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Evaluation of Matrix Metalloproteinase Inhibition by Peptide Microarray-based Fluorescence Assay on Polymer Brush Substrate and in Vivo Assessment Zhen Lei, Hongda Chen, Hua Zhang, Yaoqi Wang, Xianying Meng, and Zhenxin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15445 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

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Evaluation of Matrix Metalloproteinase Inhibition by Peptide Microarray-based Fluorescence Assay on Polymer Brush Substrate and in Vivo Assessment Zhen Lei,†,§ Hongda Chen,† Hua Zhang,† Yaoqi Wang,‡ Xianying Meng,*,‡ and Zhenxin Wang*,† †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, P. R. China.



Department of Thyroid Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P. R. China §

University of Chinese Academy of Sciences, Beijing 100049, P. R. China.

ABSTRACT: Matrix metalloproteinases (MMPs) are important biomarkers and potential therapeutic targets of tumor. In this report, a peptide microarray-based fluorescence assay is developed for MMPs inhibitors evaluation through immobilization of biotin-modified peptides on the poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush modified glass slides. After biotin is recognized with Cy3 modified avidin (Cy3-avidin), the microarrays can produce strong fluorescence signal. The biotin moieties detach from microarray while the biotin-modified peptide substrates are specially cleaved by a MMP, resulting in decreased fluorescence intensity of microarray. The decreasing level of fluorescence intensity is

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correlated with the MMP inhibition. Nine known MMP inhibitors against MMP-2 and MMP-9 are evaluated by the assay, and the quantitative determination of inhibitory potencies (IC50) are obtained, which are comparable with the literatures. Two biocompatible fluorogenic peptides containing MMP specific recognition sequences and FAM/Dabcyl fluorophore-quencher pair are designed as activatable reporter probes for sensing MMP-2 and MMP-9 activities in cell and in vivo. The peptide microarray-based results are well verified by the cell inhibition assay and in vitro fluorescence imaging, and further confirmed by the in vivo imaging of HT-1080 tumorbearing mice.

KEYWORDS: matrix metalloproteinases, inhibition, peptide microarray, polymer brush substrate, fluorescence imaging, in vivo imaging INTRODUCTION Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in degrading extracellular matrix proteins (ECM), and play vital roles in many physiological processes.1-4 The expression levels and activities of MMPs are regulated by transcription, activation

of

the

zymogens

and

inhibition

by

various

inhibitors.

The

irregular

expression/activities of MMPs may lead to several kinds of diseases, such as rheumatoid arthritis, Alzheimer's disease, tumor growth, invasion and metastasis.1-4 Because ECM degrading is viewed as essential for tumor progression, MMPs have been considered as promising targets and prognostic factors for cancer therapy.4 In the last three decades, much effort has been devoted to develop MMPs inhibitors against diseases, and some inhibitors even used for human clinical trials, such as batimastat and marimastat. However, most of tested inhibitors caused severe side effect because of lack of selectivity, and failed for further clinical trials.4 Up to date, only two

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drugs are present on the market, namely, periostat (doxycycline) used for periodontal disease and glucosemine sulfate used for osteoarthritis.4-7 Therefore, it is necessary to develop screening assays and/or techniques with high efficacy for evaluating the inhibitory potency of certain inhibitors against specific MMPs, and discovering new potential inhibitors. Various approaches have been proposed for determination of MMPs activities and inhibitions. Among them, zymography, as the earliest and most commonly used method, is quite accurate and sensitive for detecting MMPs activities in biological samples.8-9 However, it is not competitive for high-throughput screening of MMP inhibitors because of the complicated experiment process. Recently, fluorescence-based methods, especially fluorescent resonance energy transfer (FRET)-based assays, have been extensively employed for evaluating MMP functionality and inhibition.10-12 The FRET-based assays with high sensitivity are conducted in homogeneous solution without separation procedure, making them suitable for mechanism and kinetic studies. However, due to their large volume consumption of sample (0.052 mL per sample), the cost of homogeneous FRET assays are usually high when they are used to screen MMP inhibitors from a chemical library with lager numbers. Recently, Ma et al. proposed an online chromatographic assay with immobilized enzyme reactor for screening of MMP-9 inhibitors.13 With the development of genomics and proteomics, microarray-based approaches have evoked great interest among the scientists since the microarrays show the great promise in high-throughput analysis and screening.14-16 In particular, small molecule microarray (SMM) has been intensively developed for high-throughput profiling protease activities and inhibitor discovery.17-18 The SMM-based assays are realized by immobilizing enzymes on the slide and reacting with fluorescently-labeled small molecule inhibitors, or coating the substrates with fluorogenic enzyme substrate followed by spotting the mixture of proteases and inhibitors.

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Although these approaches are superior for large-scale and high-throughput screening of inhibitors, the covalent immobilization of proteases on the solid supports may result in reduced enzyme activity, leading to inaccurate results. With the advance of peptide synthesis, peptide microarray have been proven to be the promising alternative for enzymes function and inhibition analysis.19-20 The peptide microarray-based methods circumvent the fixation of enzymes, which may improve the accuracy and sensitivity of enzymes function and inhibition analysis. Herein, we develop a high-throughput peptide microarray approach for evaluation of MMPs inhibitors on the P(GMA-HEMA) brush substrates, which show high loading capacity of peptide and higher protease activity. The MMPs activities and inhibition events are determined by spotting the biotin-modified peptide substrates on the P(GMA-HEMA) brush grafted glass slide, incubating with targeted MMP and inhibitor, and labeled by Cy3-avidin. The change of fluorescence intensity of Cy3 is dependent on the MMP activity, which can be inhibited by specific compound. Nine known MMP inhibitors against MMP-2 and MMP-9 are evaluated by the assay, and the inhibitory potencies (IC50) are determined. The inhibition efficiencies of selected inhibitors have also been demonstrated by a FRET assay both in vitro and in vivo, and satisfactory results are obtained. EXPERIMENTAL SECTION Materials and reagents Recombinant human MMP-9 and MMP-2 protein were purchased from Sino Biological Inc. (Beijing, China). The biotin modified peptides were supplied by ChinaPeptides Co., Ltd (Shanghai, China), and MMP-9 and MMP-2 probes were purchased from Synpeptide Co., Ltd. (Shanghai, China) (see Table S1 in Supporting Information for details). Cy3-avidin was received from Boster (Wuhan, China), and 4-aminophenyl mercuric acetate (APMA) was purchased from Genmed Scientifics Inc. (Shanghai, China). 2-Hydroxyethyl

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methacrylate (HEMA, 97%), quercetin, caffeic acid, 1,10-phenanthroline, glucosamine sulfate and glycidyl methacrylate (GMA, ≥97%) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, USA). Doxycycline hyclate, (-)-epigallocatechin gallate (EGCG), oleic acid and resveratrol were received from Aladdin Co., Ltd. (Shanghai, China). SB-3CT was supplied by Target Molecule Corp. (Boston, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Dingguo Biotechnology Ltd. (Beijing, China). Hoechst 33342 was purchased from Invitrogen (Carlsbad, USA). The plain glass slides, commercial 3D aldehyde, 2D aldehyde and 2D epoxy substrates were provided by CapitalBio Ltd. (Beijing, China). All other chemicals were analytical grade, and Milli-Q water (18.2 MΩ cm) was used in all experiments. Peptide microarray assay on P(GMA-HEMA) brush substrate The P(GMA-HEMA) brush substrate was prepared according to our previous work (see Supporting Information for details).21 Then the biotin modified peptide substrates were spotted on three commercially available microarray substrates and the P(GMA-HEMA) brush substrate by using a SmartArrayer 136 system with the standard contact printing procedure. After incubated in vacuum at 30 °C overnight, washed and blocked, the obtained peptide microarrays were applied for MMPs activity and inhibition experiments (see Supporting Information for details). Cell inhibition Human fibrosarcoma cell line HT-1080 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 100 U mL1 penicillinstreptomycin at 37 °C in humidified air with 5% CO2. The cytotoxicities of inhibitors were measured by conventional MTT assay. Briefly, the HT-1080 cells were cultured in 96-well plate with the concentration of 5000 cells/well for 24 h. After washed by fresh culture medium, 100 µL culture medium containing MMP inhibitors of desired concentrations were added into the

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HT-1080 cells, followed by incubation for another 24 h, respectively. Then, the cells were washed with PBS (100 µL, 3 times), and treated by adding 10 µL MTT (5 mg ml1 in PBS) into 100 µL fresh medium. 4 h later, the supernatant was replaced with 100 µL dimethyl sulfoxide, and shaken for 10 min. The optical intensity (OD) at 490 nm was read by Power Wave XS 2 microplate reader. The untreated cells served as the control samples, and the cell viabilities were calculated by dividing the OD of inhibitor treated samples by OD of control samples. In the cell inhibition assay, after the HT-1080 cells were cultutred in 96-well plate for 24 h, the FBScontaining media were abandoned, 100 µL of FBS-free RPMI media with desired concentrations of MMPs inhibitors were added. Cells treated with FBS-free RPMI medium without inhibitors were used as control samples. After an additional 24 h of incubation, the conditioned medium were collected, applied to subarrays, and treated as previously described, respectively. For visually evaluating the inhibitory ability in vitro, two specific MMP-2 and MMP-9 probes were designed for cell fluorescence imaging. The cytotoxicities of the two peptide probes were measured as same as described above. HT-1080 cells were then cultured in 48-well plate with 1104 cell/well for 24 h, then washed by PBS (200 µL, twice), and treated with 5 μM MMP-9 or MMP-2 probes in the absence or presence of different concentrations of inhibitors in FBS-free medium. After incubated for 18 h, the cells were washed by PBS (200 µL, twice), and fixed with 4% paraformaldehyde for 20 min. Subsequently, the cells were washed by PBS (200 µL, 2 times), and stained with 4 μg mL1 Hoechst 33342 for 5 min. After washing, the cells in PBS were imaged by the reconstructive Nikon Ti-S fluorescent microscope (Nikon, Tokyo, Japan) with excitation wavelengths at 488 nm for green channel and 350 nm for blue channel. In vivo inhibition assay Nude mice with average age of 6~8 weeks were supplied by Vital River Company (Beijing, China). All animal experiments were approved by the Regional Ethics

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Committee of Jilin University, and complied with the guidelines for care and use of laboratory animals. The HT-1080 tumor model was obtained by subcutaneously injecting 100 µL cells suspension (5106 cell) into the nude mice. When the tumors grew to 3-5 mm in size, the mice were randomly divided into two groups, and subjected to either SB-3CT treatment (10 mg kg1 body weight, intratumorally) or control vehicle treatment (10% DMSO in PBS, intratumorally) once per day for five days successively. The MMP-2 or MMP-9 probes were injected intravenously into the mice via tail vein 30 min after the last administration of SB-3CT or control vehicle. After injected with the probe for 1, 2, 3, 4, 16 and 24 h, the mice were anesthetized with 10 wt% chloral hydrate, and in vivo fluorescence imaging was observed by the Davinch Invivo HR imaging system (Davinch K, Korea). For biodistribution analysis, the mice were sacrificed at 24 h post-injection, the tumor and primary organs were harvested, and immediately imaged by Davinch Invivo HR imaging system. For quantitative analysis, the regions of interest (ROI) were drawn over tumors and measured by the Davinch Invivo HR imaging software. The fluorescence intensity was reported as radiance photons (p/s/cm2/sr) within the ROI. The urine of mice after injected with the MMP-2 or MMP-9 probes for 24 h was collected, and diluted to same volume to conduct fluorescence spectra analysis (Ocean Optics, USA). The tumor volumes (V) were measured by a vernier caliper once a day for 6 days, and calculated as follows: V = (tumor length) × (tumor width)2/2. The relative tumor volume (V/V0) was used to represent the tumor growth, where V0 was the tumor volume when the treatment was started. Data processing and statistical analysis All the fluorescence signals of the spots on microarray have been corrected by background fluorescence intensity of substrate slide. Quantitative data were presented as mean ± standard deviation. The MMP activity was represented by the change of fluorescence intensity (ΔF%), which is calculated as follows, ΔF%

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= (F0−F)/F0×100%, where F0 and F are the mean fluorescence intensity of subarray treated with TCNB buffer or MMPs, respectively. The relative activity is ΔF% of subarray co-incubated with MMP and inhibitors with respect to that with MMP only. Comparisons between two different groups were conducted using SPSS Statistics 22 with the One-way ANOVA-test followed by the Tukey test. RESULTS AND DISCUSSION

Scheme 1. Peptide microarray-based fluorescence assay for MMP activity and inhibition analysis on polymer brush substrate. Performance of the P(GMA-HEMA) brush substrate As displayed in Scheme 1, the P(GMA-HEMA) brush substrate has been prepared on glass slide via surface-initiated atom transfer radical polymerization (SI-ATRP) of glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) monomers.21 X-ray photoelectron spectra (XPS) and water contact angle (WCA) of the polymer brush substrate are displayed in Figure S1. The WCA of the P(GMAHEMA) brush substrate is 46.9, demonstrating that the surface is hydrophilic. By analyzing the peak area ratio of [C−O−C]/[C−O−H] in the high-resolution XPS O1s spectra, the molar ratio (%) of GMA to HEMA on the polymer brush substrate is 10.8:89.2, which is consistent with

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monomer composition in feed solution (8.2:91.8 (GMA:HEMA)). The result of ellipsometric measurement indicates that the thickness of P(GMA-HEMA) brushes on glass surface is about 23.6 nm. The performance of P(GMA-HEMA) brush substrate was evaluated in respect of peptide loading capacity and protease detection efficiency. The P(GMA-HEMA) brush substrate affords a three-dimensional (3D) brush-like structure with functional epoxy groups, therefore it can be considered as a 3D epoxy substrate. For comparison, three other commercially available microarray substrates (2D epoxy, 2D aldehyde and 3D aldehyde) were also employed as platforms for peptide microarray fabrication and protease detection under similar experimental conditions. The commercial 2D epoxy and 2D aldehyde substrates are glass slides modified by highly reactive epoxide monolayer or aldehyde monolayer, respectively, while the 3D aldehyde substrates are glass slides coated by a thin layer of polysaccharide with fully activated aldehyde groups.22 Various concentrations of peptide were spotted on four substrates. As shown in Figure 1a and Figure S2a, the fluorescence intensities of two 2D substrates reach saturation at very low concentration of peptide, while those of two 3D substrates are increased as the concentration of peptide increases. The result demonstrates that the P(GMA-HEMA) brush substrate can provide high density of active sites for peptide conjugation. The loading capacity of as-prepared 3D epoxy substrate is higher than that of 2D counterpart and comparable with that of commercial 3D aldehyde substrate. Taking MMP-9 as an example, the protease detection efficiency is evaluated by four peptide microarray-based platforms. As shown in Scheme 1, MMP-9 specific biotinmodified peptide substrates are spotted on four microarray substrate, respectively, which can generate high intensity of fluorescence signal when binding with Cy3-avidin. After MMP-9 cleavage, binding capacity of microarray with Cy3-avidin is decreased because the biotin

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terminals of peptide substrates are detached from the surface, resulting in decreased fluorescence signal, i.e., the fluorescence intensity of microarray is correlated with the MMP-9 activity. As shown in Figure 1b and Figure S2b, the MMP-9 cleavage efficiencies on the two 2D substrates are lower than those of 3D substrates. In particular, the detection limit of MMP-9 (calculated by 3 times of standard deviation of control sample) on P(GMA-HEMA) brush substrate is 7 pg mL1, which is much lower than that of 3D aldehyde substrate (70 pg mL1), and those of two 2D substrate (c.a., 1 ng mL1). The microarrays made with 0.3 mg mL-1 peptide in spotting solution were used to eliminate the effect of immobilized amount of peptide on the MMP-9 proteolytic efficiencies. As shown in Figure S3, the MMP-9 proteolytic efficiency on the P(GMA-HEMA) brush substrate is still much better than those of other three commercial substrates. In addition, the substrates have similar WCAs except 2D epoxy substrate (as shown in Figure S4), demonstrating that difference of MMP-9 proteolytic efficiency is not caused by the hydrophilic natures of substrates. Based on the above results, the superiority of protease detection efficiency on P(GMA-HEMA) brush substrate may be attributed to the special brushlike 3D structure on the surface, which offers abundant active sites. The peptide substrates are conjugated along the attached polymer chains which may provide enough available space and effectively reduce steric hindrance, making the protease easily access to the specific recognition sequence and cleave the substrates. Furthermore, the sensitivity of MMP activity determination on P(GMA-HEMA) brush substrate is even higher than those on other platforms. Therefore, the P(GMA-HEMA) brush substrate is selected for evaluation of MMPs inhibition.

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Figure 1. Comparison of peptide loading capacity (a) and protease detection efficiency (b) between P(GMA-HEMA) brush substrate and three commercially available microarray substrates. For protease detection efficiency study, the concentration of peptide in spotting buffer was 0.1 mg mL1. MMPs inhibition assay and IC50 value determination As shown in Scheme 1 and described above, a peptide microarray-based fluorescence assay for MMPs activity and inhibition analysis is proposed on the P(GMA-HEMA) brush substrate. The fluorescence intensity of Cy3 is associated with the MMPs activities, which are dependent on the inhibition efficiency of inhibitor. The performance of the assay is first evaluated taking MMP-2 and MMP9 as examples, because they can degrade the main protein component of ECM. As shown in Figure S5, 200 identical cleavage reactions of MMP-2 and MMP-9 are conducted under the same experimental conditions, and the Z-factor (Z’) and coefficient of variation (CV) are calculated, respectively. The relatively high Z’ and low CV suggest the proposed assay is stable and qualified for high-throughput screening. Then, the proposed method is utilized for MMPs inhibition analysis. Nine known MMP inhibitors are selected, including two drugs launched on the market (doxycycline hyclate and glucosamine sulfate), five nature compounds (EGCG, oleic acid, caffeic acid, quercetin and

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resveratrol) and two synthetic small molecules (1,10-phenanthroline and SB-3CT). The inhibitory ability of specific inhibitor towards MMP-2 or MMP-9 is measured by the fluorescence intensity. The higher potency of inhibitor leads to lower MMPs activity, resulting in higher fluorescence intensity. The inhibitor concentration-dependent relative MMP activity is shown in Figure 2. As expected, the MMP activity is decreased with increasing concentration of inhibitor, suggesting the proposed method has the potential to qualitatively analyze the efficiencies of various inhibitors. The IC50 values of nine inhibitors for both MMP-9 and MMP-2 are determined, which are summarized in Table 1. The obtained values by this assay are comparable with the literature reported values.5,23-27 In the literatures, the inhibitions of MMP-2 or MMP-9 are normally analyzed by solution-phase methods, such as fluorescence assays or colorimetric assay. The MMPs activities are measured by monitoring the changes of fluorescence intensities of fluorophore modified peptide or protein substrates,5,23,25-27 or change of absorbance at 405 nm via the reaction between the hydrolyzed thiopeptolide substrate with DTNB (5,5'-Dithiobis-(2-nitrobenzoic acid).24 The IC50 values of inhibitors are determined by the substrate cleavage efficiencies of MMPs in the presence of inhibitors with various concentrations. Compared with solution-phase methods, the peptide microarray-based fluorescence assay has low cost of reagents and labors since sample consumption of microarray is very low and automation level of microarray fabrication and detection is relatively high. Among the tested compounds, SB-3CT is the most efficient inhibitor with the IC50 value of 623.7 nM for MMP-9 and 15.8 nM for MMP-2, respectively, which are about 100-fold lower than those of others. In addition, the other compounds exhibit similar inhibition effects on the activities of MMP-9 and MMP-2, for instance, they have close IC50 values for both of MMPs, respectively. The IC50 value of SB-3CT for MMP-2 is about 40-fold less than that for MMP-9,

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indicating the good selectivity of SB-3CT. The results are well consistent with the literature report.28 All the above results demonstrate that the proposed method is promising for high throughput screening MMPs inhibitors as well as quantitatively analyzing the inhibition potency of inhibitor.

Figure 2. Inhibition curves of various inhibitors against MMP-2 and MMP-9 (a, EGCG; b, doxycycline hyclate; c, glucosamine sulfate; d, oleic acid; e, caffeic acid; f, quercetin; g, resveratrol; h, 1,10-phenanthroline and i, SB-3CT). (j and k) Representative fluorescence microarray images of the inhibition of SB-3CT against MMP-9 and MMP-2 (j, MMP-9 and k, MMP-2). The rest of fluorescence microarray images are shown in Figure S6 in Supporting Information.

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Table 1. IC50 comparison Inhibitors

IC50, found

IC50, reported

(MMP-2/MMP-9)

(MMP-2/MMP-9)

doxycycline hyclate

100.117.0 µM/301.015.1 µM

>100 µM23

glucosamine sulfate

26.96.5 µM/30.21.5 µM

none

EGCG

13.93.0 µM/2.50.3 µM

6.0 µM/0.3 µM24

oleic acid

7.00.5 µM/5.70.7 µM

1.6 µM25/none

caffeic acid

35.26.8 µM/11.22.2 µM

12 µM/8 µM26

quercetin

6.41.4 µM/8.20.6 µM

12.4 µM/11.7 µM27

resveratrol

3.50.5 µM/1.01.1 µM

none

1,10-phenanthroline

4.70.2 µM/0.730.1 µM

none

SB-3CT

15.83.0 nM/623.772.0 nM

24 nM/500 nM5

Cell inhibition assay To gain insight into the proposed assay for in vitro inhibition analysis, the inhibitory potencies of the compounds against MMP-9 and MMP-2 in cell were investigated. Human fibrosarcoma cell line HT-1080 cells are selected as the model, since the cells have high expression levels of MMP-9 and MMP-2.29 The caffeic acid exhibits similar dose-response inhibition curves against MMP-9 and MMP-2, and the cytotoxicities of non-selective inhibitors towards HT-1080 cells are relative high under the tested concentrations except caffeic acid (data not shown). Therefore, in the cell inhibition assay, caffeic acid is chosen as the representative broad-spectrum inhibitor for MMP-2 and MMP-9, while SB-3CT is selected as the selective inhibitor for MMP-2. The cytotoxicities of the two compounds are shown in Figure S7, HT-1080 cells still show more than 90 % or 95% viability when incubated with as high as 100 µM caffeic acid or 1000 nM SB-3CT, respectively. The results indicate that caffeic acid or SB-3CT has low cytotoxicity at the tested concentrations. FBS is a complicated mixture containing growth factors, hormones and proteins. In particular, one component of FBS, α-macroglobulin, is a kind of 14 ACS Paragon Plus Environment

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MMP inhibitors. In order to eliminate the interference from endogenous inhibitors in FBS, the complete medium was replaced by FBS-free culture medium. Hence, HT-1080 cells were

Figure 3. MMP-2 and MMP-9 activities in conditioned medium of HT-1080 cells cultured with various concentrations of caffeic acid (a) and SB-3CT (b), and the corresponding fluorescence microarray images (c, caffeic acid and d, SB-3CT). incubated with desired concentrations of caffeic acid and SB-3CT in FBS-free media for 24 h, and the conditioned medium were then collected for evaluation the inhibition against cellsecreted MMP-2 and MMP-9, respectively. As shown in Figure 3, the activities of MMP-2 and MMP-9 are inhibited by both the compounds in a dose-dependent manner. Caffeic acid shows similar inhibition effect on cell-secreted MMP-2 and MMP-9. For example, the MMP-2 and MMP-9 activities in the presence of 100 µM caffeic acid are 43.1% and 33.8% of the control ones, respectively, which are consistent with the inhibition curves of pure MMPs. SB-3CT exhibits higher inhibition potency against MMP-2 than MMP-9 in culture medium, suggesting the evidence for the selectivity of SB-3CT. The results are well consistent with the results of

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purified recombinant MMPs cases, demonstrating the peptide microarray-based fluorescence assay is a robust method for evaluation of MMPs inhibitors. The inhibition effect in vitro can be confirmed visually by cell fluorescence imaging. In this case, two FRET-peptide probes (namely, MMP-2 probe and MMP-9 probe) are designed, which are comprised of three arginine residues for cell-penetrating and the MMP specific cleavage site with FAM/Dabcyl donor-acceptor fluorophores flanked on two sides for cell imaging. After coincubated with cells and cleaved by the cell-secreted MMPs, the FAM-containing peptide fragments separate from Dabcyl moieties, resulting in termination of the FRET process and fluorescence recovery of FAM. The three arginine residues can enhance the cellular internalization of FAM-containing peptide fragments since the positively charged arginine residues exhibit strongly electrostatic interaction with negatively charged cell membrane. The fluorescence intensity of FAM-containing peptide fragment stained cells is decreased with decreasing MMP activity. For testing their biocompatibilities, the cytotoxicities of MMP-9 and MMP-2 probes were also measured by MTT. As shown in Figure S8, both of the probes exhibit negligible cytotoxicity on HT-1080 cells while the concentrations of the probes are less than 10 µM. Therefore, MMP-2 and MMP-9 probes at 5 µM were chosen in the following imaging experiment. As expected, the fluorescence intensities of FAM-containing peptide fragment stained cells are decreased with increasing the concentration of inhibitors (as shown in Figure 4). In the presence of caffeic acid, both of the fluorescence intensities of MMP-2 probe and MMP-9 probe stained cells are decreased in a similar dose-dependent manner. After co-incubated with 20 nM SB-3CT, there is a significant decrease in fluorescence intensity of MMP-2 probe stained cells while the fluorescence intensity of MMP-9 probe stained cells is barely changed. In the presence of 200 nM SB-3CT in the culture medium, the fluorescence of MMP-9 probe stained

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ACS Applied Materials & Interfaces

cells can be clearly observed while the MMP-2 probe stained cells show negligible fluorescence emission. The fluorescence imaging of MMP-2 and MMP-9 probe stained cells also demonstrates that the inhibition of SB-3CT for MMP-2 is more effective than that for MMP-9. The fluorescence imaging observations are in accordance with the cell inhibition assay by peptide microarray, suggesting the proposed peptide microarray-based method is reliable.

Figure 4. Fluorescence images of HT-1080 cells treated with 5 μM MMP-2 probe (a and c) or MMP-9 probe (b and d) in the absence and presence of caffeic acid (a and b) or SB-3CT (c and d). The scale bar is 50 μm. In vivo evaluation of SB-3CT inhibition To further confirm the reliability of the peptide microarray-based fluorescence assay on MMP inhibition study, a HT-1080 tumor-bearing mice

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Figure 5. Representative in vivo fluorescence images of tumor sites in HT-1080 tumor-bearing mice injected intravenously with MMP-2 probe (a) or MMP-9 probe (c) at the indicated time points. The mice were pretreated with or without SB-3CT (10 mg kg1 body weight). The color bar is the scale of radiance photons. The corresponding fluorescence intensity analysis in the ROI of tumor sites (b, MMP-2 and d, MMP-9) (n=3). *P