Using two chemical exchange saturation transfer magnetic resonance imaging contrast agents for molecular imaging studies

Abstract

Responsive magnetic resonance imaging (MRI) contrast agents can change MR image contrast in response to a molecular biomarker. Quantitative detection of the biomarker requires an accounting of the other effects that may alter MR image contrast, such as a change in the agent's concentration, magnetic field variations, and hardware sensitivity profiles. A second unresponsive MRI contrast agent may serve as an "internal control" to isolate the detection of the molecular biomarker. Chemical exchange saturation transfer (CEST) MRI contrast agents can be selectively detected, providing the opportunity to combine a responsive CEST agent and an unresponsive CEST agent during the same MRI scan session. When two CEST MRI contrast agents are used for molecular imaging applications, the CEST agents should be designed to maximize accurate quantification of the concentrations of the two agents. From a chemical perspective, CEST agents behave like enzymes that catalyze the conversion of an unsaturated water "substrate" into a saturated water "product". The analysis of CEST agent kinetics parallels the Michaelis-Menten analysis of enzyme kinetics, which can be used to correlate the CEST effect with the concentration of the agent in solution. If the concentration of water "substrate" that is available to the CEST agent is unknown, which may be likely for in vivo MRI studies, then only a ratio of concentrations of the two CEST agents can be measured. In both cases, CEST agents should be designed with minimal T(1) relaxivity to improve concentration quantifications. CEST agents can also be designed to maximize sensitivity. This may be accomplished by incorporating many CEST agents within nanoparticles to create a large number of exchangeable protons per nanoparticle. Finally, CEST agents can be designed with rapid detection in mind. This may be accomplished by minimizing T(1) relaxivity of the CEST agent so that MRI acquisition methods have time to collect many MRI signals following a single selective saturation period. In this Account, we provide an example that shows the sensitive and rapid detection of two CEST agents in an in vivo MRI study of a mouse model of mammary carcinoma. The ratio of the concentrations of the two CEST agents was quantified with analysis methods that parallel Michaelis-Menten enzyme kinetic analysis. This example demonstrates current limitations of the method that require additional research, but it also shows that two CEST MRI contrast agents can be detected and quantitatively assessed during in vivo molecular imaging studies.

FIGURE 1

The CEST (A) two-pool model and (B) multiple-pool model. [H₂O]_DS indicates water that is directly saturated; [H₂O]_MT indicates water that undergoes chemical exchange with selectively saturated protein; [H₂O]_I indicates water that is inaccessible to the CEST agent due to biological compartmentalization; [H₂O]_UCA indicates water that exchanges protons with unsaturated CEST agent; [H₂O]_UP indicates water that exchanges protons with unsaturated proteins; [H₂O]_A indicates apparent water concentration that can be saturated by the saturated CEST agent.

FIGURE 2

The reaction schematics of (A) Michaelis–Menten enzyme kinetics and (B) CEST are similar. Terms are defined in eqs 1, 3, and 4; n_CA and n_H2O are omitted for clarity.

FIGURE 3

The (A) Lineweaver–Burke-like CEST plot, (B) Eadie–Hofstee-like CEST plot, and (C) Hanes-like CEST plot were simulated for a CEST agent that generates 5% CEST at 1 mM concentration. The thick line shows the case with no systematic error, and the thin lines represent the effect of adding +1% or −1% to the CEST measurement. (D–F) A CEST–concentration calibration can be constructed from each of these linear relationships. (G–I) The same CEST–concentration calibrations as graphs D–F, but ranging from 0 to 1 mM. These graphs show that the calibration is most accurately determined from the Hanes-like CEST plot, because the thick and thin lines are indistinguishable in graph I.

FIGURE 4

(A) The Hanes-like CEST plot and (B) CEST–concentration calibration for (C) Yb–1,4,7,10-tetraazacyclododecane-N,N′,N″-triacetic acid-N‴-orthoaminoanilide (Yb–DO3A-oAA). The molecular model was constructed using CS MOPAC Pro v. 8.0 within Chem3D Ultra v. 8.0. A metal atom was inserted in the binding pocket of the model, and metal–ligand distances were constrained during final energy minimization with the MM2 force field.

FIGURE 5

(A) The procedure for synthesizing (Yb–DOTA-Gly-pBnNCS)₆–G2PAMAM (Yb–G2). (Eu–DOTA-Gly-pBnNCS)₄₁–G5PAMAM (Eu–G5) was synthesized using the same procedure. (B) A Hanes-like CEST plot was used to generate a CEST–concentration calibration (C) for Eu–G5 and (D) for Yb–G2.

FIGURE 6

A Z-spectrum and a NMR spectrum of Tm–DOTA-Gly.

FIGURE 7

(A) A spin density weighted MR image of a MCF-7 tumor on a mouse flank, with phantoms of 40 mM (Eu–DOTA-Gly)₄₁–G5PAMAM contrast agent and water. (B) Fifty microliters of 40 mM (Eu–DOTA-Gly)₄₁–G5PAMAM was directly injected into the tumor. Twenty CEST–FISP MRI images with a range of saturation frequencies were acquired with a 20 μT saturation period for 2.25 s prior to a FISP MRI acquisition. Z-spectra were constructed from the CEST effect in each displayed image region. To account for magnetization transfer effects, the right side of the Z-spectrum of the tumor was interpolated and mirrored to the left side of the spectrum. This interpolation method is affected by the data sampling, so this analysis method only approximates magnetization transfer effects. Lorentzian line-fitting of each spectrum revealed that the CEST effects from the tumor and agent phantom both occurred at 48.4 ppm. The total acquisition required 200 s.

FIGURE 8

(Eu–DOTA-Gly)₄₁–G5PAMAM (Eu–G5) and (Yb–DOTA-Gly)₆–G2PAMAM (Yb–G2) were injected i.v. at 0.03 mmol of dendrimer per kilogram of mouse body weight into a xenograft flank mouse model of MCF-7 mammary carcinoma. (A) MR images were acquired by prepending a 20 μT saturation period for 2.25 s before a RARE-16 MR signal acquisition period. Selective saturation was applied at the PARACEST frequency of each agent. (B) The decrease in MR signal of the tumor after injection relative to the average MR signal before injection was used to measure the temporal change in the CEST effect. (C) Equation 11 was used to calculate the concentration ratio of the CEST agents.