Navegando por Palavras-chave "Gene Therapy"
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- ItemSomente MetadadadosDesenvolvimento de nanopartículas magnéticas de sílica mesoporosa como vetores não virais em magnetofecção(Universidade Federal de São Paulo (UNIFESP), 2020-05-15) Francisco, Giorgio Fernando De Avelar [UNIFESP]; Bizeto, Marcos Augusto [UNIFESP]; Universidade Federal de São PauloGene therapy is based on introduction of exogenous genetic material into the cells aiming the treatment of genetic diseases from the inactivation/substitutions of defective genes or by the introduction of genes which can express therapeutic proteins. Incompetent recombination virus based vectors are the most utilized at this transfer, but instead of the high efficiency, there are many risks associated to the possibility of the expression of remaining viral genes, promoting intense immunologic response and generation of competent replication virus by recombination which can promote oncogenesis. Thence, the development of non-viral vectors, mainly based on inorganic nanoparticles has become an interesting technological and scientific topic. The transfection process is complex, and many cell barriers must be transposed by the vector. The cell entry, generally, occurs by endocytosis, which requires ideals charge and size. After passing through cell membrane, the vector is imprisoned in cell compartments which interior has acidic medium and rich in nucleases that degrade the genetic material. Nucleic acids released at cytosol in this stage can be transferred to the cell nucleus, passing through the nucleus membrane where it will be transcript. In this work, capacity (or efficiency) of magnetofection was evaluated in order to improve the rate of cell uptake by endocytosis of vectors based in mesoporous silica nanoparticles. The magnetofection process consists in cell transfection with magnetic nanoparticles by applying an external magnetic field. Initially, it was synthesized different types of mesoporous silica nanoparticles combined with superparamagnetic nucleus of magnetite (Fe3O4), which were chemically modified with propyldiethylenotriamine to enable the conjugation with molecules of plasmid DNA responsible to promote the expression of green fluorescent protein (GFP) in HeLa cells. Among the as-prepared magnetic vectors, just one of them presented propitious characteristics to transfection study. This vector was prepared by hydrolysis of silica inorganic precursor using sodium hydroxide as catalyst around magnetic nanoparticles in the presence of micelle aggregates of surfactant hexadecyltrimethylammonium bromide, to shape the formation of silica wall pores. The as-prepared nanomaterial infrared spectrum presented the main bands of the vibrational modes of magnetite, silica and propyldiethylenotriamine group. The N2 physissortion isotherms showed an improvement on the surface area of 14 m2 /g to 254 m2 /g after the covering, with an isotherm of type IV, which is characteristic of mesoporous material. The X ray diffractometry identified the presence of magnetite as a crystal at face centered cubic form and the amorphous silica. The transmission electron microscopy showed an agglomerate of spherical particles with a well defined core-shell structure. The colloidal stability of the propyldiethylenotriamine modified nanoparticles was evaluated in dispersion made in sodium chloride solution by zeta potential (26 ± 1mV) and by determination of hydrodynamic size (516 ± 74nm) by dynamic light scattering. Finally, the magnetofection of HeLa cells with the as-prepared magnetic nanoparticles showed a higher transfection rate (5.4 ± 3.2%) when compared to the control groups presenting a good perspective of its use in gene therapy.
- ItemAcesso aberto (Open Access)Desenvolvimento de um microgel de alginato carreado com vetores adeno-associados para liberação sustentada(Universidade Federal de São Paulo (UNIFESP), 2020-01-30) Cinel, Victor Dal Posolo [UNIFESP]; Han, Sang Won [UNIFESP]; http://lattes.cnpq.br/0069955147703693; http://lattes.cnpq.br/6845346545995430; Universidade Federal de São PauloGene therapy consists in manipulating and inserting exogenous genetic material in a patient to treat diseases. Although gene transfer technologies have greatly improved, there are still several limitations such as uncontrolled vector release that leads to immediate and excessive release of vectors at the site of administration. The consequence of this uncontrolled release is the leakage of vectors to other tissues, reducing gene transfer efficiency in the desired tissues and increasing the transfer of vectors to unwanted tissues. One solution to this problem is the use of vectors encapsulated in a biodegradable gel for slow and continuous release. Droplet microfluidics enabled the production of small and homogeneous microgels as carriers. Using alginate in a microfluidic device, it is possible to produce biocompatible alginate microgels with a sustained release kinetics of molecules. Despite being a promising carrier for gene therapy, there are no studies in the literature on the encapsulation of non-integrative viral vectors using microfluidics. In this work, 1.2% alginate microgel delivery system with type 2 adeno-associated vectors (AAV) was evaluated for its use for gene therapy. To this end, microgels encapsulate with model nanoparticles and AAVs were produced by microfluidics using the competitive ligand exchange crosslinking (CLEX) method. The microgels were produced and their encapsulation and controlled release capacity were evaluated by fluorescence spectroscopy, realtime quantitative PCR and fluorescence microscopy techniques, the latter to analyze the in vitro transduction efficiency of encapsulated AAVs. For the characterization, techniques of phase contrast microscopy, scanning electron microscopy and atomic force microscopy were used. Results showed that the pure 1.2% alginate microgels, microgels loaded with nanoparticles and microgels loaded with AAVs presented an average size of 125 µm, 106 µm and 116 µm, respectively. While pure and AAV microgels were monodisperse and with a regular topography, microgels with nanoparticles were monodisperse and showed an irregular and porous topography. The average encapsulation efficiency was 70.9% for nanoparticles and 13.2% for AAVs. Release kinetics studies have shown that the microgels produced can release encapsulated nanoparticles and AAVs in a continuous manner. In vitro transduction studies with HeLa cells using microgels with encapsulated AAVs showed that despite the low encapsulation efficiency, the AAVs released from the microgels were able to transduce cells, validating this delivery system for use in gene therapy studies.
- ItemAcesso aberto (Open Access)Doping genético no esporte: Uma revisão de literatura(Universidade Federal de São Paulo (UNIFESP), 2013-02-19) Prado, Natália Vieira Nuñez do [UNIFESP]; Aguiar, Odair [UNIFESP]; D'Almeida, Vânia [UNIFESP]; http://lattes.cnpq.br/7220411418339421; http://lattes.cnpq.br/0398348332863521; http://lattes.cnpq.br/5996912655572364; Universidade Federal de São Paulo (UNIFESP)Esta pesquisa bibliográfica, que se intitula “Doping Genético no esporte – uma revisão de literatura” teve como objetivo investigar as principais características dos estudos realizados abordando o doping genético, a terapia gênica no esporte, além das principais substâncias e formas utilizadas nesta forma de dopagem. O método de pesquisa foi dado como busca em banco de dados e os descritores utilizados foram termos em português e inglês – dependendo da fonte utilizada. Ao final desta pesquisa, esperava-se obter um documento que contenha os principais aspectos relacionados ao doping genético, podendo ser utilizado em pesquisas futuras, tendo em vista as grandes competições que ocorrerão em nosso país nos próximos anos e o quão desenvolvida está a tecnologia no esporte.