Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity

Abstract

The present study describes the in vivo detection and imaging of tumour-associated MMP-7 (matrix metalloproteinase-7 or matrilysin) activity using a novel polymer-based fluorogenic substrate PB-M7VIS, which serves as a selective 'proteolytic beacon' (PB) for this metalloproteinase. PB-M7VIS is built on a PAMAM (polyamido amino) dendrimer core of 14.2 kDa, covalently coupled with an Fl (fluorescein)-labelled peptide Fl(AHX)RPLALWRS(AHX)C (where AHX stands for aminohexanoic acid) and with TMR (tetramethylrhodamine). PB-M7VIS is efficiently and selectively cleaved by MMP-7 with a k (cat)/ K (m) value of 1.9x10(5) M(-1).s(-1) as measured by the rate of increase in Fl fluorescence (up to 17-fold for the cleavage of an optimized PB-M7VIS) with minimal change in the TMR fluorescence. The K (m) value for PB-M7VIS is approx. 0.5 microM, which is approx. two orders of magnitude lower when compared with that for an analogous soluble peptide, indicating efficient interaction of MMP-7 with the synthetic polymeric substrate. With MMP-2 or -3, the k (cat)/ K (m) value for PB-M7VIS is approx. 56- or 13-fold lower respectively, when compared with MMP-7. In PB-M7VIS, Fl(AHX)RPLALWRS(AHX)C is a selective optical sensor of MMP-7 activity and TMR serves to detect both the uncleaved and cleaved reagents. Each of these can be visualized as subcutaneous fluorescent phantoms in a mouse and optically discriminated based on the ratio of green/red (Fl/TMR) fluorescence. The in vivo specificity of PB-M7VIS was tested in a mouse xenograft model. Intravenous administration of PB-M7VIS gave significantly enhanced Fl fluorescence from MMP-7-positive tumours, but not from control tumours ( P <0.0001), both originally derived from SW480 human colon cancer cells. Prior systemic treatment of the tumour-bearing mice with an MMP inhibitor BB-94 ([4-( N -hydroxyamino)-2 R -isobutyl-3 S -(thienylthiomethyl)-succinyl]-L-phenylalanine- N -methylamide), markedly decreased the Fl fluorescence over the MMP-7-positive tumour by approx. 60%. Thus PB-M7VIS functions as a PB for in vivo detection of MMP-7 activity that serves to light this optical beacon and is, therefore, a selective in vivo optical molecular imaging contrast reagent.

Near-Infrared Optical Proteolytic Beacons for In Vivo Imaging of Matrix Metalloproteinase Activity

“…The Tsien group described activatable cell-penetrating peptides consisting of a polyarginine membrane-translocating motif linked via an MMP-cleavable peptide to an appropriate masking polyanionic domain (a cleavable peptide hairpin) to deliver fluorescent labels to within tumor cells both in vitro and in vivo after cleavage by tumor-associated proteinases (3). The strategy we employed was to use a PAMAM dendrimer backbone, reference fluorophores attached directly to the dendrimer, and sensor fluorophores attached via a selective peptide linkage (4) (Figure 1). The multivalency of the dendrimer allows for adjustment in the relative amounts of sensor and reference fluorophores, provides a vehicle that is maintained in the circulation for greater than 30 minutes, and provides the opportunity to link additional agents that can alter the half-life of the reagent in circulation or provide additional functionalities.…”

“… 12 In the original procedure (4), both the intermediate and final products are diafiltered with 0.1 M NaCl, 5 mM Hepes-NaOH (pH 7.0), 1 mM EDTA. While beacons, stored with or without 20% ethanol, are stable for several weeks at 4 °C, reagents were also stable without saline, i.e., in 1 mM EDTA.…”

“… 29 Importance of timepoint for S/R analysis: Results from studies conducted with PBs in various tumor models, e.g., SW480 colon tumor xenografts (4), Min mouse adenomas (5) and LLC lung tumors (9), have revealed the importance of carrying out exploratory studies to measure the S/R ratios as a function of time after administration of the PB. Although the half-time for whole-body clearance of the PBs studied thus far are similar (on the order of 60–90 min), maximal S/R ratios in tumors have been found at different times after injection.…”

Matrix Metalloproteinase Responsive, Proximity‐Activated Polymeric Nanoparticles for siRNA Delivery

“…PAT-SPNs were treated with 50 nM MMP-7 in order to mimic the metastatic tumor microenvironment. [45] The resultant product was re-isolated and analyzed by GPC with DMF as the mobile phase. The small shift in the peak elution time of the PEG-pep-pD-pDPB after treatment with MMP-7 is consistent with the expected drop in molecular weight due to proteolytic removal of the PEG segment.…”

“…[19, 61] In our studies, we utilized 50 nM MMP, which falls at the lower end of the range of MMP concentrations that other researchers typically use to study the kinetics of cleavage of MMP-responsive biomaterial sunder conditions that mimic the tumor microenvironment. [14, 19, 45, 62] We primarily assessed cleavage of our construct in response to MMP-7, although we also studied responsiveness to MMP-2 and MMP-9. The chosen substrate VPLSLYSG can be cleaved by all three of these MMPs and has a mid-rate k cat/ K M for MMP-7 cleavage of 22,000 ± 3,000 (M -1 s -1 ) [44] , though it is anticipated that presence of the PEG corona distal to the peptide sterically limits access of MMPs to the peptide substrate and slows its cleavage.…”