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Abstract

Aim of this project is the development of novel intracellular targeted contrast agents(CA)for Magnetic Resonance Imaging (MRI). Such probes should show a specific
accumulation in targeted cells compared to non-targeted cells by specifically binding to mRNA (via an antisense peptide nucleic acid) or by specific enzymatic cleavage of the vector part. In the recent years several intracellular CA were synthesized in our group using cell penetrating peptides(CPP) to deliver such probes into cells. These CA were efficiently taken up and were able to enhance contrast in MR images in cultured cells even at low micromolar labeling concentrations and, thus, are already usable for ex vivo labeling of cells. However, a lack of targeting specificity was observed. This is most likely due to the predominantly endosomal uptake and entrapment of the CA preventing a sufficient interaction with cellular targets. A novel cysteine rich peptide was developed and extensively tested which is able to deliver cargo molecules into the entire cytosol avoiding at least partially this endosomal entrapment. These studies resulted in an international patent application. Coupling a Gd-loaded DOTA chelate to this peptide led to a highly efficient intracellular contrast agent for MRI. It combined a better internalization compared to known CPP with an unexpected high contrast enhancement of labeled cells. This is likely due to the distribution of this contrast agent complex in the
entire cytosol, resulting in the access to a larger pool of water molecules and, thereby,avoiding “relaxivity quenching” as it is observed for probes which remain entrapped in
endosomal vesicles. In parallel, we tested non-peptide delivery systems (e.g. lipid based systems like coupling
of cholesterol) for their ability to enhance cytosolic uptake of targeted imaging probes. The use of cholesterol increased the uptake efficacy further on but did not solve the problem of endosomal entrapment.