High-throughput screening of chemical exchange saturation transfer MR contrast agents

Abstract

A new high-throughput MRI method for screening chemical exchange saturation transfer (CEST) agents is demonstrated, allowing simultaneous testing of multiple samples with minimal attention to sample configuration and shimming of the main magnetic field (B(0)). This approach, which is applicable to diamagnetic, paramagnetic and liposome CEST agents, employs a set of inexpensive glass or plastic capillary tubes containing CEST agents put together in a cheap plastic tube holder, without the need for liquid between the tubes to reduce magnetic susceptibility effects. In this setup, a reference image of direct water saturation spectra is acquired in order to map the absolute water frequency for each volume element (voxel) in the sample image, followed by an image of saturation transfer spectra to determine the CEST properties. Even though the field over the total sample is very inhomogeneous due to air-tube interfaces, the shape of the direct saturation spectra is not affected, allowing removal of susceptibility shift effects from the CEST data by using the absolute water frequencies from the reference map. As a result, quantitative information such as the mean CEST intensity for each sample can be extracted for multiple CEST agents at once. As an initial application, we demonstrate rapid screening of a library of 16 polypeptides for their CEST properties, but in principle the number of tubes is limited only by the available signal-noise-ratio, field of view and gradient strength for imaging.

Figure 1

Example of a high throughput sample arrangement. A) Picture of a phantom consisting of multiple capillaries immobilized in a sample holder. B) Cartoon showing anisotropic arrangement of 18 capillaries in the holder in order to easily identify the relative position of each tube in the MR images. C) An axial T₂ weighted MR image, and D) the corresponding B₀ shift map across the FOV for this phantom.

Figure 2

Correction of B₀ inhomogeneity for a HT-CEST phantom. A) Phantom of 7 capillaries containing myo-inositol solutions (31.2 mM in PBS) with pH ranging from 5.0 to 7.8; B) Water spectra of the entire phantom under shimmed (blue line) and deshimmed (red line) conditions, C) WASSR spectra for the sample in the yellow box (pH=5.0 tube) in (A) under shimmed and deshimmed conditions; D) Water spectra for yellow box under shimmed and deshimmed conditions; E) pH = 5.0 MTR_asym curves for shimmed and deshimmed conditions before (solid line) and after (dashed line) B₀ correction; F) B₀-corrected pH dependency of myo-inositol CEST effects (shimmed, blue circles; deshimmed, red squares). Error bars estimated using the inter-voxel standard deviations of the ~60 voxels contained in each tube.

Figure 3

Spatial variability and reproducibility of the proposed HT CEST method for six samples each of protamine sulfate (0.2 mM (▲) and 0.98 mM (□) in PBS, pH = 7.3). A) Cartoon displaying the distribution of tubes. B) Mean MTR_asym for each sample, with error bars showing the standard deviations of 5 measurements, C) Mean MTR_asym of 5 repeated independent CEST experiments with a 3 hour interval between them, with error bars showing the standard deviations over the 6 samples.

Figure 4

The CEST properties of 20 mM uracil in PBS as a function of pH measured simultaneously using the high-throughput phantom. A) CEST contrast at 5.4 ppm as displayed by subtracting the CEST weighted image at −5.4ppm from CEST weighted image at +5.4ppm and B) the plot of MTR_asym as the function of pH. Error bars represent the inter-voxel standard deviations in each capillary.

Figure 5

High-throughput scanning of the CEST effect of 16 twelve-residue polypeptides. (A–C) CEST profiles of (DSSS)₃ (DTTTTT)₂, and (RT)₆. Figures D–F show the contrast at the OH (0.8 ppm, blue) NH₂ (1.8 ppm, red), and NH (3.6 ppm, green) proton frequencies, respectively. The brightness corresponds to the CEST intensity at each offset. These three-color images were overlaid to produce an ‘artificial’ RGB colormap (G) with the color corresponding to the summation of CEST effects at the three frequencies. In this procedure, more than 3 peptides therefore can be uniquely identified by their CEST ‘footprint’ in the RGB spectrum. The polypeptides were laid out as shown in the T_2W image (H) 1:(KS)₆, 2: (KSSS)₃, 3: (DSSS)₃, 4: (DSSSSS)₂ , 5: (DTT)₄, 6: (DTTT)₃, 7: (DTTTTT)₂, 8: (ETT)₄, 9: (ETTT)₃, 10: (ETTTTT)₂,11: (TK)₆, 12: (TTK)₄, 13:(TTTK)₃, 14: (TTTTTK)₂, 15: (RT)₆, 16: (RTTT)₃. The three unlabeled tubes contained only 10mM phosphate buffered saline. K = lysine, S = Serine, R = Arginine, T = Threonine, E = Glutamate, D = Aspartate.