CEST Properties Research Articles

Lanthanide‐Based Probes for Imaging Detection of Enzyme Activities by NIR Luminescence, T1‐ and ParaCEST MRI

AbstractApplying a single molecular probe to monitor enzymatic activities in multiple, complementary imaging modalities is highly desirable to ascertain detection and to avoid the complexity associated with the use of agents of different chemical entities. We demonstrate here the versatility of lanthanide (Ln3+) complexes with respect to their optical and magnetic properties and their potential for enzymatic detection in NIR luminescence, CEST and T1 MR imaging, controlled by the nature of the Ln3+ ion, while using a unique chelator. Based on X‐ray structural, photophysical, and solution NMR investigations of a family of Ln3+ DO3A‐pyridine model complexes, we could rationalize the luminescence (Eu3+, Yb3+), CEST (Yb3+) and relaxation (Gd3+) properties and their variations between carbamate and amine derivatives. This allowed the design of probes which undergo enzyme‐mediated changes detectable in NIR luminescence, CEST and T1‐weighted MRI, respectively governed by variations in their absorption energy, in their exchanging proton pool and in their size, thus relaxation efficacy. We demonstrate that these properties can be exploited for the visualization of β‐galactosidase activity in phantom samples by different imaging modalities: NIR optical imaging, CEST and T1‐weighted MRI.

Lanthanide-Based Probes for Imaging Detection of Enzyme Activities by NIR Luminescence, T1- and ParaCEST MRI.

Applying a single molecular probe to monitor enzymatic activities in multiple, complementary imaging modalities is highly desirable to ascertain detection and to avoid the complexity associated with the use of agents of different chemical entities. We demonstrate here the versatility of lanthanide (Ln3+) complexes with respect to their optical and magnetic properties and their potential for enzymatic detection in NIR luminescence, CEST and T1 MR imaging, controlled by the nature of the Ln3+ ion, while using a unique chelator. Based on X-ray structural, photophysical, and solution NMR investigations of a family of Ln3+ DO3A-pyridine model complexes, we could rationalize the luminescence (Eu3+, Yb3+), CEST (Yb3+) and relaxation (Gd3+) properties and their variations between carbamate and amine derivatives. This allowed the design of probes which undergo enzyme-mediated changes detectable in NIR luminescence, CEST and T1-weighted MRI, respectively governed by variations in their absorption energy, in their exchanging proton pool and in their size, thus relaxation efficacy. We demonstrate that these properties can be exploited for the visualization of β-galactosidase activity in phantom samples by different imaging modalities: NIR optical imaging, CEST and T1-weighted MRI.

Imaging Self-Healing Hydrogels and Chemotherapeutics Using CEST MRI at 3 T.

Imaging hydrogel-based local drug delivery to the brain after tumor resection has implications for refining treatments, especially for brain tumors with poor prognosis and high recurrence rate. Here, we developed a series of self-healing chitosan-dextran (CD)-based hydrogels for drug delivery to the brain. These hydrogels are injectable, self-healing, mechanically compatible, and detectable by chemical exchange saturation transfer magnetic resonance imaging (CEST MRI). CD hydrogels have an inherent CEST contrast at 1.1 ppm, which decreases as the stiffness increases. We further examined the rheological properties and CEST contrast of various chemotherapeutic-loaded CD hydrogels, including gemcitabine (Gem), doxorubicin, and procarbazine. Among these formulations, Gem presented the best compatibility with the rheological (G': 215.3 ± 4.5 Pa) and CEST properties of CD hydrogels. More importantly, the Gem-loaded CD hydrogel generated another CEST readout at 2.2 ppm (11.6 ± 0.1%) for monitoring Gem. This enabled independent and simultaneous imaging of the drug and hydrogel integrity using a clinically relevant 3 T MRI scanner. In addition, the Gem-loaded CD hydrogel exhibited a longitudinal antitumor efficacy of Gem over a week in vitro. Furthermore, the CD hydrogel could be visualized by CEST after brain injection with a contrast of 7.38 ± 2.31%. These natural labels on both the chemotherapeutics and hydrogels demonstrate unique image-guided local drug delivery for brain applications.

H-Bonding and intramolecular catalysis of proton exchange affect the CEST properties of EuIII complexes with HP-DO3A-like ligands.

Eu(HP-DO3A) is present in solution as a mixture of two diastereoisomers whose alcoholic groups are the source of the mobile protons for the CEST effect. The exchange is base catalyzed. Two novel EuIII complexes of HP-DO3A-like ligands containing an amino or a carboxylate functionality in the proximity of the -OH groups showed the occurrence of intramolecular catalysis of the prototropic exchange. New insights into the role of the intramolecular proton exchange on the CEST properties have been gained.

In vitro characterization of the serotonin biosynthesis pathway by CEST MRI.

The diagnosis of monoamine-related psychiatric disorders is based on the phenomenological evaluation of symptoms and behavior by trained clinicians. The CEST technique can be sensitive to monoamines such as serotonin. This study quantifies the CEST properties of the compounds in the serotonin biosynthesis pathway with the goal of developing noninvasive techniques aimed at advancing the diagnostic assessment of serotonin dysfunction. Saturation transfer-weighted images of L-tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, and melatonin phantoms were acquired over a range of saturation amplitudes and frequency offsets along with observed T1 , T2 , and B1 efficiency maps at physiological temperature and pH of 5.5, 6.7, and 7.4. The CEST and MT data were fitted to a three-pool Bloch-McConnell model of exchange to estimate the model parameters. At apH of 5.5, tryptophan, 5-hydroxytryptophan and serotonin exhibited significant CEST contrast at resonance frequency offset, Δω between 2.64 ppm and 2.71 ppm, and magnetization transfer ratio asymmetry amplitudes up to 20% per 30 mM. At apH of 7.4, all molecules exhibited significant CEST contrast between 5.11 ppm and 5.47 ppm, and magnetization transfer ratio asymmetry amplitudes up to 9.5%per 30 mM. At apH of 6.7, all studied compounds except melatonin exhibited a CEST peak from each of the preceding two pHs. At apH of 5.5, tryptophan, 5-hydroxytryptophan, and serotonin CEST contrast originates from the side chain, whereas at apH of 7.4, CEST contrast is due to the chemical exchange between water and the NH proton on the indole ring. The data in this study could be used to inform future investigations aimed at detecting and measuring in vivo serotonin.

Inert macrocyclic Eu3+ complex with affirmative paraCEST features

Highly kinetically inert paramagnetic platform shows outstanding CEST properties suitable for advantageous MRI applications.

Free-base porphyrins as CEST MRI contrast agents with highly upfield shifted labile protons.

CEST has become a preeminent technology for the rapid detection and grading of tumors, securing its widespread use in both laboratory and clinical research. However, many existing CEST MRI agents exhibit a sensitivity limitation due to small chemical shifts between their exchangeable protons and water. We propose a new group of CEST MRI agents, free-base porphyrins and chlorin, with large exchangeable proton chemical shifts from water for enhanced detection. To test these newly identified CEST agents, we acquired a series of Z-spectra at multiple pH values and saturation field strengths to determine their CEST properties. The data were analyzed using the quantifying exchange using saturation power method to quantify exchange rates. After identifying several promising candidates, a porphyrin solution was injected into tumor-bearing mice, and MR images were acquired to assess detection feasibility in vivo. Based on the Z-spectra, the inner nitrogen protons in free-base porphyrins and chlorin resonate from -8 to -13.5 ppm from water, far shifted from the majority of endogenous metabolites (0-4 ppm) and Nuclear Overhauser enhancements (-1 to -3.5 ppm) and far removed from the salicylates, imidazoles, and anthranillates (5-12 ppm). The exchange rates are sufficiently slow to intermediate (500-9000 s-1 ) to allow robust detection and were sensitive to substituents on the porphyrin ring. These results highlight the capabilities of free-base porphyrins and chlorin as highly upfield CEST MRI agents and provide a new scaffold that can be integrated into a variety of diagnostic or theranostic agents for biomedical applications.

Electronic Effects of Ligand Substitution in a Family of CoII2 PARACEST pH Probes.

We report three new Co2-based paramagnetic chemical exchange saturation transfer (PARACEST) probes with the ability to ratiometrically quantitate pH. A CoII2 complex, [LCo2(etidronate)]-, featuring tetra(carboxamide) and OH-substituted etidronate ligands with opposing pH-dependent CEST peak intensities, was previously shown to exhibit a linear correlation between log(CESTOH/CESTNH) and pH in the pH range 6.5-7.6 that provided a sensitivity of 0.99(7) pH unit-1 at 37 °C. Here, we demonstrate through a series of CF3-functionalized CoII2 complexes [(XL')Co2(etidronate)]- (X = NO2, F, Me), that modest changes in the electronic structure of CoII centers through remote ligand substitution can significantly affect the NMR and CEST properties of Co2-based PARACEST probes. Variable-pH NMR and CEST analyses reveal that the chemical shifts of the ligand protons are highly affected by the nature of the X substituent. The ratios of OH and NH CEST peak intensities at 115 and 88, 93 and 79, and 88 and 76 ppm for X = NO2, F, and Me, respectively, afford pH calibration curves with remarkably high sensitivities of 1.49(9), 1.48(7), and 2.04(5) pH unit-1 across the series. The 1.5-2-fold enhancement in pH sensitivity for the CF3-functionalized Co2 probes stems from the complete separation of the OH and NH CEST peaks. Furthermore, incorporation of electron-withdrawing CF3 groups shifts the detection window to a more acidic range of pH 6.2-7.4. Finally, the CoII2 complexes are found to be extremely robust toward substitution and oxidation in aqueous solutions. Taken together, these results highlight the unique ability of transition metal-based PARACEST probes to provide a highly sensitive concentration-independent measure of pH and demonstrate that modest ligand modifications can be a powerful tool for optimizing the pH sensing performance of these probes.

Protonation of carboxyl groups in EuDOTA-tetraamide complexes results in catalytic prototropic exchange and quenching of the CEST signal.

The CEST properties of EuDOTA-tetraamide complexes bearing pendant carboxylate and carboxyl ethyl esters were measured as a function of pH. The CEST signal from the Eu3+-bound water molecule decreased in intensity between pH 8.5 and 4.5 while the proton exchange rates (kex) increased over this same pH range. In comparison, the CEST signal in the corresponding carboxyl ester derivatives was nearly constant. Both observations are consistent with stepwise protonation of the four carboxylic acid groups over this same pH range. This indicates that negative charges on the carboxyl groups above pH 6 facilitate the formation of a strong hydrogen-bonding network in the coordination second sphere above the single Eu3+-bound water molecule, thereby decreasing prototropic exchange of protons on the bound water molecule with bulk water protons. The percentage of square antiprismatic versus twisted square antiprism coordination isomers also decreased as the appended carboxylic acid groups were positioned further away from the amide. The net effect of lowering the pH was an overall increase in kex and a quenching of the CEST signal.This article is part of the themed issue 'Challenges for chemistry in molecular imaging'.

Imaging Extracellular Lactate In Vitro and In Vivo Using CEST MRI and a Paramagnetic Shift Reagent.

Overproduction of lactate is a hallmark of cancer, yet a method to quantitatively measure lactate production by cancer cells is not straight-forward. Chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) can potentially be used to image lactate but the small difference in chemical shift of the lactate -OH proton and water proton resonances make it challenging. Like other spectroscopic methods, CEST MRI cannot discriminate intracellular lactate from extracellular lactate. Herein, we demonstrate a relatively simple way to shift the lactate -OH proton resonance far away from water by addition of the paramagnetic shift reagent, EuDO3A, while retaining the CEST properties of lactate itself. The potential of the method was demonstrated by imaging extracellular lactate excreted from lung cancer cells in tissue culture without interference from other components in the culture media and by imaging excess lactate excreted into the bladder of a mouse.

Preliminary evaluation of PARACEST MRI agents for the detection of nitric oxide synthase

Several paramagnetic chemical exchange saturation transfer magnetic resonance imaging (PARACEST MRI) agents for the potential detection of nitric oxide synthase (NOS) have been synthesized and evaluated. These agents are based on an amino acid- or dipeptide-decorated DOTAM (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid amide) chelator possessing either Tm3+ or Dy3+. The amino acid and dipeptide decorated DOTAMs were designed such that the terminal amino acid pendant group was l-arginine, which may be converted to l-citrulline by NOS. Preliminary evaluation has revealed that some of the l-arginine-decorated complexes are recognized and metabolized by the NOS. Differences in the CEST properties between Dy3+-metallated l-arginine- and l-citrulline-modified complexes suggest that these might be suitable for imaging of the NOS enzymatic activity.

Six, Seven or Eight Coordinate Fe(II) , Co(II) or Ni(II) Complexes of Amide-Appended Tetraazamacrocycles for ParaCEST Thermometry.

Fe(II) , Co(II) and Ni(II) complexes of two tetraazamacrocycles (1,4,8,11-tetrakis(carbamoylmethyl)-1,4,8,11-tetraazacyclotetradecane (L1) and 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (L2) show promise as paraCEST agents for registration of temperature (paraCEST=paramagnetic chemical exchange saturation transfer). The Fe(II) , Co(II) and Ni(II) complexes of L1 show up to four CEST peaks shifted ≤112 ppm, whereas analogous complexes of L2 show only a single CEST peak at ≤69 ppm. Comparison of the temperature coefficients (CT ) of the CEST peaks of [Co(L2)](2+) , [Fe(L2)](2+) , [Ni(L1)](2+) and [Co(L1)](2+) showed that a CEST peak of [Co(L1)](2+) gave the largest CT (-0.66 ppm (o) C(-1) at 4.7 T). NMR spectral and CEST properties of these complexes correspond to coordination complex symmetry as shown by structural data. The [Ni(L1)](2+) and [Co(L1)](2+) complexes have a six-coordinate metal ion bound to the 1-, 4-amide oxygen atoms and four nitrogen atoms of the tetraazamacrocycle. The [Fe(L2)](2+) complex has an unusual eight-coordinate Fe(II) bound to four amide oxygen atoms and four macrocyclic nitrogen atoms. For [Co(L2)](2+) , one structure has seven-coordinate Co(II) with three bound amide pendents and a second structure has a six-coordinate Co(II) with two bound amide pendents.

Label-free imaging of gelatin-containing hydrogel scaffolds

Composite hyaluronic acid (HA) hydrogels containing gelatin are used in regenerative medicine as tissue-mimicking scaffolds for improving stem cell survival. Once implanted, it is assumed that these biomaterials disintegrate over time, but at present there is no non-invasive imaging technique available with which such degradation can be directly monitored in vivo. We show here the potential of chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) as a label-free non-invasive imaging technique to monitor dynamic changes in scaffold composition in vivo. The CEST properties of the three individual hydrogel components (HA, GelinS, and polyethylene glycol diacrylate) were first measured in vitro. The complete hydrogel was then injected into the brain of immunodeficient rag2−/− mice and CEST MR images were obtained at day 1 and 7 post-transplantation. In vitro, GelinS gave the strongest CEST signal at 3.6 ppm offset from the water peak, originating from the amide protons present in gelatin. In vivo, a significant decrease in CEST signal was observed at 1 week post-implantation. These results were consistent with the biodegradation of the GelinS component, as validated by fluorescent microscopy of implanted hydrogels containing Alexa Fluor 488-labeled GelinS. Our label-free imaging approach should be useful for further development of hydrogel formulations with improved composition and stability.

Chemical Exchange Saturation Transfer Is Unaffected by Modest Changes in Pressure

ParaCEST (paramagnetic Chemical Exchange Saturation Transfer) agents offer an unparalleled opportunity to perform quantitative molecular imaging by MRI. Agents that can alter the image contrast they generate in response to changes in local environmental parameters such as pH, glucose concentration or lactate concentration can be used ratiometrically to quantitatively describe the local tissue environment. However, when performing such quantitative measurements it is important that the results are not confounded by changes in a second environmental parameter. In vivo pressure varies quite considerably, both through the respiratory cycle and from tissue to tissue (tumors in particular have high interstitial pressures). Since paraCEST agents have positive activation volumes, their exchange kinetics and therefore the CEST effect that they generate are necessarily related to pressure. The purpose of this investigation was to examine whether the relatively small changes in pressure exhibited in vivo could affect CEST sufficiently to confound attempts to quantify other local environmental parameters. The CEST properties of a rigid EuDOTA-tetraamide was examined at temperatures ranging from 288 to 319 K, at applied pressures ranging from 0 to 414 kPa and pre-saturation (B1) powers ranging from 524 to 935 Hz. At no point was pressure found to affect the CEST generated by this chelate, indicating that changes in in vivo pressure is unlikely to confound the quantitative measurement of physiologically relevant parameters by paraCEST MRI.

ParaCEST MRI contrast agents capable of derivatizationvia “click” chemistry

A comprehensive series of lanthanide chelates has been prepared with a tetrapropargyl DOTAM type ligand. The complexes have been characterized by a combination of (1)H NMR, single-crystal X-ray crystallography, CEST and relaxation studies and have also been evaluated for potential use as paramagnetic chemical exchange saturation transfer (ParaCEST) contrast agents in magnetic resonance imaging (MRI). We demonstrate the functionalization of several chelates by means of alkyne-azide "click" chemistry in which a glucosyl azide is used to produce a tetra-substituted carbohydrate-decorated lanthanide complex. The carbohydrate periphery of the chelates has a potent influence on the CEST properties as described herein.

Multifunctional Polymeric Scaffolds for Enhancement of PARACEST Contrast Sensitivity and Performance: Effects of Random Copolymer Variations

A DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid) tetraamide ligand having a single acrylamide side-chain (M1) was copolymerized with either 2-methylacrylic acid (MAA), 2-(acryloylamino)-2-methyl-1-propanesulfonic acid (AMPS) or N-isopropylacrylamide (NIPAM) to create a series of linear random copolymers using classical free radical chain polymerization chemistry. The metal ion binding properties of hydrolyzed M1 were investigated by pH potentiometry and the europium (III) complexes of the resulting heteropolymers were evaluated as PARACEST imaging agents. All polymeric agents were found to possess similar intermediate-to-slow water exchange and CEST characteristics as the parent EuDOTA-tetraamide monomer. Consistent with basic multiplexing principles, the highest molecular weight polymer, Eu-DMAA 3.1, also showed the highest CEST sensitivity with a detection limit of 20 ± 2 μM. The second arylamide component gave polymers with widely different chemical characteristics and CEST properties. In particular, the Eu-DNIPAM 4.0 and Eu-DMAA 4.1 polymers displayed different solubility characteristics as a function of pH or temperature which, in turn, affected the water exchange and CEST properties of the corresponding agents. It was concluded that introduction of hydrophobic groups into the polymer backbone reduces solvent accessibility to the Eu(3+) component, effectively slowing water exchange between the inner-sphere water coordination position at each Eu(3+) center with bulk water. The CEST properties of the heteropolymers when dissolved in plasma suggest that the more hydrophobic characteristics of these polymers could be advantageous for in vivo applications.

A paramagnetic MRI-CEST agent responsive to lactate concentration.

A paramagnetic Yb(III) complex bearing six exchangeable amide protons, [Yb(MBDO3AM)](3+), has been investigated with the aim of developing a MRI-CEST (chemical exchange saturation transfer) contrast agent responsive to the concentration of L-lactate. The complex binds the substrate quantitatively to yield [Yb(MBDO3AM)L-lactate](2+). The exchange between the free and the L-lactate-bound complex is slow on the NMR time scale, and the resonances of their corresponding amide protons are sufficiently separated (more than 10 ppm) to allow their selective irradiation. Therefore, the CEST properties of the two forms can be independently assessed. In turn, the resulting saturation transfer to the bulk water signal is dependent on the L-lactate concentration.