Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

• Type of Material: Article
• Authors: Brandon Alexander Holt, Georgia Institute of Technology; Quoc D. Mac, Georgia Institute of Technology; Gabe Kwong, Emory University
• Language: English
• Date: 2018-07-01
• Publisher: Journal of Visualized Experiments (JoVE)
• Publication Version: Final Published Version
• Copyright Statement: © 2018 Journal of Visualized Experiments.
• Final Published Version (URL): http://dx.doi.org/10.3791/57937
• Title of Journal or Parent Work: Journal of Visualized Experiments
• ISSN: 1940-087X
• Volume: 2018
• Issue: 137
• Grant/Funding Information: Q.D.M. is supported by the NSF Graduate Research Fellowships Program (Grant No. DGE-1650044).; B.A.H is supported by the National Institutes of Health GT BioMAT Training Grant under Award Number 5T32EB006343; as well as the Georgia Tech President's Fellowship.; This work was funded by an NIH Director's New Innovator Award (Award No. DP2HD091793).; G.A.K. holds a Career Award at the Scientific Interface from the Burroughs Welcome Fund.
• Keywords: Issue 137; proteases; Synthetic biomarkers; BIOMARKERS; Science & Technology - Other Topics; Science & Technology; urine diagnostics; Multidisciplinary Sciences; activity-based probes; Bioengineering; nanosensors; nanoparticles
• Research Categories: Engineering, Biomedical

Abstract

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.