<p>The subject of this thesis was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biocompatible polymers, in particular chitosan, suitable for non-viral gene therapy. At the onset of this thesis, studies had reported conflicting results on the efficiency of chitosan-based gene delivery systems. Therefore, structure-property relationships of chitosans as non-viral gene delivery systems <i>in vitro</i> and after lung administration <i>in vivo</i> were established for the first time.</p><p>Polymer-pDNA complexes (polyplexes) based on conventional high molecular weight chitosans transfected cells <i>in vitro</i> and after lung administration <i>in vivo</i>. The chitosan polyplexes were, in contrast to polyplexes formed with the "golden standard" polymer polyethylenimine (PEI), essentially non-toxic at escalating doses. However, a very high physical stability of the chitosan-pDNA complexes together with a low buffering capacity of chitosan at the slightly acidic endo/lysosomal pH resulted in a slow onset of the gene expression and also in a lower efficiency of gene expression compared to PEI polyplexes. A slow and biodegradation-dependent release of pDNA from the chitosan polyplexes was concluded to be a rate limiting step for the efficiency of high molecular weight chitosan. The optimized polyplexes of high molecular weight chitosan (around 1,000 monomer units) showed aggregated shapes and gave increased viscosity at concentrations used for <i>in vivo</i> gene delivery. To improve the pharmaceutical properties and the delivery properties of chitosan polyplexes, low molecular weight chitosans were studied. Chitosans of around 18 monomer units retained the ability to protect pDNA against DNase degradation, but were more easily dissociated than those of higher molecular weight and had an efficiency comparable to that of PEI <i>in vitro</i> and <i>in vivo</i>. The pharmaceutical advantages of low molecular weight chitosan polyplexes compared to higher molecular weights are that there is less aggregation and no increased viscosity at the concentrations used for <i>in vivo</i> gene delivery. Coupling of an oligosaccharide targeting ligand to chitosan further increased the efficiency of some oligomer polyplexes. In conclusion, biocompatible chitosan is an interesting alternative to other non-viral gene delivery systems such as PEI.</p>

In the past decades, tumors have progressively been perceived as highly integrated systems in which the genetically unstable tumor cells and the genetically stable host cells cooperate to promote tumor growth. This view suggests that, beside tumor cells (that are targeted by conventional anticancer treatments such as radio- and chemotherapy), host cells within the tumor microenvironment can be targeted by antitumor therapy. Such alternative strategies are strongly supported by the need to overcome several limitations of the conventional therapies targeting tumor cells, such as collateral toxicity due to lack of tumor selectivity, limited tumor accessibility, and the selection of treatment-resistant variants. By contrast to tumor cells, the genetically stable host cells should not develop resistance to treatments. In this context, the observation that tumor growth is fundamentally dependent on the onset of a private tumor neovasculature (tumor angiogenesis) has revolutionized the field of cancer research. Several treatments have been developed aimed to prevent tumor angiogenesis (anti-angiogenic strategies) or to erase the existent tumor vasculature (anti-vascular approaches) supporting the survival and growth of thousands of tumor cells. However, although such therapies achieved cancer cure in animal models, they turned out to be rather inefficient when tested in patients. This can be attributed to differences in the angiogenic status between fast-growing animal tumors and slow-growing human tumors at the time of clinical detection. Another reading of the above-mentioned observations is that anticancer treatments could benefit from interventions aimed at increasing their efficiency. For instance, radiotherapy could benefit from tumor reoxygenation while a decrease in tumor interstitial pressure could facilitate tumor accessibility to circulating agents. In this context, the mature vasculature is an attractive target since it controls tumor blood supply and is highly accessible for therapy. Therefore, strategies aimed at exploiting its functional reactivity by inducing vasorelaxation have the potential to improve tumor perfusion/drug delivery and oxygenation/radiosensitivity. To be exploited in the clinics, such pro-vascular approaches have to fulfill essential requirements. First, they need to achieve high selectivity for tumor vessels. It should prevent systemic toxicity as well as the stealing of the blood flow towards the peripheral vasculature. Second, vasodilation has to be transient, so that the tumor should not take advantage of an increased energetic supply to grow faster. Third, the therapeutic effects have to be achieved in several tumor types and in different host strains to gain a wide therapeutic range of applicability. Finally, vasomodulation has to be achieved with interventions relevant to the clinical situation, ensuring direct therapeutic significance. However, the therapeutic exploitation of agents modulating tumor perfusion was generally hampered by confounding effects on the systemic blood pressure. In our studies, we have documented that this lack of tumor selectivity can be overcome by identifying vasomodulatory pathways that are selectively altered within the tumor microenvironment, allowing selective vasomodulatory interventions. According to the criteria detailed above, to identify a differential tumor vascular reactivity, we had to work with mice models of mature tumor vascularization. We reasoned that preexisting host arterioles in mice, if coopted, should retain architectural characteristics (such as a muscular coat) necessary for functional reactivity but also be influenced by the tumor microenvironment at both molecular and functional levels. To gain in reproducibility, this model was developed by injecting syngeneic tumor cells in the vicinity of the saphenous arteriole (i.e., a collateral branch of the femoral artery) in the rear leg of mice. With tumor growth, this arteriole was progressively included in the tumor cortex (coopted), with side branches running deeply into tumors. This model was developed using several tumors and mice strains. It provides the unique advantage to allow the easy identification and isolation of mature tumor vessels from fast-growing animal tumors. To evaluate differential vasoreactivity in those tumor-coopted vessels, we adapted pressure myography, a device initially dedicated to the study of the reactivity of coronary arterioles (see annex 1). In our hands, the unprecedented application of pressure myography to the study of small tumor vessels proved to be very efficient. Indeed, this technique not only served us to confirm that arterioles remain sensitive to vasomodulation under tumor cooption, but also allowed us to evidence two major adaptations of host vessels to the tumor microenvironment: the acquisition of an ET-1-mediated basal constrictive tone and a defect in the vasodilatory NO pathway. Furthermore, we used pressure myography to identify and characterize vasomodulatory strategies exploiting these differential reactivities. More particularly, we showed that both BQ123 (an ETA inhibitor) and ionizing radiations (that restored a functional NO pathway) promoted the vasodilation of the tumor-coopted vessels. In vivo, we verified that these strategies fulfilled the essential requirements of pro-vascular approaches: tumor selectivity, transient effects, broad range of applicability, and therapeutic significance in clinically relevant regimens. This latter study led us to further explore the effects of radiotherapy on the status of the tumor vasculature. Hence, we showed that fractionated radiotherapy induced tumor angiogenesis, thereby providing a rationale to combine radiotherapy to anti-angiogenic therapies.

The subject of this thesis was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biocompatible polymers, in particular chitosan, suitable for non-viral gene therapy. At the onset of this thesis, studies had reported conflicting results on the efficiency of chitosan-based gene delivery systems. Therefore, structure-property relationships of chitosans as non-viral gene delivery systems in vitro and after lung administration in vivo were established for the first time. Polymer-pDNA complexes (polyplexes) based on conventional high molecular weight chitosans transfected cells in vitro and after lung administration in vivo. The chitosan polyplexes were, in contrast to polyplexes formed with the "golden standard" polymer polyethylenimine (PEI), essentially non-toxic at escalating doses. However, a very high physical stability of the chitosan-pDNA complexes together with a low buffering capacity of chitosan at the slightly acidic endo/lysosomal pH resulted in a slow onset of the gene expression and also in a lower efficiency of gene expression compared to PEI polyplexes. A slow and biodegradation-dependent release of pDNA from the chitosan polyplexes was concluded to be a rate limiting step for the efficiency of high molecular weight chitosan. The optimized polyplexes of high molecular weight chitosan (around 1,000 monomer units) showed aggregated shapes and gave increased viscosity at concentrations used for in vivo gene delivery. To improve the pharmaceutical properties and the delivery properties of chitosan polyplexes, low molecular weight chitosans were studied. Chitosans of around 18 monomer units retained the ability to protect pDNA against DNase degradation, but were more easily dissociated than those of higher molecular weight and had an efficiency comparable to that of PEI in vitro and in vivo. The pharmaceutical advantages of low molecular weight chitosan polyplexes compared to higher molecular weights are that there is less aggregation and no increased viscosity at the concentrations used for in vivo gene delivery. Coupling of an oligosaccharide targeting ligand to chitosan further increased the efficiency of some oligomer polyplexes. In conclusion, biocompatible chitosan is an interesting alternative to other non-viral gene delivery systems such as PEI.

Proopiomelanocortin (POMC) is a 241 amino acids precursor protein, which encodes various neuropeptides including corticotropin (ACTH), a-melanocyte-stimulating hormone (a-MSH), and b-endorphin (b-EP). POMC plays an important role in stress response, metabolism, energy homeostasis and anti-inflammation. Recent studies demonstrated that systemic POMC gene delivery potently suppresses the tumor growth and metastasis of B16-F10 melanoma in vitro and in vivo via inhibition of NF-£eB/COX2 pathway. However, systemic POMC expression also led to elevated urine excretion and water intake in mice. This was attributed to enhanced steroidgenesis as evidence by elevated plasma corticosteroids levels in animals and increased cortisol production in adrenal H295R cells after POMC gene delivery. Since corticosteroids are also potent anti-inflammatory agents, it remains unclear whether the ACTH-mediated cortisol synthesis also contributed to the POMC-induced tumor suppression. To address this issue, we generated a series of adenovirus vectors encoding POMC genes with mutation or deletion in ACTH domain including ACTH (K15A/R17A). Unlike the wild type POMC, gene delivery of ACTH (K15A/R17A) resulted in significantly lower cortisol production, CYP11B1 mRNA level, and glucocorticoid responsive element (GRE)-driven luciferase activities in H295R cells. ACTH (K15A/R17A) gene delivery did not affect the urination and water intake in mice. Above all, ACTH (K15A/R17A) gene delivery remained capable of inhibiting the colonies formation and invasiveness of B16-F10 melanoma cells. In summary, steroidgenesis is not essential to POMC-mediated melanoma suppression. In addition, ACTH (K15A/R17A) gene delivery may provide a better alternative for melanoma control.

No description available.

abstract: Gold nanoparticles as potential diagnostic, therapeutic and sensing systems have a long history of use in medicine, and have expanded to a variety of applications. Gold nanoparticles are attractive in biological applications due to their unique optical, chemical and biological properties. Particularly, gold nanorods (GNRs) are increasingly used due to superior optical property in the near infrared (NIR) window. Light absorbed by the nanorod can be dissipated as heat efficiently or re-emitted by the particle. However, the limitations for clinical translation of gold nanorods include low yields, poor stability, depth-restricted imaging, and resistance of cancer cells to hyperthermia, are severe. A novel high-throughput synthesis method was employed to significantly increase in yields of solid and porous gold nanorods/wires. Stable functional nanoassemblies and nanomaterials were generated by interfacing gold nanorods with a variety of polymeric and polypeptide-based coatings, resulting in unique properties of polymer-gold nanorod assemblies and composites. Here the use of these modified gold nanorods in a variety of applications including optical sensors, cancer therapeutics, and nanobiomaterials were described. / Dissertation/Thesis / Ph.D. Chemical Engineering 2012

Um problema recorrente no desenvolvimento de vacinas de DNA e terapia gênica utilizando vetores não virais é a baixa eficiência de transfecção gênica. Isso ocorre devido às diversas barreiras físicas, enzimáticas e difusionais que o DNA precisa superar para chegar ao núcleo das células. Neste trabalho tem-se por objetivo o desenvolvimento de novos vetores não virais de entrega gênica, formados por DNA plasmidial (pDNA), proteínas (protamina ou T-Rp3) e nanopartículas de ouro (NPAu) na forma de complexos ternários. Para tal, NPAu\'s foram sintetizadas por redução com citrato de sódio, apresentando diâmetros entre 20,3 e 57,3 nm e potencial zeta entre -69,0 e +43,3 mV, dependendo das condições de síntese, a saber, das quantidades de citrato de sódio adicionadas e da ordem de adição dos reagentes. Em seguida, vetores compostos por pDNA-protamina/T-Rp3-NPAu foram formados, transfectados em células HeLa cultivadas in vitro, e a atividade da enzima repórter luciferase foi medida. Deste modo, a partir de variações em proporção mássica e tamanho de nanopartículas, foi possível obter complexos utilizando protamina e ouro com uma eficiência de transfecção 33 vezes melhor do que transfecções utilizando apenas protamina. Por outro lado, complexos contendo T-Rp3 e ouro se mostraram ainda mais eficazes na entrega, apresentando níveis de transfecção próximos ao do reagente comercial Lipofectamina. Ensaios de transfecção utilizando a droga nocodazol indicaram a importância dos microtúbulos no mecanismo de entrega gênica, e ensaios com a droga cloroquina evidenciaram que as nanopartículas de ouro atuam de maneira diferenciada no escape endossomal dos vetores não virais utilizados. Visando relacionar características físico-químicas com a eficiência de transfecção, alguns destes complexos foram caracterizados por espalhamento dinâmico de luz, em que complexos com protamina apresentaram tamanhos entre 116 e 363 nm e complexos com T-Rp3 apresentaram entre 135 e 307 nm e potenciais zeta entre +7,3 e +22,5 mV e +10,6 e +27,2 mV, respectivamente, dependendo das características das NPAu\'s. / A recurrent problem in the development of DNA vaccines and gene therapy using non-viral vectors is the low efficiency of transfection. That is due to the many physical, enzymatic and diffusional barriers that DNA must overcome to reach the cell nucleus. This work aims to develop novel non-viral vectors based on plasmid DNA (pDNA), proteins (protamine or recombinant T-Rp3) and gold nanoparticles (AuNP) as ternary complexes. For such, AuNP\'s were first synthesized via sodium citrate reduction, with diameters varying from 20,3 to 57,3 nm and zeta potentials between -69,0 and +43,3 mV, depending on synthesis conditions, changing the quantities of sodium citrate added and the order of addition of reagents. Vectors formed by pDNA-protamine/T-Rp3-AuNP were then formed, transfected and luciferase activity was measured. Thus, from variations on mass ratios and gold nanoparticle sizes, it was possible to obtain complexes with protamine and gold with a transfection efficiency 33 times higher than analog complexes using only protamine. Also, complexes containing T-Rp3 and gold showed an even higher delivery efficiency, with transfection efficiency close to Lipofectamine. Assays using nocodazole indicated the importance of microtubule in the gene delivery process and, whereas assays with chloroquine showed that gold nanoparticles act in a different way over endossomal escape of used non-viral vectors. Finally, some of these complexes were characterized with dynamic light scattering. Complexes with protamine were within the size ragne of 116 to 363 nm and complexes with T-Rp3 were within the size range of 135 to 307 nm. The zeta potential varied from +7,3 to +22,5 mV and from +10,6 to +27,2 mV, respectively, depending on the gold nanoparticles used.

Um dos principais limitantes do desenvolvimento de protocolos eficientes de terapia gênica e vacinação com DNA provém da baixa eficiência de transferência gênica por parte dos vetores não-virais. Isso surge, principalmente, pela dificuldade de transporte de DNA estrangeiro do exterior para o núcleo das células alvo, devido à presença de inúmeras barreiras. O principal objetivo do trabalho realizado foi o desenvolvimento e caracterização de novos vetores não virais multifuncionais, capazes de realizar eficientemente a entrega de material genético (DNA plasmidial, pDNA) ao núcleo de células de mamífero (HeLa). Para esse fim, complexos binários (CBs) foram formados combinando-se pDNA à protamina ou proteínas recombinantes (TRP3) anteriormente desenvolvidas por nosso grupo. Foi também estudado o encapsulamento destes CBs em lipossomas catiônicos, formados pelos lipídios EPC:DOPE:DOTAP, gerando então complexos pseudo-ternários (CPTs). Os estudos com a protamina revelaram que os CPTs formados apresentavam tamanhos relativamente pequenos (< 123 nm) e com valores de potencial zeta, variando de +22,8 mV a +36,3 mV, nas várias relações mássicas (pDNA:protamina) estudadas (1:0,5; 1:0,7, 1:0,9 e 1:1). Os ensaios de transfecção mostraram que o CPT na relação 1:0,5 (CB) obteve o melhor nível de transfecção das células (17,1%) com um nível de viabilidade celular de 73,2%. Os ensaios utilizando a TRP3 mostraram que os CPTs formados adquiriram tamanhos pequenos (< 134 nm) e valores de potencial zeta entre +36,8 mV e +11,9 mV dependendo da relação mássica do CB (1:1, 1:30 e 1:60). Os ensaios de transfecção mostram que os CPTs formados com a TRP3 aumentaram o nível de transfecção em todas as relações de pDNA-TRP3 usadas (1:1; 1:30 e 1:60). Vale ressaltar que nas relações mássicas 1:30 e 1:60 houve um aumento significativo na transfecção (25,2% e 24,5%, respectivamente). Os ensaios de citotoxicidade mostram que os CBs não afetaram a viabilidade célular, entretanto quando combinada ao lipossoma foi observado aumento da citotoxicidade. Finalmente, os resultados indicam que a TRP3 associada ao lipossoma (CPTs) aumenta a eficiência de entrega de pDNA sugerindo um efeito sinérgico entre essas duas moléculas na superação das várias barreiras físicas, químicas e difusionais encontradas na célula. / One of the major challenges on the development of efficient protocols for gene therapy and DNA vaccination is the low efficiency of gene transfer by non viral vectors. This is mainly attributed to the fact that, during the traffic to target cells nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The objective of this work was the development and characterization of new multifunctional non-viral vectors, based on lipids and proteins, able to delivery efficiently the foreign pDNA (plasmid DNA) to the nucleus of mammalian cells. A model pDNA containing the reporter gene GFP was complexed to protamine or the recombinant protein (TRP3), forming binary complexes (BC). In addition, we studied the ability of the cationic liposomes (EPC:DOPE:DOTAP) to encapsulate this binary complexes to form pseudo-ternary complexes (PTCs). The studies of size (DLS) and zeta potential revealed that both proteins were able to condense pDNA to form small complexes (BCS and PTCs) (~100 nm) and positively charged (+11,9 mV a +36,8 mV), both interesting characteristics for transfections. However, the CPTs formed by TRP3 was that showed the highest transfections level (25,3%). The cytotoxicity assays indicated the BCs had a low effect on the cell viability. On the other hand, the biggest effect on cell death was found when PTCs were used. The results indicated the TRP3 associated to liposomes (PTCs) increased the delivery efficiency due to differences in the intracellular trafficking, suggesting a synergic effect between these different molecules in the vector in order to overcome the barriers found inside the cell.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-04-08T14:09:56Z No. of bitstreams: 1 michelemunkpereira.pdf: 6330020 bytes, checksum: 446922b21f5355bdcbfc26ac618f8e7c (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-04-24T03:40:43Z (GMT) No. of bitstreams: 1 michelemunkpereira.pdf: 6330020 bytes, checksum: 446922b21f5355bdcbfc26ac618f8e7c (MD5) / Made available in DSpace on 2016-04-24T03:40:43Z (GMT). No. of bitstreams: 1 michelemunkpereira.pdf: 6330020 bytes, checksum: 446922b21f5355bdcbfc26ac618f8e7c (MD5) Previous issue date: 2013-12-17 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os nanotubos de carbono multicamadas (MWCNT) e as nanofibras de celulose (NFCs) são interessantes nanomateriais (NMs) que possuem grande potencial de aplicação em áreas como tratamento de água, reforço de materiais, engenharia tecidual e entrega de moléculas terapêuticas. Em especial, os MWCNT são promissores vetores de DNA em células e embriões de mamíferos. Porém, o desenvolvimento desta área está relacionado à padronização de sistemas para avaliar o potencial impacto dos NMs na saúde humana e ambiental. O objetivo geral deste estudo foi avaliar a toxicidade de MWCNT e NFC em diferentes modelos biológicos (fibroblastos e embriões bovinos; microalgas) e o potencial de carreamento gênico de MWCNT-COOH em fibroblastos e embriões bovinos. No experimento 1, foi avaliada a viabilidade e a morfologia dos fibroblastos cultivados in vitro expostos às NFCs e aos MWCNT-COOHs. Os resultados deste experimento revelaram que em baixas concentrações (0,02-100μg ml-1) as NFC não foram citotóxicas (P = 0,06). Porém, concentrações acima de 200 μg ml-1 NFC diminuíram a viabilidade celular e causaram mudanças na morfologia das células. Para os MWCNT-COOH, a exposição dos fibroblastos a 100μg ml-1 reduziram a viabilidade (P= 0,001) e alteram a morfologia celular. No experimento 2, foram analisados os efeitos desses NMs no desenvolvimento, expressão gênica e apoptose in situ de embriões bovinos produzidos in vitro. Neste ensaio, os NMs não influenciaram o desenvolvimento (P=0,24) e o índice de apoptose embrionária (P=0,82). Contudo, os MWCNT-COOH alteraram a expressão de genes (P=0,02) relacionados à totipotência, diferenciação e estresse celular de forma mais acentuada quando comparada às NFCs. No experimento 3, o potencial ecotóxico das NFCs e dos MWCNTs foi estudado em microalgas Chlorella vulgaris e Klebsormidium flaccidum mediante análise de Potencial Zeta, viabilidade celular, atividade fotossintética e de enzimas antioxidativas, quantificação dos níveis de ATP e microscopia eletrônica. Os resultados revelaram que os MWCNTs e as NFCs afetaram a viabilidade (P<0,001), a fotossíntese (P<0,05), a atividade de enzimas antioxidativas (P<0,05), os níveis de ATP (P<0,05) e a morfologia celular das microalgas em concentrações, tempos e sistemas de cultura específicos. No experimento 4, os fibroblastos e embriões bovinos foram transfectados com MWCNT-COOH complexados ao plasmídeo contendo o gene que codifica a proteína verde fluorescente (pGFP). As análises de microscopia de fluorescência detectaram que o gene GFP foi expresso nas células e nos embriões no estágio inicial de desenvolvimento (2 a 8 células). Entretanto, a expressão de GFP não foi observada no estágio de blastocisto. A análise por PCR confirmou a presença do gene GFP nos fibroblastos (3,30% GFP+) e embriões de 2-8–células (46,67% GFP+). Em conclusão, nas condições testadas, a exposição de fibroblastos a baixas concentrações de MWCNT-COOH ou NFC não causaram impacto na viabilidade e morfologia celular. Entretanto, os MWCNTs e as NFCs foram citotóxicas para as microalgas estudadas. O uso de MWCNT-COOH como vetor de DNA em embriões bovinos mostrou-se promissora, o que abre possibilidades de geração de animais transgênicos por este método. / Multi-walled carbon nanotubes (MWCNTs) and cotton cellulose nanofibers (CNFs) are interesting nanomaterials (NMs) which possess great potential for applications in various fields such as in water treatment, reinforcement materials, tissue engineering and therapeutic molecule delivery. In particular, MWCNT have emerged as a new method for gene delivery and they can be an alternative for cell and embryos transfection. However, while engineered NMs provide great benefits, we know very little about the potential effects on human health and the environment. Thus, the objectives of this study were to evaluate the potential toxicity of MWCNT and CNF in various biological model organisms (bovine fibroblast and embryo, and green microalgaes) and whether MWCNT is able to deliver exogenous DNA molecules into bovine fibroblast and embryos. In experiment 1, we evaluated in vitro the effects of NMs on bovine fibroblast viability and morphology. The results showed that low concentrations of CNF (0.02-100μg ml-1) did not cause viability loss (P>0.05) or change in cell morphology. However, at concentrations above 200μg ml-1, NFC significantly decreased cell viability (P<0.05) and changes in cell shape. Fibroblasts exposed at concentrations above 100μg ml-1 MWCNT-COOH exhibited a reduced cell viability (P<0.05) and their cell morphology was altered. In experiment 2, we examined gene expression, apoptosis response and developmental rates of in vitro produced bovine embryos that were exposed to NMs. There was no difference (P > 0.05) in the hatching, degeneration and apoptosis rate among the control, MWCNT-COOH or cotton CNF- exposed embryos. In contrast, in embryos exposed at 0.2 μg ml-1 MWCNT-COOH showed relatively higher levels (P<0.05) of the genes associated with totipotency, differentiation and response to stress. In experiment 3, we analyzed the cytotoxic response of C. vulgaris and K. flaccidum cells (green microalgae) to NMs by investigating the zeta potential, trypan blue exclusion assay, photosynthetic activity, superoxide dismutase activity, quantification of ATP levels and microscopic investigations. NMs decreased viability, photosynthetic activity and ATP levels of microalgaes cells, depending on concentration and time. Addition of NMs further induced an increase of superoxide dismutase activity and ultrastructural damage cell. In experiment 4, we have used plasmid DNA of green fluorescent protein (pGFP) in combination with MWCNT-COOH to transfect bovine fibroblast and embryos. Detection of GFP accumulation by fluorescence microscopy examination revealed that this gene was expressed in the fibroblast and the embryo (2 to 8-cell stage). However, the expression GFP was not observed in blastocyst stage. The PCR result confirmed the presence of the pGFP gene in the transfected cells (3.30% GFP+) and embryos of 2-8–cell stage (46.67% GFP+). In conclusion, under the conditions tested, the exposures to MWCNTs or CNFs in low concentrations are not toxic to bovine fibroblast cells. However, these NMs have toxic effects in green microalgaes. Especially, this work showed that MWCNT-COOH-transfection of embryo could be a simple and suitable method to introduce foreign genes in embryos and perhaps could be also useful to generate transgenic animals.

Nous avons démontré que structurellement flexibles poly(amidoamine) (PAMAM) dendrimères sont efficaces système de livraison de siRNA in vitro et in vivo récemment. Nous voulons mener une enquête plus approfondie sur la livraison de siRNA ciblés en utilisant des dendrimères conjugués avec des ligands spécifiques ou d'anticorps, qui peuvent reconnaître les récepteurs correspondants ou des protéines exprimées à la surface des cellules. De cette façon, le siRNA peuvent être livrés spécifiquement aux cellules d'intérêt, conduisant à une délivrance ciblée, ce qui peut améliorer l'efficacité livraison et de réduire la toxicité en évitant les interactions non spécifiques et à des doses plus faibles. À cette fin, nous avons développé des dendrimères portant une chaîne PEG long et un dendron individu polyvalent. La chaîne PEG est de libérer l'encombrement stérique entre dendrimère et ligand / anticorps, tandis que le dendron multivalent fournit une plate-forme d'une conjugaison contrôlable de ligands. Par ailleurs, nous avons également conçu et synthétisé une autre dendrimères PEGylées portant un groupe thiol libre pour la préparation des anticorps / dendrimère conjugués. / We have demonstrated that structurally flexible poly(amido)amine (PAMAM) dendrimers are efficient siRNA delivery system in vitro and in vivo recently. We would like to undertake further investigation on targeted siRNA delivery using dendrimers conjugated with specific ligands or antibodies, which can recognize the corresponding receptors or proteins expressed on the cell surface. In this way, siRNA can be delivered specifically to the cells of interest, leading to targeted delivery, which can further improve the delivery efficiency and reduce the toxicity by avoiding non-specific interactions and at lower doses. To this end, we have developed dendrimers bearing a long PEG chain and an individual multivalent dendron. The PEG chain is to release the steric congestion between dendrimer and ligand/antibody, whereas the multivalent dendron provides a platform of a controllable conjugation for ligands. Besides, we also designed and synthesized another PEGylated dendrimers bearing a free thiol group for the preparation of antibody/dendrimer conjugates.