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Myocardial disease and stroke continue to be the nation's leading killer and are responsible for nearly 1 million American deaths (42%) annually. Approximately 160,000 of these losses involve individuals between the ages of 35 and 64 years (1) for whom the current early-diagnosis techniques have little effectiveness. New early-detection strategies are needed to prevent a patient's first symptomatic presentation from being the last. Although a variety of invasive approaches can be used to characterize atherosclerotic plaques and follow their changes serially (2), these techniques are primarily confined to research studies due to the increased risk, cost, and time required.
We previously proposed that early recognition and quantification of microthrombi in ruptured plaques could provide an important biomarker to justify and guide aggressive therapeutic strategies to impede disease progression (3). We also reported the use of a lipid-encapsulated, perfluorocarbon nanoparticle system for molecular MRI of fibrin, an abundant component of thrombus. This agent affords specific detection of fibrin deposits using paramagnetic gadolinium chelates on the surface (4, 5). The highly amplified paramagnetic signal provides a robust way to image targeted nanoparticles; however, routine use requires the collection of pre- and postinjection images to determine signal changes.
Chemical exchange saturation transfer (CEST) agents have exchangeable protons (NH, OH, etc.) that resonate at a chemical shift that is distinguishable from the bulk water signal. RF prepulses applied at the appropriate frequency and power level can saturate the exchangeable protons, which transfer into the bulk water pool and lead to reduced equilibrium magnetization (6). Therefore, with the use of CEST agents one can switch the image contrast “on” and “off” by simply changing the pulse sequence parameters—an ability that is unique in the realm of MRI. This can minimize the time delays and motion-induced artifacts inherent in normal pre- and postcontrast imaging protocols. Although several agents contain exchangeable protons and can produce CEST contrast (7), the chemical shifts are often very close to the bulk water signal, which makes it difficult to distinguish contrast due to saturation contrast vs. direct saturation of the bulk water. Paramagnetic ions can be utilized to shift the bound water frequency further away from the bulk water, allowing distinct saturation of the exchangeable protons (8). These paramagnetic CEST (PARACEST) agents consist of paramagnetic chelates that are specifically designed to exhibit exchangeable proton or bound water peaks, and are emerging candidates for molecular imaging applications.
The supramolecular PARACEST agents reported to date have been symmetrical chelates coupled to cationic polymers through ion-pair attractions (9, 10) or entrapped within a liposomal vesicle (11). The objectives of this study were to 1) synthesize and demonstrate an asymmetric PARACEST chelate bifunctional-modified for chemical coupling to a lipid-encapsulated nanoparticle, 2) stably incorporate very high payloads of the lipid-conjugated PARACEST chelate into the nanoparticle surfactant, 3) measure the effectiveness of this PARACEST agent in suspension at 4.7T, and 4) demonstrate the first fibrin-targeted PARACEST agent in vitro.
MATERIALS AND METHODS
Chelate Synthesis and Nanoparticle Formulation
In brief, the PARACEST chelate was based on the 1,4,7,10-tetraaza macrocycle employing N-substituted glycine ethyl ester ligating moieties and a functionalized aromatic group for lipid conjugation (Dow Chemical Co.) (Fig. 1). Europium was added at equimolar concentrations to form the PARACEST construct, Eu3+-methoxy-benzyl-DOTA. A lipophilic tail, phosphatidylethanolamine (Avanti Polar Lipids, Inc.), was coupled to the compound through a thiourea linkage. Proton NMR spectroscopy was performed on the water-soluble chelate (i.e., nonlipid conjugated) at 4.7T and revealed a distinct bound water peak at 52 ppm (data not shown), consistent with a previously reported symmetric PARACEST chelate (12).
Perfluorocarbon nanoparticles were produced in a manner similar to that described in previous reports (4, 5). The nanoparticles were prepared by emulsifying 15% (v/v) perfluorooctylbromide (PFOB; Minnesota Manufacturing and Mining), 1% (w/v) surfactant comixture, and 2.5% (w/v) glycerin and water for the balance in a microfluidizer (Microfluidics, Inc.) for 4 min at 20000 psi. The PARACEST nanoparticle surfactant was comprised of phosphatidylcholine (Avanti Polar Lipids, Inc.), biotinylated dipalmitoylphosphatidylethanolamine (Avanti Polar Lipids, Inc.), and Eu3+-methoxy-benzyl-DOTA at a molar ratio of 59:1:40, respectively. Control nanoparticles lacked the europium chelate, which was substituted with an equivalent increase in phosphatidylcholine.
Particle sizes were measured using quasi-elastic light scattering and the zeta potential, based on an electrophoretic light-scattering/laser Doppler velocimetry method, in deionized water at 25°C with a Brookhaven ZetaPlus analyzer (Brookhaven Instrument Corp.). The europium content of the emulsion was determined by standard comparator instrumental neutron activation analysis at the University of Missouri Research Reactor (MURR). Specifically, Eu3+ was quantified by measuring the 842 keV gamma ray from the beta decay of 152mEu (t1/2 = 9.31 hr) produced through neutron capture by 151Eu. The samples and comparator standards were irradiated in a thermal flux of ∼5 × 1013 n/(s*cm2) for 60 s, allowed to decay for several hours, and counted on a high-resolution gamma-ray spectrometer for 30 min. The minimum detection limit for this procedure is 2 ng of Eu3+.
PARACEST Imaging and Spectroscopy
Imaging and spectroscopy studies were performed on a 4.7T Varian Inova scanner at room temperature (23°C) using a 3-cm-diameter custom-built circular surface coil. Bulk water spectra were collected from both PARACEST and control nanoparticle samples (50 μl) using a 2-s presaturation pulse at a power level of 28 dB. The saturation frequency was stepped between ±100 ppm (relative to the bulk water frequency at 0 ppm) in 1-ppm increments and the bulk water signal was integrated using a Matlab program (The MathWorks, Inc.). The difference between the integrated signals measured at equivalent positive and negative saturation frequencies was plotted, yielding saturation contrast profiles for the PARACEST and control nanoparticles.
The effectiveness of the PARACEST nanoparticles was compared with that of control particles using a two-chamber phantom constructed with an inner 1-cm-diameter chamber that contained the undiluted nanoparticle emulsion. The outer 1.8-cm-diameter chamber contained phosphate-buffered saline (PBS). Gradient-echo images of the two-chamber phantom were collected using a 2.5-s presaturation pulse at a power level of 38 dB with a frequency offset of ±52 ppm relative to the bulk water peak. Other imaging parameters were TR = 2.52 s, TE = 4.4 ms, number of averages = 8, in-plane resolution = 156 μm by 156 μm, and slice thickness = 4 mm. Image intensity was normalized with respect to the signal from the PBS chamber, images were subtracted pixel by pixel, and ST signal enhancement was calculated. The signal enhancement of PARACEST nanoparticles was also measured following presaturation pulses of 1-, 2-, or 3-s duration at power levels of 26, 29, 32, 35, or 38 dB using low-resolution imaging (TR = 3.1 s, TE = 2.9 ms, number of averages = 2, in-plane resolution = 390 μm by 390 μm, slice thickness = 4 mm).
The concept of a fibrin-targeted PARACEST nanoparticle was studied using cylindrical plasma clots suspended in sterile saline inside plastic snap-cap tubes. The acellular clots were formed in a 5-mm-diameter plastic mold that was prepared by combining fresh dog plasma, 100 mM calcium chloride (3:1 v/v), and 5 U thrombin around a 4-0 silk suture. Clots were serially incubated with 150 μg biotinylated antifibrin antibodies (1H10) (13–15) overnight at 4°C, followed by 50 μg avidin for 1 hr at 25°C, and then 250 μl of PARACEST (N= 5) or control (N = 4) nanoparticles for 1 hr at 25°C to complete the binding. The clots were rinsed three times with sterile saline after each incubation step to remove unbound reactants. Gradient-echo images of each clot were collected with identical parameters as used for the two-chamber phantom. The clot surface was manually traced in Matlab and the contrast-to-noise ratio (CNR) was calculated relative to the standard deviation of the image intensity in air. The tracing was repeated in triplicate for each clot by a single observer and averaged.
All of the studies followed a completely randomized design. Data were analyzed by Student's t-test or analysis of variance (ANOVA). The means were separated when appropriate using the least significant difference (LSD) method and declared significant at an alpha level of 0.05 using a beta level of 0.80. Unless otherwise specified, the averages are presented as the mean ± SEM.
Incorporation of the Eu3+-methoxy-benzyl-DOTA chelate into the nanoparticle surface produced an emulsion with physical characteristics similar to those of the control nanoparticles. PARACEST and control nanoparticles had similar diameters (294 nm and 337 nm) and polydispersity (0.215 and 0.093, respectively). These nominal particle sizes are modestly larger than previously reported (4, 5) due to the lower percentage of surfactant lipids and perfluorocarbon utilized in this formulation. The apparent stability and effectiveness of these particles were unaffected by the slight increase in size. The concentration of Eu3+ in the PARACEST nanoparticle emulsion was 2.1 mM, whereas the control nanoparticles contained no detectable Eu3+. The zeta potential of the control nanoparticles was −51.4 mV in deionized water, while the PARACEST nanoparticles were 53.8 mV, indicating that the Eu3+ chelate carries a significant positive charge.
PARACEST nanoparticles displayed a marked saturation contrast effect at a presaturation frequency of 52 ppm (Fig. 2). This corresponds to saturation of the bound water peak at 52 ppm, which is transferred into the bulk water and decreases the signal acquired at 0 ppm. However, the control nanoparticles did not show any appreciable saturation contrast at this frequency.
Images of the two-chamber phantom collected with the saturation pulse at −52 ppm showed very similar signal intensities for PARACEST nanoparticles, control nanoparticles, and PBS (Fig. 3). Subtraction of the images collected with saturation at +52 ppm from the −52 ppm images revealed uniform PARACEST enhancement in the inner chamber, which was not observed with either control nanoparticles or PBS. PARACEST nanoparticles provided an image enhancement of 11.17% ± 0.01% compared to the control nanoparticles, which provided very little change in contrast (0.53% ± 0.01%, * P < 0.05).
Increasing the duration and/or power of the presaturation pulse augmented the image enhancement obtained with PARACEST nanoparticles (Fig. 4). While the best results are obtained with a very long, high-power presaturation pulse, it is evident that even a 1-s pulse with a power of 35 dB could produce adequate signal change to be reliably detected in an image. Since the PARACEST enhancement continued to increase, it is likely that the bound water peak was never completely saturated under any of our experimental conditions.
Clot images collected with presaturation at −52 ppm appeared very similar regardless of the nanoparticle treatment (Fig. 5). The clots displayed a uniform hypointensity with respect to the surrounding PBS, most likely as a result of magnetization transfer (MT) from proteins and macromolecules in the clot interacting with bulk water. These interactions widened the water peak, resulting in some direct saturation of the bulk water peak even with the presaturation pulse offset by 52 ppm. Despite the MT effect, the surface of clots treated with fibrin-targeted PARACEST nanoparticles could be clearly distinguished after image subtraction. In contradistinction, the surface of clots treated with control nanoparticles could not be discriminated from noise upon subtraction. Averaged over the nine samples, the CNR at the clot surface was significantly higher for fibrin-targeted PARACEST nanoparticles (10.0 ± 1.0) compared to the control nanoparticles (2.2 ± 0.4; * P < 0.05).
This study describes the incorporation of a bifunctional, asymmetric PARACEST chelate into the surfactant of a fibrin-targeted perfluorocarbon nanoparticle for molecular imaging. Fibrin-targeted PARACEST nanoparticles provide a contrast mechanism that may be activated or deactivated by changing the offset frequency of the presaturation pulse, which allows easy detection of the targeted agent without the need to collect images before and after contrast administration. In contradistinction to liposomal-based agents, which create contrast through a chemical shift effect (11), the contrast mechanism of the fibrin-targeted particles is due to PARACEST interactions.
The importance of detecting and treating unstable plaque before further progression to myocardial infarction or stroke occurs cannot be overstated. Despite the clinical need for this information, the cost, risk, and time required to perform invasive techniques, such as intravascular ultrasound (16), thermography (17), and coherent optical tomography (18), have dampened the general enthusiasm for their use beyond the research community. Noninvasive MRI in conjunction with fibrin-targeted molecular imaging agents (3) is positioned to be a favored modality for the detection and characterization of unstable plaque; however, the current need for pre- and postcontrast imaging offset by 30–60 min is more complicated than desirable. One can imagine that in the future it will be possible to administer a fibrin-targeted PARACEST agent to a patient at the time of presentation and to image the patient any time over the next 30 min to 2 hr without interference from circulating contrast, and without having to perform a baseline scan before treatment. The practical implication is an earlier diagnosis and treatment, which would be available at lower cost to more patients per available scanner. These clear clinical advantages fuel the mounting research and development interest in PARACEST for molecular imaging.
MT contrast is commonly used in a variety of clinical MRI applications (19). Similarly to PARACEST, MT uses a presaturation pulse to suppress some of the signals in the body. While these techniques utilize similar pulse sequences, PARACEST requires a much larger frequency offset and is asymmetric with regard to the bulk water. These differences allow detection of PARACEST agents despite the underlying MT contrast. The hypointense appearance of the clots in Fig. 5 probably reflects MT due to the interactions of water molecules with clot proteins. However, presaturation at +52 ppm and −52 ppm has identical MT effects, which allows the image subtraction to cancel out the MT contributions. No contrast is observed on the subtraction images in areas with MT but no PARACEST particles, such as the clot interior.
While the techniques used in this study clearly demonstrate PARACEST enhancement with targeted nanoparticles, optimization of the chemistry, coil, and pulse sequence is needed to truly capitalize on the strengths of this agent. The Eu3+-methoxy-benzyl-DOTA chelate displayed a bound water peak both in solution and coupled to nanoparticles. However, we did not measure the exchange rate, and thus 4.7T may not be the optimum field strength. In addition, the cationic nature of the PARACEST particles in this study is not desirable in vivo because of the potential interaction with anionic proteins and membranes, which could introduce significant formulation, pharmacokinetic, pharmacodynamic, and biosafety challenges.
We utilized a very simple surface coil that provided efficient transmission of the saturation pulses. More uniform saturation and sensitivity, however, may be required for in vivo imaging of deep tissues. Moreover, a long, low-power presaturation pulse was inserted into a standard gradient-echo imaging sequence, which effectively saturated the bound water peak but did not optimize RF deposition or imaging efficiency. Sequences utilizing multiecho acquisitions or short saturation pulse trains may shorten the imaging times and reduce RF deposition. Given the early stage of PARACEST contrast agent development, further research into the chemistry, in vivo particle clearance, and several imaging hardware issues is needed. With optimization of the agent and imaging hardware, comparisons between various agents, including CEST agents and conventional paramagnetic or superparamagnetic agents (3), could be performed for specific molecular imaging applications.
This report describes a targeted PARACEST nanoparticle agent with developmental potential for detecting fibrin in unstable atherosclerotic plaques. This agent is particularly attractive because it can be activated through the pulse sequence parameters of the MRI scanner, and thus the contrast agent can effectively be turned on and off at will. Targeted PARACEST nanoparticles may allow robust detection of contrast agents at a single time point, as opposed to comparing images collected pre- and postinjection, which would have significant practical benefits in the clinic.
The authors thank Dr. Joseph J.H. Ackerman, Professor and Chair, Department of Chemistry, and Director of the Biomedical MR Laboratory at Washington University, for technical assistance and support.