靶向E-选择素的新型荧光探针的设计合成与生物学评价

Design,synthesis and biological evaluation of a novel fluorescent probe targeting E-selectin

目的设计合成靶向E-选择素的新型荧光探针,为肿瘤转移及其靶向抗肿瘤药物相关研究提供工具分子。方法以E-选择素配体寡肽(IELLQAR)为靶向组件,使其通过连接桥与异硫氰酸荧光素(FITC)偶联,设计合成一种新型靶向E-选择素的荧光探针(IELLQAR-FITC)。合成采用固相法,首先合成了寡肽IELLQAR,再以半胱氨酸和氨基己酸为连接桥,将FITC与寡肽连接,最后,通过裂解和脱保护基,得到了目标荧光探针IELLQAR-FITC,并对该目标荧光探针进行了系列生物学评价。结果MALDI-TOF/MS分析表明,成功合成了目标荧光探针分子(相对分子质量1447.5),MTT测试结果显示该荧光探针无明显毒性,对血管内皮细胞(HUVEC)的增殖活性无明显影响。细胞黏附实验结果表明,该荧光探针具有较好的E-选择素结合能力,能与肿瘤细胞HL-60竞争性地结合HUVEC表面的E-选择素,从而抑制肿瘤细胞HL-60与HUVEC细胞的黏附;同时该荧光探针能标记HUVEC表面的E-选择素,在460~550 nm波长下荧光基团FITC的荧光清晰可见,为E-选择素的可视化和定量研究提供了分子工具。结论本文设计合成的新型靶向E-选择素的荧光探针具有合成简单、观察方便等优点,为靶向E-选择素相关研究提供了有益分子工具。

Objective To design and synthesize a novel fluorescent probe targeting E-selectin and provide a tool molecule for the research on tumor metastases and antitumor drugs,particularly,the drugs targeting the metastasis.Methods The peptide IEL LQAR,an E-selectin liigand,was used as a targeting module to design and synthesize an E-selectin-targeting probe by linking IEL LQAR with the fluorescent molecule,fluorescein isothiocyanate(FITC),via a proper linker to obtain a fluorescent peptide probe(IELLQAR-FITC).By the solid-phase synthesis method,the peptide ligand IELLQAR was synthesized at first,then the obtained IEL LQAR was connected with TITC via the linker bridge consisting of cysteine and aminohexanic acid residues,and eventually,IEL LQAR-FITC was obtained by the cleavage and removal of protecting groups.A series of biological evaluation was performed for the syn thesized fluorescent peptide probe molecule.Results The aimed fluorescent peptide probe molecule was successfully synthesized as indicated by the MALDI-TOF/MS analysis(relative molecular mass 1447.5).In the MTT test,the fluorescent peptide probe showed no significant toxicity and did not inhibit the proliferation of HUVEC cells.The results of the cell adhesion assay showed that the fluo rescent peptide probe had a great binding ability to the E-selectin protein,and could competitively bind to the E-selectin protein ex pressed on the surface of HUVEC cells,competing with HL-60 cells,thus significantly inhibiting the adhesion between the HL-60 cells and HUVEC cells.Meanwhile,the fluorescent probe could mark the E-selectin on the surface of HUVEC cells,and the fluores cence due to FITC was clearly visible at the wavelength of 460-550 nm,which provided an effective tool for visualization and quantification of E-selectin.Conclusion The present E-selectin-targeting new fluorescent probe could be simply synthesized and convenient ly detected,which provides a useful molecule tool for the E-selectin related studies.