BODIPY-functionalized nanostructured materials for intracellular glutathione sensing and mitochondria bioimaging

Abstract

[eng] In the present thesis, molecular probes and functionalized nanostructured materials were prepared for the detection and quantification of intracellular glutathione (GSH), as well as mitochondria bioimaging. For this reason, first, fluorescent probes based on a BODIPY scaffold that can detect GSH and be conjugated to the surfaces of water-dispersible nanoparticles and microchips (as carriers) were prepared. In the first chapter of this thesis, a molecular probe Bdpy1 incorporating a fluorescent BODIPY core with chlorine substituents for reaction with GSH, and a linking moiety to enable conjugation to the surface of silicon oxide nanoparticles (SONPs), was synthesized and characterized, confirming its structure. Functionalized SONPs with Bdpy1 were also characterized at the nanoscale, including their optical properties, confirming the surface functionalization and water-dispersibility. GSH sensing was evaluated in aqueous solution, conjugated to SONPs, and in HeLa cells, showing promising potential for ratiometric GSH intracellular detection. In the second chapter of this thesis, the preparation of a GSH probe Bdpy2 incorporating a fluorescent BODIPY core with diethyl malonate groups, connected via exocyclic double bonds, which provides a specific Michael addition reversible reaction between the vinyl group and GSH, and a linking moiety for conjugation processes, is reported. Bdpy1 and Bdpy2 were immobilized on silicon oxide microchips (SOμC), micro-fabricated using photolithographic techniques, to give SOμC-Bdpy1 and SOμC-Bdpy2. Both functionalized microchips were characterized using fluorescence microscopy and exhibited sensitivity to GSH, and, notably, the reversible SOμC-Bdpy2 showed less time dependency, making it more suitable for long-term intracellular GSH sensing. In vitro experiments revealed that both SOμC-Bdpy1 and SOμC-Bdpy2 were internalized in HeLa cells, showing SOμC-Bdpy2 more reliable results (due to its less time dependency) for quantifying intracellular GSH. Remarkably, the intracellular GSH measurement was monitored by SOμC-Bdpy2 for 48h, indicating the functionalized microchips capability to detect GSH amount at different time intervals. In the third chapter of this thesis, gemini imidazolium amphiphile fluorophores based on a BODIPY core were also prepared for mitochondrial imaging in living cells. These fluorophores share three key components: a) a BODIPY fluorescent moiety chosen as a spacer, b) imidazolium groups that provide cationic and hydrophilic characteristics with mitochondrial affinity, and c) hydrocarbon chains that enhance cellular internalization. However, variations in the length of these hydrocarbon chains, which form the hydrophobic part of the fluorescent dyes, and the specific functional groups conjugated to the spacer result in different hydrophilicity and photophysical properties, respectively, of the synthesized compounds. Specifically, the hydrophobic parts of these fluorescent dyes range from one carbon atomto hydrocarbon chains containing 11 or 18 carbon atoms. Additionally, differences in functional groups attached to the spacer, such as the direct conjugation of imidazolium groups or the use of a thiol functional group as a nucleophile for attaching hydrocarbon chains and imidazolium groups to the spacer, leads to variations in the optical properties of the synthesized BODIPY-gemini imidazolium amphiphiles. The chemical structure and optical properties of all synthesized fluorophores were also characterized. The self-assembly of the water soluble synthesized fluorescent dyes was also studied by critical micelle concentration (CMC) determination with fluorescence spectroscopy and surface tension measurements, as well as Cryo-TEM. In vitro experiments revealed the excellent internalization, low cytotoxicity, and exceptional labeling of mitochondria of the fluorophore containing 11 carbon atoms of hydrophobic chains attached to the BODIPY core with thioether functional groups, indicating the promising performance of this fluorescent dye for mitochondria labeling applications.

[spa] Esta tesis presenta la preparación de sondas moleculares y su conjugación a materiales nanoestructurados para la detección y cuantificación de glutatión intracelular (GSH) y para la bioimagen de mitocondrias. Se sintetizaron sondas fluorescentes basadas en la estructura de BODIPY para la detección de GSH y la conjugación a la superficie de nanopartículas y microchips de óxido de silicio dispersables en agua. En el primer capítulo, se describe la síntesis y caracterización de Bdpy1, una sonda molecular con un núcleo de BODIPY y sustituyentes cloro para la reacción con GSH. Bdpy1 se conjugó a nanopartículas de óxido de silicio (SONPs) y se evaluó para la detección de GSH en disoluciones acuosas, SONPs y células HeLa, demostrando su potencial para la detección ratiométrica de GSH intracelular. El segundo capítulo aborda la síntesis de otra sonda, Bdpy2, con un núcleo de BODIPY y grupos de malonato de dietilo. Esta sonda experimenta una reacción de adición de Michael reversible con GSH. Tanto Bdpy1 como Bdpy2 se inmovilizaron en microchips de óxido de silicio (SOμC) y se probaron para la detección de GSH. La naturaleza reversible de SOμC-Bdpy2 lo hizo más adecuado para la detección de GSH a largos intervalos de tiempo. Los estudios in vitro confirmaron la internalización de ambas sondas en células HeLa, siendo SOμC-Bdpy2 más fiable para la cuantificación de GSH. En el capítulo final, se desarrollaron fluoróforos gemini anfifílicos de imidazolio basados en el núcleo de BODIPY para la imagen de mitocondrias. Estos fluoróforos consisten en una parte de BODIPY, grupos de imidazolio y cadenas hidrocarbonadas, con variaciones en la longitud de las cadenas y los grupos funcionales que afectan su hidrofobicidad y propiedades fotofísicas. Las regiones hidrofóbicas presentan longitudes de 1, 11 o 18 átomos de carbono. Los fluoróforos sintetizados fueron caracterizados y los experimentos in vitro mostraron excelente internalización celular y etiquetado mitocondrial, especialmente con el fluoróforo que contenía cadenas de 11 carbonos unidas por grupos tioéter al núcleo de BODIPY, lo que lo convierte esta molécula en una herramienta prometedora para el marcaje de mitocondrias en células vivas.