- Email: [email protected]
Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET)
G Model
ARTICLE IN PRESS
SNB-23355; No. of Pages 7
Sensors and Actuators B xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Research Paper
Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET) Jungmi Lee, Annie Agnes Suganya Samson, Joon Myong Song ∗ College of Pharmacy, Seoul National University, Seoul 08826, South Korea
a r t i c l e
i n f o
Article history: Received 12 July 2017 Received in revised form 6 September 2017 Accepted 10 October 2017 Available online xxx Keywords: Inkjet printing FRET Matrix metalloproteinase-9 Peptide substrate Inhibitor
a b s t r a c t A peptide probe-based MMP-9 inhibitory assay was developed to assess anticancer activity of compounds on parchment paper using FRET-based inkjet printing. The fluorescent peptide containing 5-FAM (donor) and QXL520 (acceptor) was used as a substrate with the sequence of Pro-Leu-Gly-Cys-His-Ala-Arg-Lys specific to MMP-9. The suitability of the peptide as a substrate of MMP-9 was confirmed by HPLC. When both MMP-9 and the peptide were printed on the identical reaction spot of parchment paper, the peptide was cleaved and fragmented by MMP-9. The fluorescence was then recovered by 5-FAM and detected at Ex/Em = 490 ± 20 nm/520 ± 20 nm. Based on the successful performance of FRET on the parchment paper without any surface treatment, the assay platform was applied to test compounds, and a successful anticancer assay could be accomplished. This methodology holds notable strengths in versatile applications to the MMP enzyme family, as well as control of ejection volume, reduction of consumed enzyme amount, and rapid reaction time. Hence, we suggest the FRET peptide-based assay using inkjet printing as a viable replacement for sensitive and high-throughput evaluation of anticancer compounds. © 2017 Published by Elsevier B.V.
1. Introduction Enzymes as catalysts play a major role in increasing reaction rates by lowering activation energy in almost all intracellular metabolic reactions. Currently, assay kits have been exploited to mimic the intracellular reaction between enzyme and protein or peptide substrate, and applied to drug screening at the molecular level [1–3]. Matrix metalloproteinases (MMPs) are a family of secretory endopeptidases with hydrolytic activity for a diverse spectrum of extracellular proteins [4]. MMPs released from cells execute breakdown of extracellular matrix (ECM) in physiological processes, such as cell migration and angiogenesis, by degradation of collagen IV and V, and other ECM proteins [5,6]. MMP-9 is highly expressed in the metastatic cancer cell for its role of ECM remodeling and angiogenesis. Therefore, MMP-9 is deeply related to tumor progress, including invasion and metastasis [7,8]. Recently, studies dealing with determination of MMP-9 activity have been reported. Gel and in situ zymography (gelatin-based) assays were performed to detect the proteolytic activity of MMP-9. This assays had limitations in terms of fluorescence resolution. In order to overcome the limitations, several fluorescent-probe based
∗ Corresponding author. E-mail address: [email protected] (J.M. Song).
biosensors have been developed for the detection of MMP activity. The first probes capable of imaging MMP activity was developed by modifying the original protease-sensing peptide probes. Similarly, numerous peptide probes were synthesized and developed to evaluate the proteolytic activity of MMP-9. This approach includes Cy5 NIR fluorophores conjugated peptide substrate, dendrimerbased fluorogenic substrates (fluorogenic proteolytic beacon), a self-assembling homotrimeric triple helical peptide, quantum dot (QD)/BHQ-1-based FRET peptide, and genetically encoded FRETbased probes [9–12]. Anticancer activities of test compounds as MMP-9 inhibitor can be proven through fluorescence of peptide substrate composed of a fluorescent donor and an acceptor quencher [13–15]. The sequence Pro-Leu-Gly-Cys-His-Ala-Arg-Lys is specifically recognized by the enzyme MMPs and exhibits a high degree of substrate specificity for MMP-9. The peptide is cleaved efficiently at the Gly Cys bond. Studies have revealed that the peptide is also cleaved at a similar rate by MMP-1, MMP-8, MMP-9, and MMP-12 [16]. Generally, molecular assays have been applied to screen target protein, membrane receptors and ion channels, which typically generates a functional read out for drug discovery. Drug screening assays are typically performed in 96-well or 384 or 1536micro well plates. Different assay format (384 or 1536-micro well plates) are selected to minimize assay reagents and assay volume. When high throughput screening assays are performed in micro well plates, the assay volume used is as small as a few microliters.
https://doi.org/10.1016/j.snb.2017.10.051 0925-4005/© 2017 Published by Elsevier B.V.
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
G Model SNB-23355; No. of Pages 7
ARTICLE IN PRESS J. Lee et al. / Sensors and Actuators B xxx (2017) xxx–xxx
2
Recently, it has been reported that inkjet printing technology are highly suitable for printing enzymes. Assay volume consumed to perform printing based assay was further reduced to nanoliter and picoliter [17–20]. Thus, inkjet printing technology with high priority of cost-effective platform can be selected to minimize the assay volume (nL to pL), which will be highly appropriate to evaluate enzyme/inhibitor activity. In this study, a peptide containing the sequence QXL520-Pro-Leu-Gly-Cys-His-Ala-Arg-Lys (5-FAM)-NH2 based on Förster resonance energy transfer (FRET) is attempted as a substrate of MMP-9 on parchment paper. Using the FRET substrate consists of 5-FAM donor and QXL520 acceptor, an inkjet printing assay was executed to validate the anticancer activity of chemicals. The tiny ejection volume achieved by inkjet printing technology makes it possible to screen a chemical library in the drug discovery process in a cost-effective manner. Surprisingly, it is capable of working better even with 100–1000 smaller reaction volume compared to a conventional pipetting-based assay. In addition, the inkjet cartridge can be well adapted as a high-throughput module in the automation of the drug discovery process. The ability of MMP-9 to recognize the peptide substrate was confirmed by HPLC. The chromatograms revealed the distinct peak corresponding to the fragmented peptide as a result of cleavage by MMP-9. The peptide containing 5-FAM and QXL520 was applied to the reaction with MMP-9 on the parchment paper for the establishment of the inkjet printing anticancer assay. Many researcher focus on inkjet printing-based microfluidic fabrication [21,22]. But the use of intact parchment paper has a strong impact in terms of versatile applications [17]. The MMP-9 recognized the peptide sequence and cleaved the peptide into two parts. The cleavage of the peptide resulted in the disappearance of FRET, which was confirmed by the recovery of fluorescence of 5-FAM at Ex/Em = 490 ± 20 nm/520 ± 20 nm. In this work, an accurate, rapid, and cost-effective FRET peptide-based MMP-9 enzyme inhibition assay is demonstrated for the assessment of the anticancer activity of test compounds using a conventional inkjet printer. This technology will open a productive avenue in large-scale high-throughput screening to evaluate the anticancer efficacy of library compounds against target enzymes at the molecular level. 2. Materials and methods 2.1. Materials Human MMP-9 enzyme and MMP-9 peptide substrate (520 MMP FRET substrate III): QXL520-Pro-Leu-Gly-Cys-His-Ala-ArgLys (5-FAM)-NH2 was purchased from Anaspec. Batimastat and marimastat were purchased from TOCRIS Bioscience. SB-3CT was purchased from Enzo Life Science. NNGH (N-Isobutyl-N-(4methoxyphenylsulfonyl)glycyl hydroxamic acid) was purchased from Cayman. 2-propanol, PEG-400, methanol, ethanol, DMSO, and APMA (4- Aminophenylmercuric acetate) were purchased from Sigma-Aldrich, South Korea. 10 × assay buffer (50 mM Tris pH 7.5 containing 150 mM NaCl2 , 10 mM CaCl2 , 10 M ZnCl2 , 0.05% BrijTM-35, and 10 mM DTNB) was prepared. 2.2. HPLC for the evaluation of MMP-9 activity on peptide substrate The stock solutions of the peptide (0.1 nM) and MMP-9 (20 ng/mL, 60 ng/mL, and 100 ng/mL) were prepared with 1 × assay buffer solution. 100 L of MMP-9 solution (50 ng/mL, 30 ng/mL, and 10 ng/mL), and 100 L of APMA solution (1 mM) were mixed in an eppendorf tube and incubated for 1 h to activate MMP-9. 100 L of the activated MMP-9 solution and 2.6 × 10−16 mol of the peptide solution was added to 96-well plate and incubated for
2 h. Total volumes was adjusted to be 200 L by adding 1 × assay buffer. After completion of the reaction, four samples, including the control composed of only peptide, were diluted five times. Reverse phase HPLC was performed by an HP instrument (HewlettPackard, HP 1100) equipped with a diode array detector using a phenomenex luna C18 column (4.6 mm × 250 mm, 5 um) at a flow rate of 1.2 mL/min. The elution was executed with a mobile phase composed of 90% of deionized water (0.1% of TFA) and 10% of acetonitrile (0.1% of TFA) for 15 min in isocratic condition. The absorbance of peptide was detected at 220 nm. 2.3. Measurement of MMP-9 activity in 96-well plate All of the reagents were freshly prepared and the assays were carried out at room temperature. 100 L of MMP-9 (60 ng/mL, 40 ng/mL, 20 ng/mL, 10 ng/mL, and 2 ng/mL) and 100 L of APMA (1 mM) solution in 1 × assay buffer were added to five different eppendorf tubes and incubated for 1 h to activate MMP-9. 100 L of the activated MMP-9 solutions and 2.6 × 10−16 mol of the peptide solution was added into the five different wells and incubated for 2 h. Total volumes of the wells were adjusted to be 200 L by adding 1 × assay buffer. Upon the cleavage of the peptide, fluorescence of 5-FAM was measured at Ex/Em = 485 nm/525 nm wavelength using a multi plate reader (Molecular Devices, Spectra MAX M5) at a time interval of 20 min for 2 h. For the MMP-9 inhibition assay, 0.25 M of Batimastat, 0.1 M of NNGH, 0.1 M of Marimastat, and 0.1 M of SB-3CT solution were prepared by dissolving with DMSO/ethanol/distilled water. To activate MMP-9, 2.5 mL of APMA solution (1 mM) and 2.5 mL of MMP-9 solution (60 ng/mL) were mixed in a conical tube and incubated for 1 h. Consequently, 100 L of the activated enzyme solution, and stock solutions of inhibitors were added to each well. NNGH solution was added from 3 L to 24 L, batimastat solution was added from 4.5 L to 36 L, and marimastat and SB-3CT solution were added from 2 L to 16 L. Finally, 2.6 × 10−16 mol of the peptide solution was added to all of the reaction wells, and the reaction mixtures were incubated for 2 h. Total reaction volumes was adjusted to be 200 L by adding 1 × assay buffer. 100 L of 2N HCl solution was added to stop all of the reactions for endpoint reading, and their fluorescence was monitored at Ex/Em = 485 nm/525 nm. The reaction conditions optimized in 96-well plates were used in the inkjet printing. 2.4. Measurement of MMP-9 activity using inkjet printing Enzymatic reaction conditions between the MMP-9 enzyme and the peptide were optimized on the parchment paper. Solutions of MMP-9 and peptide were prepared with 1× assay buffer containing 180 L of PEG-400 and 100 L of tert-butanol per 1 mL. MMP-9 solution was activated by mixing 1 mM APMA solution and MMP-9 solution with an equivalent volume. The activated MMP-9 solutions were prepared with three different concentrations of 5 ng/mL, 10 ng/mL, and 30 ng/mL. C and K cartridges were filled with 2 mL of the activated MMP-9 solution and 2 mL of peptide of 0.1 nM, respectively. M and Y cartridges were filled with 1 × assay buffer. MMP-9 and peptide were consecutively printed on the same reaction spot, and then 1 × assay buffer was printed on the same spot to avoid drying of solution. Their fluorescence was detected at the time interval of 1 min. Fluorescence was measured at Ex/Em = 485 nm/525 nm using a fluorescence microscope. Images were analyzed using MetaMorph software (Version 7.1.3.0). The ink solutions of the activated MMP-9 (30 ng/mL) and four drugs (0.1 M of NNGH, 0.25 M of batimastat, 0.1 M of marimastat, and 0.1 M of SB-3CT) were prepared with 1× assay buffer containing 180 L of PEG-400 and 100 L of tert-butanol per 1 mL. The C, M, Y and K cartridges were filled with the activated MMP-9 (30 ng/mL), 1× assay buffer, peptide solution (0.1 nM), and drug
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
G Model SNB-23355; No. of Pages 7
ARTICLE IN PRESS J. Lee et al. / Sensors and Actuators B xxx (2017) xxx–xxx
3
Fig. 1. (a) A schematic representation of a novel peptide-based fluorescent assay to determine the MMP-9 activities/inhibition using an inkjet printer. (b) Represents the activity of MMP-9 with respect to the peptide, which was printed at an array of 3 rows × 8 columns spots using inkjet printing. The fluorescence intensity produced at reaction spots was detected using a fluorescence microscope.
solution, respectively. The value of the K cartridge (which contained drug solution) corresponding to the ejected volume was set to be 10–70 to vary the number of mole of NNGH and batimastat printed per spot area. The K value was set to be 12–84 to change the number of mole of marimastat and SB-3CT printed per spot area. Inkjet printing was performed to spots composed of an array of 3 rows x 8 columns with 3 mm diameter per spot in order to achieve the assay. The printing order to achieve the MMP-9 inhibition assay was MMP-9, inhibitor, the peptide, and 1× assay buffer. MMP-9 (30 ng/mL), MMP-9 inhibitor and the peptide (0.1 nM) solution were printed from the C, K, and M cartridges, respectively. The C value and the M value were set to be 100. The reaction components were printed 10 times at each spot. The time interval between printing each component (between enzyme and inhibitor, between inhibitor and peptide) was 15 min. The printed-paper was incubated at room temperature for 7 min, and fluorescence was acquired. The inhibitor was printed on seven different spots in each row as a function of dose. A spot in each row was used as a control without the inhibitor. At each dose, the fluorescence intensities of three different spots in a column were averaged as a value, and these averaged values over the entire range of dose were used to determine inhibitory mole 50 (IM50 ) of the inhibitor. The cartridge weight loss was measured before and after printing, and used to validate the ejection volume. Using the acquired ejection volume, the number of mole printed per spot surface area was calculated as described [17], and the resultant IM50 was determined.
3. Results and discussion 3.1. Principle of the experiment Fig. 1a outlines the principle of a novel peptide-based FRET assay to determine the MMP-9 activities/MMP-9 inhibition using an inkjet printer. Briefly, the activated MMP9/peptide (5- FAM/QXL520) or MMP-9/enzyme inhibitor/peptide (5-FAM/QXL520) were consecutively printed on the reaction spot of parchment paper [17]. The fluorescence of 5-FAM was quenched by the QXL520 present in the intact peptide. Upon the cleavage of the peptide by MMP-9, the fluorescence of 5-FAM was recovered. Since the printing system enables high-throughput analysis, this assay is capable of analyzing MMP inhibitory activities of compounds precisely based on their high-throughput spotting as a function of dose, as shown in Fig. 1b. Fig. 1b represents how the proteolytic activity of MMP-9 with respect to the peptide is detected at an array of 3 rows × 8 columns spots, including the control without the inhibitor using inkjet printing. The fluorescence at reaction spots was detected by the microscope.
3.2. HPLC for the validation of MMP-9 activity on the synthetic peptide substrate The specificity of the peptide substrate with respect to MMP-9 was investigated using HPLC. As shown in Fig. 2, increase of acti-
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
G Model SNB-23355; No. of Pages 7
ARTICLE IN PRESS J. Lee et al. / Sensors and Actuators B xxx (2017) xxx–xxx
4
Fig. 2. HPLC profile of: (a) blank; (b) only peptide; (c) cleavage of the peptide observed at 10 ng/mL of MMP-9 solution; (d) cleavage of the peptide observed at 30 ng/mL of MMP-9 solution; (e) almost full cleavage of the peptide observed at 50 ng/mL of MMP-9 solution; and (f) the surface area (black) of the HPLC profile of the peptide is generally decreasing as the concentration of enzyme increased in the reaction mixture, leading to gradual increase of the surface area of the cleaved peptide (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
vated MMP-9 concentration exhibited a gradual decrease in the peak intensity of the intact peptide due to the cleavage of the peptide, while the peak intensity of the fragmented peptide increased gradually. Peaks at 2.5 min corresponded to the intact peptide. On the other hand, peaks at 3.25 min corresponded to the fragmented peptide. The slower migration time of fragmented peptide is thought to be due to reduction of polarity in the fragmented peptide. The reduction of polarity in the sample generally provides a sample with slower migration time in reverse phase HPLC. The cleavage of the peptide was observed at 10 ng/mL of MMP-9 solution. 50 ng/mL of MMP-9 solution showed almost full cleavage of the peptide, as shown in Fig. 2c. The HPLC results suggested that the peptide substrate was cleaved efficiently by MMP-9 and possesses strong specificity for MMP-9. 3.3. MMP-9 inhibitory assay in 96-well plate After the proteolytic cleavage of the peptide by MMP-9 was confirmed by HPLC, an enzymatic reaction between MMP-9 and
the peptide was performed in the conventional microplate based on fluorescence of the fragmented peptide produced as a function of MMP-9 dose. The cleavage condition of the peptide by MMP-9 was investigated in a 96-well microplate. The peptide (2.6 × 10−16 mol) solution and activated MMP-9 (30 ng/mL, 20 ng/mL, 10 g/mL, 5 ng/mL, and 1 ng/mL) solution were incubated before the fluorescence was measured using a multi-plate reader. The fluorescence was obtained every 20 min after the reaction. As shown in Fig. 3a the relative fluorescence intensity was 15.7 in the presence of 30 ng/mL of MMP-9 and 4.09 in the presence of 1 ng/mL of MMP-9, when MMP-9 was incubated with the peptide of identical concentration for 120 min. After the investigation of cleavage condition by MMP-9, the peptide was used to screen activities of MMP-9 inhibitors. 1.5–12.0 nM of NNGH, 3.75–30.0 nM of batimastat, and 1–8 nM of marimastat and SB-3CT were consumed to test their inhibition efficacies. The proteolytic activity of MMP-9 in the presence of the inhibitor was measured at Ex/Em = 485 nm/525 nm. IC50 values of the MMP-9 inhibitors were estimated quantitatively by monitoring their fluorescence intensity versus inhibitor concen-
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
G Model SNB-23355; No. of Pages 7
ARTICLE IN PRESS J. Lee et al. / Sensors and Actuators B xxx (2017) xxx–xxx
5
Fig. 3. (a) Cleavage of the peptide by MMP-9 was measured as a function of enzyme dose and incubation time point. (b–e) MMP-9 inhibition activity determined as a function of dose of different inhibitors. The x-axis indicates the concentration of the inhibitor, and the y-axis shows the fluorescence intensity acquired as an effect of peptide hydrolysis. The relative fluorescence was monitored at Ex/Em = 485 nm/525 nm wavelength using a multi-plate reader.
tration. As shown in Fig. 3b–e, the x-axis denotes the concentration of the inhibitor, and the y-axis shows the fluorescence intensity acquired as an effect of enzymatic peptide hydrolysis. The obtained IC50 values of NNGH, batimastat, marimastat and SB-3CT were 9.2 nM, 25.7 nM, 6.3 nM and 7.2 nM, respectively. Marimastat represented the lowest IC50 value compared to other inhibitors. The inkjet printing MMP-9 inhibition assay was performed based on reaction conditions obtained in the well plate. 3.4. Measurement of MMP-9 activity using inkjet printing The proteolytic activity of MMP-9 with respect to the peptide on parchment paper was determined using inkjet printing. The peptide of 47.5 × 10−18 mol was printed on the same spot along with MMP-9 and incubated for 7 min. The fluorescence of the peptide cleaved by MMP-9 was monitored every 1 min as a function of dose of activated MMP-9 (5 ng/mL, 20 ng/mL, and 30 ng/mL). The concentration of activated MMP-9 was tested up to 30 ng/mL. This concentration has been generally used when the enzymatic reaction of MMP-9 with peptide substrate has been executed in the conventional well plate. IC50 values of MMP-9 inhibitors have been generally acquired in the concentration range of MMP-9 up to 30 ng/mL. Accordingly, it is critical to check whether the fluorescence intensity arising from the fragmented peptide on the parchment paper is produced sufficiently to determine drug inhibitory concentration at MMP-9 concentration of 30 ng/mL. As shown in Fig. 4, the fluorescence intensity of the fragmented peptide increased as a function of MMP-9 concentration and reached nearly 2000 at MMP-9 concentration of 30 ng/mL with the incubation time of 7 min. This intensity was found to be adequate to measure MMP-9 inhibitory concentration. Table 1 represents densities, ejection volumes, and ejected values of MMP-9 ink solutions obtained through inkjet printing on parchment paper. At 30 ng/mL,
Fig. 4. Inkjet printing-based MMP-9 assay. The cleavage of the peptide by MMP-9 on parchment paper was measured as a function of time and in a dose-dependent manner. The x-axis indicates the incubation time point, and the y-axis shows the fluorescence intensity acquired as an effect of peptide hydrolysis by MMP-9. Fluorescence intensity was measured at Ex/Em = 485 nm/525 nm using a fluorescence microscope.
the amount of MMP-9 spotted on the parchment paper was determined to be 14.1 fg, which is approximately 105 times smaller than the 3 ng consumed in a conventional well plate using a pipette. 3.5. MMP-9 FRET inhibition assay for the determination of IM50 using inkjet printing Fig. 5 represents MMP-9 inhibitory activities of: (a) NNGH; (b) batimastat; (c) marimastat; and (d) SB-3CT using inkjet printing.
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
G Model
ARTICLE IN PRESS
SNB-23355; No. of Pages 7
J. Lee et al. / Sensors and Actuators B xxx (2017) xxx–xxx
6
Table 1 Densities of different concentrations of MMP-9 ink solution and their respective volumes. Concentration of activated MMP-9 stock solution
MMP-9 (30 ng/mL)
MMP-9 (10 ng/mL)
MMP-9 (5 ng/mL)
Density (g/mL) Ejected volume (nL) Ejected amount of MMP-9 (fg)
1.0163 46.8 14.10
1.0141 46.9 4.69
1.0118 48.0 2.40
Fig. 5. MMP-9 inhibitory activities of NNGH, batimastat, marimastat, and SB-3CT using inkjet-printing. a-1, schematic diagram represents the printing order of ink solutions to perform the MMP-9 inhibition assay. a-2, the number of mole of inhibitor printed on reaction spots according to the K values and their respective images of fluorescence produced after cleavage of the peptide by MMP-9. b) NNGH; c) batimastat; d) marimastat; and e) SB-3CT, represents the quantitative plot of the reduction of fluorescence intensity formed on the reaction spot. Fluorescence intensity was measured at Ex/Em = 485 nm/525 nm using a fluorescence microscope, and the images were analyzed using MetaMorph software.
Table 2 Solution densities, ejection volumes, and IM50 values of MMP-9 inhibitors obtained through inkjet printing. Inhibitors
NNGH (0.1 M)
Batimastat (0.25 M)
Inhibitors
Marimastat (0.1 M)
SB-3CT (0.1 M)
Density (g/mL) Ejected volume (nL) IM50 (femto mole) R2
1.0208 43.2 17.3 0.976
1.0152 46.9 52.2 0.989
Density (g/mL) Ejected volume (nL) IM50 (femto mole) R2
1.0158 43 15.6 0.984
1.0174 46.3 17.9 0.981
Table 2 represents densities, ejection volumes, and IM50 values of NNGH, batimastat, marimastat, and SB-3CT ink obtained through the inkjet printing on the parchment paper. Fig. 5a-1 illustrates the printing order that was executed on sample arrays of 3 rows × 8 columns. Fig. 5a-2 denotes the number of mole of inhibitor printed on reaction spots according to the K values and their respective images of fluorescence arising from the cleavage of the peptide by MMP-9. At each spot, each inhibitor with different number of mole was reacted with peptide substrate of 47.5 × 10−18 mol and MMP-9 of 14.1 fg. These concentrations were found to provide the fluorescence intensity of the fragmented peptide suitable for the determination of IM50 (the number of mole needed to reduce the fluorescence intensity of the control by 50%) in the above result. When the reaction spots were excited at 490 ± 20 nm, the control without inhibitor (K value = 0) showed the brightest fluorescence intensity of nearly 2000, while the brightness of fluorescence was gradually reduced on reaction spots in the presence of the inhibitor. This tendency was observed identically in all of the tested MMP-9 inhibitors. The reduction of fluorescence brightness was quanti-
tatively plotted in Fig. 5b–e. These plots of fluorescence intensity versus the number of mole printed enabled the determination of IM50 . Table 2 represents the IM50 values of NNGH, batimastat, marimastat, and SB-3CT as 17.3 × 10−15 mol, 52.2 × 10−15 mol, 15.6 × 10−15 mol and 17.9 × 10−15 mol, respectively. The MMP-9 inhibitory activities of marimastat, NNGH, and SB-3CT were significantly larger than batimastat. The MMP-9 inhibitory activity of marimastat was the largest. This tendency was well matched with the result of the MMP-9 inhibition assay in 96-well plates. Compared to a conventional microplate, fragmentation and resultant fluorescence detection of the peptide printed on reaction spots of parchment paper require greater attention, which utilized 102 to 106 smaller amount (Table 3). This is because detection of the fragmented peptide is influenced more sensitively on the surface of parchment paper by various conditions including printing, such as enzyme activity, peptide density, and the reaction time of peptide substrate. This signifies that drug inhibitory concentration can be
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
G Model
ARTICLE IN PRESS
SNB-23355; No. of Pages 7
J. Lee et al. / Sensors and Actuators B xxx (2017) xxx–xxx Table 3 IM50 values of MMP-9 inhibitors obtained through 96 well plate and inkjet printing methods. Method
Inhibitor NNGH Batimastat Marimastat SB-3CT
96 well plate
IC50( nM) 9.2 IM50 (×10−12 mol) 1.8 −14 Inkjet printing IM50 (×10 mol) 1.7
25.7 5.1 5.2
6.3 1.3 1.6
7.2 1.4 1.8
determined on a tremendously small scale if the assay is performed using inkjet printing instead of well plate. 4. Conclusion In this study, an inkjet printing MMP-9 anticancer assay using fluorogenic peptide was performed on parchment paper based on FRET detection. The cleavage of 5-FAM/QXL520 peptide by MMP-9 enzyme resulted in the disappearance of FRET arising from the separation of chromophores, and increased the fluorescence intensity of 5-FAM significantly. Our work revealed that the peptide substrate, MMP-9 enzyme, and their respective inhibitors could be deposited on a non-fabricated paper surface with 102 to 106 smaller amount compared to pipette-based assay in a well plate, making it possible to validate fluorescence-based high throughput drug screening in an automated and reproducible manner. By the virtues of the versatile printing setup, the fluorescent peptide was used to successfully determine IM50 values of MMP-9 inhibitors, NNGH, batimastat, SB-3CT, and marimastat. This approach improves and extends universal applications of inkjet printing for enzymatic anticancer assays. Acknowledgments This work was supported by National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) (2015R1A2A1A05001842 and 2016R1A4A1010796). The authors declare no competing financial interests. We are grateful to Brain Korea 21 plus (BK 21 plus) and College of Pharmaceutical Sciences at Seoul National University for providing experimental equipment. References [1] G.A. Skarja, A.L. Brown, R.K. Ho, M.H. May, M.V. Sefton, The effect of a hydroxamic acid-containing polymer on active matrix metalloproteinases, Biomaterials 30 (2009) 1890–1897. [2] R. Salsas-Escat, P.S. Nerenberg, C.M. Stultz, Cleavage site specificity and conformational selection in type I collagen degradation, Biochemistry-US 49 (2010) 4147–4158. [3] J.A. Jacobsen, J.L. Fullagar, M.T. Miller, S.M. Cohen, Identifying chelators for metalloprotein inhibitors using a fragment-Based approach, J. Med. Chem. 54 (2011) 591–602. [4] M.M. Benjamin, R.A. Khalil, Matrix metalloproteinase inhibitors as investigative tools in the pathogenesis and management of vascular disease, EXS 103 (2012) 209–279. [5] H. Jarvelainen, A. Sainio, M. Koulu, T.N. Wight, R. Penttinen, Extracellular matrix molecules: potential targets in pharmacotherapy, Pharmacol. Rev. 61 (2009) 198–223. [6] C. Bonnans, J. Chou, Z. Werb, Remodelling the extracellular matrix in development and disease, Nat. Rev. Mol. Cell Biol. 15 (2014) 786–801. [7] K. Kessenbrock, V. Plaks, Z. Werb, Matrix metalloproteinases: regulators of the tumor microenvironment, Cell 141 (2010) 52–67. [8] A. Noel, A. Gutierrez-Fernandez, N.E. Sounni, N. Behrendt, E. Maquoi, I.K. Lund, et al., New and paradoxical roles of matrix metalloproteinases in the tumor microenvironment, Front. Pharmacol. 3 (2012) 140. [9] X. Zhang, J. Bresee, P.P. Cheney, B. Xu, M. Bhowmick, M. Cudic, et al., Evaluation of a triple-helical peptide with quenched FluorSophores for optical imaging of MMP-2 and MMP-9 proteolytic activity, Molecules 19 (2014) 8571–8588. [10] M. Stawarski, I. Rutkowska-Wlodarczyk, A. Zeug, M. Bijata, H. Madej, L. Kaczmarek, et al., Genetically encoded FRET-based biosensor for imaging MMP-9 activity, Biomaterials 35 (2014) 1402–1410.
7
[11] J.O. McIntyre, B. Fingleton, K.S. Wells, D.W. Piston, C.C. Lynch, S. Gautam, et al., Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity, Biochem. J. 377 (2004) 617–628. [12] K. Leung, Cy5.5-Aminohexanoic Acid-RPLALWRS-Aminohexanoic Acid-C-G4-PAMAM-PEG-AF750, Molecular Imaging and Contrast Agent Database (MICAD), Bethesda (MD), 2004. [13] J.R. Tauro, B.S. Lee, S.S. Lateef, R.A. Gemeinhart, Matrix metalloprotease selective peptide substrates cleavage within hydrogel matrices for cancer chemotherapy activation, Peptides 29 (2008) 1965–1973. [14] A. Dufour, N.S. Sampson, J. Li, C. Kuscu, R.C. Rizzo, J.L. Deleon, et al., Small-molecule anticancer compounds selectively target the hemopexin domain of matrix metalloproteinase-9, Cancer Res. 71 (2011) 4977–4988. [15] M.W. Ndinguri, M. Bhowmick, D. Tokmina-Roszyk, T.K. Robichaud, G.B. Fields, Peptide-based selective inhibitors of matrix metalloproteinase-mediated activities, Molecules 17 (2012) 14230–14248. [16] G.B. Fields, Using fluorogenic peptide substrates to assay matrix metalloproteinases, Methods Mol. Biol. 151 (2001) 495–518. [17] J. Lee, A.A. Samson, J.M. Song, Inkjet-printing enzyme inhibitory assay based on determination of ejection volume, Anal. Chem. 89 (2017) 2009–2016. [18] L. Wu, Z.C. Dong, F.Y. Li, H.H. Zhou, Y.L. Song, Emerging progress of inkjet technology in printing optical materials, Adv. Opt. Mater. 4 (2016) 1915–1932. [19] Y.F. Zhang, F.J. Lyu, J. Ge, Z. Liu, Ink-jet printing an optimal multi-enzyme system, Chem. Commun. 50 (2014) 12919–12922. [20] B. Creran, X.N. Li, B. Duncan, C.S. Kim, D.F. Moyano, V.M. Rotello, Detection of bacteria using inkjet-Printed enzymatic test strips, ACS Appl. Mater. Interfaces 6 (2014) 19525–19530. [21] E. Bou Chakra, B. Hannes, J. Vieillard, C.D. Mansfield, R. Mazurczyk, A. Bouchard, et al., Grafting of antibodies inside integrated microfluidic-microoptic devices by means of automated microcontact printing, Sens. Actuators B Chem. 140 (2009) 278–286. [22] H. Yang, T. Rahman, D. Du, R. Panat, Y. Lin, 3-D printed adjustable microelectrode arrays for electrochemical sensing and biosensing, Sens. Actuators B Chem. 230 (2016) 505–600.
Biographies
Jungmi Lee received her Bachelor’s degree in Chemistry Education in 2011 and Master’s degree in Chemistry in 2016 at Kyungpook National University, in Korea. Currently, she is PhD student at College of Pharmacy, Seoul National University in South Korea. Her research interest is organic synthesis, chiral separation, drug screening and diagnosis in molecule level. She has published 3 peer reviewed paper.
Annie Agnes Suganya Samson received her Master of Science degree in Biotechnology from Bharathidasan University in 2013. She was working as a Junior Research Fellow from 2013 to 2015 at Rajiv Gandhi Center for Biotechnology. Currently, she is a PhD student at College of Pharmacy, Seoul National University in South Korea. Her research interest includes cancer biology, stem cells, chemo and immunotherapy and proteomic studies. She has published 5 peer reviewed paper and 1 book chapter.
Joon Myong Song received his PhD in 1997 at Kyushu University, in Japan. He worked as a postdoctoral research fellow from 1998 to 2004 at Iowa State University, Brookhaven National Laboratory, and Oak Ridge National Laboratory in United States. At present he is a professor at College of Pharmacy, Seoul National University in South Korea. His research area includes multifunctional nanoparticle for diagnosis and therapy and high-content cell-based drug screening and diagnosis using hypermulticolor cellular imaging. He has published 104 peer reviewed papers in the top journals, 10 book chapters, and 10 patents.
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
ARTICLE IN PRESS
SNB-23355; No. of Pages 7
Sensors and Actuators B xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Research Paper
Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET) Jungmi Lee, Annie Agnes Suganya Samson, Joon Myong Song ∗ College of Pharmacy, Seoul National University, Seoul 08826, South Korea
a r t i c l e
i n f o
Article history: Received 12 July 2017 Received in revised form 6 September 2017 Accepted 10 October 2017 Available online xxx Keywords: Inkjet printing FRET Matrix metalloproteinase-9 Peptide substrate Inhibitor
a b s t r a c t A peptide probe-based MMP-9 inhibitory assay was developed to assess anticancer activity of compounds on parchment paper using FRET-based inkjet printing. The fluorescent peptide containing 5-FAM (donor) and QXL520 (acceptor) was used as a substrate with the sequence of Pro-Leu-Gly-Cys-His-Ala-Arg-Lys specific to MMP-9. The suitability of the peptide as a substrate of MMP-9 was confirmed by HPLC. When both MMP-9 and the peptide were printed on the identical reaction spot of parchment paper, the peptide was cleaved and fragmented by MMP-9. The fluorescence was then recovered by 5-FAM and detected at Ex/Em = 490 ± 20 nm/520 ± 20 nm. Based on the successful performance of FRET on the parchment paper without any surface treatment, the assay platform was applied to test compounds, and a successful anticancer assay could be accomplished. This methodology holds notable strengths in versatile applications to the MMP enzyme family, as well as control of ejection volume, reduction of consumed enzyme amount, and rapid reaction time. Hence, we suggest the FRET peptide-based assay using inkjet printing as a viable replacement for sensitive and high-throughput evaluation of anticancer compounds. © 2017 Published by Elsevier B.V.
1. Introduction Enzymes as catalysts play a major role in increasing reaction rates by lowering activation energy in almost all intracellular metabolic reactions. Currently, assay kits have been exploited to mimic the intracellular reaction between enzyme and protein or peptide substrate, and applied to drug screening at the molecular level [1–3]. Matrix metalloproteinases (MMPs) are a family of secretory endopeptidases with hydrolytic activity for a diverse spectrum of extracellular proteins [4]. MMPs released from cells execute breakdown of extracellular matrix (ECM) in physiological processes, such as cell migration and angiogenesis, by degradation of collagen IV and V, and other ECM proteins [5,6]. MMP-9 is highly expressed in the metastatic cancer cell for its role of ECM remodeling and angiogenesis. Therefore, MMP-9 is deeply related to tumor progress, including invasion and metastasis [7,8]. Recently, studies dealing with determination of MMP-9 activity have been reported. Gel and in situ zymography (gelatin-based) assays were performed to detect the proteolytic activity of MMP-9. This assays had limitations in terms of fluorescence resolution. In order to overcome the limitations, several fluorescent-probe based
∗ Corresponding author. E-mail address: [email protected] (J.M. Song).
biosensors have been developed for the detection of MMP activity. The first probes capable of imaging MMP activity was developed by modifying the original protease-sensing peptide probes. Similarly, numerous peptide probes were synthesized and developed to evaluate the proteolytic activity of MMP-9. This approach includes Cy5 NIR fluorophores conjugated peptide substrate, dendrimerbased fluorogenic substrates (fluorogenic proteolytic beacon), a self-assembling homotrimeric triple helical peptide, quantum dot (QD)/BHQ-1-based FRET peptide, and genetically encoded FRETbased probes [9–12]. Anticancer activities of test compounds as MMP-9 inhibitor can be proven through fluorescence of peptide substrate composed of a fluorescent donor and an acceptor quencher [13–15]. The sequence Pro-Leu-Gly-Cys-His-Ala-Arg-Lys is specifically recognized by the enzyme MMPs and exhibits a high degree of substrate specificity for MMP-9. The peptide is cleaved efficiently at the Gly Cys bond. Studies have revealed that the peptide is also cleaved at a similar rate by MMP-1, MMP-8, MMP-9, and MMP-12 [16]. Generally, molecular assays have been applied to screen target protein, membrane receptors and ion channels, which typically generates a functional read out for drug discovery. Drug screening assays are typically performed in 96-well or 384 or 1536micro well plates. Different assay format (384 or 1536-micro well plates) are selected to minimize assay reagents and assay volume. When high throughput screening assays are performed in micro well plates, the assay volume used is as small as a few microliters.
https://doi.org/10.1016/j.snb.2017.10.051 0925-4005/© 2017 Published by Elsevier B.V.
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
G Model SNB-23355; No. of Pages 7
ARTICLE IN PRESS J. Lee et al. / Sensors and Actuators B xxx (2017) xxx–xxx
2
Recently, it has been reported that inkjet printing technology are highly suitable for printing enzymes. Assay volume consumed to perform printing based assay was further reduced to nanoliter and picoliter [17–20]. Thus, inkjet printing technology with high priority of cost-effective platform can be selected to minimize the assay volume (nL to pL), which will be highly appropriate to evaluate enzyme/inhibitor activity. In this study, a peptide containing the sequence QXL520-Pro-Leu-Gly-Cys-His-Ala-Arg-Lys (5-FAM)-NH2 based on Förster resonance energy transfer (FRET) is attempted as a substrate of MMP-9 on parchment paper. Using the FRET substrate consists of 5-FAM donor and QXL520 acceptor, an inkjet printing assay was executed to validate the anticancer activity of chemicals. The tiny ejection volume achieved by inkjet printing technology makes it possible to screen a chemical library in the drug discovery process in a cost-effective manner. Surprisingly, it is capable of working better even with 100–1000 smaller reaction volume compared to a conventional pipetting-based assay. In addition, the inkjet cartridge can be well adapted as a high-throughput module in the automation of the drug discovery process. The ability of MMP-9 to recognize the peptide substrate was confirmed by HPLC. The chromatograms revealed the distinct peak corresponding to the fragmented peptide as a result of cleavage by MMP-9. The peptide containing 5-FAM and QXL520 was applied to the reaction with MMP-9 on the parchment paper for the establishment of the inkjet printing anticancer assay. Many researcher focus on inkjet printing-based microfluidic fabrication [21,22]. But the use of intact parchment paper has a strong impact in terms of versatile applications [17]. The MMP-9 recognized the peptide sequence and cleaved the peptide into two parts. The cleavage of the peptide resulted in the disappearance of FRET, which was confirmed by the recovery of fluorescence of 5-FAM at Ex/Em = 490 ± 20 nm/520 ± 20 nm. In this work, an accurate, rapid, and cost-effective FRET peptide-based MMP-9 enzyme inhibition assay is demonstrated for the assessment of the anticancer activity of test compounds using a conventional inkjet printer. This technology will open a productive avenue in large-scale high-throughput screening to evaluate the anticancer efficacy of library compounds against target enzymes at the molecular level. 2. Materials and methods 2.1. Materials Human MMP-9 enzyme and MMP-9 peptide substrate (520 MMP FRET substrate III): QXL520-Pro-Leu-Gly-Cys-His-Ala-ArgLys (5-FAM)-NH2 was purchased from Anaspec. Batimastat and marimastat were purchased from TOCRIS Bioscience. SB-3CT was purchased from Enzo Life Science. NNGH (N-Isobutyl-N-(4methoxyphenylsulfonyl)glycyl hydroxamic acid) was purchased from Cayman. 2-propanol, PEG-400, methanol, ethanol, DMSO, and APMA (4- Aminophenylmercuric acetate) were purchased from Sigma-Aldrich, South Korea. 10 × assay buffer (50 mM Tris pH 7.5 containing 150 mM NaCl2 , 10 mM CaCl2 , 10 M ZnCl2 , 0.05% BrijTM-35, and 10 mM DTNB) was prepared. 2.2. HPLC for the evaluation of MMP-9 activity on peptide substrate The stock solutions of the peptide (0.1 nM) and MMP-9 (20 ng/mL, 60 ng/mL, and 100 ng/mL) were prepared with 1 × assay buffer solution. 100 L of MMP-9 solution (50 ng/mL, 30 ng/mL, and 10 ng/mL), and 100 L of APMA solution (1 mM) were mixed in an eppendorf tube and incubated for 1 h to activate MMP-9. 100 L of the activated MMP-9 solution and 2.6 × 10−16 mol of the peptide solution was added to 96-well plate and incubated for
2 h. Total volumes was adjusted to be 200 L by adding 1 × assay buffer. After completion of the reaction, four samples, including the control composed of only peptide, were diluted five times. Reverse phase HPLC was performed by an HP instrument (HewlettPackard, HP 1100) equipped with a diode array detector using a phenomenex luna C18 column (4.6 mm × 250 mm, 5 um) at a flow rate of 1.2 mL/min. The elution was executed with a mobile phase composed of 90% of deionized water (0.1% of TFA) and 10% of acetonitrile (0.1% of TFA) for 15 min in isocratic condition. The absorbance of peptide was detected at 220 nm. 2.3. Measurement of MMP-9 activity in 96-well plate All of the reagents were freshly prepared and the assays were carried out at room temperature. 100 L of MMP-9 (60 ng/mL, 40 ng/mL, 20 ng/mL, 10 ng/mL, and 2 ng/mL) and 100 L of APMA (1 mM) solution in 1 × assay buffer were added to five different eppendorf tubes and incubated for 1 h to activate MMP-9. 100 L of the activated MMP-9 solutions and 2.6 × 10−16 mol of the peptide solution was added into the five different wells and incubated for 2 h. Total volumes of the wells were adjusted to be 200 L by adding 1 × assay buffer. Upon the cleavage of the peptide, fluorescence of 5-FAM was measured at Ex/Em = 485 nm/525 nm wavelength using a multi plate reader (Molecular Devices, Spectra MAX M5) at a time interval of 20 min for 2 h. For the MMP-9 inhibition assay, 0.25 M of Batimastat, 0.1 M of NNGH, 0.1 M of Marimastat, and 0.1 M of SB-3CT solution were prepared by dissolving with DMSO/ethanol/distilled water. To activate MMP-9, 2.5 mL of APMA solution (1 mM) and 2.5 mL of MMP-9 solution (60 ng/mL) were mixed in a conical tube and incubated for 1 h. Consequently, 100 L of the activated enzyme solution, and stock solutions of inhibitors were added to each well. NNGH solution was added from 3 L to 24 L, batimastat solution was added from 4.5 L to 36 L, and marimastat and SB-3CT solution were added from 2 L to 16 L. Finally, 2.6 × 10−16 mol of the peptide solution was added to all of the reaction wells, and the reaction mixtures were incubated for 2 h. Total reaction volumes was adjusted to be 200 L by adding 1 × assay buffer. 100 L of 2N HCl solution was added to stop all of the reactions for endpoint reading, and their fluorescence was monitored at Ex/Em = 485 nm/525 nm. The reaction conditions optimized in 96-well plates were used in the inkjet printing. 2.4. Measurement of MMP-9 activity using inkjet printing Enzymatic reaction conditions between the MMP-9 enzyme and the peptide were optimized on the parchment paper. Solutions of MMP-9 and peptide were prepared with 1× assay buffer containing 180 L of PEG-400 and 100 L of tert-butanol per 1 mL. MMP-9 solution was activated by mixing 1 mM APMA solution and MMP-9 solution with an equivalent volume. The activated MMP-9 solutions were prepared with three different concentrations of 5 ng/mL, 10 ng/mL, and 30 ng/mL. C and K cartridges were filled with 2 mL of the activated MMP-9 solution and 2 mL of peptide of 0.1 nM, respectively. M and Y cartridges were filled with 1 × assay buffer. MMP-9 and peptide were consecutively printed on the same reaction spot, and then 1 × assay buffer was printed on the same spot to avoid drying of solution. Their fluorescence was detected at the time interval of 1 min. Fluorescence was measured at Ex/Em = 485 nm/525 nm using a fluorescence microscope. Images were analyzed using MetaMorph software (Version 7.1.3.0). The ink solutions of the activated MMP-9 (30 ng/mL) and four drugs (0.1 M of NNGH, 0.25 M of batimastat, 0.1 M of marimastat, and 0.1 M of SB-3CT) were prepared with 1× assay buffer containing 180 L of PEG-400 and 100 L of tert-butanol per 1 mL. The C, M, Y and K cartridges were filled with the activated MMP-9 (30 ng/mL), 1× assay buffer, peptide solution (0.1 nM), and drug
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
G Model SNB-23355; No. of Pages 7
ARTICLE IN PRESS J. Lee et al. / Sensors and Actuators B xxx (2017) xxx–xxx
3
Fig. 1. (a) A schematic representation of a novel peptide-based fluorescent assay to determine the MMP-9 activities/inhibition using an inkjet printer. (b) Represents the activity of MMP-9 with respect to the peptide, which was printed at an array of 3 rows × 8 columns spots using inkjet printing. The fluorescence intensity produced at reaction spots was detected using a fluorescence microscope.
solution, respectively. The value of the K cartridge (which contained drug solution) corresponding to the ejected volume was set to be 10–70 to vary the number of mole of NNGH and batimastat printed per spot area. The K value was set to be 12–84 to change the number of mole of marimastat and SB-3CT printed per spot area. Inkjet printing was performed to spots composed of an array of 3 rows x 8 columns with 3 mm diameter per spot in order to achieve the assay. The printing order to achieve the MMP-9 inhibition assay was MMP-9, inhibitor, the peptide, and 1× assay buffer. MMP-9 (30 ng/mL), MMP-9 inhibitor and the peptide (0.1 nM) solution were printed from the C, K, and M cartridges, respectively. The C value and the M value were set to be 100. The reaction components were printed 10 times at each spot. The time interval between printing each component (between enzyme and inhibitor, between inhibitor and peptide) was 15 min. The printed-paper was incubated at room temperature for 7 min, and fluorescence was acquired. The inhibitor was printed on seven different spots in each row as a function of dose. A spot in each row was used as a control without the inhibitor. At each dose, the fluorescence intensities of three different spots in a column were averaged as a value, and these averaged values over the entire range of dose were used to determine inhibitory mole 50 (IM50 ) of the inhibitor. The cartridge weight loss was measured before and after printing, and used to validate the ejection volume. Using the acquired ejection volume, the number of mole printed per spot surface area was calculated as described [17], and the resultant IM50 was determined.
3. Results and discussion 3.1. Principle of the experiment Fig. 1a outlines the principle of a novel peptide-based FRET assay to determine the MMP-9 activities/MMP-9 inhibition using an inkjet printer. Briefly, the activated MMP9/peptide (5- FAM/QXL520) or MMP-9/enzyme inhibitor/peptide (5-FAM/QXL520) were consecutively printed on the reaction spot of parchment paper [17]. The fluorescence of 5-FAM was quenched by the QXL520 present in the intact peptide. Upon the cleavage of the peptide by MMP-9, the fluorescence of 5-FAM was recovered. Since the printing system enables high-throughput analysis, this assay is capable of analyzing MMP inhibitory activities of compounds precisely based on their high-throughput spotting as a function of dose, as shown in Fig. 1b. Fig. 1b represents how the proteolytic activity of MMP-9 with respect to the peptide is detected at an array of 3 rows × 8 columns spots, including the control without the inhibitor using inkjet printing. The fluorescence at reaction spots was detected by the microscope.
3.2. HPLC for the validation of MMP-9 activity on the synthetic peptide substrate The specificity of the peptide substrate with respect to MMP-9 was investigated using HPLC. As shown in Fig. 2, increase of acti-
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
G Model SNB-23355; No. of Pages 7
ARTICLE IN PRESS J. Lee et al. / Sensors and Actuators B xxx (2017) xxx–xxx
4
Fig. 2. HPLC profile of: (a) blank; (b) only peptide; (c) cleavage of the peptide observed at 10 ng/mL of MMP-9 solution; (d) cleavage of the peptide observed at 30 ng/mL of MMP-9 solution; (e) almost full cleavage of the peptide observed at 50 ng/mL of MMP-9 solution; and (f) the surface area (black) of the HPLC profile of the peptide is generally decreasing as the concentration of enzyme increased in the reaction mixture, leading to gradual increase of the surface area of the cleaved peptide (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
vated MMP-9 concentration exhibited a gradual decrease in the peak intensity of the intact peptide due to the cleavage of the peptide, while the peak intensity of the fragmented peptide increased gradually. Peaks at 2.5 min corresponded to the intact peptide. On the other hand, peaks at 3.25 min corresponded to the fragmented peptide. The slower migration time of fragmented peptide is thought to be due to reduction of polarity in the fragmented peptide. The reduction of polarity in the sample generally provides a sample with slower migration time in reverse phase HPLC. The cleavage of the peptide was observed at 10 ng/mL of MMP-9 solution. 50 ng/mL of MMP-9 solution showed almost full cleavage of the peptide, as shown in Fig. 2c. The HPLC results suggested that the peptide substrate was cleaved efficiently by MMP-9 and possesses strong specificity for MMP-9. 3.3. MMP-9 inhibitory assay in 96-well plate After the proteolytic cleavage of the peptide by MMP-9 was confirmed by HPLC, an enzymatic reaction between MMP-9 and
the peptide was performed in the conventional microplate based on fluorescence of the fragmented peptide produced as a function of MMP-9 dose. The cleavage condition of the peptide by MMP-9 was investigated in a 96-well microplate. The peptide (2.6 × 10−16 mol) solution and activated MMP-9 (30 ng/mL, 20 ng/mL, 10 g/mL, 5 ng/mL, and 1 ng/mL) solution were incubated before the fluorescence was measured using a multi-plate reader. The fluorescence was obtained every 20 min after the reaction. As shown in Fig. 3a the relative fluorescence intensity was 15.7 in the presence of 30 ng/mL of MMP-9 and 4.09 in the presence of 1 ng/mL of MMP-9, when MMP-9 was incubated with the peptide of identical concentration for 120 min. After the investigation of cleavage condition by MMP-9, the peptide was used to screen activities of MMP-9 inhibitors. 1.5–12.0 nM of NNGH, 3.75–30.0 nM of batimastat, and 1–8 nM of marimastat and SB-3CT were consumed to test their inhibition efficacies. The proteolytic activity of MMP-9 in the presence of the inhibitor was measured at Ex/Em = 485 nm/525 nm. IC50 values of the MMP-9 inhibitors were estimated quantitatively by monitoring their fluorescence intensity versus inhibitor concen-
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
G Model SNB-23355; No. of Pages 7
ARTICLE IN PRESS J. Lee et al. / Sensors and Actuators B xxx (2017) xxx–xxx
5
Fig. 3. (a) Cleavage of the peptide by MMP-9 was measured as a function of enzyme dose and incubation time point. (b–e) MMP-9 inhibition activity determined as a function of dose of different inhibitors. The x-axis indicates the concentration of the inhibitor, and the y-axis shows the fluorescence intensity acquired as an effect of peptide hydrolysis. The relative fluorescence was monitored at Ex/Em = 485 nm/525 nm wavelength using a multi-plate reader.
tration. As shown in Fig. 3b–e, the x-axis denotes the concentration of the inhibitor, and the y-axis shows the fluorescence intensity acquired as an effect of enzymatic peptide hydrolysis. The obtained IC50 values of NNGH, batimastat, marimastat and SB-3CT were 9.2 nM, 25.7 nM, 6.3 nM and 7.2 nM, respectively. Marimastat represented the lowest IC50 value compared to other inhibitors. The inkjet printing MMP-9 inhibition assay was performed based on reaction conditions obtained in the well plate. 3.4. Measurement of MMP-9 activity using inkjet printing The proteolytic activity of MMP-9 with respect to the peptide on parchment paper was determined using inkjet printing. The peptide of 47.5 × 10−18 mol was printed on the same spot along with MMP-9 and incubated for 7 min. The fluorescence of the peptide cleaved by MMP-9 was monitored every 1 min as a function of dose of activated MMP-9 (5 ng/mL, 20 ng/mL, and 30 ng/mL). The concentration of activated MMP-9 was tested up to 30 ng/mL. This concentration has been generally used when the enzymatic reaction of MMP-9 with peptide substrate has been executed in the conventional well plate. IC50 values of MMP-9 inhibitors have been generally acquired in the concentration range of MMP-9 up to 30 ng/mL. Accordingly, it is critical to check whether the fluorescence intensity arising from the fragmented peptide on the parchment paper is produced sufficiently to determine drug inhibitory concentration at MMP-9 concentration of 30 ng/mL. As shown in Fig. 4, the fluorescence intensity of the fragmented peptide increased as a function of MMP-9 concentration and reached nearly 2000 at MMP-9 concentration of 30 ng/mL with the incubation time of 7 min. This intensity was found to be adequate to measure MMP-9 inhibitory concentration. Table 1 represents densities, ejection volumes, and ejected values of MMP-9 ink solutions obtained through inkjet printing on parchment paper. At 30 ng/mL,
Fig. 4. Inkjet printing-based MMP-9 assay. The cleavage of the peptide by MMP-9 on parchment paper was measured as a function of time and in a dose-dependent manner. The x-axis indicates the incubation time point, and the y-axis shows the fluorescence intensity acquired as an effect of peptide hydrolysis by MMP-9. Fluorescence intensity was measured at Ex/Em = 485 nm/525 nm using a fluorescence microscope.
the amount of MMP-9 spotted on the parchment paper was determined to be 14.1 fg, which is approximately 105 times smaller than the 3 ng consumed in a conventional well plate using a pipette. 3.5. MMP-9 FRET inhibition assay for the determination of IM50 using inkjet printing Fig. 5 represents MMP-9 inhibitory activities of: (a) NNGH; (b) batimastat; (c) marimastat; and (d) SB-3CT using inkjet printing.
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
G Model
ARTICLE IN PRESS
SNB-23355; No. of Pages 7
J. Lee et al. / Sensors and Actuators B xxx (2017) xxx–xxx
6
Table 1 Densities of different concentrations of MMP-9 ink solution and their respective volumes. Concentration of activated MMP-9 stock solution
MMP-9 (30 ng/mL)
MMP-9 (10 ng/mL)
MMP-9 (5 ng/mL)
Density (g/mL) Ejected volume (nL) Ejected amount of MMP-9 (fg)
1.0163 46.8 14.10
1.0141 46.9 4.69
1.0118 48.0 2.40
Fig. 5. MMP-9 inhibitory activities of NNGH, batimastat, marimastat, and SB-3CT using inkjet-printing. a-1, schematic diagram represents the printing order of ink solutions to perform the MMP-9 inhibition assay. a-2, the number of mole of inhibitor printed on reaction spots according to the K values and their respective images of fluorescence produced after cleavage of the peptide by MMP-9. b) NNGH; c) batimastat; d) marimastat; and e) SB-3CT, represents the quantitative plot of the reduction of fluorescence intensity formed on the reaction spot. Fluorescence intensity was measured at Ex/Em = 485 nm/525 nm using a fluorescence microscope, and the images were analyzed using MetaMorph software.
Table 2 Solution densities, ejection volumes, and IM50 values of MMP-9 inhibitors obtained through inkjet printing. Inhibitors
NNGH (0.1 M)
Batimastat (0.25 M)
Inhibitors
Marimastat (0.1 M)
SB-3CT (0.1 M)
Density (g/mL) Ejected volume (nL) IM50 (femto mole) R2
1.0208 43.2 17.3 0.976
1.0152 46.9 52.2 0.989
Density (g/mL) Ejected volume (nL) IM50 (femto mole) R2
1.0158 43 15.6 0.984
1.0174 46.3 17.9 0.981
Table 2 represents densities, ejection volumes, and IM50 values of NNGH, batimastat, marimastat, and SB-3CT ink obtained through the inkjet printing on the parchment paper. Fig. 5a-1 illustrates the printing order that was executed on sample arrays of 3 rows × 8 columns. Fig. 5a-2 denotes the number of mole of inhibitor printed on reaction spots according to the K values and their respective images of fluorescence arising from the cleavage of the peptide by MMP-9. At each spot, each inhibitor with different number of mole was reacted with peptide substrate of 47.5 × 10−18 mol and MMP-9 of 14.1 fg. These concentrations were found to provide the fluorescence intensity of the fragmented peptide suitable for the determination of IM50 (the number of mole needed to reduce the fluorescence intensity of the control by 50%) in the above result. When the reaction spots were excited at 490 ± 20 nm, the control without inhibitor (K value = 0) showed the brightest fluorescence intensity of nearly 2000, while the brightness of fluorescence was gradually reduced on reaction spots in the presence of the inhibitor. This tendency was observed identically in all of the tested MMP-9 inhibitors. The reduction of fluorescence brightness was quanti-
tatively plotted in Fig. 5b–e. These plots of fluorescence intensity versus the number of mole printed enabled the determination of IM50 . Table 2 represents the IM50 values of NNGH, batimastat, marimastat, and SB-3CT as 17.3 × 10−15 mol, 52.2 × 10−15 mol, 15.6 × 10−15 mol and 17.9 × 10−15 mol, respectively. The MMP-9 inhibitory activities of marimastat, NNGH, and SB-3CT were significantly larger than batimastat. The MMP-9 inhibitory activity of marimastat was the largest. This tendency was well matched with the result of the MMP-9 inhibition assay in 96-well plates. Compared to a conventional microplate, fragmentation and resultant fluorescence detection of the peptide printed on reaction spots of parchment paper require greater attention, which utilized 102 to 106 smaller amount (Table 3). This is because detection of the fragmented peptide is influenced more sensitively on the surface of parchment paper by various conditions including printing, such as enzyme activity, peptide density, and the reaction time of peptide substrate. This signifies that drug inhibitory concentration can be
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051
G Model
ARTICLE IN PRESS
SNB-23355; No. of Pages 7
J. Lee et al. / Sensors and Actuators B xxx (2017) xxx–xxx Table 3 IM50 values of MMP-9 inhibitors obtained through 96 well plate and inkjet printing methods. Method
Inhibitor NNGH Batimastat Marimastat SB-3CT
96 well plate
IC50( nM) 9.2 IM50 (×10−12 mol) 1.8 −14 Inkjet printing IM50 (×10 mol) 1.7
25.7 5.1 5.2
6.3 1.3 1.6
7.2 1.4 1.8
determined on a tremendously small scale if the assay is performed using inkjet printing instead of well plate. 4. Conclusion In this study, an inkjet printing MMP-9 anticancer assay using fluorogenic peptide was performed on parchment paper based on FRET detection. The cleavage of 5-FAM/QXL520 peptide by MMP-9 enzyme resulted in the disappearance of FRET arising from the separation of chromophores, and increased the fluorescence intensity of 5-FAM significantly. Our work revealed that the peptide substrate, MMP-9 enzyme, and their respective inhibitors could be deposited on a non-fabricated paper surface with 102 to 106 smaller amount compared to pipette-based assay in a well plate, making it possible to validate fluorescence-based high throughput drug screening in an automated and reproducible manner. By the virtues of the versatile printing setup, the fluorescent peptide was used to successfully determine IM50 values of MMP-9 inhibitors, NNGH, batimastat, SB-3CT, and marimastat. This approach improves and extends universal applications of inkjet printing for enzymatic anticancer assays. Acknowledgments This work was supported by National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) (2015R1A2A1A05001842 and 2016R1A4A1010796). The authors declare no competing financial interests. We are grateful to Brain Korea 21 plus (BK 21 plus) and College of Pharmaceutical Sciences at Seoul National University for providing experimental equipment. References [1] G.A. Skarja, A.L. Brown, R.K. Ho, M.H. May, M.V. Sefton, The effect of a hydroxamic acid-containing polymer on active matrix metalloproteinases, Biomaterials 30 (2009) 1890–1897. [2] R. Salsas-Escat, P.S. Nerenberg, C.M. Stultz, Cleavage site specificity and conformational selection in type I collagen degradation, Biochemistry-US 49 (2010) 4147–4158. [3] J.A. Jacobsen, J.L. Fullagar, M.T. Miller, S.M. Cohen, Identifying chelators for metalloprotein inhibitors using a fragment-Based approach, J. Med. Chem. 54 (2011) 591–602. [4] M.M. Benjamin, R.A. Khalil, Matrix metalloproteinase inhibitors as investigative tools in the pathogenesis and management of vascular disease, EXS 103 (2012) 209–279. [5] H. Jarvelainen, A. Sainio, M. Koulu, T.N. Wight, R. Penttinen, Extracellular matrix molecules: potential targets in pharmacotherapy, Pharmacol. Rev. 61 (2009) 198–223. [6] C. Bonnans, J. Chou, Z. Werb, Remodelling the extracellular matrix in development and disease, Nat. Rev. Mol. Cell Biol. 15 (2014) 786–801. [7] K. Kessenbrock, V. Plaks, Z. Werb, Matrix metalloproteinases: regulators of the tumor microenvironment, Cell 141 (2010) 52–67. [8] A. Noel, A. Gutierrez-Fernandez, N.E. Sounni, N. Behrendt, E. Maquoi, I.K. Lund, et al., New and paradoxical roles of matrix metalloproteinases in the tumor microenvironment, Front. Pharmacol. 3 (2012) 140. [9] X. Zhang, J. Bresee, P.P. Cheney, B. Xu, M. Bhowmick, M. Cudic, et al., Evaluation of a triple-helical peptide with quenched FluorSophores for optical imaging of MMP-2 and MMP-9 proteolytic activity, Molecules 19 (2014) 8571–8588. [10] M. Stawarski, I. Rutkowska-Wlodarczyk, A. Zeug, M. Bijata, H. Madej, L. Kaczmarek, et al., Genetically encoded FRET-based biosensor for imaging MMP-9 activity, Biomaterials 35 (2014) 1402–1410.
7
[11] J.O. McIntyre, B. Fingleton, K.S. Wells, D.W. Piston, C.C. Lynch, S. Gautam, et al., Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity, Biochem. J. 377 (2004) 617–628. [12] K. Leung, Cy5.5-Aminohexanoic Acid-RPLALWRS-Aminohexanoic Acid-C-G4-PAMAM-PEG-AF750, Molecular Imaging and Contrast Agent Database (MICAD), Bethesda (MD), 2004. [13] J.R. Tauro, B.S. Lee, S.S. Lateef, R.A. Gemeinhart, Matrix metalloprotease selective peptide substrates cleavage within hydrogel matrices for cancer chemotherapy activation, Peptides 29 (2008) 1965–1973. [14] A. Dufour, N.S. Sampson, J. Li, C. Kuscu, R.C. Rizzo, J.L. Deleon, et al., Small-molecule anticancer compounds selectively target the hemopexin domain of matrix metalloproteinase-9, Cancer Res. 71 (2011) 4977–4988. [15] M.W. Ndinguri, M. Bhowmick, D. Tokmina-Roszyk, T.K. Robichaud, G.B. Fields, Peptide-based selective inhibitors of matrix metalloproteinase-mediated activities, Molecules 17 (2012) 14230–14248. [16] G.B. Fields, Using fluorogenic peptide substrates to assay matrix metalloproteinases, Methods Mol. Biol. 151 (2001) 495–518. [17] J. Lee, A.A. Samson, J.M. Song, Inkjet-printing enzyme inhibitory assay based on determination of ejection volume, Anal. Chem. 89 (2017) 2009–2016. [18] L. Wu, Z.C. Dong, F.Y. Li, H.H. Zhou, Y.L. Song, Emerging progress of inkjet technology in printing optical materials, Adv. Opt. Mater. 4 (2016) 1915–1932. [19] Y.F. Zhang, F.J. Lyu, J. Ge, Z. Liu, Ink-jet printing an optimal multi-enzyme system, Chem. Commun. 50 (2014) 12919–12922. [20] B. Creran, X.N. Li, B. Duncan, C.S. Kim, D.F. Moyano, V.M. Rotello, Detection of bacteria using inkjet-Printed enzymatic test strips, ACS Appl. Mater. Interfaces 6 (2014) 19525–19530. [21] E. Bou Chakra, B. Hannes, J. Vieillard, C.D. Mansfield, R. Mazurczyk, A. Bouchard, et al., Grafting of antibodies inside integrated microfluidic-microoptic devices by means of automated microcontact printing, Sens. Actuators B Chem. 140 (2009) 278–286. [22] H. Yang, T. Rahman, D. Du, R. Panat, Y. Lin, 3-D printed adjustable microelectrode arrays for electrochemical sensing and biosensing, Sens. Actuators B Chem. 230 (2016) 505–600.
Biographies
Jungmi Lee received her Bachelor’s degree in Chemistry Education in 2011 and Master’s degree in Chemistry in 2016 at Kyungpook National University, in Korea. Currently, she is PhD student at College of Pharmacy, Seoul National University in South Korea. Her research interest is organic synthesis, chiral separation, drug screening and diagnosis in molecule level. She has published 3 peer reviewed paper.
Annie Agnes Suganya Samson received her Master of Science degree in Biotechnology from Bharathidasan University in 2013. She was working as a Junior Research Fellow from 2013 to 2015 at Rajiv Gandhi Center for Biotechnology. Currently, she is a PhD student at College of Pharmacy, Seoul National University in South Korea. Her research interest includes cancer biology, stem cells, chemo and immunotherapy and proteomic studies. She has published 5 peer reviewed paper and 1 book chapter.
Joon Myong Song received his PhD in 1997 at Kyushu University, in Japan. He worked as a postdoctoral research fellow from 1998 to 2004 at Iowa State University, Brookhaven National Laboratory, and Oak Ridge National Laboratory in United States. At present he is a professor at College of Pharmacy, Seoul National University in South Korea. His research area includes multifunctional nanoparticle for diagnosis and therapy and high-content cell-based drug screening and diagnosis using hypermulticolor cellular imaging. He has published 104 peer reviewed papers in the top journals, 10 book chapters, and 10 patents.
Please cite this article in press as: J. Lee, et al., Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET), Sens. Actuators B: Chem. (2017), https://doi.org/10.1016/j.snb.2017.10.051