Navegando por Palavras-chave "Transfection"
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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)Estudo do mecanismo molecular de transfecção mediada por ultrassom(Universidade Federal de São Paulo (UNIFESP), 2010-11-24) De Paula, Daisy Maria Bentes [UNIFESP]; Han, Sang Won [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Ultrasound (US) has been widely used to improve the efficiency of non-viral vector transfection. However, the mechanism that enables the uptake of plasmid DNA in cells by US insonation is poorly understood, but it is typically attributed to sonoporation. Based on our previous results, we hypothesized that other mechanisms, such as endocytosis, are involved in this process. To explore the mechanism of plasmid DNA uptake, a plasmid vector expressing EGFP (pEGFP-N3: 4.7 kb) was used to transfect NIH3T3 cells using a therapeutic US without microbubbles and was monitored in real-time using a confocal microscope. We achieved about 40% transfection efficiency when we applied 2 W/cm2 with 20% of duty-cycle for 30 s, but 1 W/cm2 resulted in a very low level of transfection. In these experiments, the production of reactive oxygen species was augmented during the insonation but was stopped soon after turning off the US. Calcium influx was also augmented during the insonation, but its level did not return to basal levels following the 3-min observation period. However, 1 W/cm2 was not sufficient to mobilize calcium influx during the insonation, and calcium influx began 12 s after turning off the US. US insonation also changed the cell membrane potential to promote a hyperpolarization state, which returned to the normal state soon after turning off the US. The alteration of these parameters by US indicates the uptake of plasmid DNA by endocytosis. Finally, using a fluorescently labeled plasmid, we showed that this molecule enters into cells via clathrin-mediated endocytosis, not via caveolin-1.
- ItemAcesso aberto (Open Access)Influência dos agentes de transfecção na marcação de células tronco com nanopartículas para seu posterior rastreamanto na terapia celular no modelo animal de avc(Universidade Federal de São Paulo (UNIFESP), 2016-11-29) Reis, Rafael Ferreira dos [UNIFESP]; Contreras, Lionel Fernel Gamarra [UNIFESP]; http://lattes.cnpq.br/4027049192000363; http://lattes.cnpq.br/0303337319130673; Universidade Federal de São Paulo (UNIFESP)Objective: Elucidate the transfection process of superparamagneti iron oxide nanoparticles conjugated with transfection agentes in stem cell for tracking of therapeutic application in stroke. Then, superparamagnetic iron oxide nanoparticles of dextran and chitosan conjugated with protamine sulfate and poly-l-lysine. Methods: For magnetofection process, were tested different concentrations of dextran nanoparticles and chitosan nanoparticles with diferente dose of protamine and poly-l-lysine for labelling of stem cell from bone marrow rats. . Previous in vitro tests of hydrodynamic size, cytochemical analysis and physical, toxicity potential and analysis by magnetic resonance were made. Therefore, we study the cell transplant in stroke by magnetic resonance 3 T. Results: At first, we demonstrated that nanoparticles in this study are stable in RPMI culture medium when dispersed a concentration of ?gFe/m Land 6 ?g/mL of protamine sulfate. The cell labeling analysis by prussian blue that dextran nanoparticles are efficient for this purpose. Besides that, cytotoxicity assay showed that cell death caused by these nanoparticles was barely evident.). The in vitro tests showed that nanoparticle aggregation was efficient in stem cells with 10 hours of labellig used. Moreover, the oblique steel pole addition created a punctual nanoparticle accumulation in one hose?s side, being this artífice selected for subsequent in vivo studies. The magnetic resonance monitoring was effective for dextran nanoparticles identification in vitro and vivo. Therefore in stroke region after local administration, showed an important signal reduction that is good for the contrasto of the image, even 5 days. Conclusion: Taken together, our results showed the dextran nanoparticles conjugated protamine sulfate are targeting efficience, being this strategy a promisse tool for further applications of tracking and homing of stem cells.
- ItemAcesso aberto (Open Access)Modification of the linker amino acid in the cell-penetrating peptide NickFect55 leads to enhanced pDNA transfection for in vivo applications(MDPI, 2023-02-20) Porosk, Ly; Mello, Lucas; da Silva, Emerson Rodrigo [UNIFESP]; Härk, Heleri; Kurrikoff, Kaido; Arukuust, Piret; http://lattes.cnpq.br/7800589206457326Despite numerous efforts over the last three decades, nucleic acid-based therapeutics still lack delivery platforms in the clinical stage. Cell-penetrating peptides (CPPs) may offer solutions as potential delivery vectors. We have previously shown that designing a “kinked” structure in the peptide backbone resulted in a CPP with efficient in vitro transfection properties. Further optimization of the charge distribution in the C-terminal part of the peptide led to potent in vivo activity with the resultant CPP NickFect55 (NF55). Currently, the impact of the linker amino acid was further investigated in the CPP NF55, with the aim to discover potential transfection reagents for in vivo application. Taking into account the expression of the delivered reporter in the lung tissue of mice, and the cell transfection in the human lung adenocarcinoma cell line, the new peptides NF55-Dap and NF55-Dab* have a high potential for delivering nucleic acid-based therapeutics to treat lung associated diseases, such as adenocarcinoma.
- ItemSomente MetadadadosThe use of the green fluorescent protein to monitor and improve transfection in Trypanosoma cruzi(Elsevier B.V., 2000-11-01) Ramirez, Marcel Ivan [UNIFESP]; Yamauchi, Lucy Megumi [UNIFESP]; Freitas Junior, Lucio Holanda Gondim de [UNIFESP]; Uemura, Haruki; Schenkman, Sergio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Nagasaki Univ