Spatial Measurement\nand Inhibition of Calpain Activity\nin Traumatic Brain Injury with an Activity-Based Nanotheranostic Platform

Traumatic brain injury\n(TBI) is a major public health\nconcern that\ncan result in long-term neurological impairments. Calpain is a calcium-dependent\ncysteine protease that is activated within minutes after TBI, and\nsustained calpain activation is known to contribute to neurodegeneration\nand blood–brain barrier dysregulation. Based on its role in\ndisease progression, calpain inhibition has been identified as a promising\ntherapeutic target. Efforts to develop therapeutics for calpain inhibition\nwould benefit from the ability to measure calpain activity with spatial\nprecision within the injured tissue. In this work, we designed an\nactivity-based nanotheranostic (ABNT) that can both sense and inhibit\ncalpain activity in TBI. To sense calpain activity, we incorporated\na peptide substrate of calpain flanked by a fluorophore/quencher pair.\nTo inhibit calpain activity, we incorporated calpastatin peptide,\nan endogenous inhibitor of calpain. Both sensor and inhibitor peptides\nwere scaffolded onto a polymeric nanoscaffold to create our ABNT.\nWe show that in the presence of recombinant calpain, our ABNT construct\nis able to sense and inhibit calpain activity. In a mouse model of\nTBI, systemically administered ABNT can access perilesional brain\ntissue through passive accumulation and inhibit calpain activity in\nthe cortex and hippocampus. In an analysis of cellular calpain activity,\nwe observe the ABNT-mediated inhibition of calpain activity in neurons,\nendothelial cells, and microglia of the cortex. In a comparison of\nneuronal calpain activity by brain structure, we observe greater ABNT-mediated\ninhibition of calpain activity in cortical neurons compared to that\nin hippocampal neurons. Furthermore, we found that apoptosis was dependent\non both calpain inhibition and brain structure. We present a theranostic\nplatform that can be used to understand the regional and cell-specific\ntherapeutic inhibition of calpain activity to help inform drug design\nfor TBI.