Improvement of paracellular transport in the Caco-2 drug screening model using protein-engineered substrates.
Improvement of paracellular transport in the Caco-2 drug screening model using protein-engineered substrates.
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使用蛋白质工程底物改善CACO-2药物筛选模型中细胞细胞运输的改善。
DOI：
10.1016/j.biomaterials.2017.03.023
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发表时间：
2017-06
期刊：
影响因子：
14
通讯作者：
Heilshorn SC
中科院分区：
文献类型：
--
作者：
DiMarco RL;Hunt DR;Dewi RE;Heilshorn SC
在过去的二十年中，Caco-2测定作为在临床前表征期间估计药物化合物的体内口服生物利用度的体外方法在制药公司中获得了广泛的普及。尽管其受欢迎，但该测定法对细胞旁吸收的药物的转运存在严重的预测不足，所述药物通过小肠吸收细胞的细胞-细胞紧密连接被吸收。在这里，我们提出，简单地取代胶原蛋白I基质中采用的标准Caco-2测定与工程基质，我们可以控制细胞形态，从而调节细胞-细胞连接，决定细胞旁运输。具体而言，我们使用仿生工程细胞外基质（eECM），其中包含模块化的蛋白质结构域来自两个ECM蛋白质中发现的小肠，纤连蛋白和弹性蛋白。这种eECM使我们能够独立地调节呈递给Caco-2细胞的细胞粘附性RGD配体的密度以及eECM的机械刚度。我们观察到较低量的RGD配体呈递以及降低的基质硬度导致Caco-2形态比在胶原上培养的Caco-2细胞更接近于原代小肠上皮细胞。此外，这些基质导致Caco-2单层，其中肌动蛋白向顶端连接复合物的募集减少，并且紧密连接蛋白-2的表达增加，紧密连接蛋白-2是一种与较高的细胞旁通透性相关的紧密连接蛋白，其在整个小肠中高度表达。与这些形态学差异一致，已知在体内细胞旁转运的药物在该改良的Caco-2模型中表现出显著改善的转运速率。正如预期的那样，跨细胞转运药物的渗透性不受影响。因此，我们已经证明了一种提高Caco-2测定的生理准确性的方法，该方法可以很容易地被制药公司采用，而无需对其当前的测试方案进行重大更改。
The Caco-2 assay has achieved wide popularity among pharmaceutical companies in the past two decades as an in vitro method for estimation of in vivo oral bioavailability of pharmaceutical compounds during preclinical characterization. Despite its popularity, this assay suffers from a severe under-prediction of the transport of drugs which are absorbed paracellularly, that is, which pass through the cell-cell tight junctions of the absorptive cells of the small intestine. Here, we propose that simply replacing the collagen I matrix employed in the standard Caco-2 assay with an engineered matrix, we can control cell morphology and hence regulate the cell-cell junctions that dictate paracellular transport. Specifically, we use a biomimetic engineered extracellular matrix (eECM) that contains modular protein domains derived from two ECM proteins found in the small intestine, fibronectin and elastin. This eECM allows us to independently tune the density of cell-adhesive RGD ligands presented to Caco-2 cells as well as the mechanical stiffness of the eECM. We observe that lower amounts of RGD ligand presentation as well as decreased matrix stiffness results in Caco-2 morphologies that more closely resemble primary small intestinal epithelial cells than Caco-2 cells cultured on collagen. Additionally, these matrices result in Caco-2 monolayers with decreased recruitment of actin to the apical junctional complex and increased expression of claudin-2, a tight junction protein associated with higher paracellular permeability that is highly expressed throughout the small intestine. Consistent with these morphological differences, drugs known to be paracellularly transported in vivo exhibited significantly improved transport rates in this modified Caco-2 model. As expected, permeability of transcellularly transported drugs remained unaffected. Thus, we have demonstrated a method of improving the physiological accuracy of the Caco-2 assay that could be readily adopted by pharmaceutical companies without major changes to their current testing protocols.
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影响因子：
3.4
作者：
Engler, A;Bacakova, L;Discher, D
通讯作者：
Discher, D
DOI：
10.1016/0006-291x(91)91647-u
发表时间：
1991-03-29
影响因子：
3.1
作者：
ARTURSSON, P;KARLSSON, J
通讯作者：
KARLSSON, J
影响因子：
2.3
作者：
Chen S;Einspanier R;Schoen J
通讯作者：
Schoen J
影响因子：
2.5
作者：
Benitez, Patrick L.;Mascharak, Shamik;Heilshorn, Sarah C.
通讯作者：
Heilshorn, Sarah C.
影响因子：
3.7
作者：
CHO, MJ;THOMPSON, DP;SCIESZKA, JF
通讯作者：
SCIESZKA, JF