基于微流控技术的肠炎芯片模型构建及其在纳米药物疗效评估中的应用

Construction of an enteritis chip model based on microfluidic technology and its application in the evaluation of nanodrug efficacy

摘要:

为了模拟肠道的微环境及生理结构，本研究基于微流控技术构建肠炎芯片模型，同时制备一种负载五氨基水杨酸(5-aminosalicylic acid, 5-AMI)的芦荟多糖共聚物(LDG@AMI)，并利用肠炎芯片模型评价纳米药物的抗炎作用。本研究采用荧光显微镜观察芯片细胞生长情况与肠屏障(intestinal barrier protein, ZO-1)、肠黏液(intestinal mucosa protein, WGA)蛋白的表达量；借助透射电子显微镜观察LDG@AMI纳米药物的形貌；利用纳米粒度仪与傅里叶变换红外光谱仪分析LDG@AMI纳米药物的水合粒径与组成；采用紫外可见分光光度计分析LDG@AMI的溶胀性与药物释放行为；通过细胞计数试剂盒-8 (cell counting kit-8, CCK-8)实验评价LDG@AMI纳米药物的细胞安全性；使用ELISA试剂盒对肠炎芯片中的炎症因子:肿瘤坏死因子-α (tumour necrosis factor-α, TNF-α)、白细胞介素-6 (interleukin-6, IL-6)和白细胞介素-1β (interleukin-1 β, IL-1β)进行定量分析。结果表明，肠芯片细胞展示出良好的生存状态，并且其ZO-1蛋白表达正常，表明肠屏障生成。在葡聚糖硫酸钠(dextran sulfate sodium, DSS)处理肠芯片后，其渗透性升高、缺氧诱导因子-1α (hypoxia inducible factor-1α, HIF-1α)的表达水平上升，ZO-1、WGA蛋白量显著降低，证明肠炎芯片模型的构建成功。LDG@AMI纳米药物的粒径约80 nm，展现出高的溶胀率与药物缓释性，可以精准响应肠道的微环境。与5-AMI处理组相比，LDG@AMI处理的肠炎芯片展示出更高的ZO-1、WGA蛋白表达量，表明LDG@AMI纳米药物具有优异的屏障修复作用。与正常肠芯片相比，肠炎芯片模型组TNF-α、IL-1β以及IL-6炎症因子的表达量显著增加，表明DSS处理可成功诱导肠炎芯片的形成；此外，相比于未处理的肠炎芯片，LDG@AMI处理组展现出更低的TNF-α、IL-1β和IL-6表达量，进一步证明LDG@AMI纳米药物对肠炎具有优异的疗效。本研究成功构建了一种基于微流控技术的肠炎芯片模型，并通过肠炎芯片模型验证了LDG@AMI纳米药物的抗炎作用，为精准治疗肠炎开拓了新视野。

Abstract:

To simulate the intestinal microenvironment and physiological structure, this study constructed the enteritis chip model via the microfluidic technology. Meanwhile, the Aloe vera polysaccharide copolymer (LDG@AMI) loaded with 5-aminosalicylic acid (5-AMI), the nanodrug LDG@AMI was synthesized, and its anti-inflammatory effects were evaluated through the enteritis chip model. In this study, a fluorescence microscopy was employed to measure the growth conditions of cells and the expression of intestinal barrier protein (ZO-1) and intestinal mucosa protein (WGA) in the enteritis chip. In addition, the morphology, size, and composition of LDG@AMI were measured via a transmission electron microscope, a nanoparticle size analyzer, and a Fourier-transform infrared (FT-IR) spectrometer, respectively. Subsequently, the swelling and drug release properties of LDG@AMI were evaluated by ultraviolet-visible (UV-Vis) spectroscopy. Furthermore, ELISA kits were used to quantify the levels of inflammatory factors including tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1 β (IL-1β) in the enteritis chip. The results indicated that the intestine chip cells exhibited good viability and normal expression of ZO-1, which implied the formation of intestinal barrier. After the treatment with sodium dextran sulfate (DSS), the chip showcased increased permeability, elevated expression level of hypoxia inducible factor-1α (HIF-1α), and significantly declined expression levels of ZO-1 and WGA, which suggested that the enteritis chip model was successfully constructed. LDG@AMI had a particle size of about 80 nm, a high swelling rate, and sustained release properties, accurately responding to the intestinal microenvironment. The LDG@AMI-treated enteritis chip showed higher expression levels of ZO-1 and WGA than the 5-AMI-treated group, which indicated that LDG@AMI possessed the excellent ability of repairing barriers. Compared with those in the normal intestine chip, the expression levels of TNF-α, IL-1β, and IL-6 in the enteritis chip significantly increased, further confirming that the enteritis chip model was successfully constructed. Interestingly, the LDG@AMI-treated enteritis chip showed the lower levels of TNF-α, IL-1β, and IL-6 than the untreated enteritis chip, which verified that LDG@AMI possessed excellent therapeutic effect on enteritis. We successfully construct a microfluidic technology-based enteritis chip model and further evaluate the anti-inflammatory effect of LDG@AMI, expanding new horizons for precise treatment of enteritis.