Antimicrobial Peptide-Loaded Nanogels Evaluation

Mid-log phase bacteria were resuspended in PBS to a concentration of 5 × 10⁶ colony forming units (CFU)/mL. Next, 20 µL bacterial suspension was mixed with 30 µL PBS containing increasing concentrations of SAAP-148, PVA-lyophilized and redispersed SAAP-148-loaded nanogels or placebo nanogels and 50 µL of filtered, inactivated and centrifuged human plasma (Sanquin, Leiden, The Netherlands) in polypropylene V-shaped 96-well microplates. After incubation for 4 h or 24 h at 37 °C under rotation at 200 rpm using an orbital shaker, 10-fold serial dilutes were made and plated onto MH agar plates to determine the number of viable bacteria. Results are expressed as lethal concentration (LC)_99.9, i.e., the lowest concentration of SAAP-148 killing 99.9% of the inoculum.