New "multicolor" polypeptide diamagnetic chemical exchange saturation transfer (DIACEST) contrast agents for MRI

Abstract

An array of 33 prototype polypeptides was examined as putative contrast agents that can be distinguished from each other based on the chemical exchange saturation transfer (CEST) mechanism. These peptides were chosen based on predictions of the chemical exchange rates of exchangeable amide, amine, and hydroxyl protons that produce this contrast, and tested at 11.7T for their CEST suitability. Artificial colors were assigned to particular amino acid units (lysine, arginine, threonine, and serine) based on the separate resonance frequencies of these exchangeable protons. The magnitude of the CEST effect could be fine-tuned by altering the amino acid sequence, and these three exchangeable groups could be distinguished in an MR phantom based on their different chemical shifts ("colors"). These new diamagnetic CEST (DIACEST) agents possess a wide range of electrostatic charges, compositions, and protein stabilities in vivo, making them potentially suitable for a variety of biological applications such as designing MR reporter genes for imaging cells and distinguishing multiple targets within the same MR image.

Figure 1

Chemical structure of three CEST peptides with arrows denoting chemical exchange between neighboring protons. A) Gly Lys Gly Lys peptide. B) His Arg His Arg peptide. C) Asp Thr Asp Thr peptide.

Figure 2

B₀ frequency dependence of CEST contrast for PLK (30 kD, ), PLR (35 kDa, ) and PLT (7.6 kD, ) using ω₁= 2.2 μT. a) Intensity comparison of z-spectra and MTR_asym spectra . b) Expanded z spectra from a. b) MTR_asym spectra from a normalized to their highest intensity.

Figure 3

Optimization of saturation field strength and pH for the best sensitivity for the CEST peptides PLK(▲), PLR(□), and PLT(◇).a) Saturation field strength dependence of the CNR at pH = 7.3. B) Same as a, except CNR scale changed to highlight PLT. c) pH dependence of the PTE for PLR examining both NH(■) and gNH₂(□) frequencies at saturation ω₁ = 4.7 μT. c) pH dependence of the PTE for the NH (◆) and OH (◇) frequencies in PLT at saturation ω₁ = 4.7 μT.

Figure 4

Comparison of experimental vs. predicted PTE ‘s for the 12 residue peptides. Comparison for all peptides, NH PTE (•) and best fit(-). Slope =1.1, R=0.71.

Figure 5

Comparison of molar PTE values for all peptides at saturation ω₁ = 4.7 μT. One letter abbreviations are used for the amino acids (Lys=K, Arg=R, Thr=T, Ser=S, Gly=G, His=H, Asp=D, Glu=E). a) Comparison of PTE for NH color (), gNH₂ color (), OH color (). b) Comparison of gNH₂ color peptides, showing the gNH₂ (), NH , and OH () PTE. C) Comparison OH color peptides, showing the NH (), gNH₂ () and OH () PTE.

Figure 6

Phantom images using 3 different saturation frequencies. The phantom consists of 1 mm tubes inserted in a 5mm NMR tube, filled with 2.5 mg/ml peptides as outlined in (A). a) Proton density image. b) MTR_asym (±3.69 ppm) image. c) MTRasym (±1.8 ppm) image. d) MTR_asym (±0.8 ppm) image. e) Merged image from the three label channels. f) NH label from the difference between images in b, d after normalizing the maximum signal in the image. g) gNH2 channel from c after normalization the maximum signal in the image. h) OH channel from the difference between normalized images c, d.