Accumulation of Iron Oxide-Based Contrast Agents in Rabbit Atherosclerotic Plaques in Relation to Plaque Age and Vulnerability Features.

Purpose: In this study, a detailed characterization of a rabbit model of atherosclerosis was performed to assess the optimal time frame for evaluating plaque vulnerability using superparamagnetic iron oxide nanoparticle (SPION)-enhanced magnetic resonance imaging (MRI). Methods: The progression of atherosclerosis induced by ballooning and a high-cholesterol diet was monitored using angiography, and the resulting plaques were characterized using immunohistochemistry and histology. Morphometric analyses were performed to evaluate plaque size and vulnerability features. The accumulation of SPIONs (novel dextran-coated SPIONDex and ferumoxytol) in atherosclerotic plaques was investigated by histology and MRI and correlated with plaque age and vulnerability. Toxicity of SPIONDex was evaluated in rats. Results: Weak positive correlations were detected between plaque age and intima thickness, and total macrophage load. A strong negative correlation was observed between the minimum fibrous cap thickness and plaque age as well as the mean macrophage load. The accumulation of SPION in the atherosclerotic plaques was detected by MRI 24 h after administration and was subsequently confirmed by Prussian blue staining of histological specimens. Positive correlations between Prussian blue signal in atherosclerotic plaques, plaque age, and macrophage load were detected. Very little iron was observed in the histological sections of the heart and kidney, whereas strong staining of SPIONDex and ferumoxytol was detected in the spleen and liver. In contrast to ferumoxytol, SPIONDex administration in rabbits was well tolerated without inducing hypersensitivity. The maximum tolerated dose in rat model was higher than 100 mg Fe/kg. Conclusion: Older atherosclerotic plaques with vulnerable features in rabbits are a useful tool for investigating iron oxide-based contrast agents for MRI. Based on the experimental data, SPIONDex particles constitute a promising candidate for further clinical translation as a safe formulation that offers the possibility of repeated administration free from the risks associated with other types of magnetic contrast agents.

Fluorescence-based absolute quantification of near-infrared probes in tissue extracts for biodistribution analyses.

In recent years, significant progress has been made in the development of fluorescent contrast agents for clinical applications. For the development of a fluorescent probe, it is crucial to evaluate its safety profile, including biodistribution. Specific methods need to be developed for the absolute quantification of fluorescent probes in tissue specimens from animals administered with test compounds in the framework of biodistribution/efficacy/toxicity studies. Here, we describe a new method for the absolute quantification of fluorescent probes in tissue specimens from animals administered with compounds that have absorption and emission wavelength in the Near-Infrared region (600-800 nm). The protocol is based on the standard addition approach in order to minimize the interference of the matrix on the analyte signal causing inaccuracy in the absolute determination of the concentration. The measurement of the fluorescence intensity is done via a microplate reader. The method has been fully validated and applied for the quantification of a fluorescence-guided surgery targeted contrast agent in a Good Laboratory Practice (GLP) biodistribution study. Results clearly demonstrate that this procedure is fully applicable in a preclinical setting and that it overcomes common issues associated with fluorescence signal quantification in tissue extracts.

An ultrasound-guided injection method for a syngeneic orthotopic murine model of breast cancer.

Breast cancer is the most common cancer among women worldwide. For high-risk women, contrast enhanced (CE)-magnetic resonance imaging (MRI) is recommended as supplemental screening together with mammography. The development of new MRI contrast agents is an active field of research, which requires efficacy tests on appropriate preclinical pathological models. In this work, a refined method to orthotopically induce breast cancer in BALB/c mice was developed using ultrasound (US) as a guide for the precise localisation of the tumour induction site and to improve animal welfare. The method was coupled with CE-MRI to characterise the evolution of the tumoural lesion.

Nanostructured lipid carriers accumulate in atherosclerotic plaques of ApoE-/- mice.

Nanostructured lipid carriers (NLC) might represent an interesting approach for the identification and targeting of rupture-prone atherosclerotic plaques. In this study, we evaluated the biodistribution, targeting ability and safety of 64Cu-fonctionalized NLC in atherosclerotic mice. 64Cu-chelating-NLC (51.8±3.1 nm diameter) with low dispersity index (0.066±0.016) were produced by high pressure homogenization at tens-of-grams scale. 24 h after injection of 64Cu-chelated particles in ApoE-/- mice, focal regions of the aorta showed accumulation of particles on autoradiography that colocalized with Oil Red O lipid mapping. Signal intensity was significantly greater in aortas isolated from ApoE-/- mice compared to wild type (WT) control (8.95 [7.58, 10.16]×108 vs 4.59 [3.11, 5.03]×108 QL/mm2, P < 0.05). Moreover, NLC seemed safe in relevant biocompatibility studies. NLC could constitute an interesting platform with high clinical translation potential for targeted delivery and imaging purposes in atherosclerosis.

Pharmaceutical Development and Safety Evaluation of a GMP-Grade Fucoidan for Molecular Diagnosis of Cardiovascular Diseases.

The adhesion molecule P-selectin is present on the cell surface of both activated endothelium and activated platelets. The present study describes the pharmaceutical development, safety evaluation, and preclinical efficacy of a micro-dosed radiotracer. The macromolecular nanoscale assembly consisted of a natural compound made of a sulfated fucose-rich polysaccharides (fucoidan) and a radionuclide (technetium-99m) for the detection of P-selectin expression in cardiovascular diseases. After extraction and fractionation from brown seaweeds, the good manufacturing practice (GMP) production of a low molecular weight (LMW) fucoidan of 7 kDa was achieved and full physicochemical characterization was performed. The regulatory toxicology study in rats of the GMP batch of LMW fucoidan revealed no adverse effects up to 400 µg/kg (×500 higher than the expected human dose) and pseudoallergy was not seen as well. In a myocardial ischemia-reperfusion model in rats, the GMP-grade LMW fucoidan labeled with technetium-99m detected P-selectin upregulation in vivo. The present study supports the potential of using 99mTc-fucoidan as an imaging agent to detect activated endothelium in humans.

Biodistribution of Nanostructured Lipid Carriers in Mice Atherosclerotic Model.

Atherosclerosis is a major cardiovascular disease worldwide, that could benefit from innovative nanomedicine imaging tools and treatments. In this perspective, we here studied, by fluorescence imaging in ApoE-/- mice, the biodistribution of non-functionalized and RXP470.1-targeted nanostructured lipid carriers (NLC) loaded with DiD dye. RXP470.1 specifically binds to MMP12, a metalloprotease that is over-expressed by macrophages residing in atherosclerotic plaques. Physico-chemical characterizations showed that RXP-NLC (about 105 RXP470.1 moieties/particle) displayed similar features as non-functionalized NLC in terms of particle diameter (about 60-65 nm), surface charge (about -5 - -10 mV), and colloidal stability. In vitro inhibition assays demonstrated that RXP-NLC conserved a selectivity and affinity profile, which favored MMP-12. In vivo data indicated that NLC and RXP-NLC presented prolonged blood circulation and accumulation in atherosclerotic lesions in a few hours. Twenty-four hours after injection, particle uptake in atherosclerotic plaques of the brachiocephalic artery was similar for both nanoparticles, as assessed by ex vivo imaging. This suggests that the RXP470.1 coating did not significantly induce an active targeting of the nanoparticles within the plaques. Overall, NLCs appeared to be very promising nanovectors to efficiently and specifically deliver imaging agents or drugs in atherosclerotic lesions, opening avenues for new nanomedicine strategies for cardiovascular diseases.

Orthotopic induction of CH157MN convexity and skull base meningiomas into nude mice using stereotactic surgery and MRI characterization.

Meningioma in vivo research is hampered by the difficulty of establishing an easy and reproducible orthotopic model able to mimic the characteristics of a human meningioma. Moreover, leptomeningeal dissemination and high mortality are often associated with such orthotopical models, making them useless for clinical translation studies. An optimized method for inducing meningiomas in nude mice at two different sites is described in this paper and the high reproducibility and low mortality of the models are demonstrated. Skull base meningiomas were induced in the auditory meatus and convexity meningiomas were induced on the brain surface of 23 and 24 nude mice, respectively. Both models led to the development of a mass easily observable by imaging methods. Dynamic contrast enhanced MRI was used as a tool to monitor and characterize the pathology onset and progression. At the end of the study, histology was performed to confirm the neoplastic origin of the diseased mass.

Photoacoustic imaging of integrin-overexpressing tumors using a novel ICG-based contrast agent in mice.

PhotoAcoustic Imaging (PAI) is a biomedical imaging modality currently under evaluation in preclinical and clinical settings. In this work, ICG is coupled to an integrin binding vector (ICG-RGD) to combine the good photoacoustic properties of ICG and the favourable αvß3-binding capabilities of a small RGD cyclic peptidomimetic. ICG-RGD is characterized in terms of physicochemical properties, biodistribution and imaging performance. Tumor uptake was assessed in subcutaneous xenograft mouse models of human glioblastoma (U-87MG, high αvß3 expression) and epidermoid carcinoma (A431, low αvß3 expression). ICG and ICG-RGD showed high PA signal in tumors already after 15 min post-injection. At later time points the signal of ICG rapidly decreased, while ICG-RGD showed sustained uptake in U-87MG but not in A431 tumors, likely due to the integrin-mediated retention of the probe. In conclusion, ICG-RGD is a novel targeted contrast agents for PAI with superior biodistribution, tumor uptake properties and diagnostic value compared to ICG.

From design to the clinic: practical guidelines for translating cardiovascular nanomedicine.

Cardiovascular diseases (CVD) account for nearly half of all deaths in Europe and almost 30% of global deaths. Despite the improved clinical management, cardiovascular mortality is predicted to rise in the next decades due to the increasing impact of aging, obesity, and diabetes. The goal of emerging cardiovascular nanomedicine is to reduce the burden of CVD using nanoscale medical products and devices. However, the development of novel multicomponent nano-sized products poses multiple technical, ethical, and regulatory challenges, which often obstruct their road to successful approval and use in clinical practice. This review discusses the rational design of nanoparticles, including safety considerations and regulatory issues, and highlights the steps needed to achieve efficient clinical translation of promising nanomedicinal products for cardiovascular applications.

In vivo tumor targeting and biodistribution evaluation of paramagnetic solid lipid nanoparticles for magnetic resonance imaging.

The current study was performed to evaluate the in vivo efficiency of a new nano-sized contrast agent called paramagnetic Solid Lipid Nanoparticles, pSLNs, having promising relaxivity properties for Magnetic Resonance Imaging application. Good stability and stealth properties toward macrophage uptake have been demonstrated. An in vivo MRI study resulted in an improved signal enhancement in the tumor tissue particularly when folate as targeting ligand was used to decorate the nanoparticles surface. Afterward, the biodistribution of pSLNs in several organs was investigated. The accumulation of pSLNs in kidneys, femoral bones, spleen and brain was quite low while high tropism of pSLNs was found for the liver. In this regard, approaches to improve the rate of the hepatic clearance have been proposed.

Macrocyclic paramagnetic agents for MRI: Determinants of relaxivity and strategies for their improvement.

PURPOSE: To dissect the contributions to the longitudinal relaxivity (r1 ) of two commercial contrast agents (CAs), Gd-DOTA and Gd-HP-DO3A, and to synthesize/characterize a novel macrocyclic agent (Gd-Phen-DO3A) having superior r1 . METHODS: Longitudinal relaxation rates R1 of the CAs in saline with/without human serum albumin (HSA), ionized simulated body fluid (i-SBF), viscous simulated body fluid (v-SBF), and human plasma were measured. Results have been interpreted to evince the main determinants to the observed r1 values. RESULTS: In v-SBF or in the presence of HSA, r1 is enhanced for all complexes, reflecting the viscosity increase and a weak interaction with proteins. The CAs further differentiate in plasma, with a relaxivity increase (versus saline) of approximately 1, 1.5, and 2.5 mM-1 s-1 for Gd-DOTA, Gd-HPDO3A, and Gd-Phen-DO3A, respectively. R1 versus pH curves in i-SBF indicates that prototropic exchange sizably contributes to the relaxivity of Gd-HP-DO3A and Gd-Phen-DO3A. CONCLUSION: The major contributions to r1 in the physiological environment have been highlighted, namely, increased viscosity, complex-protein interaction, and prototropic exchange. The control of these terms allows the design of novel macrocyclic structures with enhanced r1 as a result of an improved interaction with plasma's macromolecules and the shift of the prototropic exchange to physiological pH. Magn Reson Med 78:1523-1532, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Hyperpolarized [1,3-13C2 ]ethyl acetoacetate is a novel diagnostic metabolic marker of liver cancer.

An increased prevalence of liver diseases such as hepatitis C and nonalcoholic fatty liver results in an augmented incidence of the most common form of liver cancer, hepatocellular carcinoma (HCC). HCC is most often found in the cirrhotic liver and it can therefore be challenging to rely on anatomical information alone when diagnosing HCC. Valuable information on specific cellular metabolism can be obtained with high sensitivity thanks to an emerging magnetic resonance (MR) technique that uses 13C labeled hyperpolarized molecules. Our interest was to explore potential new high contrast metabolic markers of HCC using hyperpolarized 13C-MR. This work led to the identification of a class of substrates, low molecular weight ethyl-esters, which showed high specificity for carboxyl esterases and proved in many cases to possess good properties for signal enhancement. In particular, hyperpolarized [1,3-13C2 ]ethyl acetoacetate (EAA) was shown to provide a metabolic fingerprint of HCC. Using this substrate a liver cancer implanted in rats was diagnosed as a consequence of an ∼4 times higher metabolic substrate-to-product ratio than in the surrounding healthy tissue, (p=0.009). Unregulated cellular uptake as well as cosubstrate independent enzymatic conversion of EAA, made this substrate highly useful as a hyperpolarized 13C-MR marker. This could be appreciated by the signal-to-noise (SNR) obtained from EAA, which was comparable to the SNR reported in a literature liver cancer study with state-of-the-art hyperpolarized substrate, [1-13C]pyruvate. Also, the contrast-to-noise (CNR) in the EAA based metabolic ratio images was significantly improved compared with the CNR in equivalent images reported using [1-13C]pyruvate.

Paramagnetic solid lipid nanoparticles as a novel platform for the development of molecular MRI probes.

Highly efficient magnetic resonance imaging (MRI) probes have been prepared by loading Gd(III) complexes on the surface of solid lipid nanoparticles (pSLNs). Applicability as molecular imaging probes is demonstrated by an in vitro model study with targeted pSLNs.

Synthesis and preliminary evaluation in tumor bearing mice of new (18)F-labeled arylsulfone matrix metalloproteinase inhibitors as tracers for positron emission tomography.

New fluorinated, arylsulfone-based matrix metalloproteinase (MMP) inhibitors containing carboxylate as the zinc binding group were synthesized as radiotracers for positron emission tomography. Inhibitors were characterized by Ki for MMP-2 in the nanomolar range and by a fair selectivity for MMP-2/9/12/13 over MMP-1/3/14. Two of these compounds were obtained in the (18)F-radiolabeled form, with radiochemical purity and yield suitable for preliminary studies in mice xenografted with a human U-87 MG glioblastoma. Target density in xenografts was assessed by Western blot, yielding Bmax/Kd = 14. The biodistribution of the tracer was dominated by liver uptake and hepatobiliary clearance. Tumor uptake of (18)F-labeled MMP inhibitors was about 30% that of [(18)F]fluorodeoxyglucose. Accumulation of radioactivity within the tumor periphery colocalized with MMP-2 activity (evaluated by in situ zimography). However, specific tumor uptake accounted for only 18% of total uptake. The aspecific uptake was ascribed to the high binding affinity between the radiotracer and serum albumin.

Synthesis of Gd and (68)Ga complexes in conjugation with a conformationally optimized RGD sequence as potential MRI and PET tumor-imaging probes.

We report the synthesis of novel chelates of Gd and (68)Ga with DPTA, DOTA, HP-DOA3, as well as with AAZTA, a novel chelating agent developed by our research group. These chelating agents were appropriately conjugated, prior to metal complexation, with DB58, an RGD peptidomimetic, conformationally constrained on an azabicycloalkane scaffold and endowed with high affinity for integrin α(ν)ß(3) . Because α(ν)ß(3) is involved in neo-angiogenesis in solid tumors and is also directly expressed in cancer cells (e.g. glioblastomas, melanomas) and ovarian, breast, and prostate cancers, these constructs could prove useful as molecular imaging probes in cancer diagnosis by MRI or PET techniques. Molecular modeling, integrin binding assays, and relaxivity assessments allowed the selection of compounds suitable for multiple expression on dendrimeric or nanoparticulate structures. These results also led us to an exploratory investigation of (68)Ga complexation for the promising (68)Ga-PET technique; the AAZTA complex 15((68)Ga) exhibited uptake in a xenograft model of glioblastoma, suggesting potentially useful developments with new probes with improved affinity.

Evidence for in vivo macrophage mediated tumor uptake of paramagnetic/fluorescent liposomes.

Dysprosium (Dy)-loaded liposomes act as excellent T(2)-susceptibility agents at high magnetic field strength. The R(2)-enhancement increases with the size of the liposomes and the concentration of entrapped paramagnetic metal complexes. Neuro-2a tumor cells are readily labeled when Dy-loaded liposomes, suitably functionalized with glutamine residues (Gln), are added to the culture medium as glutamine receptors are highly expressed in such proliferating tumor cells. By using fluorescent liposomes doped with fluorescent dyes (either incorporated in the membrane or included in the inner cavity), confocal microscopy experiments showed that targeted liposomes are taken up much more avidly than non-targeted vesicles. In vivo studies showed that glutamine-functionalized and non-functionalized liposomes accumulate in the tumor region to a similar extent. Confocal images of the excised tumor showed extensive co-localization of liposomes and macrophages in both cases. It is suggested that the loss of tumor specificity, shown by Gln-functionalized liposomes in vivo, has to be associated with the efficient removal of liposomes operated by the RES (reticulo endoplasmatic system) or tumor associated macrophages.

Matrix-assisted laser desorption ionization imaging mass spectrometry detection of a magnetic resonance imaging contrast agent in mouse liver.

The present paper describes the detection of a magnetic resonance imaging (MRI) contrast agent by matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS). The contrast agent was analyzed in both frozen and paraformaldehyde-fixed mouse livers explanted after its in vivo administration, and its identity was confirmed by fragmentation experiments. Moreover, a semiquantitative analysis was performed, evaluating its content in livers from mice sacrificed at different postadministration times. To the best of our knowledge, this is the first description of a MALDI-IMS analysis of MRI contrast agents and the first time that results obtained by MALDI-IMS are validated by both an in vivo (MRI) and an ex vivo (inductively coupled plasma atomic emission spectroscopy, ICP-AES) technique. Results shown in the present paper demonstrate the possibility of using MALDI-IMS for drug biodistribution analysis. Obviously, this application is particularly interesting in the case of unlabeled compounds, which cannot be detected by any of the other imaging techniques.

Tuning glutamine binding modes in Gd-DOTA-based probes for an improved MRI visualization of tumor cells.

Three new magnetic resonance imaging probes that target glutamine transporters have been synthesized. They consist of a Gd-DOTA-monoamide moiety (DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) linked through a six carbon atom chain to a vector represented by a glutamine residue bound through alpha-carboxylic, gamma-carboxamidic, or alpha-amino functionalities. Their uptake by HTC (rat hepatocarcinoma) and healthy rat hepatocytes has shown that the system containing the glutamine vector bound through the alpha-carboxylic group displays a markedly higher affinity for tumor cells. The observed behavior is rationalized in terms of the exploitation of an additional glutamine transporter active in hepatic tumor cells.

Paramagnetic liposomes as innovative contrast agents for magnetic resonance (MR) molecular imaging applications.

This article illustrates some innovative applications of liposomes loaded with paramagnetic lanthanide-based complexes in MR molecular imaging field. When a relatively high amount of a Gd(III) chelate is encapsulated in the vesicle, the nanosystem can simultaneously affect both the longitudinal (R(1)) and the transverse (R(2)) relaxation rate of the bulk H2O H-atoms, and this finding can be exploited to design improved thermosensitive liposomes whose MRI response is not longer dependent on the concentration of the probe. The observation that the liposome compartmentalization of a paramagnetic Ln(III) complex induce a significant R(2) enhancement, primarily caused by magnetic susceptibility effects, prompted us to test the potential of such agents in cell-targeting MR experiments. The results obtained indicated that these nanoprobes may have a great potential for the MR visualization of cellular targets (like the glutamine membrane transporters) overexpressing in tumor cells. Liposomes loaded with paramagnetic complexes acting as NMR shift reagents have been recently proposed as highly sensitive CEST MRI agents. The main peculiarity of CEST probes is to allow the MR visualization of different agents present in the same region of interest, and this article provides an illustrative example of the in vivo potential of liposome-based CEST agents.

Contrast-enhanced MRI of murine sponge model for progressive angiogenesis assessed with gadoteridol (ProHance) and gadocoletic acid trisodium salt (B22956/1).

PURPOSE: To investigate the potential value of MRI for noninvasive assessment of angiogenesis in a murine model exploiting the properties of two contrast agents, gadoteridol (ProHance) and gadocoletic acid trisodium salt (B22956/1). MATERIALS AND METHODS: Biocompatible sponges were implanted in both mice flanks. Stimulated sponges contained human recombinant basic fibroblast growth factor (bFGF) as the angiogenic agent; control sponges contained vehicle. Angiogenesis was evaluated by MRI after injection of extravascular (ProHance) or blood-pool (B22956/1) contrast agents at different times after sponge implantation. Sponges signal intensity enhancement was calculated both as the relative enhancement and the rate of relative enhancement. Results from MRI were validated by classic biochemical (hemoglobin level and protein content) and morphological (histology) assays. RESULTS: The intrinsic different properties of ProHance and B22956/1 in wash-in and wash-out kinetics were useful to detect progressive vascularization and the establishment of a functional vascular network in the implants. Moreover, MRI allowed the appreciation of differences in neovessel colonization between bFGF-treated sponges and controls. Hemoglobin level, protein content, and histology confirmed the sponge vascularization and MRI results. CONCLUSION: Contrast-enhanced MRI is a reliable tool to study vascular characteristics in animal models of angiogenesis. The different kinetic properties of contrast agents can provide evidence of different functional neovascularization aspects and levels.