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 in order to detect TBI. In this work, we engineered an activitybased 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 injured brain tissue with minimal renal filtration. A calpain-1 substrate that generates 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.
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.
Randomized mutagenesis at an endogenous chromosomal locus is a promising approach for protein engineering, functional assessment of regulatory elements, and modeling genetic variations. In mammalian cells, however, it is challenging to perform site-specific single-nucleotide substitution with single-stranded oligodeoxynucleotide (ssODN) donor templates due to insufficient homologous recombination and the infeasibility of positive selection. Here, we developed a DNA transposon based CRISPR-Cas9 regulated transcription and nuclear shuttling (CRONUS) system that enables the stable transduction of CRISPR-Cas9/sgRNA in broad cell types, but avoids undesired genome cleavage in the absence two chemical inducing molecules. Highly efficient single nucleotide alterations induced randomization of desired codons (up to 4 codons) at a defined genomic locus in various human cell lines, including human iPS cells. Thus, CRONUS provides a novel platform for modeling diseases and genetic variations.
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 can 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 are 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 glial fibrillary acidic protein (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.
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, we engineered a nanosensor that releases a measurable signal into the blood and urine in response to activity from the TBI-associated protease calpain. Readouts from our nanosensor were designed to be compatible with ELISA and lateral flow assays, clinically-relevant assay modalities. In a mouse model of TBI, we demonstrated greater sensitivity of the nanosensor with the addition of targeting ligands to hyaluronic acid. In evaluation of mice with mild or severe injuries, our nanosensor identified mild TBI with a higher sensitivity than the clinical 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.
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