Estudio fotofísico de nuevas sondas fluorescentes y su aplicación en sistemas biológicos de interés biomédico

Abstract

The main research topic of this Thesis is the study of new different probes and their application in biological processes through the innovative fluorescence microscopy technique as fluorescence lifetime imaging microscopy (FLIM). Fluorescence techniques, and particularly microscopy, represent a non-invasive method characterized by their low toxicity, providing a number of advantages including a high level of sensitivity, specificity and wide concentration range. This work can be divided into the study of two different types of fluorophores, environment-sensitive probes, including solvatochromic and fluorogenic dyes, and long-lifetime probes, such as lanthanide-based biosensors, with their biomedical applications. In the first group, we have included two different probes; one is a silicon-modified xanthene derivative (2-4-TM), that present a strong solvatochromic properties able to detect changes in the environment polarity. We studied the solvatochromic properties and discover a natural adsorption binding of this silicon-modified xanthene derivative to macrostructures. The combination of these properties allowed us to detect in real time changes in the environment polarity around the dye during the initial stages of the β-amyloid (Aβ) aggregation process, using steady-state, time resolved fluorescence spectroscopy and advanced fluorescence imaging techniques such as FLIM. By using FLIM as an excellent approach to study amyloidogenic aggregation as non-invasive detection method, we were able to establish a polarity scale to distinguish the hydrophobicity of the aggregates and differentiate between different types of pre-amyloid Aβ-42 aggregates. The early stages of the β-amyloid (Aβ) peptide aggregation process are of particular interest in understanding the origin of multiple neurodegenerative disorders, such as Alzheimer’s disease, an important public health problem affecting millions of people worldwide, and one that continues to increase dramatically. The second fluorophore included in the environment-sensitive probes is the aggregation-induced emission (AIE)-based probe, named PEMC. After study its solvatochromic properties, we found that presents preferences for nonpolar media, exhibiting AIE under specific conditions by immobilization. This allowed us to study the rate of its spontaneous incorporation into cells by fluorescence lifetime imaging and to observe its intracellular pattern produced by the AIE. Interestingly, the strong differences in fluorescence intensity and fluorescence lifetime of the different intracellular compartments facilitated selective isolation for detailed study of specific organelles, such as cytoplasm, mitochondria and peripheral F-actin structures in the plasma membrane. Since organelles in eukaryotic cells play a key role in cellular function, the importance of visualizing and monitoring the morphology and activity changes of specific organelles provide very useful information at the subcellular and molecular level that opens up opportunities for use in disease diagnosis and therapy. Respecting long-lifetime probes, we have studied and lanthanide based biothiol sensor, consisting of a small molecule that behaves as a reactive non-fluorescent Michael acceptor, which upon reaction with thiols becomes fluorescent, and an efficient Eu3+ antenna, after selfassembling with this cation in water represents a very innovative and chemically interesting achievement to detect biothiols, and specifically glutathione (GSH). The behaviour of our highly GSH-selective biosensor showed a high potential for studies in murine and human cells of the immune system (CD4+ T, CD8+ T, and B cells) by flow cytometry, being able to capture their baseline differences in intracellular GSH levels. Our biosensor was also successfully monitored intracellular changes in GSH associated with the metabolic variations governing the induction of CD4+ naïve T cells into regulatory T cells (Treg).