Luminescence Properties of Self-Aggregating TbIII-DOTA-Functionalized Calix[4]arenes.

Self-aggregating calix[4]arenes carrying four DOTA ligands on the upper rim for stable complexation of paramagnetic GdIII-ions have already been proposed as MRI probes. In this work, we investigate the luminescence properties of TbIII-DOTA-calix[4]arene-4OPr containing four propyl-groups and compare them with those of the analog substituted with a phthalimide chromophore (TbIII-DOTA-calix[4]arene-3OPr-OPhth). We show that, given its four aromatic rings, the calix[4]arene core acts as an effective sensitizer of Tb-centered luminescence. Substituents on the lower rim can modulate the aggregation behavior, which in turn determines the luminescence properties of the compounds. In solid state, the quantum yield of the phthalimide derivative is almost three times as high as that of the propyl-functionalized analog demonstrating a beneficial role of the chromophore on Tb-luminescence. In solution, however, the effect of the phthalimide group vanishes, which we attribute to the large distance between the chromophore and the lanthanide, situated on the opposite rims of the calix[4]arene. Both quantum yields and luminescence lifetimes show clear concentration dependence in solution, related to the strong impact of aggregation on the luminescence behavior. We also evidence the variability in the values of the critical micelle concentration depending on the experimental technique. Such luminescent calix[4]arene platforms accommodating stable lanthanide complexes can be considered valuable building blocks for the design of dual MR/optical imaging probes.

Molecular MR imaging of fibrosis in a mouse model of pancreatic cancer.

Fibrosis with excessive amounts of type I collagen is a hallmark of many solid tumours, and fibrosis is a promising target in cancer therapy, but tools for its non-invasive quantification are missing. Here we used magnetic resonance imaging with a gadolinium-based probe targeted to type I collagen (EP-3533) to image and quantify fibrosis in pancreatic ductal adenocarcinoma. An orthotopic syngeneic mouse model resulted in tumours with 2.3-fold higher collagen level compared to healthy pancreas. Animals were scanned at 4.7 T before, during and up to 60 min after i.v. injection of EP-3533, or of its non-binding isomer EP-3612. Ex-vivo quantification of gadolinium showed significantly higher uptake of EP-3533 compared to EP-3612 in tumours, but not in surrounding tissue (blood, muscle). Uptake of EP-3533 visualized in T1-weighted MRI correlated well with spatial distribution of collagen determined by second harmonic generation imaging. Differences in the tumour pharmacokinetic profiles of EP-3533 and EP-3612 were utilized to distinguish specific binding to tumour collagen from non-specific uptake. A model-free pharmacokinetic measurement based on area under the curve was identified as a robust imaging biomarker of fibrosis. Collagen-targeted molecular MRI with EP-3533 represents a new tool for non-invasive visualization and quantification of fibrosis in tumour tissue.

Molecular imaging of oxidized collagen quantifies pulmonary and hepatic fibrogenesis.

Fibrosis results from the dysregulation of tissue repair mechanisms affecting major organ systems, leading to chronic extracellular matrix buildup, and progressive, often fatal, organ failure. Current diagnosis relies on invasive biopsies. Noninvasive methods today cannot distinguish actively progressive fibrogenesis from stable scar, and thus are insensitive for monitoring disease activity or therapeutic responses. Collagen oxidation is a universal signature of active fibrogenesis that precedes collagen crosslinking. Biochemically targeting oxidized lysine residues formed by the action of lysyl oxidase on collagen with a small-molecule gadolinium chelate enables targeted molecular magnetic resonance imaging. This noninvasive direct biochemical elucidation of the fibrotic microenvironment specifically and robustly detected and staged pulmonary and hepatic fibrosis progression, and monitored therapeutic response in animal models. Furthermore, this paradigm is translatable and generally applicable to diverse fibroproliferative disorders.

Molecular MRI of collagen to diagnose and stage liver fibrosis.

BACKGROUND & AIMS: The gold standard in assessing liver fibrosis is biopsy despite limitations like invasiveness and sampling error and complications including morbidity and mortality. Therefore, there is a major unmet medical need to quantify fibrosis non-invasively to facilitate early diagnosis of chronic liver disease and provide a means to monitor disease progression. The goal of this study was to evaluate the ability of several magnetic resonance imaging (MRI) techniques to stage liver fibrosis. METHODS: A gadolinium (Gd)-based MRI probe targeted to type I collagen (termed EP-3533) was utilized to non-invasively stage liver fibrosis in a carbon tetrachloride (CCl4) mouse model and the results were compared to other MRI techniques including relaxation times, diffusion, and magnetization transfer measurements. RESULTS: The most sensitive MR biomarker was the change in liver:muscle contrast to noise ratio (ΔCNR) after EP-3533 injection. We observed a strong positive linear correlation between ΔCNR and liver hydroxyproline (i.e. collagen) levels (r=0.89) as well as ΔCNR and conventional Ishak fibrosis scoring. In addition, the area under the receiver operating curve (AUR0C) for distinguishing early (Ishak ≤ 3) from late (Ishak ≥ 4) fibrosis was 0.942 ± 0.052 (p<0.001). By comparison, other MRI techniques were not as sensitive to changes in fibrosis in this model. CONCLUSIONS: We have developed an MRI technique using a collagen-specific probe for diagnosing and staging liver fibrosis, and validated it in the CCl4 mouse model. This approach should provide a better means to monitor disease progression in patients.

Molecular MR imaging of liver fibrosis: a feasibility study using rat and mouse models.

BACKGROUND & AIMS: Liver biopsy, the current clinical gold standard for fibrosis assessment, is invasive and has sampling errors, and is not optimal for screening, monitoring, or clinical decision-making. Fibrosis is characterized by excessive accumulation of extracellular matrix proteins including type I collagen. We hypothesize that molecular magnetic resonance imaging (MRI) with a probe targeted to type I collagen could provide a direct and non-invasive method of fibrosis assessment. METHODS: Liver fibrosis was induced in rats with diethylnitrosamine and in mice with carbon tetrachloride. Animals were imaged prior to and immediately following i.v. administration of either collagen-targeted probe EP-3533 or non-targeted control Gd-DTPA. Magnetic resonance (MR) signal washout characteristics were evaluated from T1 maps and T1-weighted images. Liver tissue was subjected to pathologic scoring of fibrosis and analyzed for gadolinium and hydroxyproline. RESULTS: EP-3533-enhanced MR showed greater signal intensity on delayed imaging (normalized signal enhancement mice: control=0.39 ± 0.04, fibrotic=0.55 ± 0.03, p<0.01) and slower signal washout in the fibrotic liver compared to controls (liver t(1/2)=51.3 ± 3.6 vs. 42.0 ± 2.5 min, p<0.05 and 54.5 ± 1.9 vs. 44.1 ± 2.9 min, p<0.01 for fibrotic vs. controls in rat and mouse models, respectively). Gd-DTPA-enhanced MR could not distinguish fibrotic from control animals. EP-3533 gadolinium concentration in the liver showed strong positive correlations with hydroxyproline levels (r=0.74 (rats), r=0.77 (mice)) and with Ishak scoring (r=0.84 (rats), r=0.79 (mice)). CONCLUSIONS: Molecular MRI of liver fibrosis with a collagen-specific probe identifies fibrotic tissue in two rodent models of disease.

1H chemical shift magnetic resonance imaging probes with high sensitivity for multiplex imaging.

Proton-based chemical shift imaging probes were encapsulated inside nano-carriers to increase the sensivitity of the reporters. Co-encapsulation with a relaxation agent results in improved sensitivity and suppresses background signals. Simultaneous imaging of different chemical shift reporters allows multiplexed detection.

Molecular MRI of acute necrosis with a novel DNA-binding gadolinium chelate: kinetics of cell death and clearance in infarcted myocardium.

BACKGROUND: Current techniques to image cell death in the myocardium are largely nonspecific. We report the use of a novel DNA-binding gadolinium chelate (Gd-TO) to specifically detect the exposed DNA in acutely necrotic (ruptured) cells in vivo. METHODS AND RESULTS: In vivo MRI was performed in 20 mice with myocardial infarction (MI). The mice were injected with Gd-TO or Gd-DTPA at varying time points after MI. MRI was performed 2 hours after probe injection, to avoid nonspecific signal from the late gadolinium enhancement effect. Cell rupture (Gd-TO uptake) was present within 2 hours of infarction but peaked 9 to 18 hours after the onset of injury. A significant increase in the longitudinal relaxation rate (R(1)) in the infarct was seen in mice injected with Gd-TO within 48 hours of MI, but not in those injected more than 72 hours after MI (R(1)=1.24±0.08 and 0.92±0.03 s(-1), respectively, P<0.001). Gd-DTPA, unlike Gd-TO, washed completely out of acute infarcts within 2 hours of injection (P<0.001). The binding of Gd-TO to exposed DNA in acute infarcts was confirmed with fluorescence microscopy. CONCLUSIONS: Gd-TO specifically binds to acutely necrotic cells and can be used to image the mechanism and chronicity of cell death in injured myocardium. Cell rupture in acute MI begins early but peaks many hours after the onset of injury. The ruptured cells are efficiently cleared by the immune system and are no longer present in the myocardium 72 hours after injury.

Liposomes with conjugates of a calix[4]arene and a Gd-DOTA derivative on the outside surface; an efficient potential contrast agent for MRI.

Paramagnetic liposomes used as contrast agents in magnetic resonance imaging (MRI) often suffer from low efficacies because of slow water diffusion through the membrane. We present an approach to overcome this limitation by incorporation of a calix[4]arene based agent that expresses the chelates towards the bulk water.

Densely packed Gd(III)-chelates with fast water exchange on a calix[4]arene scaffold: a potential MRI contrast agent.

A pyridine-N-oxide functionalized DOTA analogue has been conjugated to a calix[4]arene and the corresponding Gd-complex was characterized with respect to its suitability as MRI contrast agent. The compound forms spherical micelles in water with a cmc of 35 microM and a radius of 8.2 nm. The relaxivity of these aggregates is 31.2 s(-1) mM(-1) at 25 degrees C and 20 MHz, which corresponds to a molecular relaxivity of 125 s(-1) mM(-1). The high relaxivity mainly originates from the short tau(M) (72.7 ns) and the size of the micelles. The interaction with bovine serum albumin (BSA) was studied and an observed relaxivity of up to 40.8 s(-1) mM(-1) (163.2 s(-1) mM(-1) per binding place) at 20 MHz and 37 degrees C was found in the presence of 2.0 mM protein.

Calix[4]arenes as molecular platforms for magnetic resonance imaging (MRI) contrast agents.

The novel amphipilic conjugate of a calix[4]arene with four Gd-1,4,7,10- tetra(carboxymethyl)-1,4,7,10-tetraazacyclododecane (DOTA) chelates has potential as a magnetic resonance imaging contrast agent, both in its monomeric and in its micellar form. The system, illustrated here with its nuclear magnetic relaxation profile, shows good relaxivities, thanks to its high rigidity.An amphiphilic conjugate 1 of a calix[4]arene with four Gd-DOTA chelates (DOTA=1,4,7,10-tetra(carboxymethyl)-1,4,7,10-tetraazacyclododecane) was prepared and the properties relevant to its application as a magnetic resonance imaging (MRI) contrast agent were investigated by NMR, dynamic light scattering (DLS), and cryo-electron microscopy (cryo-TEM). The compound aggregates in water; its critical micelle concentration (cmc) is 0.21 mM (or 0.84 mM for Gd) at 37 degrees C. The relaxivity of the aggregates at 37 degrees C and 20 MHz (18.3 s(-1) per mM Gd or 73.2 s(-1) per mM 1) is about twice that of the monomer. Nuclear magnetic relaxation dispersion (NMRD) profiles show the relaxivity of the monomer to be almost independent of the magnetic field strength up to 60 MHz. At higher concentrations, the NMRD profiles exhibit a maximum at about 20 MHz, which is typical for high molecular volumes. The average water residence lifetime is 1.20 mus at 298 K as determined by (17)O NMR. The rotational correlation time of the monomer (390 ns at 37 degrees C) is very close to the optimal value predicted for high-field contrast agents. Monomer as well as micelles are very rigid systems with negligible local contributions to the overall rotational dynamics. The binding to human serum albumin (HSA) is significant (K(A)=1.2x10(3) M(-1)) and the relaxivity of the HSA adduct at 20 MHz is 24.6 s(-1) per mM Gd or 98.5 s(-1) per mM 1.

Kinetic acidity of supramolecular imidazolium salts-effects of substituent, preorientation, and counterions on H/D exchange rates.

The deprotonation of imidazolium salts to N-heterocyclic carbenes is often a decisive step in modern catalytic reactions. Therefore, we studied the H/D exchange of the C2 H of 15 imidazolium-substituted calix[4]arenes and 11 nonmacrocyclic model compounds in methanol/water (97:3). The influence of the counterion, substitution directly on the imidazolium unit or on the preorientating calixarene backbone could be studied. The observed exchange rates might give a rational for the suitability of the imidazolium salts as precursors in the Suzuki coupling.

Second-order nonlinear optical properties of the Ag(111)/electrolyte interface in the presence of self-assembled monolayers containing conjugated pi systems. I. Alpha-functionalized terthiophene films on Ag(111).

The nonlinear optical properties of self-assembled monolayers obtained from bonding two different alpha-functionalized terthiophenes (alpha-T3) to (111) silver electrode surfaces have been investigated using second harmonic generation (SHG). The two (alpha-T3) compounds used were functionalized with alkane chains of different lengths (C8 and C4), and each was terminated with a thiol anchoring group. A nitrile group was attached to the terminal thiophene ring of the (alpha-T3) compound with the C4 chain. The orientation of the polarization of the incident beam was changed systematically and gradually between "p" and "s" orientation and the SH signal (isotropic and anisotropic contributions) analyzed in both directions ("P" and "S"). The symmetry of the system was reduced by the presence of the adlayers from C3v to C3. The dependence on the applied potential and the incident wavelength has also been studied. The relative magnitudes and phases of the various second-order tensor elements have been estimated and compared with the values for a bare surface. A resonance process in the (alpha-T3) pi moiety has been investigated, and from this, the effective "band-gap" energies of the organic semiconductor SAMs (i.e., the energy difference between the pi-pi bands) have been estimated.