Cell-Penetrating Peptides and Peptide Nucleic Acid-Coupled MRI Contrast Agents: Evaluation of Cellular Delivery and Target Binding

Molecular imaging of cells and cellular processes can be achieved by tagging intracellular targets such as receptors, enzymes, or mRNA. Seeking to visualize the presence of specific mRNAs by magnetic resonance (MR) imaging, we coupled peptide nucleic acids (PNA) with gadolinium-based MR contrast agents using cell-penetrating peptides for intracellular delivery. Antisense to mRNA of DsRed2 protein was used as proof of principle. The conjugates were produced by continuous solid-phase synthesis followed by chelation with gadolinium. Their cellular uptake was confirmed by fluorescence microscopy and spectroscopy as well as by MR imaging of labeled cells. The cell-penetrating peptide D-Tat57−49 was selected over two other derivatives of HIV-1 Tat peptide, based on its superior intracellular delivery of the gadolinium-based contrast agents. Further improved delivery of conjugates was achieved upon coupling peptide nucleic acids (antisense to mRNA of DsRed2 protein and nonsense with no natural counterpart). Significant enhancement in MR contrast was obtained in cells labeled with concentrations as low as 2.5 μM of these agents. Specific binding of the targeting PNA containing conjugate to its complementary oligonucleotide sequence was proven by in vitro cell-free assay. In contrast, a lack of specific enrichment was observed in transgenic cells containing the target due to nonspecific vesicular entrapment of contrast agents. Preliminary biodistribution studies showed conjugate-related fluorescence in several organs, especially the liver and bladder, indicating high mobility of the agent in spite of its high molecular weight. No conjugate related toxicity was observed. These results are encouraging, as they warrant further molecular optimization and consecutive specificity studies in vivo of this new generation of contrast agents.