Early detection of both primary tumors and metastatic disease remains a major challenge in the diagnosis and staging of cancer. The recognition of the role of MMPs in both the growth and metastasis of tumors has guided the development not only of therapeutic strategies utilizing synthetic, small-molecule MMP inhibitors (MMPIs), but has also catalyzed methods to detect and image tumors in vivo by means of tumor-associated proteolytic activity. These imaging approaches target MMPs involved in cancer progression via contrast agents linked to MMPIs or to MMP selective and specific substrates with sensitivity enhanced by amplification during enzymatic processing. This review draws attention to a variety of strategies utilized to image MMP activity in vivo.

Matrix metalloproteinases (MMPs) are extracellular proteolytic enzymes involved in tumor progression. We present the in vivo detection and quantitation of MMP7 activity using a specific near-infrared polymer-based proteolytic beacon, PB-M7NIR. PB-M7NIR is a pegylated polyamidoamine PAMAM-Generation 4 dendrimer core covalently coupled to a Cy5.5-labeled peptide representing a selective substrate that monitors MMP7 activity (sensor) and AF750 as an internal reference to monitor relative substrate concentration (reference). In vivo imaging of tumors expressing MMP7 had a median sensor to reference ratio 2.2-fold higher than a that of a bilateral control tumor. Ex vivo imaging of intestines of multiple intestinal neoplasia (APC Min) mice injected systemically with PB-M7NIR revealed a sixfold increase in the sensor to reference ratio in the adenomas of APC Min mice compared with control intestinal tissue or adenomas from MMP7-null Min mice. PB-M7NIR detected tumor sizes as small as 0.01 cm2, and the sensor to reference ratio was independent of tumor size. Histologic sectioning of xenograft tumors localized the proteolytic signal to the extracellular matrix; MMP7-overexpressing tumors displayed an approximately 300-fold enhancement in the sensor to reference ratio compared with nonexpressing tumor cells. In APC Min adenomas, the proteolytic signal colocalized with the endogenously expressed MMP7 protein, with sensor to reference ratios approximately sixfold greater than that of normal intestinal epithelium. PB-M7NIR provides a useful reagent for the in vivo and ex vivo quantitation and localization of MMP-selective proteolytic activity.

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.

Chemotherapeutics such as doxorubicin (DOX) and paclitaxel (PXL) have dose limiting systemic toxicities including cardiotoxicity and peripheral neuropathy. Delivery strategies to minimize these undesirable effects are needed and could improve efficacy, while reducing patient morbidity. Here DOX and PXL were conjugated to a nanodendron (ND) through an MMP9-cleavable peptide linker, producing two new therapies, ND2DOX and ND2PXL designed to improve delivery specificity to the tumor microenvironment and reduce systemic toxicity. Comparative cytotoxicity assays were performed between intact ND-drug conjugates and the MMP9 released drug in cell lines with and without MMP9 expression. While ND2DOX was found to loose cytotoxicity due to the modification of DOX for conjugation to the ND; ND2PXL was determined to have the desired properties for a prodrug delivery system. ND2PXL was found to be cytotoxic in MMP9-expressing mouse mammary carcinoma (R221-Aluc) (53%) and human breast carcinoma (MDA-MB-231) (66%) at a concentration of 50 nM (in PXL) after 48 hours. Treating ND2PXL with MMP9 prior to the cytotoxicity assay resulted in a faster response; however, both cleaved and intact versions of the drug reached the same efficacy as the unmodified drug by 96 hours in the R221A-luc and MDA-MB-231 cell lines. Further studies in modified Lewis lung carcinoma cells that either do (LLCMMP9) or do not express (LLCRSV) MMP9 demonstrate the selectivity of ND2PXL for MMP9. LLCMMP9 cells were only 20% viable after 48 hours of treatment while LLCRSV were not affected. Inclusion of an MMP inhibitor, GM6001, when treating the LLCMMP9 cells with ND2PXL eliminated the response of the MMP9 expressing cells (LLCMMP9). The data presented here suggests that these NDs, specifically ND2PXL, are non-toxic until activated by MMP9, a protease common in the microenvironment of tumors, indicating that incorporation of chemotherapeutic or cytostatic agents onto the ND platform have potential for tumor-targeted efficacy with reduced in vivo systemic toxicities.

BackgroundDrug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA.ResultsMonodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature.ConclusionsThis work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative environments, smart theranostic applications combining drug delivery with imaging of platform localization are within reach. The modular design of these USPIO nanoclusters enables future development of platforms for imaging and drug delivery targeted towards proteolytic activity in tumors and in advanced atherosclerotic plaques.