L'incorporation d'ingrédients bioactifs dans les produits alimentaires est en plein essor. Il a été démontré que ces ingrédients possèdents des propriétés biologiques importantes permettant l'amélioration de la santé et la prévention des maladies dites de civilisations. Toutefois, l'ajout de ces molécules bioactives est dans la plupart des cas impossible ou insuffisant, du fait que ces composés ne sont que peu solubles dans les systèmes aqueux et présentent i) une stabilité limitée contre les dégradations chimique ou physique, ii) une libération non contrôlée ou une faible biodisponibilité. Face à ces contraintes, les recherches actuelles visent à élaborer des systèmes d'encapsulation efficaces pour résoudre ces problèmes de formulation. Dans notre étude, deux représentants d'ingrédients alimentaires ont été choisi : le sesquiterpène nérolidol (Ner) et le flavonoïde quercétine (Quer) présentant diverses activités biologiques mais des propriétés physicochimiques problématiques. Ainsi, l'objectif de notre travail a été d'encapsuler ces composés actifs dans les cyclodextrines (CDs), les liposomes conventionnels (LCs) et le système mixte cyclodextrine-liposomes (DCLs) afin de développer des systèmes naturels et éco-compatibles ayant des applications potentielles dans les domaines alimentaires.Trois axes ont été abordés. Le premier axe a porté sur la préparation et la caractérisation des complexes d'inclusion CD/invité en solution et à l'état solide. Les techniques de spectroscopie UV-visible, Chromatographie Liquide à Haute Performance (CLHP), Carbone Organique Total (TOC), ¹H Résonance magnétique nucléaire (RMN), 2D ROESY RMN et de la modélisation moléculaire ont été utilisées comme outils pour la caractérisation des complexes obtenus. Des études de phase de solubilité ont également été réalisées. Le deuxième axe a porté sur la préparation des LCs et DCLs par la méthode d'injection éthanolique et leur caractérisation. Les préparations des LCs encapsulant la quercétine a été réalisée à partir de phospholides naturels de jaune d'oeuf (Lipoid E80) et de soja insaturés (Lipoid S100) ou saturés (Phospholipon 90H) afin d'étudier l'effet de la composition lipidique que les caractéristiques des liposomes. La formulation optimale a été par la suite appliquée pour préparer des LCs encapsulant le nérolidol et des DCLs encapsulant les deux molécules. Ce dernier est produit par l'incorporation des complexes d'inclusion HP-β-CD/Ner (à différents rapport moléculaire CD:Ner) et SBE-β-CD/Quer dans la cavité aqueuse des liposomes. Le dernier axe a été orienté vers l'évaluation de l'effet de l'encapsulation sur les propriétés physicochimiques du nérolidol et de la quercétine (libération in vitro, photostabilité, stabilité dans les milieux gastro-intestinales, stabilité de stockage) et leur activité antioxydante. Les résultats ont montré que les CSs ont été capables d'encapsuler les composés actifs étudiés, d'augmenter leur solubilité, leur photostabilité ainsi que leur activité antioxydante. En outre, les liposomes à base de Lipoid E80 ont été trouvés majoritairement de taille nanométrique et ont conféré aux molécules une efficacité d'encapsulation (EE) élevée ainsi qu'une meilleure stabilité par rapport aux deux autres types de liposomes. De plus, la taille des DCLs ains que leur EE ont été prouvées dépendante du rapport moléculaire CD:invité. Par rapport aux LCs, les DCLs ont assuré une libération prolongée du nérolidol, ont augmenté la photostabilité des composés et la stabilité de la quercétine dans les milieux biologiques. Les résultats de cette étude suggèrent que ces systèmes peuvent être considérés comme outils prometteurs pour l'optimisation des formulations alimentaires incorporant le nérolidol et/ou la quercétine. / Phytochemicals are widely distributed secondary metabolites, divided into three major classes : terpenoids, flavonoids and alkaloids. They are shown to possess important biological properties such as anti-cancer, anti-inflammatory and anti-microbial properties. Therefore, increasing the use of these bioactive molecules in food products may reduce the risk of widespread diseases referred to as "diseases of civilization". However, their low solubility, susceptibility to degradation and their rapid release reduce their bioavailability in the human body and thus their biological effect. To solve the aforementioned physicochemical drawbacks, encapsulation systems were developed to allow the incorporation of phytochemicals in food. In this study, two food ingredients : the sesquiterpene nerolidol and the flavonol quercetin were selected du to their potent biological activities but their problematic physicochemical properties.Therfore, the aim of this work was to encapsulate these molecules into cyclodextrins (CDs), conventional liposomes (CLs) and the double systeme drug-in-cyclodextrin-in-liposomes (DCLs), in order to develop nztural and biocompatible formulations that may find applications in food fields. This project was built around three main research axes. The first part dealt with the preparation and the characterization of CD/guest inclusion complexes both in solution and in solid state. Characterizations were performed with UV-visible spectroscopy, High Performance Liquid Chromatography (HPLC), Total Oragnic Carbon (TOC), ¹H NMR, 2D ROESY NMR, and molecular modeling. These investigations were complemented with phase solubility studies.The second axis addressed the preparation of CLs ans DCLs by ethanol injection method and characterization of the vesicles. CLs encapsulating quercetin were prepared from three different types of phospholipids (Lipoid E80, Lipoid S100, Phospholipon 90H) in order to study the effect of lipid composition on the characteristics of liposomes. The optimal formulation was then selected to prepare nerolidol loaded-CLs and DCLs encapsulating the two compounds. HP-β-CD/Ner (at different CD:Ner molar ratios) and SBE-β-CD/Quer inclusion complexes were used as the aqueous phase in the DCL system. The last part focused on the effect of encapsulation on the physicochemical properties of nerolidol and quercetin (in vitro release, photostability, stability in gastro-intestinal fluids, storage stability) and their antioxidant activities. Results demonstrated that CDs could successfully encapsulate bioactive compounds, enhance their solubility , photostability and antioxidant activity. Furthermore, Lipoid E80-liposomes were nanometric in size, exhibited a high entrapment efficiency and higher stability in comparison to the other formulations. Moreover, CD:guest molar ratio influenced the size of DCLs and their encapsulation efficiency. When compared to CLs, DCLs extended the release of neridol, enhanced the photostability of both compounds ans increased the stability of quercetin in biological fluids. These results could be considered as a promising tool to achieve an optimized and efficient formulation incorporating nerolidol and quercetin in food industry.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Synthesis of ternary polyurethanes (PUs) from hexamethylenediisocyanate, β- cyclodextrin and β-glycerophosphate (acid and calcium salt) was studied varying synthesis parameters such as monomer proportion, heating method (reflux and microwave), and catalyst amount. Favorable conditions were provided by microwave irradiation and use of β-glycerophosphoric acid although the results suggest that it is possible to obtain ternary PUs with the calcium salt. FTIR data indicated the existence of secondary urea linkages. After characterization of ternary PUs by FTIR spectroscopy, XRD and thermal analysis, as well as evidences that the cyclodextrin cavities remained active toward inclusion of guest molecules, the possibility of inclusion of the antibiotic ciprofloxacin was evaluated. Absence of ciprofloxacin melting peak in DSC curves indicated that it is molecularly dispersed within the polymer, possibly included in the cyclodextrin. In vitro release experiments suggested additional non-inclusion interactions, showing also that the use of dialysis membranes may mask the actual release profile. / Nesta Tese foi estudada a síntese de poliuretanos ternários (PUs) à base de diisocianato de hexametileno, β-ciclodextrina e β -glicerofosfato (na forma de ácido e de sal de cálcio), sendo avaliados diferentes parâmetros de síntese, tais como proporção monômero, método de aquecimento (refluxo e microondas), bem como a quantidade de catalisador. As condições mais favoráveis foram fornecidas pela irradiação de microondas e a utilização de ácido β -glicerofosfórico, embora os resultados sugiram que é possível obter PU ternário com o sal de cálcio. Dados de FTIR indicaram a existência de ligações de uréia secundárias na estrutura dos poliuretanos. Após a caracterização dos PU’s ternários por espectroscopia de FTIR, XRD e análise térmica, obtiveram-se evidências de que as cavidades de β-ciclodextrina permaneceram ativas para a inclusão de moléculas hóspedes, usando-se fenolftaleína (FF) como sonda. Na seqüência, foi avaliada a possibilidade de inclusão do antibiótico ciprofloxacina. A ausência do pico de fusão ciprofloxacina em curvas de DSC indicou que este fármaco está molecularmente disperso dentro do polímero, com as moléculas possivelmente incluídas nas cavidades β-ciclodextrina. Experiências de liberação in vitro sugeriram interações não-inclusivas, mostrando também que a utilização de membranas de diálise pode mascarar o perfil de liberação efetiva.

L'huile essentielle de clou de girofle (HECG) et son constituant majeur l'eugénol (Eug) sont reconnus pour leurs propriétés biologiques. Ces principes actifs naturels peuvent constituer des alternatifs aux agents antimicrobiens, antioxydants et anti-inflammatoires de synthèse dans les formulations alimentaires et pharmaceutiques. Cependant, leur utilisation est limitée en raison de leur faible solubilité aqueuse, volatilité et sensibilité à la lumière. Notre travail de thèse porte sur la préparation et la caractérisation des vésicules lipidiques encapsulant l'HECG et l'Eug ainsi que les complexes d'inclusion cyclodextrine/Eug. Dans une première étape, la méthode d'injection éthanolique est utilisée à l'échelle du laboratoire où les paramètres de préparation ont été optimisés. Des phospholipides naturels de soja saturés (Phospholipon 80H et Phospholipon 90H) et insaturés (Lipoid S100) ont été utilisés pour étudier l'effet de l'hydrogénation et de la composition des phospholipides sur les caractéristiques des liposomes. Les conditions optimales ont été par la suite appliquées pour préparer les liposomes à grande échelle par contacteur à membrane et à l'échelle pilote. Des résultats similaires en termes de taille, indice de polydispersité, potentiel zêta, morphologie et taux d'incorporation de phospholipides sont obtenus à petite et grande échelle. Ceci indique la reproductibilité de ces procédés de préparation. Par ailleurs, des complexes d'inclusion d'HP-β-CD/Eug et d'HP-β-CD/HECG sont préparés dans une solution aqueuse et ensuite incorporés dans les liposomes formant un système combiné « drug in cyclodextrin in liposomes, DCL ». Un système en double encapsulation (DCL2) a été également préparé où l'Eug ou l'HECG sont ajoutés dans la phase organique et leurs complexes d'inclusion dans la phase aqueuse. En comparant à une simple incorporation dans les liposomes, DCL et DCL2 améliorent le rendement d'encapsulation de l'Eug et possèdent des tailles plus petites. Les résultats ont montré que les liposomes et les DCLs sont stables et maintiennent l'activité anti-oxydante de l'Eug. De plus, les liposomes protègent l'Eug contre la dégradation induite par les rayons UVC. Les DCLs, dont la particularité est de maintenir une huile essentielle volatile dans un lyophilisat en dépit des pressions très basses appliquées, peuvent être considérés comme un système de vectorisation prometteur de l'HECG et de l'Eug permettant leur utilisation en tant qu'ingrédients dans les préparations cosmétiques, pharmaceutiques, et agroalimentaires / Clove essential oil (CEO) and its major constituent eugenol (Eug) are recognized for their biological properties. These molecules may constitute natural alternatives to synthetic antimicrobial, antioxidant, and anti-inflammatory agents in food and pharmaceutical formulations. However, CEO constituents are volatile, sensitive to light and possess low aqueous solubility, which may limit their wide applications. Our thesis focuses on the preparation and characterization of lipid vesicles encapsulating CEO, Eug and the inclusion complexes cyclodextrin/Eug. In a first step, the ethanol injection method is applied at laboratory scale where the preparation parameters have been optimized. Natural hydrogenated (Phospholipon 80H, Phospholipon 90H) and non-hydrogenated (Lipoid S100) soybean phospholipids were used to study the effect of hydrogenation and phospholipid composition on the characteristics of liposomes. Optimal conditions were then applied to prepare liposomes at large scale by membrane contactor and at pilot scale. Similar results in terms of size, polydispersity index, zeta potential, morphology and phospholipid loading rate were obtained at laboratory and large scale. This indicates the reproducibility of the preparation methods. In addition, HP-β-CD/Eug and HP- β-CD/CEO inclusion complexes were prepared in aqueous solution and were then incorporated into liposomes forming a combined system « drug in cyclodextrin in liposomes, DCL ». Double loaded liposomes (DCL2) were also prepared where CEO or Eug were added in the organic phase and their inclusion complexes in the aqueous phase. Compared to CEO and Eug loaded liposomes, DCL and DCL2 improved the loading rate of Eug and possessed smaller vesicles size. Results showed that both liposomes and DCLs are stable and maintain the antioxidant activity of Eug. In addition, liposomes protect Eug from degradation induced by UVC irradiation. DCLs, whose characteristic is to keep a volatile essential oil in a lyophilized form despite the very low applied pressures, could be considered as a promising carrier system of CEO and Eug permitting their use as ingredients in cosmetic, pharmaceutical and food industries

No description available.

Delivery of siRNAs for the treatment of tumors critically depends on the development of efficient nucleic acid carrier systems. The complexation of dendritic polymers (dendrimers) results in nanoparticles, called dendriplexes, that protect siRNA from degradation and mediate non-specific cellular uptake of siRNA. However, large siRNA doses are required for in vivo use due to accumulation of the nanoparticles in sinks such as the lung, liver, and spleen. This suggests the exploration of targeted nanoparticles for enhancing tumor cell specificity and achieving higher siRNA levels in tumors. In this work, we report on the targeted delivery of a therapeutic siRNA specific for BIRC5/Survivin in vitro and in vivo to tumor cells expressing the surface marker prostate stem cell antigen (PSCA). For this, polyplexes consisting of single-chain antibody fragments specific for PSCA conjugated to siRNA/maltose-modified poly(propylene imine) dendriplexes were used. These polyplexes were endocytosed by PSCA-positive 293TPSCA/ffLuc and PC3PSCA cells and caused knockdown of reporter gene firefly luciferase and Survivin expression, respectively. In a therapeutic study in PC3PSCA xenograft-bearing mice, significant anti-tumor effects were observed upon systemic administration of the targeted polyplexes. This indicates superior anti-tumor efficacy when employing targeted delivery of Survivin-specific siRNA, based on the additive effects of siRNA-mediated Survivin knockdown in combination with scFv-mediated PSCA inhibition. Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play also a prominent role in plasmid DNA delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise β cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53 deficient HCT116p53-/-/PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities.

The monitoring of pharmaceuticals, heavy metal, pH and free chlorine concentration in drinking water is important for public health and the environment. However, conventional laboratory-based analytical methods are labor-intensive, expensive, and time consuming. This thesis focuses on developing an integrated, highly sensitive, easy-to-use, and low-cost pharmaceuticals, heavy metal, pH and free chlorine sensing system for drinking water quality monitoring. A low-temperature, solution-processed modification of multi-walled carbon nanotubes (MWCNT) with β-cyclodextrin (βCD) on glassy carbon electrode is developed for detecting low levels of acetaminophen. The adsorption properties of βCD are combined with the high surface area of carbon nanotubes towards enhanced electrochemical sensing of acetaminophen with a limit of detection of 11 nM and linear range from 0.05-300 μM. Also, a systematic investigation is carried out using four types of modified MWCNT-βCD. A novel, one-step approach called Steglich esterification modified MWCNT-βCD results in large effective surface area, and fast electron transfer towards sensitive detection of acetaminophen and 17β-estradiol (E2, primary female sex hormone) in the range of 0.005–20 and 0.01–15 μM, with low detection limits of 3.3 and 2.5 nM, respectively. The similar MWCNT-βCD modified electrodes can also detect heavy metal ion (lead, Pb2+) with a limit of detection of <10 ppb. Low frequency noise behavior of these sensors are studied. A spin-coated Pd/PdO based pH sensor, and amine-modified carbon electrode-based free chlorine sensor are fabricated on a common substrate together with the pharmaceuticals and heavy metal sensors. A Wheatstone-bridge temperature sensor is fabricated based on silicon and PEDOT:PSS on another substrate. All the sensors are connected to an Arduino microcontroller based data acquisition system with a smartphone application interface. The integrated sensing system is easy-to-use, low-cost, and can provide accurate monitoring data with real drinking water samples. / Dissertation / Doctor of Philosophy (PhD) / Low-cost, easy-to-use, and sensitive monitoring system for pharmaceuticals, heavy metal, pH and free chlorine in drinking water is crucial for public health safety. In this thesis, we develop solution-based synthesis of multi-walled carbon nanotubes modified by β-cyclodextrin for electrochemical sensing of pharmaceuticals and heavy metal. The modification approaches are compared and characterized to analyze their electrochemical behavior and sensing performances. The developed sensors are highly sensitive toward the detection of acetaminophen (a widely used pain-killer) and estrogen hormone in drinking water. We also develop a modified spin-coating technique to deposit palladium/palladium oxide films for potentiometric pH sensor, a calibration-free free chlorine sensor based on modified carbon electrode, and a resistive temperature sensor. The developed pH, free chlorine and temperature sensors are highly sensitive, and stable with fast response time. All the sensors are integrated and interfaced with a custom-made and smartphone-controlled electronic readout system for accurate and on-site drinking water quality monitoring at low cost.