Abstract Th0093: Design, Synthesis and Evaluation of a Fluorescent Probe for Molecular Imaging of Plasmin Activity

Backgrounds: Once a wound is contained by fibrin, activation of plasmin promotes the removal of fibrin and stimulates angiogenesis, tissue remodeling, and tissue regeneration. Insufficient fibrin deposition or excessive plasmin-mediated fibrinolysis in early convalescence prevents injury containment, causing bleeding. Alternatively, excess fibrin deposition and/or inefficient plasmin activity later in convalescence impairs musculoskeletal repair, resulting in tissue fibrosis and osteoporosis. The ability to map plasminogen expression noninvasively and serially as a biomarker would thus have significant potential in improving novel therapies. Here, we describe the development of a selective plasmin activatable near-infrared (NIR) fluorescent imaging probe Methods: The probe was incubated with purified murine plasmin (0.01 mg/mL), thrombin (0.01 mg/mL), trypsin (0.01 mg/mL), or bovine serum albumin ([BSA] 0.1% in water), and the fluorescence increase was recorded over time. At 1 day post injury, 1 day post-injection, and 3 weeks post-injection, the WT and the Plg KO mice were used to study plasmin activation in vivo. Results: The probe should show a 10 fold increase in fluorescence compared to the initial sample fluorescence when treated with purified murine plasmin. At 5ug/mouse, there was no signal in the Plg KO or scramble probe mice. A scrambled sequence was also synthesized as a control in which the amino acid sequence was rearranged. The plasmin probe showed a signal at the fracture site 3 weeks after injury, while the signal of the scramble probe was weak. At 1d post-injury and 1d post injection, the WT and the Plg KO mice have significant signals. The signal in WT is still significantly higher than the signal in Plg KO muscle. The probe was washing out faster at the higher doses. Conclusions: The above studies demonstrate that it is feasible to design and synthesize plasminogen-specific NIRF imaging probes. Specifically, we show over 10-fold increases in NIRF signal upon plasminogen activation with the optimized probes. The probe is specific to plasminogen activity and sensitive enough to detect a small amount of enzyme in a biological system. Overall, we believe that the developed plasminogen imaging probe should be valuable. Given its broad role, the developed imaging probes could be used as a direct biomarker to assess therapeutic efficacy.