Thiolated Hyaluronic Acid: A Gateway for Targeted Killing of <i>Staphylococcus aureus</i> on the Race for Surface Colonization

Hyaluronic acid (HA) is degraded by Staphylococcal hyaluronate lyase (Hysa) and mammalian hyaluronidase (Hyal). Thiolated HA (HAMS) is used as a targeted gateway for Staphylococcus aureus killing while enhancing the previous M23 endolysin-polyphosphate (M23-PP NPs) enzyme-responsive nanoparticle formulation. Synthesis of HAMS and characterization for nuclear magnetic resonance, solubility, thiol content, pKa, and degradation by Hysa and Hyal are presented. Nanoparticles prepared via ionotropic gelation between M23-PP NPs and either HAMS or HA yield M23-PP/HAMS or M23-PP/HA NPs, respectively. Their characterization includes size, zeta potential, morphology, release profiles, safety, targeted release, and efficacy. HAMS with a thiol content of 250.18 ± 90.32 µmol g^-1, solubility of 50.99 ± 0.02 mg mL^-1, exhibits pKa values of 3.2, 4.2, and 8.8. This thiolated polymer irreversibly inhibits Hyal activity, without affecting Hysa. M23-PP/HAMS NPs (265 ± 47 nm, -25 mV) maintain their integrity for seven days at 37 °C, and HAMS coating prevents nonspecific degradation by Hyal, as confirmed by release studies. In a co-culture 'race for the surface' experiment with MC3T3 osteoblasts and S. aureus ATCC 25923, M23-PP/HAMS NPs produce 8-log bacterial killing while promoting in vitro wound healing. These findings are pivotal to the development of new enzyme-responsive excipients switchable by S. aureus.