Contrast agents activated by biological processes can be extremely useful for functional imaging of various diseases including cancer, which is typically characterized by unique microenvironment with increased concentration of proteolytic enzymes, hypoxia, and acidosis. This concept has been successfully applied for NIR optical imaging, however it remains an elusive technology for MRI, although several methods had recently been explored including activated chelating compounds for Gd and clustering of SPIO nanoparticles to increase T2 relaxivity. In this application, we will develop a novel class of activated contrast agent for MR imaging using combination of superparamegnetic iron-oxide nanoparticles (SPIO) and multiple Gd groups. When both components are localized in close proximity the resulting complex will produce strong negative T2 contrast in T1/T2-weighted MRI due to high T2 relaxivity of SPIO particles. Upon dissociation of the complex, small Gd groups will diffuse away from the SPIO core and generate positive T1 MR contrast in areas within the diffusion range of Gd but outside the diffusion range of the relatively immobile SPIO nanoparticle. Activated MR imaging agent will be synthesized using an SPIO core, matrix metalloproteinase (MMP-7) and broad-spectrum (FS-1) MMP- specific cleavable peptide linkers, and gadolinium chelates. The constructs will be characterized with DLS, EM, and element analysis by ICP-MS techniques. Proof-of-principle studies of the probe activation will be performed in a model, cell free gel system and in cultured breast cancer cells in vitro. Activation of the MR contrast agents will also be studied in preclinical models of human breast cancer. For biodistribution studies agents will be additionally labeled with multicolor fluorescent tags for SPIO and the leaving Gd group respectively. Biodistribution of the intact agent and its components will be measured using optical imaging of fluorescently labeled CA in fresh tissue sections and histological slices. A novel class of activated MR contrast agents that dramatically changes signal enhancement pattern from negative contrast enhancement (signal loss) to positive (increased signal) upon activation by proteolytic breast cancer associated MMP enzymes can provide noninvasive assessment of proteolytic activity in breast cancers. This is an important prognostic parameter as tumor aggressiveness and metastatic potential strongly correlate with the expression of proteinases. The agent consists of components that are currently approved for clinical use and therefore there are good chances for rapid translation of this imaging nanotechnology to clinic. PUBLIC HEALTH RELEVANCE: MR imaging provides an outstanding spatial resolution and soft tissue contrast, however, its ability to probe subtle features of chemical environment of a tumor is currently limited due to the lack of sensitive imaging contrast agents. In this application, we will use advances in nanotechnology to develop a novel class of MR contrast agents which will be specifically activated within the tumor microenvironment by tumor associated proteolytic enzymes and will provide significant enhancement of the tumor in MR images. These nanoscale activated MR contrast agents can improve existing imaging technology for early cancer detection and/or for noninvasive monitoring of tumor response to therapy.