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 PS + (triEG- alt -octyl) 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.