2.5. Determination of the minimum inhibitory concentration (MIC)

The MIC of DPD-L[D], mPEG-L[D], and free daptomycin were determined by the broth micro-dilution method in 96-well plates14. Since daptomycin is a calcium-dependent antibiotic, the MICs were measured by using Mueller–Hinton II broth with 50 μg/mL calcium ions15. Each preparation was added to 100 μL Mueller–Hinton II broth at sequential 2-fold dilutions, with concentrations in the range of 64–0.0625 μg/mL. A 100 μL MRSA252 aliquot (10⁶ CFU/mL) was added to each well. The plates were incubated at 37 °C for 24 h. The MIC was designated as the lowest concentration of daptomycin at which there was no visible and measurable MRSA growth after 24 h of incubation. Each experiment was performed in triplicate.