In the present study, we confirmed that MMP-14, a cell membrane-anchoring enzyme, could serve as a biomarker of vulnerable plaques. The data also demonstrated that the MMP-14 substrate-based fluorescent nanoprobe [email protected], which showed an intense fluorescence signal after activation and good biocompatibility, could be applied to screen for and monitor plaque progression in vivo.
Satisfactory imaging techniques are urgently needed to detect vulnerable plaques, which are mediated by inflammatory activities and cause lethal cerebrovascular events [30, 31]. In recent years, molecular target-based imaging of the degree of plaque inflammation has been considered a promising approach for identifying rupture-prone plaques [32, 33]. Many molecular biomarkers, such as MMPs , hypoxia-related factors, and vascular cell adhesion molecules , have been proposed. Of these, MMPs are particularly attractive, as they are proteinases that directly degrade the matrix and erode the fibrous cap and are involved in a series of other regulatory mechanisms that promote plaque instability. Seifert et al.  implanted a tapered cuff around the right common carotid artery in Apoe−/− mice to induce a model of atherosclerosis and found that the MMP-2 and MMP-9 activities were significantly higher in upstream, low shear stress-induced unstable atherosclerotic plaques than in downstream, more stable plaque phenotypes. However, many fluorogenic probes targeting MMP-2 and MMP-9 have been demonstrated to have limitations in in vivo applications. One important reason is that both MMP-2 and MMP-9 are extracellularly secreted, soluble types of MMPs. They are abundantly secreted into the bloodstream, providing high background signals, and probes are easily washed away, which thus lowers the imaging quality [36, 37]. To compensate for these drawbacks, many reported MMP-2 and MMP-9 probes were designed to carry another recognition sequence to anchor these probes to the extracellular membrane of specific cells, which increased the technical difficulty of probe synthesis, while the effects might not have been as expected . Unlike extracellular MMPs, membrane-type (MT)-MMPs are tethered to the plasma membrane via either a glycosylphosphatidylinositol linkage or a transmembrane domain . Moreover, MMP-14, also known as MT1-MMP, is directly linked to the pathogenesis of plaque vulnerability, as MMP-14 has been shown to convert MMP-2 and MMP-9 proenzymes into their active forms and to act as an attractant to facilitate monocyte/macrophage infiltration into sites of experimentally induced inflammation and established atherosclerotic lesions [5, 40]. To detect cardiac ischemia/reperfusion injury, van Duijnhoven et al.  developed a series of activatable cell-penetrating peptides (ACPPs) that were sensitive to MMP-2 and MMP-9 (ACPP-2/9) or to MMP-14 (ACPP-B). Although both ACPPs successfully detected regions of an infarcted myocardium in mouse models, the ACPP-2/9 probe showed a considerable degree of activation in all tissues, while ACPP-B was found to be tissue specific. Therefore, MMP-14 is more suitable than MMP-2 and MMP-9 as a target for plaque inflammation imaging.
In this study, the core sequence, Arg–Ile–Gly–Phe–Leu–Arg, of the peptide was adopted from previous reports. This sequence has been verified to have a high specificity and selectivity for MT-MMPs, including MT1-MMP (MMP-14), MT2-MMP (MMP-15), and MT3-MMP (MMP-16), as well as for MMP-2 and MMP-9 [25, 42]. Next, a NIR fluorescent dye, Cy5.5, and PEG 5000-wrapped AuNPs were conjugated to the N- and C-termini of the peptide, respectively. Characterisation tests demonstrated that [email protected] had a small hydrated size and homogeneous size distribution, which would allow the NPs to easily penetrate into a plaque through a damaged endothelium or the vasa vasorum. The fluorescence intensity of [email protected] was negligible in a static state because the signal could only be detected in a narrow range; however, after the activation, the signal could be amplified up to several folds, depending on the MMP-14 concentration. The biosafety assessment of [email protected] showed its low cytotoxicity and no significant injuries to organs such as the lung, heart, liver, and kidney.
MMPs not only participate in the onset and progression of atherosclerosis but also reflect plaque vulnerability , and macrophages are the most important source of MMPs in atherosclerosis. Hence, in vitro biological evaluation of the MMP-14-targeting fluorescent nanoprobe was performed using the macrophage cell line RAW 264.7. The results showed that MMP-14 was highly expressed in ox-LDL-induced foamy macrophages, at a level of 5·2-fold higher than that in normal macrophages. To verify the association of MMP-14 expression with inflammatory macrophages, we determined changes in the M1 macrophage markers CD68 and F4/80  and the classical M2 macrophage marker CD206  and confirmed that CD68 and F4/80 expression was significantly upregulated and that of CD206 was largely suppressed in ox-LDL-induced macrophages. These trends were reversed by the treatment with the nonselective MMP inhibitor GM6001 , which confirmed that MMP-14 could potentially serve as a marker for macrophage inflammatory activities. Consistent with the in vitro data, the expression of MMP-14 increased and the hyperactivity of macrophages was aggravated with plaque progression in the murine model of atherosclerosis. Cell and tissue fluorescence imaging suggested a targeting ability of [email protected] towards MMP-14, as the area of nanoprobe binding overlapped with that of MMP-14 expression. In vivo, a distinctive fluorescence signal reached a peak value within 10 min after probe injection and disappeared at 12 h post-injection. To eliminate interference from other tissues and confirm that the fluorescent signal was indeed associated with the right carotid artery, wherein plaques were induced, the right carotid artery was immediately isolated after [email protected] injection, and ex vivo studies showed that the probe was preferentially accumulated in the plaque on the right artery wall. Biodistribution studies indicated that this nanoprobe had a short circulation time and could be rapidly cleared through the liver, kidney, and intestine, which lowered the risk of possible injury to organs and tissues.
There are some limitations to this study. First, the [email protected] nanoprobe was used in a single imaging mode and only for the diagnosis of vulnerable plaques. Meanwhile, recently developed probes tend to have dual imaging modalities or be functional in both diagnosis and treatment, such as dual-functional, fluorescent-radiolabelled, composite imaging agents [47, 48], which can simultaneously detect PET and NIR fluorescence signals, or photodynamic/photothermal therapeutic drugs, which are not only used for imaging but also provide thermotherapy to kill cancer cells [49, 50]. Second, the implantation of the silicone cuff might have induced perivascular inflammation, thus causing false-positive results, such as non-atherosclerosis-related background signals for imaging. More importantly, human vessels cannot be fully mimicked by mouse vessels, in which cardio-cerebrovascular events are rare, for unknown reasons. Third, in vitro fluorescent imaging of plaque samples showed that the internal elastic membrane could also be stained by the nanoprobe, and further studies are needed to elucidate the mechanism of this off-target effect and make improvements.