Ce travail porte sur la valorisation du calix[4]arène comme complexant de métaux de transition, immobilisé sur un matériau, et comme vecteur de principe actifs, aptes à interagir avec des récepteurs cellulaires. En premier lieu, la monofonctionnalisation sélective d’un calix[4]arène, sur sa partie haute ou sur sa partie basse est réalisée. Les fonctions nitro, amine, adéhyde, et alcène ont été incorporées sur la partie basse, via un bras espaceur, et un bras acide a été incorporé sur la partie haute. Ces calix[4]arènes monofonctionnalisés se déclinent en trois séries de composés, dépendant du degré et de la nature des substituants sur la partie basse : tétrol, triméthoxy et trisbipyridyle. Le second objectif est le greffage d’un calix[4]arène pourvu d’une fonction amine, sur un polymère naturel, cellulose ou dextran. Un mode opératoire reproductible a été mis au point, qui a permis d’obtenir un calix[4]arène complexant de l’ion Cu(I), immobilisé sur cellulose en poudre ou sur dextran. L'évaluation du taux de greffage par RMN et dosage UV-Vis en présence de Cu+ est décrite pour les dextrans. Enfin, nous avons incorporé sur la partie haute du calix[4]arène d'antennes de reconnaissance cellulaire de type RGD, préparées par synthèse convergente en solution. Une chimiothèque de tripeptides RGD, modulés sur les parties C- et N-terminales, a été obtenue avec de bons rendements. Ces peptides ont fait l’objet d’une évaluation biologique (adhésion cellulaire). Nous avons ainsi préparé des calix[4]arènes, de conformation conique, antennisés par le dipeptide GD et le tripeptide RGD, destinés à élaborer des modèles de vecteurs supramoléculaire de principes actifs. / The aim of this present work is dedicated to the valorization of calix[4]arene as a transition-metal complexing agent, grafted on a polymeric material, and as a drug carrier, displaying a cell recognition peptidic sequence. We describe selective monofunctionalization of a calix[4]arene, at the upper rim or at the lower rim. Nitro, amino, aldehyde and alcene functions were incorporated on the calix[4]arene lower rim, via a spacer. In the same way, an acid arm was also incorporated on the calix[4]arne upper rim. We made those monofunctionalized calix[4]arenes available into three series, depending on the number and the nature of substituent on the three residual OH groups of the calix[4]arne carried function : tetrol, trimethoxy and trisbipyridyl. The second goal is dedicated to the grafting of an amino-fonctionalized calix[4]arene, on a natural polymer, such as cellulose or dextran. We have developped a method allowing to access to a new polymeric complexing agent. We also describe the grafting-ratio, calculated by NMR, UV-Vis and titration with Cu+. The third goal is dedicated to the grafting of the cell-recognizing RGD sequence, prepared in solution by a gram-scale convergent synthesis. A RGD analogue library, functionalized by variable groups on the C- and N- terminations, were obtained with interestly yields. Those peptides were involved into a biological evaluation (cellular adhesion). Thus, cone-conformed calix[4]arene incorporating the GD or RGD peptide sequence were elaborated as preliminary models for the access to new drug-carrier molecular devices.

The starting material 3-sulfonyl-5-methoxy glutarimide was easily prepared via stepwise [3+3] annulation of N-benzyl R-sulfonylacetamide with unsaturated ester as we previously described. We wish we could build tetrahtdronaphthridine skeleton via stretching C-chain, modifying funtional group and RCM, then complete the synthesis of key intermediate of small molecule similar to RGD.

L'objectif de cette thèse concerne la possibilité d'améliorer le diagnostic et la thérapie des gliomes par le ciblage des intégrines à l’aide du RGD et par le développement d'agents multimodaux de type alcoxyamine. L’étude de l’internalisation du RGD révèle une régulation par la densité cellulaire, paramètre histologique dans la catégorisation des gliomes. Dans notre modèle, la densité cellulaire impacte la contribution de l’endocytose clathrine-dépendante et le métabolisme mais n’influence pas le rôle du cytosquelette. La régulation de l’internalisation des peptides RGD par la densité cellulaire reste à mieux comprendre afin de perfectionner les agents utilisant ce ciblage pour l’imagerie et le diagnostic des gliomes. Dans le même temps, les propriétés multimodales des alcoxyamines ont été évaluées àdes fins théranostiques. Ces molécules s’homolysent spontanément pour libérer un nitroxyde et un radical alkylant cytotoxique pouvant en plus induire une réactivation immunitaire antitumorale. Le nitroxyde est un agent de contraste pour l’IRM rehaussée par effet Overhauser. Le fort rehaussement du signal observé à proximité du nitroxyde assure un suivi en temps réel de l’apparition de l’agent alkylant. L’adaptation des alcoxyamines pour une homolyse conditionnelle dans le gliome permettrait une action thérapeutique avec un contrôle spatial et un suivi temporel du composé cytotoxique. L’acheminement de molécules d’intérêt vers la cible est rendu difficile par la présence de barrières physiologiques. Dans ce travail, la progression de nanoparticules par la voie intratrachéale peut se substituer à celle intraveineuse avec une augmentation du temps de rétention dans le gliome. / The aim of this thesis is to improve the diagnostic and the therapy of glioma through both the integrin targeting by RGD and the development of Alkoxyamine as multimodal agent. The RGD internalization is regulated by the cellular density, a histologic parameterfor the glioma classification. In our model, the cellular density increases the contribution of both the clathrin-mediated endocytosis and the metabolism but not the one of the cytoskeletal. A better knowledge about the RGD internalization regulation by the cell density could help the MRI probe development for glioma diagnosis. Properties of alkoxyamine as multimodal agent were evaluated to perform theranostic. The spontaneous alkoxyamine homolysis give a nitroxide radical and a cytotoxic alkylating agent that could induce immune reactivation against the tumor. This nitroxide is an Overhauser enhanced MRI contrast agent. The strong signal enhancement in the nitroxide vicinity gives information in real-time about the release of the alkyl radical. Alkoxyamine adaptation for a conditional homolysis through specific glioma proteolysis activity could induce a localized alkyl therapeutic effect with a real-time monitoring. Physiological barriers limit the drug accumulation in the targeted sites. In this study, the intratracheal instillation of nanoparticles can substitute the intravenous administrationincreasing their intratumoral retention time.

München, Techn. University, Diss., 2004.

No description available.

A funcionalização de superfície nas nanocápsulas contendo doxorrubicina com o peptídeo RGD é uma estratégia promissora devido a ligação preferencial na integrina αvβ3 expressa em células tumorais. Este estudo objetivou o desenvolvimento, caracterização e estudos biológicos de nanocápsulas multiparedes com doxorrubicina e funcionalizadas com RGD. Para isso, na primeira etapa do trabalho foi realizada a síntese do peptídeo RGD. Os produtos obtidos foram caracterizados por análises de infravermelho e RMN de 1H. Na segunda etapa foram desenvolvidas formulações de nanocápsulas com doxorrubicina ou cloridrato de doxorrubicina, e, nanocápsulas multiparedes revestidas com quitosana, íons zinco, RGD ou fenilalanina. Essas suspensões foram caracterizadas através da determinação do pH, diâmetro de partícula por diferentes técnicas, potencial zeta, eficiência de encapsulação e eficiência de associação do RGD na superfície da nanopartícula. Na terceira etapa, foram realizados ensaios de viabilidade celular por MTT após 24 e 72h com as formulações desenvolvidas em células de câncer de mama (MCF7) e glioblastoma humano (U87MG). As formulações apresentaram diferentes valores de citotoxicidade e, utilizando o Gráfico de Pareto foi possível determinar os fatores que exercem maior influencia. Em células MCF7 foi a concentração de fármaco e tempo de tratamento e, nas células U87MG além desses fatores, a funcionalização mostrou-se determinante. Além disso, foi avaliada a captação das nanocápsulas funcionalizadas com RGD e fenilalanina após 24h nas células tumorais e células de queratinócitos humanos (HaCat), com diferentes níveis de expressão da integrina αvβ3. O estudo mostrou menores valores de captação nas células HaCat (sem expressão de integrina αvβ3) para as duas formulações testadas. Finalmente as nanocápsulas funcionalizadas com RGD apresentaram maior captação em células U87MG com maior expressão da integrina αvβ3. / The surface functionalization in nanocapsules containing doxorubicin with RGD peptide is a promising strategy due to preferential binding in the αvβ3 integrin expressed on tumor cells. This study aimed the development, characterization, and biological studies of multiwall nanocapsules containing doxorubicin and functionalized with RGD. For this reason, in the first stage of this study the synthesis of RGD peptide was performed and the products characterized by infrared analysis and 1H NMR. Besides, nanocapsules formulations were developed containing doxorubicin or doxorubicin hydrochloride, and multiwall nanocapsules coated with chitosan, zinc ions, RGD or phenylalanine. These suspensions were characterized by pH determination, particle diameter by different techniques, zeta potential, encapsulation efficiency, and association efficiency of RGD on the surface of the nanoparticle. Additionally, it was performed cell viability assays by MTT after 24 and 72 hours with formulations developed in breast cancer (MCF7) and human glioblastoma cells (U87MG). Formulations showed different cytotoxicity values. The Pareto chart was possible to determine factors that have more influence. In MCF7 cells was drug concentration and treatment time, and U87MG cells, besides these factors, the functionalization was decisive. Furthermore, it was performed the cellular uptake of nanocapsules functionalized with RGD or phenylalanine after 24 hours in tumor cells and human keratinocyte cells (HaCaT), with different levels of expression αvβ3 integrin. The study showed less uptake in HaCaT cells (without expression αvβ3 integrin) for the two formulations applied, and the nanocapsules functionalized with RGD showed more uptake in U87MG cells, with higher expression of integrin αvβ3.

Bovine respiratory disease complex is the most common disease that causes sig-nificant economic loss, typically in feedlot cattle. Current treatment methods are focused on reducing inflammatory responses, control of airway reactivity and improvement of pulmonary functions without potential side effects. Neutrophils are the key contributors in acute lung inflammation. However, activated neutrophils live longer and cause exces-sive tissue damage upon migration into lungs. Therefore, modulation of their migration and lifespan are attractive approaches in treatment strategies of bovine respiratory dis-ease. Nanotechnology holds significant potential to design new compounds by our ability to manipulate at the nanoscale. Helical rosette nanotubes are a class of novel, biologi-cally inspired, water soluble and metal-free nanotubes. I used helical rosette nanotubes conjugated to arginine-glycine-aspartic acid (RGD-RNT) to study their effects on neu-trophil chemotaxis, cell signaling and apoptosis. Bovine neutrophils exposed to 5% RGD-RNT reduced their migration in response to fMLP (formyl-Methionyl-Leucyl-Phenylalanine), compared to the non-treated group (P<0.001). This inhibitory effect was the same as that of groups treated with ERK1/2 inhibitor (UO126) and p38 MAPK in-hibitor (SB239063). In addition, the phosphorylated ERK1/2 and p38 MAPK for the first time were quantified by sandwich ELISA to elucidate the mechanism of neutrophil mi-gration. The phosphorylation of both the ERK1/2 and p38 was inhibited at 5 minutes by RGD-rosette nanotubes (P<0.05). Furthermore, integrin ÑvÒ3 is possibly involved in mi-gration of bovine neutrophils. Moreover, RGD-RNT did not induce apoptosis of bovine neutrophils which was inversed by pre-exposing them to LPS for 30 minutes (P<0.001). These experiments provide the first evidence that RGD-rosette nanotubes suppress phos-phorylation of ERK1/2 and p38 MAPK and inhibit chemotaxis of bovine neutrophils.

Bovine respiratory disease complex is the most common disease that causes sig-nificant economic loss, typically in feedlot cattle. Current treatment methods are focused on reducing inflammatory responses, control of airway reactivity and improvement of pulmonary functions without potential side effects. Neutrophils are the key contributors in acute lung inflammation. However, activated neutrophils live longer and cause exces-sive tissue damage upon migration into lungs. Therefore, modulation of their migration and lifespan are attractive approaches in treatment strategies of bovine respiratory dis-ease. Nanotechnology holds significant potential to design new compounds by our ability to manipulate at the nanoscale. Helical rosette nanotubes are a class of novel, biologi-cally inspired, water soluble and metal-free nanotubes. I used helical rosette nanotubes conjugated to arginine-glycine-aspartic acid (RGD-RNT) to study their effects on neu-trophil chemotaxis, cell signaling and apoptosis. Bovine neutrophils exposed to 5% RGD-RNT reduced their migration in response to fMLP (formyl-Methionyl-Leucyl-Phenylalanine), compared to the non-treated group (P<0.001). This inhibitory effect was the same as that of groups treated with ERK1/2 inhibitor (UO126) and p38 MAPK in-hibitor (SB239063). In addition, the phosphorylated ERK1/2 and p38 MAPK for the first time were quantified by sandwich ELISA to elucidate the mechanism of neutrophil mi-gration. The phosphorylation of both the ERK1/2 and p38 was inhibited at 5 minutes by RGD-rosette nanotubes (P<0.05). Furthermore, integrin ÑvÒ3 is possibly involved in mi-gration of bovine neutrophils. Moreover, RGD-RNT did not induce apoptosis of bovine neutrophils which was inversed by pre-exposing them to LPS for 30 minutes (P<0.001). These experiments provide the first evidence that RGD-rosette nanotubes suppress phos-phorylation of ERK1/2 and p38 MAPK and inhibit chemotaxis of bovine neutrophils.

Regensburg, Univ., Diss., 2009.

A funcionalização de superfície nas nanocápsulas contendo doxorrubicina com o peptídeo RGD é uma estratégia promissora devido a ligação preferencial na integrina αvβ3 expressa em células tumorais. Este estudo objetivou o desenvolvimento, caracterização e estudos biológicos de nanocápsulas multiparedes com doxorrubicina e funcionalizadas com RGD. Para isso, na primeira etapa do trabalho foi realizada a síntese do peptídeo RGD. Os produtos obtidos foram caracterizados por análises de infravermelho e RMN de 1H. Na segunda etapa foram desenvolvidas formulações de nanocápsulas com doxorrubicina ou cloridrato de doxorrubicina, e, nanocápsulas multiparedes revestidas com quitosana, íons zinco, RGD ou fenilalanina. Essas suspensões foram caracterizadas através da determinação do pH, diâmetro de partícula por diferentes técnicas, potencial zeta, eficiência de encapsulação e eficiência de associação do RGD na superfície da nanopartícula. Na terceira etapa, foram realizados ensaios de viabilidade celular por MTT após 24 e 72h com as formulações desenvolvidas em células de câncer de mama (MCF7) e glioblastoma humano (U87MG). As formulações apresentaram diferentes valores de citotoxicidade e, utilizando o Gráfico de Pareto foi possível determinar os fatores que exercem maior influencia. Em células MCF7 foi a concentração de fármaco e tempo de tratamento e, nas células U87MG além desses fatores, a funcionalização mostrou-se determinante. Além disso, foi avaliada a captação das nanocápsulas funcionalizadas com RGD e fenilalanina após 24h nas células tumorais e células de queratinócitos humanos (HaCat), com diferentes níveis de expressão da integrina αvβ3. O estudo mostrou menores valores de captação nas células HaCat (sem expressão de integrina αvβ3) para as duas formulações testadas. Finalmente as nanocápsulas funcionalizadas com RGD apresentaram maior captação em células U87MG com maior expressão da integrina αvβ3. / The surface functionalization in nanocapsules containing doxorubicin with RGD peptide is a promising strategy due to preferential binding in the αvβ3 integrin expressed on tumor cells. This study aimed the development, characterization, and biological studies of multiwall nanocapsules containing doxorubicin and functionalized with RGD. For this reason, in the first stage of this study the synthesis of RGD peptide was performed and the products characterized by infrared analysis and 1H NMR. Besides, nanocapsules formulations were developed containing doxorubicin or doxorubicin hydrochloride, and multiwall nanocapsules coated with chitosan, zinc ions, RGD or phenylalanine. These suspensions were characterized by pH determination, particle diameter by different techniques, zeta potential, encapsulation efficiency, and association efficiency of RGD on the surface of the nanoparticle. Additionally, it was performed cell viability assays by MTT after 24 and 72 hours with formulations developed in breast cancer (MCF7) and human glioblastoma cells (U87MG). Formulations showed different cytotoxicity values. The Pareto chart was possible to determine factors that have more influence. In MCF7 cells was drug concentration and treatment time, and U87MG cells, besides these factors, the functionalization was decisive. Furthermore, it was performed the cellular uptake of nanocapsules functionalized with RGD or phenylalanine after 24 hours in tumor cells and human keratinocyte cells (HaCaT), with different levels of expression αvβ3 integrin. The study showed less uptake in HaCaT cells (without expression αvβ3 integrin) for the two formulations applied, and the nanocapsules functionalized with RGD showed more uptake in U87MG cells, with higher expression of integrin αvβ3.