Figure 4:

Effect of NEM pre-treatment on AMUPol reduction, cellular viability, and DNP performance. A) Pre-treatment of cells with NEM slows cellular reduction of AMUPol. HEK293 cells were incubated with varying concentrations of NEM in PBS which was removed before the introduction of AMUPol by electroporation. Total nitroxide concentration was monitored by EPR for cells. B) AMUPol EPR spectra for cells that were pre-treated with 2.5 mM NEM (orange) or PBS (gray) at the earliest time point and after an hour of room temperature incubation. C) Pre-treatment of cells with NEM compromises cellular membrane integrity. The trypan blue permeability decreased from 90 ± 11 % for cells incubated with 0 or 1 mM NEM to 11 ± 5% after incubation in 2.5 mM NEM. This change was significant (student’s t-test, * p < 0.05, *** p < 0.001). D) Cells were unable to grow after pre-treatment with NEM. E) The DNP performance for control cells was similar across the major biomass components. Peaks for the major biomass components are indicated by arrow heads for protein, nucleotide, and lipids (green, blue, and pink, respectively) for the microwaves on and microwaves off ¹³C CP spectra. F) Despite protection of the radical from reduction by 2.5 mM NEM pre-treatment, while DNP performance was similar across the major biomass components, the DNP enhancements were low. G) NEM pre-treatment does not result in differences in the ¹³C CP spectra for the cellular biomass.