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.

Mobile proton-containing solutes can be detected by MRI by the chemical exchange saturation transfer (CEST) method. CEST sensitivity is dramatically enhanced by using, as exchanging protons, the water molecules confined inside liposomes, shifted by a paramagnetic shift reagent. The chemical shift of the intraliposomal water resonance (δIL ) is affected by the overall shape of the supramolecular system. δIL of a spherical LipoCEST acts as a sensitive reporter of the distribution of streptavidin proteins anchored at the liposome surface by biotinylated phospholipids. This finding prompted the design of a MMP-2 responsive LipoCEST agent as the streptavidin moieties can be released from the liposome surfaces when a properly tailored enzyme-cleavable peptide is inserted on the phospholipids before the terminal biotin residues. δIL reports on the overall changes in the supramolecular architecture associated to the cleavage carried out by MMP-2.

Amphiphilic Janus-dendrimers are able to self-assemble into nanosized vesicles named dendrimersomes. We recently synthesized the 3,5-C12-EG-(OH)4 dendrimer that generates dendrimersomes with very promising safety and stability profiles, that can be loaded with different contrast agents for in vivo imaging. In this contribution, nanovesicles were loaded with both the Magnetic Resonance Imaging (MRI) reporter GdDOTAGA(C18)2 and the glucocorticoid drug Prednisolone Phosphate (PLP), in order to test their effective potential as theranostic nanocarriers on murine melanoma tumour models. The incorporation of GdDOTAGA(C18)2 into the membrane resulted in dendrimersomes with a high longitudinal relaxivity (r1=39.1mM-1s-1, at 310K and 40MHz) so that, after intravenous administration, T1-weighted MRI showed a consistent contrast enhancement in the tumour area. Furthermore, the nanovesicles encapsulated PLP with good efficiency and displayed anti-tumour activity both in vitro and in vivo, thus enabling their practical use for biomedical theranostic applications.

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.

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.

Herein, a new relaxometric method for the assessment of intestinal permeability based on the oral administration of clinically approved gadolinium (Gd)-based MRI contrast agents (CAs) is proposed. The fast, easily performed and cheap measurement of the longitudinal water proton relaxation rate (R1) in urine reports the amount of paramagnetic probe that has escaped the gastrointestinal tract. The proposed method appears to be a compelling alternative to the available methods for the assessment of intestinal permeability. The method was tested on the murine model of dextran sulfate sodium (DSS)-induced colitis in comparison with healthy mice. Three CAs were tested, namely ProHance®, MultiHance® and Magnevist®. Urine was collected for 24 h after the oral ingestion of the Gd-containing CA at day 3-4 (severe damage stage) and day 8-9 (recovery stage) after treatment with DSS. The Gd content in urine measured by (1)H relaxometry was confirmed by inductively coupled plasma-mass spectrometry (ICP-MS). The extent of urinary excretion was given as a percentage of excreted Gd over the total ingested dose. The method was validated by comparing the results obtained with the established methodology based on the lactulose/mannitol and sucralose tests. For ProHance and Magnevist, the excreted amounts in the severe stage of damage were 2.5-3 times higher than in control mice. At the recovery stage, no significant differences were observed with respect to healthy mice. Overall, a very good correlation with the lactulose/mannitol and sucralose results was obtained. In the case of MultiHance, the percentage of excreted Gd complex was not significantly different from that of control mice in either the severe or recovery stages. The difference from ProHance and Magnevist was explained on the basis of the (known) partial biliary excretion of MultiHance in mice.

Mobile proton-containing solutes can be detected by MRI by the chemical exchange saturation transfer (CEST) method. CEST sensitivity is dramatically enhanced by using, as exchanging protons, the water molecules confined inside liposomes, shifted by a paramagnetic shift reagent. The chemical shift of the intraliposomal water resonance (δ

The approach to molecular imaging of enzymes by MRI typically relies upon imaging probes composed of an enzymecleavable moiety conjugated with a paramagnetic imaging reporter, such as a GdIII chelate.[1] Upon enzymatic processing, the probe is transformed into a fragment with an altered relaxivity, leading to a different capability to enhance contrast in MR images with respect to the parent species. Ideally, the unprocessed (intact) form of the probe should be completely silent while the processed (cleaved) form should have a high relaxivity (that is, high contrast enhancement). In such a way the appearance of contrast within images can be unambiguously attributed to the result of enzymatic activity and not to dynamic changes of tissue probe concentration. However, gadolinium-based agents as enzyme responsive agents are never completely silent and both forms (unprocessed and processed) contribute to the overall contrast enhancement as a function of their respective relaxivities and tissue local concentrations.[2] Exact knowledge of the total concentration of Gd is essential to translate image contrast enhancement into the molar ratio of unprocessed versus processed forms, and thus into true enzyme activity maps. Aviable solution to the concentration problem can be provided by the R2p/R1p.

// 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.

Summary Protection against oxidative damage caused by excessive reactive oxygen species (ROS) by an antioxidant network is essential for the health of tissues, especially in the cardiovascular system. Here, we identified a gene with important antioxidant features by analyzing a null allele of zebrafish ubiad1, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a nonmitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. Loss of UBIAD1 reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation in vascular cells. Surprisingly, inhibition of eNOS prevents Ubiad1-dependent cardiovascular oxidative damage, suggesting a crucial role for this enzyme and nonmitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a nonmitochondrial CoQ10-forming enzyme with specific cardiovascular protective function via the modulation of eNOS activity., Graphical Abstract Highlights ► UBIAD1 is a Golgi prenyltransferase ► UBIAD1 contributes to the nonmitochondrial pool of CoQ10 ► UBIAD1 protects cardiovascular tissues from NOS-dependent oxidative damage ► UBIAD1 is a target for therapeutic strategies by limiting the side effects of statins, UBIAD1 is identified as a nonmitochondrial CoQ10 biosynthetic enzyme required for oxidative damage protection. Through CoQ10 synthesis, UBIAD1 modulates endothelial nitric oxide synthase activity and nitric oxide signaling necessary for cardiovascular development and homeostasis.

Two novel Gd-based contrast agents (CAs) for the molecular imaging of matrix metalloproteinases (MMPs) were synthetized and characterized in vitro and in vivo. These probes were based on the PLG*LWAR peptide sequence, known to be hydrolyzed between Gly and Leu by a broad panel of MMPs. A Gd–DOTA chelate was conjugated to the N-terminal position through an amide bond, either directly to proline (compd Gd–K11) or through a hydrophilic spacer (compd Gd–K11N). Both CA were made strongly amphiphilic by conjugating an alkyl chain at the C-terminus of the peptide sequence. Gd–K11 and Gd–K11N have a good affinity for β-cyclodextrins (KD 310 and 670 µ m respectively) and for serum albumin (KD 350 and 90 µ m respectively), and can be efficiently cleaved in vitro at the expected site by MMP-2 and MMP-12. Upon MMP-dependent cleavage, the CAs lose the C-terminal tetrapeptide and the alkyl chain, thus undergoing to an amphiphilic-to-hydrophilic transformation that is expected to alter tissue pharmacokinetics. To prove this, Gd–K11 was systemically administered to mice bearing a subcutaneous B16.F10 melanoma, either pre-treated or not with the broad spectrum MMP inhibitor GM6001 (Ilomastat). The washout of the Gd-contrast enhancement in MR images was significantly faster for untreated subjects (displaying MMP activity) with respect to treated ones (MMP activity inhibited). The washout kinetics of Gd-contrast enhancement from the tumor microenvironment could be then interpreted in terms of the local activity of MMPs. Copyright © 2012 John Wiley & Sons, Ltd.

Fluorinated, arylsulfone-based inhibitors of Matrix Metalloproteinases (MMP) have been used, in the [ 18 F]-radiolabelled version, as radiotracers targeted to MMP-2/9 for Positron Emission Tomography (PET). Although they showed acceptable tumour uptake, specificity was rather low. To get further insights into the reason of low specificity, the binding interaction of these compounds with Human Serum Albumin (HSA) has been investigated. 19 F NMR spectroscopy showed that all compounds considered partition between multiple HSA binding sites, being characterized by either slow-exchange kinetics (with K a in the order of 10 5 M −1 ) and fast-exchange kinetics (with K a in the order of 10 4 M -1 ). For 2-(2-(4′-(2-fluoroethoxy)biphenyl-4-ylsulfonyl)phenyl)acetic acid ( 1a ) and 2-(2-(4′-(2-fluoroacetamido)biphenyl-4-ylsulfonyl)phenyl)acetic acid ( 1c ), these slow and fast-exchanging binding sites could be mapped to Sudlow's site I and II, respectively. It is shown that high affinity albumin binding constitutes a theoretical limitation for the specificity achievable by MMP-inhibitors as MMP-targeted PET tracers in cancer imaging, because albumin accumulating aspecifically in tumours lowers the binding potential of radiotracers.

Two novel Gd-based contrast agents (CAs) for the molecular imaging of matrix metalloproteinases (MMPs) were synthetized and characterized in vitro and in vivo. These probes were based on the PLG*LWAR peptide sequence, known to be hydrolyzed between Gly and Leu by a broad panel of MMPs. A Gd-DOTA chelate was conjugated to the N-terminal position through an amide bond, either directly to proline (compd Gd-K11) or through a hydrophilic spacer (compd Gd-K11N). Both CA were made strongly amphiphilic by conjugating an alkyl chain at the C-terminus of the peptide sequence. Gd-K11 and Gd-K11N have a good affinity for β-cyclodextrins (K(D) 310 and 670 µ m respectively) and for serum albumin (K(D) 350 and 90 µ m respectively), and can be efficiently cleaved in vitro at the expected site by MMP-2 and MMP-12. Upon MMP-dependent cleavage, the CAs lose the C-terminal tetrapeptide and the alkyl chain, thus undergoing to an amphiphilic-to-hydrophilic transformation that is expected to alter tissue pharmacokinetics. To prove this, Gd-K11 was systemically administered to mice bearing a subcutaneous B16.F10 melanoma, either pre-treated or not with the broad spectrum MMP inhibitor GM6001 (Ilomastat). The washout of the Gd-contrast enhancement in MR images was significantly faster for untreated subjects (displaying MMP activity) with respect to treated ones (MMP activity inhibited). The washout kinetics of Gd-contrast enhancement from the tumor microenvironment could be then interpreted in terms of the local activity of MMPs.

Murine melanoma B16 cells display on the extracellular side of the plasma membrane a large number of reactive protein thiols (exofacial protein thiols, EPTs). These EPTs can be chemically labeled with Gd-DO3A-PDP, a Gd(III)-based MRI contrast agent bearing a 2-pyridinedithio chemical function for the recognition of EPTs. Uptake of gadolinium up to 10(9) Gd atoms per cell can be achieved. The treatment of B16 cells ex vivo with a reducing agent such as tris(2-carboxyethyl)phosphine (TCEP) results in an increase by 850% of available EPTs and an increase by 45% of Gd uptake. Blocking EPTs with N-ethylmaleimide (NEM) caused a decrease by 84% of available EPTs and a decrease by 55% of Gd uptake. The amount of Gd taken up by B16 cells is therefore dependent upon the availability of EPTs, whose actual level in turn changes according to the extracellular redox microenvironment. Then Gd-DO3A-PDP has been assessed for the labeling of tumor cells in vivo on B16.F10 melanoma tumor-bearing mice. Gd-DO3A-PDP (or Gd-DO3A as the control) has been injected directly into the tumor region at a dose level of 0.1 μmol and the signal enhancement in MR images followed over time. The washout kinetics of Gd-DO3A-PDP from tumor is very slow if compared to that of control Gd-DO3A, and 48 h post injection, the gadolinium-enhancement is still clearly visible. Therefore, B16 cells can be labeled ex vivo as well as in vivo according to a common EPTs-dependent route, provided that high levels of the thiol reactive probe can be delivered to the tumor.

Four novel MRI Gd(III)-based probes have been synthesized and evaluated for their labeling properties on cultured cell lines K562, C6, and B16. The labeling strategy relies upon the fact that cells display a large number of reactive exofacial protein thiols (EPTs) that can be exploited as anchorage points for suitably activated MRI probes. The probes are composed of a Gd(III) chelate (based on either DO3A or DTPA) connected through a flexible linker to the 2-pyridyldithio chemical function for binding to EPTs. GdDO3A-based chelates could efficiently label cells (up to a level of 1.2 x 10(10) Gd(III) atoms/cell), whereas GdDTPA-based chelates showed poor or no cell labeling ability at all. Among the GdDO3A based compounds, that having the longest spacer (compound GdL1A) showed the best labeling efficacy. The mechanism of EPT mediated cell labeling by GdL1A involves probe internalization without sequestration of the Gd(III) chelate within subcellular structures such as endosomes.

Cells display on the outer surface of the plasma membrane a large number of protein thiols that can be reversibly labelled with suitably designed Gd(III)-based contrast agents for cell tracking by MRI.