UC San Diego

Traumatic brain injury (TBI) affects over 2.8 million people annually in the United States and leads to the hospitalization of ~300,000 patients per year. Current methods of diagnosis for TBI are either subjective and poor at discriminating mild TBI (Glasgow Coma Scale) or take extensive time and resources to run (computed tomography and magnetic resonance imaging). These diagnostic modalities also do not capture information on the biological processes driving the pathology of secondary injury, where there is a window of opportunity to prevent further damage with treatment. As a supplement to these diagnostics, the blood or cerebrospinal fluid can be sampled for biomarkers in the form of breakdown products which are released during degenerative processes after TBI. Many of these biomarkers are produced by ectopic proteases including the calcium-dependent protease calpain-1, which is implicated in cellular death and worsened prognosis after TBI. Thus, the measurement of calpain-1 activity may help to diagnose injury progression and inform patient prognosis after TBI.

To diagnose secondary injury in TBI, we developed an activity-based nanosensor for TBI (TBI-ABN) which can detect activity from the protease calpain-1. The nanosensor is comprised of a FRET peptide conjugated to a 40 kDa 8-arm PEG scaffold, and can produce a fluorescent signal once it is specifically cleaved by active calpain-1. In a mouse model of TBI, systemically administered TBI-ABNs were found to accumulate and activate in the injured brain tissue as assessed by fluorescence in brain tissue slices. Next, we investigated whether adding active targeting to components of the brain extracellular matrix, such as hyaluronic acid, could improve the sensitivity of TBI-ABNs. We conjugated hyaluronic acid-targeting peptides to TBI-ABNs and found that the activation of sensor in injured brain tissue was increased by approximately 2.8- and 6.6-fold when targeting peptides were added at moderate and high valencies, respectively, compared to non-targeted nanosensor within brain tissue homogenates. Finally, we redesigned the TBI-ABN to release a synthetic biomarker into the blood or urine after systemic administration for a minimally-invasive measurement of protease activity after TBI. The synthetic biomarker could be quantified both via fluorescence and immunoassays to detect calpain-1 activity in TBI. These nanosensors are the first demonstrations of protease activity measurement in the context of TBI and have potential both as tools to study protease activation in the injured brain and as diagnostics to identify the biological processes taking place after injury.