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

Area of Science:

• Pharmacology
• Cell Biology
• Biotechnology

Background:

• Midsize drugs are a novel class for targeting intracellular molecules.
• Assessing intracellular drug retention and membrane permeability is challenging due to a lack of rapid cytoplasm sampling techniques.

Purpose of the Study:

• To develop a straightforward and rapid method for simultaneous analysis of intracellular retention and membrane permeability.
• To introduce cytoplasm extraction by osmotic cell rupture (CyTOR) as a solution.

Main Methods:

• Developed CyTOR, a method using hypotonic solutions for rapid cytoplasm collection (within 30 s).
• Validated CyTOR using confocal microscopy with HeLa cells stained for cytoplasm and membrane markers.
• Assessed drug permeability by incubating HeLa cells with various drugs, collecting cytoplasm via CyTOR, and quantifying drug concentrations using LC-MS.

Main Results:

• CyTOR selectively extracted cytoplasm, confirmed by marker elimination times.
• Intracellular drug concentrations were accurately modeled using a theoretical function.
• Calculated permeability values and efflux ratios aligned with known drug properties.

Conclusions:

• CyTOR is a rapid and effective method for assessing membrane permeability and intracellular drug retention.
• This technique has potential for high-throughput drug screening and characterization.