Polymeric nanoparticles have gained increased popularity for drug delivery as they not only overcome the problem of limited aqueous solubility of many hydrophobic drug molecules, but also have the potential to improve the pharmacologic properties of anticancer drugs by increasing their in vivo half-life. A series of biodegradable poly(D,L-lactide-co-2-methyl-2-carboxytrimethylene carbonate), P(LA-co-TMCC), was first synthesized by Sn(Oct)2 catalyzed bulk polymerization. In order to obtain the polymer product with a better-defined composition, the polymer synthesis was improved by using organo-catalytic ring-opening copolymerization. The copolymer molar mass and composition were controlled by varying the monomer to initiator ratio and the monomer feed ratio. By grafting amine-terminated polyethylene glycol (PEG-NH2) to the carboxylate groups on the copolymer backbone, amphiphilic copolymers were formed and self-assembled to form nanoparticles with narrow size distribution. The nanoparticle size was observed to be influenced by the polymer composition and the self-assembly conditions. To gain greater insight into the stability of these nanoparticles in blood, they were tested in both fetal bovine serum and individual serum protein solutions. By encapsulating Förster resonance energy transfer (FRET) pairs and following their release by fluorescence, these micelles demonstrated strong thermodynamic and kinetic stability in the presence of serum. By incorporating functional groups (azide or furan) on the PEG chains, either cell adhesive peptides (i.e. alkyne-functionalized GRGDS) or targeting antibodies (i.e. maleimide-modified trastuzumab) were coupled to the surface of the nanoparticles through Huisgen 1,3-dipolar cycloaddition reaction or Diels-Alder chemistry, respectively. The GRGDS modified nanoparticles showed specific binding affinity to rabbit corneal epithelial cells that express αvβ1 integrin receptors.

Polymeric nanoparticles have gained increased popularity for drug delivery as they not only overcome the problem of limited aqueous solubility of many hydrophobic drug molecules, but also have the potential to improve the pharmacologic properties of anticancer drugs by increasing their in vivo half-life. A series of biodegradable poly(D,L-lactide-co-2-methyl-2-carboxytrimethylene carbonate), P(LA-co-TMCC), was first synthesized by Sn(Oct)2 catalyzed bulk polymerization. In order to obtain the polymer product with a better-defined composition, the polymer synthesis was improved by using organo-catalytic ring-opening copolymerization. The copolymer molar mass and composition were controlled by varying the monomer to initiator ratio and the monomer feed ratio. By grafting amine-terminated polyethylene glycol (PEG-NH2) to the carboxylate groups on the copolymer backbone, amphiphilic copolymers were formed and self-assembled to form nanoparticles with narrow size distribution. The nanoparticle size was observed to be influenced by the polymer composition and the self-assembly conditions. To gain greater insight into the stability of these nanoparticles in blood, they were tested in both fetal bovine serum and individual serum protein solutions. By encapsulating Förster resonance energy transfer (FRET) pairs and following their release by fluorescence, these micelles demonstrated strong thermodynamic and kinetic stability in the presence of serum. By incorporating functional groups (azide or furan) on the PEG chains, either cell adhesive peptides (i.e. alkyne-functionalized GRGDS) or targeting antibodies (i.e. maleimide-modified trastuzumab) were coupled to the surface of the nanoparticles through Huisgen 1,3-dipolar cycloaddition reaction or Diels-Alder chemistry, respectively. The GRGDS modified nanoparticles showed specific binding affinity to rabbit corneal epithelial cells that express αvβ1 integrin receptors.

Targeted delivery of drugs to specific regions of the body, or even to specific regions of the cell, promises enhanced drug efficacy and reduced systemic toxicity. By covalently coupling targeting ligands, the smart drug delivery systems are capable of targeting specific cell types exclusively through ligand-receptor interactions. The main goal of the project is to create a polymeric nanoparticle drug delivery system from synthesized biodegradable polymers and modify the polymeric nanoparticles using targeting antibodies for targeted delivery of anticancer drugs. A new biodegradable copolymer poly(2-methyl, 2-carboxy trimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG-furan) was synthesized and characterized. The copolymers self-assembled into spherical nanoparticles in aqueous environments with the hydrodynamic diameters controlled over a broad size range. Immuno-polymeric nanoparticles were created by coupling maleimide-modified anti-human epidermal growth factor receptor 2 (anti-HER2) antibodies to the self-assembled nanoparticles through Diels-Alder (DA) chemistry. This new coupling methodology was demonstrated to be relatively rapid, highly efficient and specific under mild conditions. In vitro studies showed that the immuno-nanoparticles bound specifically and efficiently with SKBR3 breast cancer cells that overexpress HER2 receptors. Anticancer drugs were incorporated into the immuno-nanoparticle system and the drug delivery via an antibody-mediated targeting mechanism was investigated in vitro. First, a protein anticancer drug, interleukin-2 (IL-2), was physically encapsulated through polymer-drug association. The IL-2 encapsulated anti-HER2 immuno-nanoparticles exhibited a cell-binding associated IL-2 release in the extracellular space upon binding with HER2-overexpressing SKBR3 breast cancer cells. Second, a small molecule hydrophobic drug, doxorubicin (DOX), was chemically conjugated on the nanoparticle surface after the antibody coupling, using the same DA chemistry. The novel formulation localized DOX in the cell nucleus of HER2-overexpressing SKBR3 breast cancer cells and remained the biological function of conjugated DOX. Compared to the nanoparticles bearing DOX or anti-HER2 antibody alone, the nanoparticles having a combination of DOX and anti-HER2 antibody exhibited the most significant cytotoxicity and specificity against SKBR3 cells relative to healthy HMEC-1 endothelial cells, demonstrating the potential of the DOX-immuno-nanoparticles as a novel platform for intracellular DOX delivery. This work provides a novel means for the delivery of combination immunotherapy/chemotherapy to more effectively treat certain malignancies.

Targeted delivery of drugs to specific regions of the body, or even to specific regions of the cell, promises enhanced drug efficacy and reduced systemic toxicity. By covalently coupling targeting ligands, the smart drug delivery systems are capable of targeting specific cell types exclusively through ligand-receptor interactions. The main goal of the project is to create a polymeric nanoparticle drug delivery system from synthesized biodegradable polymers and modify the polymeric nanoparticles using targeting antibodies for targeted delivery of anticancer drugs. A new biodegradable copolymer poly(2-methyl, 2-carboxy trimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG-furan) was synthesized and characterized. The copolymers self-assembled into spherical nanoparticles in aqueous environments with the hydrodynamic diameters controlled over a broad size range. Immuno-polymeric nanoparticles were created by coupling maleimide-modified anti-human epidermal growth factor receptor 2 (anti-HER2) antibodies to the self-assembled nanoparticles through Diels-Alder (DA) chemistry. This new coupling methodology was demonstrated to be relatively rapid, highly efficient and specific under mild conditions. In vitro studies showed that the immuno-nanoparticles bound specifically and efficiently with SKBR3 breast cancer cells that overexpress HER2 receptors. Anticancer drugs were incorporated into the immuno-nanoparticle system and the drug delivery via an antibody-mediated targeting mechanism was investigated in vitro. First, a protein anticancer drug, interleukin-2 (IL-2), was physically encapsulated through polymer-drug association. The IL-2 encapsulated anti-HER2 immuno-nanoparticles exhibited a cell-binding associated IL-2 release in the extracellular space upon binding with HER2-overexpressing SKBR3 breast cancer cells. Second, a small molecule hydrophobic drug, doxorubicin (DOX), was chemically conjugated on the nanoparticle surface after the antibody coupling, using the same DA chemistry. The novel formulation localized DOX in the cell nucleus of HER2-overexpressing SKBR3 breast cancer cells and remained the biological function of conjugated DOX. Compared to the nanoparticles bearing DOX or anti-HER2 antibody alone, the nanoparticles having a combination of DOX and anti-HER2 antibody exhibited the most significant cytotoxicity and specificity against SKBR3 cells relative to healthy HMEC-1 endothelial cells, demonstrating the potential of the DOX-immuno-nanoparticles as a novel platform for intracellular DOX delivery. This work provides a novel means for the delivery of combination immunotherapy/chemotherapy to more effectively treat certain malignancies.

No description available.

Rendre possible l'administration orale du docétaxel (Dtx), un puissant agent anticancéreux administré par voie intraveineuse, représente un défi important en cancérologie. Disposer de formulations administrables par voie orale, moins toxiques et mieux tolérées, représenterait une avancée majeure au plan clinique. Toutefois, plusieurs études ont montré que la très faible biodisponibilité du Dtx par voie orale résulte simultanément de : (i) sa faible solubilité aqueuse, (ii) son faible passage transépithélial au niveau intestinal, (iii) son efflux par les pompes d’efflux (P-gp) et son métabolisme par le cytochrome P450. Nous avons conçu une formulation capable de répondre simultanément à ces différents problèmes. Ainsi, nous avons tout d’abord fait appel aux cyclodextrines (CDs) pour augmenter la solubilité apparente du Dtx. La complexation du Dtx avec la méthyl-β-CD a permis d’augmenter la solubilité apparente du Dtx d’environ 5000 fois. Ce complexe a ensuite été associé à des nanoparticules(NPs) polymères composées d’un coeur de poly(cyanoacrylate d’alkyle) et recouvert en surface de chitosane thiolé afin de leur conférer des propriétés mucoadhésives et de diminuer localement le métabolisme. Ces NPs ont montré in vitro et ex vivo leur capacité à arriver intactes au niveau de l’intestin, d’y adhérer et de libérer le Dtx de manière contrôlée dans le temps, et finalement d’améliorer son absorption intestinale. Une évaluation de la toxicité de cette formulation vis-à-vis de la muqueuse intestinale suggère que l’encapsulation du Dtx dans les NPs assure une certaine protection de la muqueuse. Au final, la formulation orale proposée offre en perspective la possibilité de moduler la dose administrée, donc d’ajuster finement la posologie et finalement d’offrir au corps médical et aux patients les bénéfices d’une thérapie personnalisée. / Docetaxel (Dtx) is an anticancer drug widely used in therapy. However, severe allergic reactions and peripheral neurotoxicity are caused by the intravenous administration of the commercial formulation Taxotere®, requiring thus the oral administration of dexamethasone and antihistamine before infusion. In this context, there is an urgent need to design new orally administered Dtx formulations to reduce these side effects and improve the patient’s qualityof life. Dtx belongs to the Class IV of the Biopharmaceutical Classification System, which comprises substances with both low solubility in aqueous fluids and low apparent intestinal permeability. This represents a major drawback when foreseeing oral delivery. Moreover, Dtx has been shown to be substrate of biological transporters and/or metabolized in the intestinal barrier. We designed a formulation able to overcome these different problems. First of all, we solved the low solubility problem by using cyclodextrin (CDs). The complexation of Dtx with the Methyl-ß-CD allowed increasing the apparent solubility of the Dtx about 5000 times. This complex was then associated to polymeric core-shell nanoparticles (NPs) based on poly(isobutyl cyanoacrylate) coated with thiolated chitosan. Among the characteristics of this system, mucoadhesion properties are the most important for an oral administration. The presence of the positively charged chitosan chains, and the thiol groups at the surface allow NPs to adhere to the mucus layer. In vitro and ex vivo experiments showed that these NPs were able to ensure a time-controlled release of Dtx and to improve its absorption at the intestinal level. An evaluation of the local intestinal toxicity of this formulation suggests that the encapsulation of Dtx into polymeric NPs had a protective effect allowing a preservation of the mucosa integrity. The further step will be to confirm by in vivo studies if this kind of nanoparticles is able to enhance the bioavailability of Dtx allowing to display an anti-tumor activity.

O desenvolvimento de nanosensores luminescentes, não tóxicos e biocompatíveis estão sendo utilizados para marcação e visualização de células em biologia celular e imunoensaios ultrassensíveis. Neste trabalho, marcadores biológicos à base de terras raras para a detecção do 17-β-estradiol (E2), em fluido biológico(plasma) foram estudados. Os complexos precursores à base de terras raras (Eu3+ e Tb3+) foram sintetizados e caracterizados. O complexo contendo tta- de Eu3+ e liganteauxiliar TOPO [Eu(tta)3(TOPO)2] registrou a maior eficiência de luminescência dentre todos os demais compostos estudados e foi escolhido como sonda luminescente para desenvolvimento das etapas seguintes deste trabalho. Este complexo foi incorporado às matrizes poliméricas, PMMA e PHB, em diferentes concentrações de dopagens pordois métodos distintos: nanoprecipitação crioscópica e pela técnica de emulsão-difusão. As nanopartículas poliméricas de PMMA demonstraram maior eficiêncialuminescente quando dopadas com concentração de 5% do complexo precursor [Eu(tta)3(TOPO)2]. Estas nanopartículas foram funcionalizadas com 1,6 hexodiamino eposteriormente foram ligadas a um dialdeído que servirá como ponte na reação comas entidades biológicas. No entanto, devido à transferência de energia do sistemaorgânico Ac-Ac/nanossensor, as nanopartículas poliméricas luminescentes de PHBdemonstraram maior intensidade de sinal para a detecção do 17-β-estradiol por espectrofluorimetria. Pode-se concluir que as nanopartículas produzidas e testadasneste trabalho são fortes candidatas para a detecção do 17-β-estradiol em plasma sanguíneo ou outras entidades biológicas, apresentando opções diagnósticas, conduta extremamente importante para direcionamento terapêutico. / The development of luminescent, non-toxic and biocompatible nanosensors are being used for marker and imaging of cells in cellular biology and ultrasensitive immunoassays. In this work, biological markers based on rare earths for detection of 17 β -estradiol (E2), in biological fluids (Plasmon), were studied. The precursor complexes based on rare earths ( Eu3+ and Tb3+) were synthesized and characterized. The complex containing tta Eu3+ and secondary ligand TOPO- [Eu(tta)3(TOPO)2] registered the highest luminescence efficiency among all other compounds studied and was chosen as luminescent probe for the following development stages of this work. This complex was doped into polymer matrices, PMMA and PHB, at different concentrations of doping by two different methods: cryoscopic nanoprecipitation and emulsion-diffusion technique. The PMMA polymer nanoparticles demonstrated higher luminescence efficiency when doped with 5% concentration of the precursor complex [Eu(tta)3(TOPO)2]. These nanoparticles were functionalized with 1,6 diaminohexane and later they were linked to a dialdehyde that will serve as a bridge in the reaction with the biological entities . These nanoparticles were functionalized with 1,6 diaminohexane and later they were linked to a dialdehyde that can serve as a bridge in the reaction with the biological entities. Due to the energy transfer Ac- Ac/nanosensor, the PHB polymeric nanoparticles showed higher luminescence intensity signal for the detection of 17- β -estradiol by spectrofluorimetry. It can be concluded that the nanoparticles produced and tested in this work are strong candidates for the detection of 17β- estradiol in the blood plasma or others biological entities, presenting diagnostic options, an extremely important conduct for therapeutic direction.

Synthetic glycopeptides have attracted much interest in the biomedical field due to their structural similarities to the natural glycopeptides or glycoproteins. It is still difficult to synthesize glycopeptides with greater efficiency and ring opening polymerization remains an effective way to do so. Proteoglycans are a special class of glycoproteins with glycosaminoglycan chains. In this study, I tried to do controlled ring opening polymerization of Hyaluronic acid derivatives with smaller to higher molecular weight while avoiding side reactions. It is challenging to work with higher molecular weight molecules and do a click reaction in water effectively. Making nanopolymers with a desired size, studies of the characteristics, and how to build nanocarriers for drug delivery application was the focus of this work. Polymeric characteristics, e.g., modification and polymer formation were studied by nuclear magnetic resonance technique; Particle size was studied by dynamic light scattering and the loading of rhodamine B encapsulated into the polymer was measured by confocal imaging technique.

O desenvolvimento de nanosensores luminescentes, não tóxicos e biocompatíveis estão sendo utilizados para marcação e visualização de células em biologia celular e imunoensaios ultrassensíveis. Neste trabalho, marcadores biológicos à base de terras raras para a detecção do 17-β-estradiol (E2), em fluido biológico(plasma) foram estudados. Os complexos precursores à base de terras raras (Eu3+ e Tb3+) foram sintetizados e caracterizados. O complexo contendo tta- de Eu3+ e liganteauxiliar TOPO [Eu(tta)3(TOPO)2] registrou a maior eficiência de luminescência dentre todos os demais compostos estudados e foi escolhido como sonda luminescente para desenvolvimento das etapas seguintes deste trabalho. Este complexo foi incorporado às matrizes poliméricas, PMMA e PHB, em diferentes concentrações de dopagens pordois métodos distintos: nanoprecipitação crioscópica e pela técnica de emulsão-difusão. As nanopartículas poliméricas de PMMA demonstraram maior eficiêncialuminescente quando dopadas com concentração de 5% do complexo precursor [Eu(tta)3(TOPO)2]. Estas nanopartículas foram funcionalizadas com 1,6 hexodiamino eposteriormente foram ligadas a um dialdeído que servirá como ponte na reação comas entidades biológicas. No entanto, devido à transferência de energia do sistemaorgânico Ac-Ac/nanossensor, as nanopartículas poliméricas luminescentes de PHBdemonstraram maior intensidade de sinal para a detecção do 17-β-estradiol por espectrofluorimetria. Pode-se concluir que as nanopartículas produzidas e testadasneste trabalho são fortes candidatas para a detecção do 17-β-estradiol em plasma sanguíneo ou outras entidades biológicas, apresentando opções diagnósticas, conduta extremamente importante para direcionamento terapêutico. / The development of luminescent, non-toxic and biocompatible nanosensors are being used for marker and imaging of cells in cellular biology and ultrasensitive immunoassays. In this work, biological markers based on rare earths for detection of 17 β -estradiol (E2), in biological fluids (Plasmon), were studied. The precursor complexes based on rare earths ( Eu3+ and Tb3+) were synthesized and characterized. The complex containing tta Eu3+ and secondary ligand TOPO- [Eu(tta)3(TOPO)2] registered the highest luminescence efficiency among all other compounds studied and was chosen as luminescent probe for the following development stages of this work. This complex was doped into polymer matrices, PMMA and PHB, at different concentrations of doping by two different methods: cryoscopic nanoprecipitation and emulsion-diffusion technique. The PMMA polymer nanoparticles demonstrated higher luminescence efficiency when doped with 5% concentration of the precursor complex [Eu(tta)3(TOPO)2]. These nanoparticles were functionalized with 1,6 diaminohexane and later they were linked to a dialdehyde that will serve as a bridge in the reaction with the biological entities . These nanoparticles were functionalized with 1,6 diaminohexane and later they were linked to a dialdehyde that can serve as a bridge in the reaction with the biological entities. Due to the energy transfer Ac- Ac/nanosensor, the PHB polymeric nanoparticles showed higher luminescence intensity signal for the detection of 17- β -estradiol by spectrofluorimetry. It can be concluded that the nanoparticles produced and tested in this work are strong candidates for the detection of 17β- estradiol in the blood plasma or others biological entities, presenting diagnostic options, an extremely important conduct for therapeutic direction.

Les répulsifs sont des molécules naturelles ou synthétiques dont le but estd’empêcher l’approche des arthropodes afin de prévenir la transmission demaladies vectorisées. Un exemple type est celui de la transmission de Leishmaniainfantum responsable de la Leishmaniose canine, qui est contractée après la piqûre d’un chienpar un phlébotome. Divers répulsifs d’arthropodes sont commercialisés pour une applicationtopique, ils ont tous une durée d’action courte, imposant des applications répétées deformulations basiques de type solution.La formulation de molécules actives à propriétés répulsives incorporées dans des systèmesnanoparticulaires et présentant une action prolongée dans le temps a été retenue. Lesnanoparticules sont des vecteurs colloïdaux intéressants dans le domaine de la technologiepharmaceutique vu leur capacité à former des complexes avec des molécules hydrophobes,telles que la plupart des molécules répulsives (DEET, Picaridin®, IR3535®…). Le ciblage, laprotection contre la dégradation et le contrôle de la libération sont les avantages principauxapportés par les nanoparticules contenant une matière active.Les caractéristiques physico-chimiques des nanoparticules (taille et potentiel zêta) permettantleur stockage dans les couches supérieures de la peau et une accroche le long des fibrespileuses ont été déterminées. Ainsi des nanoparticules cationiques de 200 nm de diamètre ontété formulées. Cette formulation originale inspirée du procédé de nanoprécipitation a permisl’obtention en une seule étape de suspensions concentrées en matière active (concentrationsupérieure à 10%) et sans ajout d’agents stabilisants. Une corrélation entre les profils delibération et l’efficacité sur insecte modèle (la drosophile) a été vérifiée. De ce fait, lepourcentage en polymère régule la libération de la molécule active encapsulée. Une efficacitérépulsive de formulations nanoparticulaires supérieure à 15 jours a été validéeexpérimentalement. La transposition d’échelle du procédé de nanoprécipitation permetd’envisager un développement industriel pour la formulation d’un répulsif d’arthropodeinnovant à longue durée d’action. / The repellents are natural or synthetic molecules whose aim is to prevent theapproach of arthropods to avoid transmission of vector-borne diseases. A typicalexample is the transmission of Leishmania infantum responsible for canineLeishmaniasis, which is contracted after a sandfly bite on a dog. The arthropod repellentsmarketed for topical application have all a short action duration, requiring repeatedapplications of basic formulations (solution).The formulation of active molecules having repellent properties, incorporated intonanoparticle systems and having a prolonged action in time was selected. Nanoparticles arecolloidal carriers interesting in the pharmaceutical technology field due to their ability to formcomplexes with hydrophobic molecules, such as repellent molecules (DEET, Picaridin®,IR3535®...). Targeting, protection against degradation and control of the release are the mainadvantages provided by the nanoparticles containing an active ingredient.The nanoparticle physicochemical characteristics (size and zeta potential) permitting theirstorage in the upper dog skin layers and a along the hairs were determined. For that reasoncationic nanoparticles of 200 nm in diameter were formulated. This original formulationinspired from the nanoprecipitation process has allowed us to obtain one single stepconcentrated suspensions (above 10% of active molecules in the final product) and withoutstabilizer addition. A correlation between the release profiles and the effectiveness of modelinsect (Drosophila) has been verified. Therefore the percentage of polymer regulates therelease of encapsulated active molecules. Repellent efficacy of nanoparticulate formulationgreater than 15 days has been validated experimentally. The scale transposition of thenanoprecipitation process makes conceivable an industrial development for the formulation ofan innovative arthropod repellent having a long lasting effect.