Targeted Contrast Agents for Magnetic Resonance Imaging and Their Biological Evaluation
The goal of our group has been the development of new cell-specific contrast agents that are either internalized or trapped selectively in or on the target cells for structural MRI studies or tracking of cells in vivo by MRI. Target cells should be selectively labeled in the presence of other cell types without the target structure by using specific characteristics of the target cells/tissues (e.g. over-expression of proteins/enzymes, surface markers, receptors, changes of microenvironment).
For this purpose, we perform synthesis and biological evaluation of such probes in vitro and in vivo.
Since for targeted probes in particular correct interaction/binding with the cellular target and their localization inside or on the cells is highly important, one main focus is the biological evaluation in cell culture models in vitro.
In this respect, of special interest for us are uptake/binding efficacy, intracellular localization, specificity of the interaction/accumulation, intracellular transport, and biocompatibility.
For an overview of recent or ongoing projects, please go also to our project group
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Intracellular targeting (enzyme and mRNA targeting)
In recent years several, mainly intracellularly targeted CA were developed by our group. Enzymes (model: ?-galactosidase) and mRNA (model: mRNA of red fluorescent protein DsRed) were chosen as cellular targets. Although these CA efficiently enhanced the contrast in MR images of labeled cells (even when low micromolar concentrations were applied), a high unspecific background signal in non-targeted cells prevented a successful in vivo application so far. The intracellular localization of the CA (e.g. cytosol vs. endosomes) is not only crucial for a specific interaction and accumulation in the target cells but also for the efficacy of MR contrast enhancement. Even a significantly higher accumulation can be insufficient to increase contrast in MR images when the CA is predominantly located in endosomes or lysosomes.
Recently, 19F MRI and MRS are increasingly gaining an interest and relevance in biomedical and clinical research. The great advantage of 19F compared to 1H MRI is the lack of an intrinsic background signal in mammalian tissue, which allows quantitative and unambiguous detection of administered fluorine labeled probes. Accordingly, we have successfully developed a dual-modal 1H/19F MRI probe, Gd-DOMF-Gal, that can be lightened on from an off state only in the presence of the enzyme ?-galactosidase (Figure) [see also project description of ]. This enzyme is expressed by the LacZ gene, one of the most widely used reporter genes in transgenic studies. Specific activation of Gd-DOMF-Gal has been proven in vitro (phantoms, cells) and lately being investigated in in vivo studies with mice bearing ?-galactosidase expressing tumor xenografts. Another responsive 1H/19F MRI probe, which has been recently developed in this line, is sensing the activity of matrix metalloproteinase (MMP-2), an enzyme overexpressed on tumor cells.
Apart from 1H/19F MRI responsive probes, we have obtained fluorinated-fluorescent nanoparticles, which could efficiently label human mesenchymal stem cells (hMSCs) as well as CD4+ T-cells, and further optimized MR methods (G. Hagberg) for efficient detection of these labeled cells by MRI. Within this project, we have aimed at establishing a strategy that would allow an efficient assessment of information about the fate of transplanted cells in vivo, and thus provide a tool for the evaluation of efficacy of proposed cell-based therapies by 1H/19F MRI (i.e. in the treatment of urinary incontinence in collaboration with the University Hospital Tübingen). See also project description of )
Silica-based platforms for MR and multi modality imaging probes
One drawback of MRI is its relatively low sensitivity. A large number of contrast producing moieties is required to obtain a significant change in image contrast. To achieve a higher local accumulation, macromolecular or nanoparticle-based platforms can be used to increase the number of MR reporters per molecule. In collaboration with the University of Tübingen SiO2-based systems (silsesquioxanes, nonporous nanoparticles) functionalized with Gadolinium chelates were developed. The relatively small silsesquioxanes allow the coupling of up to eight chelates by retaining a pharmacokinetic profile of a low molecular weight CA. They undergo a slow degradation process under physiological conditions to mono-chelates readily excreted via the kidneys. In vivo, a rapid accumulation in the gastrointestinal tract after i.v. injection into mice was observed not present when the same concentration of Gadolinium as GdDOTA, a commercially available CA used in the clinics, was injected. Recently, such silsesquioxanes were functionalized with fluorine containing moieties instead of Gadolinium chelates for 19F MRI applications.
Silica-based nanoparticles (NPs) are much larger (~ 50 130 nm in diameter) and thus will show different biodistribution and pharmacokinetics. However, a large number of imaging reporters can be conjugated per particle and different functionalities (e.g. targeting sensors, vectors for cellular uptake) can be introduced.
NPs with a high payload of Gadolinium were synthesized and tested for their ability to enhance MR contrast in vitro. These NPs were further functionalized with fluorophores for optical imaging and a CPP to enhance cellular uptake. Such NPs can be taken up by cells and an initial in vivo study on the biodistribution after i.v. injection showed a fast accumulation particularly in lungs and liver, typical for nanoparticles. In parallel, the influence of particle size and surface charge was studied as well as the surface loading efficacy with Gadolinium chelates optimized.
In a recent project, performed in collaboration with the University of Durham (UK), we developed and characterized in vitro 1H MRI probes responsive to the metabotropic glutamate receptor (mGluR5) that is expressed in the brain. These CAs consist of Gd3+-chelate and /or a fluorophore coupled to known antagonists of mGluR5. The primary goal of this research is the non-invasive visualization of mGluR5 receptors in the brain and potentially a more direct monitoring of neuronal activity by imaging extracellular glutamate fluctuations during activation. Extending this concept, CAs targeted to N-methyl-D-aspartate (NMDA) receptor, another specific type of ionotropic glutamate receptors in the brain, were developed and tested in vitro for their ability to visualize these receptors by optical and MR imaging.
A different type of NPs (ferromagnetic metallic cobalt with a functionalized carbon coating) is used for in vivo targeting of ?-cells in pancreatic islets of Langerhans in mice. A ?-cell specific single chain antibody fragment (SCA), developed by our collaborators, was coupled to these NPs to obtain a targeted T2-CA. In combination with the unprecedented spatial resolution which was achievable at our 16.4T animal scanner or using a cryogenic coil at 9.4T as well as optimizing imaging sequences and image processing by our MRI experts, it was possible for the first time to visualize single islets in vivo in a freely breathing mouse after i.v. injection. This study particularly exemplifies the great advantage to have CA developers (chemists, biologists) and MR physicists working close together in one group. (See also project description of )
Understanding brain connectivity is important to enhance the understanding of brain function and disease. To correlate brain anatomy to the functional outcome and to follow changes e.g. with development, aging or learning, non-invasive longitudinal studies are needed. MRI is providing an excellent measure to perform dynamic investigations of brain connectivity by using MR active neuroanatomical tracers. In a collaboration with former and present members of the Dept. Logothetis we are helping to develop such novel tracer molecules by providing information about the uptake efficacy into and the transport of the probes inside neural cells along their processes. Two classes of tracers, for short term (1 - 3 days) and long term (7 - 14 days) studies, were successfully tested in vitro and in vivo.
To circumvent endosomal entrapment of our intracellular targeted CA we were also looking for new vector molecules (e.g. cell penetrating peptides, CPP) showing optimized cellular distribution. This study was leading to a novel cysteine rich peptide, CyLoP-1, which is able to transport efficiently various cargo molecules (e.g. the bioactive pro-apoptotic SmacN7-peptide) into the cytosol. When coupled to a Gadolinium chelate it is a potent MR CA strongly reducing relaxivity quenching effects due to endosomal entrapment.
In parallel, non-peptide delivery systems (e.g. lipid based systems like cholesterol) were tested for mRNA targeted imaging probes. Whilst uptake efficiency could be further increased compared to CPP conjugates the cellular distribution and thus the specificity were not altered. However, the synthesis of peptide nucleic acids, used to target mRNA, could be optimized further.
High-resolution NMR analysis of human brain tumor extracts
Recently, a collaborative project has been initiated to assess human brain tumors (gliomas) by means of 1H MRSI at 9.4T. Apart from assessing changes in important metabolites such as glutamate, glutamine, creatine or inositol, the detection (and potential quantification) of 2-hydroxyglutarate (2HG) is of high importance. An increased accumulation of this metabolite in tumor tissue is associated with mutations in the TCA cycle enzyme isocitrate dehydrogenase (IDH) that occurs frequently in grade II and III glionas. Thus, 2HG has the potential to serve as a prognostic and diagnostic biomarker.
In order to verify the detection of 2HG in human MRS spectra, tumor tissue samples were extracted and high-resolution 1H NMR spectra of the water soluble metabolites were obtained at 300 and 600 MHz. The signals for 2HG could be unambiguously identified and quantified in these spectra and were used to verify the presence of 2HG in the in vivo spectra. In addition, more than 20 different metabolites could be assigned and quantified in the NMR spectra.
In vivo visualization of nitric oxide formation in the brain using a fluorescent sensor and fiber optics
Nitric oxide (NO) is a major messenger molecule in the brain. It is a potent vasodilator playing an important role in establishing neurovascular coupling linking neuronal activity to vasodilation.
and (Research group ) plan to use fluorescent NO sensors (e.g. DAF-FM DA) to directly detect intracellular NO formation in vivo and in real-time. For this purpose, we initiated the careful characterization of the specific reaction of the sensor with NO in vitro (detection limits, kinetics etc.). Using the information from these initial experiments, the methodology will be translated and established in vivo and adapted to fiber optics. In the future, we intend to combine optogenetics and fMRI to identify the contribution of nitric oxide to the neurovascular coupling.
Molecular in vivo imaging of cellular therapeutics CeTheProbes (2008-2012)
This work was in parts performed in the frame of the joint research project of the MPI for Biological Cybernetics (coordinator), Eberhard-Karls University Tübingen, and Hannover Medical School, funded by the German Federal Ministry of Education and Research (BMBF, FKZ 01EZ0813)
1984-1990 Studies of Chemistry, University of Bremen, Germany
1990 Diploma thesis under the supervision of Prof. D. Leibfritz, University of Bremen, Germany
NMR-spectroscopic studies of metabolism of P-388 lymphoma cells
1990-1995 Ph.D. thesis under the supervision of Prof. D. Leibfritz, University of Bremen, Germany
Studies of the effects of alkylphosphocholines with multinuclear NMR-spectroscopy
1996-2000 Postdoctoral Research Scientist, Organic Chemistry (Prof. D. Leibfritz), University of Bremen, Germany
2000-2004 Research Scientist, Biochemical Laboratory of the Department of Conservative Dentistry, Periodontology and Preventive Dentistry (Prof. W. Geurtsen), Hannover Medical School, Germany
Chemical-biological interactions of components of resin-modified filling materials on human primary cells of the oral cavity
(September 2002 - Mai 2004 Deputy head of the Biochemical Laboratory)
2004-2012 Research Scientist, High-Field Magnetic Resonance Center, Max-Planck-Institute for Biological Cybernetics, Tübingen, Germany
Development of intracellular targeted contrast agents for MRI
2005-2012 Project Leader Contrast Agent Development (Chemical Biology)
Since 2012 Guest scientist