The Nature of Cell-Penetrating Poly(disulfide)s.

Cell-penetrating poly(disulfide)s (CPDs) are the most popular thiol-mediated uptake (TMU) tools for delivering attached substrates of interest (SOIs) into the cytosol. Despite their widespread use, their mode of action remains unknown. Herein, we elucidate how CPDs work using combined data from protein knockdown, µMap proteomics, coacervate assembly, mechanosensitive fluorescent membrane probes, CPD inhibitors, activators, and depolymerization. Their guanidinium sidechains first ion-pair with anionic clusters on cell surfaces. By enhancing effective CPD concentration, these non-covalent interactions enable, accelerate, and direct dynamic covalent disulfide exchange cascades with cellular thiols and disulfides. CPD bioconjugation rates match their uptake activity, exceeding those of disulfide monomers by 3000 times. Exchange with proteins capable of membrane remodeling allows CPDs to exploit these proteins' primary functions to mobilize anionic lipids (scramblases, ANO6), form toroidal elastic pores and related membrane deformations, and repair all after CPD-SOI conjugates have passed through (tetraspanins, TSPAN8, flippases, etc.). Within cells, CPDs form coacervates and integrate into biological condensates, particularly nucleoli, until they depolymerize and release the SOIs. This understanding of the nature of CPDs also clarifies TMU in general and will guide future efforts to control cellular entry for drug delivery and pathogen defense.