My research is focused on development of targeted and responsive multimodality MRI probes for non-invasive in vivo visualization of key target molecules being specific markers of distinct cellular and physiological processes. Currently, this primarily involves enzyme-activity imaging and cell tracking applications.
Enzyme-responsive MRI probes
An enormous number of biochemical processes taking place in living organisms demand the action of enzymes. These highly specific catalysts serve as indicators of diseases (e.g. stroke, brain tumors, inflammations), cellular processes and are used as essential markers of gene expression.
Aim of this project is to develop a versatile multimodal imaging platform for in vivo mapping of enzyme activity in real-time that would allow: early disease diagnosis, assessment of cellular events associated with i.e. gene expression, or monitoring neuronal stem cells via reporter gene strategy after their transplantation. Here, switched off/on fluorinated MRI probes are developed, which remain silent in the absence of enzyme and only in the presence of enzyme produce the signal detectable by MRI. Importantly such approach allows detection of enzyme activity with a large MR signal amplification and without perturbation to biological process of targeted enzyme since its complete recovery is taking place after each cycle of enzymatic conversion of probe.
In this line, 1H/19F MRI probe, Gd-DOMF-Gal, responsive to ?-galactosidase (?-gal) enzyme, expressed by the LacZ gene most widely used reporter genes in transgenic studies, was successfully developed and characterized. Accordingly, Gd-DOMF-Gal showed upon enzymatic conversion a simultaneous switching-on of the initially quenched 19F MR signal (Fig 1a) and changed the ability of the cleaved Gd3+complex (Gd-DOM) to modulate the 1H MR signal intensity of the surrounding water (Fig 1b). 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 (in collaboration with MPI for Metabolism Research, Cologne). Another responsive 1H/19F MRI probes that have been recently developed by following this research line, are sensing the activity of matrix metalloproteinase (MMP-2), an enzyme overexpressed in almost every type of human cancer and being marker of inflammation. With proposed molecular design an efficient tool was provided for monitoring enzymes activity by means of 1H /19F MRI. Such a multimodality approach for enzyme detection is expected to integrate the advantages and conquer the individual limitations of the different imaging technologies.
Figure 1. 19F MRI images (left) and the corresponding proton T1 relaxation map (right image) acquired at 7 T (~25°C). Gd-DOMF-Gal (0.96 mM) was incubated in the absence or presence of ?-gal in PBS reaction buffer at 37°C for the indicated time periods.
Keliris A., Mamedov I., Hagberg G., Logothetis N.K., Scheffler K., Engelmann J., Contrast Media & Molecular Imaging, 7(5) 478483 (2012).
Cell tracking by 19F MRI
Stem cells based therapies endow modern medicine with a powerful tool for treatment of many severe diseases such as cancer, neurodegenerative disorders, acute CNS disorders or diabetes that immensely affect the life of millions of people worldwide today. The basic idea of such therapies is that cells provided with a specified function should act as a therapeutic drug after they are transferred into a living subject (i.e. restore damaged tissues, recognize inflammation sites, fight diseases or help to cure genetic disorders). Recent years have seen a huge progress in development of these therapies, particularly in preclinical work. Despite this showcased promise of cure, the effectiveness of cell therapies is still much far from being ideal and their translation into clinical practice proved to be very difficult. This is mainly due to the current inability to adequately answer the critical questions about the fate of the transplanted cells in vivo i.e. distribution, survival. To address this, highly efficient tools are needed that would enable to follow cells in vivo. Among various imaging techniques, MRI is nowadays a modality of choice for tracking stem cells longitudinally and non-invasively in vivo. This approach requires, however, high efficiency MRI probes that do not affect the function of the stem cells and at the same guarantee their specific, quantitative and long-term detection after transplantation. Recently, after a dominance era of iron oxide nanoparticles, the use of 19F MRI probes has emerged for in vivo cell tracking in order to overcome the drawbacks of signal ambiguity often faced with 1H MRI T2/T2* contrast agents, and enable reliable cell quantification.
Aim of this project is to provide tools for reliable evaluation of efficacy of cell-based therapies in vivo through developing multimodal 1H/19F MRI-optical probes for stem cells labeling and establishment of MR methods for their efficient and long-term detection after transplantation.
In this line, we have prepared 19F PLGA nanoparticles 19F PLGA-NPs (PLGA=poly(D,L-lactide-co-glycolide) of various compositions, with and without surface modifications, loaded with perfluoro-15-crown ether (PFCE) and/or optical dye (i.e. carboxyfluorescein), and/or Gd-DOTA like complex . In our extensive in vitro studies, human mesenchymal stem cells (hMSCs) as well as mouse CD4+ T-cells were efficiently labeled with fluorinated nanoparticles without affecting their properties (i.e. proliferation kinetics, colony generation, adhesion, surface migration, and presence of stem-cell markers). Further, MR methods were optimized in vitro for efficient detection of labeled cells and estimation of method sensitivity (Dr. Gisela E. Hagberg). Here, MRI detection limit for hMSCs cell load of 0.4x1012 19F/cell was 20.000 cells (pellet) or 10.000 cells per microliter (suspension) in a 2h scan. We are now on the way with in vivo testing of established methods, for instance in the treatment of urinary incontinence with hMSCs in collaboration with the University Hospital Tübingen.