Padronização de técnica eletroforética para avaliação da complexação de ácidos nucléicos a nanoemulsões catiônicas

Abstract

Gene therapy is a promising alternative for the treatment of diseases. Among the nucleic acids used are: oligonucleotides (ON) and plasmids (pDNA). However, these systems have low potential for transfection, thus requiring vehicles to improve cellular uptake, among which the cationic nanoemulsions stand out. This raises the need to evaluate the degree of nucleation of nucleic acids to these carriers, since only qualitative, indirect and non-standard methodologies have been used. In this way, the work aims to standardize a technique capable of evaluating the degree of complexation of the formulations with two nucleic acid models (oligonucleotide and plasmid), in different charge relationships. The nanoemulsions were prepared by the spontaneous emulsification technique and characterized as droplet size distribution and zeta potential. The complexes with the nucleic acids were prepared by extemporaneous addition, at the theoretical loading ratios of [+4/-]; [+2/-]; [+0.4/-] and [+0.2/-]. The electrophoresis gel retardation technique was used to evaluate the formation of the complexes with the plasmid (1% agarose) and oligonucleotide (2% agarose). First, the amount of SYBR Gold® and sample to be added at the point of application was tested. Standard curves were then made for both the plasmid and the oligonucleotide. The formulations presented mean droplet size in the nanometric range, with Span values lower than 2 and positive zeta potential. It was defined that the concentration of agarose for the plasmid would be 1% and for the 2% oligonucleotide. The amount of sample added at the point of application was 5 μL for 5 μL of SYBR Gold®. For the evaluation of the complexation, the standardized electrophoretic technique demonstrated the appearance of bands at the lower charge ratios [+ 0,4 / -] and [+ 0,2 / -], for both nucleic acids, demonstrating that when a decrease of positive sites occurs, the electrostatic interaction is reduced and free nucleic acids are able to migrate in the gel. Any of the complexes presented a band in the load relationships [+4/-] and [+2/-], and it may be inferred that complexation occurred in these situations. In addition, although the nucleic acids had different sizes, the charge ratio limit of complexation was the same by the standardized technique.