Ultrabright Red to NIR Emitting Fluorescent Organic Nanoparticles Made from Quadrupolar Dyes with Giant Two-Photon Absorption (2PA) in the NIR Region. Confinement Effect on Fluorescence and 2PA and Tuning of Surface Properties

Abstract

Fluorescent Organic Nanoparticles (FONs) are light-emitting, molecular-based nanoparticles that can be obtained from the nanoprecipitation of dedicated hydrophobic organic dyes in water. They represent a versatile family of water-soluble fluorescent nanotools whose luminescent properties can be tuned by bottom-up molecular engineering of their composing dyes. We recently reported on a quadrupolar red-emitting dye that yields spontaneously stealthy bare FONs which do not require coating to hinder interactions with cell membranes. Its quadrupolar design also hints to the possibility that it may be a strong two-photon absorber for bioimaging purposes. In this paper, we further investigate the two-photon absorption (2PA) of this dye and resulting FONs and report on a structure-related series of extended dyes engineered to yield NIR-emitting FONs. All dyes lead to stable, small (12–20 nm in diameter), and bright FONs. The experimental study reveals that molecular confinement strongly influences the fluorescence and 2PA properties of these dyes depending on the nature of the π-extended system. As expected, extension of the π-conjugated system induces a red-shift of the absorption and emission bands as well as an increase and spectral broadening of the 2PA responses in solution. Upon aggregation of the dyes within nanoparticles, a reduction of the fluorescence quantum yield is observed whose amplitude depends strongly on the nature of the π-conjugated systems. Interestingly, the peak 2PA cross sections increase upon confinement of the shortest dye, while a spectral broadening and slight red shift of the 2PA bands of the most extended dyes is observed. Taken together, these properties allow the most extended dye to yield very bright NIR-emitting FONs (εmaxΦ = 7 × 106 M–1 cm–1, σ2 maxΦ = 4 × 105 GM). Interestingly, the nature of the π-conjugated system was also found to modulate the stealthiness of the resulting nanoparticles toward biological membranes. As a result, by fine-tuning the molecular design of the quadrupolar FON-composing dyes, we achieved NIR-absorbing, NIR-emitting, spontaneously stealthy small nanoparticles having record one- and two-photon brightness. Finally, we demonstrate that these FONs can be noncovalently surface-coated with a polycationic polymer, thanks to their highly negative surface potentials. This induces a reversal of their surface potential which in turn triggers their internalization within cells.

Stealth Luminescent Organic Nanoparticles Made from Quadrupolar Dyes for Two-Photon Bioimaging: Effect of End-Groups and Core

“…(i) A strong hydrophobic character to allow nanoprecipitation in water; (ii) a quadrupolar symmetry (D-π-A-π-D motif) favourable for 2PA; (iii) a planar, π-conjugated ethynyl bridge for red-shifted emission; and (iv) bulky triphenylamine end-groups and butyl side chains to hinder π-stacking by steric hindrance that would be deleterious to fluorescence emission. We have previously reported on the photo-physical properties, confinement effects, and stealth FONs of this chromophore in the context of modulating the extent of conjugation of the π-bridge [ 30 ]. In order to further study the structure-property relationships of this dye, we now report on chromophores designed to vary donor and acceptor strengths.…”

“…The latter, therefore, display a larger 2P brightness relative to their volume (33 GM/nm 3 ). Taken together, increasing the strength of the end-groups of our quadrupolar dyes turns out to be less efficient in terms of the ‘2P brightness vs. size’ figure of merit than the modification and lengthening of the π-linker (~100–200 GM/nm 3 [ 30 ]).…”

“…As previously described with Q 2 FONs [ 30 ], we next assessed the stealth behaviour of FONs by z-stack confocal microscopy under 1P or 2P excitation. As a positive control for internalisation, we also imaged FONs previously coated with poly(allylamine hydrochloride) (PAH), a positively charged polymer that promotes NP interactions with cellular membranes [ 50 ].…”

“…We then modulated the π-conjugation of the quadrupolar template of the composing chromophores to yield FONs of red-shifted emission. However, we found that these slight modifications of the chromophores’ chemical structure could influence the stealth behaviour of their derived FONs [ 30 ]. Here, we further explore this link between chromophore structure and FONs nano-bio interactions by presenting a structurally related series of chromophores varying in the strength of their dipolar moments.…”

“…Here, we further explore this link between chromophore structure and FONs nano-bio interactions by presenting a structurally related series of chromophores varying in the strength of their dipolar moments. To do so, starting from a dye template of known properties [ 30 ], we designed novel chromophores bearing triphenylamine end-groups either weakened or strengthened in their electron-donating character—by substitution with aldehydes or methoxy groups respectively—as well as a chromophore having a core with increased electro-acceptor strength—by introduction of a (bis)benzothiadiazole motif. We report on the photo-physical properties of these dyes, including their 2PA capacity and red to NIR emission, study the effects of nano-confinement on those properties and show that the stealth behaviour of their derived FONs can be maintained and even enhanced in the presence of these modifications.…”