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The results of a study on pollen morphology of the species of the genusPtilostemongrowing in Italy:Ptilostemon niveus, P. greuteri,P. gnaphaloides,P. casabonae,P. strictus,P. stellatusby light and scanning electron microscopy were presented and discussed including in relation to their taxonomic position. The exine shows two different ornamentation patterns: echinae and scabrae. This last ornamentation pattern together with other features was previously described in the genusPtilostemon. Of 6 species analysed,P. stellatusis the only one that shows a scabrate ornamentation of exine. Two pollen types were recognized through the exine ornamentation, length of ectocolpus and polar outline:P. stellatustype andP. niveustype. A multivariate analysis (principal component analysis and linear discriminant analysis) was carried out with the aim of examining potential morphological characters which could be used to identify taxa. The data suggest that several characteristics can be used to delimit the species in particular the exine ornamentation. The results of our studies support the actually subgeneric classification, and several features of the pollen grains analysed seem to have a palynoecological role and an important taxonomic significance. A dichotomous key based on palynological data is also given. [ABSTRACT FROM PUBLISHER]

Pollen morphology of the native Sicilian species of the genusMuscari, one of which is endemic to Sicily, belonging to the subgeneraMuscari,LeopoldiaandPseudomuscari, was studied using a light microscope and a scanning electron microscope. Pollen grains are monads, heteropolar and monosulcate; the exine is semitectate reticulate and perforate. The sulcus extends from distal to proximal face and its length is not directly proportional to the size of the granules and varies from one species to another. [ABSTRACT FROM AUTHOR]

A study on pollen morphology and seed germination behaviour ofRetama raetam(Forssk.) Webb ssp.gussonei(Webb) Greuter, endemic subspecies from Sicily and Calabria, is herein presented, aiming to preserve these populations strongly affected by anthropic pressure. [ABSTRACT FROM AUTHOR]

In this study, using light and scanning microscopes, the morphobiometric characters of pollen grains of Bupleurum fruticosum L., B. fontanesii Guss. ex Caruel, B. gerardi All., B. lancifolium Hornem., B. semicompositum L., B. tenuissimum L. have been analysed. The elaboration of qualitative and quantitative palynological data has allowed us to formulate a key of identification which characterizes six morphotypes. Pollen grains of B. fruticosum (a perennial species) have been distinguished from the grains of the other annual species examined by us on the basis of several morphobiometric characters, a condition which has confirmed the systematic arrangements suggested by several authors. The close palynological affinity between B. tenuissimum and B. semicompositum compared with B. gerardi has supported the placement of these three species in a distinct subsect. of the sect. Eubupleura Briq. or in a distinct subsect. of the sect. Isophyllum (Hoffm.) Dumort. Similarly, our palynological data on B. fontanesii have confirmed the systematic arrangement. However, these data differ from those reported by different authors who placed the three species in the sect. Aristata Godron subsect. Juncea Briq. [ABSTRACT FROM AUTHOR]

The self-assembly of peptides and peptide analogues may be exploited to develop platforms for different biomedical applications, among which CEST-MRI (chemical exchange saturation transfer magnetic resonance imaging) represents one of the most attractive techniques to be explored as a novel metal-free contrast approach in imaging acquisitions. A lysine-containing peptide sequence (LIVAGK-NH 2 , named K2) was thus modified by insertion, at the N-terminus, of a peptide nucleic acid (PNA) base, leading to a primary amine suitable for the signal generation. a-K2, c-K2, g-K2 and t-K2 peptides were synthesized and characterized. The c-K2 sequence displayed gelling properties and the Watson and Crick pairing, arising from its combination with g-K2, allowed a significant increase in the mechanical responsivity of the hydrogel. These matrices were able to generate a CEST signal around 2.5 ppm from water and, after assessing their cytocompatibility on GL261 (murine glioma), TS/a (murine breast carcinoma), and 3T3-NIH (murine fibroblasts) cell lines, their capability to work as implants for in vivo detection, was proved by intratumor injection in Balb/c mice inoculated with TS/a murine breast cancer cells.

Subcutaneous (SC) administration of monoclonal antibodies (mAbs) is a proven strategy for improving therapeutic outcomes and patient compliance. The current FDA-/EMA-approved enzymatic approach, utilizing recombinant human hyaluronidase (rHuPH20) to enhance mAbs SC delivery, involves degrading the extracellular matrix's hyaluronate to increase tissue permeability. However, this method lacks tunable release properties, requiring individual optimization for each mAb. Seeking alternatives, physical polysaccharide hydrogels emerge as promising candidates due to their tunable physicochemical and biodegradability features. Unfortunately, none have demonstrated simultaneous biocompatibility, biodegradability, and controlled release properties for large proteins (≥150 kDa) after SC delivery in clinical settings. Here, a novel two-component hydrogel comprising chitosan and chitosan@DOTAGA is introduced that can be seamlessly mixed with sterile mAbs formulations initially designed for intravenous (IV) administration, repurposing them as novel tunable SC formulations. Validated in mice and nonhuman primates (NHPs) with various mAbs, including trastuzumab and rituximab, the hydrogel exhibited biodegradability and biocompatibility features. Pharmacokinetic studies in both species demonstrated tunable controlled release, surpassing the capabilities of rHuPH20, with comparable parameters to the rHuPH20+mAbs formulation. These findings signify the potential for rapid translation to human applications, opening avenues for the clinical development of this novel SC biosimilar formulation., (© 2023 The Authors. Advanced Materials published by Wiley‐VCH GmbH.)

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., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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 1 H-nuclear magnetic relaxation dispersion (NMRD) and 17 O- R 2 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 T 1w 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.

Here we propose innovative photoacoustic imaging (PAI) contrast agents, based on the loading of Mn(iii)-, Fe(iii)- or Zn(ii)-protoporphyrin IX in serum albumin. These systems show different absorption wavelengths, opening the way to multicolor PA imaging. They were characterized in vitro for assessing stability, biocompatibility, and their optical and contrastographic properties. Finally, a proof of concept in vivo study was carried out in breast cancer bearing mice, to evaluate its effectiveness for cancer imaging., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Peptide-based hydrogels have been recently investigated as materials for biomedical applications like tissue engineering and delivery of drugs and imaging agents. Among the synthetic peptide hydrogelators, the cationic hexapeptides Ac-K1 and Ac-K2 were proposed as scaffolds for bioprinting applications. Here, we report the formulation of Ac-K1 and Ac-K2 hydrogels loaded with iopamidol, an iodinated contrast agent clinically approved for X-ray computed tomography, and more recently identified as an efficient CEST-MRI probe. Iopamidol-loaded hydrogels were soft, injectable and non-toxic both in vitro (on three tumor cell lines: GL261, TS/A and 3T3-NIH) and in vivo (in Balb/c mice inoculated with TS/A breast cancer cells). The in vitro CEST-MRI study evidenced the typical features of the CEST pattern of iopamidol, with a CEST contrast higher than 50%. Due to their injectability and good ability to retain the contrast agent, the herein investigated systems can be considered as promising candidates for the development of smart MRI detectable hydrogels.

Since the very beginnings of the chemical exchange saturation transfer (CEST) technique, poor overall sensitivity has appeared to be one of its strongest limitations for future applications. Research has therefore focused on designing systems, such as supramolecular and nanosized agents, that contain a high number of magnetically equivalent mobile spins. However, the number of mobile spins offered by these systems is still limited by their composition and surface/volume ratio. The design of compartmentalized agents, that is, systems where an aqueous inner core is separated from the MRI-detected bulk pool via a semipermeable barrier/membrane, is very much a step forward for the technique. These vesicular systems can (i) act as biocompatible and versatile carriers for dia-, para-, and hetero-nuclear CEST probes, thus offering new application options; and (ii) act as CEST probes themselves via the encapsulation of a suitable agent (e.g., a paramagnetic shift reagent) that can change the resonance frequency of the spin pool in the inner compartment only. LipoCEST agents were the pioneers in the latter category, as they are able to grant picomolar sensitivity (in terms of nanoparticle concentration), and paved the way for new applications for CEST agents, especially in the theranostic research area. The use of larger, natural vesicular systems, such as yeasts and cells, in which the huge number of intravesicular spins lowers the detection threshold to a femtomolar limit, is a further step forward in the development of compartmentalized CEST agents. Finally, interesting combinations of nanovesicular and cellular compartmentalized systems have been proposed, thus highlighting how the approach has the potential to drive CEST agents towards completing their journey to mature clinical translation., (© 2022 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Breast cancer is the leading cause of cancer-related death in women. Although many therapeutic approaches are available, systemic chemotherapy remains the primary choice, especially for triple-negative and advanced breast cancers. Unfortunately, systemic chemotherapy causes serious side effects and requires high doses to achieve an effective concentration in the tumor. Thus, the use of nanosystems for drug delivery may overcome these limitations. Herein, we formulated Poly (lactic-co-glycolic acid) nanoparticles (PLGA-NPs) containing Docetaxel, a fluorescent probe, and a magnetic resonance imaging (MRI) probe. The cyclic RGD tripeptide was linked to the PLGA surface to actively target α v β 3 integrins, which are overexpressed in breast cancer. PLGA-NPs were characterized using dynamic light scattering, fast field-cycling 1 H-relaxometry, and 1 H-nuclear magnetic resonance. Their therapeutic effects were assessed both in vitro in triple-negative and HER2+ breast cancer cells, and in vivo in murine models. In vivo MRI and inductively coupled plasma mass spectrometry of excised tumors revealed a stronger accumulation of PLGA-NPs in the RGD_PLGA group. Targeted PLGAs have improved therapeutic efficacy and strongly reduced cardiac side effects compared to free Docetaxel. In conclusion, RGD-PLGA is a promising system for breast cancer treatment, with positive outcome in terms of therapeutic efficiency and reduction in side effects.

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 (r 1 ) 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 r 1 (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 T 1 -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., (© 2022 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

Cancer stem cells (CSCs) are the main drivers of disease progression and chemotherapy resistance in breast cancer. Tumor progression and chemoresistance might then be prevented by CSC-targeted therapies. We previously demonstrated that Toll-like Receptor (TLR)2 is overexpressed in CSCs and fuels their self-renewal. Here, we show that high TLR2 expression is linked to poor prognosis in breast cancer patients, therefore representing a candidate target for breast cancer treatment. By using a novel mammary cancer-prone TLR2 KO mouse model, we demonstrate that TLR2 is required for CSC pool maintenance and for regulatory T cell induction. Accordingly, cancer-prone TLR2 KO mice display delayed tumor onset and increased survival. Transplantation of TLR2 WT and TLR2 KO cancer cells in either TLR2 WT or TLR2 KO hosts shows that tumor initiation is mostly sustained by TLR2 expression in cancer cells. TLR2 host deficiency partially impairs cancer cell growth, implying a pro-tumorigenic effect of TLR2 expression in immune cells. Finally, we demonstrate that doxorubicin-induced release of HMGB1 activates TLR2 signaling in cancer cells, leading to a chemotherapy-resistant phenotype. Unprecedented use of TLR2 inhibitors in vivo reduces tumor growth and potentiates doxorubicin efficacy with no negative impact on the host immune system, opening new perspectives for the treatment of breast cancer patients., Competing Interests: The authors declare no potential conflicts of interest., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

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 + , Ca 2+ , La 3+ , Gd 3+ ) 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 Gd 3+ detection was calculated. The effects on the emission were also analyzed in the presence of a combination of Gd 3+ 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 Gd 3+ release upon the administration of Gd-based contrast agents in murine macrophages.

Novel Mn(II)-based nanoprobes were rationally designed as high contrast enhancing agents for magnetic resonance imaging (MRI) and obtained by anchoring a Mn(II)-CDTA derivative to the surface of organo-modified silica nanoparticles (SiNPs). Large payloads of paramagnetic metal-chelates have been immobilized on biocompatible SiNPs with spherical shape and narrow size distribution of 80-90 nm, resulting in a relaxivity gain of 250% at clinical fields (0.5 T) as compared to the free chelate. Such substantial efficacy enhancement of the nanoprobes is mainly attributed to the restriction of the rotational dynamics of the conjugated complex, as revealed by comprehensive 1 H-NMR relaxometric investigations. The paramagnetic nanospheres exhibit good colloidal stability over time in biological matrices, allowing for MRI applications. High image contrast was found in T 1w -MRI images collected at 1 T on phantoms containing relatively small amounts of contrast agent (CA), for which low cellular toxicity was observed on three different cell lines. Preliminary in vivo studies on healthy mice demonstrated the efficiency of the novel Mn-based silica nanoparticle as T 1w -MRI probes, resulting in significant contrast enhancement in the liver. These findings demonstrate that these novel Mn-SiNPs are high efficacy CAs suitable for preclinical MRI applications.

Objectives: The targeting of tumor cells and their visualization with magnetic resonance imaging (MRI) is an important task in biomedicine. The low sensitivity of this technique is a significant drawback and one that may hamper the detection of the imaging reporters used.To overcome this sensitivity issue, this work explores the synergy between 2 strategies: (1) arginine, glycine, aspartic acid peptide (RGD)-functionalized giant unilamellar vesicles (GUVs) loaded with Gd complexes to accumulate large amounts of MRI contrast agent at the targeting site; and (2) the use of magnetization transfer contrast (MTC), which is a sensitive MRI technique for the detection of Gd complexes in the tumor region., Materials and Methods: Giant unilamellar vesicles were prepared using the gentle swelling method, and the cyclic RGD targeting moiety was introduced onto the external membrane. Paramagnetic Gd-containing complexes and the fluorescent probe rhodamine were both part of the vesicle membranes and Gd-complexes were also the payload within the inner aqueous cavity. Giant unilamellar vesicles that were loaded with the imaging reporters, but devoid of the RGD targeting moiety, were used as controls. U-87 MG human glioblastoma cells, which are known to overexpress the targets for RGD moieties, were used. In the in vivo experiments, U-87 MG cells were subcutaneously injected into nu/nu mice, and the generated tumors were imaged using MRI, 15 days after cell administration. Magnetic resonance imaging was carried out at 7 T, and T2W, T1W, and MTC/Z-spectra were acquired. Confocal microscopy images and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) were used for result validation., Results: In vitro results show that RGD GUVs specifically bind to U-87 MG cells. Microscopy demonstrates that (1) RGD GUVs were anchored onto the external surface of the tumor cells without any internalization; (2) a low number of GUVs per cell were clustered at specific regions; and (3) there is no evidence for macrophage uptake or cell toxicity. The MRI of cell pellets after incubation with RGD GUVs and untargeted ctrl-GUVs was performed. No difference in T1 signal was detected, whereas a 15% difference in MT contrast is present between the RGD GUV-treated cells and the ctrl-GUV-treated cells.Magnetic resonance imaging scans of tumor-bearing mice were acquired before and after (t = 0, 4 hours and 24 hours) the administration of RGD GUVs and ctrl-GUVs. A roughly 16% MTC difference between the 2 groups was observed after 4 hours. Immunofluorescence analyses and ICP-MS analyses (for Gd-detection) of the explanted tumors confirmed the specific accumulation of RGD GUVs in the tumor region., Conclusions: RGD GUVs seem to be interesting carriers that can facilitate the specific accumulation of MRI contrast agents at the tumor region. However, the concentration achieved is still below the threshold needed for T1w-MRI visualization. Conversely, MTC proved to be sufficiently sensitive for the visualization of detectable contrast between pretargeting and posttargeting images., Competing Interests: Conflicts of interest and sources of funding: The authors declare no conflicts of interest. Funding was received from the Italian Ministry of Research through FOE contribution to the Euro-BioImaging MultiModal Molecular Imaging Italian Node (www.mmmi.unito.it). Funding was also received from the University of Turin (G.F.). This research was performed in the framework of COST Action AC15209 (EURELAX)., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Malaria is a global health problem with the most malignant form caused by Plasmodium falciparum (P. falciparum). Parasite maturation in red blood cells (RBCs) is accompanied by changes including the formation of paramagnetic hemozoin (HZ) nanocrystals, and increased metabolism and variation in membrane lipid composition. Herein, MR relaxometry (MRR) was applied to investigate water exchange across RBCs' membrane and HZ formation in parasitized RBCs., Methods: Transverse water protons relaxation rate constants (R 2 = 1/T 2 ) were measured for assessing HZ formation in P. falciparum-parasitized human RBCs. Moreover, water exchange lifetimes across the RBC membrane (τ i ) were assessed by measuring longitudinal relaxation rate constants (R 1 = 1/T 1 ) at 21.5 MHz in the presence of a gadolinium complex dissolved in the suspension medium., Results: τ i increased after invasion of parasites (ring stage, mean τ i / τ i 0 = 1.234 ± 0.022) and decreased during maturation to late trophozoite (mean τ i / τ i 0 = 0.960 ± 0.075) and schizont stages (mean τ i / τ i 0 = 1.019 ± 0.065). The HZ accumulation in advanced stages was revealed by T 2 -shortening. The curves reporting R 2 (1/T 2 ) vs. magnetic field showed different slopes for non-parasitized RBCs (npRBCs) and parasitized RBCs (pRBCs), namely 0.003 ± 0.001 for npRBCs, 0.009 ± 0.002, 0.028 ± 0.004 and 0.055 ± 0.002 for pRBCs at ring-, early trophozoite-, and late trophozoite stage, respectively. Antimalarial molecules dihydroartemisinin and chloroquine elicited measurable changes in parasitized RBCs, namely dihydroartemisinin modified τ i , whereas the interference of chloroquine with HZ formation was detectable by a significant T 2 increase., Conclusions: MRR can be considered a useful tool for reporting on P. falciparum blood stages and for screening potential antimalarial molecules., (© 2020 International Society for Magnetic Resonance in Medicine.)

The set-up of reversible binding interactions between the hydrophobic region of macrocyclic GBCAs (Gadolinium Based Contrast Agents) and SO 3 - /OH containing pyrene derivatives provides new insights for pursuing relaxivity enhancements of this class of MRI contrast agents. The strong binding affinity allows attaining relaxation enhancements up to 50% at pyrene/GBCA ratios of 3 : 1. High resolution NMR spectra of the Yb-HPDO3A/pyrene system fully support the formation of a supramolecular adduct based on the set-up of hydrophobic interactions. The relaxation enhancement may be accounted for in terms of the increase of the molecular reorientation time ( τ R ) and the number of second sphere water molecules. This effect is maintained in blood serum and in vivo , as shown by the enhancement of contrast in T 1w -MR images obtained by simultaneous injection of GBCA and pyrene derivatives in mice., Competing Interests: L. L. and A. M. are Bracco Imaging S.p.A. employees. The Department of molecular biotechnologies and health sciences has received a Research Grant from Bracco Imaging S.p.A. for the topic object of this study., (This journal is © The Royal Society of Chemistry.)

A current challenge in medical diagnostics is how to obtain high MRI relaxation enhancement using Gd III -based contrast agents (CAs) containing the minimum concentration of Gd III ions. We report that in GdHPDO3A-like complexes a primary amide group located in close proximity to the coordinated hydroxyl group can provide a strong relaxivity enhancement at slightly acidic pH. A maximum relaxivity of r 1 = 9.8 mM -1 s -1 (20 MHz, 298 K) at acidic pH was achieved, which is more than double that of clinically approved MRI contrast agents under identical conditions. This effect was found to strongly depend on the number of amide protons, i.e. it decreases with a secondary amide group and almost completely vanishes with a tertiary amide. This relaxivity enhancement is attributed to an acid-catalyzed proton exchange process between the metal-coordinated OH group, the amide protons and second sphere water molecules. The mechanism and kinetics of the corresponding H + assisted exchange process are discussed in detail and a novel simultaneous double-site proton exchange mechanism is proposed. Furthermore, 1 H and 17 O NMR relaxometry, Chemical Exchange Saturation Transfer (CEST) on the corresponding Eu III complexes, and thermodynamic and kinetic studies are reported. These highlight the optimal physico-chemical properties required to achieve high relaxivity with this series of Gd III -complexes. Thus, proton exchange provides an important opportunity to enhance the relaxivity of contrast agents, providing that labile protons close to the paramagnetic center can contribute., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background and Purpose: Doxorubicin anti-cancer therapy is associated with cardiotoxicity, resulting from DNA damage response (DDR). Hepatocyte growth factor (HGF) protects cardiomyocytes from injury, but its effective use is compromised by low biodistribution. In this study, we have investigated whether the activation of the HGF receptor-encoded by the Met gene-by an agonist monoclonal antibody (mAb) could protect against doxorubicin-induced cardiotoxicity., Experimental Approach: The mAb (5 mg·kg -1 ) was injected in vivo into C57BL/6J mice, before doxorubicin (three doses of 7 mg·kg -1 ). Cardiac functions were evaluated through MRI after treatment termination. Heart histological staining and mRNA levels of genes associated with heart failure (Acta1 and Nppa), inflammation (IL-6), and fibrosis (Ctgf, Col1a2, Timp1, and Mmp9) were assessed. MAb (100 nM) was administered in vitro to H9c2 cardiomyoblasts before addition of doxorubicin (25 μM). DDR and apoptosis markers were evaluated by quantitative western blotting, flow cytometry, and immunofluorescence. Stattic was used for pharmacological inactivation of STAT3., Key Results: In vivo, administration of the mAb alleviated doxorubicin-induced cardiac dysfunction and fibrosis. In vitro, mAb mimicked the response to HGF by (a) inhibiting histone H2AX phosphorylation at S139, (b) quenching the expression of the DNA repair enzyme PARP1, and (c) reducing the proteolytic activation of caspase 3. The MET-driven cardioprotection involved, at least in vitro, the phosphorylation of STAT3., Conclusion and Implications: The MET agonist mAb provides a new tool for cardioprotection against anthracycline cardiotoxicity., (© 2020 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Moving from nano- to micro-systems may not just be a matter of scale, but it might imply changes in the properties of the systems that can open new routes for the development of efficient MRI contrast agents. This is the case reported in the present paper, where giant liposomes (giant unilamellar vesicles, GUVs) loaded with Ln III complexes have been studied as chemical exchange saturation transfer (CEST) MRI contrast agents. The comparison between nanosized liposomes (small unilamellar vesicles, SUVs) and GUVs sharing the same formulation led to differences that could not be accounted for only in terms of the increase in size (from 100-150 nm to 1-2 μm). Upon osmotic shrinkage, GUVs yielded a saturation-transfer effect three order of magnitude higher than SUVs consistent with the increase in vesicles volume. Confocal microscopy showed that the shrinkage of GUVs resulted in multilamellar particles whereas SUVs are known to yield asymmetrical, discoidal shape., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The development of an innovative and easy way to run assays for the quantitative detection of DNA present in biological fluids (i.e., blood, urine, and saliva) is of great interest for early diagnosis (e.g., tumors) and personalized medicine. Herein, a new quantitative assay based on the use of highly sensitive carboxyfluorescein-loaded liposomes as signal amplification systems is reported. The method has been tested for the detection of low amounts of DNA sequences. The reported proof of concept exploits a target DNA molecule as a linker between two complementary oligonucleotides. One oligonucleotide is biotinylated at its 3' end and binds to streptavidin-coupled magnetic beads, whereas the other one is conjugated to a cholesterol molecule incorporated in the phospholipidic bilayer of the fluorescent liposomes. In the presence of the target fragment, the correct formation of a construct takes place as witnessed by a strong fluorescence signal, amplified by dissolving lipidic nanoparticles with Triton X-100. The system is able to detect specific nucleotide sequences with a very low detection threshold of target DNA (tens of picomolar). The assay allows the detection of both single- and double-stranded DNA. Studies performed in human blood serum show the correct assembling of the probe but with a reduction of limit of detection (up to ∼1 nM). This liposome signal amplification strategy could be used not only for the detection of DNA but also for other nucleic acids (mRNA; microRNA) that are difficult to be quantified by currently available protocols., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

A theranostic nanosystem based on indocyanine green (ICG) covalently conjugated to mesoporous silica nanoparticles (MSNs) loaded with the anticancer drug mitoxantrone (MTX) is proposed as an innovative photoacoustic probe. Taking advantage of the characteristic PA signal displayed by both ICG and MTX, a PA-ratiometric approach was applied to assess the drug release profile from the MSNs. After complete in vitro characterization of the nanoprobe, a proof-of-concept study has been carried out in tumour-bearing mice to evaluate in vivo its effectiveness for cancer imaging and chemotherapeutic agent delivery.

The synthesis and characterization of a novel HPDO3A-based ligand having a C16 alkyl chain and its Eu3+, Gd3+ and Yb3+ complexes are reported. These amphiphilic paramagnetic complexes can form micelles with very good stability both in phosphate buffer and in human serum. A high number of Ln-complexes (ca. 200 molecules) are present in the micelle, providing this system with good sensitivity (μM in terms of micelle concentration) for MRI detection. Moreover, it has been found that the cell toxicity of the micelles may be reduced by adding DSPE-PEG2000 in the formulation. Both relaxometric and CEST properties of the micelles were investigated in detail. The micelles loaded with Eu- and Yb-HPDO3A complexes, similar to what was reported for the water-soluble analogs, act as pH-sensors and appear to be suitable for CEST multicolor experiments.

The new ligand HPDO3MA [(R,R,R,R)-10-(2-hydroxypropyl)-α,α',α''-trimethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid] was designed to combine and optimize the chemical properties of the macrocyclic ligands HPDO3A and DOTMA. The presence of the methyl groups on the acetic pendant arms of HPDO3A is expected to rigidify the structure of the ligand and favor an increase of the kinetic inertness of the Ln complexes. 1 H NMR spectra of Eu(HPDO3MA) displayed the presence of two pairs of diastereoisomers: SAP (square antiprismatic) and TSAP (twisted square antiprismatic) isomers (56 and 44 %, respectively). In addition, 1 H and 17 O relaxometric NMR studies of Gd(HPDO3MA) showed approximately a 10 % increase in relaxivity and a faster water exchange rate with respect to Gd(HPDO3A). Moreover, a detailed chemical exchange saturation transfer (CEST) characterization of Yb(HPDO3MA) displayed a sensitivity about two times larger than that of Yb(HPDO3A) both in phantom and in cell labeling experiments. Finally, the kinetic inertness of Yb(HPDO3MA) was measured to be twice as high as that of Yb(HPDO3A), with a dissociation half-life at physiological pH of about 2500 years., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Breast Cancer is the most diffuse cancer among women and the treatment outcome is largely determined by its early detection. MRI at fixed magnetic field is already widely used for cancer detection. Herein it is shown that the acquisition of proton T 1 at different magnetic fields adds further advantages. In fact, Fast Field Cycling Nuclear Magnetic Resonance Dispersion (FFC-NMRD) profiles have been shown to act as a high -sensitivity tool for cancer detection and staging in ex vivo murine breast tissues collected from Balb/NeuT mice. From NMRD profiles it was possible to extract two new cancer biomarkers, namely: (i) the appearance of 14 N-quadrupolar peaks (QPs) reporting on tumor onset and (ii) the slope of the NMRD profile reporting on the progression of the tumor. By this approach it was possible to detect the presence of tumor in transgenic NeuT mice at a very early stage (5-7 weeks), when the disease is not yet detectable by using conventional high field (7 T) MRI and only minimal abnormalities are present in histological assays. These results show that, NMRD profiles may represent a useful tool for early breast cancer detection and for getting more insight into an accurate tumor phenotyping, highlighting changes in composition of the mammary gland tissue (lipids/proteins/water) occurring during the development of the neoplasia.

In glioma, the acidification of the extracellular tumor microenvironment drives proliferation, angiogenesis, immunosuppression, invasion and chemoresistance. Therefore, quantification of glioma extracellular pH (pHe) is of crucial importance. This study is focused on the application of the YbHPDO3A (ytterbium 1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane) probe for in vivo glioma pHe quantification using chemical exchange saturation transfer (CEST)-MRI and its correlation with tumor metabolism assessed by immunohistochemistry. The U87 glioma mouse model was used (n = 18) and MRI performed at 4.7 T. CEST-MRI of YbHPDO3A solutions at different pH values showed two resolved CEST spectra at 71 ppm and 99 ppm, both sensitive to pH variations, allowing therefore calculation of the ratiometric curve for in vivo pH quantification. In vivo MRI sequences consisted of T 2w for tumor localization, T 2w * to assess YbHPDO3A biodistribution by exploiting its magnetic susceptibility effect and CEST for glioma pHe mapping. T 2w * images show that YbHPDO3A extravasates in tumor in regions with damaged blood-brain barrier. The pHe is calculated only in these regions. Hematoxylin/eosin histology and Ki-67, CA-IX (carbonic anhydrase 9) and NHE-1 immunohistochemical staining were performed; their expression rates were compared with the in vivo pHe values. On the basis of the cell proliferation marker Ki-67, two groups were defined: one group with a lower mitotic index (MI% < 20% = mean value) and a mean pHe value of 7.00 (low-proliferation/high-pH group) and the other with MI% > 20% and an acidic pHe of 6.6 (high-proliferation/low-pH group). CA-IX and NHE-1 were over-expressed in the high-proliferation/low-pH group (CA-IX, 92 ± 7% versus 30 ± 13%; NHE-1, 84 ± 8% versus 35 ± 11%), indicating an acidic/hypoxic microenvironment. These immunohistochemical results are consistent with our pHe mapping (Pearson correlation coefficient > 0.70) and provide evidence for the feasibility of the CEST-MRI method with the YbHPDO3A probe for glioma pHe quantification at 4.7 T. Importantly, the YbHPDO3A probe has similar chemical and biological properties to the clinically approved MRI contrast agent GdHPDO3A. This makes the method promising for a clinical translation., (© 2018 John Wiley & Sons, Ltd.)

Purpose: Magnetic resonance imaging has been used extensively to track in vivo implanted cells that have been previously labeled with relaxation enhancers. However, this approach is not suitable to track multiple cell populations, as it may lead to confounding results in case the contrast agent is released from the labeled cells. This paper demonstrates how the use of CEST agents can overcome these issues. After encapsulating paramagnetic lanthanide shift reagents, we may shift the absorption frequency of the intracellular water resonance (δ In ), thus generating frequency-encoding CEST responsive cells that can be visualized in the MR image by applying the proper RF irradiation., Methods: Eu-HPDO3A, Dy-HPDO3A, and Tm-HPDO3A were used as shift reagents for labeling murine breast cancer cells and murine macrophages by hypotonic swelling and pinocytosis. The CEST-MR images were acquired at 7 T, and the saturation transfer effect was measured. Samples at different dilution of cells were analyzed to quantify the detection threshold. In vitro experiments of cell proliferation were carried out. Finally, murine breast cancer cells were injected subcutaneously in mice, and MR images were acquired to assess the proliferation index in vivo., Results: It was found that entrapment of the paramagnetic complexes into endosomes (i.e., using the pinocytosis route) leads to an enhanced shift of the intracellular water resonance. δ In appears to be proportional to the effective magnetic moment (μ eff ) and to the concentration of the loaded lanthanide complex. Moreover, a higher shift is present when the complexes are entrapped in the endosomes. The cell proliferation index was assessed both in vitro and in vivo by evaluating the reduction of δ In value in the days after the cell labeling., Conclusion: Cells can be visualized by CEST MRI after loading with paramagnetic shift reagent, by exploiting the large ensemble of the properly shifted intracellular water molecules. A better performance is obtained when the complexes are entrapped inside the endosomes. The observed (δ In ) value is strongly correlated to the chemical nature of the probe, and to its concentration and cellular localization. Two applications of this method are reported in this paper: (1) for in vivo cell visualization and (2) for the monitoring of the cellular proliferation process, as this method is accompanied by a change in δ In that may be exploited as a longitudinal reporter of the proliferation rate., (© 2018 International Society for Magnetic Resonance in Medicine.)

Prostate Cancer (PCa) is the second most common and fifth cause of cancer-related mortality in males in Western Countries. The development of innovative tools for an early, more precise and noninvasive diagnosis is a medical need. Vascular volume (Vv) and hypoxia are two of the most important tumor hallmarks. Herein, they have been assessed in TRAMP mice by using MRI. Their quantification has been carried out by injecting autologous Red Blood Cells (RBCs), ex vivo labelled with Gd-HPDO3A or Gd-DOTP complexes, respectively. Gd-labelled-RBCs are stably confined in the intravascular space, also in presence of a very leaky tumor endothelium, thus representing efficient probes for vascular space analysis. Vv enhancement and hypoxia onset have been demonstrated to be present at early stages of PCa and their expression largely increases with tumor development. Moreover, also Diffusion weighted MRI and Amide Proton Transfer MRI have been herein applied to characterize PCa. The herein applied multiparametric MRI (mpMRI) analysis allows a detailed in vivo characterization of PCa, in which each histotype and cancer stage displays a specific MRI pattern. This provides an unprecedented opportunity to feature prostate tumor, making possible a non-invasive, precise and early diagnosis, which could direct treatments towards a more personalized medicine.