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

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.

PARACEST MRI with improved temporal resolution.

PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST) is a novel contrast mechanism for MRI. A PARACEST MRI methodology with high temporal resolution is highly desired for in vivo MRI applications of molecular imaging. To address this need, a strategy has been developed that includes a long selective saturation period before each repetition of a Rapid Acquisition with Relaxation Enhancement (RARE) pulse sequence. This strategy is suitable for the application of PARACEST contrast agents to environments with long T1 relaxation times. An alternative strategy uses short selective saturation periods before the acquisition of each k-space trajectory to maintain steady state conditions, which can be implemented with a Fast Low Angle Shot (FLASH) pulse sequence. These short saturation periods lengthen the total scan time as compared to the first approach but compensate for the loss in PARACEST contrast related to T1 relaxation. Both approaches have been demonstrated in vitro and in vivo with significantly improved temporal resolutions as compared to a conventional gradient-echo PARACEST method without sacrificing CNR efficiency. These demonstrations also adopted a strategy for measuring the PARACEST effect that only requires selective saturation at a single MR frequency, which further improves temporal resolution for PARACEST detection.

Tracking the relative in vivo pharmacokinetics of nanoparticles with PARACEST MRI.

A noninvasive assay that tracks the relative in vivo pharmacokinetics of two nanoparticles may accelerate the development of nanoparticles for biomedical applications, and may provide a method to select personalized nanomedicines for individual patients. To develop an in vivo competitive assay, two MRI contrast agents that could be selectively detected through paramagnetic chemical exchange saturation transfer (PARACEST) were conjugated to a second generation and fifth generation polyamidoamine (PAMAM) dendrimer. The CEST effects of each agent was calibrated relative to concentration. The effects of T(1) relaxivities of these dendritic PARACEST magnetic resonance imaging (MRI) contrast agents were found to be negligible relative to their CEST effects with respect to changes in image contrast, which facilitated the measurement of the ratios of their chemical exchange lifetimes. Injection of both contrast agents into a mouse model of mammary carcinoma resulted in a temporal increase in the CEST effect from each agent in the flank tumor. Although the in vivo CEST effects could not be used to determine the absolute concentrations of each agent within the tumor, the ratio of the in vivo CEST effects was used to measure the ratio of the concentrations of the agents. This result demonstrated that the relative in vivo pharmacokinetics of two nanoparticles may be evaluated using PARACEST MRI.

Synthesis and relaxometric studies of a dendrimer-based pH-responsive MRI contrast agent.

The design of effective pH responsive MRI contrast agents is a key goal in the development of new diagnostic methods for conditions such as kidney disease and cancer. A key factor determining the effectiveness of an agent is the difference between the relaxivity of the "on" state compared to that of the "off" state. In this paper, we demonstrate that it is possible to improve the pH-responsive action of a low molecular weight agent by conjugating it to a macromolecular construct. The synthesis of a bifunctional pH responsive agent is reported. As part of that synthetic pathway we examine the Ing-Manske reaction, identifying an undesirable by-product and establishing effective conditions for promoting a clean and effective reaction. Reaction of the bifunctional pH responsive agent with a G5-PAMAM dendrimer yielded a product with an average of 96 chelates per dendrimer. The relaxivity of the dendrimer conjugate rises from 10.8 mM(-1) s(-1) (pH 9) to 24.0 mM(-1) s(-1) (pH 6) per Gd(3+) ion. This more than doubles the relaxivity pH response, Deltar(1), of our agent from just 51 % for the original low molecular weight chelate to 122 % for the dendrimer.

Albumin-binding PARACEST agents.

Lanthanide complexes (Eu(3+), Gd(3+) and Yb(3+)) of two different 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tetraamide derivatives containing two (2) and four (3) O-benzyl-L-serine amide substituents were synthesized and their chemical exchange saturation transfer (CEST) and relaxometric properties were examined in the presence and absence of human serum albumin (HSA). Both Eu2 and Eu3 display a significant CEST effect from a single slowly exchanging Eu(3+)-bound water molecule, making these PARACEST complexes potentially useful as vascular MRI agents. Yb2 also showed a detectable CEST effect from both the Yb(3+)-bound water protons and the exchangeable NH amide protons, making it potentially useful as a vascular pH sensor. Fluorescence displacement studies using reporter molecules indicate that both Gd2 and Gd3 displace dansylsarcosine from site II of HSA with inhibition constants of 32 and 96 microM, respectively, but neither complex significantly displaces warfarin from site I. Water proton relaxation enhancements of 135 and 171% were observed upon binding of Gd2 and Gd3 to HSA, respectively, at 298 K and pH 7.4.