In Vivo Proton–Electron Double-Resonance Imaging of Extracellular Tumor pH Using an Advanced Nitroxide Probe

Abstract

A variable radio frequency proton–electron double-resonance imaging (VRF PEDRI) approach for pH mapping of aqueous samples has been recently developed (Efimova et al. J. Magn. Reson. 2011, 209, 227–232). A pH map is extracted from two PEDRI acquisitions performed at electron paramagnetic resonance (EPR) frequencies of protonated and unprotonated forms of a pH-sensitive probe. To translate VRF PEDRI to an in vivo setting, an advanced pH probe was synthesized. Probe deuteration resulted in a narrow spectral line of 1.2 G compared to a nondeuterated analogue line width of 2.1 G allowing for an increase of Overhauser enhancements and reduction in rf power deposition. Binding of the probe to the cell-impermeable tripeptide, glutathione (GSH), allows for targeting to extracellular tissue space for monitoring extracellular tumor acidosis, a prognostic factor in tumor pathophysiology. The probe demonstrated pH sensitivity in the 5.8–7.8 range, optimum for measurement of acidic extracellular tumor pH (pHe). In vivo VRF PEDRI was performed on Met-1 tumor-bearing mice. Compared to normal mammary glands with a neutral mean pHe (7.1 ± 0.1), we observed broader pH distribution with acidic mean pHe (6.8 ± 0.1) in tumor tissue. In summary, VRF PEDRI in combination with a newly developed pH probe provides an analytical approach for spatially resolved noninvasive pHe monitoring, in vivo.

In Vivo Application of Proton-Electron Double-Resonance Imaging

“…To see this illustration in color, the reader is referred to the web version of this article at www. liebertpub.com/ars equal to 6.75 -0.05 in agreement with the EPR-measured pK a value, 6.74 -0.05 (108). Figure 17E, F demonstrates the application of VRF PEDRI for pH e mapping of living tissue in the tumor-bearing mouse by using the NR2 radical.…”

“…The bulky ethyl substitutes at positions 2 and 5 of the radical heterocycle of the NR2 serve to protect the paramagnetic NO fragment against bioreduction, significantly enhancing its biostability. Extracellular localization of the probe has been achieved by binding the nitroxide label to hydrophilic tripeptide, glutathione, preventing the resulting NR2 probe from diffusion across the hydrophobic lipid bilayer of the biomembranes and, therefore, enforcing probe localization in extracellular aqueous volumes (108). Figure 17A-D shows the pH calibration of the NR2 probe using VRF PEDRI obtained for a 10-tube phantom (Fig.…”

Development and Application of Spin Traps, Spin Probes, and Spin Labels

“…Nitroxide-based labels allow obtaining unique information on functioning proteins, in particular on hydrogen bonding, polarity effects of microenvironment of protein sites involved in the transfer process (M€ obius et al, 2005), pH/pK determination (Voinov, Kirilyuk, & Smirnov, 2009) including extracellular tumor pH measurements in vivo (Samouilov et al, 2014), etc. There are numerous examples of SDSL combined with EPR: the study of the light-induced electron and proton transfer processes across photosynthetic membranes or ion channel formation of bacterial toxins, probing the interfacial surface properties of thiol-protected gold nanoparticles, mapping local protein electrostatic interactions using pH-sensitive thiol-specific nitroxides, and many others.…”

“…Nitroxide 5 possesses physiologically relevant рK 7.4 with 14 N HFI constant changing by $1 Gs and is relatively stable (Kirilyuk et al, 2004). Nitroxides 6 and 7 are most useful for the application in vivo and ex vivo (Bobko et al, 2012;Samouilov et al, 2014); (iii) sterically substituted pyrroline and pyrrolidine nitroxides with very high stability against reduction in biological media (8-10). Nitroxide 8 has low reduction rate by ascorbate 8 Â 10 À3 M À1 s À1 and two functional groups that can be modified to attach 8 to proteins (Morozov et al, 2012).…”

“…It can be used for obtaining information on local polarity using high-field EPR, for investigation of the surface potential and polarity of phospholipid membranes and proteins; (v) spirocyclohexane-substituted nitroxides showing high stability in reducing media, allowing PELDOR measurements at relatively high temperatures (150-300 K) and useful in the synthesis of dendrimeric contrast MRI agents (12-14) (Kathirvelu et al, 2009;Kinoshita et al, 2009;Kirilyuk et al, 2012;Meyer et al, 2015;Okazaki et al, 2007;Rajca et al, 2012). Recently, a convenient way of synthesis of 4-amino-2,5-dihydroimidazole-1-oxyls based on reaction of 5-amino-4H-imidazole-3-oxides with Grignard compounds was proposed by Kirilyuk and coworkers (Samouilov et al, 2014;Voinov et al, 2009;Scheme 9). It allows one to synthesize derivatives with different substituents at exocyclic nitrogen atom and at position 2 of heterocycle, as well as sterically hindered nitroxide radicals with high stability.…”