Objective To synthesize mitochondria-targeted caffeic acid carbon dots (CfaCDs) conjugated with triphenylphosphonium (TPP) and investigate its scavenging effect on radiation-induced reactive oxygen species (ROS).

Methods CfaCDs were synthesized via a hydrothermal method. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay was performed to evaluate the DPPH radical scavenging rate of CfaCDs, and the optimal precursor carbon source ratio (caffeic acid, arginine, and citric acid) was selected based on the highest scavenging rate. TPP groups were utilized to functionalize CfaCDs to prepare mitochondria-targeted CfaCDs-TPP. CfaCDs and CfaCDs-TPP were characterized through transmission electron microscopy, Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, fluorescence spectroscopy, and Zeta potential analysis. Human intestinal epithelial HIEC-6 cells were divided into 12 groups: control (no treatment), blank (cell counting kit-8 (CCK-8) only), CfaCDs-treated (0.3, 0.6, 0.9, 1.2 and 1.5 mg/ml), and CfaCDs-TPP-treated (0.3, 0.6, 0.9, 1.2 and 1.5 mg/ml) groups. Cytotoxicity of CfaCDs and CfaCDs-TPP were assessed via CCK-8 assay. HIEC-6 cells were divided into 12 groups: control (no treatment), irradiation-only (6 Gy), CfaCDs-treated (0.2, 0.4, 0.6, 0.8, and 1.0 mg/ml)+irradiation (6 Gy), and CfaCD-TPP-treated (0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml)+irradiation (6 Gy) groups. The fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate was used with flow cytometry to assess the scavenging effect of CfaCDs and CfaCDs-TPP on radiation-induced ROS. Pairwise comparisons between groups were performed using Student′s t-test.

Results The optimal precursor carbon source ratio (caffeic acid∶arginine∶citric acid) for CfaCDs synthesis was 5 mmol∶3 mmol∶3 mmol, which achieved the highest DPPH radical scavenging rate of 90.79%. Transmission electron microscopy analysis revealed uniformly dispersed spherical CfaCDs with a particle diameter of (2.23±0.57) nm. Ultraviolet-visible spectroscopy showed that CfaCDs and CfaCDs-TPP exhibited two absorption peaks at 300–350 nm, which corresponded to the π→π^* transitions of C=C bonds, and one absorption peak at 350−400 nm, which indicated the n→π^* transitions of C=O/C=N bonds. Fluorescence spectroscopy demonstrated the blue-to-green fluorescence emitted by CfaCDs and excitation wavelength-dependent emission wavelength characteristics exhibited by CfaCDs-TPP. Fourier transform infrared spectroscopy identified a broad peak at 3 180 cm⁻¹, which corresponded to O−H/N−H stretching vibrations, broad peaks at 1 671 and 1 393 cm⁻¹, which were attributed to C=N and C=O stretching vibrations, respectively, and a peak at 1 438 cm⁻¹ assigned to aromatic C=C stretching vibrations. In addition, peaks in the 800–700 cm⁻¹ range were ascribed to C–P stretching vibrations. Zeta potential analysis unveiled that after TPP conjugation, the surface Zeta potential of CfaCDs shifted from negative to positive. CCK-8 assay results indicate that CfaCDs and CfaCDs-TPP exhibited very low cytotoxicity even at high concentrations (1.5 mg/ml). Flow cytometry analysis revealed that in the CfaCDs-treated+irradiation group, at a high concentration (1.0 mg/ml), the fluorescence intensity of ROS in cells was significantly lower than that in the irradiation-only group (463.00±2.83 vs. 533.70±18.77; t=5.032, P<0.05). For CfaCDs-TPP-treated+irradiation groups, at various concentrations (0.4, 0.6, 0.8, and 1.0 mg/ml), the ROS fluorescence intensities were all significantly lower than those observed in the irradiation-only group (469.00±4.24 vs. 533.70±18.77, 401.50±6.36 vs. 533.70±18.77, 427.50±0.71 vs. 533.70±18.77, and 399.00±16.97 vs. 533.70±18.77), with all differences showing statistical significance (t=4.564−9.186, all P<0.05).

Conclusions Mitochondria-targeted CfaCDs-TPP were successfully synthesized. CfaCDs-TPP demonstrated excellent ROS scavenging effects and can significantly reduce radiation-induced ROS elevation.