[Gd(HB-DO3A)]: Equilibrium, Dissociation Kinetic and Structural Differences in a Simple Homolog of [Gd(HP-DO3A)] (Prohance®).

[Gd(HP-DO3A)] (gadoteridol) as an active compound of ProHance® is a widely employed contrast agent in clinical MRI scans in the last 30 years. Recent concerns about the long-term retention of gadolinium-based contrast agents (GBCAs) led to a deeper investigation of the structural features underlying the integrity of the paramagnetic metal complex. Several human and nonclinical studies have noted marked differences among the macrocyclic GBCAs, with the least retention of Gd traces and most rapid elimination consistently being reported for [Gd(HP-DO3A)]. It was deemed of interest to assess how minor structural/electronic changes associated to the ligand structure may affect basic properties of the metal complex with several [Gd(HP-DO3A)] analogues synthesized and characterized in the last years. We recently reported that the closest homolog of [Gd(HP-DO3A)], i. e.: [Gd(HB-DO3A)], in which a (±)-2-hydroxy-1-propyl pendant arm is replaced by a (±)-2-hydroxy-1-butyl moiety, showed a significantly different retention behaviour in the model interaction with collagen, despite the apparently very minor structural difference. In this paper we report a comprehensive study of the structural, thermodynamic, kinetic and relaxation properties of [Gd(HB-DO3A)], compared to the parent [Gd(HP-DO3A)] and to other closely related macrocyclic GBCAs to assess whether very minor structural changes can modulate the physico-chemical properties of Gd3+ complexes.

A Magnetic Resonance Imaging-Chemical Exchange Saturation Transfer (MRI-CEST) Method for the Detection of Water Cycling across Cellular Membranes.

Water cycling across the membrane transporters is considered a hallmark of cellular metabolism and it could be of high diagnostic relevance in the characterization of tumors and other diseases. The method relies on the response of intracellular proton exchanging molecules to the presence of extracellular Gd-based contrast agents (GBCAs). Paramagnetic GBCAs enhances the relaxation rate of water molecules in the extracellular compartment and, through membrane exchange, the relaxation enhancement is transferred to intracellular molecules. The effect is detected at the MRI-CEST (Magnetic Resonance Imaging - Chemical Exchange Saturation Transfer) signal of intracellular proton exchanging molecules. The magnitude of the change in the CEST response reports on water cycling across the membrane. The method has been tested on Red Blood Cells and on orthotopic murine models of breast cancer with different degree of malignancy (4T1, TS/A and 168FARN). The distribution of voxels reporting on membrane permeability fits well with the cells' aggressiveness and acts as an early reporter to monitor therapeutic treatments.

Seeking for Innovation with Magnetic Resonance Imaging Paramagnetic Contrast Agents: Relaxation Enhancement via Weak and Dynamic Electrostatic Interactions with Positively Charged Groups on Endogenous Macromolecules.

Gd-L1 is a macrocyclic Gd-HPDO3A derivative functionalized with a short spacer to a trisulfonated pyrene. When compared to Gd-HPDO3A, the increased relaxivity appears to be determined by both the higher molecular weight and the occurrence of an intramolecularly catalyzed prototropic exchange of the coordinated OH moiety. In water, Gd-L1 displayed a relaxivity of 7.1 mM-1 s-1 (at 298 K and 0.5 T), slightly increasing with the concentration likely due to the onset of intermolecular aggregation. A remarkably high and concentration-dependent relaxivity was measured in human serum (up to 26.5 mM-1 s-1 at the lowest tested concentration of 0.005 mM). The acquisition of 1H-nuclear magnetic relaxation dispersion (NMRD) and 17O-R2 vs T profiles allowed to get an in-depth characterization of the system. In vitro experiments in the presence of human serum albumin, γ-globulins, and polylysine, as well as using media mimicking the extracellular matrix, provided strong support to the view that the trisulfonated pyrene fosters binding interactions with the exposed positive groups on the surface of proteins, responsible for a remarkable in vivo hyperintensity in T1w MR images. The in vivo MR images of the liver, kidneys, and spleen showed a marked contrast enhancement in the first 10 min after the i.v. injection of Gd-L1, which was 2-6-fold higher than that for Gd-HPDO3A, while maintaining a very similar excretion behavior. These findings may pave the way to an improved design of MRI GBCAs, for the first time, based on the setup of weak and dynamic interactions with abundant positive groups on serum and ECM proteins.

Enzymatic Reactions Observed with Zero- and Low-Field Nuclear Magnetic Resonance.

We demonstrate that enzyme-catalyzed reactions can be observed in zero- and low-field NMR experiments by combining recent advances in parahydrogen-based hyperpolarization methods with state-of-the-art magnetometry. Specifically, we investigated two model biological processes: the conversion of fumarate into malate, which is used in vivo as a marker of cell necrosis, and the conversion of pyruvate into lactate, which is the most widely studied metabolic process in hyperpolarization-enhanced imaging. In addition to this, we constructed a microfluidic zero-field NMR setup to perform experiments on microliter-scale samples of [1-13C]fumarate in a lab-on-a-chip device. Zero- to ultralow-field (ZULF) NMR has two key advantages over high-field NMR: the signals can pass through conductive materials (e.g., metals), and line broadening from sample heterogeneity is negligible. To date, the use of ZULF NMR for process monitoring has been limited to studying hydrogenation reactions. In this work, we demonstrate this emerging analytical technique for more general reaction monitoring and compare zero- vs low-field detection.

Assessing the Therapeutic Efficacy of Proton Transport Inhibitors in a Triple-Negative Breast Cancer Murine Model with Magnetic Resonance Imaging-Chemical Exchange Saturation Transfer Tumor pH Imaging.

Proton transporters play a key role in maintaining the acidic tumor microenvironment; hence, their inhibition has been proposed as a new therapeutic treatment, although few methods can accurately assess their effect in vivo. In this study, we investigated whether MRI-CEST (Magnetic Resonance Imaging-Chemical Exchange Saturation Transfer) tumor pH imaging can be a useful tool to evaluate in vivo the therapeutic efficacy of several Proton Pump Inhibitors (PPIs) in breast cancer. Cell viability and extracellular pH assays were carried out in breast cancer cells cultured at physiological pH (7.4) or acid-adapted (pH of 6.5 and 6.8) following the exposure to inhibitors of V-ATPase (Lansoprazole, Esomeprazole) or NHE1 (Amiloride, Cariporide) at several concentrations. Next, triple-negative breast cancer 4T1 tumor-bearing mice were treated with Lansoprazole or Amiloride and MRI-CEST tumor pH imaging was utilized to assess the in vivo efficacy. Only Lansoprazole induced, in addition to breast cancer cell toxicity, a significant inhibition of proton extrusion. A significant reduction in tumor volume, prolonged survival, and increase in extracellular tumor pH after 1 and 2 weeks were observed after Lansoprazole treatment, whereas no significant changes were detected upon Amiloride treatment. Our results suggested that MRI-CEST tumor pH imaging can monitor the therapeutic efficacy of PPIs in breast cancer murine models.

Molecular Resonance Imaging of the CAIX Expression in Mouse Mammary Adenocarcinoma Cells.

The carbonic anhydrase isoform IX (hCAIX) is one of the main players in extracellular tumor pH regulation, and it is known to be overexpressed in breast cancer and other common tumors. hCA IX supports the growth and survival of tumor cells, and its expression is correlated with metastasis and resistance to therapies, making it an interesting biomarker for diagnosis and therapy. The aim of this work deals with the development of an MRI imaging probe able to target the extracellular non-catalytic proteoglycan-like (PG) domain of CAIX. For this purpose, a specific nanoprobe, LIP_PepC, was designed by conjugating a peptidic interactor of the PG domain on the surface of a liposome loaded with Gd-bearing contrast agents. A Mouse Mammary Adenocarcinoma Cell Line (TS/A) was chosen as an in vitro breast cancer model to test the developed probe. MRI results showed a high selectivity and sensitivity of the imaging probe toward hCAI-expressing TS/A cells. This approach appears highly promising for the in vivo translation of a diagnostic procedure based on the targeting of hCA IX enzyme expression.

In Situ Insonation of Alkaline Buffer Containing Liposomes Leads to a Net Improvement of the Therapeutic Outcome in a Triple Negative Breast Cancer Murine Model.

Breast cancer is characterized by an acidic micro-environment. Acidic extracellular pH gives cancer cells an evolutionary advantage, hence, neutralization of the extracellular pH has been considered as a potential therapeutic strategy. To address the issue of systemic pH alteration, an approach based on the targeted delivery of the buffering solution to the tumor region is investigated. The method relies on the use of low frequency ultrasound and sono-sensitive liposomes loaded with buffers at alkaline pH (LipHUS). After the i.v. injection of LipHUS, the application of ultrasound (US) at the sites of the pathology induces a local increase of pH that results highly effective in i) inhibiting primary tumor growth, ii) reducing tumor recurrence after surgery, and iii) suppressing metastases' formation. The experiments are carried out on a triple negative breast cancer mouse model. The results obtained demonstrate that localized and triggered release of bicarbonate or PBS buffer from sonosensitive liposomes represents an efficient therapeutic tool for treating triple-negative breast cancer. This approach holds promise for potential clinical translation.

A Ratiometric SERS Probe for Imaging the Macrophage Phenotypes in Live Mice with Epilepsy and Brain Tumor.

Macrophage performs multiple functions such as pathogen phagocytosis, antigen presentation, and tissue remodeling by polarizing toward a spectrum of phenotypes. Dynamic imaging of macrophage phenotypes is critical for evaluating disease progression and the therapeutic response of drug candidates. However, current technologies cannot identify macrophage phenotypes in vivo. Herein, a surface-enhanced Raman scattering nanoprobe, AH1, which enables the accurate determination of physiological pH with high sensitivity and tissue penetration depth through ratiometric Raman signals is developed. Due to the phenotype-dependent metabolic reprogramming, AH1 can effectively identify macrophage subpopulations by measuring the acidity levels in phagosomes. After intravenous administration, AH1 not only visualizes the spatial distribution of macrophage phenotypes in brain tumors and epileptic regions of mouse models, but also reveals the repolarization of macrophages in brain lesions after drug intervention. This work provides a new tool for dynamically monitoring the disease-associated immune microenvironment and evaluating the efficacy of immune-therapeutics in vivo.

4-Amino-TEMPO loaded liposomes as sensitive EPR and OMRI probes for the detection of phospholipase A2 activity.

This work aims at developing a diagnostic method based on Electron Paramagnetic Resonance (EPR) measurements of stable nitroxide radicals released from "EPR silent" liposomes. The liposome destabilisation and consequent radical release is enzymatically triggered by the action of phospholipase A2 (PLA2) present in the biological sample of interest. PLA2 are involved in a broad range of processes, and changes in their activity may be considered as a unique valuable biomarker for early diagnoses. The minimum amount of PLA2 measured "in vitro" was 0.09 U/mL. Moreover, the liposomes were successfully used to perform Overhauser-enhanced Magnetic Resonance Imaging (OMRI) in vitro at 0.2 T. The amount of radicals released by PLA2 driven liposome destabilization was sufficient to generate a well detectable contrast enhancement in the corresponding OMRI image.

Highlight selection of radiochemistry and radiopharmacy developments by editorial board.

BACKGROUND: The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biannual highlight commentary to update the readership on trends in the field of radiopharmaceutical development. MAIN BODY: This selection of highlights provides commentary on 21 different topics selected by each coauthoring Editorial Board member addressing a variety of aspects ranging from novel radiochemistry to first-in-human application of novel radiopharmaceuticals. CONCLUSION: Trends in radiochemistry and radiopharmacy are highlighted. Hot topics cover the entire scope of EJNMMI Radiopharmacy and Chemistry, demonstrating the progress in the research field, and include new PET-labelling methods for 11C and 18F, the importance of choosing the proper chelator for a given radioactive metal ion, implications of total body PET on use of radiopharmaceuticals, legislation issues and radionuclide therapy including the emerging role of 161Tb.

Prospects and limitations of paramagnetic chemical exchange saturation transfer agents serving as biological reporters in vivo.

The concept of using paramagnetic metal ion complexes as chemical exchange saturation transfer agents (paraCEST) for molecular imaging of various biological processes first appeared in the literature about 20 years ago. The first paraCEST agent was based on a highly shifted, inner-sphere, slowly exchanging water molecule that could be activated at a frequency far away from bulk water, a substantial advantage for selective activation of the agent alone. Many other paraCEST agent designs followed that were based on activation of exchanging -NH or -OH proton on the chelate itself. Both types of paraCEST designs are attractive for molecular imaging because the rates of water molecule or ligand proton exchange can be designed to be sensitive to a biological or physiological property such as pH, enzyme activity, or redox. Hence, the intensity or frequency of the resulting CEST signal provides a direct readout of that property. Many molecular designs have appeared in the literature over the past 20 years, mostly reported as proof-of-concept designs but, unfortunately, only a few reports have explored the limitations of paraCEST agents for imaging a biological process in vivo. As a community, we now know that the sensitivity of paraCEST agents is lower than one might anticipate based upon simple chemical exchange principles and, in general, it appears the sensitivity of paraCEST agents is even lower in vivo than in vitro. In this short review, we address some of the factors that contribute to the limited sensitivity of paraCEST agents in vivo, offer some thoughts on approaches that could lead to dramatically improved paraCEST sensitivity, and challenge the scientific community to perform more in vivo experiments designed to test these ideas.

31P ParaCEST: 31P MRI-CEST Imaging Based on the Formation of a Ternary Adduct between Inorganic Phosphate and Eu-DO3A.

Development of the field of magnetic resonance imaging (MRI) chemical exchange saturation transfer (CEST) contrast agents is hampered by the limited sensitivity of the technique. In water, the high proton concentration allows for an enormous amplification of the exchanging proton pool. However, the 1H CEST in water implies that the number of nuclear spins of the CEST-generating species has to be in the millimolar range. The use of nuclei other than a proton allows exploitation of signals different from that of water, thus lowering the concentration of the exchanging pool as the source of the CEST effect. In this work, we report on the detection of a 31P signal from endogenous inorganic phosphate (Pifree) as the source of CEST contrast by promoting its exchange with the Pi bound to the exogenous complex 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (Pibound). The herein-reported results demonstrate that this approach can improve the detectability threshold by 3 orders of magnitude with respect to the conventional 1H CEST detection (considered per single proton). This achievement reflects the decrease of the bulk concentration of the detected signal from 111.2 M (water) to 10 mM (Pi). This method paves the way to a number of biological studies and clinically translatable applications, herein addressed with a proof-of-concept in the field of cellular imaging.

In Vivo MRI-CEST Tumor pH Imaging Detects Resistance to Proton Pump Inhibitors in Human Prostate Cancer Murine Models.

The tumor microenvironment acidification confers treatment resistance; therefore, the interference with pH regulating systems is considered a new therapeutic strategy. In this study, two human prostate cancer cell lines, PC3 and LNCaP, have been treated in vitro with proton pump inhibitors (PPIs), namely Lansoprazole, Esomeprazole (V-ATPases-inhibitors), Cariporide, and Amiloride (NHE1-inhibitors). The cell viability and pH were assessed at several drug concentrations either at normoxic or hypoxic conditions. Since Esomeprazole showed the highest toxicity towards the PC3 cancer cells compared to LNCaP ones, athymic nude mice bearing subcutaneous or orthotopic PC3 tumors were treated with Esomeprazole (dose: 2.5 mg/kg body weight) for a period of three weeks-and tumor growth was monitored. MRI-CEST tumor pH imaging with Iopamidol was performed upon treatment at 3 h, 1 week (in combination with FDG-PET), and after 2 weeks for evaluating acute, early, and late responses. Although acute tumor pH changes were observed in vivo, long-term studies on both PC3 prostate cancer models did not provide any significant change in tumor acidosis or tumor growth. In conclusion, this work shows that MRI-CEST tumor pH imaging is a valuable tool for assessing the in vivo treatment response to PPIs.

Role of Transmembrane Water Exchange in Glioma Invasion/Migration: In Vivo Preclinical Study by Relaxometry at Very Low Magnetic Field.

This work shows that the longitudinal relaxation differences observed at very low magnetic fields between invasion/migration and proliferation processes on glioma mouse models in vivo are related to differences in the transmembrane water exchange basically linked to the aquaporin expression changes. Three glioma mouse models were used: Glio6 and Glio96 as invasion/migration models and U87 as cell proliferation model. In vivo proton longitudinal relaxation-rate constants (R1) at very low fields were measured by fast field cycling NMR (FFC-NMR). The tumor contribution to the observed proton relaxation rate, R1tum (U87: 12.26 ± 0.64 s−1; Glio6: 3.76 ± 0.88 s−1; Glio96: 6.90 ± 0.64 s−1 at 0.01 MHz), and the intracellular water lifetime, τin (U87: 826 ± 19 ms; Glio6: 516 ± 8 ms; Glio96: 596 ± 15 ms), were found to be good diagnostic hallmarks to distinguish invasion/migration from proliferation (p < 0.01 and 0.001). Overexpression of AQP4 and AQP1 were assessed in invasion/migration models, highlighting the pathophysiological role of these two aquaporins in water exchange that, in turn, determine the lower values in the observed R1 relaxation rate constant in glioma invasion/migration. Overall, our findings demonstrate that τin and R1 (measured at very low fields) are relevant biomarkers, discriminating invasion/migration from proliferation in vivo. These results highlight the use of FFC-NMR and FFC-imaging to assess the efficiency of drugs that could modulate aquaporin functions.

Review and consensus recommendations on clinical APT-weighted imaging approaches at 3T: Application to brain tumors.

Amide proton transfer-weighted (APTw) MR imaging shows promise as a biomarker of brain tumor status. Currently used APTw MRI pulse sequences and protocols vary substantially among different institutes, and there are no agreed-on standards in the imaging community. Therefore, the results acquired from different research centers are difficult to compare, which hampers uniform clinical application and interpretation. This paper reviews current clinical APTw imaging approaches and provides a rationale for optimized APTw brain tumor imaging at 3 T, including specific recommendations for pulse sequences, acquisition protocols, and data processing methods. We expect that these consensus recommendations will become the first broadly accepted guidelines for APTw imaging of brain tumors on 3 T MRI systems from different vendors. This will allow more medical centers to use the same or comparable APTw MRI techniques for the detection, characterization, and monitoring of brain tumors, enabling multi-center trials in larger patient cohorts and, ultimately, routine clinical use.

Effects of Cations on HPTS Fluorescence and Quantification of Free Gadolinium Ions in Solution; Assessment of Intracellular Release of Gd3+ from Gd-Based MRI Contrast Agents.

8-Hydroxypyrene-1,3,6-trisulfonate (HPTS) is a small, hydrophilic fluorescent molecule. Since the pKa of the hydroxyl group is close to neutrality and quickly responds to pH changes, it is widely used as a pH-reporter in cell biology for measurements of intracellular pH. HPTS fluorescence (both excitation and emission spectra) at variable pH was measured in pure water in the presence of NaCl solution or in the presence of different buffers (PBS or hepes in the presence or not of NaCl) and in a solution containing BSA. pKa values have been obtained from the sigmoidal curves. Herein, we investigated the effect of mono-, di-, and trivalent cations (Na+, Ca2+, La3+, Gd3+) on fluorescence changes and proposed its use for the quantification of trivalent cations (e.g., gadolinium ions) present in solution as acqua-ions. Starting from the linear regression, the LoD value of 6.32 µM for the Gd3+ detection was calculated. The effects on the emission were also analyzed in the presence of a combination of Gd3+ at two different concentrations and the previously indicated mono and di-valent ions. The study demonstrated the feasibility of a qualitative method to investigate the intracellular Gd3+ release upon the administration of Gd-based contrast agents in murine macrophages.

The interaction between iodinated X-ray contrast agents and macrocyclic GBCAs provides a signal enhancement in T1 -weighted MR images: Insights into the renal excretion pathways of Gd-HPDO3A and iodixanol in healthy mice.

PURPOSE: This work aims to investigate the supramolecular binding interactions that occur between iodinated X-ray contrast agents (CAs) and macrocyclic gadolinium (Gd)-based MRI contrast agents (GBCAs). This study provides some new insights in the renal excretion pathways of the two types of imaging probes. METHODS: The water-proton relaxivities (r1 ) of clinically approved macrocyclic and linear GBCAs have been measured in the presence of different iodinated X-ray contrast agents at different magnetic field strengths in buffer and in serum. The in vivo MRI and X-ray CT of mice injected with either Gd-HPDO3A or a Gd-HPDO3A + iodixanol mixture were then acquired to assess the biodistribution of the two probes. RESULTS: A significant increase in r1 (up to approximately 200%) was observed for macrocyclic GBCAs when measured in the presence of an excess of iodinated X-ray CAs (1:100 mol:mol) in serum. The co-administration of Gd-HPDO3A and iodixanol in vivo resulted in a marked increase in the signal intensity of the kidney regions in T1 -weighted MR images. Moreover, the co-presence of the two agents resulted in the extended persistence of the MRI signal enhancement, suggesting that the Gd-HPDO3A/iodixanol adduct was eliminated more slowly than the typical washing out of Gd-HPDO3A. CONCLUSIONS: The reported results show that it is possible to detect the co-presence of iodinated agents and macrocyclic GBCAs in contrast-enhanced MR images. The new information may be useful in the design of novel experiments toward improved diagnostic outcomes.

New tools to investigate tumor metabolism by NMR/MRI.

Changes in metabolism is an hallmark that characterizes tumour cells from healthy ones. Their detection can be highly relevant for staging the tumor and for monitoring the response to therapeutic treatments. Herein it is shown the readout of these changes can be achieved either by assessing the pH of the extracellular space in the tumour region and by monitoring real time transformations of hyperpolarized C-13 labelled substrates. Mapping pH in a MR image is possible by measuring the CEST response of an administered contrast agent such as Iopamidol that can provide accurate measurements of the heterogeneity of tumour acidosis. Direct detection of relevant enzymatic activities have been acquired by using Pyruvate and Fumarate hyperpolarized by the incorporation of a molecule of para-H2. Finally, it has been found that the tumour transformation involves an increase in the water exchange rate between the intra- and the extra-cellular compartments. A quantitative estimation of these changes can be obtained by acquiring the longitudinal relaxation times of tissue water protons at low magnetic field strength on Fast Field Cycling Relaxometers. This finding has been exploited in an application devoted to the assessment of the presence of residual tumour tissue in the margins of the resected mass in breast conservative surgery.

XNAT-PIC: Extending XNAT to Preclinical Imaging Centers.

Molecular imaging generates large volumes of heterogeneous biomedical imagery with an impelling need of guidelines for handling image data. Although several successful solutions have been implemented for human epidemiologic studies, few and limited approaches have been proposed for animal population studies. Preclinical imaging research deals with a variety of machinery yielding tons of raw data but the current practices to store and distribute image data are inadequate. Therefore, standard tools for the analysis of large image datasets need to be established. In this paper, we present an extension of XNAT for Preclinical Imaging Centers (XNAT-PIC). XNAT is a worldwide used, open-source platform for securely hosting, sharing, and processing of clinical imaging studies. Despite its success, neither tools for importing large, multimodal preclinical image datasets nor pipelines for processing whole imaging studies are yet available in XNAT. In order to overcome these limitations, we have developed several tools to expand the XNAT core functionalities for supporting preclinical imaging facilities. Our aim is to streamline the management and exchange of image data within the preclinical imaging community, thereby enhancing the reproducibility of the results of image processing and promoting open science practices.