(465c) Fluorophore-Gold Nanoparticle Contrast Agent for Specific and Sensitive MMP-14 Detection

Matrix metalloproteinases (MMPs) are a family of endo-peptidases that contain zinc ions in their structures and are highly expressed in cancer. MMPs play crucial roles in the invasion, metastasis, proliferation, and development of cancer cells. MMP-14, one of the MMPs, is a membrane bound enzyme and is expressed at a high level in several aggressive cancer types, particularly in aggressive breast cancer (e.g. MDA-MB 231). It is known to promote the cancer cell detachment from the primary site, degrades and rebuilds the extracellular matrix (ECM), and stimulates tumor angiogenesis. MMP-14 also activates other MMPs, particularly proMMP-2, and is involved in the activation of extracellular signal-regulated kinase (ERK). Thus, specifically and sensitively detecting MMP-14 is important for early diagnosis of aggressive breast cancer, and specially designed, fluorophore mediated molecular sensing may be a good way to do so.

One way to increase the specificity of the fluorophore mediated molecular sensing is designing the contrast agent to emit its fluorescence only when this agent encounters the biomarker of interest. Increasing sensitivity of the contrast agent can be done by enhancing the fluorescent emission level per fluorophore. 

Locally modulating fluorescent emission level of a fluorophore can be achieved by properly utilizing the electromagnetic (EM, plasmon) field generated by the gold nanoparticle (GNP) because GNP is small and can form a strong EM field around it. The EM field strength is highest on the GNP surface and exponentially decreases with the distance from the surface. Therefore, the fluorescent emission level can be controlled by appropriately varying the distance between the fluorophore and GNP.  When the fluorophore is placed on the surface or very close to the GNP, its fluorescence is quenched significantly.  When it is placed at an appropriate distance from the GNP, its fluorescence can be enhanced extensively. With this relationship between the EM field strength generated by a GNP and fluorescent emission level of a fluorophore, we have been developing a fluorophore contrast agent that is highly specific and sensitive for MMP-14 detection.

The fluorophore-GNP contrast agent for MMP-14 consists of a GNP, a near-infrared red (NIR) fluorophore Cypate (Cy), and spacers that link the GNP and Cy. An NIR fluorophore is chosen for a deep penetration to the tissue with a minimal background noise caused by the tissue auto-fluorescence, most of which are in the UV or visible wavelength. The GNP and Cy are linked by two different spacer types: The short spacer (SS) contains a substrate motif for MMP-14 and it is sufficiently short to place Cy close to GNP for the fluorescence quenching. When the agent encounters MMP-14, the SS is cleaved and Cy is released from the GNP, restoring its fluorescence. The long spacer (LS) is a stable, molecular sequence with a length for the Cy to be at the distance from a GNP for its maximum fluorescence. Ultimately, these two spacers will be conjugated to a single GNP together (also with an MMP-targeting molecule) so that the resulting contrast agent generates the Cy maximum fluorescence only when it encounters MMP-14.

In our process of optimizing the SS integrity, first, the length of the peptide sequence of SS was estimated by molecular simulation. Then, candidate peptide sequences possessing the MMP-14 substrate motif were conjugated to Cy (SS-Cy). Then the resulting SS-Cys were conjugated to the GNP (GNP-SS-Cy) and tested for their fluorescence levels and the cleavability of their SSs when they meet MMP-14. The fluorescence of the optimized GNP-SS-Cy was quenched up to 85% of its free form. When adding MMP-14, the fluorescence was restored up to 90%. We are currently testing the efficacy of the selected substrate motif in various cell lines. For the LS, we chose to use an amino acid sequence. The length of the optimal LS was first theoretically estimated and commercially available peptide candidates with potential length were selected. At a proper distance the fluorescence is estimated to be enhance up to 2.5 times of that of the free form. The GNPs conjugated with the Cy linked LS (GNP-LS-Cys) are currently tested for their fluorescence level.  

The resulting products of the optimized Cy-SS/LS-GNPs with targeting moieties will be able not only to detect the cancer but also to diagnose cancer type. Lately, GNPs have used for various biomedical purposes for both diagnostic and therapeutic.  In our study, the plasmon generated by the GNP was appropriately utilized to develop an optical contrast providing both enhanced specificity and sensitivity. Although this particular product is developed for MMP-14, the same concept can be used for the detection of any biomarkers that changes the spacer length, including enzymes, DNA, RNA biomarkers. In addition, GNPs are natural contrast agent for X-ray and/or CT, allowing contrast enhancement for two different imaging modalities.