Orientadores: Lucimara Gaziola de La Torre, Adriano Rodrigues Azzoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-20T20:39:39Z (GMT). No. of bitstreams: 1 Balbino_TiagoAlbertini_M.pdf: 2581790 bytes, checksum: 674a2d22b439ef811cbfb642774f6d4d (MD5) Previous issue date: 2012 / Resumo: Esta pesquisa teve como objetivo o desenvolvimento tecnológico de processo microfluídico para a obtenção de vetores não virais, baseados na complexação eletrostática entre lipossomas catiônicos (LC) e DNA plasmideal (pDNA) destinados à terapia e vacinação gênica. O desenvolvimento desse processo foi comparado ao processo convencional "bulk", que, a partir da simples mistura manual entre as soluções ou em sistema de vórtice, gera dificuldade no controle do tamanho destas estruturas e pode produzir variações nos resultados biológicos e na estabilidade coloidal. Já o processo microfluídico, que utiliza dispositivos que processam pequenas quantidades de fluidos (10-9 a 10-18 litros), permite a complexação eletrostática em regime contínuo, com o controle das condições difusionais, o que também permite melhor controle do tamanho destes complexos. Metodologicamente, o trabalho foi dividido em três principais etapas: na primeira parte, foi realizado o estudo físico-químico, estrutural e biológico dos complexos pDNA/LC obtidos por processo "bulk". Nessa etapa, verificou-se a correlação das propriedades físico-químicas e estruturais dos complexos com o processo de transfecção in vitro em células HeLa. A segunda parte do trabalho visou à otimização da produção de lipossomas catiônicos em dois dispositivos microfluídicos, com uma única e com dupla focalização hidrodinâmica, de modo a se obter lipossomas similares aos estudados na primeira parte do trabalho. Na utilização do segundo dispositivo, foi possível operar em vazões volumétricas mais altas quando comparadas ao primeiro. Por fim, na terceira parte, foi realizado o estudo da complexação entre LC e pDNA por processo microfluídico também em dois diferentes dispositivos, um similar ao utilizado na segunda parte do trabalho, com focalização hidrodinâmica única, e outro com blocos regulares nas paredes do microcanal, o que aumenta a área de contato entre os fluidos. Os complexos formados no primeiro dispositivo apresentaram melhores respostas biológicas in vitro, as quais foram similares às do processo "bulk". No segundo dispositivo, ensaios de acessibilidade de sonda de fluorescencência ao DNA indicaram alteração na associação entre LC e DNA. Dessa forma, a partir dos resultados, conclui-se que os dispositivos microfluídicos estudados são uma alternativa promissora para a formação de LC e também sua complexão com pDNA em modo contínuo, tanto pela potencialidade tecnológica quanto biológica, o que contribui para o desenvolvimento de produtos farmacêuticos que veiculam DNA e que são destinados à terapia e vacinação gênica / Abstract: This research aimed at the technological development of microfluidic process for nonviral carriers production based on the electrostatic complexation between cationic liposomes (CL) and plasmidal DNA (pDNA) for gene and vaccine therapy applications. The development of this process was compared to the conventional bulk process, in which the solutions are mixed followed by the simple hand shaking or brief vortexing, what generates difficulties on the particles sizes control and can affect the biological functionality and colloidal stability of the formulations. In contrast, microfluidic process, which uses devices that manipulate small amounts of fluids (10-9 to 10-18 liters), allows the electrostatic complexation in continuous mode, controlling diffusion conditions, which also allows the colloidal control of the obtained formulations. Furthermore, microfluidic devices have minimum dimensions and operate with low energy consumption. Methodologically, the present work was carried out in three mean steps: in the first step, the physicochemical, structural and biological characteristics of the pDNA/CL complexes obtained by the bulk process were studied. In this step, it was possible to verify the correlation of physicochemical and structural properties with the transfection phenomenon in vitro of HeLa cells. The second part of this work focused the optimization of the production of CL through two microfluidic devices, with single and double hydrodynamic focusing, to obtain similar CL to those of the first step of this work. By employing the second device, it was possible to operate at higher volumetric flow rates than the first one. Finally, in the third step, it was explored the complexation between CL and pDNA via microfluidic process also in two different microfluidic devices; the first was similar to that employed in the second part of this work, with a single hydrodynamic focusing, and a second one with patterned microchannel walls, which increase the surface contact area between the fluids. The complexes formed in the first device showed better biological results in vitro, which were similar to the complexes formed in the bulk complexation method. In the patterned device, the experiments of the DNA accessibility to fluorescent probe pointed out modifications between the pDNA and CL association in the complexes. In conclusion, we showed that the studied microfluidic devices are a promising alternative for the production of CL and the complexation with pDNA in continuous mode, because of the technological and biological potentialities, which contributes to the development of feasible processes, for the production of new pharmaceutical products for gene and vaccine therapies / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences University of Vienna, Faculty center for Pharmacy, Department of Pharmaceutical Chemistry, Laboratory of MacroMolecular Cancer Therapeutics Candidate: Barbora Kovářová Supervisor (Charles University): PharmDr. Anna Jirkovská, Ph.D. Supervisor (University of Vienna): Univ. Prof. Dipl. Ing. Dr. Manfred Ogris Co-supervisor (University of Vienna): Dr. Haider Sami, Ph.D. Title of diploma thesis: Reporter gene studies for nanoparticle mediated DNA and siRNA delivery Keywords: transfection, plasmid DNA, siRNA, nanoparticles Gene therapy is a promising field offering potential in several currently incurable diseases. Gene therapy is mediated by modulation of gene expression in specific cells by delivering exogenous nucleic acids. One of current tasks of nucleic acid delivery is exploring several synthetic vectors which would have a potential to overcome the disadvantages of commonly used viral vectors. The present study focused on different types of polyethyleneimine-based nanoparticles for plasmid DNA (pDNA) and small interfering RNA (siRNA) delivery. Integration of imaging contrast agents with gene delivery vehicles is advantageous for tracking the gene delivery process both in vivo and in vitro. Gadolinium...

Brugia malayi is one of three species of nematode known to cause lymphatic filariasis (LF) in humans. LF infects over 120 million people, causing debilitating disease. Various global programs have been launched in the past 20 years to eliminate LF. These programs have greatly scaled up the resources and efforts allocated to halting the transmission and reducing disease burden. Only a few drugs are used to treat LF, and resistance is thus a devastating possibility. Research aimed at identifying new drug targets could therefore prove essential in elimination of LF. Genetic manipulation of B. malayi has been limited to transient transfections. A transfection system allowing for stable integration of transgenic sequences into the nuclear genome of this parasite would enable more robust studies that could lead to identification of novel drug targets and vaccine candidates. The piggyBac (pB) transposon system has been successfully applied to develop a stable transfection system in a variety of species. This system involves two plasmids, a helper and a donor. The donor plasmid contains the target DNA and a selectable marker flanked by specific inverted terminal repeat (ITR) regions. The helper plasmid expresses the pB transposase that will catalyze the precise integration of any DNA report tools necessary to adapt the pB system in B. malayi. Three versions of the donor plasmid were constructed, each containing a Gaussia Luciferase (GLuc) selectable marker but differing only by the fluorescent protein expressed. The construct containing a YFP gene was used to transfect embryos via biolistics to test whether YFP and GLuc are expressed.

A leishmaniose é causada por protozoários do gênero Leishmania e no Brasil, as principais espécies causadoras da leishmaniose cutânea são Leishmania (V.) braziliensis e Leishmania (L.) amazonenses. O tratamento da leishmaniose apresenta diversas dificuldades, portanto é fundamental a descoberta de novos fármacos ativos, podendo ser detectada em células cultivadas in vitro e também em animais íntegros, através da técnica de bioimageamento. Neste trabalho, propusemo-nos a produzir linhagens de L. (V.) braziliensis e L. (L) amazonenses expressoras de luciferase e caracterizar o comportamento das linhagens mutantes em testes de sensibilidade a fármacos e de infecção in vitro e in vivo. Foi confirmada a emissão de luz pelas linhagens mutantes das duas espécies de Leishmania, em promastigotas e amastigotas. O comportamento das linhagens mutantes obtidas em relação a curvas de crescimento, sensibilidade aos fármacos tamoxifeno e anfoterina B em promastigotas, perfil de infectividade e sobrevivência em macrófagos e sensibilidade de amastigotas à anfotericina B foi comparado ao comportamento das linhagens parentais, não sendo observadas diferenças significativas. Camundongos BALB/c infectados com a linhagem expressora de luciferase de L. (L.) amazonenses desenvolveram lesões comparáveis aos animais infectados com a cepa selvagem, sendo possível quantificar a carga parasitária nesses animais por bioimageamento. Os resultados obtidos neste trabalho indicam que os parasitas mutantes expressores de luciferase obtidos podem ser utilizados em testes de sensibilidade a fármacos tanto in vitro como in vivo, representando um avanço metodológico nessa área de pesquisa. / Leishmaniasis is caused by protozoan parasites in Brazil, the main causative species of cutaneous leishmaniasis are Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonenses. The treatment of leishmaniasis presents several difficulties, and the discovery of new active drugs for the treatment of leishmaniasis is therefore fundamental. The enzyme luciferase is a reporter widely used in screening tests for new drugs. This enzyme catalyzes the oxidation of luciferase in the presence of ATP emitting light that can be detected in cultured cells in vitro as well as in intact animals, using the technique of bioimaging. In this work, we sought to produce strains of L. (V.) braziliensis and L. (L) amazonenses expressing luciferase and characterize the behavior of these mutant strains in drug susceptibility tests and in in vitro and in vivo infections. Production of light was detected in mutants of both species, in all life cycle stages. Mutant strains were compared to their corresponding parental lines as to their growth pattern, infectivity and survival profile in macrophages and sensitivity to amphotericin B and tamoxifen. No significant differences were observed for these parameters. BALB/c mice infected with the luciferase expressing line of L. (L.) amazonenses developed lesions comparable to those in animals infected with the wild-type strain. The parasite load in these animals was quantified through bioimaging. The results obtained of this study indicate that the mutant parasites expressing luciferase can be used for drug susceptibility testing in vitro and in vivo, representing a methodological advance in this area of research.

The topic of this thesis is electrophysiological characterization of Kir2.1 membrane channel. Inward rectifier potassium channel Kir2.1 is located in muscular, heart and nerve cells and its dysfunction causes various diseases. Practical part of this stage is focused on cultivation of the HEK293T cell line that is used to transfection of the plasmid Kir2.1 and subsequent measurement of the ionic current through the electrophysiological method patch-clamp in whole-cell mode.

Advances in molecular medicine have produced a large amount of information about genes that translate to therapeutic molecules when expressed in living cells. There is an increasing interest in nonviral methods for gene delivery, to address all concerns on non-toxic, easy, and possibly efficient delivery systems. In this paper we introduced a new attractive approach for non-viral transferring of genetic materials on demand. By using lipofectin reagent (1:1 molar ratio of DOPE:DOTMA. DOPE: L-α-doleoyl posphatidylethanolamine; DOTMA: N-[1-(2,3-dideyloxy) propyl]-n,n,n-trimethylammonium chloride), the lipid/DNA complexes (lipoplexes) can be electrostatically adsorbed on the gold microelectrode surface. The resulting lipoplexes molecules can be subsequently removed from the surface by applying -1.0 V (vs. Ag/AgCl) in physiological phosphate buffer medium (pH 7.4). This electrochemically controlled-release process has been extensively examined by gel electrophoresis (GE), electrochemical quartz crystal microbalance (EQCM), infrared spectroscopy (IR), and square wave voltammetry (SWV) techniques. The lipoplex composition has been addressed for efficient gene delivery protocol, based on their different charge ratios. The results from different techniques coincided, as also verified by the repetitive control experiments. This in-vitro electrically - triggered release protocol for genetic material offers the current gene delivery arsenal a new, simple, and non-viral alternative.

Functionalized gold nanoparticles (AuNPs) provide an excellent scaffold for numerous biological applications. In these systems, the gold core imparts stability to the assembly, while the monolayer allows tuning of surface characteristics such as charge and hydrophobicity. The nano-scale size and tunable surface properties have made them an ideal candidate for manipulating protein-protein/protein-nucleic acid interactions, and delivery of therapeutics. In this thesis work, it has been demonstrated how the surface coating plays an important role in achieving a desired goal. Using organic synthesis as a tool, the monolayer was tailored to afford useful particles with biocompatibility and the ability to respond in the cellular environment. The recognition units present on the periphery of particles dictates/controls their interactions with biomolecular or cell surfaces. As described here, these engineered particles exhibited a number of bio-applications, including folding of a peptide into an α-helix, binding with DNA, and cellular delivery of genes and proteins.

Arrays of silicon lances were made using photolithographic and STS DRIE Bosch techniques. Arrays consist of a 10 mm square grid pattern of lances measuring 100 m tall and having a 3 mm diameter, each lance being spaced 30 mm apart. The tips of lances are pointed, allowing easier penetration through plant cell walls. A nanoinjector device was also made to accept the silicon lance arrays and perform nanoinjections. A nanoinjection consisted of 2 silicon lance arrays, with lances oriented towards each other, being moved into and out of a plant cotyledon placed between them. Prior to the nanoinjection, polar molecules in solution can be attracted to the lances through a process utilizing the nanoinjector device’s ability to control the electrical current between the 2 lance arrays. During the nanoinjection the displacement between the lances, the force exerted on the plant cotyledon and the electrical current between the lance arrays are controlled. Once the lances are inserted into the cells, the electrical current between the lance arrays is reversed, repelling the molecular load from the lance array. Propidium iodide (PI) and Cotton Leaf Crumple Virus (CLCrV) were used as molecular loads in nanoinjections. The nanoinjector also records and outputs data from the nanoinjection for analysis. Nanoinjections were performed on Arabidopsis and Cotton cotyledons. Changes in the force applied during a nanoinjection and varying the number of repeated nanoinjections on the same cotyledon were observed. Too much force or too many repeated injections causes physical damage to the cotyledon. An optimal force and number of repeated injections can be performed without causing physical damage to the cotyledon. Successful transfection of PI and CLCrV was not observed in a relatively small number of performed nanoinjection procedures on either Arabidopsis or Cotton cotyledons. Possible interacting variables and recommendations for further work are discussed.

La dualité hydrophobe/hydrophile des molécules amphiphiles est à l’origine de leur autoassemblage en solution, sous forme de nombreuses structures supramoléculaires, telles que les micelles. Ce travail de thèse présente la formation, la caractérisation et l’étude de nouvelles micelles diacétylèniques photopolymérisables. Une première partie décrit ainsi lasynthèse de nouvelles micelles cationiques et l’étude de leur utilisation en tant qu’agent de transfert de gènes. Dans une seconde partie, nos travaux présentent l’étude de micelles polydiacétylèniques, porteuses de têtes polaires octaéthylèneglycol, comme potentiel système de délivrance de médicament. Les propriétés d’encapsulation de ces micelles ont été évaluées en présence d’un dérivé fullerène fluorescent. Puis, l’incorporation d’une sonde membranaire dans leur couronne lipophile a permis de réaliser une étude de leurs propriétés de délivrance in vitro. Une étude préliminaire de leur biodistribution in vivo a également été réalisée par tomographie à émission monophotonique grâce à la chélation d’un isotoperadioactif sur la surface des micelles. Enfin une dernière partie présente l’analyse de deux nouveaux auto-assemblages tubulaires obtenus au cours de nos travaux. / The hydrophobic/hydrophilic duality of amphiphilic molecules is at the origin of their selfassembly in solution, into many supramolecular structures such as micelles. This thesis presents the formation, characterization and study of new photopolymerizable diacetylenic micelles. The first part describes the synthesis of new cationic micelles and the study of theiruse as gene transfer agent. In the second part, our work presents the study of polydiacetylene micelles, bearing polar heads octaétylèneglycol as potential drug delivery system. The encapsulation properties of these micelles were first evaluated in the presence of a fluorescent fullerene derivative. Then, the incorporation of a membrane probe in their lipophilic shell has allowed a study of their in vitro delivery properties. Finally a study of their in vivo biodistribution was also carried out by single photon emission tomography through the chelation of a radioactive isotope on micelle surface. Finally the last part presents theanalysis of two new tubularself-assemblies, obtained during our work.

La conception de biomatériaux actifs est actuellement encouragée par le manque de thérapies régénératives efficaces pour des tissus endommagés présentant une faible capacité d’autoréparation. Les progrès récents concernant les techniques de préparation de matériaux structurés (électrospinning, microfluidique) ainsi que la découverte du fort potentiel régénératif des cellules souches ont suscité un regain d’intérêt pour des projets collaboratifs à l’interface entre biologie et sciences des matériaux. Une approche prometteuse de régénération tissulaire repose donc sur la combinaison de cellules souches et de biomatériaux implantables. Des biomatériaux innovants, injectables et servants à la fois de support aux cellules et de réservoir de molécules actives telles que des protéines ou des agents de thérapie génique (Matrice Génétiquement Activée) ont été développés. Se plaçant plus particulièrement dans le contexte de l’ingénierie du cartilage, ce travail a pour objectif de développer une stratégie complémentaire concernant l’orientation de la différenciation de cellules souches mésenchymateuses (CSM) grâce au mécanisme d’interférence ARN.La principale difficulté rencontrée lors de l’utilisation d’acides nucléiques pour induire la différenciation des CSM reste leur faible capacité à traverser les membranes cellulaires, due à leur nature hydrophile et leur charge négative. De plus, les acides nucléiques sont dégradés très facilement par les nucléases extracellulaires, ce qui rend nécessaire l’utilisation d’un vecteur. Les vecteurs non-viraux sont d’excellents candidats pour des applications in vivo en raison de leur faible coût de production et leur faible immunogénicité. Toutefois, la plupart des systèmes de vectorisation trouvés dans la littérature présentent un manque de reproductibilité associé à une cytotoxicité vis-à-vis des cellules primaires. Nous souhaitions donc développer un système de transfection synthétique à la fois efficace et biocompatible. Pour cela, nous nous sommes basés sur les résultats encourageants concernant l’utilisation des micelles de complexes polyioniques (PIC) pour la transfection des cellules dendritiques. Ces micelles sont formées par complexation de deux polyélectrolytes : un copolymère à blocs double-hydrophiles (CBDH) avec un bloc anionique et un homopolymère cationique. Dans ce travail, nous avons évalué le polyoxyde d’éthylène – b – polyacide méthacrylique en tant que CBDH et la poly-L-lysine ou le polyéthylènimine en tant que polycation. L’influence des caractéristiques des composantes (asymétrie du CBDH, nature du polycation, taille des blocs, ratio de charges…) sur les propriétés physico-chimiques des micelles formées (taille, charge de surface) a d’abord été étudiée. Puis, la possibilité de complexation d’un siRNA au sein des micelles ainsi que leur stabilité en conditions physiologiques ont été évaluées. La formulation des micelles a été conçue pour permettre une dissociation des objets à un pH comparable à celui des endosomes ; ceci a été vérifié par diffusion dynamique de la lumière. Une analyse par cytométrie en flux avec un siRNA marqué TAMRA ont démontré l’internalisation effective des micelles dans les CSM. Plus important encore, l’inhibition spécifique d’un gène cible, Runx2, a été démontrée à un niveau comparable à celui d’un vecteur commercial standard, la Lipofectamine2000®. La seconde partie de la thèse a consisté en l’élaboration de microparticules. A cet effet, nous avons préparé des microsphères de collagène par un dispositif de microfluidique, et ce à partir de diverses sources de collagène (murin, porcin, bovin). Des expériences préliminaires démontrent qu’il est possible d’imprégner les micelles dans les microsphères. De même, de premiers résultats encourageants ont été obtenus quant à la capacité du système globale à assurer l’adhésion cellulaire et permettre une transfection efficace des CSM dans un environnement 3D par les micelles PIC vectorisant un siRNA anti-Runx2. / The relative lack of efficient regenerative therapies for damaged tissues with low capacity for self-repair is one major motivation for the design of new active biomaterials. Recent progress in hierarchical materials processing techniques (electrospinning, microfluidics…) and the demonstration of the strong regenerative potential of stem cells have prompted renewed interest for collaborative projects at the biology / materials science interface. The combination of stem cells and active implantable materials has emerged as a high potential approach for the regeneration of damaged tissues. In particular, injectable cell carriers also acting as a reservoir for active molecules like proteins or gene therapy agents (Gene Activated Matrices) bring about innovative solutions to current issues in the field of tissue engineering. In the context of cartilage regeneration, the main objective of this work was to investigate a complementary strategy to orient mesenchymal stem cell (MSC) fate by the use of RNA interference. One major difficulty to reach high transfection levels and efficiently direct MSC differentiation comes from the low ability of nucleic acids (NA) to cross cellular membranes, largely due to their hydrophilicity and negative charge. This, along with a strong susceptibility to extracellular nucleases, calls for efficient gene delivery vectors. Their low production cost and low immunogenic potential make non viral vectors good candidates for in vivo applications. Besides, most systems reported in the literature show reproducibility and cytotoxicity issues with primary cells that we intended to address to achieve a safe and efficient synthetic vector for MSC. Based on previous encouraging results on the transfection of dendritic cells, we chose to investigate tripartite polyionic complex (PIC) micelles. Their formation is based on the polyelectrolyte complexation of a polyanionic double-hydrophilic block copolymer (DHBC) with a cationic homopolymer. In this work, we investigated polyethylene oxide – b – polymethacrylic acid as the DHBC and Poly-L-Lysine or Polyethyleneimine as the polycation. One major part of the work was to study the influence of micelles components characteristics (block size, DHBC asymmetry, polycation nature and molecular weight, polyelectrolyte charge ratios, etc.) on the physical characteristics (dimensions, surface charge) of the obtained nanoparticles. We then studied the ability of micelles to stably complex siRNA at high loading levels, and their stability in physiological conditions. Importantly, the PIC micelles’ formulation was designed to allow for pH-triggered disassembly in acidic conditions similar to those found in endosomes, as assessed by light scattering measurements. These nanoparticles were shown to be efficiently internalized inside MSC by flow cytometry using a fluorescently labeled SiRNA-TAMRA. Most importantly, they were shown to efficiently down-regulate Runx2 mRNA in MSC, at levels similar to those reached with the gold standard Lipofectamine2000®. The second major step for the development of a GAM suited for cartilage regeneration was to elaborate injectable microparticles. To this purpose, we prepared collagen microspheres through a microfluidic-based process and with different collagen sources (murine, bovine, and porcine). Preliminary experiments show that micelles can be efficiently loaded into the microspheres. First encouraging results were also obtained regarding the ability of the created GAM to support cell adhesion, and to allow for the efficient transfection of MSC in this 3D environment, thanks to an anti-runX2 siRNA vectorized with PIC micelles. This proof-of-concept study has demonstrated that the main elements of the nano-in-micro system are ready and mostly meet the assigned requirements. This opens the way for further work to assess the ability of this GAM to effectively improve MSC chondrogenesis and ultimately cartilage repair.