Design and Synthesis of Antisense Peptide Nucleic Acid Conjugated MR Contrast Agents [Design und Synthese von Antisense Peptid-Nukleinsäure konjugierten MR Kontrastmitteln]

Magnetic Resonance Imaging (MRI) is one of the most important diagnostic tools available in medicine. The specificity and sensitivity of MRI can be further enhanced by the introduction of contrast agents. As many clinically valuable targets reside inside the cell membrane, therefore, developing efficient intracellular targeted MR contrast agent is required. The objective of the present project is to construct novel targeted intracellular MR contrast agents aiming to image mRNA transcription by MRI. The first part of this thesis takes an effort to search for an optimal vector for the intracellular delivery of MR contrast agents. Eight intracellular MR contrast agents, which conjugate different cell-penetrating peptides (CPP) with FITC and Gd(III) complexes, were synthesized by a continuous solid phase synthesis scheme. The key intermediates and final products were characterized by ESI-MS. Relaxivities of these MR contrast agents were measured at a frequency of 123 MHz and a magnetic field of 3 T. The comparison studies of the uptake and toxicity on NIH/3T3 cells suggest that D-Tat57-49 contrast agent could label cells sufficient to enhance significantly relaxation rates R1 and R2 for MR measurements, thus D-Tat57-49 peptide proves to be a useful CPP for the development of new intracellular MR contrast agents. The second part of this thesis describes the design and synthesis of antisense MR contrast agents, which conjugate PNA with CPP, Gd-DOTA and FITC. The intracellular uptake was confirmed by fluorescence spectroscopy, fluorescence microscopy and MR imaging on NIH/3T3 mouse fibroblasts as well as on transgenic dsRed cells. A subtoxic labeling concentration of 0.5 µM is sufficient to enhance significantly MR imaging contrast. The intracellular Gd(III) contents are at the range of 10-9~10-8 mol Gd/107 cells. An in vitro PNA-DNA binding assay confirmed that there is a significant higher specificity of the dsRed antisense contrast agent in comparison to its non-sense counterpart. However, no specific accumulation of the antisense dsRed CA in comparison to the non-sense CA could be detected in the target containing dsRed cells. Fluorescence microscopy studies have showed an exclusive endosomal localization of the contrast agents. Thus, further modifications of the contrast agents are required to achieve the release from endosomes or a direct uptake into the cytosol