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Updated: May 5, 2026
Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics
Published on: July 16, 2018
Rational design of matrix metalloproteinase-13 activatable probes for enhanced specificity
Lei Zhu1, Ying Ma, Dale O Kiesewetter
1Center for Molecular Imaging and Translational Medicine (CMITM), School of Public Health, Xiamen University , Xiamen 361005, China.
Abstract:
Because of the important roles that matrix metalloproteinases (MMPs) play in tumor invasion and metastasis, various activatable optical probes have been developed to visualize MMP activities in vitro and in vivo. Our recently developed MMP-13 activatable probe, l-MMP-P12, has been successfully applied to image the expression and inhibition of MMPs in a xenografted tumor model [Zhu, L., et al. (2011) Theranostics 1, 18-27]. In this study, to further optimize the in vivo behavior of the proteinase activatable probe, we tracked and profiled the metabolites by a high-resolution liquid chromatography-mass spectrometry (LC-MS) system. Two major metabolites that contributed to the fluorescence recovery were identified. One was specifically cleaved between glycine (G(4)) and valine (V(5)) by MMP, while the other one was generated by nonspecific cleavage between glycine (G(7)) and lysine (K(8)). To visualize the MMP activity more accurately and specifically, a new probe, D-MMP-P12, was designed by replacing the l-lysine with d-lysine in the MMP substrate sequence. The metabolic profile of the new probe, D-MMP-P12, was further characterized by in vitro enzymatic assay, and no nonspecific metabolite was found by LC-MS. Our in vivo optical imaging also demonstrated that D-MMP-P12 had a tumor-to-background ratio (TBR, 5.55 ± 0.75) significantly higher than that of L-MMP-P12 (3.73 ± 0.31) 2 h postinjection. The improved MMP activatable probe may have the potential for drug screening, tumor diagnosis, and therapy response monitoring. Moreover, our research strategy can be further extended to study other protease activatable probes.
Insights
A new optical probe, D-MMP-P12, was developed to specifically detect matrix metalloproteinases (MMPs) by minimizing nonspecific cleavage. This improved probe offers enhanced accuracy for in vivo tumor imaging and potential applications in cancer diagnostics and therapy monitoring.
Area of Science:
- Biomedical Engineering
- Molecular Imaging
- Cancer Biology
Background:
- Matrix metalloproteinases (MMPs) are crucial in tumor invasion and metastasis.
- Activatable optical probes are used to visualize MMP activity in vitro and in vivo.
- Previous MMP-13 probe (l-MMP-P12) showed limitations due to nonspecific cleavage.
Purpose of the Study:
- To optimize in vivo behavior of proteinase activatable probes.
- To design a more accurate and specific MMP activatable probe.
- To evaluate the performance of the new probe (D-MMP-P12) compared to the previous one.
Main Methods:
- Metabolite profiling using high-resolution liquid chromatography-mass spectrometry (LC-MS).
- Design and synthesis of a new probe (D-MMP-P12) with d-lysine substitution.
- In vitro enzymatic assays and in vivo optical imaging in a xenografted tumor model.
Main Results:
- Identified two major metabolites from l-MMP-P12, one from specific and one from nonspecific MMP cleavage.
- D-MMP-P12 showed no nonspecific metabolites in vitro.
- D-MMP-P12 achieved a significantly higher tumor-to-background ratio (5.55 ± 0.75) than l-MMP-P12 (3.73 ± 0.31) in vivo.
Conclusions:
- D-MMP-P12 is a more accurate and specific MMP activatable probe.
- The improved probe has potential for drug screening, tumor diagnosis, and therapy response monitoring.
- The research strategy can be extended to other protease activatable probes.
