Synthesis and characterization of a cell-permeable bimodal contrast agent targeting β-galactosidase

Aneta Keliris, Thomas Ziegler, Ritu Mishra, Rolf Pohmann, Martin G. Sauer, Kamil Ugurbil, Jörn Engelmann

Research output: Contribution to journalArticle

39 Scopus citations

Abstract

Noninvasive monitoring of intracellular targets such as enzymes, receptors, or mRNA by means of magnetic resonance imaging (MRI) is increasingly gaining relevance in various research areas. A vital prerequisite for their visualization is the development of cell-permeable imaging probes, which can specifically interact with the target that characterizes the cellular or molecular process of interest. Here, we describe a dual-labeled probe, Gd-DOTA-k(FR)-Gal-CPP, designed to report the presence of intracellular β-galactosidase (β-gal) enzyme by MRI. This conjugate consists of a galactose based core serving as cleavable spacer, incorporated between the cell-penetrating peptide D-Tat49-57 and reporter moieties (Gd-DOTA, fluorescein (FR)). We employed a facile building block approach to obtain our bimodal probe, Gd-DOTA-k(FR)-Gal-CPP. This strategy involved the preparation of the building blocks and their subsequent assembly using Fmoc-mediated solid phase synthesis, followed by the complexation of ligand 14 with GdCl 3. Gd-DOTA-k(FR)-Gal-CPP showed a considerably higher relaxivity enhancement (16.8 ± 0.6 mM-1 s-1, 123 MHz, ∼21 °C) relative to the commercial Gd-DOTA (4.0 ± 0.12 mM-1 s-1, 123 MHz, ∼21 °C). The activation of Gd-DOTA-k(FR)-Gal- CPP was based on a cellular retention strategy that required enzymatic cleavage of the delivery vector from galactose moiety following the cell internalization to achieve a prolonged accumulation of the reporter components (Gd-DOTA/FR) in the β-gal expressing cells. Cellular uptake of Gd-DOTA-k(FR)-Gal-CPP in β-gal expressing C6/LacZ and enzyme deficient parental C6 rat glioma cells was confirmed by fluorescence spectroscopy, MR imaging and ICP-AES measurements. All methods showed higher accumulation of measured reporters in C6/LacZ cells compared to enzyme deficient parental C6 cells. Fluorescence microscopy of cells labeled with Gd-DOTA-k(FR)-Gal-CPP indicated a predominantly vesicular localization of the green fluorescent conjugate around cell nuclei. This cellular distribution was most likely responsible for the observed non-specific background signal in the enzyme deficient C6 cells. Even though the specific accumulation of our bimodal probe has to be further improved, it could be already used for cell imaging by MRI and optical modalities.

Original languageEnglish (US)
Pages (from-to)2529-2540
Number of pages12
JournalBioorganic and Medicinal Chemistry
Volume19
Issue number8
DOIs
StatePublished - Apr 15 2011

Keywords

  • Cellular imaging
  • Gadolinium complex
  • Intracellular MRI contrast agent
  • Solid phase synthesis
  • β-d-Galactopyranose

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