Ce travail démontre l'utilité des propriétés photothermiques dans le proche Infrarouge (NIR) des complexes de nickel-bis(dithiolène) neutres pour des applications en science des matériaux et biotechnologies. Des complexes Ni-bis(dithiolène) neutres hydrophiles et hydrophobes ont été élaborés et leur effet photothermique, sous irradiation laser NIR, a été quantifié pour la première fois. Grâce à cette propriété remarquable et à leur grande stabilité sous irradiation, il a été mis en évidence, que ces complexes pouvaient trouver des applications pour la thérapie photothermique, la libération contrôlée de médicament mais également pour le développement de nanomatériaux photosensibles à un stimulus externe. En effet, cet effet photothermique, sous irradiation laser NIR, permet de détruire des cellules cancéreuses, d'augmenter la perméabilité de nanoparticules polymériques ou de liposomes contenant des principes actifs et de désagréger des métallogels à base de complexes Ni-bis(dithiolène). Enfin, nous avons montré que les complexes [Ni(R2-timdt)2]0 ayant une haute efficacité photothermique sont de nouveaux candidats très prometteurs pour ce type d'applications. / The photothermal properties in the near Infrared (NIR) of neutral nickel-bis(dithiolene) complexes were used in materials science and for the first time in biotechnology. Hydrophilic and hydrophobic neutral Ni-bis(dithiolene) complexes were developped and for the first time, their photothermal effect under NIR laser irradiation was quantified. Thanks to this remarkable property and their high stability under irradiaton, it has been demonstrated that these complexes could find applications to photothermal therapy, controlled drug delivery but also in the developpement of stimuli responsive nanomaterials. Indeed, this photothermal effect, under NIR laser irradiation, allow to destroy malignant cells, increase the permeability of polymeric nanoparticles or liposomes containing active ingredients and disintegrate métallogels based on Ni-bis(dithiolene) cores. Finally, we showed that the [Ni(R2-timdt)2]0 complexes having higher phothothermal activity are very promising new candidates for these applications.

Polymere Nanopartikel sind ein vielversprechender Ansatz für die Diagnose und Therapie von Krankheiten. Sie ermöglichen den Einsatz von schwerlöslichen oder instabilen Wirkstoffen. Ein weiterer Vorteil ist die Möglichkeit das Targetings, durch gezielte Modifikationen des Nanopartikels wird der Wirkstoff zum Zielort transportiert und kann dort in der gewünschten Form freigesetzt werden; dadurch könnten bei erhöhter Wirksamkeit die Nebenwirkungen von Medikamenten reduziert werden. Ziel dieser Arbeit war die Untersuchung von physikalischen und biochemischen Eigenschaften von Nanopartikeln bestehend aus einem abbaustabilen Polystyren- Kern und einer biologisch abbaubaren Schale aus Polybutylcyanoacrylat. Es werden Methoden beschrieben, um die Größe, Struktur und den Abbau dieser Wirkstoffträger zu untersuchen. Die untersuchten Nanopartikel zeigen RAYLEIGH-Streuung, sowohl Größe als auch Abbau können durch Messung des Absorptionsspektrums bestimmt werden. Weiterhin konnten diese Eigenschaften mit Hilfe von dynamischer und statischer Lichtstreuung sowie Neutronenkleinwinkelstreuung untersucht werden. Bei letzterer Methode konnte gezeigt werden, dass die Schale größtenteils abgebaut werden kann, während der Kern intakt bleibt. In einem weiteren Teil der Arbeit wurde die Überwindung der Blut-Hirn-Schranke durch polymere Nanopartikel untersucht. Dabei wurde der fluoreszierende Thioflavine als Modellwirkstoffe eingesetzt. Das Durchdringen der Blut-Hirn-Schranke konnte nur mit Nanopartikeln erreicht werden, an deren Oberfläche ein Apolipoprotein E-Peptid gekoppelt war. Es konnte gezeigt werden, das die Nanopartikelschale im Gehirn abgebaut wird, der Wirkstoff freigesetzt wird und an Amyloid β, einem Marker der Alzheimer-Krankheit, bindet.

A via oral é considerada preferencial para a administração de fármacos, sobretudo no tratamento de doenças crônicas. Entretanto, princípios ativos administrados por essa via podem apresentar biodisponibilidade variável e/ou limitada. Diversos tipos de sistemas de liberação vêm sendo desenvolvidos com o objetivo de melhorar esse parâmetro, dentre os quais se destacam as nanopartículas de poli (alquil-cianoacrilato) (PACA). Pelo exposto, no presente trabalho foram desenvolvidas nanopartículas de poli(n-butilcianoacrilato) (PBCA) contendo aciclovir (ACV), revestidas por N,N,N-trimetilquitosana (TMQ), um promissor promotor de absorção. A TMQ foi sintetizada com elevado rendimento e grau de quaternização de aproximadamente 73%. As nanopartículas de PBCA foram obtidas com rendimento adequado e apresentaram características físico-químicas semelhantes às descritas na literatura. Após o revestimento, foi observado um aumento no diâmetro médio, bem com uma inversão nos valores de potencial zeta. Essas observações podem indicar a ocorrência do revestimento. A partir das análises de DSC, pôde-se comprovar a eficiência do revestimento das nanopartículas pelo derivado sintetizado, já que o comportamento das nanopartículas de PBCA-TMQ foi diferente daquele obtido para a mistura física entre os constituintes da formulação. Nessa mesma perspectiva, análises de FTIR foram conduzidas e a ocorrência do revestimento foi corroborada. Além disso, as análises morfológicas por Microscopia de Força Atômica (AFM) revelaram que as nanopartículas revestidas apresentam baixa tendência à agregação, o que pode ser um indicativo de estabilidade para a formulação desenvolvida. Em relação aos ensaios de citotoxicidade, foi evidenciado que as nanopartículas de PBCA não apresentaram toxicidade significativa frente às células Caco-2, ao passo que a formulação revestida mostrou um efeito tóxico dose-dependente influenciado pelo grau de quaternização. Além disso, as nanopartículas desenvolvidas foram capazes de diminuir, reversivelmente, a Resistência Elétrica Transepitelial (RET) da monocamada de células. A fim de quantificar o fármaco associado às nanopartículas, foi desenvolvido e validado método analítico por espectrofotometria derivada com detecção no UV. Tal método mostrou-se capaz de eliminar a interferência dos excipientes, permitindo a quantificação do ACV na formulação de nanopartículas com precisão e exatidão adequadas. Assim, a porcentagem de fármaco associado às nanoestruturas pode ser calculada, obtendo-se um valor satisfatório. De maneira semelhante, foi desenvolvido e validado método por CLAE para a quantificação do fármaco nos ensaios de permeação. A metodologia proposta mostrou-se adequada considerando-se as recomendações da RE 899/03. Por meio dos ensaios de permeabilidade em células Caco-2, foi constatado que a formulação desenvolvida aumentou em 3 vezes o valor de Permeabilidade aparente (Papp) do fármaco em estudo. Além disso, as nanopartículas revestidas foram capazes de propiciar a liberação controlada do ACV nos ensaios de liberação in vitro utilizando meios com diferentes valores de pH (1,2; 6,8 e 7,4). / The oral route is considered for the administration of drugs, especially in the treatment of chronic diseases. However, drugs administered by this route may have variable and/or limited bioavailability. Various types of delivery systems have been developed with the goal of improving this parameter, among which stand out the nanoparticles of poly (alkylcyanoacrylate) (PACA).Such nanomaterials have been coated to improve stability in the gastrointestinal tract, promote greater solubility or enhance permeation. Therefore, in this work were developed nanoparticles of poly (n-butilcianoacrilato) (PBCA) containing acyclovir (ACV), coated with N,N,N-trimethylchitosan (TMC), a promising absorption promoter. The TMC was synthesized with high-yield and approximately 73% of quaternization. The PBCA nanoparticles presented physico-chemical characteristics similar to those described in the literature. After the coating, it was observed an increase in the average diameter, and a inversion on the values of zeta potential. These observations may indicate the occurrence of coating. DSC analysis could proved the efficiency of the coating of nanoparticles, since the behavior of nanoparticles of PBCA-TMC was different from those obtained for the physical mixture between the constituents of the formulation. In this same perspective, FTIR analyses were conducted and the occurrence of coating was corroborated. In addition, morphological analyses by Atomic Force Microscopy (AFM) showed that nanoparticles coated presented low tendency to aggregate, which can be an indication of stability for the formulation developed. In relation to cytotoxicity assays, it was evidenced that the PBCA nanoparticles showed no significant toxicity against the Caco-2 cells, whereas the coated formulation showed a dose-dependent toxic effect influenced by the degree of quaternization. In addition, the nanoparticles developed were able to decrease, reversibly, Transepitelial Electric Resistance (TEER) of the monolayer. In order to quantify the drug associated with nanoparticles, was developed and validated analytical method by derivative spectrophotometry with UV detection. This method was able to eliminate the interference of excipients, allowing the quantification of ACV in the formulation of nanoparticles with appropriate precision and accuracy. Thus, the percentage of drug associated with nanostructures can be calculated, obtaining a satisfactory value. Similarly, has been developed and validated HPLC method for the quantification of drug permeation tests. The proposed methodology was appropriate considering the recommendations of the RE 899/03. Through the permeability assays in Caco-2 cells, it has been found that the formulation developed increased by 3 times the value os Apparent Permeability (Papp) of ACV. In addition, the nanoparticles were able to provide controlled release of ACV in vitro using media with different pH values (1.2; 6.8 and 7.4).

O câncer de mama constitui o segundo tipo de câncer mais frequente no mundo e o mais comum entre as mulheres, representando uma das principais causas de morte. O tamoxifeno é um fármaco antiestrogênico utilizado para o tratamento deste tipo de câncer desde 1971 e ainda é o mais utilizado nos casos de tumores mamários que expressam receptores de estrógeno. Apesar de apresentar resultados significativamente positivos, seu efeito antiestrogênico não se restringe apenas ao sítio tumoral causando, com isso, efeitos colaterais graves que podem deixar sequelas. A proposta deste trabalho foi desenvolver sistemas de liberação nanoparticulados à base de PLA e PLA-PEG para veiculação do tamoxifeno, como uma estratégia para o potencial aumento da segurança e da eficácia deste fármaco através de um possível direcionamento passivo ao sítio de ação, devido à permeabilidade vascular aumentada destas regiões tumorais. As nanopartículas foram preparadas pela técnica de nanoprecipitação e apresentaram diâmetro médio inferior a 200 nm para a maioria das formulações. Foram avaliados três estabilizantes, o poloxamer 407, o poloxamer 188 e o polissorbato 80, este último proporcionou maior eficiência de encapsulação, 86,7% e 100%, nas nanopartículas de PLA e PLA-PEG, respectivamente. Quanto à composição das nanopartículas de PLA-PEG, o polímero utilizado inicialmente (PLA(1000)-PEG(750)) apresentou distribuição de tamanho heterogênea, perfil multimodal e alto índice de polidispersividade. Assim, este polímero foi substituído pelo PLA(5000)-PEG(1000), que apresentou distribuição de tamanho uniforme, perfil monomodal e baixo índice de polidispersividade. A caracterização por microscopia eletrônica de varredura comprovou a homogeneidade no tamanho de partícula, mostrando seu formato esférico. As análises de espectrofotometria no infravermelho e calorimetria diferencial exploratória sugeriram que não ocorreu nenhum tipo de interação ou reação entre o fármaco e os demais componentes das formulações. Dois métodos analíticos para a determinação do tamoxifeno foram validados com sucesso por CLAE e espectroscopia UV-vis. O perfil de liberação in vitro do tamoxifeno a partir das nanopartículas de PLA apresentou característica sustentada e alcançou 50% em 180 h, tendo sido totalmente liberado após 288 h. Já as nanopartículas de PLA(5000)-PEG(1000) liberaram apenas 16,9% do fármaco após 216 h. A liberação do fármaco a partir das nanopartículas foi muito mais lenta comparada ao tamoxifeno não encapsulado, evidenciando a vantagem da incorporação do fármaco em nanopartículas compostas por PLA e PLA-PEG. No estudo do perfil de concentração plasmática em ratas Wistar, não foi possível detectar o fármaco e seu principal metabólito pelo método por CLAE desenvolvido, sugerindo que os sistemas nanoparticulados tenham extravasado rapidamente para os órgãos. / Breast cancer is the second most frequent type of cancer in the world and it is the most common among women, representing a major cause of death. Tamoxifen is an antiestrogen drug used in the treatment of this type of cancer since 1971 and it is the most employed drug in the treatment of breast cancer subtypes that expresses estrogen receptors. Despite presenting significantly positive results, its antiestrogen effect is not restricted to the tumour site, causing, as consequence, severe side effects. The purpose of this work was to develop nanostructured drug delivery systems based on PLA and PLA-PEG loaded with tamoxifen, as a strategy to potentially increase the safety and efficacy of this drug through a possible passive accumulation the site of action, due to the enhanced vascular permeability of tumour sites. Nanoparticles were prepared by the nanoprecipitation technique and presented average diameter smaller than 200 nm for the majority of the formulations. Three stabilizing adjuvants were analysed, poloxamer 407, poloxamer 188 and polysorbate 80 and the last one yielded the highest encapsulation efficiency, 86.7% and 100%, for the PLA and PLA-PEG nanoparticles, respectively. Regarding the PLA-PEG nanoparticles composition, the first polymer employed was (PLA(1000)-PEG(750)), which presented heterogeneous particle size distribution, multimodal profile and high polydispersity index. So, it was replaced by PLA(5000)-PEG(1000), which exhibited uniform particle size distribution, monomodal profile and low polydispersity index. The characterization by scanning electron microscopy confirmed the homogeneity of particles size, evidencing their spherical shape. Infrared spectrophotometry and differential scanning calorimetry analysis suggested that any interaction or reaction had occurred between the drug and the other components of the formulations. Two analytical methods for tamoxifen quantification were successfully validated by HPLC and UV-vis spectroscopy. In vitro tamoxifen release profile from PLA nanoparticles presented sustained release and reached 50% in 180 h, being completely released after 288 h, whereas PLA(5000)-PEG(1000) nanoparticles released only 16.9% of tamoxifen after 216 h. Drug release from nanoparticles was much slower compared to the non-encapsulated tamoxifen, showing the advantage of nanoparticles composed of PLA and PLA-PEG. In the plasmatic concentration profile study carried out in Wistar rats, it was not possible to detect tamoxifen or its main metabolite by the HPLC method, suggesting that nanoparticles quickly extravased to organs.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-04-05T18:07:31Z No. of bitstreams: 1 Almeida, Kessiane Belshoff de [Dissertação, 2014].pdf: 1092616 bytes, checksum: ecf1d02bbd15139a6b65760edc3d1bb8 (MD5) / Made available in DSpace on 2017-04-05T18:07:31Z (GMT). No. of bitstreams: 1 Almeida, Kessiane Belshoff de [Dissertação, 2014].pdf: 1092616 bytes, checksum: ecf1d02bbd15139a6b65760edc3d1bb8 (MD5) / A nanoencapsulação de substâncias lipofílicas e sua posterior incorporação em formulações tópicas semissólidas oferece uma alternativa tecnologicamente viável para modular a permeação do ativo, melhorar sua distribuição na superfície da pele, reduzir sua toxicidade e conferir proteção frente a fatores extrínsecos. O polímero poli (ácido lático-co-glicolídeo) (PLGA) tem sido amplamente empregado na preparação de nanossistemas carreadores de fármacos em função de suas pronunciadas características de biocompatibilidade e biodegradabilidade. O presente estudo objetivou desenvolver um hidrogel para incorporação do óleo essencial de Cymbopogon citratus (OECc) encapsulado em nanopartículas poliméricas e avaliar sua atividade frente ao vírus Herpes simplex (HSV) tipos 1 e 2. Inicialmente, procedeu-se a extração de OECc por hidrodestilação, seguindo-se sua caracterização química por Cromatografia Gasosa de Alta Resolução acoplada ao Espectrômetro de Massas (CGAR-EM). Nanopartículas contendo OECc (NPOE) e nanopartículas branco (NP) foram preparadas pela técnica de emulsificação-difusão do solvente empregando PLGA, como polímero, e álcool polivinílico (PVA) como estabilizante. A distribuição de tamanho e o potencial zeta das partículas foram avaliados, respectivamente, pelas técnicas de espalhamento de luz dinâmico e mobilidade eletroforética, e sua eficiência de encapsulação determinada após extração com solvente, empregando metodologia analítica desenvolvida por espectrofotometria UV-Vis. Após preparo e caracterização, NPOE foram incorporadas a gel hidrofílico de Carbopol® Ultrez 10 NF (HNPOE), a fim de investigar a estabilidade da formulação nanoestruturada frente ao armazenamento e o perfil de liberação in vitro do óleo a partir da nanopartícula. Adicionalmente, hidrogel base (HB), hidrogel contendo o óleo livre (HOE) e o mesmo contendo nanopartículas branco (HNP) foram desenvolvidos como controles. A atividade inibitória das formulações frente HSV-1 e -2 sensíveis ao aciclovir foi avaliada por redução do título viral em placa, utilizando-se células Vero como sistema hospedeiro, após prévia determinação da citotoxicidade das amostras. O rendimento do processo extrativo foi de 0,37% e a análise qualitativa do óleo por CGAR-EM exibiu o citral como seu constituinte químico majoritário, correspondendo a 89,57 % da área relativa do cromatograma. Partículas em escala nanométrica e potencial zeta negativo foram obtidas, com um conteúdo de OECc de 58,59 ± 0,85 mg/g e eficiência de encapsulação de 28,48% ± 0,40%. Os hidrogéis desenvolvidos exibiram características apropriadas para aplicação cutânea, as quais se mantiveram inalteradas durante os 60 dias de armazenamento a 4 °C. Cabe salientar que as formulações HNPOE e HOE apresentaram redução significativa (p<0,05, t-Student) no conteúdo de óleo volátil em relação ao teor inicial, permanecendo subsequentemente constante durante o estudo de estabilidade. A análise do perfil de liberação in vitro de OECc a partir das formulações demonstrou padrão bifásico, com burst inicial e posterior fase de liberação sustentada, onde NPOE seguiu modelo cinético de Hixon-Crowell, e HNPOE e HOE modelo de Higuchi. Além disso, as mesmas exibiram comportamento anômalo, dependente dos mecanismos de difusão e erosão polimérica. Na avaliação da atividade antiviral, HNPOE foi capaz de inibir mais eficientemente ambas as estirpes virais em concentração não citotóxica de óleo, inferior às empregadas nas demais formulações. Estes resultados evidenciam o potencial do nanogel em proteger, modular a liberação e otimizar a atividade do óleo essencial frente ao vírus Herpes simplex / The nanoencapsulation of lipophilic substances and their subsequent incorporation into semisolid topical formulations offers a technologically feasible alternative to modulate the permeation of active, improve its distribution on the surface of the skin, reduce their toxicity and confer protection against extrinsic factors. The polymer poly (lactic acid-co-glycolide) (PLGA) has been widely employed in the preparation of drug carriers nanosystems due to their pronounced biocompatibility and biodegradability. The present study aimed to develop semisolid hydrogel incorporating of Cymbopogon citratus essential oil (CcEO) associated with polymeric nanoparticles and assess their activity against Herpes simplex virus (HSV) types 1 and 2. Initially, it was proceeded to extract the CcEO by hydrodistillation, following their chemical characterization by High-Resolution Gas Chromatography coupled to Mass Spectrometer (HRGC-MS). Nanoparticles containing CcEO (NPEO) and white nanoparticles (NP) were prepared by emulsification-diffusion of the solvent using PLGA as polymer and polyvinyl alcohol (PVA) as a stabilizer. The size distribution and zeta potential of the particles were determined, respectively, by dynamic light scattering and electrophoretic mobility, and its encapsulation efficiency was given by solvent extraction technique, using analytical methodology developed by UV-Vis spectrophotometry. After preparation and characterization, NPEO were incorporated into a hydrophilic gel Carbopol® Ultrez 10 NF (HNPEO) in order to investigate the stability of the nanostructured formulation across the storage and in vitro release profile of the oil from the nanoparticle. In addition, hydrogel base (HB), hydrogel containing free oil (HEO) and the same unloaded nanoparticles (HNP) were developed as controls. The inhibitory activity of the formulations against HSV-1 and -2 sensitive to acyclovir was assessed by viral titer reduction using Vero cells as a host system after prior determination of the cytotoxicity of the samples. The yield of the extraction process was 0.37% and the qualitative analysis of the oil by HRGC-MS showed the citral as its major chemical constituent, accounting for 89.57% of the chromatogram relative area. Particles on the nanometer scale and negative zeta potential were obtained with CcEO content of 58.59 ± 0.85 mg/g and encapsulation efficiency of 28.48 ± 0.40%. The developed hydrogels exhibit suitable characteristics for cutaneous application, which remained unchanged during the 60 days of storage at 4°C. Is worth emphasizing that the HNPEO and HEO formulations showed a significant reduction (p<0.05, Student's t test) in the content of volatile oil in relation to the initial rate, subsequently remaining constant during the stability study. The analysis of the in vitro release profile CcEO from the formulations showed a biphasic pattern with an initial burst and subsequent sustained release phase, which followed NPEO kinetic model Hixon-Crowell, and HNPOE and HOE Higuchi model. Moreover, they exhibited anomalous behavior, dependent on the mechanisms of diffusion and polymer erosion. In the evaluation of antiviral activity, HNPEO was able to more efficiently inhibit both viral strains in the non-cytotoxic oil concentration lower than used in the other formulations. These results highlight the potential of nanogel in protecting, modulating the release profile and improve the activity of the essential oil against Herpes simplex virus

Depuis plusieurs années, la vectorisation de molécules est considérée comme la stratégie la plus prometteuse pour améliorer la pénétration cutanée des principes actifs et pour cibler et contrôler leur libération, augmentant ainsi l'efficacité thérapeutique des traitements tout en limitant leurs effets secondaires. Les nanoparticules polymériques ont fait l'objet de nombreuses études car elles possèdent une très grande stabilité et une capacité supérieure aux autres vecteurs à libérer les principes actifs de façon prolongée. Récemment, le laboratoire de Pharmacie Galénique de Besançon a montré, in vivo, que les nanoparticules polymériques de diamètre inférieure ou égale à 100 nm pénétraient spécifiquement dans la peau inflammée de la souris, alors qu'aucune pénétration n'était observée dans la peau saine. Le premier objectif de cette thèse était de confirmer ces hypothèses dans la peau inflammée d'un autre animal, le porc. Les résultats de notre étude in vivo confirment l'absence de pénétration des nanoparticules polymériques dans la peau saine du porc et montrent une pénétration taille dépendante dans la peau inflammée. Le second objectif poursuivi était de vérifier ces résultats chez l'Homme. Pour cela, une preuve de concept a été mise en place dans le service de Dermatologie du CHRU de Besançon. Les premiers résultats de cette étude clinique semblent confirmer l'absence de pénétration des nanoparticules polymérique de I 00 nm dans la peau des volontaires sains et dans la peau non lésée des patients souffrant de dermatite atopique. A l'inverse, une forte pénétration des nanoparticules est observée au niveau des plaques d'eczéma des patients. Si les résultats de l'étude clinique se confirment, nous prévoyons d'encapsuler un anti­inflammatoire ou un immunosuppresseur pour vérifier l'intérêt thérapeutique de ces vecteurs en médecine humaine dans le traitement de la dermatite atopique. Parallèlement, une évaluation pourrait être réalisée en médecine vétérinaire pour le traitement de cette pathologie fréquente chez le chien, et dont le traitement actuel repose sur l'administration per os de corticoïdes à l'origine de nombreux effets secondaires. / For several years now, nanocarriers have been considered as the most promising strategy to improve skin penetration of active ingredients. Moreover, these carriers are more efficient at targeting and controlling drug release into the skin, which leads to increased treatment efficiency and reduced side effects. Polymeric nanoparticles have been the object of an increasing number of studies due to their good physicochemical stability and prolonged release of active ingredients which is superior to any other carriers. Recently, the Laboratory of Pharmaceutical Engineering at Besançon proved in vivo, that polymeric nanoparticles with a diameter smaller or equivalent to I 00 nm specifically penetrated in inflamed skin of mice whilst no penetration was observed in healthy skin. The first aim ofthis thesis was to confirm this hypothesis on another animal's inflamed skin, in instance the pig. Our results confirm the poor penetration of polymeric nanoparticles in healthy skin ofpigs and show various degree ofpenetration depending on the size of the nanoparticles into the inflamed skin area. The second objective ofthis work was to evaluate the skin penetration of our polymeric nanoparticles in humans. A proof of concept has been developed in the Department of Dermatology at Besancon University Hospital. The first results ofthis clinical trial tend to confim1 the greater penetration ofour carrier, specifically in inflamed skin. In fact, no penetration of polymeric nanoparticles with a size close to 100 nm was observed in healthy skin ofvolunteers or in the non-inflamed skin of patients suffering from atopic dennatitis. Conversely, a high penetration ofthese carriers was observed in the skin lesions of patients with atopic dermatitis. If the results ofthis clinical trial are confirmed, we plan to load an anti-inflammatory or an immunosuppressive drug into the nanoparticles to evaluate the therapeutic value ofthese nanocarriers in human medicine in the treatment ofatopic dermatitis. Meanwhile, a similar study may be undertaken in veterinary medicine for the treatment of atopic dermatitis in dogs which is a common disease whose current treatment is based on the oral administration of corticosteroids and cause many undesirable side effects.

Les nanoparticules polymériques fluorescentes apparaissent comme des outils importants pour l'imagerie en temps réel des processus biologiques au niveau moléculaire et cellulaire. L’objectif de mon projet de doctorat a été d’optimiser les nanoparticules polymériques fluorescentes pour l’imagerie biologique. Premièrement, nous avons pu, en faisant varier la chimie des polymères, obtenir un très bon contrôle de leur taille. Ceci a permis de mettre en évidence l’importance de la taille des NPs pour des applications intracellulaires avec une taille maximale de 23 nm pour une distribution dans tout le cytosol. Deuxièmement, nous avons pu montrer que la simple adsorption d’un amphiphile PEGylé de type Pluronic permet la stabilisation des nanoparticules dans des milieux biologiques. Le nombre de molécules incorporées et leur stabilité ont été étudiés en combinant des techniques de FRET et de FCS. Les meilleures formulations résultent en une stabilité des nanoparticules in vivo, ce qui a permis leur imagerie en tant que particules individuelles dans les vaisseaux sanguins du cerveau de souris. Troisièmement, le transfert d’énergie entre différents fluorophores encapsulés dans les NPs a été étudié et optimisé. / Fluorescent polymeric nanoparticles appear as important tools for real-time imaging of biological processes at the molecular and cellular level. The objective of my PhD project was to optimize fluorescent polymeric nanoparticles for biological imaging. First, by varying the chemistry of the polymers, we have been able to obtain a very good control of their size. This made it possible to highlight the importance of NPs size for intracellular applications with a maximum size of 23 nm for optimal distribution throughout the cytosol. Secondly, we have shown that simple adsorption of a PEGylated amphiphiles pluronic family allows the stabilization of nanoparticles in biological media. The number of incorporated molecules and their stability has been studied by combining FRET and FCS techniques. The best formulations result in nanoparticle stability in vivo, which allowed their imaging as individual particles in the blood vessels of the mouse brain. Third, energy transfer among different fluorophores encapsulated in NPs has been studied and optimized.

The delivery of small interfering RNAs (siRNA) is an efficient method for gene silencing through the induction of RNA interference (RNAi). It critically relies, however, on efficient vehicles for siRNA formulation, for transfection in vitro as well as for their potential use in vivo. While polyethylenimines (PEIs) are among the most studied cationic polymers for nucleic acid delivery including small RNA molecules, polypropylenimines (PPIs) have been explored to a lesser extent. Previous studies have shown the benefit of the modification of small PEIs by tyrosine grafting which are featured in this paper. Additionally, we have now extended this approach towards PPIs, presenting tyrosine-modified PPIs (named PPI-Y) for the first time. In this study, we describe the marked improvement of PPI upon its tyrosine modification, leading to enhanced siRNA complexation, complex stability, siRNA delivery, knockdown efficacy and biocompatibility. Results of PPI-Y/siRNA complexes are also compared with data based on tyrosine-modified linear or branched PEIs (LPxY or PxY). Taken together, this establishes tyrosine-modified PPIs or PEIs as particularly promising polymeric systems for siRNA formulation and delivery.

L’utilisation de nanoparticules (NPs) dans divers domaines industriels est de plus en plus fréquente ce qui génère leur propagation dans l’environnement. Selon leur persistance, mobilité, bioaccumulation et toxicité, des risques inconnus pour la santé et pour des écosystèmes peuvent en résulter. En effet, la caractérisation et la quantification sont des défis analytiques très complexes en raison de la nature dynamique (petite taille, grande réactivité et instabilité) des nanomatériaux. L'objectif de cette étude est donc de caractériser par ultracentrifugation analytique (AUC) des nanoparticules polymériques (Allosperse® dites allosphères) qui sont destinées à des fins agricoles. Pour y parvenir, différentes NPs métalliques (argent, quantum dot), oxydes métalliques (dioxyde de titane, oxyde de zinc) et NPs de polystyrène ont d’abord été mesurés par AUC à l’aide des différents systèmes de détection (absorbance, fluorescence et interférence). Dans le cas des allosphères, un grand nombre d'essais préliminaires ont été réalisés afin d'optimiser la vitesse d'ultracentrifugation, le temps d'ultracentrifugation, le nombre de numérisations et la concentration de l'échantillon. Un protocole optimisé a été utilisé pour la détermination du diamètre hydrodynamique (dh) des NPs. Les différentes analyses qui ont été réalisées dans cette étude révèlent que l’AUC permet de déterminer la taille de très petites NPs. Par ailleurs, une étude du comportement de ces allosphères pour des pH entre 4-8, des forces ioniques de 0 à 500 mM, en présence ou absence de matière organique naturelle a été entreprise. Les travaux ont montré que le dH était d’environ 7,0 nm avec de petites augmentations à faible pH, ou à très grande force ionique ou dureté. Ces résultats indiquent la grande stabilité physique et chimique des allosphères qui auront, ainsi, une grande mobilité dans les sols. La diffusion de lumière dynamique et la spectroscopie de corrélation de fluorescence ont été utilisées afin de valider les résultats obtenus par l’AUC. / The use of nanoparticles (NPs) in numerous industrial fields is becoming more common, which increases their propagation in the environment. Their generally unknown persistence, mobility, bioaccumulation and toxicity all contribute to increased risks to human health and to ecosystems. Unfortunately, their characterization and quantification are complex analytical challenges due in large part to their dynamic nature (small size, high reactivity and instability). The objective of this study was to characterize polymeric nanoparticles (Allosperse®), which are intended for the dispersion of the nanopesticides using analytical ultracentrifugation (AUC). To achieve this goal, the sizes of various metallic nanoparticles (nAg, QD), metallic oxides (nTiO2, nZnO) and polystyrene nanoparticles (nPS) were first determined by AUC using different detectors (absorbance, fluorescence and interference). In the case of polymeric nanoparticles, a number of preliminary tests were carried out in order to optimize the speed and duration of the ultracentrifugation, the number of scans and the concentration of the NPs for the determination of their hydrodynamic diameter (dh). The analysis indicated that the AUC was able to measure the sizes of the smallest nanoparticles. In addition, evaluations of the behavior of these nanoparticles between pH 4-8, ionic strengths from 0 to 500 mM, in the presence and absence of natural organic matter (NOM) showed that they had a dh of about 7.0 nm with small increases at low pH or for large ionic strengths or hardness. These results strongly demonstrated a high physical and chemical stability of allosphères, which implied that they would have a high mobility in soils. Dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS) were used to validate the results obtained by the AUC.

Selon les dernières estimations de l'OMS, on enregistre chaque année dans le monde 498.9 millions de nouveaux cas d'infections sexuellement transmissibles (IST) dont 276.4 millions sont dus au parasite Trichomonas vaginalis (T. vaginalis). Au niveau du tractus génital, la colonisation et l’irritation de la muqueuse vaginale par T. vaginalis favorisent la survenue de complications infectieuses. Ces infections associées peuvent conduire à des infections chroniques et avoir à terme des conséquences graves (stérilité, rupture prématuré du placenta, mort prématurée du nourrisson). De plus, ces infections vaginales représentent des facteurs qui favorisent les infections par le Virus d’Immunodéficience Humaine (VIH-1).A l’heure actuelle, la lutte contre ce type d’infections consiste à agir tant au niveau curatif, que préventif. Ainsi, l’objectif de ce projet est de développer de nouvelles formulations pour la prévention et le traitement des pathogènes qui colonisent les muqueuses vaginales. Dans ce contexte, la formulation que nous proposons est composée de metronidazole inclut dans un hydrogel thermogélifiant à base de pluronic® F127 et de chitosane. Il a été montré que cet hydrogel conserve ces propriétés physiques à une température physiologique même après dilution dans les fluides vaginaux. Ces hydrogels sont stables et permettent une libération prolongée du metronidazole. La formulation n’a montré aucune toxicité envers les cellules HeLa ni envers la muqueuse vaginale porcine. L’efficacité de cette formulation a été prouvée envers T. vaginalis et présente un effet protecteur envers les cellules HeLa en présence de T. vaginalis. L’ensemble des résultats suggère donc la capacité́ de cette formulation à constituer une double barrière, physique et pharmacologique, protectrice de la muqueuse vaginale vis-à-vis de T. vaginalis. / According to the latest WHO estimates, 498 millions of new cases of sexually transmitted infections (STIs) are recorded annually in the world, including 276.4 million due to the parasite Trichomonas vaginalis (T. vaginalis). In the genital tract colonization and irritation of the vaginal mucosa by T. vaginalis promote the occurrence of infectious complications. Associated infections can lead to chronic infections and eventually have serious consequences (infertility, premature placental abruption, premature death). In addition, these vaginal infections can promote infection by Human Immunodeficiency Virus (HIV-1).Currently, the fight against these infections is to act at curative and preventive level. Thus, the objective of this project is to develop new formulations for the prevention and treatment of pathogens that colonize the vaginal mucosa. In this context, we propose a formulation composed of metronidazole and chitosan include in a thermogelling hydrogel of pluronic F127®.It was shown that the hydrogel retains its physical properties even at a physiological temperature and after dilution in the vaginal fluids. These hydrogels are stable and allow a sustained release of the metronidazole. The formulation showed no toxicity against HeLa cells or porcine vaginal mucosa. The effectiveness of this formulation has been proven against T vaginalis and has a protective effect on HeLa cells in the presence of T. vaginalis. The overall results therefore suggest the ability of this formulation to form a double barrier, physical and pharmacological, than protect vaginal mucosa against T. vaginalis.