Abstract: Sonodynamic inactivation (SDI) of pathogens has an important advantage when compared to optical excitation-based protocols due to the deeper penetration of ultrasound (US) excitation in biological media or animal tissue. Sonosensitizers (SS) are compounds or systems that upon US stimulation in the therapeutic window (frequency = 0.8–3 MHz and intensity < 3 W/cm2) can induce damage to vital components of pathogenic microorganisms. Herein, we report the synthesis and application of conjugated polymer nanoparticles (CPNs) as an efficient SS in SDI of methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae and Candida tropicalis. A frequent problem in the design and testing of new SS for SDI is the lack of proper sonoreactor characterization which leads to reproducibility concerns. To address this issue, we performed dosimetry experiments in our setup. This enables the validation of our results by other researchers and facilitates meaningful comparisons with different SDI systems in future studies. On a different note, it is generally accepted that the mechanisms of action underlying SS-mediated SDI involve the production of reactive oxygen species (ROS). In an attempt to establish the nature of the cytotoxic species involved in our CPNs-based SDI protocol, we demonstrated that singlet oxygen (1O2) does not play a major role in the observed sonoinduced killing effect. SDI experiments in planktonic cultures of optimally growing pathogens using CPNs result in a germicide effect on the studied pathogenic microorganisms. The implementation of SDI protocols using CPNs was further tested in mature biofilms of a MRSA resulting in ～40 % reduction of biomass and ～70 % reduction of cellular viability. Overall, these results highlight the unique and unexplored capacity of CPNs to act as sonosensitizers opening new possibilities in the design and application of novel inactivation protocols against morbific microbes.
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