J Engelmann

Synthesis and Characterization of a Cell-Permeable Bimodal Contrast Agent Targeting β-Galactosidase

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-Tat(49-57) 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), 123MHz, ∼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.

CyLoP-1: A Novel Cysteine-Rich Cell-Penetrating Peptide for Cytosolic Delivery of Cargoes

Cell-penetrating peptides (CPPs) may have impli-cations in biomedical sciences by improving the delivery of a wide variety of drugs through the membrane barrier. CPPs are generally taken up by endocytotic pathways, and vesicular encapsulation is a limiting factor in the area of intracellular targeting. A novel, cationic cysteine-rich CPP, CyLoP-1, has been developed exhibiting distinguished diffused cytosolic distribution along with endosomal uptake at low micromolar concentrations. Comparative uptake analysis with known CPPs showed CyLoP-1 as a promising delivery vector to access the cytosol in a variety of cell types. In addition to the positively charged residues, the presence of cysteines and tryptophans proved to be essential to maintain its functionality. Also, the oxidation status of the cysteines played an important role for the uptake efficiency of CyLoP-1, with the disulfide-containing form being more effective. The distinct feature of CyLoP-1 to enter the cytosol was further explored by the covalent attachment of cargoes of different nature and sizes. In particular, induction of caspase-3 activity (indicating apoptosis) by a CyLoP-1-SmacN7 conjugate proved successful delivery of the pro-apoptotic cargo to its site of action in the cytosol. Efficient intracellular delivery into the entire cytosol already at low micromolar concentrations makes CyLoP-1 a promising candidate for cytosolic delivery of cargoes of small sizes. Thus, this peptide might prove to be useful for efficient transmembrane delivery of agents directed to cytosolic targets.

A smart 19F and 1H MRI probe with self-immolative linker as a versatile tool for detection of enzymes

Here we report on a dual-modal (19) F and (1) H MRI paramagnetic probe with a self-immolative linker, Gd-DOMF-Gal. The enzymatic conversion of this probe by β-galactosidase resulted in a simultaneous turning on of the fluorine signal and changed ability of the Gd(3+) complex to modulate the (1) H MR signal intensity of the surrounding water molecules. A versatile imaging platform for monitoring a variety of enzymes by (19) F and (1) H MRI using this molecular design is proposed.

Synthesis, characterization and examination of Gd[DO3A-hexylamine]-functionalized silica nanoparticles as contrast agent for MRI-applications

Spherical, nonporous and monodisperse silica nanoparticles (NPs) with a diameter of about 100 nm were synthesized and covalently functionalized with lanthanoid(III) (Ln=Gd or Y) chelate complexes, which serve as contrast agents (CAs) for magnetic resonance imaging (MRI). The materials were fully characterized after each synthetic step by different analytical methods, such as dynamic light scattering, scanning electron microscopy, DRIFT and NMR spectroscopy, thermogravimetry and elemental analysis, as well as zetapotential measurements. High surface concentrations of Gd(III) complexes (up to 50 μmol g(-1)) were determined by ICP-AES and T(1)-measurements, respectively. MRI experiments show the typical concentration-dependent increase of the longitudinal relaxation rate. T(1)-weighted images of samples with more than 25 μg NPs per 100 μL agar display a clear contrast enhancement in the agar layer. The transverse relaxivities r(2) of the materials are significantly higher than r(2) of the corresponding free Gd(III) complexes in water and medium, whereas the longitudinal relaxivities r(1) are slightly increased. Due to the high loading of Gd(III) complexes, the relaxivities per particle are remarkably high (up to 2.78×10(5) mM(-1) s(-1) for r(1)). Thus, new hybrid materials, based on nonporous silica NPs with high local relaxivity values were synthesized, which can serve as very effective CAs for MRI.

Responsive imaging probes for metabotropic glutamate receptors

The design, synthesis and evaluation of eight contrast agents for metabotropic glutamate receptors is reported. Each of the contrast agents contains a selective mGluR5 binding moiety linked to a ‘DOTA’-derived gadolinium complex. The potential of these systems was evaluated in vitro for application as responsive MR imaging probes. The targeting moieties mGluR5antagonists based on aromatic alkyne and dipyridyl/heterobiaryl amide derivatives integrated in a modular fashion, involving linkage to the macrocyclic DOTA ligand to allow specific binding to the mGluR5 receptors. Signal intensity enhancements of up to 27% were observed by MRI in primary astrocyte suspensions and the reversibility of probe binding to the receptor sites, induced by added glutamate, was demonstrated using optical emission and the antagonistic activity of complexes was defined by calcium binding assays.

Investigation of a calcium-responsive contrast agent in cellular model systems: feasibility for use as a smart molecular probe in functional MRI.

Responsive or smart contrast agents (SCAs) represent a promising direction for development of novel functional MRI (fMRI) methods for the eventual noninvasive assessment of brain function. In particular, SCAs that respond to Ca(2+) may allow tracking neuronal activity independent of brain vasculature, thus avoiding the characteristic limitations of current fMRI techniques. Here we report an in vitro proof-of-principle study with a Ca(2+)-sensitive, Gd(3+)-based SCA in an attempt to validate its potential use as a functional in vivo marker. First, we quantified its relaxometric response in a complex 3D cell culture model. Subsequently, we examined potential changes in the functionality of primary glial cells following administration of this SCA. Monitoring intracellular Ca(2+) showed that, despite a reduction in the Ca(2+) level, transport of Ca(2+) through the plasma membrane remained unaffected, while stimulation with ATP induced Ca(2+)-transients suggested normal cellular signaling in the presence of low millimolar SCA concentrations. SCAs merely lowered the intracellular Ca(2+) level. Finally, we estimated the longitudinal relaxation times (T1) for an idealized in vivo fMRI experiment with SCA, for extracellular Ca(2+) concentration level changes expected during intense neuronal activity which takes place upon repetitive stimulation. The values we obtained indicate changes in T1 of around 1-6%, sufficient to be robustly detectable using modern MRI methods in high field scanners. Our results encourage further attempts to develop even more potent SCAs and appropriate fMRI protocols. This would result in novel methods that allow monitoring of essential physiological processes at the cellular and molecular level.

MR spectroscopy for in vivo assessment of the oncometabolite 2-hydroxyglutarate and its effects on cellular metabolism in human brain gliomas at 9.4T.

To examine in vivo metabolic alterations in the isocitrate dehydrogenase (IDH) mutated gliomas using magnetic resonance spectroscopy (MRS) at magnetic field 9.4T.

Multifunctional silica nanoparticles for optical and magnetic resonance imaging.

Abstract The surface of spherical, nonporous silica nanoparticles (SiO2-NPs) was modified with gadolinium (Gd) complexes, fluorophores, and cell-penetrating peptides to achieve multifunctionality on a single particle. The Gd surface concentrations were 9–16 μmol/g resulting in nanomaterials with high local longitudinal and transversal relaxivities (~1×105 and ~5×105 /mm/s/NP, respectively). Rapid cellular uptake was observed in vitro; however, larger extracellular agglomerates were also formed. In vivo administration revealed a fast distribution throughout the body followed by a nearly complete disappearance of fluorescence in all organs except the lungs, liver, and spleen after 24 h. Such NPs have the potential to serve as efficient multimodal probes in molecular imaging.

In vivo visualization of single native pancreatic islets in the mouse

The purpose of this study was to investigate the potential of a novel targeted contrast agent (CA) for the in vivo visualization of single native pancreatic islets, the sites of insulin production, in the pancreas of mice using magnetic resonance imaging (MRI). The CA for intravenous administration was composed of the β-cell-specific single-chain antibody fragment, SCA B1, and ferromagnetic carbon-coated cobalt nanoparticles. MRI experiments were performed at 7, 9.4 and 16.4 T in excised organs (pancreas, liver, kidney, spleen), at 7 T in mice fixed in formalin and at 9.4 and 16.4 T in living mice. Image contrast in untreated control animals was compared with images from mice treated with unspecific and specific CA. For the validation of MRI results, selected pancreases were subjected to immunohistochemical staining and numerical contrast simulations were performed. Ex vivo results and the outcome of immunohistochemistry suggest that islets are marked only by the CA containing SCA B1. Strong accumulation of particles was found also in other investigated organs owing to the uptake by the reticuloendothelial system, but the contrast in the MR images is clearly distinguishable from the islet specific contrast in pancreases and numerical predictions. In vivo experiments based on averaged dynamic sampling with 66 × 66 × 100 µm³ and triggered acquisition with 90 × 90 × 200 µm³ nominal resolution resulted in similar particle contrast to in in vitro measurements. The newly developed CA and MRI strategies have the potential to be used for studying mouse diabetes models by visualizing single native pancreatic islets.

MR contrast agent composed of cholesterol and peptide nucleic acids: Design, synthesis and cellular uptake

A new mRNA targeting contrast agent consisting of three main functional domains, (i) gadolinium based magnetic resonance reporter part, (ii) antisense peptide nucleic acids targeted to mRNA, and (iii) cholesterol as the delivery vector, was developed and synthesized. The new contrast agent showed efficient cellular uptake and significant contrast enhancement at very low labeling concentrations (0.5 microM). However, after uptake into cells the agent was located predominantly in endosomes like a similar cell penetrating peptide conjugated probe. Our results indicate that this newly developed contrast agent could be used for the labeling of cells for optical as well as magnetic resonance imaging.