Department of Bio-system Pharmacology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
Sci Rep. 2017 Dec 13;7(1):17484. doi: 10.1038/s41598-017-17459-y.
Various physiological and pathological processes are accompanied with the alteration of pH at extracellular juxtamembrane region. Accordingly, the methods to analyze the cell surface pH have been demanded in biological and medical sciences. In this study, we have established a novel methodology for cell surface pH imaging using poly(ethylene glycol)-phospholipid (PEG-lipid) as a core structure of ratiometric fluorescent probes. PEG-lipid is a synthetic amphiphilic polymer originally developed for the cell surface modification in transplantation therapy. Via its hydrophobic alkyl chains of the phospholipid moiety, PEG-lipid is, when applied extracellularly, spontaneously inserted into the plasma membrane and retained at the surface of the cells. We have demonstrated that the PEG-lipid conjugated with fluorescein isothiocyanate (FITC-PEG-lipid) can be used as a sensitive and reversible cell-surface-anchored pH probe between weakly alkaline and acidic pH with an excellent spatiotemporal resolution. The remarkably simple procedure for cell-surface labeling with FITC-PEG-lipid would also be advantageous when considering its application to high-throughput in vitro assay. This study further indicates that various probes useful for the investigation of juxtamembrane environments could also be developed by using PEG-lipid as the core structure for bio-membrane anchoring.
各种生理和病理过程伴随着细胞外近膜区域 pH 值的改变。因此，在生物和医学科学中需要分析细胞表面 pH 值的方法。在本研究中，我们建立了一种使用聚乙二醇-磷脂（PEG-脂质）作为比率荧光探针的核心结构进行细胞表面 pH 值成像的新方法。PEG-脂质是一种最初为移植治疗中的细胞表面修饰而开发的合成两亲聚合物。通过其磷脂部分的疏水烷基链，PEG-脂质在细胞外被自发插入质膜并保留在细胞表面。我们已经证明，与异硫氰酸荧光素（FITC-PEG-脂质）偶联的 PEG-脂质可在弱碱性和酸性 pH 值之间用作敏感且可逆的细胞表面锚定 pH 探针，具有优异的时空分辨率。用 FITC-PEG-脂质进行细胞表面标记的过程非常简单，这在考虑将其应用于高通量体外测定时也具有优势。这项研究进一步表明，通过使用 PEG-脂质作为生物膜锚定的核心结构，也可以开发出用于研究近膜环境的各种探针。
