Fluorescent enhanced small molecular dye for NIR-II imaging

Objectives: Within the last five years, the development of new fluorophores emitting in the second near-infrared widow (NIR-II) at wavelengths ranging from 1,000-1,700 nm has allowed visualization of deep anatomical features with an unprecedented degree of clarity. However, the majority of NIR-II fluorophores suffer from low quantum yields, ~0.01-1.4% compared with the visible or NIR-I (~750-1000 nm) regions ~80% and ~10%. In this study, we demonstrate that changing the functional groups from carboxylic to sulfonic acid results in a completely water-soluble organic NIR-II dye (CH-4T) that readily forms supramolecular assemblies with plasma proteins to produce a brilliant increase in fluorescent brightness. Human serum albumin(HSA) fluorescent enhanced CH-4T was used for brain vessel, lymph nodes, tumor PET and NIR-II fluorescent dual mode imaging. It shows a cheerful prospect in clinical application.

Methods: CH-4T was synthesized by CH1055, excess taurine dehydration condensation. After HPLC purification, pure CH-4T was dissolved in HSA PBS solution. For dual mode tumor imaging, CH-4T was mixed with NODAGA conjugated HSA. The dye-protein mixture was heated to 70 ^oC for 10mins before injecting into mice or before labeling with ⁶⁴Cu. Optical characterization was measured the same as our previous work¹. The brain vessel NIR-II fluorescent imaging of 4 hair removed C57BL/6 mice were gotten by tail vein injection of CH-4T/HSA heated probe. The popliteal and sacral lymph nodes were imaged in nude mice in a prone position with fluorophores (CH-4T/PBS and CH-4T/HSA heated) injected in both footpads. CH-4T/HSA-NODAGA-⁶⁴Cu heated probe was iv injected into the U87MG bearing nude mice (4 per group). NIR-II and PET-CT imaging were gotten at the time point 1h, 4h, 12h, 1d, 2d, 3d, 4d.

Results: CH-4T was synthesized with 93% yield. HSA and FBS show ~17-fold and ~34-fold fluorescence enhancement before heating. After heating, HSA and FBS show ~100-fold enhancement with the quantum yield around 11%. Mouse cerebral vasculature imaging was gotten with sub-10-μm resolution after CH-4T/HSA heated(HT) complex iv injection. Popliteal and sacral lymph nodes were imaged in nude mice in a prone position with CH-4T/PBS injected in left footpad and CH-4T/HSA HT in right. The lymph node-to-background ratios are 4, 2, 32, 17 respectively. ~3 times U87MG tumor-to-background ratio was achieved in NIR-II imaging with 3d PI of CH-4T/HSA-NODAGA HT. PET imaging showed the accumulation of CH-4T/HSA-NODAGA ⁶⁴Cu HT in tumor 12h PI. 1. Antaris, A.L. et al. A small-molecule dye for NIR-II imaging. Nat Mater 15, 235-242 (2016).

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