Search

One possibility for the non-invasive imaging of encapsulated cell grafts is to label the lumen of cell embedding capsules with a redox-responsive probe, as an increased extracellular reducing potential can be considered as a marker of hypoxia-induced necrosis. A Gd(III)-HPDO3A-like chelate has been conjugated to glycol-chitosan through a redox-responsive disulphide bond to obtain a contrast agent for Magnetic Resonance Imaging (MRI). Such a compound can be interspersed with fibroblasts within the lumen of alginate-chitosan capsules. Increasing reducing conditions within the extracellular microenvironment lead to the reductive cleavage of the disulphide bond and to the release of gadolinium in the form of a low molecular weight, non-ionic chelate. The efflux of such chelate from capsules is readily detected by a decrease of contrast enhancement in T 1 -weighted MR images.

[Image: see text] A recently developed synthetic protocol allowed for the functionalization of the active peptide A9 with a fluorogenic probe, which is useful for studying biomolecular interactions. Essentially, a nucleophilic attack on a halo-substituted benzofurazan is selectively performed by a cysteine sulfhydryl group. The process is assisted by the basic catalysis of activated zeolites (4 Å molecular sieves) and promoted by microwave irradiation. Fluorescence studies revealed that a donor–acceptor pair within the peptide sequence was introduced, thus allowing a deeper investigation on the interaction process between the peptide ligand and its receptor fragment. The obtained results allowed us to come full circle for all the currently understood structural determinants that were found to be involved in the binding process.

International audience; Dysregulated remodeling of the extracellular matrix (ECM) can lead to excessive accumulation of ECM proteins (primarily collagen, elastin/ tropoelastin, fibronectin and fibrin) resulting in tissue fibrosis. In many pathologies, changes in the molecular pattern of ECM components have been related to the progression and severity of fibrosis. Thus, magnetic resonance imaging (MRI) probes sensing specific ECM components hold promise for accurate staging of fibrotic diseases. This paper focuses on gadolinium-based contrast agents (GBCA) targeted to ECM components, including structural proteins and enzymes. According to available examples, they can be grouped into: 1) GBCA conjugated to targeting vectors that recognize and noncovalently bind to specific sites on the molecular target; 2) GBCA carrying a reactive chemical function able to bind covalently to the complementary chemical function of the molecular target; and 3) enzyme-responsive probes, whose relaxivity and pharmacokinetics change after enzymatic processing. Pros and cons of each approach are discussed.

International audience; Dysfunctional elastin turnover plays a major role in the progression of atherosclerotic plaques. Failure of tropoelastin cross-linking into mature elastin leads to the accumulation of tropoelastin within the growing plaque, increasing its instability. Here we present Gd4-TESMA, an MRI contrast agent specifically designed for molecular imaging of tropoelastin within plaques. Gd4-TESMA is a tetrameric probe composed of a tropoelastin-binding peptide (the VVGS-peptide) conjugated with four Gd(III)-DOTA-monoamide chelates. It shows a relaxivity per molecule of 34.0 ± 0.8 mM-1 s-1 (20 MHz, 298 K, pH 7.2), a good binding affinity to tropoelastin (KD = 41 ± 12 μM), and a serum half-life longer than 2 h. Gd4-TESMA accumulates specifically in atherosclerotic plaques in the ApoE-/- murine model of plaque progression, with 2 h persistence of contrast enhancement. As compared to the monomeric counterpart (Gd-TESMA), the tetrameric Gd4-TESMA probe shows a clear advantage regarding both sensitivity and imaging time window, allowing for a better characterization of atherosclerotic plaques.

Presentation at Open Repository 2021

An efficient synthesis of vinyl‐[1‐13C]pyruvate has been reported, from which 13C hyperpolarized (HP) ethyl‐[1‐13C]pyruvate has been obtained by means of ParaHydrogen Induced Polarization (PHIP). Due to the intrinsic lability of pyruvate, which leads quickly to degradation of the reaction mixture even under mild reaction conditions, the vinyl‐ester has been synthesized through the intermediacy of a more stable ketal derivative. 13C and 1H hyperpolarizations of ethyl‐[1‐13C]pyruvate, hydrogenated using ParaHydrogen, have been compared to those observed on the more widely used allyl‐derivative. It has been demonstrated that the spin order transfer from ParaHydrogen protons to 13C, is more efficient on the ethyl than on the allyl‐esterdue to the larger J‐couplings involved. The main requirements needed for the biological application of this HP product have been met, i. e. an aqueous solution of the product at high concentration (40 mM) with a good 13C polarization level (4.8 %) has been obtained. The in vitro metabolic transformation of the HP ethyl‐[1‐13C]pyruvate, catalyzed by an esterase, has been observed. This substrate appears to be a good candidate for in vivo metabolic investigations using PHIP hyperpolarized probes., 13C hyperpolarized ethyl‐[1‐13C]pyruvate by means of PHIP‐SAH: Vinyl‐[1‐13C]pyruvate has been prepared on laboratory scale through a new synthesis route. After its hydrogenation with p‐H2 and magnetization transfer (via MFC), polarized ethyl‐[1‐13C]pyruvate has been obtained in aqueous solution at high concentration (40 mM) and good polarization level (4.8 %). In the end, the in‐vitro conversion of HP ethyl‐[1‐13C]pyruvate into [1‐13C]pyruvate catalysed by an esterase, has been observed.

A green and efficient method for preparing lanthionine peptides by a highly chemoselective and stereochemically controlled procedure is presented. It involves anS-alkylation reaction, promoted by activated molecular sieves, on chiral cyclic sulfamidates, bothN-protected and unprotected. Of note, the reaction yield was high also for cyclic sulfamidates bearing a free amine group, while other strategies failed to achieve a ring-opening nucleophilic reaction withN-unprotected substrates. To prove the feasibility of the procedure, the synthesis of a thioether ring B mimetic of the natural lantibiotic haloduracin β was performed.

Cell scaffolds are often used in cell transplantation as they provide a solid structural support to implanted cells and can be bioengineered to mimic the native extracellular matrix. Gadolinium fluoride nanoparticles (Gd-NPs) as a contrast agent for Magnetic Resonance Imaging (MRI) were incorporated into poly(lactide-co-glycolide)/chitosan scaffolds to obtain Imaging Labelled Cell Scaffolds (ILCSs), having the shape of hollow spherical/ellipsoidal particles (200&ndash, 600 &mu, m diameter and 50&ndash, 80 &mu, m shell thickness). While Gd-NPs incorporated into microparticles do not provide any contrast enhancement in T1-weighted (T1w) MR images, ILCSs can release Gd-NPs in a controlled manner, thus activating MRI contrast. ILCSs seeded with human mesenchymal stromal cells (hMSCs) were xenografted subcutaneously into either immunocompromised and immunocompetent mice without any immunosuppressant treatments, and the transplants were followed-up in vivo by MRI for 18 days. Immunocompromised mice showed a progressive activation of MRI contrast within the implants due to the release of Gd-NPs in the extracellular matrix. Instead, immunocompetent mice showed poor activation of MRI contrast due to the encapsulation of ILCSs within fibrotic capsules and to the scavenging of released Gd-NPs by phagocytic cells. In conclusion, the MRI follow-up of cell xenografts can report the host cell response to the xenograft. However, it does not strictly report on the viability of transplanted hMSCs.

Numerous studies on molluscs have been carried out to clarify the physiological roles of haemolymph serum proteins and haemocytes. However, little is known about the presence and functional role of the serum metabolites. In this study, Nuclear Magnetic Resonance (NMR) was used to assess whether changes of the metabolic profile of Mytilus galloprovincialis haemolymph may reflect alterations of the physiological status of the organisms due to environmental stressors, namely copper and temperature. Mussel haemolymph was taken from the posterior adductor muscle after a 4-day exposure to ambient (16 °C) or high temperature (24 °C) and in the absence or presence (5 μg/L, 20 μg/L, or 40 μg/L) of sublethal copper (Cu2+). The total glutathione (GSH) concentration in the haemolymph of both control and treated mussels was minimal, indicating the absence of significant contaminations by muscle intracellular metabolites due to the sampling procedure. In the 1H-NMR spectrum of haemolymph, 27 metabolites were identified unambiguously. The separate and combined effects of exposure to copper and temperature on the haemolymph metabolic profile were assessed by Principal Component Analysis (PCA) and Ranking-PCA multivariate analysis. Changes of the metabolomic profile due to copper exposure at 16 °C became detectable at a dose of 20 μg/L copper. Alanine, lysine, serine, glutamine, glycogen, glucose and protein aliphatics played a major role in the classification of the metabolic changes according to the level of copper exposition. High temperature (24 °C) and high copper levels caused a coherent increase of a common set of metabolites (mostly glucose, serine, and lysine), indicating that the metabolic impairment due to high temperature is enforced by the presence of copper. Overall, the results demonstrate that, as for human blood plasma, the analysis of haemolymph metabolites represents a promising tool for the diagnosis of pollutant-induced stress syndrome in marine mussels.

The redox microenvironment within a cell graft can be considered as an indicator to assess whether the graft is metabolically active or hypoxic. We present a redox-responsive MRI probe based on porous silica microparticles whose surface has been decorated with a Gd-chelate through a disulphide bridge. Such microparticles are designed to be interspersed with therapeutic cells within a biocompatible hydrogel. The onset of reducing conditions within the hydrogel is paralleled by an increased clearance of Gd, that can be detected by MRI.

A one-pot, high-yield procedure for synthesizing lanthionine-containing peptides was developed. It relies on the S-alkylation of cysteine-containing peptides with chiral cyclic sulfamidates. The key feature of this approach is the use of mild reaction conditions (only activated molecular sieves are employed as the catalyst), leading to good chemoselectivity and excellent stereochemical control. The potential of the new methodology has been investigated by synthesizing the thioether ring of a natural lantibiotic, Haloduracin β.

An efficient and rapid procedure for synthesizing S-linked glycopeptides is reported. The approach uses activated molecular sieves as a base to promote the selective S-alkylation of readily prepared cysteine-containing peptides, upon reaction of appropriate glycosyl halides. Considering the very mild conditions employed, the chemoselective linkage of the electrophilic sugar with a peptide sulfhydryl group occurred in satisfactory yield, allowing the incorporation of mono and disaccharide moieties. The sugar-peptide conjugates obtained from α-d-glycosyl derivatives adopt a β-S-configuration, indicating the high stereoselectivity of the substitution reaction.

Molecular imaging requires the specific accumulation of contrast agents at the target. To exploit the superb resolution of MRI for applications in molecular imaging, gadolinium chelates, as the MRI contrast agents (CA), have to be conjugated to a specific vector able to recognize the epitope of interest. Several Gd(III)-chelates can be chemically linked to the same binding vector in order to deliver multiple copies of the CA (multimers) in a single targeting event thus increasing the sensitivity of the molecular probe. Herein three novel bifunctional agents, carrying one functional group for the bioconjugation to targeting vectors and four Gd(III)-AAZTA chelate functions for MRI contrast enhancement (AAZTA = 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid), are reported. The relaxivity in the tetrameric derivatives is 16.4 ± 0.2 mMGd–1 s–1 at 21.5 MHz and 25 °C, being 2.4-fold higher than that of parent, monomeric Gd(III)-AAZTA. These compounds can be used as versatile building blocks to insert pre...

// Cecilia Roux 1, 2 , Chiara Riganti 3 , Sammy Ferri Borgogno 1, 2 , Roberta Curto 1, 2 , Claudia Curcio 1, 2 , Valeria Catanzaro 4 , Giuseppe Digilio 4 , Sergio Padovan 5 , Maria Paola Puccinelli 6 , Monica Isabello 6 , Silvio Aime 2, 7 , Paola Cappello 1, 2, 7, * and Francesco Novelli 1, 2, 7, * 1 Center for Experimental Research and Medical Studies, Citta della Salute e della Scienza di Torino, 10126 Turin, Italy 2 Department of Molecular Biotechnology and Health Sciences, University of Turin, 10126 Turin, Italy 3 Department of Oncology, University of Turin, 10126 Turin, Italy 4 Department of Science and Technologic Innovation, Universita del Piemonte Orientale “A. Avogadro”, 15121 Alessandria, Italy 5 Institute for Biostructures and Bioimages (CNR) c/o Molecular Biotechnology Center, 10126 Turin, Italy 6 Clinical Biochemistry Laboratory, Citta della Salute e della Scienza di Torino, 10126 Turin, Italy 7 Molecular Biotechnology Center, University of Turin, 10126 Turin, Italy * These authors have contributed equally to this work Correspondence to: Francesco Novelli, email: franco.novelli@unito.it Paola Cappello, email: paola.cappello@unito.it Keywords: PDAC; amino acid; circulating biomarkers; pancreatitis; diagnosis Received: March 24, 2017 Accepted: August 04, 2017 Published: August 24, 2017 ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is becoming the second leading cause of cancer-related death in the Western world. The mortality is very high, which emphasizes the need to identify biomarkers for early detection. As glutamine metabolism alteration is a feature of PDAC, its in vivo evaluation may provide a useful tool for biomarker identification. Our aim was to identify a handy method to evaluate blood glutamine consumption in mouse models of PDAC. We quantified the in vitro glutamine uptake by Mass Spectrometry (MS) in tumor cell supernatants and showed that it was higher in PDAC compared to non-PDAC tumor and pancreatic control human cells. The increased glutamine uptake was paralleled by higher activity of most glutamine pathway-related enzymes supporting nucleotide and ATP production. Free glutamine blood levels were evaluated in orthotopic and spontaneous mouse models of PDAC and other pancreatic-related disorders by High-Performance Liquid Chromatography (HPLC) and/or MS. Notably we observed a reduction of blood glutamine as much as the tumor progressed from pancreatic intraepithelial lesions to invasive PDAC, but was not related to chronic pancreatitis-associated inflammation or diabetes. In parallel the increased levels of branched-chain amino acids (BCAA) were observed. By contrast blood glutamine levels were stable in non-tumor bearing mice. These findings demonstrated that glutamine uptake is measurable both in vitro and in vivo . The higher in vitro avidity of PDAC cells corresponded to a lower blood glutamine level as soon as the tumor mass grew. The reduction in circulating glutamine represents a novel tool exploitable to implement other diagnostic or prognostic PDAC biomarkers.

Inflammation is signaled by the overexpression of epitopes on the vascular endothelium that primarily aim at recruiting immune cells into the inflamed area. The intravascular localization of these biomarkers makes them suitable targets for the MRI visualization of inflammation. Phospholipid-based nanosystems appear excellent candidates in virtue of their good biocompatibility, ability to deliver a high number of imaging units at the target site, and for the easy functionalization with targeting vectors. In this work, phospholipid-based micelles (hydrodynamic diameter of 20 nm) loaded with the amphiphilic Gd(III)-complex Gd-DOTAMA(C18)2 were vectorized with a small peptide able to specifically bind VCAM-1 receptors. The micelles displayed a high longitudinal relaxivity (36.4 s-1mmolGd-1 at 25 °C and 0.7 T). A 1H- and 17O-water relaxometry study indicated that the paramagnetic complex embedded in the nanoparticles adopted two isomeric conformations, likely reflecting the well-known square antiprismatic (SAP) and twisted square antiprismatic (TSAP) configurations typically observed in DOTA-like lanthanide complexes. Interestingly, the TSAP structure, showing a much faster exchange rate for the water molecule coordinated to the metal ion, was the most abundant, thus explaining the high relaxivity of the micellar agent. The systemic administration of the micelles into a lipopolysaccharide-induced murine model of acute inflammation successfully demonstrated the ability of the targeting agents to detect the diseased area by T1 contrast enhanced MRI.

OBJECTIVES: The aim of this study was to evaluate 4 nonionic x-ray iodinated contrast agents (CAs), commonly used in radiographic procedures, as novel chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) agents by assessing their in vitro exchange properties and preliminary in vivo use as tumor enhancing agents. MATERIALS AND METHODS: The CEST properties, as function of pH (range, 5.5-7.9) and of radio frequency conditions (irradiation field strength range of 1-9 ?T and time of 1-9 seconds), have been determined at 7 T and 310 K for 4 x-ray CAs commonly used in clinical settings, namely, iomeprol, iohexol, ioversol, and iodixanol. Their in vivo properties have been investigated upon intravenous injection in a murine HER2+ breast tumor model (n = 4 mice for each CA) using both computed tomography (CT) and MRI modalities. RESULTS: The prototropic exchange rates measured for the 4 investigated iodinated molecules showed strong pH dependence with base catalyzed exchange rate that was faster for monomeric compounds (20-4000 Hz in the pH range of 5.5-7.9). Computed tomography quantification showed marked (up to 2 mg I/mL concentration) and prolonged accumulation (up to 30 minutes postinjection) inside tumor regions. Among the 4 agents we tested, iohexol and ioversol display good CEST contrast properties at 7 T, and in vivo results confirmed strong and prolonged contrast enhancement of the tumors, with elevated extravasation fractions (74%-91%). A strong and significant correlation was found between CT and CEST-MRI tumor-enhanced images (R = 0.70, P < 0.01). CONCLUSIONS: The obtained results demonstrate that iohexol and ioversol, 2 commonly used radiographic compounds, can be used as MRI perfusion agents, particularly useful when serial images acquisitions are needed to complement CT information.