Dextran-conjugated tetrathiatriarylmethyl radicals as biocompatible spin probes for EPR spectroscopy and imaging

Abstract

Tetrathiatriarylmethyl (TAM) radicals represent soluble paramagnetic probes for biomedical electron paramagnetic resonance (EPR)-based spectroscopy and imaging. There is an increasing demand in the development of multifunctional, biocompatible and targeted trityl probes hampered by the difficulties in derivatization of the TAM structure. We proposed a new straightforward synthetic strategy using click chemistry for the covalent conjugation of the TAM radical with a water-soluble biocompatible carrier exemplified here by dextran. A set of dextran-grafted probes varied in the degrees of Finland trityl radical loading and dextran modification by polyethelene glycol has been synthesized. The EPR spectrum of the optimized macromolecular probe exhibits a single narrow line with high sensitivity to oxygen and has advantages over the unbound Finland trityl of being insensitive to interactions with albumin. In vivo EPR imaging of tissue oxygenation performed in breast tumor-bearing mouse using dextran-grafted probe demonstrates its utility for preclinical oximetric applications.

Keywords: Electron paramagnetic resonance; Spin probes; Trityl radicals.

Figure 1

Representative structures of TAM radicals.

Figure 2

¹³C NMR spectrum of azidified dextran. Ratio of integrals of the peaks 9 and 1 gives the fraction of modification of sugar units by azide groups. Acquisition parameters: 100 MHz, D₂O, time delay=15 s, 8000 scans. Insert: schematic structure of azidified dextran showing α−1,6 glycosidic linkages between glucose monomers with branches from α−1,3 linkages. The numbering of the carbon in the dextran structure corresponds to the corresponding peak numbering in the NMR spectrum.

Figure 3

HPLC chromatograms of (A) TAM mono-propargyl ester radical and (B) dextran grafted with TAM radicals and PEG chains. Inserts: the UV spectrum of the main peak for each chromatogram (See SI for details).

Figure 4

L-band (1.2 GHz) EPR spectra of Dextran-TAM_3.6PEG_6.4 (A) and Dextran-TAM_6.5PEG_3.5 (B) spin probes, and the corresponding dependences of their linewidth on oxygen concentration, (C) and (D). The spectra were measured for 0.5 ml samples of 65 µM solutions of the probes in deoxygenated 100 mM phosphate buffer in the absence (red lines) and presence (black lines) of 500 µM bovine serum albumin. The values of the peak-to-peak linewidth, ΔH_pp, are shown near the spectra. The linear fits of the linewidth dependences on oxygen yield the spectral sensitivities to oxygen being equal to 2.7 mG/(% O₂) (C) and 2.6 mG/(% O₂) (D). The spectrometer settings were as follows: sweep time, 30 sec, sweep width, 480 mG; modulation amplitude, 30 mG; modulation frequency, 100 kHz; power attenuation, 15 dB.

Figure 5

In vivo 4D (1-spectral-3-spatial) rapid scan 800 MHz EPR image of oxygen distribution in a breast tumor of a PyMT tumor-bearing mouse. An image acquisition was started 25 minutes after intratissue injection of 20 µl solution of 0.75 mM probe in 10 mM phosphate buffered saline; tumor volume, 214 mm³. Data acquisition parameters were as follows: acquisition time, 16.5 minutes; number of projections, 2546; rapid scan frequency, 9.4 kHz; and maximum gradient, 3G/cm. An integral intensity threshold of 30% was implemented to remove low signal-to-noise data. A. A two-dimensional slice (xz-plane) of the image is shown. B. A histogram of pO₂ distribution within the entire image.

Scheme 1

Schematic representation of the synthesis of dextran-conjugated TAM radicals using a click chemistry approach. The dextran grafting by the dFTr radical was achieved by copper-catalyzed azide-alkyne cycloaddition (CuAAC) of a TAM mono-propargyl ester followed by addition of excess of alkyne-PEG. Varying the trityl radical/dextran ratio allows for the tuning of the radical loading onto the polymer. Incorporation of PEG chains enhances the solubility of the grafted dextran and prevents spin-spin interactions between trityl radicals.

Scheme 2

Synthesis of TAM mono propargyl ester (See SI for details).