Desenvolvimento de processo microfluídico para incorporação de DNA em lipossomas catiônicos destinados a terapia e vacinação gênica = : Development of microfluidic process for DNA incorporation into cationic liposomes for gene therapy and vaccination / Development of microfluidic process for DNA incorporation into cationic liposomes for gene therapy and vaccination

AUTOR(ES)
FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

22/07/2012

RESUMO

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.

ASSUNTO(S)

lipossomas transfecção microfluidica nanotecnologia liposomes transfection microfluidics nanotechnology

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