Synthesis and characterization of water-soluble dendronized dye systems as fluorescent labels for bioimaging applications

Abstract

Perylene bisimides (PBIs) possess characteristics such as high chemical and photophysical stability, outstanding fluorescence properties, and availability of monofunctionalized derivatives. These features make them ideal candidates for an application as fluorescent label. However, their insolubility in water and the resulting aggregation tendency leading to fluorescence quenching have limited the full potential of these functional dyes. To overcome these limitations, several polyglycerol- (PG) dendronized monofunctionalized PBIs are presented as site-specific labels for cellular bioimaging. The general structure of the biolabels consists of a hydrophilic and sterically demanding PG dendron, the fluorescent PBI core, and a linker with a functional group. The PG dendron introduces water solubility and suppresses the aggregation tendency of the fluorophores. The monofunctional linker serves as a coupling-active unit for the site-specific conjugation of biomolecules or other components. To study the effect of steric shielding, the PBI labels were synthesized in different dendron generations ([G low] vs. [G high]). To additionally evaluate the impact of electrostatic shielding, the hydroxylated dendron headgroups of the labels were modified with ionically charged sulfate groups (OH vs. SO4-). In the first project, a series of hydroxylated and sulfated [G1]- to [G3]-dendronized PBIs with a poly(ethylene glycol) (PEG) linker was synthesized, optically characterized, and studied as site-specific labels by conjugation to an antibody. The photophysical properties of the labels could be improved by increasing the steric bulk and amount of charge of the attached dendron, resulting in highly fluorescent PBIs with fluorescence quantum yields (FQYs) up to 100%. Receptor-binding, cytotoxicity, and cellular uptake studies confirmed the suitability of the PBIs as site-specific labels. In the second project, [G1]- to [G3]-dendronized PBIs were used for the synthesis of fluorescent polymer nanoparticles consisting of linear dendronized polyols (LDPs). The enhanced dendritic shielding effect of the various [Gn]-PBIs was evidenced by an increase in FQY of their respective LDPs from 7.8 to 23%. The incorporation of two different fluorophores into one polymer backbone led to large Stokes shifts caused by the occurrence of Förster resonance energy transfer (FRET). The fluorophore-conjugated LDPs showed no cytotoxic side effects and were successfully employed in cellular bioimaging studies. In the third project, single-walled carbon nanotubes (SWNTs) were functionalized with linear polymers equipped with neutral or charged [G2]-dendronized PBIs, leading to fluorescent polymer-SWNT complexes. The polymer wrapping improved the cytocompatibility of the nanotubes and enabled the direct imaging of their cellular uptake via the PBI and SWNT emission using the 1st and 2nd optical windows. Charged complexes showed superior SWNT dispersibility, intracellular fluorescence intensity, and cellular uptake over their neutral counterparts. A final complementary study affirmed the concept of dendritic site isolation of fluorophores using the example of a PG-dendronized near-infrared (NIR) cyanine derivative. The dye exhibited a pH-driven turn-on/turn-off fluorescence mechanism and showed good staining properties in the imaging of macrophages. In summary, the introduction of (charged) sterically demanding PG dendrons and monofunctionalized linkers on core-unsubstituted PBIs led to water-soluble, highly fluorescent, and site-specific biolabels. These results demonstrate the versatile potential of dendronized PBIs as fluorescent labels and promise their diverse application in future bioimaging studies.