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...

<ol>
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Dr. Kwong is co-founder and serves as consultant to Glympse Bio, which is developing products related to the research described in this paper. This study could affect his personal financial status. The terms of this arrangement have been reviewed and approved by Georgia Tech in accordance with its conflict of interest policies

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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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">0.2 &#181;m syringe filters</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">4652</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">18G needle</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">89134-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">15 mL conicals</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">89039-670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">250 mL Erlenmeyer flask</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">89000-362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Stir bar</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">58949-006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Hot Plate/Magnetic Stirrer</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">97042-634</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glacial acetic acid</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">97064-482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:171px;">Thermo Fisher</td>
			<td style="width:233px;">A13100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Iron (III) chloride hexahydrate</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">236489</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Iron (II) chloride tetrahydrate</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">44939</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Epichlorohydrin</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">45340-500ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">DMF</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">D4551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Ammonium Hydroxide</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">320145-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium Hydroxide pellets</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">221465-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">EDTA</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">E9884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium Borate</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">B9876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">L-Cysteine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">168149-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris-HCl</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">T5941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris base</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">T6066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">PBS tablets</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Dextran</td>
			<td style="width:171px;">Pharmacosmos</td>
			<td>5510 0020 9006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amicon 15 mL 10k filters, 24 pk</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:233px;">UFC901024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amicon 15 mL 30k filters, 24 pk</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:233px;">UFC903024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amicon 15 mL 100k filters, 24 pk</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:233px;">UFC910024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Zetasizer Nano ZS</td>
			<td style="width:171px;">Malvern Panalytical</td>
			<td style="width:233px;">NanoZS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Slide-A-Lyzer Dialysis Cassette</td>
			<td style="width:171px;">LifeTech</td>
			<td style="width:233px;">66130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Dynabeads MyOne Tosylactivated</td>
			<td style="width:171px;">LifeTech</td>
			<td style="width:233px;">65501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">SIA</td>
			<td style="width:171px;">Life Tech</td>
			<td style="width:233px;">22349</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">PEG 20k</td>
			<td style="width:171px;">Laysan Bio</td>
			<td style="width:233px;">MPEG-SH-20K-1g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Fluorescein Antibody [2A3]</td>
			<td style="width:171px;">GeneTex</td>
			<td style="width:233px;">GTX10257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Hiload 16/600 superdex 200</td>
			<td style="width:171px;">GE Healthcare</td>
			<td style="width:233px;">45-002-490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Plate Reader</td>
			<td style="width:171px;">Fisher</td>
			<td style="width:233px;">BTCYT5M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BD Insulin Syringes</td>
			<td style="width:171px;">Fisher</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 ...

This method can help answer key questions in protea mix and diagnostics, such as how to design probes that can detect the progression of diseases by measuring proteates'activity in vivo. The main advantage of this method is that the diagnositics developed will be noninvasive, and can actually amplify the detection of biological signals. This method can be applied to numerous diseases, because many proteuses are actually major drivers of pathogenesis.In the chemical fume hood, transfer one ml of 30%ammonium hydroxide to a 15 ml conical tube. Place this tube into an ice bucket and let it chill for 30 minutes. Wash a 250 ml Ehrlenmeyer flask with deionized water.Then add 10 ml of double distilled water. Submerge the flask in an ice bath resting on a heat plate. Using a plastic pipette, begin the flow of nitrogen gas into the double distilled water in the flask.Bubble for 15 minutes to deoxygenate. Next, weigh out 4.5 g of dextrine in a 15 ml conical tube. Add water such that the final volume of the mixture including the dextrine is 20 ml.Vortex vigorously to dissolve the dextrine. Weigh out 78.5 mg of iron 3 chloride hexahydrate and add this to the dextrine solution. Vortex to dissolve.Using a 0.2 um filter, filter the resulting solution. After this, transfer 9 ml of the deoxygenated water to the 15 ml conical tube containing the 1 ml of chilled ammonium hydroxide. Return the conical tube to the ice bucket to chill.Weigh out 73 mg of iron 2 chloride tetrahydrate and dissolve it in the remaining 1 ml of deoxygenated double distilled water. Use a 0.2 um filter to filter the solution. Then, transfer the dextrine iron 3 solution into the 250 ml Ehrlenmeyer flask.Deoxygenate with nitrogen gas for 15 minutes on ice, while mixing at 1, 600 rpm. Cap the flask with a rubber septum. Using an 18 gauge needle, puncture the septum to flow nitrogen gas into the flask.Insert a separate 18 gauge needle, which will be the flow outlet. Next, use a 1 ml syringe to add 467 ul of the iron 2 solution to the dextrine iron 3 solution. Stir at 1, 600 rpm.Add the chilled dilute ammonium hydroxide solution dropwise to initiate the nucleation process. Make sure each drop mixes well before adding another. Stop the flow of nitrogen gas and remove the rubber septum.Remove the ice bath and replace it with a bath of warm water. Heat the solution to 75 degrees Celsius and then incubate for 75 minutes. Remove the flask from the hot plate.Doubly filter the solution through a 0.2 um filter and then a 0.1 um filter to remove any coarse particles. Using 100 kDa molecular weight cutoff concentrators, buffer exchange the particles into double distilled water to remove excess dextrine. If the flow through remains dark after two to three spins, replace the filters as they may have broken.Next, resuspend the iron oxide nanoparticles in double distilled water at a concentration of 10 mg per ml. Add 1.6 volumes of 5 molar sodium hydroxide, then add 0.65 volumes of epichlorohydrine. Using a plate shaker, rigorously mix at room temperature for 12 hours.Use a 20 ml syringe with an 18 gauge needle to transfer the iron oxide nanoparticle solution into a dialysis membrane with a 50 kDa molecular weight cutoff. Place the dialysis membrane into 4 liters of double distilled water. Incubate at room temperature overnight.After determining the concentration, bring the iron oxide nanoparticles to a concentration of 5 mg per ml. Add ammonium hydroxide to reach 20%and shake at room temperature for at least 12 hours to amenate the surface of the iron oxide nanoparticles. Then, buffer exchange using 30 kDa molecular weight cutoff filters.Use dynamic light scattering to determine the hydrodynamic radius of the iron oxide nanoparticles as outlined in the text protocol. After synthesizing a peptide substrate, aliquot 0.5 mg of iron oxide nanoparticles into the 10 kDa molecular weight cutoff spin filter tube. Buffer exchange into coupling buffer using a 10 kDa molecular weight cutoff spin filter as outlined in the text protocol.Dissolve SIA in DMF to reach a concentration of approximately 30 mg per ml. Then, add SIA to the iron oxide nanoparticles at a mole ratio of 500 to one. After incubation and buffer exchange, bring the final product to a concentration of 1 mg per ml.Mix the peptide of interest with 20 kDa thiol terminated polyethylene glycol, and then mix this peptide glycol solution with iron oxide nanoparticles. Cover the tubes in foil to prevent photo bleaching the fluorescent molecules. And incubate overnight at room temperature on a plate shaker set the to the highest speed.Next, add L cystine at a molar ratio of 500 to one to the iron oxide nanoparticles. Incubate for one hour at room temperature on a shaker set to the highest speed. After this, make an 18 ul nanoparticle solution using PVS, and a 200 nanomolar concentration of peptide.Add 2 ul of the protease of interest. Incubate in a plate reader for one hour at 37 degrees Celsius taking flourescence measurements every one to two minutes to monitor cleavage. Immediately after injecting the prepared nanosensor solution via tail vein injection, place the mice into metabolic cages, and note the time of injection for each mouse.Combine 2 ul of urine with 5 ul of prepared magnetic beads. Using PVS, bring the final total volume to 50 ul. Incubate at room temperature for 60 minutes.Next, wash twice using 50 ul of PVS each time, while using a magnetic separator to collect the magnetic beads after each wash. Elute twice with 32.5 ul of 5%glacial acetic acid. Use 35 ul of 2 molar tris to neutralize the pool dilution and achieve a final ph around 7.After this, use a plate reader at appropriate excitation and emission wavelengths to quantify the urine flourescence. Calculate the concentration of fluorescent reporter against a ladder of known concentrations of free flourophor. In this study, activity based nanosensors are developed by conjugating protease substrates to a nanoparticle core.The average diameter of iron oxide nanoparticles is observed to be between 40 and 50 nm. After pecolation, the circulation half life for this size range is seen to be approximately 6 hours. After successful peptide conjugation, spectral absorbance analysis reveals a distinct absorbance peak at the maximum excitation wavelength of the fluorescent reporter.The ability of the probe to sense protease activity is then tested by incubating an aliquot of nanosensors with recombinant proteases. It is typical to observe substrate cleavage velocities that follow Michaelis-Menten kinetics. Nanosensors coated with a near infrared fluorescent reporter are then used to visualize the pharmacokinetics in mouse models of cancer by whole animal fluorescent imaging.A fluorescent signal is seen to localize in the urine of mice, bearing LS 174 t colorectal, 60 to 90 minutes after intravenous administration of the nanosensors. Significantly lower fluorescent signals are observed in the bladders of control mice. It should be noted that the signal observed in the liver arises because particles are eventually scavenged by monocites and and macrophages in the ridiculoendophilial system, which are commonly found in the liver, spleen, and lymph nodes.Once mastered, this technique can be done in four days, if it's performed properly. While attempting this procedure, it's important to remember that flourescence is the primary readout, so it's important to keep the peptide substrates with fluorescent markers in the dark. Following this procedure, other methods like biodistribution studies and in viva imaging can be performed in order to answer additional questions, like localization of particles to various organs.After its development, this technique paved the way for researchers in the field of diagnostics to explore cancer biomarkers in mice. After watching this video, you should have a good understanding of how to synthesize and characterize iron oxide nanoparticles, use these to build nanosensors, then use the nanosensors in vivo. Don't forget that working with the chemicals involved in a nanoparticle synthesis can be extremely hazardous, and precautions such as personal protective equipment and working within a fume hood should always be taken while performing this procedure.

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16.0K ビュー.  Georgia Tech College of Engineering and Emory School of Medicine. This method can help answer key questions in protea mix and diagnostics, such as how to design probes that can detect the progression of diseases by measuring proteates'activity in vivo. The main advantage of this method is that the diagnositics developed will be noninvasive, and can actually amplify the detection of biological signals. This method can be applied to numerous diseases, because many proteuses are actually major drivers of pathogenesis.In the chemical fume hood, transfer ...