Detection of Matrix Metalloproteinase Activity Using a Fluorogenic

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Bioconjugate Chem. 2010, 21, 1378–1384

“One-Step” Detection of Matrix Metalloproteinase Activity Using a Fluorogenic Peptide Probe-Immobilized Diagnostic Kit Ju Hee Ryu,†,‡ Aeju Lee,†,‡ Seulki Lee,‡ Cheol-Hee Ahn,§ Jong Woong Park,| James F. Leary,⊥ Sangjin Park,‡ Kwangmeyung Kim,‡ Ick Chan Kwon,‡ In-Chan Youn,*,‡ and Kuiwon Choi*,‡ Biomedical Research Center, Korea Institute of Science and Technology, Seongbuk-Gu, Seoul, South Korea, Research Institute of Advanced Materials (RIAM), Department of Materials Science and Engineering, Seoul National University, San 56-1 Sillim, Gwanak, Seoul, 151-744, South Korea, Department of Orthopaedic Surgery, College of Medicine, Korea University, Seoul, South Korea, and Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana. Received January 5, 2010; Revised Manuscript Received March 5, 2010

Matrix metalloproteinases (MMPs) have been shown to be abundant in pathological conditions such as cancer, osteoarthritis (OA), and rheumatoid arthritis (RA). The extent of MMPs detected in biological samples provides important clinical information for diagnosis, prognosis, and therapeutic monitoring of various diseases relating with MMPs. Herein, we developed a new high-throughput MMP diagnostic kit (MMP-D-KIT) based on a 96well plate by immobilizing MMP-13 specific fluorogenic peptide probes (MMP peptide probe), which is a pair consisting of a near-infrared (NIR) fluorophore (Cy5.5) and a quencher (BHQ-3), onto the biocompatible glycol chitosan (GC) polymer anchored 96-well plate. When MMP enzymes were simply added and incubated in a MMP-D-KIT, the fluorescence of each well was recovered and the fluorescence intensity showed distinct difference within minutes through NIR fluorescence imaging system. The fluorescence was recovered not only by MMP-13 activity, but also by other MMPs activity. Furthermore, recovery of NIR fluorescent signals in MMP-D-KIT was proportional to concentrations of immobilized MMP peptide probe-GC conjugates and, importantly, MMP concentration. The MMP-D-KIT is most specific for target MMP, compared with other enzymes including caspase-3 and 20s proteasome. Additionally, the MMP-D-KIT was used to detect MMP activity in biological samples such as synovial fluid from 12 OA patients (grades 1-4 based on the Kellgren-Lawrence grading scale). It was found that the fluorescence intensity measured using MMP-D-KIT decidedly correlates with the progression of OA. The MMP-D-KIT could be applicable in detecting MMP activities in various biological samples and evaluating the effects of MMP inhibitors in a rapid and easy fashion.

INTRODUCTION Matrix metalloproteinases (MMPs) are a class of secretory proteinases that are capable of degrading extracellular matrix (ECM) components in physiological and pathophysiological turnover of tissues (1). In particular, MMPs have been shown to be overexpressed in pathological situations such as cancer, atherosclerosis (2, 3), rheumatoid arthritis (RA), and osteoarthritis (OA) (4). The amount of MMP expression has been found tobeassociatedwithtumorstage,invasiveness,andmetastasis(5-7). Therefore, the detection of MMP activity in biological samples provides important information for diagnosis, prognosis, and therapeutic monitoring of various diseases. Numerous attempts have been made to detect activities of MMPs in biological samples by natural or synthetic substratebased methods (8-10). Natural substrate-based methods included radiolabeled gelatin and zymography. The radiolabeled gelatin method detects only degraded products small enough to be soluble in 20% trichloroacetic acid (Mr < 5000), which consequently deteriorate the reliability of the assay (11). In * To whom correspondence should be addressed. (Dr. I. C. Youn) Tel: +82-2-958-5913; Fax: +82-2-958-5909; E-mail: [email protected]. (Dr. K Choi) Tel: +82-2-958-5921; Fax: +82-2-958-5909; E-mail: [email protected]. † These authors contributed equally to this paper. ‡ Korea Institute of Science and Technology. § Seoul National University. | Korea University. ⊥ Purdue University.

addition, zymography, the most commonly used natural substrate method, is superior in its detection of MMPs in terms of sensitivity. Gelatin zymography can detect MMP-2 and -9 at low level, but might not be suitable to detect MMP-13 activity. Nonetheless, they are unsuitable for rapid high-throughput screening of potential MMP inhibitors because of its timeconsuming and cumbersome to perform and, furthermore, limitation in specificity (12). Some synthetic substrate-based methods include the use of internally quenched fluorogenic substrates that comprise a fluorophore, a quencher, and short peptide substrates. In normal condition, the emitting light from the fluorophore is absorbed into the quencher by resonance energy transfer. Once target enzymes like MMPs cleave specific peptide substrates, separating fluorophore from the quencher, the extent of fluorescence increases remarkably. In order to maximize this phenomenon, great efforts have been made to prepare an efficient pair of a fluorophore and a quencher (13, 14). Advantages of using internally quenched fluorogenic substrates include increased selectivity and applicabilty for high-throughput analysis and allow for rapid and continuous measurement of enzyme activity (15, 16). Previously, we developed a highly efficient fluorogenic probe for the visual detection of MMP-13 in an OA-induced rat model (17). A pair of a fluorophore (Cy5.5) and a quencher (black hole quencher-3; BHQ-3) showed low fluorescence background and significant fluorescent signal recovery, enabling a highly sensitive visual detection in vitro and in vivo. Herein, we rationally designed a new MMP activity diagnostic kit (MMP-D-KIT) for high-throughput screening of the bioac-

10.1021/bc100008b  2010 American Chemical Society Published on Web 06/24/2010

Technical Notes

tivity of MMPs in a rapid and simple fashion. To construct the MMP-D-KIT, first, MMP-13 specific fluorogenic peptide probe (MMP peptide probe) consisted of NIR fluorophore, MMP-13 specific peptide, and black hole quencher were chemically conjugated to glycol chitosan (GC) polymer, resulting in MMP peptide probe-GC conjugates. Second, MMP peptide probe-GC conjugates were effectively immobilized onto the maleic anhydride group-terminated 96-well plate, resulting in MMPD-KIT. On the basis of this polymer surface modification method, MMP peptide probes were successfully chemically conjugated onto the plate surface, wherein MMP peptide probes can maintain their excellent fluorogenicity against target MMP13, due to the three-dimensional support of biocompatible GC polymers (18-22). Further, we investigated whether this new MMP-D-KIT can effectively monitor MMP activity using an NIR fluorescence imaging system, when MMP enzymes or synovial fluids from OA patients were simply added into the MMP-D-KIT.

EXPERIMENTAL METHODS Preparation of MMP-13 Specific Fluorogenic Peptide Probe (MMP Peptide Probe). MMP peptide probe was prepared by conjugating NIR fluorochrome, Cy5.5 (ex/em; 675/ 695 nm) and black hole quencher-3 (BHQ-3, abs. 650 nm) to MMP-13 specific substrate (Gly-Pro-Leu-Gly-Val-Arg(pbf)-GlyLys(boc)-Gly-Gly) (23), wherein the cleavage site between Gly and Val was synthesized using standard solid-phase Fmoc peptide chemistry (Peptron, Daejeon, Korea). First, Cy5.5 mono N-hydroxysuccinamide ester (8.4 µmol; GE Healthcare, Piscateway, NJ) was chemically conjugated to the N-terminus of the MMP-13 specific peptide (4.0 µmol) in anhydrous dimethylformamide (DMF, 200 µL; Sigma) containing N-methyl morpholine (NMM, 52 µmol; Sigma) and 4-dimethylaminopyridine (2.5 µmol; Sigma) at room temperature in the dark with shaking for 2 h. The Cy5.5-MMP-13 specific peptide was precipitated by adding ethyl ether (Sigma). The precipitate was washed with ethyl ether and dried in vacuum, and the side chain protecting groups were removed by incubating in 1 mL of trifluoroacetic acid (TFA; Sigma)/distillated water (DW)/anisole (Sigma) (95:2.5:2.5, v/v). The TFA was evaporated on the rotary evaporator and an additional 1 mL of DW containing 0.1% TFA/ acetonitrile containing 0.1% TFA (1:1, v/v) was then added to the product in the rotary evaporator. The dissolved product in the rotary evaporator was purified by C18 semipreparative reversed-phase HPLC; 22% to 40% acetonitrile containing 0.1% TFA versus DW containing 0.1% TFA over 20 min at a flow rate of 4.0 mL/min. Purity (>95%) was confirmed by HPLC. The appropriate fractions were collected and lyophilized. Next, the BHQ-3 mono N-hydroxysuccinamide ester (0.90 µmol; Biosearch Technologies, Inc., Novato, CA) was coupled to the primary amine of the lysine of the Cy5.5-Gly-Pro-Leu-Gly-ValArg-Gly-Lys-Gly-Gly (0.90 µmol) in anhydrous DMF (30 µL) containing NMM (9.7 µmol) and 4-dimethylaminopyridine (1.0 µmol) at room temperature in the dark with shaking overnight. The product was purified by C18 semipreparative reversed-phase HPLC; 30% to 70% acetonitrile containing 0.1% TFA versus DW containing 0.1% TFA over 20 min at a flow rate of 4.0 mL/min. Purity (>95%) was confirmed by HPLC. The appropriate fractions were collected and lyophilized. Synthesis of MMP Peptide Probe-GC Conjugates. Darkquenched MMP peptide probe of Cy5.5-MMP-13-substrateBHQ-3 was chemically conjugated to the GC polymer, as follows. MMP peptide probe (0.88 µmol) dissolved in dimethyl sulfoxide (DMSO, 100 µL; Sigma)/phosphate buffered saline (PBS, 100 µL, pH 6.0; Sigma) was mixed with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC, 5.2 µmol; Sigma) and N-hydroxysulfosuccinimide (Sulfo-NHS, 5.2

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µmol; Pierce, Rockford, IL) dissolved in PBS (100 µL, pH 6.0) for 15 min. The mixture was then added to GC (250 kDa, 0.04 µmol; Sigma) and dispersed in PBS (14 mL, pH 7.4). The reaction was performed at room temperature in the dark with shaking overnight. Unreacted MMP peptide probes were removed for 3 days by dialysis (molecular weight cutoff ) 12-14K, Spectrum Laboratories, Inc., Rancho Dominguez, CA). The product was subsequently lyophilized. UV Measurements of MMP Peptide Probe-GC Conjugates. In order to obtain a standard curve for calculation of the amounts of MMP peptide probes conjugated to GC polymer, UV absorbance of MMP peptide probe-GC conjugates (1.10 µM) was recorded from 450 to 800 nm using UV/vis spectrophotometer (Optizen 2120, Mecasys, Daejeon, Korea). MMP peptide probes at various concentrations (1.5, 2.6, 5.5, 11.0, and 22.0 µM, respectively) were served as standard. Standard curve was achieved using the UV absorption spectra data at 690 nm. Enzyme Specificity of the MMP Peptide Probe-GC Conjugates. The MMP-13 specific fluorogenic property of the MMP peptide probe-GC conjugates (20 nM) was examined by incubating in the reaction buffer (50 mM Tris · HCl, 10 mM CaCl2 · 2H2O, 0.15 M NaCl, 0.05% Brij35, pH 7.8) containing 15 nM of activated MMP-2, MMP-3, MMP-7, MMP-9, MMP13 (R&D Systems, Inc., Minneapolis, MN), and MMP-13 with inhibitor (N-hydroxy-1,3-di-(4-methoxybenzenesulfonyl)-5,5dimethyl-[1,3]-piperazine-2-carboxamide) (Calbiochem, San Diego, CA), respectively. MMP-2, MMP-3, MMP-7, and MMP13 enzymes were activated by incubating them with 2.5 mM of p-aminophenyl mercuric acid in 0.1% NaOH buffer for 1 h at 37 °C before initiation of the assay. Fluorescence intensity was monitored using a spectrofluorometer (F-7000 Fluorescence Spectrophotometer, Hitach, Tokyo, Japan) every 10 min at 37 °C using a 1 mL of cuvette. The excitation wavelength was set at 675 nm and emission spectra recorded from 676 to 800 nm. The same experimental conditions were applied to various concentrations of activated MMP-13 (0.5, 1.9, 3.8, 7.5 nM) in the presence of a fixed concentration of MMP peptide probe-GC conjugates (20 nM). Immobilization of MMP Peptide Probe-GC Conjugates onto 96-well Plate (MMP-D-KIT). MMP peptide-GC conjugates (10 µM) in PBS/DMSO (7:3, 100 µL) were directly immobilized on each well of maleic anhydride-activated 96well plates (Pierce) and another MMP peptide-GC conjugates (10 µM) in PBS/DMSO (7:3, 100 µL) were placed on each well of usual 96-well plates. After incubating for 12 h at room temperature while shaking, the MMP peptide-GC conjugate solutions from maleic anhydride-activated 96-well plate and usual 96-well plate were diluted 10-fold, respectively, and UV absorbance was measured. The difference of UV absorbance between the MMP peptide-GC conjugate solutions from maleic anhydride-activated 96-well plate and usual 96-well plate were calculated to concentration of immobilized MMP probes, comparing with standards of known concentration of MMP peptide-GC conjugates. Various concentrations of MMP peptide-GC conjugates (0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, 10, 20, and 40 µM) in PBS/DMSO were directly immobilized on each well of maleic anhydride-activated 96-well plates. After incubating for 12 h at room temperature while shaking, the MMP peptide-GC conjugate solution was removed from each well. The plates were blocked by addition of 0.5% bovine serum albumin in PBS for 1 h at room temperature, after which the plates were washed three times with washing buffer (50 mM Tris · HCl, 10 mM CaCl2 · 2H2O, 0.15 M NaCl, 0.05% Brij35, pH 7.8). Fluorescence Quantitative Analysis of the MMP-D-KIT. Activated MMP-13 enzyme (15 nM) was added to each well and incubated for 1 h at 37 °C. After the incubation, MMP-D-

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KIT was imaged using a Kodak Image Station 4000MM. The NIR fluorescent images of MMP-D-KIT were obtained using a 12-bit CCD camera (Kodak, Japan) equipped with a special C-mount lens and a Cy5.5 bandpass emission filter (680 to 720 nm; Omega Optical, Brattleboro, VT). Simultaneously, the emission spectrum of each sample at the excitation wavelength (675 nm) was obtained to quantify the NIR fluorescence intensity. To confirm that visual detection of the MMP-D-KIT was dependent on the amount of MMPs, 96-well plates immobilized of MMP peptide probe-GC conjugates (10 µM) were prepared under the same experimental conditions. Serial dilutions of activated MMP-13 enzyme (1.0, 1.8, 3.7, 7.5, 15, 30 nM) was added to each well and incubated for 1 h at 37 °C. To confirm the specificity of the MMP-D-KIT, it was incubated with activated MMP-13 (15 nM), activated MMP-13 (15 nM) with inhibitor, caspase-3 (70 nM; R&D Systems), and 20s proteasome (1 nM, Boston Biochem, Cambridge, MA). 20s proteasome in working buffer (50 mM HEPES, pH 7.6, 100 mM NaCl, 1 mM dithiothreitol) was activated by adding sodium dodecyl sulfate (0.035%) for 20 min at 37 °C. Activated 20s proteasome and caspase-3 in working buffer (25 mM HEPES, pH 7.5, 0.1% CHAPS, 10 mM dithiothreitol) were added to each MMP-D-KIT and incubated for 1 h at 37 °C, and the fluorescence image and intensity were measured using Kodak Image Station and fluorescence spectofluorometer. Data were obtained from three samples per group and expressed as the mean ( standard deviation. Screening the MMP Activity from Osteoarthritis (OA) Patients. Twelve OA patients were divided into four groups according to the severity of OA based on the Kellgren-Lawrence grading scale: grade 1, doubtful narrowing of joint space and possible osteophytic lipping; grade 2, definite osteophytes, definite narrowing of joint space; grade 3, moderate multiple osteophytes, definite narrowing of joints space, some sclerosis and possible deformity of bone contour; grade 4, large osteophytes, marked narrowing of joint space, severe sclerosis, and definite deformity of bone contour (24). The severity of OA was separately scored and statistically confirmed by four physicians using the cross-validation method. The synovial fluid (3-21 mL) was harvested from the knee joint of OA patients and aliquoted in 2 mL samples and stored at -70 °C before use. Finally, each sample was diluted 10-fold in TCNB buffer solution, and it was added to the MMP-D-KIT and incubated for 1 h at 37 °C to measure the MMP activity. Therefore, TCNB buffer was regarded as controls. After the incubation, MMPD-KIT was imaged using a 12-bit CCD camera (Kodak, Japan) equipped with a special C-mount lens and a Cy5.5 bandpass emission filter (680 to 720 nm; Omega Optical, Brattleboro, VT). The fluorescence intensity was measured using a fluorescence spectofluorometer. Data were obtained from three synovial fluids per group and expressed as the mean ( standard deviation.

RESULTS AND DISCUSSION In order to prepare a high-throughput MMP-D-KIT, first, MMP peptide probes were chemically conjugated to GC polymer (MMP peptide probe-GC conjugates), which act as polymeric linker between MMP peptide probe and 96-well plate. Second, MMP peptide probe-GC conjugates were directly immobilized onto the maleic anhydride group-terminated 96well plate, wherein the maleic anhydride group likely reacts with primary amine of GC, resulting in MMP-D-KIT (Figure 1). The MMP fluorogenic peptide probe (MMP peptide probe) was prepared by conjugating near-infrared (NIR) fluorophore, Cy5.5, and black hole quencher, BHQ-3, to the MMP-13 specific peptide substrate of GPLGVRGKGG, in resulting Cy5.5-MMP13 substrate-BHQ-3 (17, 23). The MMP peptide probe of Cy5.5-

Ryu et al.

Figure 1. Schematic diagram of MMP diagnostic kit (MMP-D-KIT) for “one step” detection of MMP acitivity. First, MMP peptide probe consisted of Cy5.5-MMP-13 substrate-BHQ-3 was conjugated to GC polymer (MMP peptide probe-GC conjugates). Second, MMP peptide probe-GC conjugates were immobilized on each well of maleic anhydride-terminated 96-well plates (MMP-D-KIT). The MMP-D-KIT was quenched before treatment of MMPs, resulting in no fluorescent signals, but active MMP could recover the NIR fluorescent signal.

MMP-13 substrate-BHQ-3 was optimized by modeling the X-ray crystal structure of hMMP-13 (17), wherein a short peptide sequence, GPLGVRGKGG, was specifically cleaved by MMP-13 (the MMP-13 specific cleavage site is between G and V). This MMP peptide probe showed a minimal NIR fluorescence signal at normal condition, because black hole quencher, BHQ-3, is able to quench emitting fluorophores of Cy5.5, resulting in significantly low fluorescent backgrounds without MMP-13 enzyme. However, it was reported that the darkquenched fluorogenic MMP peptide probe presented strong fluorescent signal by the addition of target MMP-13 (17). To give a biocompatible and three-dimensional support of MMP peptide probes on the surface of 96-well plate, we introduced a polymer support of GC polymer (Mw ) 250 kDa) by chemical coupling reaction between MMP peptide probes and GC polymer, in resulting MMP peptide probe-GC conjugates. It has been reported that this three-dimensional support of biocompatible polymers may minimize the steric hindrance of MMP peptide probes against MMP enzymes on the solid surface (20-22). The MMP peptide probes in the flexible polymer conjugates may freely access the target MMP enzymes with high specificity and sensitivity. The optimal reaction ratio of MMP peptide probes in the GC conjugate was confirmed with UV absorbance, wherein most MMP peptide probes were successfully chemically conjugated to GC polymer. UV absorbance of serial dilutions of MMP fluorogenic probes (1.5 to 11 µM) and MMP peptide probe-GC conjugate (1.10 µM) were observed at 690 nm (Figure 2A). The standard curve was plotted according to the MMP peptide probe concentration, and this graph showed a linear relationship (R2 > 0.999) (Figure 2B). On the basis of the UV measurement, the UV absorbance of MMP peptide probe-GC conjugate (1.10 µM) corresponds to that of MMP peptide probe (10.6 µM) in the standard curve, indicating that approximately 10 molecules of MMP peptide probe were conjugated to 1 molecule of GC polymer. Furthermore, when the MMP peptide-GC conjugate was incubated with different MMP enzymes (MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13), it showed different NIR fluorescent signals at 695 nm over the reaction time at 37 °C

Technical Notes

Figure 2. Characterization of MMP peptide probe-GC conjugates. (A) UV absorbance of MMP peptide probe-GC conjugates (1.10 µM) and various concentrations of MMP peptide probes (1.5, 2.6, 5.5, 11.0 µM). (B) Standard curve for calculation of the amounts of MMP peptide probes conjugated to GC polymer. (C) Fluorescence intensity of MMP peptide probe-GC conjugates in solution (20 nM) in the presence of various stimuli (MMP-2, -3, -7, -9, -13, and MMP-13 with inhibitor) as a function of time following incubation for 80 min at 37 °C. (D) Fluorescence emission spectra of MMP peptide probe-GC conjugates in the presence of various concentrations of MMP-13 following incubation for 60 min at 37 °C. Inset: standard curve of MMP peptide probe-GC conjugates.

(Figure 2C). No detectable change was found in the fluorescence signal of polymer conjugates treated by MMP-3 and MMP-7, which meant that MMP-3 and MMP-7 were less effective in cleaving MMP peptide probe-GC conjugates than any other tested MMP enzymes. A small amount of recovery of the NIR fluorescent intensity occurred in the polymer conjugates treated by MMP-9. Noticeably, 25-fold and 22-fold increases in NIR fluorescent intensity occur in the polymer conjugates treated by MMP-2 and MMP-13 after 80 min of incubation, respectively. This result indicates that MMP-13 specific peptide probe-GC conjugates are very specific to target MMP-2 and MMP-13. It was also reported that some overlapping substrate specificity in MMPs was observed, due to the similar homology of MMP enzymes (25). As a control experiment, NIR fluorescent signal of the polymer conjugates was not observed by adding MMP-13 inhibitor, indicating the higher specificity of MMP peptide probe-GC conjugates against MMP-13. To test the sensitivity of the polymer conjugates against target enzyme of MMP-13, MMP peptide probe-GC conjugates were incubated with various concentrations of MMP-13 for 1 h at 37 °C (Figure 2D). At the lower MMP-13 concentration of 1.9 nM, substantial NIR fluorescent signal was observed, indicating the higher sensitivity of the polymer conjugates against active MMP-13. Also, the polymer conjugates showed the linear proportional fluorescent signals according to MMP-13 concentrations (R2 ) 0.95) (inset image in Figure 2D). Approximately 2.4-, 3.1-, 3.7-, and 6.4-fold increases in NIR fluorescent intensity occur according to the 0.5, 1.9, 3.8, 7.5 nM of active MMP-13, respectively. It suggests that chemically conjugated MMP peptide probes in the GC polymer conjugates still maintained their fluorogenic enzyme activity against target MMP-13 protease with the higher specificity and sensitivity.

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Figure 3. Immobilization of MMP peptide probe-GC conjugates onto 96-well plates. (A) Bright and NIR fluorescent images of 96-well plates containing MMP peptide probe-GC conjugates in the immobilized phase (MMP-D-KIT) and those in the solution, respectively. (B) Bright and NIR fluorescent images of MMP-D-KIT where various concentrations of MMP peptide probe-GC conjugates were immobilized. (C) Graph of corresponding fluorescence intensity of MMP-D-KIT versus the concentration of MMP peptide probe-GC conjugates. Inset: standard curve of MMP concentration.

To prepare the MMP-D-KIT, MMP peptide probe-GC conjugates (10 µM) were directly immobilized onto the maleic anhydride group-terminated 96-well plate by simply adding the polymer conjugate solution. At 12 h postreaction, unreacted polymer conjugates were removed from the plate, and the reaction was blocked by addition of 0.5% bovine serum albumin in PBS for 1 h. The freshly prepared MMP-D-KIT was completely washed with 50 mM Tris · HCl washing buffer and further used for in vitro assay. As shown in Figure 3A, at the normal state without MMP-13, MMP-D-KIT presented a completely quenched background, compared to MMP peptide probe-GC conjugate solution with the same MMP peptide probe concentration. It is deduced that surface immobilized MMP peptide probe-GC conjugates may present an additional intermolecular quenching effect of MMP peptide probes on the 96-well plate surface, due to the short distance between MMP peptide probes at the two-dimensional solid surface, compared to the polymer conjugates in solution. In particular, MMP-DKIT recovered 90% of NIR fluorescent intensity by adding active MMP-13 (15 nM), compared to the MMP peptide probe-GC conjugate solution. It means that the surfaceimmobilized MMP peptide probes on the GC polymer layer can be freely exposed to the target enzymes. Therefore, this excellently quenched and highly reactive MMP-D-KIT may be applicable in detecting target MMP activity in a rapid and easy fashion. Before treatment of active MMP-13, the different MMPD-KIT, treated with different MMP peptide probe-GC conjugate concentrations from 1 µM to 40 µM, presented the same quenched state at normal condition (Figure 3B), due to the complete quenching effect of MMP peptide probes. However, at 1 h postincubation with active MMP 13 (15 nM), the NIR fluorescence signal proportionally increased according to the MMP peptide probe-GC conjugate solution concentration. On the basis of the fluorescence spectrofluo-

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Figure 4. Fluorescence quantitative analysis of the MMP-D-KIT. (A) Bright and NIR fluorescent images of MMP-D-KIT in which various concentrations of MMP-13 were treated. (B) Graph of corresponding fluorescence intensity of MMP-D-KIT versus MMP concentration. (C) Bright and NIRF images of MMP-D-KIT where various proteases (MMP-13, MMP-13 with inhibitor, caspase-3, and 20s proteosome) were treated. (D) NIR fluorescent intensity of various proteases-treated MMP-D-KIT (MMP-13, MMP-13 with inhibitor, caspase-3, and 20s proteosome).

rometer data, a plot of average fluorescence intensity versus probe concentration revealed a hyperbola curve (Figure 3C). The fluorescence signal was further replotted against the logarithm of MMP peptide probe concentration, and it showed a linear relationship, with a good correlation coefficient (R2 ) 0.97, inset image in Figure 3C). This result indicated that the concentration of MMP peptide probes in the MMP-DKIT can be easily controlled by adding MMP peptide probe solution with a predetermined concentration. In order to assay the extremely small enzyme activity, we optimized the polymer conjugate concentration to 10 µM, wherein the nanomolar MMP activity could be detectable with the higher specificity and sensitivity. Under this optimized condition, recovery of NIR fluorescent signals in the detection kit was proportional to the nanomolar MMP concentration (Figure 4A). At the nanomolar MMP concentration in the range 1-30 nM, the MMP-D-KIT exhibited distinguishable color differences in a 96-well plate after 1 h postincubation at 37 °C, enabling facile visual comparison of relative bioactivity of MMP. The MMP-D-KIT for “one-step” detection of MMP is capable of measuring up to 1 nM (0.5 ng/mL) of MMP13. There are other detection methods such as radiolabeled gelatin and zymography for MMP-2 and MMP-9. Interestingly, the detection limit of MMP-D-KIT is similar with that of radiolabeled gelatin assay or gelatin zymograpy assay for MMP-2 and MMP-9 (27). Furthermore, detection of MMP using MMP-D-KIT is a rapid and simple process compared with other detection methods. As predicted, control MMPD-KIT without active MMP-13 gave no detectable NIR fluorescent signal. This proportional relationship between NIR fluorescent signal and MMP concentration was quantitatively confirmed by a spectrofluorometer measurement. A plot of average fluorescence intensity against MMP concentrations demonstrated a MMP concentration-dependent hyperbola curve

Ryu et al.

Figure 5. Application of MMP-D-KIT to synovial fluids from OA patients. (A) X-ray data of OA patients. (B) NIR fluorescent image of OA patient’s synovial fluid-treated MMP-D-KIT. (C) Fluorescent intensity of OA patient’s synovial fluid-treated MMP-D-KIT. TCNB buffer serves as control.

(Figure 4B). The fluorescence signal was further replotted against a logarithm of MMP enzyme concentration, and it shows a linear relationship with a good correlation coefficient (R2 ) 0.96, inset image in Figure 4B). On the basis of the optimal MMP activity assay, our new MMP-D-KIT could assay the nanomolar MMP activity by “one-step” fluorescence imaging without any additional procedure. Furthermore, our MMP-D-KIT is most specific for target MMP-13, compared with the other proteases including caspase-3 and 20s proteasome (Figure 4C,D). Within 1 h after protease addition, the MMP-D-KIT containing MMP-13 exhibited distinguishable change in the NIR fluorescence signal, wherein an 80fold increase in NIR fluorescence intensity was observed, compared to the MMP-D-KIT without MMP-13. As a control experiment, the wells containing 20s proteasome and caspase-3 exhibited little to no detectable fluorescence change after protease reaction. The near-recovery of fluorescence signal of the MMP-13 treated detection kit was inhibited in the presence of the MMP inhibitor, suggesting that the detection kit has a reasonable specificity for MMP-13. For clinical application, the MMP-D-KIT was used to detect MMP-13 activity in synovial fluid from OA patients (Figure 5A). It was previously reported that significantly high MMP13 activity was monitored in synovial fluid harvested from the knee joint of OA patients (26). In order to assay the OA gradedependent MMP activity, OA patients were divided into four different groups based on the original Kellgren-Lawrence radiographic grading scale, which emphasizes patellofemoral joint space narrowing and presence of osteophytes (grades 1-4, with 1 being early stage of OA and 4 severe OA). When the 10-fold diluted synovial fluid (100 µL) with different OA grades (1-4 grades) were simply incubated in the MMP-D-KIT at 37 °C, each showed distinct NIR fluorescent signal differences at 1 h postincubation, indicating OA grade-dependent MMP activities. TCNB buffer serving as control exhibited little to no detectable fluorescence change. Interestingly, the synovial fluid sample of early-stage OA patients (grade 1) presented the strongest NIR fluorescent signal,

Technical Notes

compared to those of other OA patients (grades 2-4), wherein 50-fold increased NIR fluorescent signal was observed (Figure 5B,C). As the OA grades increased from 2 to 3, the NIR fluorescent signals rapidly decreased, indicating the decrease of MMP-13 activity in late-stage OA patients. The results suggested that the expression of MMP-13 in OA patients may be closely related to the development stages of OA patients. Therefore, our results confirmed the diagnostic feasibility of our MMP-D-KIT for detection and visualization of overexpressed MMP-13 activity in OA patients. We are currently conducting experiments to analyze MMP activity in synovial fluids from a statistically significant number of OA patients with the MMP-D-KIT. In our experimental design, we carried out measurements of MMP activity with synovial fluid of OA patients and TCNB buffer used as a control group, because it is ethically difficult to harvest synovial fluid from individuals without any clinical sign of arthritis. It would be meaningful to compare MMP activity in the synovial fluid of OA patients with that of individuals without any sign of arthritis. In summary, this study showed that the new fluorogenic peptide-immobilized MMP detection kit based on a 96-well plate could monitor protease activity in a rapid and easy fashion. Fluorogenic peptide probe-GC conjugates were easily immobilized on a 96-well plate, and fluorogenic peptide probes were freely exposed to target MMP-13 enzyme in three-dimensional support GC polymer layer. When target MMP enzyme was simply added and incubated in the MMP detection kit, recovery of NIR fluorescent signals within minutes was proportional to the MMP concentration. The MMP detection kit was also applied to detect MMP activity in synovial fluids from OA patients and showed the fluorescence intensity to correlate with the stage of OA development. The detection kit can be utilized to detect MMP activity in various biological samples and evaluate the effects of MMP inhibitors in a rapid and easy fashion. Additionally, the detection kit can be extended to other proteases simply by changing the specific peptide substrate linker between the fluorophore and black hole quencher.

ACKNOWLEDGMENT This work was financially supported by the Real-Time Molecular Imaging Project, 2009K 001594, and GRL Program, Pioneer Research Center (2009-0081523) of MEST and by grant to the Intramural Research Program (Theragnosis) of KIST, by a grant of by Advanced Medical Technology Cluster for Diagnosis and Prediction at Kyungpook National University from MOCIE.

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