微流控肠-肝-乳腺癌芯片的构建及其体外药物PK-PD分析

目的:基于微流控技术构建肠-肝-乳腺癌多器官芯片并将其用于药物的体外药物代谢动力学-药物效应动力学(PK-PD)研究。方法:利用微流控技术构建包含4层聚二甲基硅氧烷(PDMS)基板和2层聚甲基丙烯酸甲酯(PMMA)盖板的多器官芯片;分别使用Cell Tracker Red/Green和Hoechst对生长21 d的人结肠癌细胞株Caco-2细胞层和生长3 d的人脐静脉内皮细胞株HUVEC细胞层进行染色,考察细胞间的连接情况,通过测定2 g·L^-1荧光素钠和33.28 mg·L^-1普萘洛尔跨细胞层的透过率来验证所建肠模块的功能;通过比较125μmol·L^-1环磷酰胺经过常规孔板中人肝癌细胞株Hep G2细胞和芯片肝模块处理48 h后对人乳腺癌细胞株MCF-7细胞的抑制率来考察肝模块的代谢水平;通过检测芯片中Hep G2细胞分泌白蛋白情况验证肝模块的合成功能;将Caco-2细胞层,HUVEC细胞层,Hep G2细胞层,MCF-7细胞层及透析膜组装在芯片上,在芯片上层通道中通入含55 mg·L^-1普萘洛尔的培养液4 h后换成正常培养液,检测72 h内各个时间点下层循环培养液中普萘洛尔的质量浓度,绘制药-时曲线;在芯片上层循环液中分别通入含125μmol·L^-1环磷酰胺,5μmol·L^-1紫杉醇,50μmol·L^-1卡培他滨的培养液,研究3种抗肿瘤药物在芯片上对MCF-7细胞层的72 h抑制率,并将该结果与96孔板结果进行比较。结果:构建的芯片运行良好,Caco-2和HUVEC细胞层均连接紧密,荧光素钠和普萘洛尔在细胞层间的透过率证明构建的肠模块具有良好的吸收转运功能;125μmol·L^-1环磷酰胺经孔板上的HepG2细胞代谢后对MCF-7的抑制率22.12%,未被代谢的环磷酰胺对MCF-7的抑制率1.84%;而125μmol·L^-1环磷酰胺从芯片肝模块上层注入后对MCF-7的抑制率提升至32.13%,而从芯片肝模块下层注入后对MCF-7的抑制率7.23%;测得普萘洛尔质量浓度在芯片上随时间变化的趋势与体内基本一致;125μmol·L^-1环磷酰胺在孔板上对MCF-7的抑制率比芯片上要低,而5μmol·L^-1紫杉醇和50μmol·L^-1卡培他滨在孔板上对MCF-7的抑制率则高于芯片结果。结论:构建的肠-肝-乳腺癌多器官芯片具有肠的吸收转运功能、肝的代谢功能;该芯片能够反映普萘洛尔在体内的药代动力学特性,同时可用于紫杉醇和卡培他滨的药效学研究。

Construction of A Microfluidic Intestine-liver-breast Cancer Chip and Analysis of Its Application in PK-PD in Vitro of Drugs

Objective: A multi-organ chip of intestine-liver-breast cancer was constructed based on microfluidic technology and used for pharmacokinetics-pharmacodynamics(PK-PD) study of drugs in vitro.Method: A multi-organ chip comprising a 4-layer polydimethylsiloxane(PDMS) substrate and a 2-layer poly(methyl methacrylate)(PMMA) cover was constructed by microfluidic technology.The connection between cells was investigated by staining the 21-day-grown human colon cancer cell line Caco-2 cell layer and the 3-day-grown human umbilical vein endothelial cell line HUVEC cell layer with CellTracker Red/Green and Hoechst,respectively.The transmission rates of 2 g·L^-1 fluorescein sodium and 33.28 mg·L^-1 propranolol acrossing the cell layer were employed to verify the function of the constructed intestinal module.The metabolic level of the liver module was investigated by comparing the inhibition rate of 125 μmol·L^-1 cyclophosphamide against human breast cancer cell line MCF-7 cells treated with human hepatoma cell line HepG2 cells in a conventional well plate and chip liver module for 48 h.The secretion of albumin by HepG2 cells in the chip was detected to verify the synthesis function of hepatic module.Caco-2 cell layer,HUVEC cell layer,HepG2 cell layer,MCF-7 cell layer and dialysis membrane were assembled on the chip,the culture medium containing 55 mg·L^-1 propranolol was injected into the upper channel of the chip for 4 h,and then changed into the normal culture solution.The mass concentration of propranolol in the lower circulating culture medium at each time point within 72 h was determined,and the drugtime curve was drawn.The culture medium containing 125 μmol·L^-1 cyclophosphamide,5 μmol·L^-1 paclitaxel,50 μmol·L^-1 capecitabine was injected into the circulating fluid in the upper layer of the chip,in order to study the inhibition rates of the three anti-tumor drugs on the MCF-7 cell layer on the chip within 72 h,and the results were compared with those of the 96-well plate.Result: The constructed chip performed well.The Caco-2 and HUVEC cell layers were tightly connected.The transmission of fluorescein sodium and propranolol between the cell layers demonstrated that the constructed intestinal module had good absorption and transport function.The inhibition rate of MCF-7 by 125 μmol·L^-1 cyclophosphamide after metabolism of HepG2 cells on the well plate was 22.12%,and the inhibition rate of MCF-7 by the unmetabolized cyclophosphamide was 1.84%.The inhibition rate of MCF-7 increased to 32.13% after injected 125 μmol · L-1 cyclophosphamide from the upper layer of the chip liver module,and the inhibition rate of MCF-7 after injection from the lower layer of the chip liver module was 7.23%.The mass concentration of propranolol on the chip changed with time,which was basically consistent with that in vivo.The inhibition rate of MCF-7 on the plate with 125 μmol·L^-1 cyclophosphamide was lower than that on the chip,and the inhibition rates of MCF-7 on the plate with 5 μmol·L^-1 paclitaxel and 50 μmol·L^-1 capecitabine were higher than those on the chip.Conclusion: The constructed multi-organ chip of intestine-liver-breast cancer has the absorption and transport function of the intestine and the metabolic function of the liver.The chip can reflect the pharmacokinetic properties of propranolol in vivo,and can be used for pharmacodynamic studies of paclitaxel and capecitabine.