Non-biased fluorescent dyes as markers of drugs for optical in cellulo and in vivo imaging

Projektinformationen

NoBiasFluors

ID Finanzhilfevereinbarung: 872331

DOI

10.3030/872331

Projekt abgeschlossen

EK-Unterschriftsdatum 23 Oktober 2019

Startdatum 1 Januar 2020

Enddatum 31 Dezember 2024

Finanziert unter

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Gesamtkosten

€ 699 200,00

EU-Beitrag

€ 671 600,00

Koordiniert durch

FRIEDRICH-ALEXANDER-UNIVERSITAET ERLANGEN-NUERNBERG
Germany

Periodic Reporting for period 2 - NoBiasFluors (Non-biased fluorescent dyes as markers of drugs for optical in cellulo and in vivo imaging)

Berichtszeitraum: 2023-01-01 bis 2024-12-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

Fluorescence imaging of biological processes in live animals is an excellent non-invasive bioanalytical tool. Due to the intrinsic properties of biological tissues, only red-fluorescing dyes can be used for the imaging. Currently available dyes of this type exhibit important disadvantages including their clear bias towards particular intracellular organelles, for example mitochondria. Therefore, distribution of drug~dye conjugates in live animals is often dominated by the dye, rather than by the drug. The problem is especially prominent if the drug is a low-molecular weight compound. The majority of currently used in clinics drugs are compounds of such type. NoBiasFluors worked on solving this problem by developing non-biased red-fluorescing dyes suitable for in vivo imaging of distribution of low molecular weight drugs. NoBiasFluors findings will allow substantial expanding our knowledge on the mode of action of typical drugs.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

We conducted a comprehensive screening of various pH-sensitive fluorescent dyes to evaluate their potential in forming supramolecular assemblies aimed at reducing toxicity and enhancing water solubility. In addition to physicochemical improvements, we assessed the capability of these newly formed assemblies to selectively target and fluorescently label acidic intracellular organelles, particularly lysosomes and mitochondria.

Furthermore, we investigated the self-assembly behavior of the supramolecular structures, focusing on how the molecular architecture of each dye influences its aggregation properties. Our findings indicate that specific structural features of the fluorescent compounds play a crucial role in the formation of well-defined assemblies.

As part of this study, we also explored the subcellular localization bias of a range of fluorescent dyes. Notably, we identified a series of red-emitting BODIPY derivatives that exhibit minimal organelle-specific bias. These non-biased dyes are particularly suitable for the fluorescent tagging of aminoferrocene-based anticancer prodrugs and active pharmaceutical compounds with molecular weights below 1000 Da.

We further synthesized conjugates of these non-biased BODIPY dyes with aminoferrocenes to examine their intracellular distribution, uptake, and activation profiles. This approach enabled a detailed evaluation of the mechanisms underlying drug activation and localization, thereby contributing valuable insights into the intracellular behavior and therapeutic potential of aminoferrocene-based drug candidates.

We used the selected aminoferrocene-dye conjugates to explore their distribution and transformation in mice. We observed activation of aminoferrocenes and their accumulation in specific tissues. This study provided important information on behavior of aminoferrocenes in vivo.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

The research conducted in this project represents a significant advancement in understanding the behavior of fluorescent probes within living cells and in vivo in murine models. Through extended monitoring of fluorescence responses—spanning over 48 hours—in both cultured cells and whole animals, we gained critical insights into the intracellular migration patterns and specific accumulation tendencies of the studied dyes. This long-term observation revealed dynamic processes governing dye distribution, localization, and retention within cellular compartments.

Importantly, the stability of the fluorescent signal over time, along with a notable increase in fluorescence intensity, was closely linked to the pH-sensitive nature of the dyes. This pH-dependent enhancement in fluorescence served as a valuable indicator of the dyes' responsiveness to the acidic microenvironments within cells, particularly within organelles such as lysosomes. These findings provided essential data for the validation and optimization of the current project and highlight the potential of these fluorescent systems for real-time, non-invasive imaging applications in biological and medical research.

We demonstrated that non-biased red fluorescing dyes can be alternative markers of low molecular weight drugs and prodrugs. These dyes can substitute radioactive markers in future.

Molecules 2020, 25, 4397