线粒体靶向的咖啡酸碳点的合成及其活性氧清除作用研究

Synthesis of caffeic acid carbon dots targeted by mitochondria and study of free radical scavenging activity

    摘要
  • 摘要:
    目的 合成线粒体靶向的碳点，探讨其对辐射诱导的活性氧（ROS）的清除作用。
    方法 采用水热法合成咖啡酸碳点（CfaCDs），采用1,1-二苯基-2-苦基肼（DPPH）实验评估CfaCDs对DPPH自由基的清除率，筛选DPPH自由基清除率最高的CfaCDs及其前驱体碳源（咖啡酸、精氨酸和柠檬酸）的最佳比例。采用三苯基膦（TPP）基团对CfaCDs进行功能化修饰，制备线粒体靶向的CfaCDs-TPP。通过透射电子显微镜、傅里叶变换红外光谱、紫外可见光谱、荧光光谱、和Zeta电位等技术对其进行表征。将人肠上皮细胞HIEC-6分为12组：对照组（不给药）、空白组（只加入CCK-8）、不同浓度（0.3、0.6、0.9、1.2、1.5 mg/ml）CfaCDs给药组和不同浓度（0.3、0.6、0.9、1.2、1.5 mg/ml）CfaCDs-TPP给药组，采用细胞计数试剂盒8（CCK-8）实验检测CfaCDs和CfaCDs-TPP的细胞毒性，使用流式细胞仪检测2′,7′-二氯二氢荧光素二乙酸酯荧光探针，评估CfaCDs和CfaCDs-TPP对辐射诱导活性氧（ROS）的清除效果。组间两两比较采用Student t 检验。
    结果 水热法合成CfaCDs时，前驱体碳源咖啡酸、精氨酸和柠檬酸的最佳投料比例为5 mmol∶3 mmol∶3 mmol，该条件下合成的CfaCDs的自由基清除率最高。透射电子显微镜分析结果显示，CfaCDs呈现均匀分散的球形形貌，粒径为（2.23±0.57） nm。紫外可见吸收光谱测定结果显示，CfaCDs和CfaCDs-TPP在300~350 nm范围内出现两个吸收峰，为C=C键的π→π跃迁，而在350~400 nm范围内出现一个吸收峰，为C=O/C=N键的n→π跃迁。荧光光谱测定结果显示，CfaCDs具有蓝色至绿色的荧光，而CfaCDs-TPP则表现出激发波长依赖的发射波长特性。傅里叶变换红外光谱分析结果显示，3180 cm−1处的宽峰对应于O-H/N-H的伸缩振动，1671 cm−1和1393 cm−1处的宽峰分别归属于C=N和C=O键的伸缩振动，1438 cm−1处的峰则为苯环C=C键的伸缩振动。此外，700~800 cm−1范围的峰归因于C–P键的伸缩振动。Zeta电位分析结果表明，经过TPP偶联后，CfaCDs表面的Zeta电位由负转为正。CCK-8实验结果显示，CfaCDs和CfaCDs-TPP在较高浓度（1.5 mg/ml）下均表现出极低的细胞毒性。流式细胞分析结果显示，CfaCDs给药组在高剂量时（1.0mg/mL），细胞中ROS的平均荧光强度与照射组相比明显降低533.70±18.77对463.00±2.83；t=5.032，P<0.05；CfaCDs-TPP给药组在不同浓度时（0.4、0.6、0.8、1.0 mg/ml），细胞中ROS的荧光强度与照射组相比均明显降低 533.70±18.77对469.00±4.24、533.70±18.77对401.5±6.36、533.70±18.77对427.5±0.71、533.70±18.77对399.00±16.97，差异均有统计学意义（t=4.564~9.186，均p<0.05）。
    结论 CfaCDs-TPP对ROS具有良好的清除效果，能够明显降低辐射诱导的ROS的升高。

     

    Abstract:
    Objective To synthesize mitochondria-targeted carbon dots and investigate their free radical scavenging effect against radiation-induced radicals.
    Methods Caffeic acid carbon dots (CfaCDs) were synthesized by hydrothermal method. The free radical scavenging ability of CfaCDs was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. CfaCDs with the highest free radical scavenging efficiency were selected for functionalization with triphenylphosphine (TPP) to prepare mitochondria-targeted CfaCDs-TPP. These were characterized by transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, UV-Vis spectroscopy, fluorescence spectroscopy, and Zeta potential analysis. Human intestinal epithelial cells (HIEC-6) were divided into 12 groups: control group (no treatment), blank group (only CCK-8), different concentrations (0.3, 0.6, 0.9, 1.2, 1.5 mg/ml) of CfaCDs treatment groups, and different concentrations (0.3, 0.6, 0.9, 1.2, 1.5 mg/ml) of CfaCDs-TPP treatment groups. Cell toxicity was assessed using the CCK-8 assay. To evaluate the scavenging effect on radiation-induced reactive oxygen species (ROS), HIEC-6 cells were also divided into the same 12 groups, and ROS levels were measured using flow cytometry with the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Statistical comparisons between groups were performed using Student′s t-test.
    Results The optimal molar ratio for synthesizing CfaCDs by hydrothermal method was 5 mmol∶3 mmol∶3 mmol of caffeic acid, arginine, and citric acid, respectively, which resulted in the strongest free radical scavenging ability. CfaCDs exhibited uniform spherical morphology with a diameter of 2.23±0.57 nm. The UV-Vis absorption spectra of CfaCDs and CfaCDs-TPP showed two peaks in the range of 300–350 nm, attributed to the π→π* transition of C=C bonds, and one peak in the range of 350–400 nm, attributed to the n→π* transition of C=O/C=N bonds. CfaCDs displayed blue-to-green fluorescence, whereas CfaCDs-TPP showed excitation-wavelength-dependent emission characteristics. FTIR analysis revealed a broad peak around 3180 cm−1 corresponding to O-H/N-H stretching vibrations, while peaks at 1671 cm−1 and 1393 cm−1 were attributed to C=N and C=O stretching vibrations, and the peak at 1438 cm−1 was related to the C=C stretching vibration in the benzene ring. Additionally, peaks in the 700–800 cm−1 range were attributed to C–P bond stretching vibrations. Zeta potential analysis showed that after TPP conjugation, the Zeta potential of CfaCDs shifted from negative to positive. Both CfaCDs and CfaCDs-TPP exhibited very low cytotoxicity at higher concentrations. Flow cytometry results indicated that in the CfaCDs treatment group at a high dose (1.0 mg/mL), the average fluorescence intensity of ROS in cells significantly decreased compared to the radiation-only group 533.70±18.77 vs. 463.00±2.83; t=5.032, P<0.05. CfaCDs-TPP, at various concentrations (0.4, 0.6, 0.8, 1.0 mg/mL), also significantly reduced the fluorescence intensity of ROS in cells 533.70±18.77 vs. 469.00±4.24; 533.70±18.77 vs. 401.5±6.36; 533.70±18.77 vs. 427.5±0.71; 533.70±18.77 vs. 399.00±16.97, with all differences being statistically significant (t=4.564−9.186, all P<0.05).
    Conclusion CfaCDs-TPP exhibits excellent free radical scavenging ability and significantly reduces radiation-induced ROS levels.

     

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