Dissertations / Theses on the topic 'Protein encapsulation'

Els extractes comercials de polifenols són molt utilitzats para formular aliments. La seva efectivitat, però, depèn de la seva capacitat per mantenir l’estabilitat i biodisponibilitat dels ingredients actius. L’encapsulació és una estratègia que permet aconseguir-ho. S’han investigat dos sistemes per encapsular un extracte ric en procianidines: emulsions aigua-en-oli-en-aigua (W1/O/W2) i microcàpsules sòlides (obtingudes per atomizació d’emulsions W1/O/W2). Per produir les emulsions s’utilitzà emulsificació amb membranes (ME) premix i, en particular, l’estudi es centrà en com la capa interfacial (O–W2) afecta l’estabilitat de les emulsions i l’alliberament de procianidines. Mitjançant ME premix es produïren emulsions O/W i W1/O/W2 estabilitzades amb un fracció proteica de sèrum de llet (WPI) i amb complexos WPI-carboximetil cel•lulosa (WPI–CMC), WPI-goma aràbiga (WPI–GA) i WPI–Quitosà (WPI–Chi). Mesures d’absorció mostraren que els complexos WPI–polisacàrid formen interfases de major gruix però menor densitat que les formades únicament per WPI. Les emulsions W1/O/W2 enriquides amb procianidines i estabilitzades amb complexos WPI–CMC, WPI–GA, o WPI–Chi retingueren al menys el 70% de las procianidines inicials en acabar el procés de ME premix. L’estabilitat de l’emulsió va dependre del tipus d’interfase i del pH de la fase W2. La velocitat d’alliberament de procianidines es va correlacionar amb el gruix de la interfase formada per WPI–polisacàrid: les interfases de major gruix disminueixen la velocitat d’alliberament. Totes les emulsions W1/O/W2 varen permetre produir microcàpsules de procianidines després d’una etapa d’atomització i recuperar l’estructura pròpia d’una emulsió doble en rehidratar-se. La composició de la interfase determinà la mida de partícula de les emulsions W1/O/W2 després de la rehidratació. En particular, el complex WPI–CMC fou capaç d’estabilitzar les gotes d’oli durant les distintes etapes de la producció de microcàpsules, malgrat que va retenir moderadament la migració de procianidines a través de la interfase O–W2. .<br>Los extractos comerciales de polifenoles son muy utilizados para formular alimentos. Su efectividad, sin embargo, depende de su capacidad para mantener la estabilidad y biodisponibilidad de los ingredientes activos. La encapsulación es una estrategia que permite conseguirlo. Se han investigado dos sistemas para encapsular un extracto rico en procianidinas: emulsiones agua-en-aceite-en-agua (W1/O/W2) y microcápsulas sólidas (obtenidas por atomización de emulsiones W1/O/W2). Para producir las emulsiones se empleó emulsificación por membranas (ME) premix y, en particular, el estudio se centró en cómo la capa interfacial (O–W2) afectó la estabilidad de las emulsiones y la liberación de procianidinas. Con ME premix se produjeron emulsiones O/W y W1/O/W2 estabilizadas con un fracción proteica de suero de leche (WPI) y con complejos WPI-carboximetil celulosa (WPI–CMC), WPI–goma arábiga (WPI–GA) y WPI–Quitosano (WPI–Chi). Medidas de adsorción mostraron que los complejos WPI–polisacáridos forman interfases de mayor espesor pero menor densidad que las formadas únicamente por WPI. Las emulsiones W1/O/W2 enriquecidas en procianidinas y estabilizadas con complejos WPI–CMC, WPI–GA, o WPI–Chi retuvieron al menos el 70% de las procianidinas iniciales tras el proceso de ME premix. La estabilidad de la emulsión dependió del tipo de interfase y del pH de la fase W2. La velocidad de liberación de procianidinas se correlacionó con el espesor de la interfase formada por WPI–polisacáridos: las interfases de mayor espesor disminuyen la velocidad de liberación. Todas las emulsiones W1/O/W2 permitieron producir microcápsulas de procianidinas después de una etapa de atomización y recuperar la estructura propia de una emulsión doble tras la rehidratación. La composición de la interfase determinó el tamaño de partícula de las emulsiones W1/O/W2 después de la rehidratación. En particular, el complejo WPI–CMC fue capaz de estabilizar las gotas de aceite durante las distintas etapas de la producción de microcápsulas pese a que retuvo, moderadamente, la migración de procianidinas a través de la interfase O–W2.<br>Commercial extracts rich in polyphenols are extensively used to formulate foods. Nevertheless, their effectiveness depends on preserving the stability and bioavailability of the active ingredients. Encapsulation is a strategy to meet these requirements. We have investigated two encapsulation systems to entrap a procyanidin-rich extract: water-in-oil-in-water (W1/O/W2) emulsions and solid microcapsules (spray dried W1/O/W2 emulsions). While premix membrane emulsification (ME) was used to produce these emulsions, we focused on how the interfacial layer (O–W2) affected emulsion stability and procyanidin release. Premix ME enabled to produce single and double emulsions stabilized with whey protein isolate (WPI), and WPI–Carboxymethyl cellulose (WPI–CMC), WPI–Gum Arabic (WPI–GA) and WPI–Chitosan (WPI–Chi) complexes. Adsorption measurements showed that WPI–polysaccharides interfaces form thicker but less dense layers than only WPI. In O/W emulsions, different interfacial structures made of WPI and CMC led to large differences in lipid oxidation. The negatively charged droplets of the emulsions stabilized with WPI–MC would attract positively charged transition metals, promoting lipid oxidation. Procyanidin-loaded W1/O/W2 emulsions stabilized by WPI–CMC, WPI–GA, or WPI–Chi complexes retained at least 70% of the initial procyanidins at the end of premix ME. Emulsion stability depended on the interfacial layer and the pH of the W2 phase. The release rate of procyanidins was correlated to the interfacial thickness of the WPI–polysaccharide layer: thicker layers lowered the release rate. All the W1/O/W2 emulsions were able to produce procyanidin-loaded microcapsules after spray drying and could all recover their W1/O/W2 emulsion structure upon rehydration. The interfacial composition affected particle size of W1/O/W2 emulsions after microcapsule rehydration. Particularly, WPI–CMC complex was able to truly stabilize the W1/O droplets during the different stages of microcapsule production although it moderately retained the migration of procyanidins through the O–W2 interface. For each type of encapsulation system, a tailor-made hydrophilic emulsifier is required to comply with the type of protection needed, the addenda used and the delivery conditions.

Using protein design, a series of metal binding proteins have been designed, allowing the local factors that contribute to metal affinity and thermostability to be studied. Those proteins with the highest metal binding affinities had the lowest apo-form Tm and the largest ÄTm upon metal binding. In this thesis, major steps have been taken toward applying the engineered protein to MR imaging. The progress of magnetic resonance imaging is hindered by low specificity and rapid elimination of FDA-approved MRI contrast agents. The engineered protein contrast agent has been conjugated to a cancer-specific targeting peptide and encapsulated in albumin microspheres to provide tandem passive and active tumor targeting. Also, a simple, high-yield purification method has been developed.

One of the most challenging goals in nanoparticle research is to develop successful protocols for the large-scale, simple and possibly low-cost preparation of morphologically pure nanoparticles with enhanced properties. The work presented in this thesis was focused on the synthesis, characterisation and testing of magnetic nanoparticles and their potential applications. There are a number of magnetic nano-materials prepared for specific applications such as metal oxide nanoparticles encapsulated with various porous materials including Fe₃O₄/Fe₂O₃ coated with soft bio-organic materials such as glycol chitosan and bovine serum albumin and hard materials such as silica (SiO₂) and zinc sulphide (ZnS). The preparation of these materials was achieved principally by bottom-up methods with different approaches including micro-emulsion, precipitation, electrostatic and thermolysis processes. The thesis also presents the uses of various analytical techniques for characterising different types of nano-materials including Attenuated Total Reflection Fourier Transformer Infrared Vibrational Spectroscopy (ATR-FTIR), Ultraviolet Visible- Near Infrared (UV-Vis-NIR) Spectroscopy, Zeta Potentiometric Surface Charge Analysis, Superconducting Quantum Interference Device (SQUID) and Vibration Sample Magnetometry (VSM) for magnetic analysis and powder X-Ray Diffraction (XRD) for crystallographic pattern analysis. There are many applications of magnetic nanoparticles, including nano-carriers for biological and catalytic reagents. The magnetic nanoparticles can facilitate separation in order to isolate the carriers from solution mixtures as compared to many inefficient and expensive classic methods, which include dialysis membrane, electrophoresis, ultracentrifugation, precipitation and column separation methods. There are six key chapters in this thesis: the first chapter introduces the up-to-date literature regarding magnetic nano-materials. The uses of magnetic nano-materials in drug binding and for protein separation are discussed in the second and third chapters. The fourth chapter presents the use of magnetic nanoparticle in conjunction with a photo-catalytic porous overlayer for the photo-catalytic reduction of organic molecules. The fifth chapter describes different analytical techniques used for the characterisation of nanoparticles and the underlying principles and the experimental details are also given. The sixth chapter summarises the results and provides an overview of the work in a wider context of future applications of magnetic nanoparticles.

Les aliments fonctionnels connaissent aujourd’hui un intérêt grandissant. Pour leur formulation, l'encapsulation de bioactifs représente une voie intéressante et les protéines de lactosérum (PS) présentent de bonnes potentialités en tant qu'agents d’encapsulation. Des travaux antérieurs ont démontré que deux PS, la beta-lactoglobuline (BLG) et la lactoferrine (LF), peuvent former spontanément des co-assemblages par coacervation complexe, une technologie d'encapsulation connue. Cette thèse étudie la coacervation BLG-LF pour l'encapsulation d'un bioactif modèle, la vitamine B9. En testant une gamme de pH et de ratios molaires, les conditions optimales de coacervation B9-PS sont obtenues dans l'eau, à pH 5,5, avec un ratio molaire LF:B9:BLG de 1:5:10, permettant d’atteindre des rendements de coacervation de 45 à 55%, et d’encapsulation de B9 de 98 %.L’échelle de production des coacervats est augmentée avec succès du µL au L, avec des solutions protéiques de qualité commerciale et un mélangeur statique. Les rendements de coacervation et d’encapsulation sont conservés, avec l’encapsulation d’environ 4 mg de B9/g coacervats. Par ailleurs, les coacervats montrent un effet protecteur de la forme native de B9 vis-à-vis des UV, de l’oxydation et pendant la lyophilisation. Une étude in vivo chez le rat démontre une augmentation de la biodisponibilité de B9 lorsque administrée sous forme de coacervats. Les coacervats apparaissent stables lorsqu’ils sont resuspendus en gouttelettes dans du lait. Ce travail permet d’approfondir les connaissances sur la coacervation hétéroprotéique et s<br>Encapsulation of bioactives is relevant for the development of functional foods. Food proteins as encapsulating agents could match the objective of industries to develop “clean label” products. Moreover, whey proteins (WP) exhibit good potentialities as encapsulating agents. Previous works have demonstrated that the WPs, beta-lactoglobulin (BLG) and lactoferrin (LF), are able to spontaneously co-assemble by complex coacervation. This study explores the ability of BLG-LF coacervates as a potential carrier for the encapsulation of a model bioactive, vitamin B9. Throughout screening experiments, we determined the domains where B9-WP coacervation occured according to a tested range of pH, proteins and vitamin concentrations and molar ratios. Optimal conditions for coacervation were found in water, at pH 5.5, with LF:B9:BLG molar ratio of 1:5:10, affording coacervation yields of 45 to 55% and B9 encapsulation up to 98%.Coacervation was scaled-up from laboratory to bench scale using commercial-grade protein sources and static mixing. Final efficiencies were obtained with coacervates containing 4 mg of B9/g coacervates. Under degradative conditions (UV light irradiation, oxidation, freeze-drying), WP coacervates provided good protective properties limiting chemical degradation of native B9. In vivo oral administration of B9-WP coacervates in rats enhanced the plasmatic concentrations of B9 compared to unencapsulated B9. In addition, good physical stability over time was found after incorporated and resuspension of formed coacervates in milk. The combined results of this thesis p

Les gradients de concentration de peptides ou de protéines, ou leur relargage localisé, jouent un rôle primordial dans les voies de communication inter-cellulaire. L'activation locale de cellules est étudiée in vitro à l'aide de sources artificielles contraignantes ou invasives de protéines (pipettes, dispositifs microfluidiques). Des méthodes plus douces et moins invasives sont très demandées. À cet effet, nous avons développé deux types de capsules capables de libérer des macromolécules sous l'effet d'une stimulation extérieure. Le premier système emploie des liposomes additionnés d'un amphiphile à azobenzène, tensioactif ou polyélectrolyte, capable de générer des pores à travers la membrane sous l'effet de la lumière. Les temps de dissolution des assemblages lipides/tensioactifs et les cinétiques de relargage (perméabilité) sous irradiation lumineuse ont été étudiées par diffusion dynamique de la lumière et fluorescence. Le second système repose sur des capsules à cœur aqueux et à coque polymère, formées par polyaddition interfaciale. Nous avons montré que l'inclusion de chaînes thermosensibles dans la membrane (polyNIPAM, par ex.) rendait la stabilité de la capsule dépendante de la température. Nous avons démontré sur des capsules millimétriques, chargées avec du dextrane ou des protéines, que cela permettait le contrôle du relargage. L'utilisation de polymère à UCST en milieu aqueux nous a permis d'obtenir les premières capsules thermosensibles capables de libérer leur contenu par élévation de la température au dessus d'une valeur critique. Cela ouvre une voie prometteuse au développement d'un système biocompatible de libération de protéines<br>Concentration gradients and local delivery of peptides or proteins play a crucial role in intercellular communication. In vitro, the effects of local activation of cells are studied with constrained or invasive artificial protein sources (pipettes, microfluidics). Milder and remotely-triggered techniques for the release of encapsulated biomolecules are highly in demand. To this aim we developed two classes of capsules able to release macromolecules upon an external stimulation. The first system is based on liposomes sensitized with azobenzene-containing amphiphiles (surfactants or polyelectrolytes) that can open pores in the membrane upon exposure to light. The dissolution time of lipids/surfactants assemblies and rate of release (permeability) under light irradiation has been assessed by dynamic light scattering and fluorescence measurements. The second system is a model of capsules with an aqueous core and a polymer shell, formed by interfacial polyaddition. We showed that inclusion of temperature-responsive chains in the membrane, e.g. polyNIPAM, confers temperature-dependant stability to the capsules; we demonstrated with millimeter-sized capsules loaded with dextran or proteins that this can be used to trigger the release. Using chains with UCST in water, we obtained the first temperature-sensitive capsules able to release their content upon increasing the temperature above a threshold. This represents a promising route to the biocompatible delivery of proteins

Cell-free protein synthesis (CFPS) is a powerful protein expression platform where protein synthesis machinery is borrowed from living organisms. Target proteins are synthesized in a reaction tube together with cell extract, amino acids, energy source, and DNA. This reaction is versatile, and dynamic optimizations of the reaction conditions can be performed. The "œopen" nature of CFPS makes it a compelling candidate for many technologies and applications. This dissertation reports new and innovative applications of CFPS including 1) enzyme encapsulation in a virus-like particle, 2) detection of endocrine disrupting chemicals in the presence of blood and urine, and 3) expression of a multi-disulfide bond therapeutic protein. Two major limitations of enzymes are their instability and recycling difficulty. To overcome these limitations, we report the first enzyme encapsulation in the CFPS by immobilizing in a virus-like particle using an RNA aptamer. This technique allows simple and fast enzyme production and encapsulation We demonstrate, for the first time, the Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting endocrine disrupting chemicals (EDCs) in human blood and urine samples. Current living cell-based assays can take a week to detect EDCs, but RAPID requires only 2 hours. It utilizes the versatile nature of CFPS for biosensor protein complex production and EDC detection. Biotherapeutic protein expression in E. coli suffers from inclusion body formation, insolubility, and mis-folding. Since CFPS is not restricted by a cell wall, dynamic optimization can take place during the protein synthesis process. We report the first expression of full-length tissue plasminogen activator (tPA) using CFPS. These research works demonstrate the powerful and versatile nature of the CFPS.

In food and pharma industry, fish oils are highly demanded due to the many associated health benefits of omega-3 fatty acids. However, the delivery of fish oil through food is a major challenge as this oil is susceptible to oxidation and to produce off-flavors. To prevent these undesired effects, microencapsulation techniques are used. Typically oil encapsulation is carried out by two steps (i) emulsion preparation (ii) drying. The main objective is to study the effect of the emulsification method, emulsion formulation, membrane type and microcapsule formulation on relevant physic-chemical parameters of fish oil microcapsules.The approach of the project is to combine, for the first time, the advantages of using a low energy emulsification technique (membrane emulsification) and spray drying to obtain food-grade fish oil microcapsules. The results show a clear improvement in the oil encapsulation efficiency (OEE) when decreasing the droplet size of the emulsion and increasing the amount of wall material. The combination of a polysaccharide with a protein has been found to improve protection against oxidation during storage of the microcapsules, while the addition of denatured proteins as a part of the microcapsule wall material enhances OEE but does not improve the mechanical strength of the microcapsules.<br>El aceite de pescado es altamente valorado en la industria alimentaria por su demostrada actividad en la prevención y tratamiento de numerosas patologías, asociada a su contenido en ácidos grasos omega-3. La incorporación de aceite de pescado en alimentos presenta algunas dificultades relacionadas con su rápida oxidación y su característico aroma y sabor. La encapsulación del aceite de pescado retrasa la oxidación y permite enmascarar sus propiedades sensoriales. Tradicionalmente, la encapsulación se lleva a cabo combinando una etapa de emulsificación seguida de secado por atomización. El objetivo principal del trabajo es estudiar el efecto del método de emulsificación y la formulación de la emulsión y las microcápsulas en los parámetros físico-químicos más relevantes de las microcápsulas. En este proyecto se combina por primera vez la emulsificación por membranas con el secado por atomización para obtener microcápsulas de aceite de pescado aplicables a la industria alimentaria. Los resultados muestran una clara mejora en la eficiencia de encapsulación del aceite cuando se reduce el tamaño de gota de la emulsión y se incrementa la cantidad de material de pared de las microcápsulas. La combinación de un polisacárido con una proteína para la formación de la pared mejora la estabilidad oxidativa de las microcápsulas durante el almacenamiento. Por otra parte la adición de proteínas desnaturalizadas para reforzar las paredes de las microcápsulas ha resultado en una mejora de la eficiencia de encapsulación de aceite pero no ha mejora su resistencia mecánica

La thérapie cellulaire encapsulée présente un grand potentiel pour délivrer des niveaux soutenus de protéines thérapeutiques aux patients et améliorer la gestion des maladies chroniques. Cependant, le saut translationnel vers le succès clinique est entravé par le manque de lignée cellulaire allogénique adaptée aux applications thérapeutiques humaines, ainsi que le manque d'un dispositif d'encapsulation polyvalent, biocompatible, et facile à administrer, à récupérer ou à remplacer. Dans ce travail, nous présentons le développement d'un dispositif de macroencapsulation biocompatible, cargo-agnostique et doté de caractéristiques optimisées pour l'administration de protéines. Ce dispositif est compatible avec les cellules adhérentes et en suspension, et peut être administré et récupéré sans procédures chirurgicales lourdes. Nous avons montré que différentes lignées cellulaires produisant différentes protéines thérapeutiques peuvent être combinées dans le dispositif. Parallèlement, nous avons généré une lignée cellulaire de myoblastes humains immortalisés (IHM) stable qui peut être modifiée génétiquement pour sécréter diverses protéines fonctionnelles. Nous avons encapsulé ces deux composants et évalué leur combinaison à la fois in vitro et in vivo pour la délivrance de protéines à long terme. Nous avons démontré la capacité des cellules IHM sécrétant une cytokine, encapsulées dans notre dispositif et implantées dans la peau humaine, à mobiliser et à activer les cellules présentatrices d'antigènes, ce qui pourrait potentiellement servir de stratégie adjuvante efficace dans les thérapies d'immunisation contre le cancer. Enfin, nous avons étudié l'efficacité de l'administration soutenue de faibles doses d'anti-CTLA-4 humain délivré par nos cellules IHM encapsulées dans notre dispositif, dans la région péritumorale de souris hCTLA4 knock-in greffées par voie sous-cutanée avec des cellules d'adénocarcinome du côlon MC38. Dans l'ensemble, nos résultats montrent que notre technologie peut contribuer aux thérapies cellulaires contre le cancer et, potentiellement, contre les troubles métaboliques et les maladies dues à une déficience en protéines<br>Encapsulated cell therapy (ECT) holds a great potential to deliver sustained levels of highly potent therapeutic proteins to patients and improve chronic disease management. Yet the translational leap to clinical success has been hindered by the scarcity of an allogeneic cell line suitable for human therapeutic applications along with an adapted versatile and biocompatible encapsulation device that is scalable, and easy to administer, retrieve, or replace. Here, we report on the development of a biocompatible, cargo-agnostic, macroencapsulation device with optimized features for protein delivery. Such device is compatible with adherent and suspension cells, and can be administered and retrieved without burdensome surgical procedures. We showed that different cell lines producing different therapeutic proteins can be combined in the device. In parallel, we have generated a stable, immortalized human myoblast (IHM) cell line that can be genetically engineered to secrete diverse functional proteins. We performed ECT using these two components and assessed their combination both in vitro and in vivo for long term, sustained protein delivery. We demonstrated the ability of cytokine-secreting IHM cells encapsulated in our device and implanted in human skin to mobilize and activate antigen-presenting cells, which could potentially serve as an effective adjuvant strategy in cancer immunization therapies. Finally we investigated the efficacy of sustained, low-dose human anti-CTLA-4 delivered by genetically engineered IHM encapsulated in our device in the peritumoral region of human CTLA4 knock-in mice subcutaneously engrafted with MC38 colon adenocarcinoma cells. Overall, our results support that our technology may contribute to cell therapies for cancer, and potentially metabolic disorders, and protein-deficient diseases

Orientador: Rosiane Lopes da Cunha<br>Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos<br>Made available in DSpace on 2018-08-23T01:16:34Z (GMT). No. of bitstreams: 1 Kuhn_KatiaRegina_D.pdf: 3588047 bytes, checksum: 56035122a0c4826c829bf99675b3f00c (MD5) Previous issue date: 2013<br>Resumo: A encapsulação de compostos bioativos vem sendo utilizada como uma alternativa para minimizar a degradação destes ingredientes durante o processamento, armazenamento e/ou processo digestivo, permitindo o aumento da vida de prateleira dos alimentos e a liberação controlada destes compostos. Nesse sentido, o objetivo geral deste trabalho foi produzir microgéis a partir da extrusão de emulsões O/A contendo isolado protéico de soro de leite (IPS) e gelana em uma solução gelificante de cloreto de cálcio visando a encapsulação e liberação controlada de óleo e hidrolisado protéico da linhaça. Na primeira parte deste estudo, a influência das condições de homogeneização (pressão e número de passagens) no preparo de emulsões estabilizadas por IPS foi avaliada com o intuito de obter sistemas mais estáveis e com menor oxidação lipídica. Todos os sistemas foram estáveis à cremeação e um aumento da pressão de homogeneização para 800 bar e do número de passagens para até 3 vezes, diminuiu o tamanho médio de gota das emulsões. Condições extremas de homogeneização levaram à formação de agregados protéicos de alta massa molecular (>200 kDa), favorecendo o aumento na viscosidade das emulsões. Com o aumento da pressão, uma distribuição de tamanho de gotas bimodal, indicando que coalescência pode ter ocorrido, e um aumento na formação de produtos primários da oxidação foram observados. Na segunda etapa do trabalho, avaliou-se o potencial do isolado protéico da linhaça (IPL) como agente emulsificante em sistemas puros e mistos com proteínas do soro de leite preparados sob alta pressão de homogeneização. As emulsões estabilizadas por IPL ou IPSIPL mostraram-se cineticamente instáveis e o aumento da concentração de IPL e das condições de homogeneização melhoraram a estabilidade dos sistemas puros, o que foi atribuído à sua maior viscosidade. No entanto, a maior estabilidade foi obtida com a adição de IPS nas emulsões contendo menor concentração de IPL (0,14% m/v) e utilizando condições mais drásticas de homogeneização. Por último, microgéis de IPS e gelana foram produzidos a partir da gelificação iônica de emulsões visando a encapsulação de compostos bioativos da linhaça. Os microgéis foram avaliados quanto à estabilidade, resistência e liberação destes compostos bioativos através da simulação in vitro do processo digestivo. Os resultados mostraram que óleo e hidrolisado protéico da linhaça foram encapsulados e que os microgéis resistiram às condições gástricas, mas foram desintegrados no meio intestinal. Além disso, a adição de hidrolisado diminuiu o tamanho das partículas e parece ter auxiliado na encapsulação do óleo de linhaça. Sendo assim, os microgéis produzidos poderiam ser utilizados para a proteção e liberação controlada dos compostos bioativos encapsulados<br>Abstract: The encapsulation of bioactive compounds has been used as an alternative to minimize degradation of these ingredients during processing, storage and/or digestive process, allowing increased shelf life of foods and the controlled release of these compounds. In this way, the general purpose of this work was to produce microbeads from extrusion of the O/W emulsions containing whey protein isolate (WPI) and gellan into a gelling solution of calcium chloride aiming the encapsulation and controlled release of oil and protein hydrolysate from flaxseed. In the first part of this study, the influence of homogenization conditions (pressure and number of passes) in the preparation of emulsions stabilized by WPI was assessed in order to obtain more stable systems and decreased lipid oxidation. All the systems were stable to creaming and an increase of homogenization pressure to 80 MPa and the number of passes up to 3 times, decreased the mean droplet size of the emulsions. Extreme homogenization conditions led to the formation of high molecular weight protein aggregates (>200 kDa), favoring the increase in viscosity of the emulsions. Increasing pressure, a bimodal droplets size distribution, indicating droplets coalescence, and an increase in the formation of primary oxidation products were observed. In the second step of the work, the potential of flaxseed protein isolate (FPI) as an emulsifying agent was evaluated in pure systems and mixed with whey proteins prepared under high pressure homogenization. The emulsions stabilized by FPI or WPI-FPI were kinetically unstable and the increase of FPI concentration and homogenization conditions improved the stability of pure systems, which was attributed to its higher viscosity. However, the greatest stability was achieved with the WPI addition in the emulsions containing the lowest FPI concentration (0.14% w/v) and using more drastic homogenization conditions. Finally, WPI-gellan microgels were produced by ionic gelation of the emulsions aiming the encapsulation of bioactive compounds from flaxseed. Microgels were evaluated in relation to stability, resistance and release of these bioactive compounds by simulating in vitro digestion process. The results showed that oil and protein hydrolysate from flaxseed were encapsulated and that microbeads resisted to gastric conditions, but were disintegrated in intestinal medium. Furthermore, the hydrolysate addition decreased the particle size and seems to have contributed for the flaxseed oil encapsulation. Thus, microbeads produced could be used for protection and controlled release of the encapsulated bioactive compounds<br>Doutorado<br>Engenharia de Alimentos<br>Doutora em Engenharia de Alimentos

In novel drug delivery system, the encapsulation of therapeutic cells in microparticles has great promises for the treatment of a range of health con- ditions. Therefore, the encapsulation material and technology are of great importance to the validity and efficiency of the advanced medical therapy. Several unsolved challenges in regards to versatile microparticle synthesis ma- terials and methods form the main obstacle for a translation of novel cell therapy concepts from research to clinical practice. Thiol-ene based polymer systems have emerged and gained great popular- ity in material development in general and in biomedical applications specif- ically. The thiol-ene platform is broad and therefore of interest for a variety of applications. At the same time, many aspects of this material platform are largely unexplored, for example material and manufacturing technology developments for microfluidic applications . In this Ph.D. thesis, thiol-ene materials are explored for use in cell encap- sulation. The marriage of these two technology fields breeds the possibility for a novel microfluidic cell encapsulation approach using a novel encapsulation material. To this end, several new manufacturing technologies for thiol-ene and thiol-ene-epoxy droplet microfluidic devices were developed. Moreover, core-shell microparticle synthesis for cell encapsulation based on a novel co- synthesis concept using a thiol-ene based material was developed and inves- tigated. Finally, a thiol-ene-epoxy system was also used for the formation of microwells and microchannels that improve protein analysis on microarrays. The first part of the thesis presents the background and state-of-the-art technologies in regards to cell therapy, microfluidics, and thiol-ene based ma- terials. In the second part of the thesis, a novel manufacturing approach of thiol-ene-epoxy material as well as core-shell particle co-synthesis in micro- fluidics using thiol-ene based material are presented and characterized. The third part of the thesis presents the cell viability studies of encapsulated cells using the novel encapsulation material and method. In the final part of the thesis, two applications of thiol-ene-epoxy gaskets for protein detection mi- croarrays are presented.<br>Inkapsling av levande celler i mikrokapslar för terapeutiska ändamål är mycket lovande för frmatida behandling av många olika sjukdomar. Emeller- tid är en behandlings effektivitet i hög grad beroende av vilka material som används för inkapsling och vilken teknisk lösning som används för att ska- pa mikrokapslarna. För närvarande återstår det många utmaningar för att omvandla grundforskningresultat till klinisk verklighet, vilken kräver mer än- damålsenliga tillvägagångssätt för att tillverka mikrokapslar i material som är kompatibla med användningsområdena. De senaste åren har tiol-en baserade polymerer har blivit mycket använda för materialutveckling i stort och för biomedicinska tillämpningar i synnerhet. Med tiol-en kemi kan en mycket stor mängd helt olika syntetiska material framställas, vilket gör tiol-ener intressanta för en mängd applikationer. För närvarande är dock mycket inom denna materialklass outforskat, t.ex. inom material och tillverkningmetodik för mikrofluidiktillämpningar. I denna avhandling används tiol-ener för cellinkapsling. Sammanslagning av dessa teknologier möjliggör en ny typ av cellinkapsling med nya materi- alegenskaper. En mängd olika tillverkningssätt där tiol-en eller tiol-en-epoxi används för droplet-mikrofluidiksystem utvecklades. Core-shell mikrokapsel- syntes för cell-inkapsling baserat på en ny metod för samtidig syntes av både core och shell utvecklades och karaktäriserades. Slutligen utvecklades ett tiol- en-epoxi system för enkel integrering med proteinmikroarrayer på objektsglas. I avhandlingens första del presenteras bakgrund och dagens bästa teknolo- gier för terapeutisk cellinkapsling, mikrofluidik och tiol-en baserade material. I avhandlingens andra del presenteras en ny tillverkningsmetod för mikro- strukturerade tiol-en-epoxi artiklar och samtidig syntes av core och shell för mikrokapslar med användande av mikrofluidik. I den tredje delen presenteras cellöverlevandsstudier för de celler som inkapslats med de nya materialen och de nyutvecklade metoderna. I den avslutande delen beskrivs två specifika fall där tiol-en-epoxi komponenter används för proteindetektion och mikroarrayer.<br><p>QC 20171122</p>

L'Imagerie par Résonance Magnétique (IRM) est une technique prometteuse largement répandue dans les milieux hospitaliers. Elle est non invasive, présente une bonne résolution spatiale et permet de visualiser en profondeur dans un organisme vivant. Elle possède cependant quelques défauts, dont sa faible sensibilité. Pour palier ce problème, il est possible d'utiliser des espèces hyperpolarisables telles que le xénon. Cependant, n'étant spécifique d'aucun récepteur biologique, le xénon nécessite d'être vectorisé. Pour ce faire, des auteurs ont proposé son encapsulation dans une cage moléculaire capable de reconnaître la cible biologique à imager. Les meilleurs candidats à ce jour sont les cryptophanes.Nous nous sommes fixés comme objectif dans cette thèse de concevoir et de synthétiser de nouvelles cages plus adaptées pour les applications en IRM 129Xe ainsi que des biosondes pertinentes pour se rapprocher d'applications in vivo. Dans une première partie de ma thèse, nous nous sommes intéressés au développement de nouvelles cages afin d'étudier et d'affiner les propriétés d'encapsulation du xénon au sein des cryptophanes. Dans les parties suivantes, nous nous sommes concentrés sur la conception de biosondes par fonctionnalisation de cryptophanes déjà décrits pour diverses applications d'intérêt biologique. D'une part, nous avons évalué la possibilité de détecter des métaux de manière plus spécifique et plus sensible grâce à l'IRM xénon hyperpolarisé. D'autre part, nous avons travaillé sur la conception de biosondes bimodales, afin de coupler des techniques complémentaires d'imagerie médicale.

This thesis has developed an innovative technology, electrospraying, that allows biodegradable microparticles to deliver pharmaceuticals that aid bone regeneration. The establishment, characterisation and optimisation of the technique are a step forward in developing an affordable and safe alternative to the products used currently in the clinical setting for the treatment of musculoskeletal disorders. The researcher has also investigated electrospraying as a coating technique on biodegradable structures that are used to replace damaged tissues, in order to provide localised and efficient drug delivery in the site of the defect to help tissue reconstruction.

Nous avons cherché à évaluer l'impact de conditions opératoires pour la préparation d'agrégats protéiques et d'émulsions stabilisées par des protéines en vue de la protection de l'α-tocopherol, servant de modèle de molécules d'intérêt, hydrophobes et sensibles. Les matrices protéiques ont été formées à partir d'un concentrat de protéines de lactosérum (6 wt% de WPC, pH 6.5 et 65 à 75°C), en présence ou absence de 4% α-tocopherol. Le mélange (65°C -15 min) des protéines en solution sans ou avec α-tocopherol a donné lieu à la formation de particules avec modification de charge (de -42 à -51 mV) et de taille (de 183 à 397 nm). Ces paramètres ont diminué davantage sous l'effet d'homogénéisation sous haute pression à 1200 bar que à 300 bar, alors qu'une meilleure protection de l'α-tocopherol a été observée après 8 semaines conservation. Les mécanismes impliqués dans la formation des matrices protéiques correspondantes ont été décrits sur la base de procédés de dénaturation-agrégation de protéines sériques, à partir de résultats obtenus par calorimétrie différentielle à balayage (DSC), spectrofluorescence, diffusion multiple de la lumière et électrophorèse SDS-PAGE. Les matrices lipidiques ont été préparées à partir de phases aqueuses contenant (6 wt% or 3 wt% de WPC) et lipidiques (20 %) en présence ou absence de lécithines (1.5%) avec ou sans α-tocopherol (4%), et par application d'une première étape de dispersion (65°C - 15 min) suivie d'une homogénéisation sous pression à 300 ou 1200 bar. Les nanoparticles lipidiques formées à plus haute pression étaient de taille et concentration protéique de surface plus faibles et de degré d'encapsulation de l'α-tocopherol plus faible (près de 15 %). L'analyse par DSC en modes balayage et isothermes des particules lipidiques a montré que plus leur taille est faible, plus le sur-refroidissement est important, l'apparition des cristaux de matière grasse plus retardée, et leur développement à 4°C moins important. Ces effets sont accentués dans les gouttelettes contenant l'α-tocopherol. La diffraction aux grands et petits angles de rayons X (synchrotron Soleil), couplée à la DSC, a montré la co-existence des polymorphes 2Lα, 2Lβ' et 2Lβ dans toutes les émulsions, mais à des proportions différentes. Les cristaux 2Lβ étaient plus développés dans les gouttelettes de plus petite taille et contenant du tocopherol en présence de lécithins, celles qui présentaient la plus forte dégradation chimique d'α-tocopherol pendant une conservation à long-terme<br>We investigated effects of processing conditions for the preparation of protein aggregates and protein-stabilized lipid droplets, as matrix carriers of sensitive lipophilic bioactive compounds, with α-tocopherol as a model. Protein-based matrices were formed from whey protein concentrate (6 wt% WPC, pH 6.5 and 65 to 75°C), in presence or absence of 4% α-tocopherol. Mixing the protein solutions without or with α-tocopherol (65°C for 15 min) led to changes in particle surface charges (from -42 to -51 mV) and sizes (from 183 to 397 nm). These parameters decreased more under further high pressure homogenisation at 1200 bar than 300 bar, in parallel with increased vitamin protection over 8 week's storage. Molecular mechanisms involved in formation of corresponding α-tocopherol-loaded protein matrix were described on the basis of heat- and high-pressure-induced whey protein denaturation and aggregation, as evidenced by differential scanning calorimetry (DSC), spectrofluorescence, multi-light scattering and SDS-PAGE electrophoretic patterns. Lipid-based matrices were developed from aqueous phases (80 wt%) containing WPC (6 wt% or 3 wt%) and lipid phases (20 wt%) in presence or absence of lecithins and/or 4% α-tocopherol, and by using a first dispersion step (65°C for 15 min) followed with HPH at 300 or 1200 bar. Our results showed that increasing HPH was accompanied by formation of lipid nanoparticles with decreasing size and protein surface concentration with an increase in α-tocopherol degradation (up to 15 wt% for 1200 bar). DSC in scanning and isothermal modes showed that reduction in lipid droplet size was accompanied by retardation in crystalline fat development under storage at 4°C, with further reduction in crystalline fat development along with further increase in supercooling for lipid droplets containing α-tocopherol. Fat polymorphism observed using time-resolved synchrotron X-ray scattering at wide and small angles (WAXS and SAXS) coupled with DSC, showed co-existence of 2Lα, 2Lβ' and 2Lβ polymorphs in all the emulsions, but at different proportions. It was observed that 2Lβ polymorphs were more prominent in lipid droplets with lower size and containing α-tocopherol in presence of lecithins that were shown to present the lowest long-term stability of α-tocopherol against chemical degradation

Les nanoémulsions (NEs) huile/eau peuvent être utilisées en tant que systèmes de délivrance des médicaments pour l’encapsulation des substances actives hydrophobes afin d’améliorer leur stabilité et leur biodisponibilité. Néanmoins, leur stabilisation nécessite l’utilisation de concentrations plus importantes de tensioactifs par rapport aux émulsions conventionnelles en raison de l’augmentation de la surface spécifique. La plupart des tensioactifs synthétiques couramment utilisés dans la formulation des émulsions sont potentiellement irritants, voire toxiques. Cela entrave l'application thérapeutique des NEs en particulier pour les traitements à long terme. L'objectif de cette thèse est alors de formuler des NEs pharmaceutiques huile/eau stabilisées par un biopolymère, la beta-lactoglobuline (beta-lg), à la place des tensioactifs synthétiques.Les NEs ont été préparées par homogénéisation à haute pression (HHP). La composition de la formulation et les conditions du procédé ont été optimisées afin d’obtenir des gouttelettes nanométriques dans des NEs stables. Les résultats ont montré que les NEs les plus stables, avec une taille de gouttelettes &lt; 200 nm, ont été obtenues quand 5 m/m% de l’huile ayant la viscosité la plus faible ont été utilisés en tant que phase huileuse, 95 m/m% de la solution de beta-lg à une concentration de 1 m/m% ont été utilisés en tant que phase aqueuse et 4 cycles d’HPH de 100 MPa ont été appliqués. Cette formulation a été stable contre les phénomènes de croissance de gouttelettes pendant au moins 30 jours grâce à un film interfacial quasiment purement élastique. La gomme xanthane, un polysaccaride naturel, a été ajoutée à la formulation optimale à une concentration de 0,5 m/m% en tant qu’agent épaississant. Cela a permis d’obtenir une texture crémeuse avec un comportement rhéofluidifiant. Dans cette dernière formulation, la vitesse de migration des gouttelettes a été considérablement réduite et la stabilité des NEs a été améliorée.Les effets du procédé d’HPH sur les différents niveaux de structure de la protéine ont été évalués à l’aide de méthodes spectroscopiques, chromatographiques et électrophorétiques. L’influence de ce traitement sur ses propriétés interfaciales et émulsionnantes a également été étudiée. L’efficacité émulsionnante optimale a été obtenue quand les conditions d’HPH n’ont pas altéré la structure de la beta-lg, ni ses propriétés interfaciales. Néanmoins, un traitement d’HHP excessif (300 MPa/5 cycles) a induit des modifications structurelles, principalement une transformation des feuillets beta en structures désordonnées, une large perte dans le cœur hydrophobe, et une agrégation importante par des liaisons disulfure intermoléculaires. La beta-lg modifiée par l’HHP a montré une hydrophobie de surface plus importante conduisant à une vitesse d’adsorption à l’interface huile/eau plus élevée et une formation plus précoce d’un film interfacial. La dénaturation de la protéine par ce traitement à haute pression, qui a été effectuée avant le processus d’émulsification, n'a pas modifié de façon significative l'efficacité émulsionnante. La réduction de l’efficacité a été probablement plutôt induite par la dénaturation simultanée avec l’émulsification sous conditions d’écoulement très turbulent.L’intérêt de la formulation développée en tant que véhicule pour un modèle de substance active hydrophobe a été étudié avec l’isotrétinoïne (IT), usuellement utilisé pour le traitement de l’acné sévère. La formulation développée a permis d’encapsuler 0,033 m/m% d’IT sans aucune modification de la stabilité du système. Environ 10 % de l’IT ajoutée ont été solubilisés dans la phase aqueuse en association avec la protéine libre en excès. L’IT encapsulée dans les gouttelettes huileuses a été plus stable contre la photo-isomérisation que celle associée à la protéine libre. La formulation développée apparait prometteuse en tant que système de délivrance de l’IT pour une application cutanée<br>Oil-in-water nanoemulsions can be used as drug delivery systems for the encapsulation of hydrophobic active substances in order to increase their solubility and their bioavailability. However, due to their higher specific area, their stabilization requires higher surfactant concentrations compared to conventional emulsions. Most of the synthetic surfactants commonly used in emulsion formulation are potentially irritant and even toxic, which hinders the therapeutic application of nanoemulsions especially during long-term treatment. The objective of this thesis is thus to formulate pharmaceutical oil/water nanoemulsions stabilized by a biopolymer, beta-lactoglobulin (beta-lg), instead of synthetic surfactants. Nanoemulsions were prepared by high pressure homogenization (HPH). The formulation composition and the process conditions were optimized in order to obtain nanometric droplets within stable nanoemulsions. The results showed that the most stable nanoemulsions, with droplet size inférieure à 200 nm, were obtained when 5 w/w% of the oil with the lowest viscosity value was used as the oily phase, 95 w/w% of beta-lg solution at a concentration of 1 w/w% was used as the aqueous phase, and 100 MPa of homogenization pressure was applied for 4 cycles. This formulation was stable against droplet growth phenomena during 30 days at least, thanks to a quasi purely elastic interfacial film. Xanthan gum, a natural polysaccharide, was added to the optimal formulation as a texturizing agent at a concentration of 0.5 w/w%. This allowed obtaining a cream texture with a shear thinning behavior. In this formulation, the migration rate of droplets was considerably reduced and the nanoemulsions stability was enhanced.The effects of the homogenization process on the different levels of the protein structure were assessed by spectroscopic, chromatographic and electrophoretic methods. The influence of this treatment on its interfacial and emulsifying properties was also investigated. The optimal emulsifying efficiency was obtained when the homogenization conditions did alter neither the structure of beta-lg nor its interfacial properties. However, an excessive HPH treatment (300 MPa/5 cycles) introduced structural modifications, mainly from beta-sheets into random coils, wide loss in lipocalin core, and protein aggregation by intermolecular disulfide bridges. HPH modified beta-lg displayed higher surface hydrophobicity inducing a higher adsorption rate at the O/W interface and an earlier formation of an elastic interfacial film. Structural and interfacial properties modifications by HPH denaturation appeared qualitatively similar to that of the heat denaturation with, however, differences in extent. Protein denaturation by a high pressure treatment that was performed before the emulsification process did not alter significantly its emulsifying efficiency. The reduction in the efficiency was rather induced by the simultaneous denaturation with the emulsification under high turbulent flow.The efficiency of the developed formulation as a vehicle for a model hydrophobic active substance was studied using isotretinoin, usually used for the treatment of severe acne. The developed formulation was able to encapsulate 0.033 w/w of isotretinoin without any modification on the system stability. About 10 % of the added isotretinoin was solubilized in the aqueous phase associated with the free protein in excess. Isotretinoin encapsulated in the oily droplets was more stable against photo isomerization than the one associated to the excess protein in the aqueous phase. The developed formulation seems promising as a drug delivery system of isotretinoin for a dermal application

Made available in DSpace on 2014-10-09T12:33:56Z (GMT). No. of bitstreams: 0<br>Made available in DSpace on 2014-10-09T14:03:58Z (GMT). No. of bitstreams: 0<br>Dissertação (Mestrado)<br>IPEN/D<br>Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Dans ce travail, le comportement des protéines de pois vis-à-vis de la gélification enzymatique par la transglutaminase microbienne (MTGase) a été évalué à l’état natif et après dénaturation (réduction chimique ou thermo-dénaturation). L’application finale concernait la formation de microparticules protéiques permettant d’encapsuler la riboflavine, choisie comme molécule active hydrophile modèle. Le procédé d’extraction des fractions protéiques de pois a été optimisé de manière à affecter le moins possible la structure des protéines et de récupérer des fractions natives riches en albumines (Alb) et en globulines (Glob), ou leur mélange. La mise en place des méthodes de suivi de la réaction enzymatique a permis de mettre en évidence leur complémentarité ainsi que leurs limites. Deux nouvelles méthodes de suivi de la réticulation enzymatique ont été développées. L’une basé sur la RMN permet la détermination simultanée de la quantité du fragment glutamine-lysine, produit de la réaction enzymatique, et le degré de réticulation ; l’autre méthode, basée sur les techniques de mesure de taille (SDS-PAGE et DLS), permet de visualiser les liaisons intramoléculaires. L’étude du traitement enzymatique appliqué aux fractions Alb et Glob de pois à l’état natif et dénaturé, ainsi qu’en mélange natif, a montré que la réaction enzymatique est fortement liée à la structure et à la conformation des protéines. Contrairement à la fraction Alb, la fraction Glob constitue un bon substrat pour la MTGase et la réticulation met en jeu des sous-unités constitutives des globulines différentes pour chaque condition de traitement. Néanmoins, la fraction Alb peut être utilisée en tant que booster de réaction enzymatique ce qui peut faire l’objet d’une voie innovante d’amélioration de la susceptibilité des protéines vis-à-vis de la MTGase. Le mécanisme semble basé sur un phénomène d’affinité sélective. Les bonnes propriétés mécaniques et de capacité de rétention d’eau du gel de la fraction protéique de pois totale ont été exploitées pour produire des microparticules à partir de la dispersion de la solution protéique sous forme d’émulsion suivie d’une gélification enzymatique par la MTGase. Les microparticules ont été pratiquement insolubles dans les milieux gastro-intestinaux en absence d’enzymes et lentement dégradable en présence d’enzymes. La libération de la riboflavine est gouvernée par un phénomène de diffusion en absence d’enzyme et de dégradation de support en présence d’enzymes selon des cinétiques compatibles avec des applications nutraceutiques<br>In this work, pea proteins behavior toward enzymatic gelation by microbial transglutaminase (MTGase) was studied at native state and after denaturation (chemical reduction or thermal denaturation). The final application was the formation of protein microparticules to encapsulate riboflavin, chosen as hydrophilic active molecule model. The extraction process of the pea protein fractions has been optimized in such a way to minimize as possible protein denaturation and recover native fractions rich in albumin (Alb) and globulin (Glob) or a mixture of both.The setting up of the enzymatic reaction monitoring methods has brought out their complementarity as well as their limits. Two new monitoring methods of enzymatic cross-linking reaction have been developed. The first one, based on the NMR, allows to the simultaneous determination of the glutamine-lysine isopeptide bond, product of the enzymatic reaction, and the degree of crosslinking; the second method, based on size measuring techniques (SDS-PAGE and DLS), permit to view the intramolecular links. The study of enzymatic treatment applied to pea Alb and Glob at the native and denatured states, as well as thier native mixture showed that the enzymatic reaction is strongly related to the structure and conformation of proteins. Unlike Alb, the Glob fraction is a good substrate to transglutaminase and crosslinking reaction involves different subunits constituting globulins for each treatment condition. However, the Alb can be used as a booster of enzyme reaction which can be an innovative way for improving the proteins susceptibility toward transglutaminase treatment. The mechanism seems to be based on a selective affinity phenomenon. The good mechanical properties and water holding capacity of total pea proteins gel have been exploited to produce microparticles from a water-in-oil emulsion followed by enzymatic gelation. The produced microparticles were practically insoluble in gastrointestinal media in the absence of enzymes and slowly degradable in the presence of enzymes. The release mechanisms of riboflavin in digestive environments are governed by a diffusion phenomenon in the absence of enzymes and by support degradation phenomenon in the presence of enzymes according to kinetics compatible with nutraceutical applications

L'encapsulation de protéines pose de nombreuses difficultés liées à leurs propriétés physicochimiques, leur sensibilité aux conditions environnementales et opératoires. La structure 3D spécifique de chaque protéine étant directement liée à son activité biologique, un contrôle de l'intégrité protéique est indispensable pour assurer l'efficacité et la sécurité des systèmes formulés. Dans cet optique, l'encapsulation par émulsion multiple a été appliquée à une protéine modèle : la myoglobine (Mb). Le but de cette thèse a été l'étude du procédé d'encapsulation à travers 3 objectifs. Le premier fut d'étudier l'influence de paramètres de formulation sur les propriétés des particules obtenues et sur la conformation de la Mb. Les mesures en spectrométrie UV/Vis. et le calcul des principaux rapports d'absorbance ont constitué une méthode de contrôle fiable et rapide. Le second fut de valider la pertinence et de montrer les limites de cette méthode en la comparant à une seconde méthode utilisant des mesures conductimétriques. Enfin, l'étape de solidification des particules par élimination du solvant ainsi que le changement de solvant ont été étudiés.. Des microsphères creuses de 15μm avec un rendement d'encapsulation de 36% ont pu être obtenues tout en maintenant la conformation native de la Mb. L'emploi de polymère de masse moléculaire élevée et un taux d'élimination de solvant trop rapide sont 2 paramètres altérants significativement la protéine. Ces travaux ouvrent la voie au développement de transporteurs d'O2 utilisables par exemple dans le cas de pathologies musculaires<br>Protein encapsulation results in several problems related to their physicochemical properties, their sensitivity to environmental conditions and operating procedures. The specific 3D structure being directly linked to their biological activity, monitoring of protein integrity is crucial to ensure the efficacy and security of formulations. In this context, the multiple emulsion method was applied to a model protein: myoglobin (Mb). The aim of this thesis was to study the encapsulation process through 3 objectives. The first was to study the influence of formulation parameters on the particle properties and the conformation of Mb. Measurements by UV/Vis. spectrometry and calculation of key absorbance ratios established a reliable and rapid method for protein monitoring. The second was to validate the pertinence and show the limits of this method by comparing it to a second one using conductimetry measurements. Finally, the particle solidification by solvent removal and the solvent exchange were studied. The process maintains the Mb native conformation in Hollow microspheres of 15μm in diameter and an encapsulation efficiency of 36% were obtained, while keeping intact the Mb native conformation. The use of high molecular weight polymer and a fast solvent removal rate are 2 parameters inducing significant protein alteration. This work paves the way for the development of O2 carriers for muscular diseases for example

Nowadays, scientific and political communities are focused on ways to better preserve and manage the natural resources of our planet. In order to reduce consumption of fossil resources, and to develop more environmentally friendly industrial processes, the industry of flavors and fragrances became interested in developing new bio-based encapsulating materials. In the present work, maltodextrins have been chosen as main component of the matrix, and pea protein isolate and a modified starch were selected as compatibilizing additives. The incorporation of volatile odorant compounds and the elaboration of the new bio-based delivery systems were performed, all in one single step, by low temperature twin-screw extrusion. The physicochemical, thermal and morphological properties of these matrices were studied, as well as the encapsulation efficiency and the release profile of the active compounds. These investigations have led to a better understanding of the impact of the formulations and of the incorporation of the active compound on the process parameters. The interactions between the wall and the encapsulated materials were also analyzed. The characteristics of the new bio-based delivery systems and the established extrusion process conditions were found to be very promising to be employed in the field of perfumery.

<p>Spray drying is a method with a high potential in the preparation of protein particles suitable for pulmonary delivery. However, surface induced denaturation of bio-molecules during atomization and subsequent drying can be substantial and it is therefore important to develop new formulation concept for concurrent encapsulation and stabilization of proteins during spray drying. Hence, with an overall objective to increase the knowledge of the formation of particulate systems for systemic administration of proteins by spray drying, the first part of this thesis, systematically investigated the particle formation by droplet size and particle size measurements. It was described how specific properties, such as the solubility and the crystallization propensity of the solute, can affect the product, e.g. the particle size, internal structures, and possibly particle density. A new method using atomic force microscopy (AFM) for the assessment of the effective particle density of individual spray-dried particles was demonstrated. In the second part, two different formulation concepts for encapsulation of protein during spray drying were developed. Both systems used non-ionic polymers for competitive adsorption and displacement of protein from the air/water interface during spray drying. The aqueous two-phase system (ATPS) of polyvinyl alcohol (PVA) and dextran, and the surface-active polymers, hydroxypropyl methylcellulose (HPMC) and triblock co-polymer (poloxamer 188) used for in situ coating, proved efficient in encapsulation of a model protein, bovine serum albumin (BSA). Inclusion of polymeric materials in a carbohydrate matrix also influenced several particle properties, such as the particle shape and the surface morphology, and was caused by changes in the chemical composition of the particle surface and possibly the surface rheology. In addition, powder performance of pharmaceutical relevance, such as dissolution and flowability, were affected.</p>

Des copolymères biodégradables amphiphiles constitués d'un bloc hydrophobe d'acide polylactique (PLA) et d'un bloc hydrophile de monométhoxypolyoxyéthylène (MPOE) ont été synthétisés dans le but de préparer des nanoparticules injectables furtives permettant une libération contrôlée dans le sang des principes actifs encapsulés. En tirant parti des propriétés amphiphiles des copolymères et en particulier des différences de solubilité des deux blocs en phase aqueuse et organique, les procédés d'émulsion/évaporation (simple émulsion, huile-dans l'eau) et de double émulsion huile-dans l'eau-dans l'huile adoptés conduisent à la formation de nanosphères constituées d'un cœur dense hydrophobe de PLA, recouvertes d'une brosse hydrophile de MPOE. Cette dernière a pour but d'empêcher l'adsorption des protéines (opsonisation), phénomène qui déclenche la réponse immunitaire, et protéger ainsi les nanoparticules de l'élimination de la circulation sanguine. Les propriétés de surface des nanosphères ont été étudiées, notamment du point de vue de la charge, et comparées à celles de nanosphères de PLA non recouvertes de MPOE. Suivant les procédés de simple ou double émulsion appliqués, il a été possible d'encapsuler efficacement un principe actif hydrophobe (Cyclosporine A, médicament aux propriétés immunosuppressives) et une protéine modèle (albumine du sérum humain). Les taux d'encapsulation et charges en principes actifs ont été étudiés en fonction de divers paramètres (concentration en polymère, charge théorique en principe actif, taux de MPOE dans la matrice) et les cinétiques de libération ont été suivies in vitro. Ainsi, nous avons montré qu'il était possible de retarder la libération des principes actifs lorsqu'ils sont incorporés dans une matrice biodégradable. Enfin, nous avons mis en évidence l'efficacité de la brosse protectrice de MPOE en surface des nanosphères vis-à-vis de l'adsorption des protéines plasmatiques et de la capture par des cellules phagocytes humaines (polymorphonucéaires).

La technique de nanoprécipitation est une méthode simple et reproductible pour la synthèse de nanocapsules à coeur huileux recouvertes d’une enveloppe de polymères hydrophiles réticulés (polysaccharides, glycopolymères vinyliques…) en une seule étape. Grâce à leur biocompatibilité, leur biodégradabilité et leur activité biologique adaptables, les protéines constituent une autre grande famille de biopolymères d’intérêt pour des applications dans le domaine de l’encapsulation. Cependant, la production de nanocapsules protéiques par nanoprécipitation n’a jamais été décrite. Dans ce contexte, l’objectif principal de ce travail de thèse a été l’évaluation du potentiel d’une famille de protéines, les Suckerines, pour le procédé de nanoprécipitation. Les Suckerines sont une famille de protéines issues des dents décorant les ventouses du calamar géant Humboldt avec de prometteuses applications dans le domaine biomédical. Ces protéines possèdent une structure modulaire de type copolymère à bloc capable de former des feuillets bêta conférant de bonnes propriétés mécaniques. Les suckerines étant solubles dans une solution tampon composée d’acide acétique (pH 3) mais fortement agrégées dans les conditions de pH (valeurs comprises entre 5 et 10) classiquement utilisées pour la préparation de nanocapsules à coeur huileux par basculement de solvant, nous avons finalement choisi d’explorer la nanoprécipitation des protéines par salt shifting et donc la préparation de nanoparticules protéiques. L’utilisation du persulfate d’ammonium comme agent de coacervation et précurseur de radicaux et du tris(2,2′ bipyridyl)dichlororuthenium(II) hexahydrate a permis de produire des nanoparticules de suckerine de tailles modulables (100-185 nm de diamètre). Ces nanoparticules présentent des structures secondaires type feuillets bêta qui sont à l’origine du module de Young très élevé observé pour ces nano-objets (de l’ordre de grandeur du GPa). Une protéine de fusion, soluble en milieu aqueux à pH 7 a spécialement été conçue par voie de recombinaison dans le but de générer des nanocapsules protéiques par nanoprécipitation. Cette protéine (suckerine-soie) est formée d’un bloc central de peptide dérivé de suckerine de calamar promouvant une stabilité structurelle et deux blocs terminaux issus de fibroïnes de soie qui permettent à la protéine de fusion d’être soluble à un pH physiologique. Ce design moléculaire a permis la fabrication de nanocapsules remplies respectivement de hexadécane ou de miglyol avec une enveloppe de suckerine-soie et de tailles de l’ordre de grandeur de 190 à 250 nm. Finalement, aspirant à encapsuler un principe actif anti-cancéreux dans les nanocapsules à base de glycogène, nous avons développé un protocole où la méthode de nanoprécipitation est utilisée pour produire des nanoparticules de prodrogue entourés de glycogène<br>The nanoprecipitation technique is a reliable route to synthesize oil filled nanocapsules with shells made of hydrophilic polymers such as polysaccharides and vinyl based glycopolymers in a one pot procedure. Thanks to their biocompatibility, biodegradability and tunable biological activity, proteins are another promising class of materials for encapsulation purposes. However, the generation of proteinaceous nanocapsules by nanoprecipitation has never been reported. In this context, the main objective of this PhD was to evaluate the potential of a family of proteins, the Suckerins, in nanoprecipitation processes. Suckerins are a family of proteins found in the sucker ring teeth of the giant Humboltd squid with promising biomedical applications. These proteins possess a modular, block copolymer like structure capable of forming β-sheets responsible for good mechanical properties. The suckerin proteins are not soluble at a pH range between 5 and 10, a requirement of the nanoprecipitation technique. However, they can be solubilized using aqueous buffers at pH 3 containing acetic acid. Other ways of precipitating the protein were explored in this manuscript with salt shifting using ammonium persulphate as coacervation agents being capable of generating 100 nm nanoparticles. These nanoparticles presented the β sheet secondary structure which resulted in Young modulus in the GPa range. A fusion protein that could be solubilized in aqueous solutions at pH 7, and therefore be used in the nanoprecipitration process, was recombinantly produced. The protein (suckerin silk) is formed by a central squid suckerin-derived peptide block that provides structural stability and both termini from silk fibroins that make the modular protein highly soluble at physiological pH. This molecular design allowed the fabrication of hexadecane and miglyol filled nanocapsules with suckerin silk shells and sizes in the range 190 – 250 nm. Finally, aiming to encapsulate an anti cancer drug in glycogen nanocapsules we developed a protocol where the nanoprecipitation process is used to generate glycogen coated prodrug nanoparticles

La Nanotecnologia es considera una de les tecnologies clau del segle XXI. A cavall d’àrees com la Física, Química, Biologia, Medicina i electrònica, les aplicacions nanotecnològiques en el camp de les ciències naturals desperta especial interès. La nanomedicine en particular, explora el potencial de gran varietat de nanomaterials en medicina, tant en diagnòstic com en tractament de malalties. Són camps importants de la nanomedicine el “drug delivery”, teràpia gènica i cel•lular, diagnòstic molecular i imaging. Respecte el tractament de malalties i drug delivery, les teràpies basades en proteïnes han tingut un impacte important en multitud de malalties en les últimes dècades. Molts fàrmacs de naturalesa proteica i peptídica com enzims i anticossos han estat aprovats i són teràpies efectives contra malalties com la diabetis, el càncer i les malalties d’emmagatzematge lisosomal (LSD de l’anglès). Les proteïnes terapèutiques ofereixen avantatges sobre fàrmacs convencionals com millor especificitat, més activitat i menys toxicitat. No obstant, limitacions en l’ús de proteïnes terapèutiques, com la susceptibilitat a degradació enzimàtica, vida mitja en circulació curta i permeabilitat baixa en membranes afecten severament la seva eficàcia, estabilitat, i al cap i a la fi, la seva capacitat terapèutica. L’ús d’estratègies nanotecnològiques, com l’encapsulació de proteïnes o la modificació química d’aquestes, ofereix expectatives prometedores per superar les limitacions de les teràpies proteiques i millorar la seva eficàcia terapèutica. En aquesta tesi, s’ha estudiat l’aplicació de diverses estratègies nanotecnològiques per a l’encapsulació i recobriment de proteïnes per tal de millorar-ne el valor terapèutic. La tesi s’ha centrat en dues proteïnes, ambdues amb valor terapèutic pel tractament de malalties, però degut a diferències entre elles en mida, complexitat estructural i activitat enzimàtica, les estratègies emprades s’han de tractar per separat. El Factor de Creixement Epidèrmic (EGF) és una proteïna de 6 kDa amb activitat mitogènica que molts estudis han descrit que juga un paper important en la curació de malalties com l’úlcera pèptica. L’EGF pot augmentar la curació de ferides a l’estómac i al duodè, No obstant, l’ambient desfavorable per proteïnes a la llum gastrointestinal, amb pH extrems i presència de proteases, pot induir la degradació de l’EGF i provocar que perdi activitat biològica. Proposem una estratègia per co-encapsular l’EGF en nanopartícules de PLGA conjuntament amb NvCI, un inhibidor de carboxipeptidases digestives, per tal de protegir l’EGF de la degradació per proteases i augmentar-ne la capacitat terapèutica. L’EGF i l’NvCI s’encapsulen amb èxit en nanopartícules de PLGA i mantenen la seva activitat biològica. La Velaglucerasa alfa és una versió recombinant de la -glucocerebrosidasa humana, i un fàrmac usat en teràpia de substitució enzimàtica (ERT) pel Síndrome de Gaucher (GD), una malaltia d’emmagatzematge lisosomal (LSD). Les LSD són malalties que presenten un enzim lisosomal defectuós, GBA en el cas de GD, provocant acumulació de substrat i afectant el funcionament normal dels lisosomes. Les ERT consisteixen en l’administració d’una versió funcional de l’enzim defectuós perquè arribi al lisosoma i restauri l’activitat enzimàtica. La Velaglucerasa és una proteïna de 63 kDa amb activitat enzimàtica intrínseca. Polymer Masked-Unmasked Protein Therapy (PUMPT) és una estratègia que consisteix en la conjugació d’una proteïna amb un polímer biodegradable. La conjugació de Velaglucerasa amb el polímer Poli(àcid L-glutàmic) (PGA) permet emmascarar i preservar l’activitat enzimàtica de la Velaglucerasa fins que s’allibera al lisosoma, on restaura parcialment els nivells d’activitat GBA. La conjugació amb PGA confereix a la Velaglucerasa una major estabilitat en plasma sanguini humà. Desenvolupar noves estratègies capaces d’encapsular o recobrir proteïnes grans amb activitat biològica sensible a les condicions ambientals presenta expectatives prometedores per a millorar la capacitat terapèutica d’aquestes.<br>Nanotechnology is considered one of the key technologies of the 21st century. At the meeting point of many different areas such as physics, chemistry, biology, medicine and electronics, nanotechnological applications in the field of natural sciences are of special interest. Nanomedicine in particular, explores the potential of a huge variety of nanomaterials in medicine, covering both disease diagnosis and treatment. Prominent fields in nanomedicine include drug delivery, cell and gene therapy, molecular diagnostics and imaging. Regarding disease treatment and drug delivery, protein-based therapeutics have had an important impact in a myriad of human disease for the last decades. Many drugs of peptide and protein nature such as enzymes and antibodies have been approved and become effective therapies for diseases like diabetes, cancer and Lysosomal Storage Diseases (LSD). Protein therapeutics offer significant advantages over conventional drugs including higher specificity, greater activity and lower toxicity. However, limitations in the use of therapeutic proteins, such as sensitivity to enzymatic degradation,short circulation half-lives in the bloodstream and poor membrane permeability; severely affect protein efficiency, stability and at the end, their therapeutic capacity. The use of nanotechnologial strategies, such as protein encapsulation and chemical modification of proteins, offer promising expectations to overcome the limitations of protein therapies and improve their therapeutic efficacy. In this thesis, it has been studied the application of several nanotechnological strategies for protein encapsulation and coating in order to improve the therapeutic value of those proteins. Two proteins were focused, both with therapeutic value in disease treatment, but because of their differences regarding size, structural complexity and enzymatic activity, strategies on each of them need to be addressed particularly. Epidermal Growth Factor is a 6 kDa protein with mitogenic activity that has been described in many studies to have an important role in the healing of diseases like peptic ulcers. EGF can increase the healing rate of sores in lining of the stomach and the duodenum. However, the harmful environment for proteins found in the gastrointestinal lumen, with extreme pH and the presence of proteases, may induce EGF degradation and a subsequent loss of its biological activity. We suggest a strategy for EGF co-encapsulation in PLGA nanoparticles with NvCI, a digestive carboxypeptidase inhibitor, in order to protect it from protease degradation and thus increasing its therapeutic capacity. EGF and NvCI can be successfully encapsulated in PLGA nanoparticles, maintaining its biological activity. Velaglucerase alfa is a recombinant version of the human -glucocerebrosidase (GBA), a protein drug used in Enzyme Replacement Therapy (ERT) for Gaucher Disease (GD), a Lysosomal Storage Disorder (LSD). LSD are diseases that present a defective lysosomal enzyme, GBA in the case of GD, causing accumulation of their substrate and impairing the normal function of lysosomes. ERT consists in administrating a functional version of the defective enzyme intending its delivery into the lysosome to restore the enzymatic activity. Velaglucerase is a 63 kDa protein with intrinsic enzymatic activity, and because of its structural complexity encapsulation in PLGA nanoparticles is unable to preserve enzymatic activity. Polymer Masked-Unmasked Protein Therapy (PUMPT) is a strategy that consists in conjugating a protein with a biodegradable polymer. Conjugation of Velaglucerase with the biocompatible polymer Poly(L-Glutacic acid) is able to mask and preserve the enzyme activity of Velaglucerase until its delivery into the lysosome, where it partially restores the GBA activity levels. PGA conjugation also grants Velaglucerase a higher stability in human blood plasma. Developing new nanotechnological methods capable of encapsulating or coating large proteins whose biological activity is sensitive to environmental conditions offers promising expectations in order to improve their therapeutic capacity.

An aqueous droplet in a solution of lipids in oil acquires a lipid monolayer coat, and two such droplets adhere to form a bilayer at their interface. Networks of droplets have been constructed in this way that function as light sensors, batteries and electrical circuits by using membrane proteins incorporated into the bilayers. However, the droplets have been confined to a bulk oil phase, which precludes direct communication with physiological environments. Further, the networks typically have been assembled manually, which limits their scale and complexity. This thesis addresses these limitations, and thereby enables prospective medical and technological applications for droplet networks. In the first part of the work, defined droplet networks are encapsulated within mm-scale drops of oil in water to form structures called multisomes. The encapsulated droplets adhere to one another and to the surface of the oil drop to form interface bilayers that allow them to communicate with each other and with the surrounding aqueous environment through membrane pores. The contents of the droplets can be released by changing the pH or temperature of the surrounding solution. Multisomes have potential applications in synthetic biology and medicine. In the second part of the work, a three-dimensional printing technique is developed that allows the construction of complex networks of tens of thousands of heterologous droplets ~50 µm in diameter. The droplets form a self-supporting material in bulk oil or water analogous to biological tissue. The mechanical properties of the material are calculated to be similar to those of soft tissues. Membrane proteins can be printed in specific droplets, for example to establish a conductive pathway through an otherwise insulating network. Further, the networks can be programmed by osmolarity gradients to fold into designed shapes. Printed droplet networks can serve as platforms for soft devices, and might be interfaced with living tissues for medical applications.

El principal objectiu fou produir sistemes d’encapsulació basats en emulsions emprant ingredients i tecnologies sostenibles. S’han combinat amb èxit la concentració per osmosi directa i la emulsificació per membranes en la valorització d’extracte de polifenol de garrofa. Especies d’insectes amb potencial per ser comercialitzades s’han valorat com a emulsificants. Fraccions proteiques de larva de mosca de soldat negre (BSFPC, Hermetia illucens) i d’escarabat búfal (LMPC, Alphitobius diaperinus) s’han utilitzat per estabilitzar emulsions simples i múltiples mitjançant emulsificació amb membranes dinàmiques de porositat controlada. BSFPC presenta millors resultats que la proteïna de sèrum de llet (WPI) per l’estabilització d’emulsions d’oli de llimona amb una fracció d’oli del 40%. Per emulsions d’oli de gira-sol, BSFCP es comporta de forma similar a WPI. En emulsions W1/O/W2, LMPC es comporta de manera similar a WPI i a la proteïna de pèsol (PPI) durant cicles de congelació-descongelació, en medi àcid i bàsic i durant l’emmagatzematge a diferents condicions. A més, mostra millors propietats que WPI i PPI per estabilitzar emulsions a 90ºC, indicant els avantatges d’utilitzar-les en aliments que han de passar per tractaments tèrmics. En canvi, no poden igualar l’estabilitat de les emulsions amb WPI quan es varia el gradient de pressió osmòtica entre les dues fases aquoses. S’ha comprovat l’eficàcia de les proteïnes d’insecte per encapsular un polifenol comercial en sistemes sòlids a partir d’emulsions W1/O/W2 assecades per atomització o liofilització. En el procés de fraccionament de proteïnes s’ha comprovat que solvents verds, com l’etanol, isopropanol i 2-metiltetrahidrofurà, es poden utilitzar alternativament a l’hexà en el desgreixat de molturats d’insecte. El rendiment d’extracció i les propietats emulsificants de les proteïnes resultants confirmen l’eficàcia d’aquests solvents. El resultats demostren que els concentrats de proteïna d’insectes utilitzats són una bona alternativa a les proteïnes làctiques o de plantes per la producció de sistemes d’encapsulació basats en emulsions.<br>El principal objetivo fue producir sistemas de encapsulación basados en emulsiones utilizando ingredientes y tecnologías sostenibles. Se han combinado con éxito la concentración por ósmosis directa y la emulsificación por membranas en la valorización de extracto de polifenol de algarroba. Especies de insectos con potencial para ser comercializadas se han valorado como emulsificantes. Fracciones proteicas de larva de mosca de soldado negro (BSFPC, Hermetia illucens) y de escarabajo búfalo (LMPC, Alphitobius diaperinus) se han utilizado para estabilizar emulsiones simples y múltiples mediante emulsificación con membranas dinámicas de porosidad controlada. BSFPC presenta mejores resultados que la proteína de suero de leche (WPI) para la estabilización de emulsiones de aceite de limón con una fracción de aceite del 40%. En emulsiones de aceite de girasol, BSFCP se comporta de forma similar a WPI. En emulsiones W1/O/W2, LMPC actúa como WPI y la proteína de guisante (PPI) durante los ciclos de congelación-descongelación, en medio ácido y básico y durante el almacenamiento en diferentes condiciones. Además, muestra mejores propiedades que WPI y PPI para estabilizar emulsiones a 90ºC, indicando la ventaja de utilizarlas en alimentos que tienen que pasar por tratamientos térmicos. En cambio, no pueden igualar la estabilidad de las emulsiones con WPI cuando se varía el gradiente de presión osmótica entre las dos fases acuosas. Se ha comprobado la eficacia de las proteínas de insecto para encapsular un polifenol comercial en sistemas sólidos a partir de emulsiones W1/O/W2 secadas por atomización o liofilización. En el proceso de fraccionamiento de proteínas se ha comprobado que solventes verdes, como el etanol, isopropanol y 2-metiltetrahidrofurano, se pueden utilizar alternativamente al hexano en el desengrasado de molturados de insecto. Los resultados demuestran que los concentrados de proteína de insectos utilizados son una buena alternativa a las proteínas lácteas o de plantas para la producción de sistemas de encapsulación basados en emulsiones.<br>The focus of the thesis is to produce emulsion-based encapsulation systems using both sustainable ingredients and technologies. Specifically, valorisation of an agri-food by product (carob pulp polyphenol) was proved feasible coupling forward osmosis for concentration and membrane emulsification for encapsulation. Insect powder defatting by solvent extraction was investigated using green solvents (ethanol, isopropanol, and 2-methyltetrahydrofuran), paying special attention to defatting yield and the techno-functional properties of the resulting protein fractions, particularly emulsifying ability. Single and double emulsions stabilised with sustainable protein sources from insects, black soldier fly (Hermetia illucens) and lesser mealworm (Alphitobius diaperinus) larvae, have been successfully produced for the first time by a low-energy high-throughput emulsification technology based on dynamic membranes of tunable pore size (DMTS). H. illucens protein concentrate showed superior ability to stabilize higher lemon oil fraction (40 wt%) compared to whey protein isolate. A commercial polyphenol extract was encapsulated in W1/O/W2 emulsions stabilised with A. diaperinus protein concentrate. These emulsions displayed a comparable stability under freeze-thaw cycles, storage conditions, acidic, and alkaline conditions than the ones stabilized with WPI, and better than the ones stabilized with pea protein. However, they were less able to withstand osmotic pressure differences compared to whey protein. Insect protein stabilized W1/O/W2 emulsions showed less changes in droplet size distribution at the highest temperature tested (90ºC) than the ones stabilized with whey or pea protein, pointing out the benefit of using insect proteins in emulsions that need to undergo heat treatment. Solid microcapsules were successfully produced from refined polyphenol loaded W1/O/W2 emulsions stabilized with insect protein by spray drying or freeze drying. The results demonstrate the insect protein concentrates assessed are a promising sustainable ingredient to replace diary and plant proteins in the emulsion-based encapsulation systems.

Le glioblastome (GBM) est la forme de cancer du cerveau la plus courante et la plus meurtrière. Sa nature diffusive entraine une impossibilité d’élimination complète par chirurgie. Une récidive de la tumeur chez ≥ 90% des patients peut être provoqué par des cellules GBM résiduelles se trouvant près du bord de la cavité de résection. Un implant pouvant libérer de manière durable la protéine SDF-1α, qui se lie aux récepteur CXCR4 à la surface des cellules GBM, peut être utile pour induire le recrutement des cellules GBM résiduelles, permettre leur élimination sélective et finalement réduire la récurrence de la tumeur. Dans ce travail, le SDF-1α a été initialement encapsulé dans des nanoparticules à base d'acide poly-lactique-co-glycolique (PLGA). Une efficacité d'encapsulation élevée (76%) a pu être obtenue en utilisant un processus simple de séparation de phase. Les nanoparticules chargées de SDF-1α ont ensuite été incorporées dans un scaffold à base de chitosan par électrofilage pour obtenir des implants nanofibreux imitant la structure de la matrice extracellulaire du cerveau. Une étude de libération in vitro a révélé que l'implant pouvait fournir une libération prolongée de SDF-1α jusqu'à 35 jours, utile pour établir un gradient de concentration de SDF-1α dans le cerveau et induire une attraction des cellules GBM. Une étude de biocompatibilité in vivo à 7 jours a révélé des signes d'inflammation locale sans aucun signe visible de détérioration clinique chez les sujets animaux. Une étude à 100 jours visant à confirmer l'innocuité in vivo des implants avant de passer aux études d'efficacité dans un modèle de résection GBM approprié est actuellement en cours<br>Glioblastoma (GBM) is the most common and lethal form of brain cancer. The diffusive nature of GBM means the neoplastic tissue can not be removed completely by surgery. Often, residual GBM cells can be found close to the border of the resection cavity and these cells can multiply to cause tumor recurrence in ≥90% of GBM patients. An implant that can sustainably release chemoattractant molecules called stromal cell-derived factor-1α (SDF-1α), which bind selectively to CXCR4 receptors on the surface of GBM cells, may be useful for inducing chemotaxis and recruitment of the residual GBM cells. This may then give access to selective killing of the cells and ultimately reduce tumor recurrence. In this work, SDF-1α was initially encapsulated into poly-lactic-coglycolicacid (PLGA)-based nanoparticles. A high encapsulation efficiency (76%) could be achieved using a simple phase separation process. The SDF-1α-loaded nanoparticles were then incorporated into a chitosan-based scaffold by electrospinning to obtain nanofibrous implants that mimic the brain extracellular matrix structure. In vitro release study revealed that the implant could provide sustainedSDF-1α release for 5 weeks. The gradual SDF-1αrelease will be useful for establishing SDF-1α concentration gradients in the brain, which is critical for the chemotaxis of GBM cells. A 7-day in vivo biocompatibility study revealed evidence of inflammation at the implantation site without any visible signs of clinical deterioration in the animal subjects. A long-term study (100 days) aiming to confirm the in vivo safety of the implants before proceeding to efficacy studies in a suitable GBM resection model is currently underway

Nutraceuticals face significant challenges in delivery due to poor solubility of some bioactives, and instability during processing. The major whey protein in bovine milk, ß-lactoglobulin (ß-Lg), has versatile properties and the potential for the development of novel delivery vehicles for bioactives. The aim of this study is to develop a solid and reproducible method to produce ß-Lg nanoparticles and investigate their application to the encapsulation of bioactives. Temperature and pH were found to be the key operating parameters to produce and control the size of nanoparticles. Monodisperse and spherical ß-Lg nanoparticles in the size range of 200-300 nm were produced at optimum conditions of pH (6.0) and heat load (heating incubation 75 °C for 45 mins). ß-Lg nanoparticles exhibited high colloidal stability (zeta potential: -37.42 mV) and high yield of aggregation (93%). The hydrogen bonding and hydrophobic interactions played a predominant role in the microstructure of these nanoparticles. Caffeine was bound to these nanoparticles with 13.54% as maximum encapsulation efficiency at 50:1 molecular ratio and the binding followed a Langmuir type isotherm. Only partial release of caffeine happened at gastric conditions (about 36%) whilst total release happened at intestinal conditions. Also, resveratrol-ß-Lg nanoparticles were produced at 181.8 nm using this method. Heat degradation and isomerisation of resveratrol occurred when heating at 75°C which led to reduced antioxidant activity. Resveratrol-beta-lactoglobulin nanoparticles exhibited an ability to improve antioxidant activity at this temperature and at pasteurisation condition. Lastly, whey protein nanoparticles could be produced following the same method with smaller size and lower yield of aggregation. Further studies should be carried out to explore other applications of ß-Lg/whey protein nanoparticles as a novel nanovehicle of bioactives in food products.

Introduction and hypothesis: Peritoneal dialysis (PD) is an important option for renal replacement therapy. Peritoneal sclerosis (PS) limits PD duration due to loss of ultrafiltration (UF) capacity, while about 3% of PD patients experience a condition termed encapsulating peritoneal sclerosis (EPS). In many fibrotic diseases reduced Extracellular matrix (ECM) breakdown due to lowered matrix metalloproteinase (MMP) activity occurs, often from over-expression of tissue inhibitors of MMP (TIMPs) that underlie fibrotic remodeling. Furthermore, recent application of 2D gel proteomics on peritoneal dialysis effluent (PDE) samples has identified several proteins that are elevated in patients with membrane damage. These observations have led to the hypothesis that: changes in proteins in PDE samples, in particular those associated with ECM breakdown have value as non-invasive biomarkers of PS and the switch to EPS. To test this hypothesis, PDE samples from 3 patient cohorts was analysed for ECM proteolytic activity. A range of ECM processing proteins and 3 proteins identified from previous proteomic studies of patients developing EPS (intellectin-1, dermatopontin and collagen α1 (I)) were analysed in PDE samples. Methods: Three patient cohorts were studied: two were from Sheffield Kidney Institute (SKI) that consisted of 32 spot PDE samples (SKI-1) that included 1 EPS patient & 51 PDE & plasma samples collected during a peritoneal equilibrium test (PET) with multiple dwell times in patients who did not have EPS (SKI-2). The third cohort consisted of 209 samples from the Global Fluid Study (GFS) including sequential samples from 12 EPS & 42 matched controls patients. MMP activity was assessed using the ENZchek assay system. Plasmin activity was assessed by using cleavage of the V0882 substrate. TIMPs, MMPs, intelectin-1, dermatopontin, and collagen (α1) I were quantified by commercial ELISA in PDE and plasma samples. PDE cytology (macrophages, leukocytes, fibroblasts and mesothelial cells) was performed to determine if changes in any protein could be associated with changes in cell types. Clinical data were recovered from either the peritoneal dialysis database (PDDB) at Sheffield or the GFS archives. The analysis was performed using Microsoft Excel 2010 software, SPSS, and Graphpad prism (prism 5.01 for windows). Results: Plasmin activity in PDE samples decreases with long duration of PD therapy. Minimal MMP activity was found in all PDE samples. In the SKI-1 cohort, MMP-1, -9, & -13 were almost undetectable with only MMP-2 & -3 being measurable with levels of ((mean±SD) 46±37 & 2.1±2.2 ng/mL respectively). In contrast TIMP-1 and TIMP-2 and to lesser extent TIMP-3 had significant levels in PDE samples from commencing PD (109±88, 17±12, and 0.28±0.33 ng/mL respectively). All TIMPs & MMP-2 were raised in the single patient who had a diagnosis of EPS. In samples from the GFS cohort, there was a rapid 6 fold increases in TIMP-1 within 100 days of the diagnosis of EPS, which when normalised to TIMP-2 levels was a good predictor of EPS. Calculation of the plasma to dialysate transfer rate by reference to that of circulating proteins with no peritoneal production and of known molecular weight (albumin, beta2microglobulin (B2M), transferrin, IgG, and creatinine) demonstrated that TIMPs & MMPs (especially TIMP-1 and MMP-2) have significant peritoneal production. Plasma levels for TIMP-1,-2, MMP-2,-3, and intelectin-1 (mean±SD) were 121±27, 85±16, 176±35, 11±5, and 374±136 ng/mL in healthy individuals respectively. Plasma levels in PD patients for TIMP-1,-2, MMP-2,-3, and intelectin-1 (mean±SD) were 297±78, 158±33, 309±112, 42±28, and 749 ±722 ng/mL respectively. None of the proteins identified by proteomics as predictors of EPS were able to be validated by ELISA. However TIMP-1,-2, MMP-2, intelectin-1, and collagen (α1) I in PDE samples had significant correlations with the loss of ultrafiltration and thus membrane damage. PDE cytology showed that peritoneal fibroblast and leukocyte numbers increase with time on PD, while peritoneal macrophage decreases with time on PD. There were no significant changes in mesothelial cells. Conclusions: Negligible MMP activity in PDE samples results from high TIMP levels which could underlie the development of PS. The rapid increase in TIMP-1 within 100 days of EPS development offers value as a diagnostic tool or a late biomarker. Plasma levels of TIMP-1,2, MMP-2,3, and intelectin-1 are higher in patients on PD compare to healthy individuals. The increase in peritoneal fibroblasts may be a source of TIMP-1.

Ce travail de thèse a permis la mise au point d'un procédé d'encapsulation de bactéries probiotiques dans des matrices ne contenant que des ingrédients laitiers. L'étude à l'échelle laboratoire puis, le dimensionnement du procédé à l'échelle pilote ont permis la production de microparticules stables dans des milieux aqueux et résistantes à des conditions simulant l'estomac. La nature des protéines présentes mais également leurs proportions ont influencé la localisation des bactéries dans les microparticules : à la surface pour une matrice uniquement composée de caséine et à l'intérieur pour une matrice composée à la fois de caséines et de protéines solubles. L'interprétation de ces résultats par une étude à l'échelle moléculaire des interactions qui s'établissent entre les bactéries probiotiques et les protéines laitières a été possible grâce à l'utilisation de la microscopie à force atomique. Les protéines solubles interagissent de façon spécifique avec les bactéries alors que les caséines interagissent de façon non spécifique. De même, la présence de pili à la surface de la bactérie est favorable à la l'établissement d'interactions fortes entre LGG et les protéines solubles. Ce travail a donc permis d'expliquer des résultats obtenus à l'échelle macroscopique (taux d'encapsulation et de survie dans des conditions gastriques) grâce à des observations microscopiques et une étude à l'échelle nanoscopique des interactions entre les différents composants. Cette approche multi-échelle a élucidé certains mécanismes régissant l'encapsulation de bactéries probiotiques dans des matrices laitières<br>In this work thesis, an encapsulation process for probiotic bacteria using only milk proteins is developed. The laboratory scale followed by a pilot and an industrial scale development of the process allow the production of stable and resistant microparticles both in aqueous and gastric media. The nature and quantities of proteins added is found to influence the bacterial location in the microparticles. The bacteria are located mostly at the surface when only caseins are present. Oppositely, well encapsulated bacteria are observed when addition of whey proteins are performed. A molecular study of interactions established between milk proteins and bacteria is possible with the use of atomic force microscopy. Whey proteins are found to specifically interact with bacteria whereas the caseins establish non-specific links. In addition, the presence of piliated bacteria is found favorable to establish strong and long interactions between proteins and bacteria. This work permits the interpretation of results obtained at a macroscale (encapsulation rate and bacterial survival in stomach conditions) thanks to microscopic observations and nanoscopic interactions study. This multiscale approach permits the elucidation of mechanisms driving the probiotic encapsulation in milk matrices

The master's thesis is focused on study of specific protective proteins from animal products. Two different types of antimicrobial egg white proteins were studied in detail - antimicrobial protein ovotransferrin (conalbumin) and enzyme lysozyme. Ovotransferrin belongs to transferrin group of proteins and exhibits activities similar to milk protective protein lactoferrin. The main effects of ovotransferrin are antiviral, anticancer and immunomodulatory. Antimicrobial activity of ovotransferrin based on the possibility to bind iron is still a subject of interest. For comparison the second egg protein lysozyme (N-acetyl muramidglycan hydrolase) was used. Lysozyme is a hydrolytic enzyme which primary attack cell wall of bacteria. In the theoretical part of the thesis an overview of the specific antimicrobial proteins in selected animal products was introduced mainly focused on ovotransferrin and lysozyme. The experimental part of this work was focused on optimization of methods for the determination of antimicrobial activity, protein concentration and purity. For quantitative analysis of total proteins, optimized Hartree – Lowry spectrophotometric method was used. For the determination of molecular weight and purity SDS-PAGE was used and stained by Coomassie Brilliant Blue G250 and silver. In experimental part the real sample of egg white was compared with samples of lyophilized antimicrobial proteins and therapeutical pills supplied by industrial partner. Protein composition and purity of these preparative has been determined. Antimicrobial activity of ovotransferrin was studied on cultures of G+ bacterium Bacillus subtilis and for comparison on G– E. coli. Ovotransferrin showed antimicrobial effect only at very high concentrations of about 75 mg/ml (Bacillus subtilis) and 50 mg/ml (E coli) even with addition of high amount (100 mM) of hydrogen carbonate ions. The inhibitory effect was most evident in liquid media. On the other hand, lysozyme exhibited significant inhibitory activity from 0.3 mg/ml on gram positive bacteria. Inhibitory effect on E. coli was not observed. Another part of study was focused on isolation of ovotransferrin from egg white using gel permeation chromatography on Sephadex G100. As mobile phases 0.1 M phosphate buffer and 0.05 M Tris-HCl buffer were tested. By SDS-PAGE the purity of ovotransferin comparing to standard was evaluated. Finally, the encapsulation of ovotransferrin and lysozyme was tested. Ovotransferrin and lysozyme was encapsulated into liposome and chitosan particles. Particles stability, distribution and average size distribution were studied by dynamic light scattering and zeta potential measurement. The stability of particles in the model physiological conditions was studied too.

Ce travail de thèse a permis d’étudier l’influence de l’ajout d’ingrédients fonctionnels laitiers sur l’encapsulation de L. rhamnosus GG (LGG). Deux ingrédients laitiers (ß-lactoglobuline et membrane des globules gras du lait - MFGM) ont été identifiés comme étant capables d’adhérer fortement à LGG par l’intermédiaire de ses pili. Le rôle clé de ces adhésions dans la localisation spatiale des bactéries dans la matrice laitière a été mis en évidence, ainsi que le rôle des constituants de la matrice dans sa structuration. Cela a permis de sélectionner, in vitro, une matrice d’encapsulation capable de protéger de manière efficace les bactéries des conditions gastriques et de libérer les bactéries vivantes au niveau de l’intestin. En parallèle, la MFGM dans l’encapsulation des bactéries s’est révélée prometteuse. Ce travail a également démontré l’importance primordiale du choix de la matrice d’encapsulation. En effet, une compétition entre l’adhésion de LGG aux cellules intestinales et l’adhésion de LGG à certains composants de la matrice laitière a été démontrée. Les deux phénomènes impliquent probablement les mêmes mécanismes : adhésion aux pili glycosylés de LGG. Pour terminer, un procédé de séchage par atomisation a été développé pour encapsuler LGG. Il permet une bonne survie des bactéries après séchage et la production de microparticules présentant des propriétés fonctionnelles innovantes liées à la température du milieu de réhydratation<br>The aim of this work was to understand how functional dairy components influence L. rhamnosus GG (LGG) encapsulation. First, two dairy components (-lactoglobulin and milk fat globule membrane - MFGM) able to strongly adhere to LGG through their pili are identified. The key role of these adhesions on bacteria spatial location in the matrix is highlighted, as well as the role of matrix dairy components in their structuration. This allowed to select, in vitro, a matrix able to protect bacteria in gastric conditions and to release them viable in the intestine. Simultaneously, the use of MFGM in bacteria encapsulation has proven to be promising. This work demonstrated the importance of the matrix choice in the encapsulation procedure. Results demonstrated that adhesion between LGG and dairy matrix may compete with adhesion of LGG to epithelial intestinal cell. The two phenomena likely involve the same mechanisms: adhesion to glycosylated pili of LGG. To finish, a spray drying encapsulation process is developed to encapsulate bacteria. It leads to a high bacteria survival after drying and the production of microparticles with innovative properties depending on rehydration temperature

Encapsulation d'adn dans des liposomes contenant du lactosylceramide dont le sucre terminal est reconnu specifiquement par des recepteurs presents sur la membrane plasmique des cellules visees, c. A. D. , les hepatocytes et les cellules endotheliales du foie et egalement les lymphocytes de la rate. Injection par voie intraveineuse des liposomes. Role de l'endocytose, dans leur internalisation. Modele genetique constitue du gene de la preproinsuline i de rat insere dans des vecteurs retroviraux permettant l'expression du gene dans des celules non insulogenes

L’ingénierie protéique servant autrefois à comprendre les relations structures-fonctions des protéines connait un tournant majeur depuis plusieurs années. L’ingénierie protéique évolue pour créer des nouvelles fonctions protéiques : c’est la naissance de l’ingénierie protéique moderne. L’objectif de ma thèse a consisté à mettre en place et caractériser deux approches indépendantes d’ingénierie protéique dans le domaine du vaccin et du diagnostic. Le premier projet consistait à générer des ligands protéiques à partir d‘échafaudages moléculaires (des alternatifs aux anticorps) en couplant le ribosome display au NGS et en développant des outils d’analyses bio-informatiques. Des sélections contre des cibles protéiques d’origine bactérienne et virale ont conduit à l’identification de ligands Affibodies affins (µM au nM). Leur caractérisation a validé leur potentiel comme outil de recherche et de réactif diagnostique. Ces études ont permis de valider la plateforme de génération des ligands mise en place, en augmentant l’exploration de l’espace de diversité des interactions des ligands. Le second projet portait sur le développement d’une plateforme de présentation et de vectorisation à partir de particules d’encapsuline. Elles ont été génétiquement modifiées pour présenter de manière répétée à leur surface l’ectodomaine de la protéine de matrice M2 (M2e) du virus Influenza A H1N1 tout en encapsulant une protéine hétérologue : l’eGFP. Les nanoparticules modifiées sont correctement formées et encapsulent l’eGFP. Des souris immunisées par ces particules induisent une réponse anticorps spécifique contre l’épitope M2e et l’eGFP. L’utilisation de ces nanoparticules comme plateforme vaccinale de présentation et de vectorisation est prometteuse et ouvre la voie pour d’autres applications en biotechnologie<br>In the past, protein engineering used to understand function and structure relationship. But since few years, protein engineering was used to create new protein functions: modern protein engineering was born. The aim of my thesis was to set up and characterize two approaches of protein engineering in diagnostic and vaccine field. The first project was to generate artificial binder using protein scaffolds as an alternative to antibodies by coupling ribosome display (RD) to NGS and developing bio-informatics tools. Screening and selection against bacterial and viral targets have led to affibody binder’s identification with an affinity range from µM to nM. Their characterization has validated their potential as research tools and protein reagents for diagnostic assay. Coupling ribosome display to high throughput sequencing as means to directly identify selected binder coding sequences, enormously enhance binder discovery depth. The second project was to generate an innovative nanocarrier based on encapsulin nanoparticle, for customized peptide display and cargo protein vectorization. Encapsulin particles from T.maritima were genetically modified for simultaneous display of the matrix protein 2 ectodomain of the influenza H1N1 A virus and heterologous protein eGFP packaging. Genetically engineered encapsulin nanoparticles were well-formed and abled to efficiently load eGFP. Immunogenicity studies revealed antibody responses against both the surface epitope and the loaded cargo protein. Taken together, this display system is a versatile tool for rational vaccine design and paves the way for new applications in the research fields of vaccine, antimicrobial research and other biotechnological applications

Tuberculosis (TB) is an infectious, deadly disease, caused by Mycobacterium tuberculosis (M.tb). In 2010, there were 8,8 million incident cases of TB globally. South Africa currently has the third highest TB incident cases worldwide. In an attempt to address the challenges facing TB chemotherapy, among which frequent dosing and long duration of therapy resulting in poor patient compliance, a novel poly(DL-lactic-co-glycolic) acid (PLGA) nanoparticulate drug delivery system (DDS) encapsulating anti-TB drugs was developed. It is hypothesised that this nanoparticulate DDS will address the challenges mentioned by enabling decreased dosing frequency, shortening duration of therapy and minimising adverse side effects. Therefore, favourable modification of pharmacodynamic (PD) and pharmacokinetic (PK) properties of the conventional anti-TB drugs was demonstrated. Furthermore, the nanoparticles will provide a platform for drug delivery to macrophages that serve as hosts for M.tb. The study design was based on determining specific physicochemical properties of the nanoparticulate DDS to elucidate the hypothesis. Spray-dried PLGA nanoparticles were prepared using the double emulsion solvent evaporation technique. In vivo analysis of macrophage uptake and possible immunological response in mice were evaluated. In vitro protein-binding assays of PLGA nanoparticles encapsulating anti-TB drugs isoniazid (INH) and rifampicin (RIF) were performed with subsequent in vivo tissue distribution assays to support protein-binding data generated. Finally, PK/PD analyses were conducted to evaluate the effect of nanoencapsulation on the anti-TB drugs. These involved in vitro assays to determine if sufficient drug was released from the nanoparticles to exhibit minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC). Furthermore, in vivo drug distribution and drug release kinetics assays of encapsulated RIF, INH, pyrazinamide (PZA) and ethambutol (ETB) in a mouse model were performed. The results confirmed that the PLGA nanoparticles (<250 nm, low positive zeta potential) were taken up by macrophages in vivo with no significant immunological effect. Furthermore the nanoparticles were present in the brain, heart, kidneys, lungs, liver and spleen for up to 7 days following once-off oral dosing at 13.23± 0.11%, 16.81± 0.11%, 54.89± 0.95%, 15.61± 1.15%, 48.48± 2.28% and 5.73± 0.21%, respectively. This was further confirmed by drug analysis demonstrating the presence of INH, RIF and ETB at different time points up to 7 days in the lungs, kidneys, liver and spleen. However, PZA was not detected. Nanoencapsulated RIF and INH exhibited MICs and MBCs in vitro over 14 days and these drugs were also observed in plasma for up to 7 days post once-off oral dosing. ETB and PZA were observed up to 3 days. From the results generated, it can be concluded that the nanoparticles were taken up by macrophages without eliciting an immune response. This provides a platform for drug delivery to specific sites. Furthermore, the nanoparticulate DDS exhibited sustained drug release in vitro and in vivo over a number of days above the MIC for the drugs analysed. Sustained drug distribution was also observed. It can therefore be concluded that the hypothesised reduction in dose frequency and duration of therapy for this DDS is a possibility<br>Thesis (PhD (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013

Orientadores: Maria Helena Andrade Santana, Ricardo de Lima Zollner<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica<br>Made available in DSpace on 2018-07-27T03:55:49Z (GMT). No. of bitstreams: 1 Victorino_IgorRicardodeSouza_M.pdf: 6760377 bytes, checksum: 05fc12e1f97a8b092dbd91b47b06441d (MD5) Previous issue date: 2000<br>Resumo: Os lipossomas têm sido utilizados como imunoadjuvantes antigênicos, em estudos que enfatizam a sua atuação no sistema imunológico. Extratos peptídicos ou protéicos de várias fontes foram encapsulados em lipossomas ou associados à sua superficie, e aplicados em imunoterapias e vacinas. Apesar disso, poucos são os estudos voltados para a performance do processo de preparação desses lipossomas, eficiência da associação proteína/lipídio e estabilidade das vesículas. Este trabalho trata da avaliação do encapsulamento das proteínas: Albumina de Soro Bovino (BSA), Mioglobina (Mio) e Cito cromo C (Cit C), simulando extratos alergênicos provenientes de fungos e ácaros, no que se refere à faixa de peso molecular das suas proteínas. Os lipossomas foram preparados com os lipídios L-a-distearoilfosfatidi1colina (DSPC) e Colesterol (Col) na razão molar 70:30 respectivamente. As proteínas foram encapsuladas individualmente e em forma de misturas de composições molares BSA:Mio:Cit C de 20:40:40, 40:20:40 e 40:40:20, respectivamente. Além disso, a mistura de proteínas de composição 40:20:40 (BSA:Mio:Cit C) foi também associada covalentemente à superficie das vesículas. Os lipossomas foram caracterizados pelo teor de fósforo e diâmetro médio. O desempenho do encapsulamento foi analisado através dos perfis e eficiências de encapsulamento das proteínas individuais e em misturas, além da estabilidade das vesículas em tensoativo penta etileno glicol mono-n-dodecil éter (C12Es), e de plasma humano. Para as misturas, analisou-se a exclusão de proteínas durante o encapsulamento e a associação à superficie dos lipossomas. Os resultados indicam que a eficiência de encapsulamento depende mais das interações proteína:lipídio que do tamanho das proteínas. As eficiências de encapsulamento variaram entre 0,03% e 3,55% para as proteínas individuais e entre 1,04% e 3,94% para as misturas. Não houve alteração na estabilidade dos lipossomas em C12Es com a presença das proteínas. Em plasma, essas vesículas permaneceram estáveis por aproximadamente 20 horas. Na associação à superficie dos lipossomas, predominou a presença de BSA em relação às outras proteínas<br>Abstract: Liposomes has been studied as antigenic immunoadjuvants with emphasis on their performance in the imunological system. Peptides or proteins of various sources were encapsulated in liposomes or associated to their surface, and applied in immunotherapy and vaccines. In spite of this, there are few studies about the performance of preparation process ofthese liposomes, efficacy ofthe association proteinllipid and the stability ofvesic1es. This work concems with the evaluation of the encapsulation of the proteins Bovine Serum Albumin (BSA), Myoglobin (Myo) and Cythochrome C (Cyt C), simulating allergen extracts from molds and mites, related to the range of molecular weight of their proteins. Liposomes were prepared with lipid L-a-disteraoylphosphatidylcholine (DSPC) and Cholesterol (Chol) at molar ratio 70:30 respectively. The proteins were encapsulated individualyand in mixtures with molar composition BSA:Myo:Cyt C of20:40:40, 40:20:40 and 40:40:20 respectively. Furthermore, the mixture 40:20:40 (BSA:Myo:Cyt C) was also covalent1y associated to the surface of vesic1es. Liposomes were characterized by their phosphate contents and mean diameter. The performance of the protein encapsulation was evaluated through the profiles and efficiency of the encapsulation and throught the stability of vesic1es in the presence of the surfactant penta ethylene glycol mono-n-dodecyl ether (C12ES) and human plasma. For the mixtures, the exclusion of proteins duringentrapment or association to the liposome surface was also evaluated. The experimental results indicate that the efficacy of encapsulation is more dependent of the interactions proteinllipid than the size of the proteins. The efficiencies of encapsulation changed between 0.03% and 3,55% for individual proteins and were between 1,04% and 3,94% for the mixtures. The presence ofproteins does not altered the stability of liposomes in C12ES . In human plasma the vesic1es remained with stability about 20 hours. For the mixtures, the presence ofthe BSA protein predominated in the vesicles<br>Mestrado<br>Desenvolvimento de Processos Biotecnologicos<br>Mestre em Engenharia Química

Ce travail porte sur l'étude de la microencapsulation d'huile de lin par coacervation complexe des protéines du lactosérum et de la gomme d'acacia. Il se focalise sur la coacervation des protéines du lactosérum et de la gomme d'acacia en milieu aqueux ainsi que sur les mécanismes impliqués dans la formation des microcapsules par coacervation complexe. La coacervation complexe est un phénomène de séparation de phase associative induit par des interactions électrostatiques entre deux polymères. Le couple de polymères le plus utilisé en coacervation complexe est le système gélatine/gomme d'acacia. Cependant, pour des raisons sanitaires et religieuses, l'utilisation de la gélatine devient controversée pour les applications alimentaires. Un substituant intéressant à la gélatine est constitué par les protéines du lactosérum ainsi que leur composant majoritaire, la bêta-lactoglobuline. L'étude de la composition du coacervat du système protéines du lactosérum/gomme d'acacia a été réalisée lors de ce travail. L'électrophorèse capillaire sur gel a été employée afin de quantifier la bêtalactoglobuline et l'alpha-lactalbumine dans le coacervat. L'influence du ratio protéine/polysaccharide et du pH sur la composition du coacervat a été étudiée. Bien que le procédé d'encapsulation par coacervation complexe soit connu depuis de nombreuses années, les mécanismes conduisant à la formation des microcapsules restent peu décrits. Le procédé d'encapsulation par coacervation complexe conduisant à la formation des microcapsules est composé de plusieurs étapes nécessitant chacune d'être examinée. L'étape d'émulsification a lieu en régime d'écoulement intermédiaire. La modélisation en régime turbulent rend compte des résultats expérimentaux et pourrait être utilisée pour une transposition d'échelle. Le suivi in situ du procédé d'encapsulation par coacervation complexe a été réalisé pour la première fois lors de cette étude. Il a été réalisé au moyen d'une sonde vidéo immergée dans le réacteur agité. Cette technique a permis de relever quatre étapes successives induites par l'abaissement du pH. Les influences des paramètres physico-chimiques (ratio protéine/polysaccharide et concentration totale en biopolymères) et des paramètres liés à l'étape de coacervation sur la formation des microcapsules ont aussi été étudiées au moyen de la sonde vidéo. Coacervation complexe, encapsulation, protéines du lactosérum, électrophorèse capillaire, suivi in situ, émulsification<br>This work deals with the study of linseed oil microencapsulation by complex coacervation of whey proteins and acacia gum. It focuses on the coacervation of whey proteins and acacia gum in aqueous medium as well as the mechanisms involved in the formation of microcapsules by complex coacervation. Complex coacervation is an associative phase separation phenomenon induced by electrostatic interactions between two polymers. The most widely used pair of polymers in complex coacervation is the system gelatin / acacia gum. However, the use of gelatin is a matter of controversy for food applications. An interesting alternative to gelatin consists of whey proteins and their major component, beta-lactoglobulin. An investigation of the composition of the coacervate system whey protein / acacia gum was carried out during this work. Capillary gel electrophoresis was used to quantify beta-lactoglobulin and alphalactalbumin in the coacervate. The influence of protein / polysaccharide ratio and pH on the composition of the coacervate was studied. Although the encapsulation process by complex coacervation has been known for many years, the mechanisms leading to the formation of microcapsules are not so much described. The encapsulation process by complex coacervation leading to the formation of microcapsules includes several stages that were examined. The emulsification step takes place in the intermediate flow regime. The modeling in turbulent regime accounted for experimental results and might be used for scaling-up the process. In situ monitoring of the encapsulation process by complex coacervation was performed for the first time in this study. It was carried out using a video probe immersed in a stirred reactor. This technique identified four successive steps induced by lowering the pH: the emulsification of the oil, the formation of the coacervate, the adsorption of the coacervate on the oil droplets, the formation of an encapsulation shell. The influence of physico-chemical parameters (protein / polysaccharide ratio and total concentration of biopolymers) and parameters related to the coacervation step on the formation of microcapsules were also studied by using the video probe

A method was developed to produce and incorporate a network of discrete, genipin-crosslinked gelatin microfibers around a pancreatic islet within a barium alginate microcapsule. This technique allows for the encapsulation of a porous fibrous matrix without the geometrical restrictions required for cellular aggregate seeding. Microfibers were produced from a novel vortex-drawn extrusion system with an alginate support matrix. Optimization culminated in a hydrated fiber diameter of 22.3 ± 0.4 μm, a 98% reduction in cross sectional area, while making the process more reliable and less labour intensive. The optimized microfibers were encapsulated at 40 vol% within 294 ± 4 μm 1.6% barium alginate microparticles by an electrostatic-mediated dropwise extrusion system. Pancreatic islets extracted from Sprague Dawley rats were encapsulated within the microparticles, and analyzed over a 21-day preliminary in vitro study. Acridine orange and propidium iodide fluorescent viability staining and light microscopy indicated a significant increase in viability for the fiber-laden particles relative to fiber-free control particles at days 7, 14, and 21. The fiber-laden system also reduced the incidence of disrupted islet cohesion from 31% to 8% at day 21, and showed evidence of islet-fiber adhesion. Preliminary investigations into insulin secretion and metabolic activity showed no significant difference between test and control groups. Further investigation into benefits of islet encapsulation within an extracellular matrix fiber network will be the subject of future studies with this body of work serving as a foundation. The system developed in this investigation could be developed into a modular scaffold system for tissue engineering beyond the field of islet research.<br>Thesis (Master, Chemical Engineering) -- Queen's University, 2011-08-18 15:05:50.917

The overall goal of this research was to develop a plant protein-based microcapsule capable of carrying, protecting and delivering flaxseed oil within the food and gastrointestinal environment. Specifically, the research aimed to: a) screen a variety of plant proteins and pre-treatment conditions based on their emulsifying properties for use as a wall material; b) develop and optimize encapsulation protocols for entrapping flaxseed oil; and c) study the oxidative stability and delivery of entrapped oils from capsules under different environmental and simulated gastrointestinal conditions. In Chapter 3 and 4, the emulsifying and physicochemical properties of legume and oilseed protein isolates, respectively produced from isoelectric precipitation and salt extraction were investigated. Findings in Chapter 3 indicated that both the legume source and method of production showed significant effects on the emulsifying and physicochemical properties of chickpea (ChPI), faba bean (FbPI), lentil (LPI), pea (PPI), and soy (SPI) protein isolates. The emulsion capacity (EC) values ranged between 476-542 g oil/g protein with LPI showing the highest capacity. Isoelectric-precipitated ChPI and LPI displayed higher emulsion activity index (EAI) (~46.2 m2/g), (emulsion stability index) ESI (~84.9 min) and (creaming stability) CS (98.6%), which were comparable to those of SPI. In Chapter 4, findings indicated that both protein source and method of production had significant effects on the physicochemical and emulsifying properties of canola (CaPI) and flaxseed protein isolates (FlPI). CaPI showed significantly higher EC (~515.6 g oil/g protein) than FlPI (~498.9 g oil/g protein). EAI for FlPI was found to be higher (~40.1 m2/g) than CaPI (~25.1 m2/g) however, ESI values of CaPI and FlPI were similar. Creaming stability of emulsions stabilized by CaPI and FlPI ranged between 86.1 and 96.6%. CaPI and FlPI were shown to have emulsion forming properties; however their stability was low. In Chapter 5, ChPI and LPI-stabilized emulsions were optimized based on pH, protein concentration and oil content for their ability to form and stabilize oil-in-water emulsions using response surface methodology. Droplet charge was shown to be only affected by pH, while droplet size and creaming index were affected by protein concentration, oil content and pH. Optimum conditions for minimal creaming (no serum separation after 24 h), small droplet size (<2 μm), and high net droplet charge (absolute zeta potential (ZP) value >40 mV) were identified as: 4.1% protein, 40.0% oil, and pH 3.0 or 8.0, regardless of the plant protein used for emulsion preparation. Flaxseed oil was microencapsulated by freeze (Chapter 6) or spray (Chapter 7) drying employing ChPI or LPI and maltodextrin. Effects of emulsion formulation (oil, protein and maltodextrin levels) and protein source (ChPI vs. LPI) on the physicochemical characteristics, oxidative stability, and release properties of the resulting capsules were investigated. Optimized capsule designs were found to have high encapsulation efficiencies, low surface oil, and afforded protection against oxidation over a 25 d room temperature storage study relative to free oil. Microcapsules were also able to deliver 84.2% of the encapsulated oil in the simulated gastrointestinal environments.

The overarching goal of this research was to formulate an encapsulated powder using a modified lentil protein isolate-maltodextrin mixture to encapsulate flaxseed oil by freeze drying. The primary objectives were: a) to examine the physicochemical and emulsifying properties of lentil protein isolates with different degrees of hydrolysis; b) to design and test the physicochemical properties of encapsulated flaxseed oil using a wall material with native, heat treated and partially hydrolyzed lentil proteins in combination with maltodextrin; and c) test the oxidative stability of encapsulated flaxseed oil with the capsule design with the lowest surface oil and highest encapsulation efficiency versus free oil. During the first study, the physicochemical and emulsifying properties of lentil protein isolates (LPI) were investigated as a function of their degree of hydrolysis (DH of 4, 9 and 20%) following exposure to trypsin/heat. Interfacial tension, surface characteristics (charge and hydrophobicity) and intrinsic fluorescence were determined and related to changes in the emulsification activity (EAI) and stability indices (ESI) of unhydrolyzed (u-LPI) and hydrolyzed LPI (h-LPI) in a flaxseed oil-water emulsion. Most importantly surface hydrophobicity declined from ~30 to ~24 for the u-LPI and h-LPI (DH 4-20%), respectively. The changes in physicochemical properties induced by hydrolysis had a detrimental effect on EAI and ESI values, which declined from ~51 to ~47 m2 g-1 and ~12 to ~ 11 min for u-LPI and h-LPI (DH 4-20%), respectively. In the second study, the physicochemical properties of encapsulated flaxseed oil within lentil protein-based maltodextrin microcapsules were investigated using native (n-LPI), pre-treated (heated, un-hydrolyzed (u-LPI); and heated, hydrolyzed (h-LPI)) lentil protein isolates and as a function of oil load (10.0, 20.0 and 30.0% of total solids). The moisture, water activity, surface oil and encapsulation efficiency (EE) were assessed, along with droplet size and emulsion morphology. Light microscopy imaging of the emulsions, showed that the h-LPI had slightly larger oil droplets than the n-LPI and u-LPI, which both appeared similar. Microcapsules prepared from h-LPI showed significantly higher surface oil and lower EE than both the n-LPI and u-LPI materials. The microcapsules prepared using n-LPI with 10.0% oil loading were found to have the lowest surface oil content (~3.7%) and highest EE (~62.8%) for all formulations, and were subjected to an oxidative storage stability test over a 30 d period vs. free oil. The encapsulation process however induced autooxidation leading the production of a greater amount of primary oxidative products than free oil. Findings indicate that future studies are necessary to enhance the stability of the flaxseed oil through the encapsulation process.

2-Methacryloyloxyethyl Phosphorylcholine is an interesting biocompatible monomer. An improved method for the synthesis of poly(MPC) and its copolymers using Reversible Addition-Fragmentation chain Transfer (RAFT) has been discussed in the first part of the thesis. Previous reports related to the synthesis of MPC homopolymers and copolymers in aqueous medium are found to be less effective because of the hydrolysis of chain transfer agent in water. Hydrolysis of chain transfer agent results in the loss of active chain ends thereby, reducing control over polymerization and increasing the polydispersity of resulting polymers. Therefore, in this work MPC polymers were synthesized by RAFT using methanol as solvent. This method of synthesis produced polymers having controlled molecular weights as well as narrow polydispersities. In the second part of the work, methoxydiethylene glycol methacrylate (MeODEGM)-MPC based thermo-responsive core-shell nanogels were synthesized for use in protein encapsulation and release. The size of the nanogels was controlled by varying the concentration of cross-linker. The nanogels were synthesized using an acid degradable crosslinker which helped in the release of encapsulated protein at acidic pH. The effect of various parameters on encapsulation efficiency of proteins was studied and it was found that apart from the size of protein, the cross-linker concentration of nanogel also affected the amount of protein encapsulated.<br>Chemical Engineering

More and more newly registered drugs are proteins. Although many of them suffer from instabilities in aqueous media, the most common way of protein drug administration still is the injection of a solution. Numerous protein drugs require frequent administration, but suitable controlled release systems for proteins are rare. Chapter 1 presents current advances in the field of controlled delivery of particulate protein formulations. While the main focus lies on batch crystallized proteins, amorphous particulate proteins are also discussed in this work. The reason is that, on the one hand precipitated protein particles hold some of the advantages of crystalline proteins and on the other hand the physical state of the protein may simply be unknown for many drug delivery systems or semi-crystalline particles have been used. Crystallization and precipitations methods as well as controlled delivery methods with and without encapsulation in a polymeric delivery system are summarized and critically discussed. In chapter 2 a novel way of protein crystal encapsulation by electrospinning is introduced. Electrospinning of proteins has been shown to be challenging via the use of organic solvents, frequently resulting in protein unfolding or aggregation. Encapsulation of protein crystals represents an attractive but largely unexplored alternative to established protein encapsulation techniques because of increased thermodynamic stability and improved solvent resistance of the crystalline state. We herein explore the electrospinning of protein crystal suspensions and establish basic design principles for this novel type of protein delivery system. Poly-ε-caprolactone (PCL) is an excellent polymer for electrospinning and matrix-controlled drug delivery combining optimal processability and good biocompatibility. PCL was deployed as a matrix, and lysozyme was used as a crystallizing model protein. By rational combination of lysozyme crystals with a diameter of 0.7 or 2.1 μm and a PCL fiber diameter between 1.6 and 10 μm, release within the first 24 h could be varied between approximately 10 and 100%. Lysozyme loading of PCL microfibers between 0.5 and 5% was achieved without affecting processability. While relative release was unaffected by loading percentage, the amount of lysozyme released could be tailored. PCL was blended with poly(ethylene glycol) and poly(lactic-co-glycolic acid) to further modify the release rate. Under optimized conditions, an almost constant lysozyme release over 11 weeks was achieved. Chapter 3 takes on the findings made in chapter 2 and further modifies the properties of the nonwovens as protein crystal delivery system. Nonwoven scaffolds consisting of poly-ε-caprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA) and polidocanol (PD), and loaded with lysozyme crystals were prepared by electrospinning. The composition of the matrix was varied and the effect of PD content in binary mixtures, and of PD and PLGA content in ternary mixtures regarding processability, fiber morphology, water sorption, swelling and drug release was studied. Binary PCL/PD blend nonwovens showed a PD-dependent increase in swelling of up to 30% and of lysozyme burst release of up to 45% associated with changes of the fiber morphology. Furthermore, addition of free PD to the release medium resulted in a significant increase of lysozyme burst release from pure PCL nonwovens from approximately 2% to 35%. Using ternary PCL/PD/PLGA blends, matrix degradation could be significantly improved over PCL/PD blends, resulting in a biphasic release of lysozyme with constant release over 9 weeks, followed by constant release with a reduced rate over additional 4 weeks. Based on these results, protein release from PCL scaffolds is improved by blending with PD due to improved lysozyme desorption from the polymer surface and PD-dependent matrix swelling. Chapter 4 gives deeper insight on lysozyme batch crystallization and shows the influences of the temperature on the precipitation excipients. Yet up to now protein crystallization in a pharmaceutical useful scale displays a challenge with crystal size and purity being important but difficult to control parameters. Some of these influences are being discussed here and a detailed description of crystallization methods and the achieved crystals are demonstrated. Therapeutic use of such protein crystals may require further modification of the protein release rate through encapsulation. Silk fibroin (SF) harvested from the cocoons of Bombyx mori is a well-established protein suitable for encapsulation of small molecules as well as proteins for controlled drug delivery. This novel polymer was deployed for as carrier for the model drug crystals. Lysozyme again was used as a crystallizable protein and the effect of process- as well as formulation parameters of batch crystallization on crystal size were investigated using statistical design of experiments. Lysozyme crystal size depended on temperature and sodium chloride and poly(ethylenglycol) concentration of precipitant solution. Under optimized conditions, lysozyme crystals in a size range of approximately 0.3 to 10 µm were obtained. Furthermore, a solid-in-oil-in-water process for encapsulation of lysozyme crystals into SF was developed. Using this process, coating of protein crystals with another protein was achieved for the first time. Encapsulation resulted in a significant reduction of dissolution rate of lysozyme crystals, leading to prolonged release over up to 24 hours<br>Immer mehr neu zugelassene Arzneimittel sind Proteine. Obwohl viele von ihnen in wässrigen Milieus sehr instabil sind, werden die meisten Proteine immer noch als Parenteralia formuliert. Kontrollierte Freisetzungssysteme sind selten, und das obwohl zahlreiche Proteine regelmäßig verabreicht werden müssen. Kapitel 1 befasst sich mit den aktuellen Fortschritten im Bereich der kontrollierten Darreichungsformen für Proteinpartikel. Das Hauptaugenmerk liegt klar auf den „batch“ kristallisierten Proteinen, aber auch allgemeine ‚amorphe‘ Proteinpartikel werden diskutiert. Das liegt daran, dass zum einen präzipitierte Proteinpartikel ebenfalls einige Vorteile der Proteinkristalle haben und zum anderen wurde in einigen Fällen der physikalische Zustand der Proteine nicht bestimmt oder es wurden zumindest semi-kristalline Proteinpartikel verwendet. In diesem Kapitel werden Kristallisations- und Präzipitationsmethoden sowie kontrollierte Freisetzungssysteme mit und ohne vorherige Einbettung in ein Polymersystem zusammengefasst und kritisch diskutiert. Kapitel 2 stellt eine neue Methode der Proteinkristalleinbettung durch Electrospinning vor. Electrospinning von Proteinen hat sich aufgrund des häufigen Einsatzes von organischen Lösemitteln als schwierig heraus gestellt und führt häufig zum Entfalten oder Denaturieren des Proteins. Aufgrund höherer thermodynamischer Stabilität und verbesserter Kompatibilität mit organischen Lösemitteln haben sich Proteinkristalle hier als attraktive wenngleich bisher ziemlich unbeachtete Alternative heraus gestellt. In diesem Kapitel wird das das Electrospinning von Proteinkristallsuspensionen erforscht und grundlegende Bedingungen für diese neuartige Art der Proteinverkapselung etabliert. Poly-ε-caprolacton (PCL) eignet sich hervorragend für das Electrospinning und zeichnet sich durch matrixkontrollierte Freisetzung, einfache Handhabbarkeit sowie gute Biokompatibilität aus. PCL wurde als Matrix und Lysozym als Modelprotein eingesetzt. Mittels Kombination zweier Lysozymkristalldurchmesser, 0,7 oder 2,1 µm, und der Variation des PCL-Fadendurchmessers zwischen 1,6 und 10 µm konnte die Initialfreisetzung innerhalb der ersten 24 Stunden zwischen ca. 10 und 100 % modifiziert werden. Die Beladung der PCL Mikrofäden mit Lysozym zwischen 0,5 und 5 % konnte ohne Beeinträchtigung des Herstellungsprozesses erreicht werden. Hierdurch konnte die gesamt freigesetzte Menge an Lysozym variiert werden, während die relative freigesetzte Menge konstant blieb. Um weitere Modifikationen an der Freisetzungsrate vorzunehmen, wurden Poly(ethylen glycol) und Poly(lactic-co-glycolic acid) (PLGA) dem PCL beigemischt. Dadurch konnte unter optimierten Bedingungen eine Freisetzungsdauer von über 11 Wochen erreicht werden. Kapitel 3 greift die Entdeckungen des zweiten Kapitels auf und nimmt weitere Modifikationen an den Fasermatten vor. Die mit Proteinkristallen beladenen Fasermatten wurden wieder durch Electrospinning hergestellt und bestehen aus PCL, PLGA und Polidocanol (PD). Die Zusammensetzung der Matrix wurde variiert und die Auswirkungen des PD-Gehalts in binären Mischung, sowie des PD- und PLGA-Gehalts in ternären Mischsystemen auf die Verarbeitbarkeit, Fadenmorphologie, Wassersorption, Quellung und Wirkstofffreisetzung untersucht. Binäre Fasermatten aus einer Mischung aus PCL/PD zeigten PD-abhängige Zunahme der Quellung von bis zu 30 % und eine Zunahme der Initialfreisetzung von bis 45 %. Dieser Anstiege werden einer Veränderung der Fasermorphologie zugeschrieben. Das Hinzufügen von freiem PD zum Freisetzungsmedium von reinen PCL Fasermatten führte zu einer Zunahme der Initialfreisetzung von ca. 2 % auf etwa 35 %. Durch den Einsatz von ternären Mischsystem, bestehend aus PCL/PD/PLGA, kam es zu einem signifikant erhöhten Matrixabbau gegenüber den PCL/PD Mischungen. Das führte wiederrum zu einem zweiphasigen Freisetzungsverhalten, das durch eine konstante Freisetzungsrate innerhalb von 9 Wochen, sowie eine anschließende langsamere, ebenfalls konstante Freisetzung über weitere 4 Wochen gekennzeichnet war. Aufgrund dieser Ergebnisse kann gesagt werden, dass die Proteinfreisetzung aus PCL Matrices durch das Hinzufügen von PD aufgrund von verbesserter Lysozymdesorption vom Polymer sowie PD-abhängiger Matrixquellung verbessert werden konnte. Kapitel 4 bringt tiefere Erkenntnisse bezüglich der Lysozym Batchkristallisation und zeigt den Einfluss der Temperatur auf die verwendeten Fällungsreagenzien. Bis heute stellt die Kristallisation von Proteinen in einem pharmazeutisch sinnvollen Maßstab eine Herausforderung dar, wobei die Kristallgröße und die Reinheit wichtige und dennoch schwer kontrollierbare Parameter sind. Einige dieser Einflüsse werden hier näher beleuchtet und Kristallisationsmethoden sowie die erhaltenen Kristalle näher beschrieben. Lysozym wurde erneut als kristallisierbares Protein eingesetzt und die Einflüsse der Prozess- sowie Formulierungsparameter der Batch-Kristallisation mit Hilfe von statistischen Experimentendesigns (Design of Experiment, DoE) untersucht. Die Kristallgröße wurde durch die Temperatur sowie die Natriumchlorid- und Poly(ethylenglycol)konzentration der Fällungslösung bestimmt. Unter kontrollierten Bedingungen konnten Kristallgrößen in einem Bereich zwischen ca. 0.3 bis 10 µm erreicht werden. Darüber hinaus wurde ein „solid-in-oil-in-water“ (Feststoff-in-Öl-in-Wasser) Verfahren entwickelt mit dem Lysozymkristalle in Seidenfibroin eingebettet werden konnten. Mit Hilfe dieses Verfahrens wurden erstmals Proteinkristalle mit einem anderen Protein überzogen. Diese Einbettung brachte eine signifikante Auflösungsverzögerung mit einer verlängerten Freisetzung über 24 Stunden

<p>The design and application of effective drug carriers is a fundamental concern in the delivery of therapeutics for the treatment of cancer and other vexing health problems. Traditionally utilized chemotherapeutics are limited in efficacy due to poor bioavailability as a result of their size and solubility as well as significant deleterious effects to healthy tissue through their inability to preferentially target pathological cells and tissues, especially in treatment of cancer. Thus, a major effort in the development of nanoscopic drug delivery vehicles for cancer treatment has focused on exploiting the inherent differences in tumor physiology and limiting the exposure of drugs to non-tumorous tissue, which is commonly achieved by encapsulation of chemotherapeutics within macromolecular or supramolecular carriers that incorporate targeting ligands and that enable controlled release. The overall aim of this work is to engineer a hybrid nanomaterial system comprised of protein and silica and to characterize its potential as an encapsulating drug carrier. The synthesis of silica, an attractive nanomaterial component because it is both biocompatible as well as structurally and chemically stable, within this system is catalyzed by self-assembled elastin-like polypeptide (ELP) micelles that incorporate of a class of biologically-inspired, silica-promoting peptides, silaffins. Furthermore, this methodology produces near-monodisperse, hybrid inorganic/micellar materials under mild reaction conditions such as temperature, pH and solvent. This work studies this material system along three avenues: 1) proof-of-concept silicification (i.e. the formation and deposition of silica upon organic materials) of ELP micellar templates, 2) encapsulation and pH-triggered release of small, hydrophobic chemotherapeutics, and 3) selective silicification of templates to potentiate retention of peptide targeting ability.</p><br>Dissertation