<p>The success of gene medicine ultimately depends on the efficient intracellular delivery and sustained expression of nucleic acid therapeutics, yet nonviral gene delivery performed with cationic polymer carriers has been chronically hindered by the slow release of nucleic acid payloads at their targets, as well as the transient nature of exogenous transgene expression. Polymer-nucleic acid nanocomplexes made with passive gene carriers using traditional bulk methods have proven inadequate for most translational applications. The objective of this work is to improve nonviral gene delivery through the selection, formulation, and application of improved nanoparticles. </p><p> After screening a number of number of cationic polymer delivery systems ranging from natural to synthetic, high molecular weight to low, binary and ternary, we identified a bioreducible linear poly(amido amine) able to give sustained, robust expression of both DNA and RNA through serial dosing. We next turned our attention to the process of nanocomplex assembly. Traditional assembly via bulk mixing is poorly controlled, and the poor quality of these nanocomplexes is a significant impediment to both the establishment of robust structure-function relationships and the advancement of nonviral gene delivery. So, we developed an emulsion-based microfluidic nanomanufacturing platform to better control the self-assembly process, and thus the physical properties of nanocomplexes. Confined mixing within picoliter droplets generates self-assembled nanocomplexes that are more uniform and more effective. This microfluidic nanomanufacturing approach possesses broad utility in the production of polymer-nucleic acid nanocomplexes; we demonstrated that its benefits extend to multiple gene carriers, a range of nucleic acid payloads, and translationally relevant cell types. Then, we applied the improved nanomanufactured particles to begin to address an unmet clinical need, namely the lack of a safe and ethical source of cells to treat neurodegenerative diseases. Nonviral cellular reprogramming strategies eliminate the integration of viral DNA sequences and represent a potentially safer alternative to viral transdifferentiation methods to generate therapeutic cells. Using nanomanufactured polymer-nucleic acid nanocomplexes, we improved the efficiency of the nonviral cellular reprogramming of fibroblasts directly to functional induced neuronal cells. </p><p> Nonviral gene therapy will continue to demand more sophisticated delivery systems to continue to progress. Microfluidic nanomanufacturing represents a reproducible and scalable platform to synthesize more uniform and effective nanocomplexes that not only improves their functional performance, but may also help establish clearer structure-function relationships that will inform future gene carrier design. Complementing the innovative chemical and biological approaches to create multifunctional nanoparticles, this study indicates that microfluidic nanomanufacturing can serve as a parallel physical strategy to both optimize the properties of polymer-nucleic acid nanocomplexes and improve their performance in applications with important clinical implications.</p> / Dissertation

Les nanotubes de carbone (CNTs) sont une nouvelle forme allotropique du carbone, décrits pour la première fois à l’échelle atomique en 1991 par Iijima. Dans ce travail de thèse, les MWCNTs portant des charges cationiques ont été fonctionnalisés, avec pour objectif d’étudier leur aptitude à complexer des acides nucléiques pour obtenir un système de délivrance génétique. Initialement, nous avons fonctionnalisé les MWCNTs avec des dendrons portant à leur extrémité des groupes ammonium ou guanidinium et leur aptitude à complexer des acides nucléiques a été évaluée par électrophorèse en gel d’agarose. En outre, nous avons fonctionnalisé et caractérisé les MWCNTs avec un peptide ciblant les mitochondries et leur habilité à se localiser à l’intérieur de ces dernières a été étudié par différentes techniques microscopiques. Ensuite, nous avons doublementfonctionnalisé les CNTs avec un dendron de deuxième génération et avec le peptide de ciblage. La capacité de ce conjugué à complexer l’ADN a finalement été confirmée par électrophorèse en gel d’agarose. / Carbon nanotubes (CNTs) are a new allotropic form of carbon described at the atomic level in 1991 by Iijima. During my thesis, carbon nanotubes bearing cationic moieties have been functionalized, in order to study their ability to complex the genetic material to obtain a gene delivery system. Initially we have functionalized MWCNTs with dendrons bearing at their termini ammonium or guanidinium groups. Their ability to complex the genetic material has been evaluated through agarose gelelectrophoresis. Moreover, we have functionalized and characterized MWCNTs with a targeting peptide for mitochondria and their ability to localize inside this organelle was studied by different microscopic techniques. Then, we have double-functionalized MWCNTs with a dendron of second generation and with the targeting peptide and the ability of this conjugate to complex DNA was confirmed by agarose gel electrophoresis.

abstract: Gold nanoparticles have emerged as promising nanomaterials for biosensing, imaging, photothermal treatment and therapeutic delivery for several diseases, including cancer. We have generated poly(amino ether)-functionalized gold nanorods (PAE-GNRs) using a layer-by-layer deposition approach. Sub-toxic concentrations of PAE-GNRs were employed to deliver plasmid DNA to prostate cancer cells in vitro. PAE-GNRs generated using 1,4C-1,4Bis, a cationic polymer from our laboratory demonstrated significantly higher transgene expression and exhibited lower cytotoxicities when compared to similar assemblies generated using 25 kDa poly(ethylene imine) (PEI25k-GNRs), a current standard for polymer-mediated gene delivery. Additionally, sub-toxic concentrations of 1,4C-1,4Bis-GNR nanoassemblies were employed to deliver expression vectors that express shRNA ('shRNA plasmid') against firefly luciferase gene in order to knock down expression of the protein constitutively expressed in prostate cancer cells. The roles of poly(amino ether) chemistry and zeta-potential in determining transgene expression efficacies of PAE-GNR assemblies were investigated. The theranostic potential of 1,4C-1,4Bis-GNR nanoassemblies was demonstrated using live cell two-photon induced luminescence bioimaging. The PAE class of polymers was also investigated for the one pot synthesis of both gold and silver nanoparticles using a small library poly(amino ethers) derived from linear-like polyamines. Efficient nanoparticle synthesis dependent on concentration of polymers as well as polymer chemical composition is demonstrated. Additionally, the application of poly(amino ether)-gold nanoparticles for transgene delivery is demonstrated in 22Rv1 and MB49 cancer cell lines. Base polymer, 1,4C-1,4Bis and 1,4C-1,4Bis templated and modified gold nanoparticles were compared for transgene delivery efficacies. Differences in morphology and physiochemical properties were investigated as they relate to differences in transgene delivery efficacy. There were found to be minimal differences suggestion that 1,4C-1,4Bis efficacy is not lost following use for nanoparticle modification. These results indicate that poly(amino ether)-gold nanoassemblies are a promising theranostic platform for delivery of therapeutic payloads capable of simultaneous gene silencing and bioimaging. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2014

L'oxyde de graphène (GO) a attiré un intérêt croissant comme vecteur potentiel pour la délivrance de gènes, en particulier pour l’inhibition de gènes spécifiques. Le but principal de ce travail est le développement de nouvelles plateformes complexant de petits ARN interférents (siRNA) et la rationalisation des interactions supramoléculaires entre la surface du GO et l'ARN double brin. L'étude s'est concentrée d'abord sur la synthèse de GO avec divers groupes oxygénés, puis sur la fonctionnalisation covalente du GO avec des amines et des polymères. De plus, j'ai étudié les facteurs qui pourraient affecter la structure double hélice du siRNA. Enfin, la question que je me suis posé: « l’oxyde de graphène peut-il devenir une plateforme appropriée pour la complexation d’acides nucléiques ?» a été résolue à l’aide d’expériences biologiques prouvant la capacité du GO à délivrer du siRNA dans les cellules. / Graphene oxide (GO) has attracted increasing interest as a prominent potential vector in gene delivery and in particular in gene silencing. The main goal of this work is to develop novel platforms to complex small interfering RNA (siRNA) molecules and to rationalize the supramolecular interactions between GO surface and the double strand RNA. The study focused first on the synthesis of GO with various oxygenated groups, subsequently chemically covalently modified with amines and polymers. Moreover, I investigated on the factors that could affect the double helix siRNA structure. Finally, the question of the thesis, « Can graphene oxide be a suitable platform for complexation of nucleic acids? » could be answered from the biological tests proving the ability of graphene derivatives as a carrier of siRNA into the cells.

Electric field mediated gene delivery modalities have preferable safety profiles with the ability to rapidly transfect cells in vitro and in vivo with high efficiency. However, the current state of the art has relied on trial and error studies that target the average cell within a population present in treated tissue to derive electric pulse parameters. This results in fixed gene electrotransfer (GET) parameters that are not universally optimum. Slow progress towards the validation of a mechanism that explains this phenomena has also hindered its advancement in the clinic. To date, GET methods utilizing feedback control as a means to optimize doses of electric field stimulation have not been investigated. However, with modern electric components the electric characteristics of tissue exposed to electric pulses can be measured in very short time scales allowing for a near instantaneous assessment of the effect these pulses have on cells and tissue. This information is ideal for use in optimizing GET parameters to ensure the conditions necessary for gene delivery can be created regardless of anisotropic tissue architecture and electrode geometry. Bioimpedance theory draws parallels between cell structures and circuit components in an attempt to use circuit theory to describe changes occurring at a cellular and tissue level. In short, a reduction in tissue impedance indicates a reduction to the opposition of current flow in a volume conductor indicating new pathways for current. It has been purported these new pathways exist in the cell membrane and indicate a degree of membrane permeability/destabilization that either indicates or facilitates the uptake of exogenous molecules, such as nucleic acids or plasmid DNA. This study evaluated the use of relative impedance changes from 10 Hz – 10 kHz that occur in tissue before and after GET to indicate relative increase in tissue and membrane permeability. An optimum reduction in impedance was then identified as an indicator of the degree of membrane permeability required to significantly enhance exogenous DNA uptake into cells. This study showed the use of impedance-based feedback control to optimize GET pulse number in real time to target 80% or 95% reduction in tissue impedance resulted in an 12 and 14 fold increase in transgene expression over controls and a 6 and 7 fold increase in transgene expression over fixed pulse open loop protocols.

siRNA therapy is an emerging technique that reduces protein expression in cells by degrading their mRNAs via the RNA interference pathway (RNAi). Diseases such as cancer often proliferate due to increased protein expression and siRNA therapy offers a new method of treatment for those diseases. Although siRNA therapy has shown success in vitro, it often fails in vivo due to instability in the blood stream. To overcome this limitation, delivery vehicles are necessary for successful transfection of siRNA into target cells and cationic polymers have been widely studied for this purpose. However, complexes between siRNA and delivery vehicles made from cationic polymers exhibit stability issues in the blood stream which results in toxicity and low transfection. This work hypothesizes that improvement of vehicle/siRNA complex stability will improve siRNA transfection efficiency. To test this, the contributions and outcomes of poly(ethylene glycol) [PEG] shell and hydrophobic core modification to a polyethylenimine (PEI) based tethered nanoassemblies (TNAs) were examined. Initially, hydrophobic modification of palmitate (PAL) to the core of the TNA yielded improved transfection efficiency due to an enhanced endosomal escape capability. However, this modification also reduced the TNA/siRNA complex stability. This indicated that the core hydrophobicity must be balanced in order increase stability while increasing transfection efficiency. Additionally, TNAs made from PEG and PEI did not cause transfection in our initial study. The PEG shell density was found to be too great and thereby reduced transfection efficiency. Reducing the PEG density by lowering PEG molecular weight, reducing attachment percentage, and removing small PEI impurities from the synthesis stock increased overall transfection efficiency and unimolecularity of the TNA complexes. This indicated that the shell composition of the TNA must be tuned in order to improve particle design. Further study of the hydrophobically modification to TNAs yielded unintended effects on the transfection efficiency evaluation assay. These particles exhibited an siRNA independent reduction in the reporter protein used to observe transfection, or a false positive effect, that was not previously observed. It was found that this false positive was influence mainly by the hydrophobic group rather than the cationic polymer backbone. Cellular stress was observed in cells dosed with the hydrophobically modified TNAs which lead to over ubiquitination and rapid degradation of the luciferase protein. This demonstrated that core components of TNAs could cause cellular stress and influence interaction outside of the TNA. Overall, this work demonstrates that hydrophobic core and PEG shell modification require balancing and consideration to improve properties of future cationic polymer based siRNA delivery vehicle design.

Gene therapy involves the use of nucleic acids, either DNA or RNA for the treatment, cure, or prevention of human diseases. Synthetic cationic polymers are promising as a tool for gene delivery because of their high level of design flexibility for biomaterial construction and are capable of binding and condensing DNA through electrostatic interactions. Our lab has developed a novel polymer (poly (polyethylene glycol-dodecanoate) (PEGD), a polyester of polyethylene glycol (PEG) and dodecanedioic acid (DDA). PEGD is a linear viscous polymer that self-assembles into a vesicle upon immersion in an aqueous solution. A copolymer of dodecanedioc acid and polyethylene glycol (PEG) was synthesized at a 1:1 ratio. Furmaric (FA) or itaconic acid (IA) was used to suppress DDA in the PEGD copolymer at an 80:20 ratio (DDA: furmaric/itaconic acid) to form the PEGDF/I variant. PEGDF/I are then modified through the Michael addition of Protamine Sulfate (PEGDF/I-PS) and Cys-Arg8 (PEGDF/I-CA) peptide to the carbon-carbon double bond on the polymer backbone to introduce a positive charge. The modified PEGDF/I polymers were capable of binding and condensing DNA. Transfection of HEK 293 cells with pTurboGFP plasmid using modified PEGDF/I polymers was successful but showed varied efficiency. The PEGDF/I-CA polymer had around 30% transfection efficiency and was shown to be non-cytotoxic.

Orientadores: Adriano Rodrigues Azzoni, Anete Pereira de Souza / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T06:15:51Z (GMT). No. of bitstreams: 1 Toledo_MarceloAugustoSzymanskide_D.pdf: 15660446 bytes, checksum: 8e64c5b4455cf458c2eb0d9b8e030e70 (MD5) Previous issue date: 2013 / Resumo: Apesar de seguros e simples de produzir, o uso de vetores não virais como o DNA plasmidial (DNAp) em estudos de terapia gênica e vacinação por DNA tem sido limitado pela baixa eficiência quando comparados aos vetores virais. Essa limitação provém principalmente da reduzida capacidade de superar as barreiras físicas, enzimáticas e difusionais encontradas durante o tráfego intracelular para o interior do núcleo das células alvo. Dentro deste contexto, o presente trabalho demonstra a utilização de cadeias leves modificadas de Dineína (Lc8 e Rp3) como vetores não-virais de entrega gênica. A escolha de cadeias leves de Dineína justifica-se pela possibilidade de utilizar o transporte retrógrado celular mediado por complexos motores de Dineína para facilitar o tráfego de material genético exógeno através do citoplasma em direção à periferia nuclear. Através da adição de pequenos domínios peptídicos, ricos em aminoácidos polares positivos (arginina e lisina), ao N-terminal de cadeias leves de Dineína foi possível conferir a estas proteínas a habilidade de interagir com material genético condensando-o em partículas. Ensaios de transfecção demonstraram que tais partículas apresentam elevada eficiência de entrega do material genético exógeno ao núcleo de células HeLa, eficiência esta superior àquela apresentada pelo peptídeo protamina, amplamente estudado como vetor não-viral de entrega gênica. A formação de complexos ternários utilizando-se DNA plasmidial, cadeias leves de Dineína modificadas e lipídios catiônicos apresentou eficiência de entrega superior àquelas apresentadas na ausência do lipídio. Adicionalmente, complexos de entrega formados apenas com DNA plasmidial e cadeias leves de Dineína modificadas apresentaram baixo efeito citotóxico em células HeLa, característica esta de grande relevância uma vez que a toxicidade dos vetores de entrega gênica atua como importante fator limitante em sua aplicação clínica. O mecanismo envolvido no processo de entrega gênica mediado por cadeias leves de Dineína modificadas também foi estudado, podendo ser observado que (1) a entrada dos complexos de entrega na célula é altamente dependente do processo de endocitose, (2) a eficiência de entrega observada depende da rede de microtúbulos e (3) parte significativa dos complexos de entrega é degradada na via de endossoma/lisossomo celular. Os vetores não-virais de entrega gênica descritos no presente estudo associam elevada eficiência de transfecção, baixa toxicidade celular e relativo baixo custo de produção, uma vez que as cadeias leves de Dineína recombinantes são produzidas em sistema heterólogo utilizando-se Escherichia coli. Ressalta-se ainda a possibilidade de adição de novos domínios peptídicos às cadeias leves de Dineína modificadas, agregando novas funções/capacidades que poderiam resultar em maior eficiência de entrega gênica através da otimização dos processos de internalização celular ou escape endossomal. A abordagem de se utilizar a via de transporte retrógrado celular para o desenvolvimento de vetores não-virais para entrega gênica é pouco explorada pela comunidade científica e o presente estudo apresenta-se entre os poucos da área, esperando assim contribuir para o desenvolvimento de vetores não-virais mais eficientes e seguros / Abstract: The use of non viral vectors such as plasmidial DNA (pDNA) in gene therapy and DNA vaccination protocols has been limited due to its low transfection efficiency when compared to viral vectors. This limitation occurs mainly due to the physical, enzymatic and diffusion barriers faced during the transport of the genetic material to the nucleus of target eukaryotic cells. Regarding this subject, the present work demonstrates the feasibility of using modified Dynein light chains (Lc8 and Rp3) as non viral vectors for gene delivery. The use of Dynein light chains relies on the possibility to exploit the Dynein based cellular retrograde transport in order to improve the exogenous genetic material transport across the citosol towards the nuclear periphery. By adding small peptide domains, based in positively charged aminoacids (arginine and lysine) to the N-terminal of Dynein light chains, the resulting recombinant proteins were able to interact and condense genetic material into delivery particles. Transfection assays demonstrated that these particles are highly efficient to delivery plasmidial DNA to nucleus of HeLa cells when compared to the transfection efficiency presented by protamine, a well characterized non viral vector peptide. Ternary complexes formed by modified Dynein light chains, pDNA and a cationic lipid showed even higher transfection efficiency. Additionally, the light chain based non viral delivery vectors presented low citotoxic effect to HeLa cells, a valuable feature as toxicity is regarded as one of the main concerns on delivery vectors development. The mechanism by which the modified Dynein light chain based vectors mediates gene delivery was also investigated and we could observe that (1) the internalization process deeply relies on endocytosis, (2) it depends on the microtubule network and (3) a significant fraction of the delivery complexes are trapped and degraded in the endocytic pathway. The non viral vectors developed in the present study combine high transfection efficiency, low toxicity and relative low production cost, as all modified proteins were produced in Escherichia coli prokaryotic host. Its noteworthy that additional peptide domains can be further associated to the delivery vectors described providing it with new abilities such as higher internalization or endosomal escape capacity. The approach to use the cellular retrograde transport in order to develop non viral vectors is poorly exploited by the scientific community and the present study stands among few in the field hopefully contributing to the development of more efficient and safer non viral vectors for gene delivery / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Les carbon dots (CDs), dernier membre de la famille des nanoparticules (NPs) carbonées à avoir été découverts, sont des particules hydrophiles quasi-sphériques qui sont généralement présentées comme biocompatibles et seulement faiblement toxiques. Ils sont facilement accessibles par voie de synthèse et peuvent être tout aussi aisément modifiés par modification chimique des groupes fonctionnels présents à leur surface. Leurs propriétés de fluorescence intrinsèques les rendent intéressants pour de nombreuses applications, notamment dans le domaine biomédical où les CDs trouvent des applications en tant que vecteurs de principes actifs, comme d’autres NPs développées dans ce domaine. Les travaux développés au cours de cette thèse visaient premièrement à identifier les propriétés physicochimiques intrinsèques des CDs qui conditionnent leur toxicité. Pour cela, le profil toxicologique d’une large collection de CDs présentant des propriétés de taille, charge et chimie de surface a été établi en utilisant des modèles in vitro et in vivo. Nous avons pu montrer ainsi que, bien que la taille des NPs joue un rôle important dans leur toxicité, elle ne constitue pas à elle seule un facteur prédictif de cette dernière. En effet, la charge et la chimie de surface régissent de façon importante les interactions entre les NPs et l’environnement biologique dans lequel elles sont introduites et, donc, leur toxicité. Le deuxième objectif de cette thèse était d’étudier le potentiel qu’offrent les CDs dans le domaine de la délivrance de gènes. Nous avons pu montrer la supériorité de CDs préparés à partir d’acide citrique et de bPEI600 sur tous les autres CDs cationiques préparés et évalués dans notre étude. Une approche systématique nous a permis d’améliorer, pas à pas, l’efficacité des CDs jusqu’à surpasser celle du bPEI25k, un agent de référence dans le domaine.Dans l’ensemble, nos études ont ouvert de nouvelles perspectives dans le domaine des NPs en apportant des éléments de compréhension des mécanismes de leur toxicité et en identifiant des conditions de production qui permettent d’optimiser leur propriétés en tant qu’agent de transfection. / Carbon dots (CDs) are the latest member of the family of carbon nanoparticles (NPs) to be discovered. They were isolated for the first time in 2004, during electrophoresis purification of carbon nanotubes. In addition to their nanometric size, these objects are almost spherical and hydrophilic, and are generally presented as biocompatible and very weakly toxic NPs. They are fairly easily accessible by synthesis and can be conveniently modified by reaction of the functional groups present on their surface (amines, carboxylic acids, alcohols, etc.). Finally, they exhibit intrinsic fluorescence properties, are relatively resistant to photobleaching, and can be excited by multi-photon irradiation. Thus, like the other members of the family of carbon NPs (graphene, nanodiamonds, fullerenes, nanotubes), CDs have remarkable properties which are the subject of intense research for applications in fields as different as those of electronics, catalysis, energy storage, imaging, and medicine. In the latter area, CDs can find applications as drug delivery systems, like other NPs successfully developed in this field. The work developed during this thesis had two distinct objectives. The first one was to identify the intrinsic physicochemical properties responsible for the toxicity of NPs. For this, the toxicological profile of a large collection of CDs produced in the laboratory and exhibiting various size, charge, and surface chemistry was characterized using in vitro lung models and mice. We found that although the size of the NPs plays an important role it is not, by itself, a predictive element of the toxicity of the NPs. The charge and the surface chemistry largely effect the interactions between the NPs and the biological medium systems and, therefore, their intrinsic toxicity. The second objective of this thesis was to assess the potential of CDs in the field of drug delivery as synthetic gene carriers. We were able to show the superiority of NPs prepared from citric acid and bPEI600 over all other cationic CDs produced in the laboratory. A systematic evaluation has allowed us, step by step, to improve the efficiency of these transfection agents, to exceed that of bPEI25k, a gold standard for in vitro transfection, without significant toxicity. Overall, this work opens up new horizons in NPs research that may provide 1-a better understanding of the toxicological mechanisms of NPs, especially their determinants, and 2-identification of the relationship between the CDs synthesis methods and the efficiency of these NPs as DNA transfection reagents.

Efficient delivery of genetic material to T-lymphocytes is key in gene therapy using T-lymphocytes with chimeric antigen receptors. Current procedures require the use of potentially dangerous viral vectors or large amount of input material. The diploma thesis therefore focuses on exploring new approaches for gene transfer into T-lymphocytes: use of safe virus-like particles (VLPs) derived from mouse polyomavirus in combination with the amphipathic cationic peptide LAH4. LAH4 has the potential to increase the efficiency of DNA and viral vector transport into cells. The system which combines VLPs and the LAH4 peptide was optimized for the delivery of reporter gene (encoding GFP and luciferase) to the model T-cell line Jurkat. It has been found that Jurkat cells cannot be efficiently transduced by DNA packed into VLPs. When cells were transfected only with DNA and LAH4, consistent results were not obtained, and the transfection efficiency ranged from 0.5 to 19%. The diploma thesis also analysed the effect of phosphorylation of viral structures on gene transfer. The impact of treatment of virus particles by alkaline phosphatase on the infectivity of the virus was studied and it was necessary to analyse the effect of the reaction components. Sublytic concentration of Triton-X100 in the reaction buffer...