新型肿瘤归巢穿膜肽的固相合成及体外活性研究

Solid-phase synthesis and in vitro activity of a new tumor homing penetrating peptide

目的:采用固相法人工合成异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的新型肿瘤归巢穿膜肽t Ly P-1(CGNKRTR),并于体外检测其肿瘤归巢及穿膜特性。方法:采用Na-芴甲氧羰基(Fmoc)法固相合成t Ly P-1,并用FITC标记N端。高效液相色谱及质谱仪检测其纯度及分子量;激光共聚焦显微镜及流式细胞仪观察其肿瘤归巢及穿膜能力;CCK8(cell counting kit-8)评价其细胞毒性。结果:合成的FITC-t Ly P-1纯度为99.14%,分子量为1 334.7;激光共聚焦显微镜可见FITCt Ly P-1在MDA-MB-231组细胞内分布多于HUVEC组,流式细胞仪检测MDA-MB-231组细胞内荧光强度值高于HUVEC组;CCK8检测不同浓度的FITC-t Ly P-1对细胞活性没有明显影响(P>0.05)。结论:采用固相法成功合成FITC标记的新型肿瘤归巢穿膜肽t Ly P-1,具有乳腺癌MDA-MB-231肿瘤靶向性和穿膜性,能作为一种潜在的肿瘤靶向药物运输载体。

Objective：To synthesize a new tumor homing penetrating peptide with fluorescein isothiocyanate（FITC）by solid-phase synthesis method,tLyP-1（CGNKRTR）,and to investigate its tumor homing and cell-penetrating properties in vitro. Methods：The FITC-tLyP-1 was solid-phase synthesized by the Na-fluorene methoxyl carbonyl method（Fmoc）,and its N-terminal was labeled by FITC. The purity and molecular weight of the FITC-tLyP-1 were determined using HPLC and MALDI-TOF-MS. The tumor homing and cell-penetrating properties of the FITC-tLyP-1were examined by confocal laser scanning microscopy and flow cytometry. The cytotoxicity of the FITC-tLyP-1 was evaluated by CCK8 assay. Results：The purity of FITC-tLyP-1 was 99.14%,and molecular weight was1 334.7. The confocal laser scanning microscopy showed that intracellular distribution of FITC-tLyP-1 in MDA-MB-231 group was more than that of HUVEC group. The flow cytometry showed that the intracellular fluorescence intensity in MDA-MB-231 group was higher than that of HUVEC group. The CCK8 assay indicated that different concentrations of FITC-tLyP-1 had no significant effects on cell activity（P〈0.05）. Conclusion：A new tumor homing penetrating peptide with FITC（FITC-tLyP-1）is successfully synthesized by solid-phase synthesis method. FITC-tLyP-1 has the tumor homing and cell-penetrating properties in vitro,which can be used as a potential carrier for tumor targeting and drug transporting.