Noncharged and Charged Monodendronised Perylene Bisimides as Highly Fluorescent Labels and their Bioconjugates

Abstract

A series of water-soluble, hydroxylated and sulphated, polyglycerol (PG) dendronised, monofunctional perylene bisimides (PBIs) were synthesised in three generations. Their photophysical properties were determined by absorption and emission spectroscopy and their suitability as potential biolabels examined by biological in vitro studies after bioconjugation. It could be shown that the photophysical properties of the PBI labels can be improved by increasing the sterical demand and ionic charge of the attached dendron. Thereby, charged labels show superior suppression of aggregation over charge neutral labels owing to electrostatic repulsion forces on the PG-dendron. The ionic charges also enabled a reduction in dendron generation while retaining the labels' outstanding fluorescence quantum yields (FQYs) up to 100 %. These core-unsubstituted perylene derivatives were successfully applied as fluorescent labels upon bioconjugation to the therapeutic antibody cetuximab. The dye-antibody conjugates showed a strongly enhanced aggregation tendency compared to the corresponding free dyes. Biological evaluation by receptor-binding, cellular uptake, and cytotoxicity studies revealed that labelling did not affect the antibody's function, which renders the noncharged and charged dendronised PBIs suitable candidates as fluorescent labels in biological imaging.

Hydrogen-bonded perylene bisimide J-aggregate aqua material

“…While at room temperature no significant photoluminescence could be detected, the formation of J-aggregates upon heating is accompanied by a fluorescence turn-on in the far-red region of the spectrum ( λ em = 647 nm). Such aggregation-induced emission phenomenon58 resembles the one observed for classical cyanine dye J-aggregates,59 and might be rationalized by Kasha's exciton coupling theory 29,60…”

“…1a) are considered archetypal dyes that combine outstanding absorption and fluorescence properties, thermal and photochemical robustness, and the strong tendency to form well-defined aggregates 25–28. Therefore, water-soluble PBIs have not only been extensively studied in the monomeric form for biological29–32 and biomedical33–36 applications, but also in functional supramolecular architectures 25,37–39. As an example, supramolecular hydrogels based on self-assembled PBIs have been reported40–42 showing applications as separation membranes,21,43 for photooxidation44 and for photocatalysis 45,46.…”

“…It is noteworthy that in all reported examples, PBI self-assembly is driven by an interplay of π–π-interactions and the hydrophobic effect. This affords a typical cofacial π-stacking arrangement that compromises the fluorescence properties of these dyes 29,32,48. On the contrary, the highly desired slipped-stack packing arrangement with J-type coupling and red-shifted absorption and fluorescence bands49,50 remains as a challenging goal in the hydrogel state.…”

Fluorescent Polymer—Single‐Walled Carbon Nanotube Complexes with Charged and Noncharged Dendronized Perylene Bisimides for Bioimaging Studies

“…Perylene bisimides (PBIs) possess excellent properties for bioimaging purposes such as high chemical and photophysical stability, outstanding fluorescence properties, and a long emission wavelength . They have been versatilely employed as fluorescent labels, membrane markers, or anti‐inflammatory agents . The striking photostability of PBI expressed in long fluorescence lifetimes of ≈4–5 ns and monoexponential fluorescence decays over at least 16 ns becomes obvious in comparison to other conventional fluorescent probes such as cy5 (0.91 ns), alexa fluor 647 (1.04 ns), and rhodamine B (1.74 ns) with significantly lower lifetimes.…”

“…However, a major drawback of PBI‐based dyes is their aggregation tendency in aqueous solution caused by the polyaromatic scaffold of the PBI cores resulting in fluorescence quenching. Charged groups have been introduced in the bay‐regions or imide positions to overcome the aggregation behavior and preserve their fluorescence properties with FQYs up to 100%. However, charged dyes inevitably raise the concern of nonspecific binding through electrostatic interactions with ionic cell components when being applied in bioimaging .…”