Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method'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.
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.
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.
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. Please click here to view a larger version of this figure.
Institutional approval from Institutional Animal Care and Use Committee (IACUC) at the researcher'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 Physiological Research Laboratory (PRL) at one's institution. All animal work was approved by IACUC at Georgia Tech (protocol: A14100).
1. Iron Oxide Nanoparticle (IONP) Synthesis
NOTE: Safety: The entire Iron Oxide Nanoparticle synthesis should be performed using personal protective equipment and underneath a chemical fume hood.
2. Peptide Design, Conjugation to IONP, and In Vitro Validation
3. Administration of Nanosensors and Urine Detection of Cancer
NOTE: For more details on the example model, see our previous report13.
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 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 spherical iron oxide nanoparticles crosslinked together by an outer layer of dextran.13 A solution of IONPs will appear translucent with no precipitate and light brown in color. After successful peptide conjugation, spectral absorbance analysis will reveal a distinct absorbance peak at the maximum excitation wavelength of the fluorescent reporter. In the case of FITC, this will be centered around 488 nm (see Figure 2b). A typical peptide conjugation reaction will result in a peptide to IONP molar ratio of 20 – 50 after FPLC purification. To test the ability of the probe to sense protease activity, an aliquot of nanosensors can be incubated with recombinant proteases and monitored by fluorimetry. Cleavage activity will result in the release of fluorescent reporters – which are otherwise homoquenched on the surface of the nanoparticle – into solution and increase sample fluorescence. It is typical to observe substrate cleavage velocities that follow Michaelis-Menten kinetics (see Figure 2c).
Figure 2: Validating the Structure and Function of Nanosensors In vitro. (a) A DLS graph representing the size distribution of an IONP population post-synthesis. (b) The absorbance spectrum of IONPs post-conjugation with FITC-labeled MMP-9 substrates. (c) The in vitro cleavage assay showing that MMP9 steadily cleaves the peptide presented by the nanosensor over the course of an hour. A protease concentration of 0.5 mg/mL was used in PBS (1% BSA) buffer. Please click here to view a larger version of this figure.
To apply this platform in mouse models of cancer, it is typical to use nanosensors coated with a near-infrared fluorescent reporter to allow visualization of pharmacokinetics by whole-animal fluorescent imaging (Figure 3a). In mice bearing LS174T colorectal tumors, a fluorescent signal will localize into the urine within 60-90 minutes after intravenous administration of nanosensors due to MMP9 cleavage of peptide substrates on the nanosensors. By contrast, there will be lower fluorescent signals in the bladder of control group mice (see Figure 3a). It should be noted that the signal observed in the liver arises because particles are eventually scavenged by monocytes and macrophages in the reticuloendothelial system, which are commonly found in the liver, spleen and lymph nodes. Therefore, the signal coming from the liver in Figure 3a is due to clearance of the nanoparticles.13 When quantifying urine fluorescence by immunoprecipitation, a significantly elevated signal will be observed in mice bearing LS174T tumors compared to healthy controls (see Figure 3b).
Figure 3: Detecting Colorectal Cancer in a Mouse Model. (a) In vivo images of tumor and control group mice, observing IONP localization to sites of disease, as well as urine localization due to tumor progression. (b) Urine fluorescence quantification of tumor in control mice in the LS174T tumor model. Error bars are plotted as standard deviation (*p < 0.05 by two-tailed paired t test). Please click here to view a larger version of this figure.
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 "pharmacokinetic switch", because cleaved peptide products are smaller than the renal size filtration limit of 5 nm23 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 smaller will be rapidly cleared by kidneys and confound urine signals. We use pegylated IONPs because they are FDA-approved, well-tolerated, and have a circulating half-life of 3 – 5 hours.13 Another important consideration in formulating nanosensors is the PEG to peptide substrate molar ratio.24,25 Increasing the ratio will reduce non-specific cleavage by off-target proteases through steric shielding of peptides26, but potentially decrease on-target activity as well. Conversely, a lower PEG to peptide ratio will increase protease accessibility and cleavage of peptides, which will amplify detection signals but may also increase background noise. In vivo, a lower surface concentration of PEG may also increase nanosensor uptake by the reticuloendothelial system (RES) and decrease circulation time.
To apply this technology to diagnose other diseases, proteases that are upregulated in a disease-state should be identified from the literature17. Discovery efforts should focus on extracellular proteases because these nanosensors are not designed to penetrate cells to sense intracellular activity. These nanosensors are also engineered to sense endoproteases that cleave in the middle of the peptide, which provides many candidate diseases to target. By targeting extracellular endoproteases alone we have developed diagnostics for liver fibrosis, multiple types of cancer, and thrombosis13,19,20,21. This platform can be further designed to probe for exoprotease activity after endoprotease cleavage in the context of disease26,27. It is also important to design a peptide substrate that is specific for the protease of interest. This could be accomplished by screening candidate peptide sequences with the in vitro cleavage assay (steps 2.10 – 2.11). Potential non-specific protease cleavage should also be tested during peptide design. For example, in the case of MMP9, we tested substrate specificity with thrombin, a coagulation protease found at high concentrations in blood. To use activity nanosensors in vivo, the optimal urine collection time should be determined by monitoring kinetics of signal production by imaging or discrete sampling of the urine after administration of nanosensors. Mice can also be infused with sterile saline to overhydrate animals and increase urine production. Practically, this technology can be extended to any disease with upregulated or downregulated extracellular protease activity. A strength of this platform is that it allows for the amplification of target protease signal, so even if only a fraction of the disease-site protease population is surveyed, a significant read-out can be measured. The current platform is engineered to sense extracellular endoproteases. When applying this platform to other diseases, one needs to identify the protease biology of that disease, such that this platform can be tuned to sense proteases of interest.
This core technology can be further engineered with cleavage reporters that can be detected with different analytic platforms to increase the number of applications. For example, cleavage reporters that contain ligands for antibody binding can be detected by paper tests for low cost point-of-care diagnostics19. Additionally, labeling substrates with mass reporters allows multiple proteases to be simultaneously detected from urine by mass spectrometry13. In conclusion, this method describes formulating activity-based nanosensors to sense protease activity in vivo as a noninvasive urinary biomarker of disease.
The authors have nothing to disclose.
This work was funded by an NIH Director'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'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.
0.2 µm syringe filters | VWR | 4652 | |
18G needle | VWR | 89134-024 | |
15 mL conicals | VWR | 89039-670 | |
250 mL Erlenmeyer flask | VWR | 89000-362 | |
Stir bar | VWR | 58949-006 | |
Hot Plate/Magnetic Stirrer | VWR | 97042-634 | |
Glacial acetic acid | VWR | 97064-482 | |
Albumin from Bovine Serum (BSA) | Thermo Fisher | A13100 | |
Iron (III) chloride hexahydrate | Sigma | 236489 | |
Iron (II) chloride tetrahydrate | Sigma | 44939 | |
Epichlorohydrin | Sigma | 45340-500ML-F | |
DMF | Sigma | D4551 | |
Ammonium Hydroxide | Sigma | 320145-500ML | |
Sodium Hydroxide pellets | Sigma | 221465-500G | |
EDTA | Sigma | E9884 | |
Sodium Borate | Sigma | B9876 | |
L-Cysteine | Sigma | 168149-100G | |
Tris-HCl | Sigma | T5941 | |
Tris base | Sigma | T6066 | |
PBS tablets | Sigma | P4417 | |
Dextran | Pharmacosmos | 5510 0020 9006 | |
Amicon 15 mL 10k filters, 24 pk | Millipore | UFC901024 | |
Amicon 15 mL 30k filters, 24 pk | Millipore | UFC903024 | |
Amicon 15 mL 100k filters, 24 pk | Millipore | UFC910024 | |
Zetasizer Nano ZS | Malvern Panalytical | NanoZS | |
Slide-A-Lyzer Dialysis Cassette | LifeTech | 66130 | |
Dynabeads MyOne Tosylactivated | LifeTech | 65501 | |
SIA | Life Tech | 22349 | |
PEG 20k | Laysan Bio | MPEG-SH-20K-1g | |
Fluorescein Antibody [2A3] | GeneTex | GTX10257 | |
Hiload 16/600 superdex 200 | GE Healthcare | 45-002-490 | |
Plate Reader | Fisher | BTCYT5M | |
BD Insulin Syringes | Fisher | NC0872854 |