Visualization of MMP-2 Activity Using Dual-Probe Nanoparticles to Detect Potential Metastatic Cancer Cells

Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes capable of degrading extracellular matrix components. Previous studies have shown that the upregulation of MMP-2 is closely related to metastatic cancers. While Western blotting, zymography, and Enzyme-Linked Immunosorbent Assays (ELISA) can be used to measure the amount of MMP-2 activity, it is not possible to visualize the dynamic MMP-2 activities of cancer cells using these techniques. In this study, MMP-2-activated poly(lactic-co-glycolic acid) with polyethylenimine (MMP-2-PLGA-PEI) nanoparticles were developed to visualize time-dependent MMP-2 activities. The MMP-2-PLGA-PEI nanoparticles contain MMP-2-activated probes that were detectable via fluorescence microscopy only in the presence of MMP-2 activity, while the Rhodamine-based probes in the nanoparticles were used to continuously visualize the location of the nanoparticles. This approach allowed us to visualize MMP-2 activities in cancer cells and their microenvironment. Our results showed that the MMP-2-PLGA-PEI nanoparticles were able to distinguish between MMP-2-positive (HaCat) and MMP-2-negative (MCF-7) cells. While the MMP-2-PLGA-PEI nanoparticles gave fluorescent signals recovered by active recombinant MMP-2, there was no signal recovery in the presence of an MMP-2 inhibitor. In conclusion, MMP-2-PLGA-PEI nanoparticles are an effective tool to visualize dynamic MMP-2 activities of potential metastatic cancer cells.


Introduction
Cancer is one of the major diseases affecting humans throughout the world. Over the past decades, many research groups have studied cancer cell mutations related to proliferation, survival, and metastasis. Recently, it has been recognized that the tumor microenvironment (TME), in addition to fibroblasts and the extracellular matrix (ECM), is an important factor influencing tumor progression [1]. It has been shown that constant interactions between the tumor and TME influence the growth and metastasis of cancer [2].
Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that are capable of degrading ECM components. They are present as pro-MMP forms in healthy individuals and play near-infrared (NIR) fluorescence dye was procured from Lumiprobe (Hannover, Germany), and Black Hole Quencher-3 (BHQ-3) was purchased from Bioresearch Technologies (Petaluma, CA, USA). Matrix metalloproteinase-2 peptide substrate was a customized product from Peptron (Daejeon, Korea).

Preparation of PLGA-PEI Nanoparticles
PLGA-PEI nanoparticles were synthesized via a nanoprecipitation method [19]. Briefly, PLGA (6.5 mg) was dissolved in 650 µL of acetonitrile. RhoB lipid (6.5 µL) was added and thoroughly mixed by repeated pipetting. PEI (6.3 mg) was dissolved in 65 µL of deionized water and gently vortexed. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 65 mg) and N-hydroxysuccinimide (NHS, 65 mg) were dissolved in 1.3 mL of phosphate-buffered saline (PBS, pH 7.4). The EDC/NHS solution was added to PEI in a dropwise manner, followed by the addition of deionized water to achieve a volume of 6.5 mL. The reaction was performed for 20 min at room temperature. After 20 min, PLGA containing RhoB lipid was added to the PEI solution in a dropwise manner and the reaction mixture was stirred overnight at 400 rpm at room temperature under dark conditions. To remove the unconjugated ingredients, the solution was dialyzed against deionized water for a day (molecular weight cut-off, (Molecular weight cut-off) MWCO = 10 kDa).

Conjugation of MMP-2-Activated Peptide Sensor with PLGA-PEI Nanoparticles
To conjugate the MMP-2-activated peptide sensor with the PLGA-PEI nanoparticles to produce MMP-2-PLGA-PEI nanoparticles, 3.5 mg of the MMP-2-activated peptide sensor was dissolved in 200 µL of dimethyl sulfoxide (DMSO)/PBS (1:1, v/v). Then, EDC (32.5 mg) and NHS (32.5 mg) were dissolved in 100 µL of PBS and added to the solution of the MMP-2-activated peptide sensor in a dropwise manner. After reacting for 20 min at room temperature, the sensor mixture was added to the PLGA-PEI nanoparticle solution and incubated for 12 h at room temperature under dark conditions. Dialysis against deionized water was performed for a day (MWCO = 10 kDa). The final product was centrifuged (13,500 rpm, 15 min), and the MMP-2-PLGA-PEI nanoparticles in the pellet were lyophilized.

Size, Stability, and Morphology Characterization of MMP-2-PLGA-PEI Nanoparticles
The size and zeta potential of the MMP-2-PLGA-PEI nanoparticles were confirmed by a Zetasizer Nano (Malvern Instruments, Worcestershire, UK). For size measurements, the nanoparticles were resuspended in water or PBS. The stability of each group was confirmed under the same conditions at 37 • C for up to 7 days. For scanning electron microscopy (SEM) images, a drop of the samples was spotted on a silicon template and dried at room temperature. The dried samples were sputtered with platinum to increase the contrast and signal-to-noise ratio. For transmission electron microscopy (TEM) images, a drop of the sample was spotted on a grid and stained with 0.01% tungsten solution for 5 min. The sample was dried in a desiccator.

NIR Fluorescence Signal Recovery of MMP-2-PLGA-PEI Nanoparticles by Active Recombinant Enzyme
To confirm the NIR fluorescence signal recovery, 10 nM of the MMP-2-activated peptide sensor or MMP-2-PLGA-PEI nanoparticles were incubated with active recombinant MMP-2 (50 nM) with or without the inhibitor (10 nM). The fluorescence signal was detected every 10 min at 37 • C. TCNB buffer was used as a control.

NIR Fluorescence Signal Recovery of MMP-2-PLGA-PEI Nanoparticles by Culture Media and Cell Lysate
Cell culture media and cell lysates from HaCat (MMP-2-positive) and MCF-7 (MMP-2-negative) cells were used to confirm the fluorescence recovery of the MMP-2-PLGA-PEI nanoparticles. The cells were washed with PBS and then lysed at 4 • C with a lysis buffer containing 1% Triton X-100, protease inhibitor cocktail, phosphatase inhibitor cocktail, and PBS. The lysates were centrifuged and the collected supernatants were used as the cell lysate. MMP-2-PLGA-PEI nanoparticles (10 nM) were incubated with 400 µL of either the culture media or cell lysate, and the fluorescence signals were detected every 10 min at 37 • C. Fresh media and lysis buffer were used as controls.

In Vitro Cytotoxicity of MMP-2-PLGA-PEI Nanoparticles
MCF-7 and HaCat cells were seeded into 6-well plates at 2 × 10 5 cells/well in complete media and incubated for 24 h. The cell media was changed to serum-free media and the cells were incubated for a further 24 h. Subsequently, the cells were treated with five different concentrations (0 to 160 µg/mL) of nanoparticles for 30 min and then detached using 0.25% trypsin. Next, they were washed and stained with trypan blue. Nonstained viable cells were counted using a hemocytometer.

Confocal Microscopy
Cells (4 × 10 4 /well) were seeded in 24-well plates in complete media and cultured for 24 h, followed by a further incubation in serum-free media. After incubation for 3 h and 24 h, MMP-2-PLGA-PEI nanoparticles (80 µg/mL) were added to the plates for 30 min. The cells were washed twice with PBS, fixed in 4% paraformaldehyde for 15 min at room temperature, and washed again twice with PBS. After addition of the mounting medium containing 4',6-diamidino-2-phenylindole (DAPI), the cells were imaged using a Zeiss LSM 710 Confocal laser scanning microscope (Carl Zeiss).

Statistical Analysis
Data are presented as mean ± SD. Statistical differences were determined by two-way Analysis of Variance, ANOVA (n = 3).

Statistical analysis
Data are presented as mean ± SD. Statistical differences were determined by two-way Analysis of Variance, ANOVA (n = 3).

Characterization of the MMP-2-PLGA-PEI Nanoparticles
The MMP-2-PLGA-PEI nanoparticles were composed of PLGA-PEI, which entrapped the RhoB lipid. The MMP-2-activated peptide sensor was conjugated onto the surface of the PLGA-PEI nanoparticles. The nanoparticles successfully recovered fluorescence signals from MMP-2-positive cells ( Figure 1B).
Time-dependent recovery of fluorescent signals of the MMP-2-activated peptide sensor and MMP-2-PLGA-PEI nanoparticles was confirmed by incubation with active recombinant MMP-2 with or without the MMP-2 inhibitor. The NIR fluorescence signals demonstrated that the MMP-2-activated peptide sensor and MMP-2-PLGA-PEI nanoparticles recovered the fluorescence signal with 19.1-and 15.6-fold enhancement, respectively. However, there was no recovery in the presence of an MMP-2 inhibitor ( Figure 2C). To confirm the fluorescence signal recovery of MMP-2-PLGA-PEI nanoparticles at a cellular level, the sensors were incubated with cell culture media and cell lysates. Cell culture media from both HaCat and MCF-7 cells were collected after 3 h and 24 h incubation. The culture media was incubated again with the MMP-2-PLGA-PEI nanoparticles for 0, 30, and 60 min, and the recovered fluorescence intensities were measured. When the results of 0 min and 60 min incubation experiments were compared, it was found that the culture media from HaCat cells showed 1.4-and 3.18-fold increased MMP-2 fluorescence intensity at 3 h and 24 h, respectively. The culture media from MCF-7 cells showed 1.02-and 1.22-fold increased MMP-2 fluorescence intensity at 3 h and 24 h, respectively ( Figure 2D). This result clearly demonstrated that the MMP-2-PLGA-PEI nanoparticles could detect the active MMP-2 enzymes secreted from these cells, differentiating HaCat from MCF-7. HaCat and MCF-7 cell lysates were prepared using the lysis buffer described above. It was evident from a comparison of the results of the 0 min and 60 min incubation experiments that the HaCat and MCF-7 cell lysates showed 1.17-fold and 1.14-fold increased MMP-2 fluorescence intensities, respectively. This result suggested that there was a substantially smaller difference in the MMP-2 signals between these cells. This was because most active MMP-2 enzymes are secreted outside of the cells and do not stay inside the cells [13,20]. Interestingly, the MMP-2-PLGA-PEI nanoparticles detected higher MMP-2 enzyme activities inside the HaCat cells (p < 0.01) than the MMP-2 activity inside the MCF-7 cells (p < 0.05) ( Figure 2E).
The expression of MMP-2 is closely related to metastasis and prognosis [21,22]. In tumor angiogenesis, MMP-2 and MMP-9 have been shown to promote the "angiogenic switch" [23]. In general, MMP-9 is known to induce the release of vascular endothelial growth factor (VEGF) in the ECM. MMP-2 activity is necessary to activate pro-MMP-9 [24]. Therefore, the measurement of active MMP-2 could be a promising approach to early diagnosis of cancer metastasis. MMP-2 is secreted into the ECM as a proenzyme and gets activated. For this reason, active MMP-2 expression was low in the cell lysates of both cell lines ( Figure 2E). In contrast, a clear increase in MMP-2 activity was observed for HaCat cell culture media. This is especially noteworthy considering that the concentration of active MMP-2 would be very low given the volume of culture media used (10 mL in a 100 mm dish) ( Figure 2D). These results suggest that the MMP-2-PLGA-PEI nanoparticles can be a promising tool for an accurate and sensitive detection of active MMP-2 at a low concentration.
The MMP-2-PLGA-PEI nanoparticles showed a mono-dispersed size in the aqueous solution, as confirmed by dynamic light scattering (DLS). The MMP-2-PLGA-PEI nanoparticles presented a size of 204.2 ± 69.4 nm with zeta potential of 60.1 ± 7.73 mV ( Figure 3A). To determine their stability, the MMP-2-PLGA-PEI nanoparticles were re-suspended in water or PBS. Both samples were incubated at 37 • C, and the hydrodynamic diameter of the nanoparticles was analyzed after 1, 2, 4, 8, 12, and 24 h by DLS ( Figure 3B). The morphology of the MMP-2-PLGA-PEI nanoparticles was confirmed by TEM and SEM imaging. The TEM images showed the presence of PEI on the surface of the nanoparticles ( Figure 3C). The SEM images confirmed the spherical shape of the nanoparticles in aqueous solutions. Nanomaterials 2018, 8, 119 8 of 12

Confirmation of MMP-2 Gene Expression, Cellular Cytotoxicity, and Cell Internalization
Based on previous research, MCF-7 and HaCat cells were selected as candidate MMP-2-negative and -positive cells, respectively [25,26]. A comparison of the MMP-2 mRNA expression of the two cell lines showed that the HaCat cells strongly expressed MMP-2 whereas the MCF-7 cells did not ( Figure 4A). Next, the cytotoxicity of MMP-2-PLGA-PEI nanoparticles on MCF-7 and HaCat cells was investigated. Each cell line was treated with a different concentration of MMP-2-PLGA-PEI nanoparticles (0 to 160 μg/mL), and cell survival was assessed after incubation for 30 min. It was found that the MMP-2-PLGA-PEI nanoparticles were not toxic to MCF-7 and HaCat cells even at a concentration of 160 μg/mL, as shown in Figure 4B. Confocal microscopy using RhoB confirmed that there was no significant difference in the internalization of MMP-2-PLGA-PEI nanoparticles between 3 and 24 h incubation ( Figure 4C). In this study, biodegradable and biocompatible PLGA nanoparticles were modified by a cationic agent (PEI) to enhance cell permeability [27]. As PEI has a strong positive charge in aqueous solutions, the MMP-2-PLGA-PEI nanoparticles were easily attached to the cell surface and induced cell internalization within 30 min with no significant cytotoxicity ( Figures 4B and 4C). These results suggest that the MMP-2-PLGA-PEI nanoparticles can be efficiently applied not only for sensing MMP-2, but also for the delivery of therapeutic agents such as small interfering MMP-2 genes.

Confirmation of MMP-2 Gene Expression, Cellular Cytotoxicity, and Cell Internalization
Based on previous research, MCF-7 and HaCat cells were selected as candidate MMP-2-negative and -positive cells, respectively [25,26]. A comparison of the MMP-2 mRNA expression of the two cell lines showed that the HaCat cells strongly expressed MMP-2 whereas the MCF-7 cells did not ( Figure 4A). Next, the cytotoxicity of MMP-2-PLGA-PEI nanoparticles on MCF-7 and HaCat cells was investigated. Each cell line was treated with a different concentration of MMP-2-PLGA-PEI nanoparticles (0 to 160 µg/mL), and cell survival was assessed after incubation for 30 min. It was found that the MMP-2-PLGA-PEI nanoparticles were not toxic to MCF-7 and HaCat cells even at a concentration of 160 µg/mL, as shown in Figure 4B. Confocal microscopy using RhoB confirmed that there was no significant difference in the internalization of MMP-2-PLGA-PEI nanoparticles between 3 and 24 h incubation ( Figure 4C). In this study, biodegradable and biocompatible PLGA nanoparticles were modified by a cationic agent (PEI) to enhance cell permeability [27]. As PEI has a strong positive charge in aqueous solutions, the MMP-2-PLGA-PEI nanoparticles were easily attached to the cell surface and induced cell internalization within 30 min with no significant cytotoxicity ( Figure 4B,C). These results suggest that the MMP-2-PLGA-PEI nanoparticles can be efficiently applied not only for sensing MMP-2, but also for the delivery of therapeutic agents such as small interfering MMP-2 genes.

Evaluation of Cellular Active MMP-2 Using MMP-2-PLGA-PEI Nanoparticles
To confirm the MMP-2-specific recovery of MMP-2-PLGA-PEI nanoparticles, HaCat and MCF-7 cells were incubated with the MMP-2-PLGA-PEI nanoparticles. The representative confocal images show that active MMP-2 was highly expressed in HaCat cells, which is an MMP-2-positive cell line, whereas active MMP-2 was barely detectable in MCF-7 cells ( Figure 5A). Furthermore, the active MMP-2 signals became stronger with the increasing MMP-2-PLGA-PEI incubation time. To confirm the time-dependent active MMP-2 expression, HaCat cells were pre-incubated for 3 and 24 h. The level of active MMP-2 was approximately twice as high in 24 h precultured HaCat cells compared with 3 h precultured HaCat cells ( Figure 5B). This result clearly showed the ability of the nanoparticles to differentiate the MMP-2-positive cells from the MMP-2-negative cells. Moreover, the comparison with RhoB signals allowed us to visualize dynamic MMP-2 activities from cancer cells and their surroundings. The activity of MMP-2 and the location of the nanoparticles were confirmed at the same time using dual image probes (Supplementary Figure S2). For future research based on the results of this study, the activity of MMP-2 using these nanoparticles might be visualized by in vivo experiments, which will be helpful for an early diagnosis of cancer and improvement of the molecular diagnostic technology to distinguish between malignant and nonaggressive or benign tumors.

Evaluation of Cellular Active MMP-2 Using MMP-2-PLGA-PEI Nanoparticles
To confirm the MMP-2-specific recovery of MMP-2-PLGA-PEI nanoparticles, HaCat and MCF-7 cells were incubated with the MMP-2-PLGA-PEI nanoparticles. The representative confocal images show that active MMP-2 was highly expressed in HaCat cells, which is an MMP-2-positive cell line, whereas active MMP-2 was barely detectable in MCF-7 cells ( Figure 5A). Furthermore, the active MMP-2 signals became stronger with the increasing MMP-2-PLGA-PEI incubation time. To confirm the time-dependent active MMP-2 expression, HaCat cells were pre-incubated for 3 and 24 h. The level of active MMP-2 was approximately twice as high in 24 h precultured HaCat cells compared with 3 h precultured HaCat cells ( Figure 5B). Thi result clearly showed the ability of the nanoparticles to differentiate the MMP-2-positive cells from the MMP-2-negative cells. Moreover, the comparison with RhoB signals allowed us to visualize dynamic MMP-2 activities from cancer cells and their surroundings. The activity of MMP-2 and the location of the nanoparticles were confirmed at the same time using dual image probes (Supplementary Figure S2). For future research based on the results of this study, the activity of MMP-2 using these nanoparticles might be visualized by in vivo experiments, which will be helpful for an early diagnosis of cancer and improvement of the molecular diagnostic technology to distinguish between malignant and nonaggressive or benign tumors.

Conclusions
This research demonstrated that MMP-2-PLGA-PEI nanoparticles were able to accurately detect MMP-2 activity of cancer cells. The use of an MMP-2-activated peptide sensor and RhoB lipid incorporated into PLGA-PEI nanoparticles allowed the detection of the active MMP-2 expression and the monitoring of the exact location of the nanoparticles. In our future research, we aim to focus on multisensor conjugated nanoparticles, including therapeutic agents.