Figure 2.

(a) Plasma stability. heMAMP was incubated with plasma at 37 °C up to 3 h and the relaxation time was monitored at 0, 30, 60, 90, 120, and 180 min. There was no significant relaxation time change over 3 h (n = 3, means with standard error of the mean (SEM)). (b) Cytotoxicity of heMAMP. RAW 264.7, and HEK 293 cells (1.5 × 10⁴ cells per well) were incubated with heMAMP in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) at different concentrations for 16 h at 37 °C. The MTT assay demonstrated no significant toxicity of heMAMP up to 5 mM for both cell lines (n = 3, means with SEM). (c) Dynamic R1 ratios of heMAMP and MPO-Gd after activation by MPO. heMAMP demonstrated more efficient activation with >3 times higher R1 ratio compared with MPO-Gd when incubated with MPO, glucose oxidase (GOX), and glucose over 180 min at 40 °C (n = 3, *p = 0.033, Kolmogorov–Smirnov test). (d) Binding to bovine serum albumin (BSA) after activation by MPO. When incubated with MPO, GOX, glucose, and BSA for 1 h at 40 °C, heMAMP showed about 1.5-fold higher binding efficacy compared to MPO-Gd (n = 3). (e) Biodistribution and retention of heMAMP at 3 h and day 7 determined by inductively coupled plasma mass spectrometry (ICP-MS) using a mouse model of complete Freund’s adjuvant (CFA)-induced paw inflammation (n = 3 for each time point). (f) Blood half-life of heMAMP determined by ICP-MS with the two-phase decay model. The fast-phase blood half-life of heMAMP is 1.2 min and the slow phase is 31.7 min (n = 5). A total of 0.1 mmol/kg of heMAMP was intravenously administered as shown in Figure 2e,f.