Development of enzyme-responsive multimodal probes

Goal: The aim of this project is to develop a versatile multimodal imaging platform for monitoring the enzyme activity that would allow assessment of cellular events associated with gene expression and monitoring neuronal stem cells after their transplantation.

An enormous number of biochemical processes taking place in living organisms demands the action of enzymes. These highly specific essential catalysts serve as indicators of diseases (e.g. stroke, brain tumors), cellular processes and are used as essential markers of gene expression. Hence, real-time non-invasive in vivo mapping of enzyme activity can provide a means for the assessment of disease processes, evaluation of novel therapies (e.g. gene and neuronal stem cell therapies), and also better understanding of biochemical events vital for sustaining life. More specifically, we are developing probes that upon interaction with β-galactosidase enzyme, expressed by LacZ reporter gene, produce the characteristics detectable by ¹H/¹⁹F MRI and optical imaging modalities. Such a multimodality approach for enzyme detection is expected to integrate the advantages and conquer the individual limitations of the different imaging technologies.

Methods

Dual-modal β-gal targeting imaging probes were obtained in a multi-step synthesis and fully characterized by analytical methods. Proton T₁, T₂ relaxation times in probe solutions and cellular relaxation rates of labeled cells were determined at 123 MHz and 300 MHz. ¹⁹F T₁, T₂ relaxation times and ¹⁹F images were acquired at 282 MHz. Cellular uptake of probes in C6/LacZ (β-gal containing) and C6 (no target) glioma cells was investigated by fluorescence spectroscopy, microscopy and MR measurements of labeled cells.

Results

Our initial work towards developing β-gal activable dual-modal probes focused on cell-permeable MR/optical probes whose activation by enzyme was based on a cellular retention strategy of imaging reporters in the β-gal expressing cells. This retention was achieved via enzymatic cleavage of a delivery vector following Gd-DOTA-k(FITC)-Gal-CPP probe internalisation. Although higher accumulation of imaging reporters in C6/LacZ cells compared to the C6 cells was shown [1], providing the proof of principle for the proposed strategy, a non-specific background signal in the β-gal-deficient cells was also observed due to predominantly vesicular localisation of unconverted probe restricting its fast efflux from these cells.To provide further advances and overcome the problem of non-specific background signal we are currently focusing on developing of β-gal responsive probes that show turn- on signal only upon enzymatic conversion. The first representative of this series, dual-modal ¹⁹F and ¹H MRI probe (Gd-DOMF-Gal) [2] with a self-immolative linker was synthesized. The efficient enzymatic conversion of Gd-DOMF-Gal by β-gal resulted in the simultaneous turning-on of the initially quenched ¹⁹F MRI 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). With this molecular design we provided an efficient tool for monitoring β-gal activity by means of ¹H and ¹⁹F MRI.

Conclusions

The presented here results indicate that our bimodal probes could be specifically activated in the presence of targeted enzyme. Further, Gd-DOMF-Gal as ¹H/¹⁹F MRI probe with its remarkable characteristics will be now tested in vivo. Further developments towards novel probes responsive to other enzymes are also in progress.

References:

Keliris A., Ziegler T., Mishra R., Pohmann R., Sauer M., Ugurbil K., Engelmann J.: Biorganic & Medicinal Chemistry 19(8) 2529-2540 (2011).

Keliris A., Mamedov I., Hagberg G., Logothetis N.K., Scheffler K., Engelmann J., Contrast Media & Molecular Imaging, under revision.

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 β-gal in PBS reaction buffer at 37°C for the indicated time periods.

Development of catecholamine-sensing MRI probes

Goal: The goal of this project is to develop a new class of catecholamine-sensing MRI-detectable probes (contrast agents). The ability of such probes to affect the relaxation times in their surroundings is reversibly modulated in response to the dynamic changes in extracellular levels of catecholamine (dopamine or norepinephrine) during task dependent neuronal activity.

Catecholamines such as dopamine or norepinephrine are principal neurotransmitters that act as the chemical messengers of information between different brain cells. These communication agents mediate various central nervous system functions such as cognition, motor control, emotion, reward or memory processing. The ability to detect the fluctuation in catecholamine concentration would provide means for direct monitoring of neuronal processing, but also can be of critical importance for understanding the dysfunctions in catecholamine systems connected with several neurologic and neuropsychiatric disorders (e.g. Parkinson’s disease, depression). In this line, we are currently developing dopamine-responsive MRI probes based on the lanthanide complexes with specific recognition sites. The presence of catecholamine would induce changes in the observed MR signal as the result of modulation of parameters governing the relaxivity of contrast agents such as hydration number (model A) or rotational correlation time (model B).