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Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2022)

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.

    Cover page: Engineered nanosensors for detecting protease activity in traumatic brain injury
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2021)
    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.
      Cover page: Targeting the Extracellular Matrix in Traumatic Brain Injury Increases Signal Generation from an Activity-Based Nanosensor
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2023)
      Current screening and diagnostic tools for traumatic brain injury (TBI) have limitations in sensitivity and prognostication. Aberrant protease activity is a central process that drives disease progression in TBI and is associated with worsened prognosis; thus direct measurements of protease activity could provide more diagnostic information. In this study, a nanosensor is engineered to release a measurable signal into the blood and urine in response to activity from the TBI-associated protease calpain. Readouts from the nanosensor were designed to be compatible with ELISA and lateral flow assays, clinically-relevant assay modalities. In a mouse model of TBI, the nanosensor sensitivity is enhanced when ligands that target hyaluronic acid are added. In evaluation of mice with mild or severe injuries, the nanosensor identifies mild TBI with a higher sensitivity than the biomarker GFAP. This nanosensor technology allows for measurement of TBI-associated proteases without the need to directly access brain tissue, and has the potential to complement existing TBI diagnostic tools.
        Cover page: An Activity-Based Nanosensor for Minimally-Invasive Measurement of Protease Activity in Traumatic Brain Injury.
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2020)
        Currently, traumatic brain injury (TBI) is detected by medical imaging; however, medical imaging requires expensive capital equipment, is time- and resource-intensive, and is poor at predicting patient prognosis. To date, direct measurement of elevated protease activity has yet to be utilized to detect TBI. In this work, we engineered an activity-based nanosensor for TBI (TBI-ABN) that responds to increased protease activity initiated after brain injury. We establish that a calcium-sensitive protease, calpain-1, is active in the injured brain hours within injury. We then optimize the molecular weight of a nanoscale polymeric carrier to infiltrate into the injured brain tissue with minimal renal filtration. A calpain-1 substrate that generates a fluorescent signal upon cleavage was attached to this nanoscale polymeric carrier to generate an engineered TBI-ABN. When applied intravenously to a mouse model of TBI, our engineered sensor is observed to locally activate in the injured brain tissue. This TBI-ABN is the first demonstration of a sensor that responds to protease activity to detect TBI.
          Cover page: An Activity-Based Nanosensor for Traumatic Brain Injury.
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2024)
          Traumatic brain injury (TBI) is a major public health concern that can result in long-term neurological impairments. Calpain is a calcium-dependent cysteine protease that is activated within minutes after TBI, and sustained calpain activation is known to contribute to neurodegeneration and blood-brain barrier dysregulation. Based on its role in disease progression, calpain inhibition has been identified as a promising therapeutic target. Efforts to develop therapeutics for calpain inhibition would benefit from the ability to measure calpain activity with spatial precision within the injured tissue. In this work, we designed an activity-based nanotheranostic (ABNT) that can both sense and inhibit calpain activity in TBI. To sense calpain activity, we incorporated a peptide substrate of calpain flanked by a fluorophore/quencher pair. To inhibit calpain activity, we incorporated calpastatin peptide, an endogenous inhibitor of calpain. Both sensor and inhibitor peptides were scaffolded onto a polymeric nanoscaffold to create our ABNT. We show that in the presence of recombinant calpain, our ABNT construct is able to sense and inhibit calpain activity. In a mouse model of TBI, systemically administered ABNT can access perilesional brain tissue through passive accumulation and inhibit calpain activity in the cortex and hippocampus. In an analysis of cellular calpain activity, we observe the ABNT-mediated inhibition of calpain activity in neurons, endothelial cells, and microglia of the cortex. In a comparison of neuronal calpain activity by brain structure, we observe greater ABNT-mediated inhibition of calpain activity in cortical neurons compared to that in hippocampal neurons. Furthermore, we found that apoptosis was dependent on both calpain inhibition and brain structure. We present a theranostic platform that can be used to understand the regional and cell-specific therapeutic inhibition of calpain activity to help inform drug design for TBI.
            Cover page: Spatial Measurement and Inhibition of Calpain Activity in Traumatic Brain Injury with an Activity-Based Nanotheranostic Platform.
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2020)
            Currently, traumatic brain injury (TBI) is detected by medical imaging; however, medical imaging requires expensive capital equipment, is time- and resource-intensive, and is poor at predicting patient prognosis. To date, direct measurement of elevated protease activity has yet to be utilized to detect TBI. In this work, we engineered an activity-based nanosensor for TBI (TBI-ABN) that responds to increased protease activity initiated after brain injury. We establish that a calcium-sensitive protease, calpain-1, is active in the injured brain hours within injury. We then optimize the molecular weight of a nanoscale polymeric carrier to infiltrate into the injured brain tissue with minimal renal filtration. A calpain-1 substrate that generates a fluorescent signal upon cleavage was attached to this nanoscale polymeric carrier to generate an engineered TBI-ABN. When applied intravenously to a mouse model of TBI, our engineered sensor is observed to locally activate in the injured brain tissue. This TBI-ABN is the first demonstration of a sensor that responds to protease activity to detect TBI.
              Cover page: An Activity-Based Nanosensor for Traumatic Brain Injury
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