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

Abstract

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.

Keywords: PLGA-PEI nanoparticles; active matrix metalloproteinase-2; imaging sensor; metastasis.

Figure 1

Schematic illustration of the matrix metalloproteinase-2 (MMP-2)-activated peptide sensor and MMP-2-activated poly(lactic-co-glycolic acid) with polyethylenimine (MMP-2-PLGA-PEI) nanoparticles. (A) Chemical structure of the MMP-2-activated peptide sensor. The sensor consisted of a near-infrared fluorescence dye (Cy5), MMP-2 substrate peptide, and a dark quencher (BHQ-3); (B) Schematic diagram of the MMP-2-PLGA-PEI nanoparticles. The fluorescence signal was recovered only in the presence of MMP-2-positive cancer cells.

Figure 2

In vitro characterization of the MMP-2-activated peptide sensor and MMP-2-PLGA-PEI nanoparticles. (A) Analysis of MMP-2-activated peptide sensor fluorescence recovery by SDS-PAGE (upper panel; inset box: M-marker, B-TCNB buffer, E-Enzyme) and HPLC analysis results with MMP-2 enzyme (lower panel) and without MMP-2 enzyme (upper panel) indicated by the red dotted box. The MMP-2-activated peptide sensor was incubated with active recombinant MMP-2 for 30 min; (B) Signal recovery after incubation of the MMP-2-activated peptide sensor in the presence of various recombinant MMPs (MMP-1, -2, -3, -7, -9, -13) for 80 min at 37 °C; (C) MMP-2 fluorescence signal recovery from the MMP-2-activated peptide sensor, MMP-2-PLGA-PEI nanoparticles, and inhibitor-treated MMP-2-activated peptide sensor during incubation with recombinant MMP-2 for 80 min at 37 °C; (D) Analysis of MMP-2-PLGA-PEI fluorescence recovery after incubation with cell culture media; (E) MMP-2-PLGA-PEI fluorescence recovery of the cell lysate from MCF-7 and HaCat cells. Cell culture media and cell lysates were collected after culturing in fresh media for 3 h and 24 h. MMP-2 fluorescence signal recovery was confirmed at 0, 30, and 60 min. (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.005).

Figure 3

Characterization of MMP-2-PLGA-PEI nanoparticles. (A) Size distribution in aqueous solutions; (B) Stability tests at 37 °C in water and physiological solution (PBS, pH 7.4); (C) Transmission emission microscopy (TEM) images of the nanoparticles; (D) Scanning electron microscopy (SEM) images of the nanoparticles.

Figure 4

Confirmation of MMP-2 expression, cellular cytotoxicity, and MMP-2-PLGA-PEI nanoparticle uptake. (A) MMP-2 mRNA level in MCF-7 and HaCat cells was analyzed by RT-PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control; (B) In vitro cell cytotoxicity analysis for MCF-7 (cyan) and HaCat (pink) cells after a 30 min incubation with PLGA-PEI nanoparticles; (C) Confocal microscopy analysis of cellular uptake of Rhodamine B lipid PLGA-PEI nanoparticles. Results are presented as mean ± SD (n = 3) (Scale bar: 10 µm).

Figure 5

MMP-2 fluorescence signal recovery according to cell line and culture time. (A) Activity of MMP-2 in MCF-7 and HaCat cells by confocal imaging using MMP-2-PLGA-PEI nanoparticles. After culturing in fresh media for 24 h, the cells were incubated with MMP-3-PLGA-PEI nanoparticles for 30 min. Mean signal intensities were calculated by the imaging software (n = 3); (B) Cell culture time–dependent activity of MMP-2 in HaCat cells. Cells were precultured in serum-free media for 3 h and 24 h, respectively, and then incubated with MMP-2-PLGA-PEI nanoparticles for 30 min. Mean signal intensities were calculated by the imaging software (n = 3). (** indicates p < 0.01.) (Scale bar: 10 µm).