Discipline: Technology and Engineering
Subcategory: Biomedical Engineering
Session: 2
Room: Marriott Balcony A
Alexander Edmonson - Hampton University
Co-Author(s): Dr. Jerald Dumas, Hampton University, Hampton, VA; Ron Sanders, Hampton University, Hampton, VA
Matrix Metalloproteinases 9 (MMP-9) is one of the most studied proteinases for its connection to many diseases such as cancer. MMP-9 is responsible for cleaving proteins from the extracellular matrix (ECM) which then insights for ECM remodeling and cancer [1]. This ECM remodeling is accomplished through one of the two zinc ions of MMP-9 that is crucial for proteolytic activity. This proteolytic activity is able to degrade gelatin, thus MMP-9 is in the gelatinase subgroup as gelatinase B [2,3]. Current fluorogenic assays used to measure MMP-9 activity typically require the preparation of a solution containing a fluorogenic substrate. The fabrication of opto-active polyurethane (PUR) cast would enhance such assays by: 1.) embedding the fluorogenic substrate in an extracellular matrix (ECM) surrogate (i.e., PUR), 2.) providing local proteolytic activity data, and 3.) a more high throughput system with easy storage. DQ-gelatin produces fluorescent products when degraded by proteases such as MMP-9. In this study, we conjugated DQ-gelatin within a PUR network and studied the fluorescent signal of the resulting opto-active PUR (OA-PUR) casts when exposed to recombinant MMP-9. Overall, we hypothesize that these OA-PUR casts will provide a sufficient fluorescent signal that will indicateMMP-9 activity. OA-PUR casts were fabricated by mixing DQ-gelatin, polycaprolactone (molecular weight 300-PCL 300), and HDI-trimer. To ensure even distribution of the DQ-gelatin, a speedmixer was used to mix at 1500 rpm for one minute. Subsequently, the reactive mixture was transferred into a 12 well plate to form a film in each respective well. Two concentrations of OA-PUR casts were studied: 39 (low) and 446 (high) ?g DQ-gelatin/mL PUR cast. The OA-PUR casts were allowed to cure at room temperature for overnight and MMP-9 (5 ?g/mL) was added to each well control PUR casts and OA-PUR experimental casts (n = 3). Fluorescence of the samples were read every 15 minutes for 4 hours on a Biotek plate reader. One way ANOVA (Turkey?s multiple comparison test) indicated that the difference in the mean fluorescent intensity (arbitrary units) between high concentration OA-PUR and control/low concentration groups was significant (p < 0.0001). These results suggest that the high concentration of OA-PUR is capable of detecting MMP-9 activity. There was no significant difference between the control and low concentration OA-PUR group, suggesting that 39 ?g DQ-gelatin/mL is below the threshold of detection. Current results are promising as OA-PUR was able to detect MMP-9 activity, supporting our hypothesis. Future work includes determining the minimum detection limit of MMP-9 and the reusability of the OA-PUR platform. Implications of this research may be able to lead to detecting cancer or other diseases in patients as we also plan to apply platform to study other proteases in cells/tissue.
Funder Acknowledgement(s): NSF RIA [Award Number:1700351] and NSF PREM [Award Number:1827820].
Faculty Advisor: Dr. Jerald Dumas, jerald.dumas@hamptonu.edu
Role: I assisted with the fabrication of test samples and conducted calculations as well as interpret the data.