Hydrophilic–Hydrophobic Sequence Regulation in Alternating Polysulfoniums Enables “Wake-and-Kill” Eradication of Antibiotic-Tolerant Persisters and Their Biofilms

Phenotypically tolerant persister bacteria can survive antibiotic treatment by entering a metabolically dormant state and are widely recognized as major contributors to infection relapse. To address this challenge, we systematically investigated how the alternating hydrophilic-hydrophobic sequence pattern of polysulfoniums modulate the membrane potential and respiratory activity in dormant bacterial cells. While rifampicin and ampicillin at 25-100 × MIC (mininum inhibitory concentration) were ineffective against persister populations, the <b>PS</b>^<b>+</b><b>(triEG-<i>alt</i>-octyl)</b> alternating polymer significantly reactivated the electron transport chain (ETC) in persister cells, achieving >9-log reductions in viability at 8-16 μg/mL (2-4 × MIC) via precise sequence regulation of hydrophilic and hydrophobic segments. Integration of nanoparticle-assisted delivery with NIR-triggered release enabled efficient penetration of persister-dominated biofilms, resulting in ∼90% biomass clearance and >99.9% elimination of embedded persister cells. These findings highlight the sequence modulation of cationic polymers that offers a highly effective "wake-and-kill" strategy for the eradication of persisters and their associated biofilms.