細胞を破裂させて低・中分子薬の「入りやすさ」と「出やすさ」を評価～がん細胞の薬剤取り込み量を簡便かつ直接計測可能に～

2025-10-03

2025-10-02 九州大学

九州大学の研究チームは、細胞を意図的に破裂させて低・中分子薬の「細胞への入りやすさ」と「出やすさ」を定量的に評価する新手法を開発した。従来の膜透過性評価法は分子の細胞内取り込みを正確に測定できず、薬剤候補のスクリーニングに課題があった。本手法では破裂細胞からの薬剤放出をリアルタイムで解析し、膜透過性を高感度に評価できる。これにより、抗がん剤や抗ウイルス薬など、低・中分子医薬品候補の膜透過性に基づく最適化が可能となる。研究成果は創薬研究の効率化や新薬開発の加速に寄与すると期待される。

図1. 開発したCyTOR法のコンセプト図

<関連情報>

迅速浸透圧細胞質抽出法を用いた小中型薬物の細胞膜透過性の定量 Quantifying Cell Membrane Permeability of Small to Mid-Size Drugs Using Quick Osmotic Cytoplasm Extraction

Serena Igari,Daiki Sakai,Chenchen Liu,Kohei Torikai,Nobuaki Matsumori,and Takayuki Kawai
Analytical Chemistry Published: October 1, 2025
DOI:https://doi.org/10.1021/acs.analchem.5c01064

Abstract

Midsize drugs are emerging as a novel modality for targeting intracellular molecules. However, a straightforward method for simultaneously analyzing intracellular retention and membrane permeability has been lacking due to the absence of a rapid cytoplasm sampling technique. To address this issue, we developed cytoplasm extraction by osmotic cell rupture (CyTOR), a simple method that enables cytoplasm collection within 30 s by rupturing cells with a hypotonic solution. The concept of CyTOR was first demonstrated using confocal microscopy. HeLa cells were stained with calcein AM (a cytoplasm marker) and CellMask (a membrane marker) and then ruptured with deionized water. The cytoplasm marker was eliminated within 5 s, while the membrane marker persisted for over 1 min, confirming the selective cytoplasm extraction by CyTOR. To assess drug permeability, HeLa cells were incubated with three small-molecule drugs (propranolol, cimetidine, and atenolol) and one midsize drug (cyclosporin A) for 1–360 min. Cytoplasm was collected using CyTOR, and drug concentrations were quantified using liquid chromatography–mass spectrometry. Intracellular drug concentrations were plotted against the incubation time and fitted to the function y = a(1 – e^–bt) based on our proposed theoretical model. The calculated apparent influx/efflux permeability values and efflux ratios demonstrated distinct drug properties consistent with conventional reports. These results highlight the potential of CyTOR as a rapid, high-throughput method for assessing membrane permeability and intracellular drug retention.