Real-Time Monitoring of Pharmacokinetics of Mitochondria-Targeting Molecules in Live Cells with Bioorthogonal Hyperspectral Stimulated Raman Scattering Microscopy
Real-Time Monitoring of Pharmacokinetics of Mitochondria-Targeting Molecules in Live Cells with Bioorthogonal Hyperspectral Stimulated Raman Scattering Microscopy
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DOI：
10.1021/acs.analchem.9b02838
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发表时间：
2020-01-07
影响因子：
7.4
通讯作者：
Huang, Zhiwei
中科院分区：
文献类型：
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作者：
Bae, Kideog;Zheng, Wei;Huang, Zhiwei
活细胞中线粒体的动态在细胞代谢、早期凋亡和细胞分化等生物事件中发挥着关键作用。三苯基膦 (TPP) 是线粒体研究中常用的线粒体靶向剂。然而，由于在生物环境中跟踪和量化小分子的困难，人们对 TPP 在靶向线粒体过程中的细胞内行为缺乏了解。在这里，我们报告了与合成拉曼标签相关的高光谱受激拉曼散射（SRS）显微镜的实用性，用于实时可视化和定量活细胞内亚细胞水平的 TPP 动力学。凭借合成的基于芳基二炔的拉曼标签的众多优点，例如优异的光稳定性、可忽略的背景干扰以及SRS信号对TPP浓度的线性依赖性，我们成功建立了定量模型，将线粒体膜电位与活细胞内TPP的关键药代动力学参数相关联。该模型表明，线粒体膜电位的降低会导致 TPP 的摄取率和细胞内浓度显着降低。此外，基于多重 SRS 图像同时突出显示细胞蛋白质和脂质而无需进一步标记，我们发现 TPP 摄取对宿主细胞几乎没有细胞毒性。生物正交高光谱SRS显微成像揭示，TPP在线粒体网络重构过程中可以保持与线粒体的稳定亲和力，展示了其在实时监测与活体生物宿主相关的小分子药代动力学方面的巨大潜力，从而在不久的将来促进线粒体靶向成像探针和疗法的发展。
The dynamics of mitochondria in live cells play a pivotal role in biological events such as cell metabolism, early stage apoptosis, and cell differentiation. Triphenylphosphonium (TPP) is a commonly used mitochondria-targeting agent for mitochondrial studies. However, there has been a lack of understanding in intracellular behaviors of TPP in the course of targeting mitochondria due to the difficulty in tracking and quantifying small molecules in a biological environment. Here, we report the utility of hyperspectral stimulated Raman scattering (SRS) microscopy associated with a Raman tag synthesized for real-time visualization and quantitation of TPP dynamics within live cells at the subcellular level. With the myriad of merits offered by a synthesized aryl-diyne-based Raman tag such as excellent photostability, negligible background interferences, and a linear dependence of the SRS signal on the TPP concentration, we successfully establish a quantitative model to associate the mitochondrial membrane potential with the key pharmacokinetic parameters of TPP inside the live cells. The model reveals that reduction in the mitochondrial membrane potential leads to significant decreases in both the uptake rate and intracellular concentrations of TPP. Further, on the basis of the multiplexed SRS images concurrently highlighting the cellular proteins and lipids without further labeling, we find that the TPP uptake causes little cytotoxicity to the host cells. The bioorthogonal hyperspectral SRS microscopy imaging reveals that TPP can maintain stable affinity to mitochondria during the restructuring of mitochondrial networking, demonstrating its great potential for real-time monitoring of pharmacokinetics of small molecules associated with live biological hosts, thereby promoting the development of mitochondria-targeting imaging probes and therapies in the near future.