Multilayer Spheroids To Quantify Drug Uptake and Diffusion in 3D
Multilayer Spheroids To Quantify Drug Uptake and Diffusion in 3D
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DOI：
10.1021/mp500002y
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发表时间：
2014-07-01
影响因子：
4.9
通讯作者：
Morgan, Jeffrey R.
中科院分区：
文献类型：
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作者：
Achilli, Toni-Marie;McCalla, Stephanie;Morgan, Jeffrey R.
需要新的药物摄取和扩散的定量体外模型来帮助评估药物毒性/功效以及新的用于药物发现的更具预测性的模型。我们报告了一个三维（3D）多层球体模型和一个新的算法来定量研究荧光钙黄绿素通过间隙连接细胞间通讯（GJIC）的摄取和向内扩散。当与钙黄绿素-AM（外排转运蛋白P-糖蛋白（Pgp）的底物）孵育时，来自各种细胞类型的球状体随时间推移积累钙黄绿素。在过表达Pgp（HEK-MDR）的球状体中蓄积减少，在存在Pgp抑制剂（维拉帕米、洛哌丁胺、环孢菌素A）的情况下蓄积增加。钙黄绿素的向内扩散在缺乏GJIC（OVCAR-3，SK-0 V-3）的球状体中是可忽略的，并且在GJIC（甘珀酸）的抑制剂的存在下减少。除了抑制Pgp，维拉帕米和洛哌丁胺，但不是环孢素A，抑制向内扩散的钙黄绿素，这表明他们也抑制GJIC。维拉帕米抑制Pgp和GJIC的量效曲线相似（IC_（50）：8 μ M）。该方法适用于许多不同的细胞类型，并可作为定量3D模型，更准确地复制体内药物摄取和扩散的障碍。
There is a need for new quantitative in vitro models of drug uptake and diffusion to help assess drug toxicity/efficacy as well as new more predictive models for drug discovery. We report a three-dimensional (3D) multilayer spheroid model and a new algorithm to quantitatively study uptake and inward diffusion of fluorescent calcein via gap junction intercellular communication (GJIC). When incubated with calcein-AM, a substrate of the efflux transporter P-glycoprotein (Pgp), spheroids from a variety of cell types accumulated calcein over time. Accumulation decreased in spheroids overexpressing Pgp (HEK-MDR) and was increased in the presence of Pgp inhibitors (verapamil, loperamide, cyclosporin A). Inward diffusion of calcein was negligible in spheroids that lacked GJIC (OVCAR-3, SK-OV-3) and was reduced in the presence of an inhibitor of GJIC (carbenoxolone). In addition to inhibiting Pgp, verapamil and loperamide, but not cyclosporin A, inhibited inward diffusion of calcein, suggesting that they also inhibit GJIC. The dose response curves of verapamil's inhibition of Pgp and GJIC were similar (IC50: 8 mu M). The method is amenable to many different cell types and may serve as a quantitative 3D model that more accurately replicates in vivo barriers to drug uptake and diffusion.