Síntese e caracterização de nanopartículas de quitosana para aplicação em transfecção celular

Abstract

The approach of genetic engineering in animals involves several types of technologies directed to the production of Genetically Modified Animals (AGMs). An essential step for the production of AGM is the in vitro transport of genetic material into somatic cells. However, the techniques developed for gene loading have not been efficient due to the failures in transfection and transgene integration that are related to several factors. Currently, new alternatives have emerged for the efficient delivery of exogenous DNA, such as the use of nanocarriers, due to their ability to interact with genetic material and cellular internalization. The aim of this study is to synthesize and characterize the of nanoparticles (NPs) of chitosan for transfection application. The synthesis of NP of chitosan was performed by the ionic gelation method. The plasmid used was PEF-GFP cloned in E. coli stbl3 and purified following the QIAprep® Spin Maxprep kit protocol. NPs were characterized by the DLS technique that revealed a variation of sizes according to the proportions used from nanoparticles with 117.7nm to 313.9nm. Among the treatments tested, the ratio of 6/1/1 with free NP varied from 124.7 to 142.7 nm for the transfection assays. The nanocomplex containing plasmid DNA / TPP / NPs of chitosan had a diameter varying from 387.2nm and 284.4nm and the nanocomplex DNA plasmid / chitosan NPs presented a diameter varying from 351,9nm and 369,7nm. Several transfection assays were performed altering production of NPs with and without TPP, exposure time, pH, Zeta potential and use of DMEM or PBS / SFS media. The treatment in which the nanocomplexes were prepared with TPP using PBS / SFB, pH 6.5 and the HEK 293 cells were exposed for 24 hours to the nanocomplexes showed a better transfection efficiency (8.25%) in relation to the other treatments. Therefore, the results demonstrated that the pH and time of exposure to the nanocomplexes influence the cellular transfection rate using chitosan NP.