Magnetic Nanoparticles for the Detection of Matrix Metalloproteinase-2 Activity in Tumor Models

Introduction: In their lifetime, around one in every seven men will get prostate cancer. This disease can be very serious, however if diagnosed can be treated. The goal of this project was to develop bioprocess-sensitive “smart” magnetic nanoparticles (MNP) which respond to overexpressed protease activity in the tumor microenvironment – and thus to tumor aggressiveness – and which can be monitored and quantified non-invasively over the entire tumor volume. This was done by using a conjugated fluorescent marker that’s rate of release correlated with the activity of an enzyme linked to cancer aggressiveness.

Materials and Methods: The expression of matrix metalloproteinase-2 (MMP-2), an important tumor-associated protease, has been correlated with increasing metastatic potential in prostate cancer and is therefore an attractive target for MNP targeting. We successfully conjugated a near-infrared fluorescent marker via an MMP-2 cleavable peptide linker. This was done through a series of steps, first we attached a matrix metalloproteinase-2 (MMP-2) cleavable peptide with a fluorescent marker (FAM-GPLGVRGC) on the surface of aminated magnetic nanoparticles through NHS-amine chemistry. Thereafter, we exposed the resulting probe to various proteases of the MMP-family, mainly MMP-2, 7 and 13. The experimental data revealed that our probe demonstrated significantly higher selectivity towards MMP-2, as measured by the fluorescent intensity of the cleaved fluorescent marker.

Results and Discussion: The MNP response to the different MMP proteases indicated whether the specific nanoparticles could differentiate each protease as shown by the raw results of the experiments conducted in lab, the fluorescent response of each proteases was measured over time. The fluorescence was then measured using a microplate reader set at excitation/emission wavelengths of 495/525 nm and then set to take measurements every two minutes. This yielded promising results as seen in data, as after 160 minutes, a notable fluorescence intensity was only seen from the MMP-2 protease. This seems to affirm the correct conjugation of the peptide linker for fluorescence and the nano-particles seem to now can differentiate the MMP-2 specifically.

Conclusions: A conjugated near-infrared fluorescent marker via an MMP-2 cleavable peptide linker displayed sensitivity to MMP-2 activity; and the rate of release of the fluorescent marker correlated strongly with MMP-2 activity. In addition, the probe displayed significantly higher specificity towards MMP-2 compared to other members of the MMP family, specifically MMP-7,9, and 14.

Acknowledgements: Graduate Student Tareq Anani helped with data processing and MNP creation. Funding sources include Auburn University Biomedical Engineering Lab.