération tissulaire repose donc sur la combinaison de cellules souches et de biomatériaux implantables. Des biomatériaux innovants, injectables et servants à la fois de support aux cellules et de réservoir de molécules actives telles que des protéines ou des agents de thérapie génique (Matrice Génétiquement Activée) ont été développés. Se plaçant plus particulièrement dans le contexte de l’ingénierie du cartilage, ce travail a pour objectif de développer une stratégie complémentaire concernant l’orientation de la différenciation de cellules souches mésenchymateuses (CSM) grâce au mécanisme d’interférence ARN.La principale difficulté rencontrée lors de l’utilisation d’acides nucléiques pour induire la différenciation des CSM reste leur faible capacité à traverser les membranes cellulaires, due à leur nature hydrophile et leur charge négative. De plus, les acides nucléiques sont dégradés très facilement par les nucléases extracellulaires, ce qui rend nécessaire l’utilisation d’un vecteur. Les vecteurs non-viraux sont d’excellents candidats pour des applications in vivo en raison de leur faible coût de production et leur faible immunogénicité. Toutefois, la plupart des systèmes de vectorisation trouvés dans la littérature présentent un manque de reproductibilité associé à une cytotoxicité vis-à-vis des cellules primaires. Nous souhaitions donc développer un système de transfection synthétique à la fois efficace et biocompatible. Pour cela, nous nous sommes basés sur les résultats encourageants concernant l’utilisation des micelles de complexes polyioniques (PIC) pour la transfection des cellules dendritiques. Ces micelles sont formées par complexation de deux polyélectrolytes : un copolymère à blocs double-hydrophiles (CBDH) avec un bloc anionique et un homopolymère cationique. Dans ce travail, nous avons évalué le polyoxyde d’éthylène – b – polyacide méthacrylique en tant que CBDH et la poly-L-lysine ou le polyéthylènimine en tant que polycation. L’influence des caractéristiques des composantes (asymétrie du CBDH, nature du polycation, taille des blocs, ratio de charges…) sur les propriétés physico-chimiques des micelles formées (taille, charge de surface) a d’abord été étudiée. Puis, la possibilité de complexation d’un siRNA au sein des micelles ainsi que leur stabilité en conditions physiologiques ont été évaluées. La formulation des micelles a été conçue pour permettre une dissociation des objets à un pH comparable à celui des endosomes ; ceci a été vérifié par diffusion dynamique de la lumière. Une analyse par cytométrie en flux avec un siRNA marqué TAMRA ont démontré l’internalisation effective des micelles dans les CSM. Plus important encore, l’inhibition spécifique d’un gène cible, Runx2, a été démontrée à un niveau comparable à celui d’un vecteur commercial standard, la Lipofectamine2000®. La seconde partie de la thèse a consisté en l’élaboration de microparticules. A cet effet, nous avons préparé des microsphères de collagène par un dispositif de microfluidique, et ce à partir de diverses sources de collagène (murin, porcin, bovin). Des expériences préliminaires démontrent qu’il est possible d’imprégner les micelles dans les microsphères. De même, de premiers résultats encourageants ont été obtenus quant à la capacité du système globale à assurer l’adhésion cellulaire et permettre une transfection efficace des CSM dans un environnement 3D par les micelles PIC vectorisant un siRNA anti-Runx2. / The relative lack of efficient regenerative therapies for damaged tissues with low capacity for self-repair is one major motivation for the design of new active biomaterials. Recent progress in hierarchical materials processing techniques (electrospinning, microfluidics…) and the demonstration of the strong regenerative potential of stem cells have prompted renewed interest for collaborative projects at the biology / materials science interface. The combination of stem cells and active implantable materials has emerged as a high potential approach for the regeneration of damaged tissues. In particular, injectable cell carriers also acting as a reservoir for active molecules like proteins or gene therapy agents (Gene Activated Matrices) bring about innovative solutions to current issues in the field of tissue engineering. In the context of cartilage regeneration, the main objective of this work was to investigate a complementary strategy to orient mesenchymal stem cell (MSC) fate by the use of RNA interference. One major difficulty to reach high transfection levels and efficiently direct MSC differentiation comes from the low ability of nucleic acids (NA) to cross cellular membranes, largely due to their hydrophilicity and negative charge. This, along with a strong susceptibility to extracellular nucleases, calls for efficient gene delivery vectors. Their low production cost and low immunogenic potential make non viral vectors good candidates for in vivo applications. Besides, most systems reported in the literature show reproducibility and cytotoxicity issues with primary cells that we intended to address to achieve a safe and efficient synthetic vector for MSC. Based on previous encouraging results on the transfection of dendritic cells, we chose to investigate tripartite polyionic complex (PIC) micelles. Their formation is based on the polyelectrolyte complexation of a polyanionic double-hydrophilic block copolymer (DHBC) with a cationic homopolymer. In this work, we investigated polyethylene oxide – b – polymethacrylic acid as the DHBC and Poly-L-Lysine or Polyethyleneimine as the polycation. One major part of the work was to study the influence of micelles components characteristics (block size, DHBC asymmetry, polycation nature and molecular weight, polyelectrolyte charge ratios, etc.) on the physical characteristics (dimensions, surface charge) of the obtained nanoparticles. We then studied the ability of micelles to stably complex siRNA at high loading levels, and their stability in physiological conditions. Importantly, the PIC micelles’ formulation was designed to allow for pH-triggered disassembly in acidic conditions similar to those found in endosomes, as assessed by light scattering measurements. These nanoparticles were shown to be efficiently internalized inside MSC by flow cytometry using a fluorescently labeled SiRNA-TAMRA. Most importantly, they were shown to efficiently down-regulate Runx2 mRNA in MSC, at levels similar to those reached with the gold standard Lipofectamine2000®. The second major step for the development of a GAM suited for cartilage regeneration was to elaborate injectable microparticles. To this purpose, we prepared collagen microspheres through a microfluidic-based process and with different collagen sources (murine, bovine, and porcine). Preliminary experiments show that micelles can be efficiently loaded into the microspheres. First encouraging results were also obtained regarding the ability of the created GAM to support cell adhesion, and to allow for the efficient transfection of MSC in this 3D environment, thanks to an anti-runX2 siRNA vectorized with PIC micelles. This proof-of-concept study has demonstrated that the main elements of the nano-in-micro system are ready and mostly meet the assigned requirements. This opens the way for further work to assess the ability of this GAM to effectively improve MSC chondrogenesis and ultimately cartilage repair.

De nombreux composés bioactifs hydrophobes sont actuellement mis en avant en raison de leurs propriétés nutritionnelles et fonctionnelles. Une attention particulière est, en conséquence, portée à leur incorporation en tant qu'ingrédients dans des aliments fonctionnels. Cependant, la majorité de ces composés bioactifs sont caractérisés par une faible solubilité en milieu aqueux, une dégradation au cours des procédés de transformation ainsi qu'une absorption limitée au niveau du tractus gastro-intestinal. La micelle de caséines, grâce à ses propriétés fonctionnelles uniques, peut être considérée comme un support d’encapsulation naturel pour ces molécules bioactives hydrophobes. En effet, une des originalités de cette suprastructure est sa dynamique dans le lait se caractérisant par des échanges réversibles de protéines et de minéraux entre le sérum et la structure micellaire interne en fonction des conditions physicochimiques, et notamment avec la température. En particulier, un stockage du lait à 4°C permet la dissociation sélective de la caséine β de la phase micellaire vers la phase soluble et un retour à température ambiante permet sa réintégration. L’objectif de cette thèse est de développer une nouvelle stratégie d’encapsulation de molécules bioactives hydrophobes dans les micelles de caséines via cette dynamique de la caséine β. Dans un premier temps, l’optimisation de la dissociation de la caséine β de la micelle de caséines a été réalisée en modifiant la température et le pH, tout en portant une attention particulière au maintien de l’intégrité des micelles déplétées en caséines β. Un procédé de séparation physique de la caséine β solubilisée a été optimisé par microfiltration à l’échelle pilote. Une étude de la concentration micellaire critique de la caséine β a permis de vérifier son état monomérique à l’issue de cette séparation. Une étude de la cinétique d’interaction entre la caséine β monomérique et deux composés bioactifs hydrophobes, la curcumine et la vitamine D3, a ensuite été réalisée par résonance plasmonique de surface et par spectroscopie de fluorescence. La curcumine a été choisie pour la suite de l’étude au vu de sa bonne affinité pour la caséine β. Le complexe caséine β monomérique-curcumine a ensuite été encapsulé dans des micelles de caséines préalablement déplétées en caséines β. Les résultats de ces travaux montrent l’efficacité de cette stratégie d’encapsulation qui peut présenter un intérêt particulier pour la vectorisation de molécules bioactives hydrophobes afin d’assurer leur protection dans des produits laitiers pauvres en matière grasse.De plus, au cours de ce projet, une méthode de caractérisation des propriétés morphologiques et nano-mécaniques des micelles de caséines par microscopie à force atomique en milieu liquide a été développée. Cette méthode représente un outil intéressant de compréhension de la structure micellaire dans son environnement natif et offre la possibilité d’évaluer l’impact de certaines modifications sur les propriétés de la micelle de caséines, comme sa déplétion en caséine β ou sa réticulation. / In the last years, the number of studies highlighting the nutritional and functional properties of several hydrophobic bioactives has markedly increased. Special attention is consequently paid to their addition as ingredients to food. However, most of these hydrophobic compounds display a low aqueous solubility, poor stability during processing and low absorption in the gastrointestinal tract. Casein micelles exhibiting unique set of properties can be considered as a natural nanocarrier for these molecules. Actually, changes in environmental factors namely pH and temperature induce the dissociation of caseins and minerals from the colloidal phase to the soluble phase. Particularly, a selective dissociation of β-casein occurs at low temperatures. This effect is reversed with an increase in temperature, with a transfer of β-casein from the serum to the micelles when equilibrated at room temperature. The aim of this study is to develop a novel encapsulation strategy to incorporate hydrophobic bioactive compounds into casein micelles using the β-casein reversible dissociation. First, the β-casein dissociation from casein micelles was optimized by temperature and pH modifications while preserving the integrity of the β-casein depleted casein micelles. The separation of dissociated β-caseins from casein micelles was carried out by microfiltration at a pilot scale. The β-casein critical micelle concentration was concurrently evaluated to ensure the monomeric state of -casein after separation. Secondly, the binding kinetic between monomeric β-casein and two hydrophobic compounds, curcumin and vitamin D3, was investigated by surface plasmon resonance and fluorescence spectroscopy. Curcumin was then selected thanks to its high affinity to -casein β. The complex monomeric β-casein – curcumin was encapsulated in β-casein depleted casein micelles. The results of this study show the efficiency of this encapsulation strategy of hydrophobic bioactive compounds, which could be used to protect such molecules in low fat dairy products.Besides, during this project, a novel strategy was developed in order to evaluate the casein micelle topography and nanomechanical properties by atomic force microscopy in liquid environment. This method opens a new line of investigation to better understand the casein micelle structure in its native environment but also investigate the impact induced by the modification of physico-chemical conditions on its topography and elastic properties.

Neue Technologien der additiven Fertigung läuten auch im Bereich der Medizin, Medizintechnik und der biomedizinischen Forschung eine neue Ära ein: Über verschiedene Fertigungsverfahren wie lithographische Techniken oder Laser-Sintering ist es bereits jetzt möglich, aus biokompatiblen Materialien entsprechend eines spezifischen Designs und ohne zusätzlich nötige Fertigungsmasken dreidimensionale Implantate in anatomischer oder patienten-individueller Geometrie herzustellen. Großes Potential ergibt sich daraus beispielsweise für Lösungen zu orthopädischen Fragestellungen: Hierzu gehören u.a. die potentielle Korrektur von Fehlbildungen im mund-, kiefer- und gesichtschirurgischen Bereich, der Ersatz größerer Knochendefekte sowie der Ausgleich von kleineren Gelenkdefekten. Letztere beschreiben die Anwendung am Gelenkknorpel-Knochen-Übergang, um Eingriffen zur Implantation einer Totalendoprothese zu entgehen oder diese ggf. hinauszuzögern. Aus diesem Grund finden für entsprechende zukünftige, klinische Konzepte, sowie in den entsprechenden Forschungsbereichen unterschiedlichste Fertigungsmethoden Anwendung. Die additive Fertigung hat nicht nur das Design und die aufbauende Fabrikation von patientenindividuellen Titanimplantaten (Gander et al. 2015) durch Spritzgussverfahren revolutioniert, sondern auch den schnellen Aufbau von Strukturen aus thermoplastischen Polymeren (Probst et al. 2010) und weicheren Materialien durch extrusionsbasierte Techniken ermöglicht. Diese über Rapid Prototyping erhaltenen, individuellen Objekte können im Vergleich zu konventionellen Verfahren sehr schnell und ohne Materialausschuss auch in Kleinserien und Einzelanfertigungen individuell gefertigt werden. Die essentielle Aufgabe für den klinischen Einsatz liegt hier in der Herstellung patientenindividueller Implantate. Allerdings sind die Technologien ebenso hinsichtlich spezifischer Gewebemodelle für forschungsrelevante Fragestellungen, die mittel- bis langfristig zu Erfolgen für die biomedizinische Patientenversorgung beitragen können, von großer Bedeutung. Um hierbei eine Lücke zwischen den individualisierten, bezüglich ihrer äußeren Geometrie spezifisch designten Implantaten und einer intrinsischen Funktionalität zu schaffen, greift die biomedizinische Forschung auf die Kombination von Ansätzen aus dem Tissue Engineering (Langer & Vacanti, 1993) und den Vorteilen der additiven Fertigung sowie der damit verbundenen Konstruktion von anatomischen Geometrien zurück. Das Forschungsfeld und die Methoden der sog. Biofabrikation (Groll et al. 2016; Moroni et al. 2018) bringen die Vorteile und Konzepte dieser doch unterschiedlichen Wissenschaftsfelder zusammen, indem sie biologische Komponenten in den Fabrikationsprozess einbeziehen. [... aus der Einleitung]

This doctoral thesis was conducted in the framework of a co-supervised PhD between the Department of Agriculture, Forestry and Food Sciences of the University of Turin and the Unit of Pharmacognosy, Bioanalysis and Drug Discovery of the Faculty of Pharmacy at the Université libre de Bruxelles. The present manuscript was conceived as a thesis of articles and is composed of 9 different scientific publications. The general introduction of the work was issued from a published narrative review, while the result and discussion part is composed by eight chapters based on different scientific articles issued from the PhD project. The cocoa bean shell (CBS) is the external tegument that covers the cocoa bean, and is one of the major by-products in cocoa industry. It is normally discarded or underutilized, which could result in economical and environmental issues. However, CBS represents a notable source of polyphenols and methylxanthines (theobromine and caffeine) which can give it different biofunctionalities such as antioxidant and antidiabetic properties, among others. It also contains high amounts of dietary fiber (about 50% w/w), minerals, vitamins, and proteins. CBS has low-fat content, and interesting cocoa-aroma compounds. All this could make CBS useful as a food ingredient, and source of biofunctional compounds. The first part of the experimental work of this thesis is devoted to the chemical characterization of CBS and the establishment of its polyphenolic and volatile organic compound (aroma) profiles. The utilization of such profiles, determined by both complete characterization methods and screening methods, was also proposed for authentication purposes of CBS depending on its geographical origin and variety. In a second step, the utilization of CBS as a low-cost food ingredient for functional food production was envisaged. CBS-based beverages and biscuits were proposed as model foods. The influence of the CBS addition to the model foods was evaluated from both technological and nutritional points of view. Changes on the physicochemical characteristics of the model foods were assessed as well as their content in compounds of interest and potential biofunctionalities. Moreover, these studies served also to evaluate the effect of the different food matrices on the bioaccessibility and intestinal permeability of the bioactive compounds contained in CBS. In the third and last part of this work, a different utility was given to the study of the cocoa by-product. The antimicrobial potential of CBS was assessed against different bacterial and fungal strains and a metabolomic strategy was applied in order to individualize the putative active compounds against the Streptococcus mutans proliferation. This work was a contribution for the valorization of a high add-value product such as the CBS, and a step towards a zero-waste cocoa industry within the frame of sustainable circular economy. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

<p>The development of low-cost, and reliable platforms for on-site detection of pathogenic agents, and toxic environmental traces is still a critical need for real-time monitoring of potential environmental pollution and imminent outbreaks. The biosensors market is projected to attain 31.5 billion by 2024. In this landscape, colorimetric and electrochemical devices continue to have significant relevance, with paper-based platforms leading the point-of-care (POC) segment for pathogen detection and environmental monitoring.</p> <p>Despite the true potential of biosensors in general, they have witnessed a slow rate in commercialization, mainly due to cost restrictions, and concerns related to their reliability and repeatability once scaled-up. This research evaluates the implementation of printing techniques as a strong approach for the fabrication of paper-based and flexible electrochemical biosensors. The results obtained demonstrated the ability to control and predict the variables affecting the sensing performance, achieving high precision of the printing parameters, and allowing optimization, and iterations since very early stages of prototype development.</p> <p>Besides the novel fabrication approach, this work introduces the use of truncated aptameric DNA sequences for whole cell detection of E. coli O157:H7 and heavy metals (Hg2+ and As3+), providing evidence of high stability and robustness under harsh conditions. Results obtained demonstrate their equal or even superior performance when compared to antibodies.</p> <p>We established the use of aptamer-functionalized multilayered label particles (PEI-grafted gold decorated polystyrene) with high stability as label particles. These particles address the well known drawback of non-selective aggregation typical of traditional naked Gold nanoparticles. The outstanding stability of these multilayered labels was demonstrated when used in an enhanced version of the lateral flow assay for detection of E. coli O157:H7 (state of the art for paper-based colorimetric detection of whole cell bacteria), and in a multiplexed paper-based microfluidic device for dual detection of Mercury and Arsenic. This work sets the foundation of the development of a next generation of health care and environmental monitoring devices that are portable, sensitive, quantitative, and can reliably detect multiple targets with one single test.</p>