A Platform for Specific Delivery of Lanthanide–Scandium Mixed-Metal Cluster Fullerenes into Target Cells

Lanthanides (Ln) find broad applications as contrast agents in medical imaging techniques such as magnetic resonance imaging (MRI). MRI is one of the most powerful, noninvasive imaging procedures, which is able to create images of tissues, organs and diseases in vivo. Every year, about six million patients undergo MRI studies of which 30% are performed using Gd-based contrast agents (CAs), which significantly reduce the spin-lattice relaxation time T1 of water protons leading to an increase of the signal intensity and improved contrast. Although medical applications of lanthanides in imaging are dominated by Gd , other lanthanides can be also used as MRI CAs (e.g. , Dy is considered as an efficient contrast agent for high-field MRI) and X-ray CAs. Radioisotopes of lanthanides (especially Lu) are employed as therapeutic radiopharmaceuticals. Direct administration of Ln ions in vivo is not possible because of their toxicity in the free ion form. In MRI, organic chelates of Gd and other lanthanides are used to circumvent this problem. The toxicity of Gd is thus substantially decreased and its solubility in biological fluids is improved, albeit some negative phenomena still remain (e.g. , fibrosis effects in kidneys). The encapsulation of Ln ions into the hollow carbon cages with formation of endohedral metallofullerenes (Ln-EMF) might be a more advantageous solution for a CAs, because (1) carbon cages protect Ln ions against external chemical exposure and their release into the body, and (2) the water proton relaxivity r1 (the effect on 1/T1) of Ln-EMFs and especially Gd-EMFs is remarkably higher compared with organic chelates. Among the other potential medical applications of Ln-EMFs, their use as X-ray CAs or radiolabeling of Ln-EMFs for imaging and therapy can be mentioned. Combination of different lanthanides, for example, Gd/Lu or Ho/Lu, in one EMF can be used for a design of multimodal contrast media. The distribution of standard CAs is usually restricted to the blood stream and the interstitial space. As a result, even contrast-enhanced imaging techniques still suffer from insufficient image resolution of morphological structures. The diagnostic problems, such as the limited possibility to exactly determine the actual tumor size and volume, to secure the metastases existence, and to distinguish the tumor tissues from healthy ones, have dramatic consequences for surgery and radiation therapy. The development of “molecular imaging” (MI) as an academic discipline has opened the way for further development in diagnostic imaging procedures. MI can be defined as imaging measurement of the cellular processes on the molecular level. The strategy is based on the targeting of specific proteins, in particular cells or cell parts, and coupling of this targeting with imaging techniques, which can be a promising way to enhance the contrast to differentiate between the tissues. Reported applications of Ln-EMFs for imaging are mostly limited to the use of their water-soluble derivatives as standard nonspecific MRI agents. Their use for MI-related techniques is very rare, however available results show high potential of Ln-EMFs in MI. 18] For instance, in vitro studies of a GdSc2N@C80-based BioShuttle system specifically designed to target human MDA-MB-231 breast adenocarcinoma cells showed that at a concentration of only 1/20 of the typical clinical dose, the sensitivity of this system was more than 500-fold higher than that of the commercial MRI-CA, Gd-DTPA (Gdbased complex with diethylene triamine pentaacetic acid), while a cell viability assay did not reveal any cell toxicity of the BioShuttle system. In this work, we introduce a Ln-EMF-based BioShuttle system as a platform for intracellular delivery of Ln-EMFs into c-myc mRNA-expressing cells suitable as an MI and potentially a therapeutic agent. In particular, we describe a synthesis of MI probe c-myc-antisense-Gd@BioShuttle comprised of (1) a Gdcontaining nitride cluster fullerene as an imaging component, (2) an address module (nuclear localization sequence), and (3) a transmembrane carrier peptide. Facile transport of this system into the target cells is demonstrated by in vitro studies. Gd-containing mixed-metal cluster fullerenes were produced by the Kr tschmer–Huffman method modified in our group. In this work, we used melamine (C3H6N6; organic base with high nitrogen content of 66% by mass) as a new selective nitrogen source. The graphite rods packed with a Gd/Sc/graphite/melamine mixture were evaporated in 200 mbar helium atmosphere with a current of 100 A. MS data analysis of the fullerene extract showed formation of EMFs with the mixed-metal nitride clusters with the general formula GdxSc3 xN@C2n (x= 0–3, 39 n 44). Isolation of these mixed-metal nitride cluster fullerenes (NCFs) was accomplished by one-step HPLC. The analysis of the chromatogram and mass spectra proved the formation of NCFs as the major products of the reaction [a] A. Svitova, Dr. A. A. Popov, Prof. Dr. L. Dunsch Department of Electrochemistry and Conducting Polymers Leibniz Institute of Solid State and Material Research Helmholtzstrasse 20, 01069 Dresden (Germany) E-mail : [email protected] [b] Dr. K. Braun Department of Medical Physics in Radiology German Cancer Research Center INF 280, 69120 Heidelberg (Germany) E-mail : [email protected] 2012 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.