In Vivo Mapping of Enzyme Activity using SWIR-emitting, Self-illuminating Quantum Dot Sensors

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Project Summary/Abstract Enzymes play a critical role in biological processes, and measurements of enzyme activity in cells and tissues are foundational to our understanding of physiology and pathophysiology. Measuring enzyme activity in vivo provides context within the intact living tissue, enabling organ- or organism-level understanding of the spatial and temporal differences in enzyme activity. However, most research regarding enzyme activity has been lim- ited to in vitro and ex vivo methods due to our inability to observe enzyme activity with high sensitivity and high resolution in deep tissues. Fluorescence-based techniques offer a non-invasive, high resolution look at molec- ular or morphological information but are often hampered by limited tissue penetration. Imaging in optical tis- sue windows in the short-wave infrared (SWIR; 900-1700 nm) enables deeper tissue penetration and improved resolution than visible or NIR light due to reduced absorption and scattering by biological materials. Even at ideal wavelengths, photoluminescence imaging requires incident light to excite the fluorophore, resulting in light attenuation both going in and out of the tissue. Luciferase-based bioluminescence imaging eliminates the need for external excitation, thereby reducing the distance light must traverse through tissue as well as elimi- nating autofluorescence. While bioluminescence is preferred for many in vivo imaging applications, there are no reports of SWIR-emitting luciferases, limiting this approach to less preferred visible wavelengths or NIR probes emitting < 750 nm. In this proposal, bioluminescence energy transfer (BRET) from luciferases to SWIR- emitting semiconductor quantum dots (QDs) generate self-illuminating SWIR-emitting contrast agents. We pro- pose the development of the first enzyme-sensing, SWIR-emitting, self-illuminating quantum dots (QDs). Multiplexing reference and enzyme-cleavable probes for ratiometric in vivo imaging will calibrate the enzyme activity to the local concentration of probes and is facilitated by our hyperspectral SWIR preclinical im- ager. We will test our sensor by visualizing the activity of MMP-16, a cell-surface biomarker of liver disease progression that is correlated with fibrosis, hepatitis, and hepatocellular carcinoma (HCC). To test our hypothe- sis that dual probe imaging with self-illuminating, SWIR-emitting contrast agents will enable spatial and tem- poral enzyme activity maps, we will design, characterize, and optimize the BRET probes in vitro. In parallel, we will use tissue phantoms to compare the signal-to-noise ratios (S/N) and image resolution using BRET vs. pho- toluminescence excitation mechanisms. Finally, we will demonstrate the functionality of our sensor in phan- toms and in a mouse model of liver cirrhosis. Successful completion of this project would result in a sensor ca- pable of reporting MMP-16 activity deep in the liver with high resolution and high sensitivity. Demonstrating this new way to take non-invasive, high resolution images of enzyme activity in deep tissues will enable a wide ar- ray of new studies into the role of enzymes in pathophysiology by the scientific community.