Red-emitting fluorescent probes based on sulfur-substituted barbituric acid derivatives: synthesis, properties and biomedical applications

Abstract

Fluorescence bioimaging has emerged as a vital tool for tracking dynamic biochemical processes and monitoring disease biomarkers in vivo. This technique has demonstrated significant clinical and commercial potential in fluorescence-guided surgery (FGS) for tumor treatment, attributed to its real-time visualization capabilities and high biocompatibility. To achieve precise delineation of tumor boundaries and complete tumor resection, the rational design and selection of contrast agents are critical. Herein, we report a novel red-emitting fluorophore, TTS, based on thiobarbituric acid (TBA) derivatives and tetraphenylethylene. The design strategy, synthetic route, optical properties, and cytotoxicity of TTS are systematically described. Benefiting from its highly twisted molecular conformation and enhanced intramolecular charge transfer (ICT) effect, TTS exhibits superior aggregation-induced emission (AIE) characteristics with emission wavelengths extending into the NIR-I region (700-900 nm), as well as a reactive oxygen species (ROS)-generating capability. Systematic in vivo evaluations further demonstrate that TTS nanoparticles (NPs), which were formulated with DSPE-PEG2000 encapsulation, can accurately illuminate osteosarcoma tissues and effectively guide surgical resection through the enhanced permeability and retention (EPR) effect. This novel fluorophore not only boosts the development of AIE-based medical materials but also benefits intraoperative fluorescence imaging for clinical applications.

Scheme 1. Schematic illustration of TTO/TTS structures and in vivo imaging in mouse models.

Scheme 2. Synthetic routes to TTO and TTS.

Fig. 1. (A) UV-vis absorption spectra of TTS in various organic solvents. (B) Fluorescence emission spectra of TTS in various organic solvents. (C) Normalized fluorescence emission spectra of TTS versus water fraction (f_w) in water/tetrahydrofuran (THF) mixtures. (D) Plot of PL peak intensities of TTO and TTS versus water fraction (f_w) in water/THF mixtures. I₀ and I represent the PL intensities of the compounds in water/THF mixtures with f_w = 10% and f_w > 10%, respectively.

Fig. 2. Fluorescence intensity enhancement of DCF at 524 nm versus light irradiation time of TTO, TTS, TTO + TBPA, TTS + TBPA and methylene blue.

Fig. 3. (A) HOMO–LUMO plots of TTO and TTS calculated by the DFT/ωB97XD/def2-TZVP. (B) Energy diagrams of TTO and TTS as calculated using TD-DFT.

Fig. 4. (A) Dynamic light scattering (DLS) diameter of TTO NPs; inset: transmission electron microscopy (TEM) image of the NPs. (B) DLS diameter of TTS NPs; inset: TEM image of the NPs. (C) UV-vis absorption and fluorescence emission spectra of TTO NPs. (D) UV-vis absorption and fluorescence emission spectra of TTS NPs.

Fig. 5. Fluorescence imaging and average radiance of TTO and TTS in the LM8 osteosarcoma subcutaneous tumors model at different time points. The area of radiance sampling is denoted by black circles.

Fig. 6. Preoperative and postoperative fluorescence images of the mouse bearing LM8 osteosarcoma subcutaneous tumors 12 hours after administering 160 μmol L⁻¹ of TTO and ex vivo fluorescence images of the same mouse. The actual position of tumor is denoted by a black circle.

Fig. 7. Preoperative and postoperative fluorescence images of the mouse bearing LM8 osteosarcoma subcutaneous tumors 12 hours after administering 160 μmol L⁻¹ of TTS and ex vivo fluorescence images of the same mouse. The actual position of tumor is denoted by a black circle.