Temporal imaging of drug dynamics in live cells using stimulated Raman scattering microscopy and a perfusion cell culture system.
Temporal imaging of drug dynamics in live cells using stimulated Raman scattering microscopy and a perfusion cell culture system.
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DOI：
10.1039/d2cb00160h
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发表时间：
2022-08-31
影响因子：
4.1
通讯作者：
Graham, Duncan
中科院分区：
文献类型：
--
作者：
Tipping, William J.;Merchant, Andrew S.;Fearon, Rebecca;Tomkinson, Nicholas C. O.;Faulds, Karen;Graham, Duncan
受激拉曼散射（SRS）显微镜是一种强大的技术，用于在生物相容性成像条件下可视化活细胞中药物和小分子的细胞摄取和分布。在药物分子内使用生物正交基团，包括炔和腈，使得能够以最小扰动的方式直接检测过多的生物活性分子。在生理条件下实时检测药物摄取和分布到活细胞中的进展有限，尽管它为临床前药物开发提供了一致的潜力。SRS显微镜已应用于药物7 RH的细胞动力学研究，7 RH是dicoidin结构域受体1（DDR 1）的有效抑制剂，并在体外阻止细胞粘附，增殖和迁移。各种哺乳动物细胞模型对7 RH的摄取显示不依赖于DDR 1表达。使用灌注室，实现了对活癌细胞的反复治疗，使得能够使用SRS显微镜实时可视化7 RH摄取，之后在多模式成像实验中使用市售荧光标记物评估相同细胞群体的活力。在相同的活细胞群中使用顺序灌注和延时成像研究了7 RH治疗与化疗剂顺铂组合的效果，以证明该方法的应用。SRS显微镜还发现，随着7 RH处理浓度的增加，乳腺癌细胞模型中的细胞粘附和迁移受到有效抑制，因此代表了这种表型测定的新型读出方法。在生理条件下直接评估药物-细胞相互作用为临床前药物开发过程提供了巨大的潜力。使用灌注细胞培养系统能够在相同的活细胞群中进行药物摄取和细胞活力分析的多模式成像。
Stimulated Raman scattering (SRS) microscopy is a powerful technique for visualising the cellular uptake and distribution of drugs and small molecules in live cells under biocompatible imaging conditions. The use of bio-orthogonal groups within the drug molecule, including alkynes and nitriles, has enabled the direct detection of a plethora of bioactive molecules in a minimally perturbative fashion. Limited progress has been made towards real-time detection of drug uptake and distribution into live cells under physiological conditions, despite the accordant potential it presents for preclinical drug development. SRS microscopy has been applied to the study of cellular dynamics of the drug 7RH, which is a potent inhibitor of dicoidin domain receptor 1 (DDR1) and prevents cellular adhesion, proliferation and migration in vitro. The uptake of 7RH into a variety of mammalian cell models was shown to be independent of DDR1 expression. Using a perfusion chamber, the recurrent treatment of live cancer cells was achieved, enabling 7RH uptake to be visualised in real-time using SRS microscopy, after which the viability of the same cellular population was assessed using commercially available fluorescent markers in a multimodal imaging experiment. The effect of 7RH treatment in combination with the chemotherapeutic, cisplatin was investigated using sequential perfusion and time-lapse imaging in the same live cell population, to demonstrate the application of the approach. SRS microscopy also identified potent inhibition of cellular adhesion and migration in breast cancer cell models with increasing 7RH treatment concentrations, thus representing a novel read-out methodology for phenotypic assays of this kind. The direct assessment of drug–cell interactions under physiological conditions offers significant potential for the preclinical drug development process. Multimodal imaging of drug uptake and cell viability analysis in the same live cell population is enabled using a perfusion cell culture system.
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