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

Abstract

The exuberant expression of proteinases by tumor cells has long been associated with the breakdown of the extracellular matrix, tumor invasion, and metastasis to distant organs. There is both epidemiological and experimental data that support a causative role for proteinases of the matrix metalloproteinase (MMP) family in tumor progression. Optical imaging techniques provide an extraordinary opportunity for non-invasive “molecular imaging” of tumor-associated proteolytic activity. The application of optical proteolytic beacons for the detection of specific proteinase activities associated with tumors has several potential purposes: 1) Detection of small, early-stage tumors with increased sensitivity due to the catalytic nature of proteolytic activity, 2) Diagnosis and Prognosis to distinguished tumors that require particularly aggressive therapy or those that will not benefit from therapy, 3) Identification of tumors appropriate for specific anti-proteinase therapeutics and optimization of drug and dose based on determination of target modulation, and 4) as an indicator of efficacy of proteolytically-activated pro-drugs. This chapter describes the synthesis, characterization, and application of reagents that use visible and near infrared fluorescence resonance energy transfer (FRET) fluorophore pairs to detect and measure MMP-referable proteolytic activity in tumors in mouse models of cancer.

New tools for the quantitative assessment of prodrug delivery and neurotoxicity

“…Paclitaxel was attached to the N-terminus of the peptide using published procedures to give ND PXL (Majoros et al, 2006). Our MMP-beacons, originally on a polyamidoamine (PAMAM) scaffold, include a two-color FRET probe with sensor and reference dyes for quantifying MMP activation (McIntyre et al, 2004; McIntyre and Matrisian, 2009; McIntyre et al, 2010). For these studies, novel beacons were made from AVRWLLTAC peptide attached to the ND as will be described in detail elsewhere (Scherer et al, 2015).…”

“…Phantoms were prepared with either the sensor or reference dye bound to PAMAM, (McIntyre et al, 2004; McIntyre et al, 2010), or ND PXL -ND PB (not protease treated) or MMP9-treated ND PXL -ND PB , or ND PB without ND PXL , each in matrigel and injected subcutaneously into nu/nu mice, to create a localized phantom (Fig. 2A).…”

“…Using a design based on our proteolytic beacons (McIntyre et al, 2004; McIntyre and Matrisian, 2009; McIntyre et al, 2010), we recently demonstrated a PXL prodrug that is not toxic to tumor cells in vitro until activated by MMP9 (Samuelson et al, 2013). In these studies, we report the synthesis of a similar MMP9-activated PXL prodrug, ND PXL , constructed on a clickable NanoDendron (ND) scaffold and an analogous proteolytic beacon, ND PB .…”

New radiotracers for imaging of vascular targets in angiogenesis-related diseases

“…Currently, PET or SPECT imaging of MMPs is still at a preliminary stage and no related clinical study has been reported yet. Compared with blooming studies in fluorescence imaging of MMPs [135], PET or SPECT imaging of MMPs enabled the researchers to observe the MMP distribution or activation in deeper tissues, but usually resulted in lower target-to-background ratio. In order to improve the PET or SPECT tracers for imaging of MMP, chemical modification of existing MMPIs to decrease the non-specific binding or using anti-MMP antibodies could be alternative strategies.…”

Intravital microscopy: new insights into metastasis of tumors

“…The injectable probes that have been used to measure protease activity in tumors include autoquenched protease substrates that fluoresce upon cleavage (Weissleder et al, 1999), proteolytic beacons that lose FRET upon cleavage (e.g. McIntyre et al, 2010;Scherer et al, 2008) and fluorescently labeled activatable cell-penetrating peptides (ACCPs) that translocate into cells upon cleavage (Olson et al, 2009;Olson et al, 2010). The disadvantage of these probes is that chemical knowledge is required to synthesize them, although recent commercialization of some of these probes has overcome this problem.…”