Synthesis of fluorinated curcumin derivatives for detecting amyloid plaques by 19F-MRI.

The most prominent pathophysiological hallmark of Alzheimer's disease is the aggregation of amyloid-ß (Aß) peptides into senile plaques. Curcumin and its derivatives exhibit a high affinity for binding to Aß fibrils, effectively inhibiting their growth. This property holds promise for both therapeutic applications and diagnostic molecular imaging. In this study, curcumin was functionalized with perfluoro-tert-butyl groups to create candidate molecular probes specifically targeted to Aß fibrils for use in 19F-magnetic resonance imaging. Two types of fluorinated derivatives were considered: mono-substituted (containing nine fluorine atoms per molecule) and disubstituted (containing eighteen fluorine atoms). The linker connecting the perfluoro moiety with the curcumin scaffold was evaluated for its impact on binding affinity and water solubility. All mono-substituted compounds and one disubstituted compound exhibited a binding affinity toward Aß fibrils on the same order of magnitude as reference curcumin. The insertion of a charged carboxylate group into the linker enhanced the water solubility of the probes. Compound Curc-Glu-F9 (with one L-glutamyl moiety and a perfluoro-tert-butyl group), showed the best properties in terms of binding affinity towards Aß fibrils, water solubility, and intensity of the 19F-NMR signal in the Aß oligomer bound form.

Novel Nanogels Loaded with Mn(II) Chelates as Effective and Biologically Stable MRI Probes.

Here it is described nanogels (NG) based on a chitosan matrix, which are covalently stabilized by a bisamide derivative of Mn-t-CDTA (t-CDTA = trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid). the Mn(II) complex acts both as a contrast medium and as a cross-linking agent. These nanogels are proposed as an alternative to the less stable paramagnetic nanogels obtained by electrostatic interactions between the polymeric matrix and paramagnetic Gd(III) chelates. The present novel nanogels show: i) relaxivity values seven times higher than that of typical monohydrated Mn(II) chelates at the clinical fields, thanks to the combination of a restricted mobility of the complex with a fast exchange of the metal-bound water molecule; ii) high stability of the formulation over time at pH 5 and under physiological conditions, thus excluding metal leaking or particles aggregation; iii) good extravasation and accumulation, with a maximum contrast achieved at 24 h post-injection in mice bearing subcutaneous breast cancer tumor; iv) high T1 contrast (1 T) in the tumor 24 h post-injection. These improved properties pave the way for the use of these paramagnetic nanogels as promising magnetic resonance imaging (MRI) probes for in vitro and in vivo preclinical applications.

Engineered Magnetic Nanocomposites to Modulate Cellular Function.

Magnetic nanoparticles (MNPs) have various applications in biomedicine, including imaging, drug delivery and release, genetic modification, cell guidance, and patterning. By combining MNPs with polymers, magnetic nanocomposites (MNCs) with diverse morphologies (core-shell particles, matrix-dispersed particles, microspheres, etc.) can be generated. These MNCs retain the ability of MNPs to be controlled remotely using external magnetic fields. While the effects of these biomaterials on the cell biology are still poorly understood, such information can help the biophysical modulation of various cellular functions, including proliferation, adhesion, and differentiation. After recalling the basic properties of MNPs and polymers, and describing their coassembly into nanocomposites, this review focuses on how polymeric MNCs can be used in several ways to affect cell behavior. A special emphasis is given to 3D cell culture models and transplantable grafts, which are used for regenerative medicine, underlining the impact of MNCs in regulating stem cell differentiation and engineering living tissues. Recent advances in the use of MNCs for tissue regeneration are critically discussed, particularly with regard to their prospective involvement in human therapy and in the construction of advanced functional materials such as magnetically operated biomedical robots.

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.

High Relaxivity with No Coordinated Waters: A Seemingly Paradoxical Behavior of [Gd(DOTP)]5- Embedded in Nanogels.

Nanogels (NGs) obtained by electrostatic interactions between chitosan and hyaluronic acid and comprising paramagnetic Gd chelates are gaining increasing attention for their potential application in magnetic resonance bioimaging. Herein, the macrocyclic complexes [Gd(DOTP)]5-, lacking metal-bound water molecules (q = 0), were confined or used as a cross-linker in this type of NG. Unlike the typical behavior of Gd complexes with q = 0, a remarkable relaxivity value of 78.0 mM-1 s-1 was measured at 20 MHz and 298 K, nearly 20 times greater than that found for the free complex. A careful analysis of the relaxation data emphasizes the fundamental role of second sphere water molecules with strong and long-lived hydrogen bonding interactions with the complex. Finally, PEGylated derivatives of nanoparticles were used for the first in vivo magnetic resonance imaging study of this type of NG, revealing a fast renal excretion of paramagnetic complexes after their release from the NGs.

An efficient MRI agent targeting extracellular markers in prostate adenocarcinoma.

PURPOSE: Prostate cancer (PCa) is the most widespread tumor affecting males in Western countries. We propose a novel MRI molecular tetrameric probe based on the heptadentate gadolinium (Gd)-AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid) that is able to in vivo detect PCa through the recognition of the fibrin-fibronectin (FB-FN) complex. METHODS: The peptide CREKA (Cys-Arg-Glu-Lys-Ala), targeting the FB-FN complex in the reactive stroma of the tumor, was synthesized by solid phase peptide synthesis (SPPS) and conjugated to the tetramer dL-(Gd-AAZTA)4 . The resulting probe was characterized by 1 H relaxometry, tested in vitro on FB clots and in vivo on an orthotopic mouse model of PCa. RESULTS: CREKA-dL-(Gd-AAZTA)4 showed a remarkable relaxivity of 18.2 m MGd-1s-1 (0.47 T, 25°C) because of the presence of 2 water molecules (q = 2) in the inner coordination sphere of each Gd3+ ion, whose rotational motion (τR ) is lengthened as the result of the relatively high molecular weight. The probe displayed a detectable affinity for plasma-derived FB clots. On intravenous injection of the probe in an orthotopic mouse model of PCa, a significant increase in the prostate T1 contrast (~40%) was observed. The MRI signal appears statistically higher either with respect to the one observed for the control probes and to the one detected when CREKA-dL-(Gd-AAZTA)4 was administered to healthy animals. CONCLUSIONS: This study demonstrated the ability of the CREKA-dL-(Gd-AAZTA)4 probe to specifically localize in prostate tumor after injection. The high relaxivity of the probe allows the reduction of the injected dose to 20 µmolGd /kg, yielding a good in vivo contrast enhancement in the region of prostate tumor.

MRI visualization of neuroinflammation using VCAM-1 targeted paramagnetic micelles.

The detection of neuroinflammatory processes using innovative and non-invasive imaging techniques is of great help to deeply investigate the onset and progression of neurodegenerative diseases. Since Vascular Cell Adhesion Molecule (VCAM-1) is over expressed at the blood brain barrier in the event of neuroinflammation, the goal of this work was the testing of MRI detectable micelles targeted towards VCAM-1 to visualize inflamed regions in a mouse model of acute neuroinflammation. The developed probe allowed for the early detection of the disease, with higher T1 signal enhancement and more precise localization in comparison to untargeted micelles or to the clinically approved contrast agent MultiHance. Moreover, the relatively long blood half-life of the nanosystem (ca. 6.3 h) guaranteed a good accumulation in the inflamed regions, paving the way to future diagnostic/theranostic applications, implying the loading of neuroprotective or even anti-cancer drugs inside the core of the micelles.

Paramagnetic Phospholipid-Based Micelles Targeting VCAM-1 Receptors for MRI Visualization of Inflammation.

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(-1)mmolGd(-1) at 25 °C and 0.7 T). A (1)H- and (17)O-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.

Innovative Design of Ca-Sensitive Paramagnetic Liposomes Results in an Unprecedented Increase in Longitudinal Relaxivity.

Bioresponsive MRI contrast agents sensitive to Ca(II) fluctuations may play a critical role in the development of functional molecular imaging methods to study brain physiology or abnormalities in muscle contraction. A great challenge in their chemistry is the preparation of probes capable of inducing a strong signal variation that could be detected in a robust way. To this end, the incorporation of small molecular weight bioresponsive agents into nanocarriers can improve the overall properties in a few ways: (i) the agent can be delivered into the tissue of interest, increasing the local concentration; (ii) its biokinetic properties and retention time will improve; (iii) the high molecular weight and size of the nanocarrier may cause additional changes in the MRI signal and raise the chances for their detection in functional experiments. In this work, we report the preparation of the new class of liposome-based, Ca-sensitive MRI agents. We synthesized a novel amphiphilic ligand which was incorporated into the liposome bilayer. A remarkable increase of ∼420% in longitudinal relaxivity r1, from 7.3 mM(-1) s(-1) to 38.1 mM(-1) s(-1) at 25 °C and 21.5 MHz in the absence and presence of Ca(II), respectively, was achieved by the most active liposomal formulation. To the best of our knowledge, this is the highest change in r1 observed for Ca-sensitive agents at physiological pH and can be explained by simultaneous Ca-triggered increase in hydration and reduction of local motion of Gd(III) complex, which can be followed at low magnetic fields.

Successful entrapping of liposomes in glucan particles: an innovative micron-sized carrier to deliver water-soluble molecules.

Glucan particles (GPs) are monodisperse microspheres derived from baker's yeast and represent an interesting class of microcarriers for theranostic applications as they show a high affinity toward immune system cells. The typical loading strategy was to harness the ability of the molecule to be loaded to interact with nano-/microassembled systems through electrostatic or hydrophobic forces. However, small water-soluble chemicals could not be steadily retained by the leaky shell of GPs. In this work, we propose an alternative loading approach for small water-soluble compounds that is based on their entrapment in the aqueous core of liposomes that are directly formed into the microparticles through the reverse phase evaporation method (REV). The construct obtained may act as biocompatible carrier to deliver and release, even in a triggerable way, bioactive compounds.

A versatile fluorinated azamacrocyclic chelator enabling 18F PET or 19F MRI: a first step towards new multimodal and smart contrast agents.

Macrocyclic chelators play a central role in medical imaging and nuclear medicine owing to their unparalleled metal cation coordination abilities. Their functionalization by fluorinated groups is an attractive design, to combine their properties with those of 18F for Positron Emission Tomography (PET) or natural 19F for Magnetic Resonance Imaging (MRI), and access potential theranostic or smart medical imaging probes. For the first time, a compact fluorinated macrocyclic architecture has been synthesized, based on a cyclen chelator bearing additional pyridine coordinating units and simple methyltrifluoroborate prosthetic groups. This ligand and its corresponding model Zn(ii) complex were investigated to evaluate the 18F-PET or 19F MRI abilities provided by this novel molecular structure. The chelator and the complex were obtained via a simple and high-yielding synthetic route, present excellent solvolytic stability of the trifluoroborate groups at various pH, and provide facile late-stage 18F-radiolabeling (up to 68% radiochemical yield with high activity) as well as a satisfying detection limit for 19F MRI imaging (low mM range).

Prussian Blue Staining to Visualize Iron Oxide Nanoparticles.

Iron deposits in cells and tissues can be detected by ex vivo histological examination through the Prussian blue (PB) staining. This practical, inexpensive, and highly sensitive technique involves the treatment of fixed tissue sections and cells with acid solutions of ferrocyanides that combine with ferric ion forming a bright blue pigment (i.e., ferric ferrocyanide). The staining can be applied to visualize iron oxide nanoparticles (IONPs), versatile magnetic nanosystems that are used in various biomedical applications and whose localization is usually required at a higher resolution than that enabled by in vivo tracking techniques.

On the Road to Precision Medicine: Magnetic Systems for Tissue Regeneration, Drug Delivery, Imaging, and Theranostics.

Magnetic systems have always been considered as attractive due to their remarkable versatility [...].

Ultrasound boosts doxorubicin efficacy against sensitive and resistant ovarian cancer cells.

Ovarian cancer (OC) is characterised by the highest mortality of all gynaecological malignancies, frequent relapses, and the development of resistance to drug therapy. Sonodynamic therapy (SDT) is an innovative anticancer approach that combines a chemical/drug (sonosensitizer) with low-intensity ultrasound (US), which are both harmless per sé, with the sonosensitizer being acoustically activated, thus yielding localized cytotoxicity often via reactive oxygen species (ROS) generation. Doxorubicin (Doxo) is a potent chemotherapeutic drug that has also been recommended as a first-line treatment against OC. This research work aims to investigate whether Doxo can be used at very low concentrations, in order to avoid its significant side effects, as a sonosensitiser under US exposure to promote cancer cell death in Doxo non-resistant (A2780/WT) and Doxo resistant (A2780/ADR) human OC cell lines. Moreover, since recurrence is an important issue in OC, we have also investigated whether the proposed SDT with Doxo induces immunogenic cell death (ICD) and thus hinders OC recurrence. Our results show that the sonodynamic anticancer approach with Doxo is effective in both A2780/WT and A2780/ADR cell lines, and that it proceeds via a ROS-dependent mechanism of action and immune sensitization that is based on the activation of the ICD pathway.

Hard-Shelled Glycol Chitosan Nanoparticles for Dual MRI/US Detection of Drug Delivery/Release: A Proof-of-Concept Study.

This paper describes a novel nanoformulation for dual MRI/US in vivo monitoring of drug delivery/release. The nanosystem was made of a perfluoropentane core coated with phospholipids stabilized by glycol chitosan crosslinked with triphosphate ions, and it was co-loaded with the prodrug prednisolone phosphate (PLP) and the structurally similar MRI agent Gd-DTPAMA-CHOL. Importantly, the in vitro release of PLP and Gd-DTPAMA-CHOL from the nanocarrier showed similar profiles, validating the potential impact of the MRI agent as an imaging reporter for the drug release. On the other hand, the nanobubbles were also detectable by US imaging both in vitro and in vivo. Therefore, the temporal evolution of both MRI and US contrast after the administration of the proposed nanosystem could report on the delivery and the release kinetics of the transported drug in a given lesion.

Lestaurtinib inhibits Citron kinase activity and medulloblastoma growth through induction of DNA damage, apoptosis and cytokinesis failure.

Introduction: Medulloblastoma (MB), the most common malignant pediatric brain tumor, is currently treated with surgery followed by radiation and chemotherapy, which is accompanied by severe side effects, raising the need for innovative therapies. Disruption of the microcephaly-related gene Citron kinase (CITK) impairs the expansion of xenograft models as well as spontaneous MB arising in transgenic mice. No specific CITK inhibitors are available. Methods: Lestaurtinib, a Staurosporine derivative also known as CEP-701, inhibits CITK with IC50 of 90 nM. We therefore tested the biological effects of this molecule on different MB cell lines, as well as in vivo, injecting the drug in MBs arising in SmoA1 transgenic mice. Results: Similar to CITK knockdown, treatment of MB cells with 100 nM Lestaurtinib reduces phospho-INCENP levels at the midbody and leads to late cytokinesis failure. Moreover, Lestaurtinib impairs cell proliferation through CITK-sensitive mechanisms. These phenotypes are accompanied by accumulation of DNA double strand breaks, cell cycle block and TP53 superfamily activation in vitro and in vivo. Lestaurtinib treatment reduces tumor growth and increases mice survival. Discussion: Our data indicate that Lestaurtinib produces in MB cells poly-pharmacological effects extending beyond the inhibition of its validated targets, supporting the possibility of repositioning this drug for MB treatment.

In Humanized Sickle Cell Mice, Imatinib Protects Against Sickle Cell-Related Injury.

Drug repurposing is a valuable strategy for rare diseases. Sickle cell disease (SCD) is a rare hereditary hemolytic anemia accompanied by acute and chronic painful episodes, most often in the context of vaso-occlusive crisis (VOC). Although progress in the knowledge of pathophysiology of SCD have allowed the development of new therapeutic options, a large fraction of patients still exhibits unmet therapeutic needs, with persistence of VOCs and chronic disease progression. Here, we show that imatinib, an oral tyrosine kinase inhibitor developed for the treatment of chronic myelogenous leukemia, acts as multimodal therapy targeting signal transduction pathways involved in the pathogenesis of both anemia and inflammatory vasculopathy of humanized murine model for SCD. In addition, imatinib inhibits the platelet-derived growth factor-B-dependent pathway, interfering with the profibrotic response to hypoxia/reperfusion injury, used to mimic acute VOCs. Our data indicate that imatinib might be considered as possible new therapeutic tool for chronic treatment of SCD.

Micro/Nanosystems for Magnetic Targeted Delivery of Bioagents.

Targeted delivery of pharmaceuticals is promising for efficient disease treatment and reduction in adverse effects. Nano or microstructured magnetic materials with strong magnetic momentum can be noninvasively controlled via magnetic forces within living beings. These magnetic carriers open perspectives in controlling the delivery of different types of bioagents in humans, including small molecules, nucleic acids, and cells. In the present review, we describe different types of magnetic carriers that can serve as drug delivery platforms, and we show different ways to apply them to magnetic targeted delivery of bioagents. We discuss the magnetic guidance of nano/microsystems or labeled cells upon injection into the systemic circulation or in the tissue; we then highlight emergent applications in tissue engineering, and finally, we show how magnetic targeting can integrate with imaging technologies that serve to assist drug delivery.

Imaging of Inflammation in Spinal Cord Injury: Novel Insights on the Usage of PFC-Based Contrast Agents.

Labeling of macrophages with perfluorocarbon (PFC)-based compounds allows the visualization of inflammatory processes by 19F-magnetic resonance imaging (19F-MRI), due to the absence of endogenous background. Even if PFC-labeling of monocytes/macrophages has been largely investigated and used, information is lacking about the impact of these agents over the polarization towards one of their cell subsets and on the best way to image them. In the present work, a PFC-based nanoemulsion was developed to monitor the course of inflammation in a model of spinal cord injury (SCI), a pathology in which the understanding of immunological events is of utmost importance to select the optimal therapeutic strategies. The effects of PFC over macrophage polarization were studied in vitro, on cultured macrophages, and in vivo, in a mouse SCI model, by testing and comparing various cell tracking protocols, including single and multiple administrations, the use of MRI or Point Resolved Spectroscopy (PRESS), and application of pre-saturation of Kupffer cells. The blood half-life of nanoemulsion was also investigated by 19F Magnetic Resonance Spectroscopy (MRS). In vitro and in vivo results indicate the occurrence of a switch towards the M2 (anti-inflammatory) phenotype, suggesting a possible theranostic function of these nanoparticles. The comparative work presented here allows the reader to select the most appropriate protocol according to the research objectives (quantitative data acquisition, visual monitoring of macrophage recruitment, theranostic purpose, rapid MRI acquisition, etc.). Finally, the method developed here to determine the blood half-life of the PFC nanoemulsion can be extended to other fluorinated compounds.

Biodegradable polyelectrolyte/magnetite capsules for MR imaging and magnetic targeting of tumors.

Rationale: The tireless research for effective drug delivery approaches is prompted by poor target tissue penetration and limited selectivity against diseased cells. To overcome these issues, various nano- and micro-carriers have been developed so far, but some of them are characterized by slow degradation time, thus hampering repeated drug administrations. The aim of this study was to pursue a selective delivery of magnetic biodegradable polyelectrolyte capsules in a mouse breast cancer model, using an external magnetic field. Methods: Four different kinds of magnetic polyelectrolyte capsules were fabricated via layer-by-layer assembly of biodegradable polymers on calcium carbonate templates. Magnetite nanoparticles were embedded either into the capsules' shell (sample S) or both into the shell and the inner volume of the capsules (samples CnS, where n is the number of nanoparticle loading cycles). Samples were first characterized in terms of their relaxometric and photosedimentometric properties. In vitro magnetic resonance imaging (MRI) experiments, carried out on RAW 264.7 cells, allowed the selection of two lead samples that proceeded for the in vivo testing on a mouse breast cancer model. In the set of in vivo experiments, an external magnet was applied for 1 hour following the intravenous injection of the capsules to improve their delivery to tumor, and MRI scans were acquired at different time points post administration. Results: All samples were considered non-cytotoxic as they provided more than 76% viability of RAW 264.7 cells upon 2 h incubation. Sample S appeared to be the most efficient in terms of T2-MRI contrast, but the less sensitive to external magnet navigation, since no difference in MRI signal with and without the magnet was observed. On the other side, sample C6S was efficiently delivered to the tumor tissue, with a three-fold T2-MRI contrast enhancement upon the external magnet application. The effective magnetic targeting of C6S capsules was also confirmed by the reduction in T2-MRI contrast in spleen if compared with the untreated with magnet mice values, and the presence of dense and clustered iron aggregates in tumor histology sections even 48 h after the magnetic targeting. Conclusion: The highlighted strategy of magnetic biodegradable polyelectrolyte capsules' design allows for the development of an efficient drug delivery system, which through an MRI-guided externally controlled navigation may lead to a significant improvement of the anticancer chemotherapy performance.