Quantitation of Cell Membrane Permeability of Cyclic Peptides by Single-Cell Cytoplasm Mass Spectrometry

Takayuki Kawai, Yasuhiro Mihara, Makiko Morita, Masahiko Ohkubo, Taiji Asami, Tomonobu M. Watanabe

研究成果: ジャーナルへの寄稿学術誌査読

5 被引用数 (Scopus)


Cyclic peptides (CPs) have attracted attention as next-generation drugs because they possess both cell-permeable potential as small molecules and specific affinity similar to antibodies. As intracellular molecules are important targets of CPs, quantitation of the intracellular retention and transmembrane permeability of CPs is necessary for drug development. However, permeated CPs within cells cannot be directly assessed by conventional permeability assays using methods such as artificial membranes and cell monolayers. Here, we propose a new approach using single-cell cytoplasm mass spectrometry (SCC-MS). After cells were incubated with CPs, the cytoplasm was directly collected from a single cell using a microneedle followed by nanoelectrospray ionization mass spectrometry detection of the CPs. The height of the CP peak was plotted against time and fitted with a simple function, y = a(1 - e-bx), to calculate the apparent permeability coefficient (Papp) for both the influx and efflux directions. MCF-7 cells were selected as model cancer cells and cultured with cyclosporin A (CsA) and its demethylated analogs (dmCsA-1, -2, and -3) as model CPs. Papp values (10-6 cm/s) obtained from cells incubated with 50 μM CPs ranged from 0.017 to 0.121 for influx and 0.20 to 1.48 for efflux. The higher efflux ratio was possibly caused by efflux transporters such as P-glycoprotein, a well-known receptor of CsA. The equilibrated intracellular concentration of CPs was estimated to be as low as 4.1-6.8 μM, which showed good consistency with the high efflux ratio. SCC-MS is promising as a reliable permeability assay for next-generation CP-based pharmaceuticals.

ジャーナルAnalytical Chemistry
出版ステータス出版済み - 2月 23 2021

!!!All Science Journal Classification (ASJC) codes

  • 分析化学


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