基于GSH响应共价成环的AIE自组装探针的构建及体外评价

Construction and in vitro evaluation of AIE self-assembled probe based on GSH response covalent cyclization

目的构建基于谷胱甘肽(GSH)响应共价成环的聚集诱导发光(AIE)自组装探针并进行体外评价。方法通过固相多肽合成法制备含2-氰基-6-氨基苯并噻唑-半胱氨酸(CBT-Cys)缩合基序的多肽序列,再经点击化学反应与AIE分子偶联,构建了一种基于GSH响应共价成环的AIE自组装探针(记作探针1),同时以缺乏Cys结构的探针2为对照。测试探针的吸收和发射光谱,分析探针对GSH的特异性及响应后的粒径和结构变化,考察不同pH值、温度、探针浓度和GSH浓度对探针荧光强度的影响,并评价探针对肿瘤细胞HeLa、HepG2和MDA-MB-231的毒性。结果经GSH响应后,探针1的荧光增强约6倍,探针2的荧光增强约2倍;探针1转化为二聚体,粒径约896.1 nm,探针2缺乏成环基序,仅转化为单体,粒径约427.4 nm。在pH值为7.0、温度为37℃条件下,探针1的荧光强度显著高于探针2。两种探针对肿瘤细胞HeLa、HepG2和MDA-MB-231的毒性均较低。结论探针1分子中的二硫键经GSH还原后,分子因失去亲水序列导致荧光开启(第1次聚集),并因含有CBT-Cys成环基序随即生成AIE二聚体(第2次聚集)。与探针2单次聚集相比,探针1实现了双重AIE效应,显著增强了荧光强度,并在生理环境下具有较好的适用性,为体内原位生成共价成环探针提供了思路,后期有望用于体内肿瘤成像与治疗。

Objective To construct an aggregation induced emission(AIE)self-assembled probe based on glutathione(GSH)response covalent cyclization and evaluate it in vitro.Methods The peptide sequence containing the 2-cyano-6-aminobenzothiazole-cysteine(CBT-Cys)condensation sequence was synthesized by the solid-phase peptide synthesis method.After coupling with an AIE molecule by click chemical reaction,an AIE self-assembled probe 1 based on GSH response covalent cyclization was constructed,and probe 2 lacking Cys structure was used as the control.The absorption and emission spectra of probes were tested and the specificity of probes to GSH was analyzed.The hydrodynamic diameter and structure of the probes after response were compared.The effects of different pH values,temperatures,probe concentrations,and GSH concentrations on fluorescence intensity were investigated.The toxicity of probes to tumor cells such as HeLa,HepG2 and MDA-MB-231 was evaluated.Results After GSH response,the fluorescence of probe 1 was enhanced by about 6 times and that of probe 2 was enhanced by about 2 times;probe 1 was converted into a dimer with a hydrodynamic diameter of about 896.1 nm.Probe 2 lacked a cyclization motif and was converted into a monomer with a hydrodynamic diameter of about 427.4 nm.The fluorescence intensity of probe 1 was significantly higher than that of probe 2 at pH=7.0 and 37℃,and the toxicity of probes to tumor cells(HeLa,HepG2 and MDA-MB-231)was low.Conclusions After the disulfide bond of probe 1 was reduced by GSH,the probe molecule lost the hydrophilic sequence,resulting in fluorescence turn-on(the first aggregation),and probe 1 immediately generates an AIE dimer(the second aggregation)because it contains a CBT-Cys cyclization sequence,which realizes the dual AIE effect compared with the single aggregation of probe 2,and significantly enhances the fluorescence emission.Probe 1 has better applicability in physiological environments,which provides an idea for in-situ generation of covalent cycling probes in vivo and is expected to be used in tumor imaging and treatment in the later stages.