The applicant proposes to use the T1/T2 quenching effect for imaging protease activity, and he has demonstrated in preliminary results a combined T2 and T1 agent (Dysprosium and Gadolinium connected by a PEG chain) which has very small modulation to the MR signal intensity, due to the offsetting effect of the T2 and T1 agents. By design, the T2 agent is attached to the PEG chain through a trypsin cleavage site, which can be broken by a target enzyme (e.g. trypsin). Once detached from the PEG chain, the T2 agent loses its modulation effect on the MR signal, resulting in an intensity increase reflecting the protease activity. The applicant ultimately seeks to apply this methodology to image the activity of MMP-7 in mouse tumors, since it is over-expressed in several cancers.
This Career Award focussed on developing new imaging agents that could improve the diagnosis of human diseases. Several new imaging agents were developed during the period 2005-2010, which will have a significant impact on diagnosing diseases at an early stage. The two most signifcant results are described below. The first one is focussed on imaging hydrogen peroxide, with a new contrast agent termed the peroxalate nanoaprticles, the second result focusses on imaging bacterial infections with maltodextrin based contrast agents. These developments are described below. Several other contributions to biotechnology were also made with Career funding, however because of space limitations these will not be discussed. The overproduction of hydrogen peroxide is implicated in the development of numerous diseases and there is currently great interest in developing contrast agents that can image hydrogen peroxide, in vivo. In this proposal, we demonstrate that nanoparticles formulated from peroxalate esters and fluorescent dyes can image hydrogen peroxide in vivo with high specificity and sensitivity. The peroxalate nanoparticles image hydrogen peroxide by performing a three component chemiluminescent reaction in vivo between hydrogen peroxide, peroxalate esters and fluorescent dyes. The peroxalate nanoparticles have several attractive properties for in vivo imaging, such as, tunable wavelength emission (460-630 nm), nanomolar sensitivity for hydrogen peroxide and excellent specificity for hydrogen peroxide over other reactive oxygen species. The peroxalate nanoparticles were capable of imaging hydrogen peroxide in the peritoneal cavity of mice, during an LPS-induced inflammatory response. We anticipate numerous applications of peroxalate nanoparticles for in vivo imaging of hydrogen peroxide, given their high specificity, sensitivity, and deep tissue imaging capability. The second probe we developed with Career funding focussed on imaging bacterial infections. The diagnosis of bacterial infections remains a major challenge in medicine. Although numerous contrast agents have been developed to image bacteria, their clinical impact has been minimal because they are unable to detect small numbers of bacteria in vivo, and cannot distinguish infections from other pathologies such as cancer and inflammation. In this proposal, we developed a new family of contrast agents, termed maltodextrin based imaging probes (MDPs), which can detect bacteria in vivo with a sensitivity two order of magnitude higher than previously reported, and, for the first time, detect bacteria via a bacteria-specific mechanism that is independent of host response and secondary pathologies. MDPs are composed of a fluorescent dye conjugated to maltohexaose, and are rapidly internalized through the bacterial-specific maltodextrin transport pathway, endowing the MDPs with a unique combination of high specificity and sensitivity for bacteria. We show that MDPs selectively accumulate within bacteria at millimolar concentrations, and are a thousand-fold more specific for bacteria than mammalian cells. In addition, we demonstrate that MDPs can image as few as 105 CFUs in vivo and can discriminate between active bacteria and inflammation induced by either LPS or metabolically inactive bacteria.
