Mapeamento do processo de nucleação e crescimento do peptídeo beta amilóide pelo complexo luminescente cis-[Ru(phen)2(Apy)2]2+

Abstract

Amyloid beta (Ab) is a peptide fragment highly prone to aggregation that deposits in the extracellular medium and is directly associated with the development and progression of a number of nervous system diseases, in particular, Alzheimer's disease (AD). With this in mind, the objective of this work is to obtain a luminescent Ru(II) complex capable of mapping in real time the aggregation process of the amyloid beta peptide through light stimulus. The cis-[Ru(phen)2(Apy)2]2+ complex was synthesized for this purpose. The complex showed an intense and wide absorption band covering the region 350 to 600 nm with a maximum of 450 nm (molar absortivity = 9800 mol-1.L .cm-1) and broad emission with maximum at 655 nm, exhibiting a Stokes shift in the order of 4800 cm-1 and phosphorescence lifetime of 129 ns. The interaction of the complex with the Aβ1-40 peptide was analyzed by the luminescent responses of the complex throughout the course of aggregation. Interaction studies through changes in luminescence intensity were conducted. Luminescent images and changes in luminescence lifetimes during the aggregation process were obtained by confocal microscopy using the FLIM technique (Fluorescence Lifetime Imaging). The complex proved to be a good marker for the Ab peptide aggregation process. With the fluorescence lifetimes obtained from the luminescent images, it was possible to evaluate the interaction between the complex and Ab1-40. Ab1-40 images, with samples being prepared without the complex, have an average bi exponential lifetime of 3.5 ns. On the other hand, the images of the complex Ab1-40 have an average tri exponential lifetime of 4.8 ns, with a significant contribution of a third long lifetime of around 7 ns. Once the interaction between the complex and Ab1-40 was confirmed, the possible sites of interaction were investigated. Three fragments of the peptide were studied: Ab1-28, Ab11-22 and Ab29-40. The images of FLIM without and with complex proved to be quite different, as well as their lifetimes. After determining the interaction of the complex with the peptide, the ability of the complex to influence the peptide aggregation process was investigated. Studies of circular dichroism at different stages of aggregation showed a significant difference in the ellipticity of the beta-sheet signals formed throughout the aggregation, suggesting a possible inhibitory effect by the complex. Studies with transmission electron microscopy were also performed with the fragments Ab1-40, Ab1-28, Ab11-22 and Ab29-40. Studies with Ab1-40 in the 1:5 complex:amyloid ratio did not show significant change over the control group. On the other hand, studies in the 1:1 complex:amyloid ratio show smaller aggregates, again highlighting the possibility of an inhibitory effect of aggregation.