Endothelial cells require functional FLVCR1a during developmental and adult angiogenesis.

The Feline Leukemia Virus Subgroup C Receptor 1a (FLVCR1a) is a transmembrane heme exporter essential for embryonic vascular development. However, the exact role of FLVCR1a during blood vessel development remains largely undefined. Here, we show that FLVCR1a is highly expressed in angiogenic endothelial cells (ECs) compared to quiescent ECs. Consistently, ECs lacking FLVCR1a give rise to structurally and functionally abnormal vascular networks in multiple models of developmental and pathologic angiogenesis. Firstly, zebrafish embryos without FLVCR1a displayed defective intersegmental vessels formation. Furthermore, endothelial-specific Flvcr1a targeting in mice led to a reduced radial expansion of the retinal vasculature associated to decreased EC proliferation. Moreover, Flvcr1a null retinas showed defective vascular organization and loose attachment of pericytes. Finally, adult neo-angiogenesis is severely affected in murine models of tumor angiogenesis. Tumor blood vessels lacking Flvcr1a were disorganized and dysfunctional. Collectively, our results demonstrate the critical role of FLVCR1a as a regulator of developmental and pathological angiogenesis identifying FLVCR1a as a potential therapeutic target in human diseases characterized by aberrant neovascularization.

Mini-Laparoscopic Repair of Apical Pelvic Organ Prolapse (POP) by Lateral Suspension with Mesh.

BACKGROUND: The aim of the present study is to analyze the feasibility, safety and learning curve of Mini- Laparoscopic Lateral suspension (LLS) for the treatment of apical and anterior defects following pelvic organ prolapse. METHODS: This is a cohort study on a retrospective series of 35 consecutive patients who underwent Mini-LLS for symptomatic POP between January 2014 and July 2016. All 35 patients were operated at the Gynaecological Unit in S. Chiara Hospital by two senior surgeons (S. Tateo and L. Mereu) and by a team with optimal skills in laparoscopic surgery. Patients were divided in two groups according to two different chronological phases: phase 1 identified the initial 12 cases, phase 2 the last 23 cases. We collected pre-, peri- and post-operative information to analyze the surgical outcomes and learning curve after Mini-LLS procedures. RESULTS: The mean LLS-Overall Time (OT) was 107.6 min (range, 185- 63 min). None of the patients had intra-operative complications. No conversion to laparotomy was necessary. The mean post-operative hospital stay was 58 hours in total (SD +/-22). Only in 3 cases (8.6 %) post-operative grade I complications were observed. Recurrence of POP was observed in 3 cases (8.6 %) during a mean follow up of 18 months. The mean OT decreased with experience, in particular after the first 12 cases (phase 1: 113.54 minutes versus phase 2: 104.43 minutes). In consequence, the reduction of time per procedure was statistically significant considering the Cusum Time (CT) (P < .05). CONCLUSIONS: Mini-LLS with mesh is a safe and reproducible technique with good anatomical results, low complication rates and a short learning curve.

The release of Doxorubicin from liposomes monitored by MRI and triggered by a combination of US stimuli led to a complete tumor regression in a breast cancer mouse model.

The work aimed at developing a novel MRI-based theranostic protocol for improving the anticancer efficacy of a Doxil-like liposomal formulation. The goal was achieved stimulating the intratumor release of the drug from the nanocarrier and favoring its diffusion in the lesion by the sequential application of low-intensity pulsed ultrasound. The protocol was tested on mice bearing a syngeneic breast cancer model. The combination of acoustic waves with different characteristics allowed for: i) the release of the drug and the co-encapsulated MRI agent (Gadoteridol) from the liposomes in the vessels of the tumor region, and ii) the extravasation of the released material, as well as intact liposomes, in the tumor stroma. The MR-T1 contrast enhancement measured in the tumor reported on the delivery and US-triggered release of Doxorubicin. The developed protocol resulted in a marked increase in the intratumor drug concentration that, in turn, led to the complete regression of the lesion. The protocol has a good clinical translatability because all the components of the theranostic agent (Doxorubicin, liposomes, Gadoteridol) are approved for human use.

GdDOTAGA(C18)2: an efficient amphiphilic Gd(iii) chelate for the preparation of self-assembled high relaxivity MRI nanoprobes.

A new amphiphilic GdDOTA-like complex functionalized with two octadecyl chains was synthesised and incorporated into the bilayer of liposomes and dendrimersomes. (1)H NMR relaxometric studies and in vivo MRI experiments on mice bearing a syngeneic melanoma tumour have shown a great improvement in performance.

Novel stable dendrimersome formulation for safe bioimaging applications.

Dendrimersomes are nanosized vesicles constituted by amphiphilic Janus dendrimers (JDs), which have been recently proposed as innovative nanocarriers for biomedical applications. Recently, we have demonstrated that dendrimersomes self-assembled from (3,5)12G1-PE-BMPA-G2-(OH)8 dendrimers can be successfully loaded with hydrophilic and amphiphilic imaging contrast agents. Here, we present two newly synthesized low generation isomeric JDs: JDG0G1(3,5) and JDG0G1(3,4). Though less branched than the above-cited dendrimers, they retain the ability to form self-assembled, almost monodisperse vesicular nanoparticles. This contribution reports on the characterization of such nanovesicles loaded with the clinically approved MRI probe Gadoteridol and the comparison with the related nanoparticles assembled from more branched dendrimers. Special emphasis was given to the in vitro stability test of the systems in biologically relevant media, complemented by preliminary in vivo data about blood circulation lifetime collected from healthy mice. The results point to very promising safety and stability profiles of the nanovesicles, in particular for those made of JDG0G1(3,5), whose spontaneous self-organization in water gives rise to a homogeneous suspension. Importantly, the blood lifetimes of these systems are comparable to those of standard liposomes. By virtue of the reported results, the herein presented nanovesicles augur well for future use in a variety of biomedical applications.

Preparation and in vitro characterization of chitosan nanobubbles as theranostic agents.

Theranostic delivery systems are nanostructures that combine the modality of therapy and diagnostic imaging. Polymeric micro- and nanobubbles, spherical vesicles containing a gas core, have been proposed as new theranostic carriers for MRI-guided therapy. In this study, chitosan nanobubbles were purposely tuned for the co-delivery of prednisolone phosphate and a Gd(III) complex, as therapeutic and MRI diagnostic agent, respectively. Perfluoropentane was used for filling up the internal core of the formulation. These theranostic nanobubbles showed diameters of about 500nm and a positive surface charge that allows the interaction with the negatively charged Gd-DOTP complex. Pluronic F68 was added to the nanobubble aqueous suspension as stabilizer agent. The encapsulation efficiency was good for both the active compounds, and a prolonged drug release profile was observed in vitro. The effect of ultrasound stimulation on prednisolone phosphate release was evaluated at 37°C. A marked increase on drug release kinetics with no burst effect was obtained after the exposure of the system to ultrasound. Furthermore, the relaxivity of the MRI probe changed upon incorporation in the nanobubble shell, thereby offering interesting opportunity in dual MRI-US experiments. The ultrasound characterization showed a good in vitro echogenicity of the theranostic nanobubbles. In summary, chitosan drug-loaded nanobubbles with Gd(III) complex bound to their shell might be considered a new platform for imaging and drug delivery with the potential of improving anti-cancer treatments.

Sonosensitive theranostic liposomes for preclinical in vivo MRI-guided visualization of doxorubicin release stimulated by pulsed low intensity non-focused ultrasound.

The main goal of this study was to assess the theranostic performance of a nanomedicine able to generate MRI contrast as a response to the release from liposomes of the antitumor drug Doxorubicin triggered by the local exposure to pulsed low intensity non focused ultrasounds (pLINFU). In vitro experiments showed that Gadoteridol was an excellent imaging agent for probing the release of Doxorubicin following pLINFU stimulation. On this basis, the theranostic system was investigated in vivo on a syngeneic murine model of TS/A breast cancer. MRI offered an excellent guidance for monitoring the pLINFU-stimulated release of the drug. Moreover, it provided: i) an in vivo proof of the effective release of the liposomal content, and ii) a confirmation of the therapeutic benefits of the overall protocol. Ex vivo fluorescence microscopy indicated that the good therapeutic outcome was originated from a better diffusion of the drug in the tumor following the pLINFU stimulus. Very interestingly, the broad diffusion of the drug in the tumor stroma appeared to be mediated by the presence of the liposomes themselves. The results of this study highlighted either the great potential of US-based stimuli to safely trigger the release of a drug from its nanocarrier or the associated significant therapeutic improvement. Finally, MRI demonstrated to be a valuable technique to support chemotherapy and monitoring the outcome. Furthermore, in this specific case, the theranostic agent developed has a high clinical translatability because the MRI agent utilized is already approved for human use.

A novel SWCNT platform bearing DOTA and ß-cyclodextrin units. "One shot" multidecoration under microwave irradiation.

The functionalization of single-walled carbon nanotubes (SWCNTs) via microwave-assisted grafting reactions enables efficient multidecoration in a single step. A novel water-soluble SWCNT platform was prepared via the simple 1,3-dipolar cycloaddition of azomethine ylides under dielectric heating. Thanks to a single grafting reaction the CNT surface binds in a 1 : 1 ratio an amino acidic ß-cyclodextrin (ß-CD) derivative and the DOTAMA moiety (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid monoamide). This novel "one shot" synthesis, compared with multistep functionalizations, preserves the SWCNT's structural integrity (TEM images). Besides thermogravimetric analyses, the determination of the amount of ß-CD and DOTA moieties grafting onto the SWCNT's surface was performed on the basis of phenolphthalein and gadolinium complexation, respectively.

Quantitative assessment of cancer vascular architecture by skeletonization of high-resolution 3-D contrast-enhanced ultrasound images: role of liposomes and microbubbles.

The accurate characterization and description of the vascular network of a cancer lesion is of paramount importance in clinical practice and cancer research in order to improve diagnostic accuracy or to assess the effectiveness of a treatment. The aim of this study was to show the effectiveness of liposomes as an ultrasound contrast agent to describe the 3-D vascular architecture of a tumor. Eight C57BL/6 mice grafted with syngeneic B16-F10 murine melanoma cells were injected with a bolus of 1,2-Distearoyl-sn-glycero-3-phosphocoline (DSPC)-based non-targeted liposomes and with a bolus of microbubbles. 3-D contrast-enhanced images of the tumor lesions were acquired in three conditions: pre-contrast, after the injection of microbubbles, and after the injection of liposomes. By using a previously developed reconstruction and characterization image processing technique, we obtained the 3-D representation of the vascular architecture in these three conditions. Six descriptive parameters of these networks were also computed: the number of vascular trees (NT), the vascular density (VD), the number of branches, the 2-D curvature measure, the number of vascular flexes of the vessels, and the 3-D curvature. Results showed that all the vascular descriptors obtained by liposome-based images were statistically equal to those obtained by using microbubbles, except the VD which was found to be lower for liposome images. All the six descriptors computed in pre-contrast conditions had values that were statistically lower than those computed in presence of contrast, both for liposomes and microbubbles. Liposomes have already been used in cancer therapy for the selective ultrasound-mediated delivery of drugs. This work demonstrated their effectiveness also as vascular diagnostic contrast agents, therefore proving that liposomes can be used as efficient "theranostic" (i.e. therapeutic 1 diagnostic) ultrasound probes.

Advances in metal-based probes for MR molecular imaging applications.

The role of MRI in the armory of diagnostic modalities for the medicine of the forthcoming years largely depends on how chemistry will provide advanced tools to meet the medical needs. This review aims at outlining the most innovative approaches that have been undertaken in the recent history of MRI contrast agents for tackling the challenges of sensitivity and specificity required by the new generation of contrast agents that should allow the visualization of pathological processes occurring on cellular and molecular scale (the so-called Molecular Imaging). Most of the classes of MRI agents clinically approved or currently under investigation in a preclinical phase exploit peculiar magnetic properties of metals. The conventional agents acting as T(1) or T(2)/T(2)* relaxation enhancers are primarily based on the paramagnetic or the superparamagnetic properties of Gd(III)-, Mn(II)- and iron oxides systems. Recently, there has been a renewed interest towards paramagnetic lanthanide complexes with an anisotropic electronic configuration thanks to their ability to induce strong effect on the resonance frequency of the spins dipolarly coupled with them. Such systems, formerly mainly used as shift reagents, have now attracted much attention in the emerging field of Chemical Exchange Saturation Transfer (CEST) MRI agents.

Development and validation of a smoothing-splines-based correction method for improving the analysis of CEST-MR images.

Chemical exchange saturation transfer (CEST) imaging is an emerging MRI technique relying on the use of endogenous or exogenous molecules containing exchangeable proton pools. The heterogeneity of the water resonance frequency offset plays a key role in the occurrence of artifacts in CEST-MR images. To limit this drawback, a new smoothing-splines-based method for fitting and correcting Z-spectra in order to compensate for low signal-to-noise ratio (SNR) without any a priori model was developed. Global and local voxel-by-voxel Z-spectra were interpolated by smoothing splines with smoothing terms aimed at suppressing noise. Thus, a map of the water frequency offset ('zero' map) was used to correctly calculate the saturation transfer (ST) for each voxel. Simulations were performed to compare the method to polynomials and zero-only-corrected splines on the basis of SNR improvement. In vitro acquisitions of capillaries containing solutions of LIPOCEST agents at different concentrations were performed to experimentally validate the results from simulations. Additionally, ex vivo investigations of bovine muscle mass injected with LIPOCEST agents were performed as a function of increasing pulse power. The results from simulations and experiments highlighted the importance of a proper 'zero' correction (15% decrease of fictitious CEST signal in phantoms and ex vivo preparations) and proved the method to be more accurate compared with the previously published ones, often providing a SNR higher than 5 in different simulated and experimentally noisy conditions. In conclusion, the proposed method offers an accurate tool in CEST investigation.

[DOTA-bis(amide)]lanthanide complexes: NMR evidence for differences in water-molecule exchange rates for coordination isomers.

Two derivatives of 1,4,7,10-tetraazacyclododecane with trans-acetate and trans-amide side-chain ligating groups have been prepared and their complexes with lanthanide cations examined by multinuclear NMR spectroscopy. These lanthanide complexes exist in aqueous solution as a mixture of slowly interconverting coordination isomers with 1H chemical shifts similar to those reported previously for the major (M) and minor (m) forms of the tetraacetate ([Ln(dota)]-) and tetraamide ([Ln(dtma)]3+) complexes. As in the [Ln(dota)]- and [Ln(dtma)]3+ complexes, the m/M ratio proved to be a sensitive function of lanthanide size and temperature. An analysis of 1H hyperfine shifts in spectra of the Yb3+ complexes revealed significant differences between the axial (D1) and non-axial (D2) components of the magnetic susceptibility tensor anisotropy in the m and M coordination isomers and the energetics of ring inversion and m <==> M isomerization as determined by two-dimensional exchange spectroscopy (EXSY). (17)O shift data for the Dy3+ complexes showed that both have one inner-sphere water molecule. A temperature-dependent (17)O NMR study of bulk water linewidths for solutions of the Gd3+ complexes provided direct evidence for differences in water exchange rates for the two coordination isomers. The bound-water lifetimes (tauM298) in the M and m isomers of the Gd3+ complexes ranged from 1.4-2.4 micros and 3-14 ns, respectively. This indicates that 1) the inner-sphere water lifetimes for the complexes with a single positive charge reported here are considerably shorter for both coordination isomers than the corresponding values for the [Gd(dtma)]3+ complex with three positive charges, and 2) the difference in water lifetimes for M and m isomers in these two series is magnified in the [Gd[dota-bis(amide)]] complexes. This feature highlights the remarkable role of both charge and molecular geometry in determining the exchange rate of the coordinated water.

High-Relaxivity contrast agents for magnetic resonance imaging based on multisite interactions between a beta-cyclodextrin oligomer and suitably functionalized GdIII chelates.

The results reported in this work show that tightly assembled adducts formed by trisubstituted GdIII complexes and a beta-CD multimer (Poly-beta-CD, d.p. ca. 12) may represent very interesting candidates for novel MRI applications wherein a high number of paramagnetic ions endowed with high relaxivity (per GdIII ion) are necessary. The relaxivities found for the paramagnetic adducts represent a remarkable step forward on the relaxivity scale. However, a detailed investigation of the determinants of the relaxation enhancement in these systems shows that their relaxivities are still limited by a nonoptimal tauR and a relatively long exchange lifetime of the coordinated water(s). Moreover, the exchange rate of the water molecule(s) coordinated to the GdIII ion further decreases upon binding to the Poly-beta-CD. It is suggested that this finding is related to the structural properties of the supramolecule, which brings a high density of hydroxyl groups into the proximity of the "guest" complexes, and this yields an overall reinforcement of the hydrogen-bonding network involving the coordinated water(s). On the other hand, such a tight arrangement appears responsible for an enhanced contribution to the observed relaxivity arising from water molecules in the second coordination sphere of the metal center.

Ternary Gd(III)L-HSA adducts: evidence for the replacement of inner-sphere water molecules by coordinating groups of the protein. Implications for the design of contrast agents for MRI.

Two novel gadolinium(III) chelates based on the structure of the heptadentate macrocyclic 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) ligand have been synthesized and their relaxometric and luminescent properties thoroughly investigated. They contain two water molecules in the inner coordination sphere in fast exchange with the bulk solvent and bear either a p-bromobenzyl or a p-phosphonatomethylbenzanilido substituent for promoting further interaction with macromolecular substrates. Upon interaction with human serum albumin the expected relaxation enhancement is not observed owing to displacement of the two inner-sphere water molecules of the complexes by a donor atom (likely from a carboxylate group) on the protein and possibly the phosphate anion of the buffered solution, respectively. We modeled the observed behavior by measuring the decrease of the relaxation rate of the water protons upon addition of malonate anion to aqueous solutions of the complexes. Conversely, no change in the hydratation state of the Gd(III) center for both complexes has been observed when the substrate for the formation of the macromolecular adduct is represented by poly-beta-cyclodextrin.

Contrast agents for magnetic resonance angiographic applications: 1H and 17O NMR relaxometric investigations on two gadolinium(III) DTPA-like chelates endowed with high binding affinity to human serum albumin.

The relaxometric properties of two Gd(III) DTPA-like complexes (DTPA = diethylenetriamine-N,N',N,N"'-pentaacetic acid) bearing different substituents for binding to human serum albumin (HSA) are compared. In spite of the structural differences of the recognition synthon and of the residual electric charge, the two chelates display an analogous binding affinity for the serum protein. Upon formation of the adducts with HSA, the exchange rates of the coordinated water appear slowed down by an amount corresponding to ca. 50% of the rates found for the free complexes. The relaxivity of [Gd(BOM)3DTPA (H2O)]2- is significantly higher than that of MS-325 either in the free complex or in the macromolecular adduct. Finally, the effect of pH on the stability of the HSA adducts and on the values of their relaxivities has been investigated.

NMR relaxometric investigations of solid lipid nanoparticles (SLN) containing gadolinium(III) complexes.

This work deals with the preparation and relaxometric investigations of solid lipid nanoparticles (SLN) containing [Gd-DTPA(H2O)]2- and [Gd-DOTA(H2O)]-. These paramagnetic chelates are commonly used as contrast agents (CA) for magnetic resonance imaging (MRI) owing to their ability to strongly increase the tissue water proton relaxation rate. The amount of gadolinium(III) (Gd(III)) complex included in the SLN has been evaluated and, on this basis, it has been found that the longitudinal relaxivity of these Gd(III) chelates apparently does not vary, at physiological pH, following their inclusion in SLN. We are unable to establish whether this is due to the free exchange of water from the inner compartment containing the Gd(III) chelate to the bulk water or whether the observed relaxation rate is essentially determined by a fraction of the complex which is close to the surface of the SLN in a region easily accessible to the bulk water. At acidic pH values, the relaxivity of the paramagnetic SLN containing the less thermodynamically and kinetically stable [Gd-DTPA(H2O)]2- markedly increases. This effect may be ascribed to an increased immobilization and/or to an enhanced hydration of the complex on SLN.

NMR and luminescence studies on the formation of ternary adducts between HSA and Ln(III)-malonate complexes.

At physiological pH and in the presence of an excess of malonate ligand (MAL), the lanthanide ions (Ln=Eu(III), Gd(III) and Tb(III)) are under the form of [Ln(MAL)2(H2O)4]-. Upon addition of human serum albumin (HSA), formation of two different ternary adducts of stoichiometry HSA-Ln(MAL)x(H2O)q (x=2, q=2; x=2, q=4) is detected. On the basis of the reasonable assumption that the binding strength for the two sites on the protein are inversely proportional to the hydration state of the metal ion, stability constants of 4.0.103 M-1 and 3.5.102 M-1 have been evaluated for the system with q=2 and q=4, respectively. Whereas for the stronger binding site it is suggested that the protein provides two or three donor atoms to the coordination cage of the Ln(III) ion, in the case of the weaker binding site it is likely that it corresponds to a simple electrostatic interaction between the negatively charged [Ln(MAL)2(H2O)4]- and positively charged groups on the surface of the protein.

A new ytterbium chelate as contrast agent in chemical shift imaging and temperature sensitive probe for MR spectroscopy.

A paramagnetic Yb(III) complex that is the prototype of a novel class of probes for MRI and MRS has been developed. The complex displays highly shifted 1H resonances that are characterized by short relaxation times and, as such, may prove to be a valuable alternative in applications that currently require fluorine-containing probes. Selective excitation of the paramagnetically shifted resonances allows the spatial distribution of the complex to be mapped. This communication reports the images that were obtained by selectively exciting the most intense methyl group (-14.2 ppm at 27 degrees C) for complex concentrations ranging from 0.003-0.1 M. Spectroscopically, the complex may be used as a temperature probe since the proton chemical shifts exhibit a strong temperature dependence. In human serum the chemical shift difference of a selected pair of proton resonances was observed to follow a gradient of -0.42 +/- 0.01 ppm/degrees C. Furthermore, since the chemical shift of the methyl resonance displays a temperature coefficient of -0.04 +/- 0.01 ppm/degrees C, it should be possible to use the image phase for thermal mapping.