Targeting the Extracellular Matrix in Traumatic Brain Injury Increases Signal Generation from an Activity-Based Nanosensor

[Image: see text] Traumatic brain injury (TBI) is a critical public health concern and major contributor to death and long-term disability. After the initial trauma, a sustained secondary injury involving a complex continuum of pathophysiology unfolds, ultimately leading to the destruction of nervous tissue. One disease hallmark of TBI is ectopic protease activity, which can mediate cell death, extracellular matrix breakdown, and inflammation. We previously engineered a fluorogenic activity-based nanosensor for TBI (TBI-ABN) that passively accumulates in the injured brain across the disrupted vasculature and generates fluorescent signal in response to calpain-1 cleavage, thus enabling in situ visualization of TBI-associated calpain-1 protease activity. In this work, we hypothesized that actively targeting the extracellular matrix (ECM) of the injured brain would improve nanosensor accumulation in the injured brain beyond passive delivery alone and lead to increased nanosensor activation. We evaluated several peptides that bind exposed/enriched ECM constituents in the brain and discovered that nanomaterials modified with peptides that target hyaluronic acid (HA) displayed widespread distribution across the injury lesion, in particular colocalizing with perilesional and hippocampal neurons. Modifying TBI-ABN with HA-targeting peptide led to increases in activation in a ligand-valency-dependent manner, up to 6.6-fold in the injured cortex compared to a nontargeted nanosensor. This robust nanosensor activation enabled 3D visualization of injury-specific protease activity in a cleared and intact brain. In our work, we establish that targeting brain ECM with peptide ligands can be leveraged to improve the distribution and function of a bioresponsive imaging nanomaterial.