摘要/Abstract
绿色荧光蛋白(GFP)生色团由于良好的生物相容性、低暗毒性和光稳定性而备受关注, 然而, 将GFP生色团用于光动力治疗鲜有报道. 以N-丁基吩噻嗪-3-甲醛、甘氨酸叔丁酯盐酸盐和亚胺酯为原料, 通过[2+3]环加成反应合成了一种吩噻嗪荧光蛋白生色团类新型光敏剂(Ptz-FP), Ptz-FP是一种不含重原子、由S原子促进的具有优异单线态氧产生效果的光敏剂, 可用于光动力治疗. 在二甲基亚砜(DMSO)溶液中, Ptz-FP光敏剂的吸收波长位于436 nm, 此处的摩尔消光系数为1.4×104 L•mol–1•cm–1, 发射波长在626 nm, Stokes位移高达190 nm, 荧光量子产率为1.5%. 以Ru(bpy)3Cl2为参比, 测得光敏剂Ptz-FP在甲醇溶液中单线态氧产率为33.1%. 通过噻唑蓝(MTT)法测试了光敏剂Ptz-FP的暗毒性和光毒性, 结果表明其具有较低的细胞毒性(≥89.9%)和较高的光毒性, 能在30 min内杀死50%以上的A-549细胞. 此外, 通过活性氧检测试剂盒DCFH-DA在15 min内成功检测到A-549细胞中活性氧的产生, 并由AO/EB活死细胞染色剂监测到A-549细胞凋亡过程. 最后, 光敏剂Ptz-FP成功应用于双光子荧光成像. 光敏剂Ptz-FP有望为今后荧光成像指导的荧光蛋白生色团类光敏剂的开发奠定基础.
关键词: 荧光蛋白生色团, 含S原子的光敏剂, 光动力治疗, 双光子荧光成像
The green fluorescent protein (GFP) chromophore has attracted much attention due to its good biocompatibility, low dark cytotoxicity and high photostability. However, there are rare reports about GFP chromophore analogues in photodynamic therapy (PDT). In this paper, a new photosensiter phenothiazine fluorescent protein (Ptz-FP) chromophore was synthesized by [2+3] cycloaddition reaction using N-butylphenothiazine-3-carbaldehyde, tert-butyl glycine hydrochloride and imine ester as raw materials. Ptz-FP was a heavy-atom free photosensitizer that promoted by S atom and showed excellent singlet oxygen generation which can be used in PDT. In dimethyl sulfoxide (DMSO) solution, the absorption wavelength of Ptz-FP photosensitizer was at 436 nm, the molar extinction coefficient was 1.4×104 L•mol–1•cm–1, the emission wavelength was at 626 nm, stokes shift was 190 nm, and the fluorescence quantum yield was 1.5%. The singlet oxygen yield of Ptz-FP was 33.1% using Ru(bpy)3Cl2 as a reference in methanol solution. The dark cytotoxicity and phototoxicity of the photosensitizer Ptz-FP were tested by methyl thiazolyl tetrazolium (MTT) assay. The results showed that it had negligible dark toxicity (≥89.9%) and excellent phototoxicity. Besides that, DCFH-DA (ROS indicator) successfully detected the production of reactive oxygen in A-549 cells within 30 min, and the apoptosis process of A-549 cells was monitored by the AO/EB stain assay. Finally, the photosensitizer Ptz-FP was successfully used in two-photon fluorescence imaging. Therefore, the photosensitizer Ptz-FP is expected to lay a foundation for FP chromophore based photosensitizers on fluorescence imaging guided PDT in the future.
Key words: fluorescent protein chromophore, containing S atom photosensitizer, photodynamic therapy, two-photon fluorescence imaging
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