Quantifying Cell Membrane Permeability of Small to Mid-Size Drugs Using Quick Osmotic Cytoplasm Extraction

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.