Extracellular Matrix Targeted Activity-Based Nanosensors to Visualize Protease Activity in Traumatic Brain Injury

Ectopic protease activity is a driver of disease progression in traumatic brain injury (TBI), contributing to cell death, extracellular matrix degradation, and vascular dysregulation. However, currently there are few tools available to detect protease activity with spatial localization in the injured brain. We have developed an activity-based nanosensor to detect the proteolytic activity of calpain-1, a calcium-dependent protease that is highly expressed in the brain and whose activity is correlated with worsened TBI outcomes in human patients. We hypothesized that the addition of targeting ligands that bind to the brain extracellular matrix (ECM) to our calpain-1 activity-based nanosensor would increase nanosensor bioavailability in the injured brain and therefore its activation. To test this hypothesis, we synthesized nanomaterials modified with several peptides that bind to brain ECM. When we evaluated the distribution of these peptide-modified nanomaterials in the injured brain after systemic administration in a mouse model of TBI, we found that nanomaterials modified with peptides that bind to hyaluronic acid broadly distributed in perilesional brain tissue. In particular, these nanomaterials localized to neurons and endothelial cells in the injured brain, which are known sources of calpain-1. Hyaluronic acid binding peptides were added to our activity-based nanosensor for calpain-1 at several valencies, administered intravenously to a mouse model of TBI, and tissue analyzed for bulk nanosensor activation. We observed that the activation of nanosensor in injured brain tissue increased in a ligand valency-dependent manner. Robust activation of nanosensor mediated by active targeting to brain ECM enabled its visualization in intact, cleared brains using light sheet microscopy. This 3D reconstruction of nanosensor activation revealed the extent and spatial distribution of calpain-1 activity in the perilesional cortex and hippocampus post-TBI. The goal of future work is to evaluate the activity of multiple proteases, the temporal dynamics of protease activation, and correlation of protease activity with long-term neurodegeneration.