通过酸敏四肽构建载吉西他滨免疫纳米微粒的方法  

The method for construction of gemcitabine immunized nanoparticles through GFLG-nitrophenyl ester peptide

作　　者：李佳佳[1] 李雅晴[1] 练国达[1] 陈少杰[1] 曾林涓[2] 李若梦 邹金茂 黄开红[1] 陈茵婷[1] LI Jiajia 1, LI Yaqing 1, LIAN Guoda 1, CHEN Shaojie 1, ZENG Linjuan 2, LI Ruomeng 1,ZOU Jinmao 1, HUANG Kaihong 1, CHEN Yinting 1(1. Department of Gastroenterology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China;2. Department of Oncology, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong 519000, Chin)

机构地区：[1]中山大学孙逸仙纪念医院消化内科,广州510120 [2]中山大学附属第五医院肿瘤科,广东珠海519000

出　　处：《中国科技论文》2018年第6期714-720,共7页China Sciencepaper

基　　金：高等学校博士学科点专项科研基金资助项目(20130171120093);国家自然科学基金资助项目(81672408;81572396)

摘　　要：通过酸敏四肽构建靶向载吉西他滨(gemcitabine,Gem)免疫纳米微粒,为载药纳米合成提供新思路。Gem和IONP(iron oxide nanoparticles)通过酸敏四肽链接,形成Gem-IONP-NPs;将其与CD44v6单链抗体(single chain antibody fragment,scFv)偶联,形成scFv-Gem-IONP-NPs。高效液相色谱仪检测Gem释放,细胞免疫荧光验证抗体偶联,MTS法检测Gem、Gem-IONPNPs和scFv-Gem-IONP-NPs细胞杀伤能力,流式细胞学检测各组细胞凋亡情况,皮下移植瘤模型验证各组体内抑瘤作用。结果表明:纳米微粒在肿瘤组织酸性环境下释放更多Gem,细胞免疫荧光证实抗体偶联成功,细胞毒性及凋亡细胞增加、移植瘤重量降低证实靶向组杀伤作用强于非靶向组,具有统计学差别(P<0.05)。酸敏四肽偶联吉西他滨及单链抗体,可成功制备胰腺癌靶向的免疫纳米微粒,增强抗瘤作用。Gemcitabine（Gem）nanoparticles were constructed by GFLG-nitrophenyl ester peptide,and their targeted anti-tumor effects were verified,providing a new idea for the synthesis of drug loaded nanoparticles.To synthetize the complex of GemIONP-NPs,we combine the iron oxide nanoparticles（IONP）and gemcitabine according to a certain ratio and through the chemical reaction induced by apolypeptide（9-luorenylmethoxycarbonyl-（Fmoc）-Gly-Phe-Leu-Gly（GFLG）-nitrophenyl ester peptide）.Then,the single chain antibody fragment of CD44 v6（scFv）,which targets at the pancreatic cancer cells,was linked to it to gain the complex of scFv-Gem-IONP-NPs.The dissolution curve of drug release was detected by high performance liquid chromatography（HPLC）,the targeted function of scFv was evidenced using near-infrared fluorescent（NIRF）imaging.And the cytotoxicity and the apoptotic cells caused by the group of Gem,Gem-IONP-NP and scFv-Gem-IONP-NPs were tested by MTS method and ANNEXIN V/PI dye flow cytometry analysis.The anti-tumor effect was verified by the subcutaneous transplantation model in vivo.The results show that the nanoparticle complex releases more Gem in the mild acidic environment.When the scFv-GemIONP-NPs complexes are uptaken by pancreatic cancer cells Panc-1,the fluorescence of the scFv can be found in cell membrane and cytoplasma under the fluorescence microscopy.The scFv-Gem-IONP-NPs group received a more powerful synergetic effect than the other groups,including the Gem and Gem-IONP-NPs.The results are illustrated adequately through the increasing cytotoxicity,the number of apoptotic cells and the weight of the tumors.Generally,we successfully developed a targeted nanoparticle of gemcitabine using the GFLG-nitrophenyl ester peptide and scFv.The targeting and cell killing effects on the pancreatic cancer were confirmed.

关 键 词：消化系统疾病 胰腺癌 CD44V6 吉西他滨 酸敏四肽 靶向纳米微粒 

分 类 号：R576[医药卫生—消化系统]