The aims of this thesis are to design and synthesize non-viral cationic lipid vectors based on spermine, for the intracellular delivery of siRNA (short interfering RNA) and the subsequent siRNA mediated gene silencing. Two parameters were varied: the type of fatty acid and the cationic head-group. Among the symmetrical spermine conjugates, N4,N9-dierucoyl spermine (DES) resulted in higher siRNA delivery compared to N4,N9-dioleoyl spermine (DOS), while enhanced green fluorescent protein (EGFP) silencing in HeLa cells showed that the unsaturated fatty acid conjugates are more efficient than the saturated fatty acid ones, and cell viability was 75%-85% for conjugates with chain length ≥ 18. Two cationic lipids with guanidine head-groups, N1,N12-diamidino-N4,N9-dioleoylspermine and N1,N12-diamidino-N4-linoleoyl-N9-oleoylspermine, were more efficient in EGFP gene silencing compared to cationic lipids with shorter C12 (lauroyl) and very long C22 (erucoyl) chains, with cell viability (64%-83% for chain length ≥ 18). Changing the cationic headgroup to guanidine did not offer a significant advantage in gene silencing over the conjugates with terminal primary amine groups. The asymmetrical N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS) resulted in the best gene silencing, while LigOS (with one lignoceroyl 24:0 chain) resulted in the best siRNA delivery. Conjugates with two unsaturated fatty chains generally resulted in better EGFP gene silencing, while conjugates with one saturated chain and one unsaturated chain resulted in better siRNA delivery. Increasing the chain length also resulted in increased siRNA delivery (cell viabilities of asymmetrical > 74%, LinOS 88%). siRNA lipoplexes prepared using mixtures of LinOS with either cholesterol or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) resulted in increased siRNA delivery, and enhanced EGFP silencing, with LinOS/Chol mixture (1:2 molar ratio) resulting in the highest siRNA delivery and the best gene silencing (EGFP reduced to 20%). Temperature studies of intracellular entry showed that the majority of lipoplexes are internalized by endocytosis, however the majority of gene-silencing occurs due to lipoplexes internalized via another mechanism.

The study of drug delivery for the treatment of illnesses and injuries is an important area of pharmaceutical technology. A relatively new area of drug delivery being explored is gene therapy, which utilizes the idea that genes can be used as an alternative treatment. The exploration of gene delivery brought major advancements in the treatment of cancers and tumors as well as many challenges. In this study, the challenges of maintaining a stable vehicle for delivery, delivering genes into the cells, and the efficacy of the gene delivery vehicle were explored. Seven co-polymers of 12% (w/v) poly (D, L-lactic glycolide) (PDLG) were used to find a biodegradable polymer composition as an implant that temporarily controls the delivery of the genes. Of the formulations tested, 65/35 DL 3A and 50/50 DLG 4A were observed to show degradation time frames that best fit our purposes. Also, nanoparticles have been used to aid in the targeting of drugs to desired cells in delivery. One drawback of using nanoparticles is the potential toxic side effects they might cause. Zinc oxide nanoparticles coated with poly (vinyl pyrrolidone) (PVP) used as drug carriers were observed to have an effect on cell viability in previous studies. The cytotoxic effects of ZnO nanoparticles and PVP have on NIH 3T3 mouse fibroblast cells were investigated to see if there is a direct correlation between the level of PVP and zinc nanoparticles to the amount of cell death. It was found that an increase in concentration of ZnO nanoparticles correlates to a decrease in viability of the cells. It was also noted that the method of cell death is likely to be apoptosis. To confirm the efficacy of gene therapy through transfection, the transfection of the serum response factor (SRF) gene plasmid DNA and short interfering RNA (siRNA) were investigated. The efficiency of the transfection method were tested for both twodimensional and three-dimensional transfection of the SRF plasmid and siRNA. Experiments with two-dimensional transfection of the SRF plasmid and siRNA were successful, and transfer of the gene in the three-dimensional environment was observed with promising results with the siRNA.

Nanodiamonds (NDs) are the most biocompatible member of the carbon nanofamily which are widely researched for diagnostic and therapeutic applications. Unlike other carbon nanomaterials, the surface of NDs is innately reactive, hence capable of conjugating various chemical moieties for targeted actions. This work focuses on utilizing the surface reactivity of NDs for gene therapeutics and addressing the challenges associated with its application in the biological environment. Pristine carboxylated NDs were functionalized with basic amino acids (lysine and lysyl-histidine) through covalent conjugation via a three carbon chain linker. Amino acid functionalized NDs were characterized by infrared spectroscopy, thermogravimetry and size and zeta potential measurements. Lysine conjugation was evident through a marked change in the zeta potential of ND dispersion from negative to a high positive value (-54.6 mV to +26.3 mV). The thermogram of lysine functionalized NDs (Lys-NDs) revealed a significant weight loss from 150ᵒC to 700ᵒC confirming the functionalization through loss of amino acid conjugates from the surface and total loading was calculated as 1.97 mmols/g. Lys-NDs also showed optimum binding with pDNA and siRNA at weight ratios of 1:1 and 1:20 (pDNA/siRNA:ND), respectively. Functionalization of NDs with lysine contributed to limiting aggregation and enhancing the colloidal stability of ND dispersions in biological milieu. The aqueous dispersion of lys-NDs showed minimum sedimentation and remained stable over a period of 25 days. Average sizes under 100 nm and zeta potentials higher than +20 mV indicate a preservation of the cationic surface throughout the testing period. Moreover, size distributions and zeta potentials changed significantly upon incubation of lys-NDs with blood serum suggesting an interaction with biomolecules, mainly proteins and a possible formation of a protein corona. Cellular internalization of bare lys-NDs and their diamoplexes (i.e. complexes of NDs with nucleic acids) was assessed through scanning transmission X-ray microscopy and flow cytometry. Functional efficiency of lysine NDs was determined by flow cytometry monitoring the GFP knockdown through anti-GFP siRNA delivery. Results reveal a promising GFP knockdown of ~17% upon treating the cells with NDs/siRNA diamoplexes at a ratio of 20:1. Subsequent analyses regarding the effect of NDs to prevent cellular proliferation and to cause cellular apoptosis confirmed that they are innately biocompatible at a wide range of concentrations. Unlike lysine NDs, lysyl-histidine functionalization was limited and the surface loading of this conjugate on NDs was very low. Therefore, they were unable to bind pDNA and siRNA even at high weight ratios and hence demand design modifications. Overall this work demonstrates a novel approach of functionalizing NDs with basic amino acids capable of enhancing colloidal stability and delivering of therapeutic genes into mammalian cells. It represents an important step in the development of safe and efficient gene therapy for inherited and acquired diseases.

O processo de interferência de RNA refere-se ao silenciamento pós transcricional seqüência-específico de genes em animais e plantas capaz de ser promovido por dsRNA homólogo à seqüência do gene silenciado. Este processo pode ser aplicado como terapia, que apresenta como vantagens a especificidade pelos alvos escolhidos, a possibilidade de tratar uma enorme gama de doenças genéticas, além do fato de ser muito potente e eficaz. Contudo, o principal desafio consiste em manter a estabilidade dos siRNAs nos fluidos biológicos, visto que estes são bastante susceptíveis à excreção renal e a degradação por RNAses. Com isso, reforça-se a necessidade de sistemas de liberação adequados, que sejam capazes de manter a estabilidade dos siRNAs por tempo suficiente para que atinjam os órgãos alvo da terapia e promover sua liberação sustentada. De particular interesse são determinadas proteínas e peptídeos de transdução (PTDs) que podem ser ligados a fármacos hidrofílicos e assim tornam possível com que estes atravessem membranas. Neste sentido, muitos sistemas carreadores não-virais tem sido estudados para a veiculação de siRNA, sendo de cunho inovador o desenvolvimento de sistemas de liberação nanoestruturados baseados em cristais líquidos funcionalizados com peptídeos de transdução de membrana para a veiculação tópica de siRNAs. Desta forma, nanopartículas de cristais líquidos de fase hexagonal contendo ou não os aditivos catiônicos polietileimina (PEI) e oleilamina (OAM) foram funcionalizadas com peptídeos de transdução de membranas TAT (TAT) ou penetratin (PNT). Os sistemas obtidos foram complexados com siRNA por interação eletrostática e caracterizados através de medidas de tamanho de partícula/ polidispersividade, potencial zeta e eficiência de complexação. A citotoxicidade dos sistemas foi avaliada em fibroblastos L929 pelo ensaio do MTT e por citometria de fluxo e a avaliação da transfecção in vitro foi realizada por citometria de fluxo e por microscopia de fluorescência. Os sistemas contendo PEI ou OAM apresentavam potencial zeta positivo e foram capazes de complexar o siRNA adicionado na concentração de 10 ?M. Os estudos em culturas celulares demonstraram que os sistemas contendo ácido oleico (AO) foram mais eficientes quanto à transfecção em células de fibroblastos L929 e esta eficiência de transfecção foi aumentada com a funcionalização com o peptídeo TAT. A partir daí, os sistemas selecionados foram avaliados quanto a penetração cutânea in vivo. Os sistemas nanodispersos formados por MO/AO/PEI proporcionaram uma maior liberação de siRNA na pele e a eficiência de supressão de TNF-alfa em modelo animal de inflamação cutânea foram maiores que formulações controle. Com isso, demonstrou-se que os sistemas desenvolvidos são promissores para a futura aplicação na terapia gênica tópica de doenças cutâneas inflamatórias. / The RNA interference process refers to the sequence-specific posttranscriptional silencing of genes in animals and plants capable of being promoted by dsRNA that are homologous to the sequence of the silenced gene. This process can be applied as therapy, which presents advantages such as the specificity to the chosen targets, the possibility to treat a variety of genetic diseases, besides being very potent and efficacious. However, the major hurdle consists in keeping the siRNAs stability in the biological fluids, because they are susceptible to renal clearance and degradation by RNAses. Thus, there is the need for suitable delivery systems, capable of maintaining the stability of siRNAs for sufficient time so they can reach the target organ in the therapy and promote sustained release. Of particular interest are certain proteins and peptides transduction domains (PTDs) that can be connected to hydrophilic drugs and thus make it possible to cross cell membranes. Within this context, many non-viral vectors have been studied for siRNA vehiculation which makes innovative the present study because it aims at the development of nanostructured delivery systems based on liquid crystals functionalyzed with membrane transduction peptides for the topical vehiculation of siRNAs. Thus, hexagonal phase liquid crystal nanoparticles containing or not the cationinc polyethylenimine (PEI) and oleylamine (OAM) were functionalyzed with membrane transduction peptides TAT (TAT) or penetratin (PNT). The obtained systems were complexed with siRNA by eletrostatic interaction and characterized for particle size, polidispersity, zeta potential and complexation efficiency. The cytotoxicity of the formulations was performed with L929 fibroblasts by MTT assay and flow cytometry and the in vitro transfection was evaluated by flow cytometry and fluorescence microscopy. The systems containing PEI or OAM presented positive zeta potential and could complex siRNA at the concentration of 10 ?M. The cell culture studies demonstrated that the systems containing oleic acid (OA) were the most efficient to transfect L929 cells and the transfection efficiency was enhanced with the functionalization with the TAT peptide. Thereafter, the selected systems were evaluated for the in vivo skin penetration. The nanosdispersed systems composed of MO/OA/PEI functionalyzed with TAT resulted in a higher siRNA penetration and release in the skin, promoting higher TNF alfa supression in animal model of cutaneous inflammation, compared to the control formulations. Hence, we demonstrated that the developed systems are promising for the treatment of inflammatory skin diseases.

As células da granulosa (CG) são constituintes do ambiente folicular de suma importância para o desenvolvimento, maturação e aquisição da competência oocitária, desempenhando funções como a esteroidogênese, expressão de receptores de LH (LHR) e síntese de inúmeras proteínas essenciais. Contudo, a funcionalidade e ação destas células são dependentes de alguns fatores derivados do oócito, como o GDF9 e o BMP15. A ação desses fatores, por sua vez, depende da sinalização via receptor BMPRII, já identificado em células da granulosa de mamíferos, porém com suas funções ainda pouco conhecidas na espécie bovina. O silenciamento gênico por lipofecção consiste num método de transfecção eficiente e pouco agressivo para as células, representando uma importante ferramenta para o estudo funcional de genes e proteínas celulares. O objetivo do presente trabalho foi, primeiramente, otimizar as condições de lipofecção em CG bovinas e, a partir desse protocolo, estabelecer uma metodologia de silenciamento gênico para o BMPRII usando a técnica de RNA de interferência, a fim de possibilitar a futura utilização de tal estratégia como ferramenta para o estudo funcional do BMPRII e outros genes de interesse. Para tanto, no primeiro experimento referente à otimização das condições de lipofecção em CG bovinas, onde as células da granulosa, obtidas de ovários oriundos de abatedouro foram tratadas com diferentes quantidades dos lipofectores Lipofectamine® RNAiMAX (1, 2 e 3µl) ou Lipofectamine™ 2000 (1, 2 e 3µl) e dos indicadores de transfecção siGLO® (30, 50, 75 e 100nM) ou plasmídeo transgênico FUGW (100, 200, 300, 400, 600 e 900 nM) durante 24 e 48 horas de cultivo. A melhor eficiência de lipofecção foi observada às 24 horas de cultivo com 2µl de Lipofectamine™ 2000 + 100nM de siGLO®. Num segundo experimento, a partir das condições de lipofecção determinadas, foram testadas diferentes concentrações do siBMPRII (0 a 500 pmol) durante 24 h de cultivo. Ao final desse período, as células de cada grupo foram avaliadas quanto à abundância relativa mRNA BMPRII por PCR em tempo real. Todas as concentrações resultaram em uma redução semelhante de transcritos, sendo possível adotar a menor concentração (100 pM), a qual foi utilizada para determinar o tempo mínimo de cultivo necessário para obter eficiência no processo de silenciamento. As células foram tratadas por 0, 6, 12, 18 e 24 h e também avaliadas quanto aos transcritos de BMPRII. Os tempos de incubação que proporcionaram maior redução do mRNA para o BMPRII foram 18 e 24 horas. A melhor concentração e tempo de incubação com o siRNA foram avaliados por Western Blotting, confirmando a redução da expressão de BMPRII também em nível de proteína. Em conclusão, este trabalho possibilitou o estabelecimento de uma metodologia eficiente para o silenciamento gênico por lipofecção do BMPRII em CG bovinas, a qual poderá ser utilizada como ferramenta para o estudo funcional de diversos genes de interesse. / Granulosa cells (GC) are part of the folicular environment and are important for the development, maturation and acquisition of developmental competence of oocytes, performing functions such as steriodogenesis, expression of LH receptors (LHR) and synthesis of several important proteins. The function of these cells is dependent on some oocyte-derived factors, such as GDF9 and BMP15. Signaling of these factors involves the activation of the BMPRII receptor identified in granulosa cells, however, its function is not well established in bovine cells. Gene silencing is an important tool for functional studies of different cellular proteins. The aim of the present study was to develop a protocol for gene silencing using the RNA interference technique by lipofecction to knockdown BMPRII expression, for future functional studies on the role of this receptor and other genes of interest in bovine granulosa cells. For this purpose, the first experimente aimed to optimize lipofection conditions in bovine granulosa cells, collected from abbattoir ovaries and cultured in vitro. Lipofecction was tested for different concentrations of two diferent lipofection agents (1, 2 and 3 µl Lipofectamine® RNAiMAX or Lipofectamine™ 2000) and two transfection indicators (30, 50, 75 and 100nM siGLO® or 100, 200, 300, 400, 600 and 900 nM transgenic plsamid FUGW) for 24 and 48 h in culture. Best lipofection efficiency was observed at 24 h culture with 2µl Lipofectamine™ 2000 + 100nM siGLO®, which was used for the next experiment. The second experiment tested different BMPRII-siRNA concentrations (0 to 500 pM) for 24 h. At the end of culture the cells were be assessed for the relative abundance for BMPRII by real time PCR. All concentrations similarly reduced transcript abundance relative to control (0 pM). For the third experiment, the lowest effective concentration was used (100 pM) to test different culture periods with the BMPRII-siRNA and determine the minimum period to obtain transcript reduction. Cells will be treated for 0, 6, 12, 18 and 24 h and assessed for BMPRII transcripts. Greater reduction in BMPRII transcripts was observed after 18 and 24 h. The best concentration (100 pM) and time (24 h) were confirmed for knockdown of the BMPRII protein by western blotting. In conclusion, this work has provided the establishment of a reliable method for gene silencing using lipofection for the BMPRII in bovine granulosa cells, which may be used for functional studies for this gene and others of interest.

As células da granulosa (CG) são constituintes do ambiente folicular de suma importância para o desenvolvimento, maturação e aquisição da competência oocitária, desempenhando funções como a esteroidogênese, expressão de receptores de LH (LHR) e síntese de inúmeras proteínas essenciais. Contudo, a funcionalidade e ação destas células são dependentes de alguns fatores derivados do oócito, como o GDF9 e o BMP15. A ação desses fatores, por sua vez, depende da sinalização via receptor BMPRII, já identificado em células da granulosa de mamíferos, porém com suas funções ainda pouco conhecidas na espécie bovina. O silenciamento gênico por lipofecção consiste num método de transfecção eficiente e pouco agressivo para as células, representando uma importante ferramenta para o estudo funcional de genes e proteínas celulares. O objetivo do presente trabalho foi, primeiramente, otimizar as condições de lipofecção em CG bovinas e, a partir desse protocolo, estabelecer uma metodologia de silenciamento gênico para o BMPRII usando a técnica de RNA de interferência, a fim de possibilitar a futura utilização de tal estratégia como ferramenta para o estudo funcional do BMPRII e outros genes de interesse. Para tanto, no primeiro experimento referente à otimização das condições de lipofecção em CG bovinas, onde as células da granulosa, obtidas de ovários oriundos de abatedouro foram tratadas com diferentes quantidades dos lipofectores Lipofectamine® RNAiMAX (1, 2 e 3µl) ou Lipofectamine™ 2000 (1, 2 e 3µl) e dos indicadores de transfecção siGLO® (30, 50, 75 e 100nM) ou plasmídeo transgênico FUGW (100, 200, 300, 400, 600 e 900 nM) durante 24 e 48 horas de cultivo. A melhor eficiência de lipofecção foi observada às 24 horas de cultivo com 2µl de Lipofectamine™ 2000 + 100nM de siGLO®. Num segundo experimento, a partir das condições de lipofecção determinadas, foram testadas diferentes concentrações do siBMPRII (0 a 500 pmol) durante 24 h de cultivo. Ao final desse período, as células de cada grupo foram avaliadas quanto à abundância relativa mRNA BMPRII por PCR em tempo real. Todas as concentrações resultaram em uma redução semelhante de transcritos, sendo possível adotar a menor concentração (100 pM), a qual foi utilizada para determinar o tempo mínimo de cultivo necessário para obter eficiência no processo de silenciamento. As células foram tratadas por 0, 6, 12, 18 e 24 h e também avaliadas quanto aos transcritos de BMPRII. Os tempos de incubação que proporcionaram maior redução do mRNA para o BMPRII foram 18 e 24 horas. A melhor concentração e tempo de incubação com o siRNA foram avaliados por Western Blotting, confirmando a redução da expressão de BMPRII também em nível de proteína. Em conclusão, este trabalho possibilitou o estabelecimento de uma metodologia eficiente para o silenciamento gênico por lipofecção do BMPRII em CG bovinas, a qual poderá ser utilizada como ferramenta para o estudo funcional de diversos genes de interesse. / Granulosa cells (GC) are part of the folicular environment and are important for the development, maturation and acquisition of developmental competence of oocytes, performing functions such as steriodogenesis, expression of LH receptors (LHR) and synthesis of several important proteins. The function of these cells is dependent on some oocyte-derived factors, such as GDF9 and BMP15. Signaling of these factors involves the activation of the BMPRII receptor identified in granulosa cells, however, its function is not well established in bovine cells. Gene silencing is an important tool for functional studies of different cellular proteins. The aim of the present study was to develop a protocol for gene silencing using the RNA interference technique by lipofecction to knockdown BMPRII expression, for future functional studies on the role of this receptor and other genes of interest in bovine granulosa cells. For this purpose, the first experimente aimed to optimize lipofection conditions in bovine granulosa cells, collected from abbattoir ovaries and cultured in vitro. Lipofecction was tested for different concentrations of two diferent lipofection agents (1, 2 and 3 µl Lipofectamine® RNAiMAX or Lipofectamine™ 2000) and two transfection indicators (30, 50, 75 and 100nM siGLO® or 100, 200, 300, 400, 600 and 900 nM transgenic plsamid FUGW) for 24 and 48 h in culture. Best lipofection efficiency was observed at 24 h culture with 2µl Lipofectamine™ 2000 + 100nM siGLO®, which was used for the next experiment. The second experiment tested different BMPRII-siRNA concentrations (0 to 500 pM) for 24 h. At the end of culture the cells were be assessed for the relative abundance for BMPRII by real time PCR. All concentrations similarly reduced transcript abundance relative to control (0 pM). For the third experiment, the lowest effective concentration was used (100 pM) to test different culture periods with the BMPRII-siRNA and determine the minimum period to obtain transcript reduction. Cells will be treated for 0, 6, 12, 18 and 24 h and assessed for BMPRII transcripts. Greater reduction in BMPRII transcripts was observed after 18 and 24 h. The best concentration (100 pM) and time (24 h) were confirmed for knockdown of the BMPRII protein by western blotting. In conclusion, this work has provided the establishment of a reliable method for gene silencing using lipofection for the BMPRII in bovine granulosa cells, which may be used for functional studies for this gene and others of interest.

O processo de interferência de RNA refere-se ao silenciamento pós transcricional seqüência-específico de genes em animais e plantas capaz de ser promovido por dsRNA homólogo à seqüência do gene silenciado. Este processo pode ser aplicado como terapia, que apresenta como vantagens a especificidade pelos alvos escolhidos, a possibilidade de tratar uma enorme gama de doenças genéticas, além do fato de ser muito potente e eficaz. Contudo, o principal desafio consiste em manter a estabilidade dos siRNAs nos fluidos biológicos, visto que estes são bastante susceptíveis à excreção renal e a degradação por RNAses. Com isso, reforça-se a necessidade de sistemas de liberação adequados, que sejam capazes de manter a estabilidade dos siRNAs por tempo suficiente para que atinjam os órgãos alvo da terapia e promover sua liberação sustentada. De particular interesse são determinadas proteínas e peptídeos de transdução (PTDs) que podem ser ligados a fármacos hidrofílicos e assim tornam possível com que estes atravessem membranas. Neste sentido, muitos sistemas carreadores não-virais tem sido estudados para a veiculação de siRNA, sendo de cunho inovador o desenvolvimento de sistemas de liberação nanoestruturados baseados em cristais líquidos funcionalizados com peptídeos de transdução de membrana para a veiculação tópica de siRNAs. Desta forma, nanopartículas de cristais líquidos de fase hexagonal contendo ou não os aditivos catiônicos polietileimina (PEI) e oleilamina (OAM) foram funcionalizadas com peptídeos de transdução de membranas TAT (TAT) ou penetratin (PNT). Os sistemas obtidos foram complexados com siRNA por interação eletrostática e caracterizados através de medidas de tamanho de partícula/ polidispersividade, potencial zeta e eficiência de complexação. A citotoxicidade dos sistemas foi avaliada em fibroblastos L929 pelo ensaio do MTT e por citometria de fluxo e a avaliação da transfecção in vitro foi realizada por citometria de fluxo e por microscopia de fluorescência. Os sistemas contendo PEI ou OAM apresentavam potencial zeta positivo e foram capazes de complexar o siRNA adicionado na concentração de 10 ?M. Os estudos em culturas celulares demonstraram que os sistemas contendo ácido oleico (AO) foram mais eficientes quanto à transfecção em células de fibroblastos L929 e esta eficiência de transfecção foi aumentada com a funcionalização com o peptídeo TAT. A partir daí, os sistemas selecionados foram avaliados quanto a penetração cutânea in vivo. Os sistemas nanodispersos formados por MO/AO/PEI proporcionaram uma maior liberação de siRNA na pele e a eficiência de supressão de TNF-alfa em modelo animal de inflamação cutânea foram maiores que formulações controle. Com isso, demonstrou-se que os sistemas desenvolvidos são promissores para a futura aplicação na terapia gênica tópica de doenças cutâneas inflamatórias. / The RNA interference process refers to the sequence-specific posttranscriptional silencing of genes in animals and plants capable of being promoted by dsRNA that are homologous to the sequence of the silenced gene. This process can be applied as therapy, which presents advantages such as the specificity to the chosen targets, the possibility to treat a variety of genetic diseases, besides being very potent and efficacious. However, the major hurdle consists in keeping the siRNAs stability in the biological fluids, because they are susceptible to renal clearance and degradation by RNAses. Thus, there is the need for suitable delivery systems, capable of maintaining the stability of siRNAs for sufficient time so they can reach the target organ in the therapy and promote sustained release. Of particular interest are certain proteins and peptides transduction domains (PTDs) that can be connected to hydrophilic drugs and thus make it possible to cross cell membranes. Within this context, many non-viral vectors have been studied for siRNA vehiculation which makes innovative the present study because it aims at the development of nanostructured delivery systems based on liquid crystals functionalyzed with membrane transduction peptides for the topical vehiculation of siRNAs. Thus, hexagonal phase liquid crystal nanoparticles containing or not the cationinc polyethylenimine (PEI) and oleylamine (OAM) were functionalyzed with membrane transduction peptides TAT (TAT) or penetratin (PNT). The obtained systems were complexed with siRNA by eletrostatic interaction and characterized for particle size, polidispersity, zeta potential and complexation efficiency. The cytotoxicity of the formulations was performed with L929 fibroblasts by MTT assay and flow cytometry and the in vitro transfection was evaluated by flow cytometry and fluorescence microscopy. The systems containing PEI or OAM presented positive zeta potential and could complex siRNA at the concentration of 10 ?M. The cell culture studies demonstrated that the systems containing oleic acid (OA) were the most efficient to transfect L929 cells and the transfection efficiency was enhanced with the functionalization with the TAT peptide. Thereafter, the selected systems were evaluated for the in vivo skin penetration. The nanosdispersed systems composed of MO/OA/PEI functionalyzed with TAT resulted in a higher siRNA penetration and release in the skin, promoting higher TNF alfa supression in animal model of cutaneous inflammation, compared to the control formulations. Hence, we demonstrated that the developed systems are promising for the treatment of inflammatory skin diseases.

Les « siRNA » sont des molécules double brin d’acide ribonucléique capables d’inhiber l’expression d’un gène spécifique, présentant ainsi un fort potentiel thérapeutique pour les maladies génétiques, les cancers et les infections virales. Cependant, son utilisation in vivo est restreinte par sa sensibilité à la dégradation enzymatique. Le projet de thèse consiste à créer un système de vectorisation des siRNA pour des applications in vivo. Nous avons synthétisé des copolymères à blocs amphiphiles biocompatibles et biodégradables capable de s’auto-assembler en diverses structures et d’encapsuler les siRNA. Les propriétés physico-chimiques des assemblages formées et l’évaluation cellulaire préliminaire est réalisée / Amphiphilic block copolymers are molecules composed of hydrophilic and hydrophobic segments having the capacity to spontaneously self-assemble into a variety of supramolecular structures like micelles and vesicles. Here, we propose an original way to self-assemble amphiphilic block copolymers into a supported bilayer membrane for defined coating of nanoparticles. The heart of the method rests on a change of the amphiphilicity of the copolymer that can be turned off and on by varying the polarity of the solvent. In this condition, the assembly process can take advantage of specific molecular interactions in both organic solvent and water. The higher gene silencing activity of the copolymer-modified complexes over the complexes alone shows the potential of this new type of nanoconstructs for biological applications, especially for the delivery of therapeutic biomolecules.

Background:In angiosperms, the endosperm plays a crucial placenta-like role in that not only is it necessary for nurturing the embryo, but also regulating embryogenesis through complicated genetic and epigenetic interactions with other seed compartments and is the primary tissue in which genomic imprinting occurs.Results:We observed a gradual increase of paternal siRNA expression in the early stages of kernels and an expected 2:1 maternal to paternal ratio in 7-DAP endosperm via sequencing of small interfering RNA (siRNA) transcriptomes in developing kernels (0, 3 and 5 days after pollination (DAP)) and endosperms (7, 10 and 15 DAP) from the maize B73 and Mo17 reciprocal crosses. Additionally, 460 imprinted siRNA loci were identified in the endosperm, with the majority (456/460, 99.1%) being maternally expressed at 10 DAP. Moreover, 13 out of 29 imprinted genes harbored imprinted siRNA loci within their 2-kb flanking regions, a significant higher frequency than expected based on simulation analysis. Additionally, gene ontology terms of "response to auxin stimulus", "response to brassinosteroid stimulus" and "regulation of gene expression" were enriched with genes harboring 10-DAP specific siRNAs, whereas those of "nutrient reservoir activity", "protein localization to vacuole" and "secondary metabolite biosynthetic process" were enriched with genes harboring 15-DAP specific siRNAs.Conclusions:A subset of siRNAs subjected to imprinted expression pattern in maize developing endosperm, and they are likely correlated with certain imprinted gene expression. Additionally, siRNAs might influence nutrient uptake and allocation processes during maize endosperm development.

<p>The aryl hydrocarbon receptor (Ah-receptor) is a member of the bHLH-PAS protein family. The Ah-receptor is a ligand dependent transcription factor, which activates a wide range of genes, most notably the xenobiotica metabolising genes, CYP1A1 and CYP1A2. The biological function of the Ah-receptor is still unknown and an endogenous ligand has yet not been identified. A possible Ah-receptor ligand is 6-formylindolo[3,2-b]carbazole (FICZ). FICZ has a high affinity for the Ah-receptor and is rapidly metabolised by CYP1A1, CYP1A2 and aldehydeoxidase (AOX). To try to trap FICZ or other possible endogenous Ah-receptor ligands, the metabolising enzymes CYP1A1, CYP1A2 and AOX were blocked. This was achieved through chemical blockage of CYP1A1 and CYP1A2 by ellepticin and through silencing with siRNA directed against CYP1A1 and CYP1A2. Successful blockage would be seen as an increase in Ah-receptor dependent XRE-luciferase activity. Chemical blockage of AOX with tungstate did not affect FICZ-dependent XRE-luciferase activation which could indicate that HepG2 cells lack AOX. The chemical blockage of CYP1A1 and CYP1A2 with ellepticin modified the XRE-luciferase response, but did not completely block Ah-receptor activation. In addition it is possible that ellepticin is a ligand for the Ah-receptor. The blockage of CYP1A1 by siRNA was successful; a silencing of CYP1A1 mRNA by at least 50 percent was detected. However due to lack of time it was not tested if the blockage of CYP1A1 and CYP1A2 was sufficient to trap Ah-receptor ligands.</p>