Name: nanosensors to detect protease activity in vivo for noninvasive diagnostics
Uploaded: 2018-07-16T14:30:00
Description: Watch this Scientific Journal Video about Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics at JoVE.com

This work was funded by an NIH Director&#39;s New Innovator Award (Award No. DP2HD091793). 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&#39;s Fellowship. G.A.K. holds a Career Award at the Scientific Interface from the Burroughs Welcome Fund. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Institutional approval from Institutional Animal Care and Use Committee (IACUC) at the researcher&#39;s institution is necessary to carry out the following animal experiments. Additionally, standard animal care facilities (e.g., housing chambers, sterile animal hoods, isofluorane chambers for anesthetization, and CO2 chambers for ethical endpoint euthanization) are necessary to properly carry out these experiments. Special training and assistance with these facilities can be provided by the Physiologi...

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This method describes the development of activity-based nanosensors consisting of protease substrates conjugated to a nanoparticle core. The event of proteolytic cleavage is dubbed the &#34;pharmacokinetic switch&#34;, because cleaved peptide products are smaller than the renal size filtration limit of 5 nm23&#160;and filter into urine to produce a noninvasive signal. Therefore, it is important to use nanoparticles or carriers with a hydrodynamic radius that is larger than 5 nm, as anything smalle...

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and have significant control over many biological processes, including homeostasis, allostasis, and disease1. An altered state of protease activity has been correlated to a variety of diseases, including cancer and cardiovascular disease, making proteases attractive candidates for development into clinical biomarkers2,3. Moreover, protease activity is functionally linked to distinct pathogeneses, patient outcomes, and prognosis of disease4. Broadly, biosensors have been developed to detect various biological phenomena and diseases, such as cancer, neurodegenerative disease, and electron transfer processes5,6,7,8,9. More specifically, substrate-based protease sensors have been developed to detect protease activity, and include fluorogenic probes for diagnostic imaging10 and isotopically labeled peptide substrates for in vitro detection by mass spectrometry11. In addition, activity-based probes have been developed, which contain substrate-like regions that bind or modify the target protease12. With this method, the target protease is irreversibly inhibited when the active site is modified, and analysis requires harvesting of tissue, which limits in vivo applications. However, it is important to sense protease activity in vivo, because regulation of protease activity is heavily dependent on the context of other biological activities such as the presence of endogenous inhibitors.
The goal of this work is to describe the formulation of activity-based nanosensors that detect protease activity in vivo by producing a measurable signal in urine. This platform is used as a noninvasive diagnostic to discriminate complex diseases such as cancer by using dysregulated protease activity as a functional biomarker. Our nanosensor platform consists of iron oxide nanoparticles (IONP) conjugated to protease substrates. These substrates are terminated by a fluorescent reporter which is released when proteases cleave the substrate. These IONPs circulate in vivo, localize to disease sites, and expose substrates to active disease-associated proteases. After cleavage, fluorescent reporters are released and, due to their small size, are filtered into urine, while uncleaved substrates on the IONP remain in the body. Therefore, an increase in protease activities in vivo will result in higher concentrations of reporter in urine (Figure 1). Since our platform is a urine test, no imaging platform is required and diagnostic signals are enriched in urine.
This platform can be engineered to detect a variety of diseases including cancer, fibrosis, and thrombosis13,14. Here we describe the design of nanosensors to detect elevations in Matrix metallopeptidase 9 (MMP9) activity as a biomarker of colorectal cancer. Colorectal cancer is the second leading cause of cancer death in the United States, with an estimated 136,800 new cases and 50,300 deaths in 2014 alone15. Colorectal tumor cells produce MMP9, which has been shown to drive malignant progression , matrix degradation, as well as metastasis16. Additionally, we identified a suitable peptide substrate (PLGVRGK) for MMP9 from the literature17. This platform may be used for early cancer detection and low-cost point-of-care diagnostics13,14,18,19,20,21.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57937/57937fig1.jpg" /> 
Figure 1: Schematic of Nanosensor Activity In vivo. Nanosensors circulate through the body and localize to sites of disease. Then, disease-related proteases cleave peptide substrates presented by IONPs. The size of cleaved fragments allows for renal clearance, causing them to localize in the urine. After the animal urinates, these peptide fragments can be analyzed by their reporter molecule. <a href="https://www.jove.com/files/ftp_upload/57937/57937fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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			<td height="21" style="height:21px;width:375px;">0.2 &#181;m syringe filters</td>
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			<td height="21" style="height:21px;width:375px;">Tris-HCl</td>
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			<td height="21" style="height:21px;width:375px;">BD Insulin Syringes</td>
			<td style="width:171px;">Fisher</td>
			<td style="width:233px;">NC0872854</td>
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nanosensors to detect protease activity in vivo for noninvasive diagnostics

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.

The majority of the population of the IONPs is around the average diameter, which ranges from 40 - 50 nm. After pegylation, this size range has a circulation half-life of approximately 6 hours13&#160;in vivo (see Figure 2a). If one wants to select for a particular size range, one can use size exclusion chromatography to isolate IONP fractions with different diameters. The nanoparticles by TEM will appear as individual spherica...

Watch this Scientific Journal Video about Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics at JoVE.com

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Research

JoVE Journal

Bioengineering

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