Carmen Burtea

Molecular imaging of αvβ3 integrin expression in atherosclerotic plaques with a mimetic of RGD peptide grafted to Gd-DTPA†

AIMS The integrin alpha v beta3 is highly expressed in atherosclerotic plaques by medial and intimal smooth muscle cells and by endothelial cells of angiogenic microvessels. In this study, we have assessed non-invasive molecular magnetic resonance imaging (MRI) of plaque-associated alpha v beta3 integrin expression on transgenic ApoE-/- mice with a low molecular weight peptidomimetic of Arg-Gly-Asp (mimRGD) grafted to gadolinium diethylenetriaminepentaacetate (Gd-DTPA-g-mimRGD). The analogous compound Eu-DTPA-g-mimRGD was employed for an in vivo competition experiment and to confirm the molecular targeting. The specific interaction of mimRGD conjugated to Gd-DTPA or to 99mTc-DTPA with alpha v beta3 integrin was furthermore confirmed on Jurkat T lymphocytes. METHODS AND RESULTS The mimRGD was synthesized and conjugated to DTPA. DTPA-g-mimRGD was complexed with GdCl3.6H2O, EuCl3.6H2O, or with [99mTc(CO)3(H2O)3]+. MRI evaluation was performed on a 4.7 T Bruker imaging system. Blood pharmacokinetics of Gd-DTPA-g-mimRGD were assessed in Wistar rats and in c57bl/6j mice. The presence of angiogenic blood vessels and the expression of alpha v beta3 integrin were confirmed in aorta specimens by immunohistochemistry. Gd-DTPA-g-mimRGD produced a strong enhancement of the external structures of the aortic wall and of the more profound layers (possibly tunica media and intima). The aortic lumen seemed to be restrained and distorted. Pre-injection of Eu-DTPA-g-mimRGD diminished the Gd-DTPA-g-mimRGD binding to atherosclerotic plaque and confirmed the specific molecular targeting. A slower blood clearance was observed for Gd-DTPA-g-mimRGD, as indicated by a prolonged elimination half-life and a diminished total clearance. CONCLUSION The new compound is potentially useful for the diagnosis of vulnerable atherosclerotic plaques and of other pathologies characterized by alpha v beta3 integrin expression, such as cancer and inflammation. The delayed blood clearance, the significant enhancement of the signal-to-noise ratio, and the low immunogenicity of the mimetic molecule highlight its potential for an industrial and clinical implementation.

Contrast Agents: Magnetic Resonance

Even though the intrinsic magnetic resonance imaging (MRI) contrast is much more flexible than in other clinical imaging techniques, the diagnosis of several pathologies requires the involvement of contrast agents (CAs) that can enhance the difference between normal and diseased tissues by modifying their intrinsic parameters. MR CAs are indirect agents because they do not become visible by themselves as opposed to other imaging modalities. The signal enhancement produced by MRI CAs (i.e., the efficiency of the CAs) depends on their longitudinal (r1) and transverse (r2) relaxivity (expressed in s(-1) mmol(-1) 1), which is defined as the increase of the nuclear relaxation rate (the reciprocal of the relaxation time) of water protons produced by 1 mmol per liter of CA. Paramagnetic CAs (most of them complexes of gadolinium) are frequently used in clinics as extracellular, hepatobiliary or blood pool agents. Low molecular weight paramagnetic CAs have similar effects on R1 and R2, but the predominant effect at low doses is that of T1 shortening (and R1 enhancement). Thus, organs taking up such agents will become bright in a T1-weighted MRI sequence; these CAs are thus called positive contrast media. The CAs known as negative agents influence signal intensity mainly by shortening T2* and T2, which produces the darkening of the contrast-enhanced tissue. These CAs are generally composed of superparamagnetic nanoparticles, consisting of iron oxides (magnetite, Fe3O4, maghemite, gammaFe2O3, or other ferrites). Iron oxide nanoparticles are taken up by the monocyte-macrophage system, which explains their potential application as MRI markers of inflammatory and degenerative disorders. Most of the contemporary MRI CAs approved for clinical applications are non-specific for a particular pathology and report exclusively on the anatomy and the physiological status of various organs. A new generation of MRI CAs is progressively emerging in the current context of molecular imaging, agents that are designed to detect with a high specificity the cellular and molecular hallmarks of various pathologies.

Peptidic Targeting of Phosphatidylserine for the MRI Detection of Apoptosis in Atherosclerotic Plaques

Molecular and cellular imaging of atherosclerosis has garnered more interest at the beginning of the 21st century, with aims to image in vivo biological properties of plaque lesions. Apoptosis seems an attractive target for the diagnosis of vulnerable atherosclerotic plaques prone to a thrombotic event. The aim of the present work was to screen for apoptosis peptide binders by phage display with the final purpose to detect apoptotic cells in atherosclerotic plaques by magnetic resonance imaging (MRI). A phosphatidylserine-specific peptide identified by phage display was thus used to design an MRI contrast agent (CA), which was evaluated as a potential in vivo reporter of apoptotic cells. A library of linear 6-mer random peptides was screened in vitro against immobilized phosphatidylserine. Phage DNA was isolated and sequenced, and the affinity of peptides for phosphatidylserine was evaluated by enzyme-linked immunosorbent assay. The phosphatidylserine-specific peptide and its scrambled homologue were attached to a linker and conjugated to DTPA-isothiocyanate. The products were purified by dialysis and by column chromatography and complexed with gadolinium chloride. After their evaluation using apoptotic cells and a mouse model of liver apoptosis, the phosphatidylserine-targeted CA was used to image atherosclerotic lesions on ApoE(-/-) transgenic mice. Apoptotic cells were detected on liver and aorta specimens by the immunostaining of phosphatidylserine and of active caspase-3. Sequencing of the phage genome highlighted nine different peptides. Their alignment with amino acid sequences of relevant proteins revealed a frequent homology with Ca2+ channels, reminiscent of the function of annexins. Alignment with molecules involved in apoptosis provides a direct correlation between peptide selection and utility. The in vivo MRI studies performed at 4.7 T provide proof of concept that apoptosis-related pathologies could be diagnosed by MRI with a low molecular weight paramagnetic agent. The new CA could have real potential in the diagnosis and therapy monitoring of atherosclerotic disease and of other apoptosis-associated pathologies, such as cancer, ischemia, chronic inflammation, autoimmune disorders, transplant rejection, neurodegenerative disorders, and diabetes mellitus. The phage display-derived peptide could also play a potential therapeutic role through anticoagulant activity by mimicking the role of annexin V, the endogenous ligand of phosphatidylserine.

Development of a Magnetic Resonance Imaging Protocol for the Characterization of Atherosclerotic Plaque by Using Vascular Cell Adhesion Molecule-1 and Apoptosis-Targeted Ultrasmall Superparamagnetic Iron Oxide Derivatives

Objective—Acute ischemic events are often caused by the disruption of lipid-rich plaques, which are frequently not angiographically visible. Vascular cell adhesion molecule-1 and apoptotic cell-targeted peptides studied during our previous work were conjugated to ultrasmall superparamagnetic iron oxide (USPIO) (USPIO-R832 for vascular cell adhesion molecule-1 targeting; USPIO-R826 for apoptosis targeting) and assessed by magnetic resonance imaging. Methods and Results—Apolipoprotein E knockout mice were injected with 0.1 mmol Fe/kg body weight and were imaged on a 4.7-T Bruker magnetic resonance imaging until 24 hours after contrast agent administration. Aortic samples were then harvested and examined by histochemistry, and the magnetic resonance images and histological micrographs were analyzed with ImageJ software. The plaques enhanced by USPIO-R832 contained macrophages concentrated in the cap and a large necrotic core, whereas USPIO-R826 produced a negative enhancement of plaques rich in macrophages and neutral fats concentrated inside the plaque. Both USPIO derivatives colocalized with their target on histological sections and were able to detect plaques with a vulnerable morphology, but each one is detecting a specific environment. Conclusion—Our vascular cell adhesion molecule-1 and apoptotic cell targeted USPIO derivatives seem to be highly promising tools for atherosclerosis imaging contributing to the detection of vulnerable plaques. They are able to attain their target in low doses and as fast as 30 minutes after administration.

Magnetic resonance imaging of inflammation with a specific selectin-targeted contrast agent.

E‐selectin‐targeted contrast enhancement of blood vessels in inflamed tissues was investigated with a new contrast agent, Gd‐DTPA‐B(sLex)A, which was recently obtained by grafting a synthetic mimetic of sialyl‐Lewisx, an E‐selectin ligand, onto Gd‐DTPA. The pharmacokinetics, biodistribution, and potential to image inflammation by MRI of this E‐selectin‐targeted contrast agent were evaluated. The inhibition (by 15–34%) produced by Gd‐DTPA‐B(sLex)A on Sialyl Lex‐PAA‐biotin binding to E‐selectin confirmed the specific interaction of the new contrast agent with this adhesion molecule. Gd‐DTPA‐B(sLex)A was tested at a dose of 0.1 mmol/kg b.w. on mice and rats in a fulminant hepatitis model induced by the co‐administration of D‐galactosamine and E. coli lipopolysaccharide. A significant and prolonged contrast enhancement between blood vessels and liver parenchyma was obtained in pathological conditions, which attests to the specificity of the agent for E‐selectin. The prolonged vascular residence (48.9 min in hepatitis vs. 29.8 min in healthy animals), as evidenced by the pharmacokinetic characterization, suggests that Gd‐DTPA‐B(sLex)A interacts with the specific receptors expressed during inflammation. The biodistribution of the compound indicates its retention in inflamed liver by both specific mechanisms and nonspecific accumulation due to the necrotic lesions. The same mechanisms are invoked to account for its retention in the spleen. Magn Reson Med 53:800–807, 2005.

How to quantify iron in an aqueous or biological matrix: a technical note

Iron oxide (nano)particles are powerful contrast agents for MRI and tags for magnetic cellular labeling. The need for quantitative methods to evaluate the iron content of contrast media solutions and biological matrixes is thus obvious. Several convenient methods aiming at the quantification of iron from iron oxide nanoparticle-containing samples are presented.

Magnetic Resonance Molecular Imaging of Vascular Cell Adhesion Molecule-1 Expression in Inflammatory Lesions Using a Peptide-Vectorized Paramagnetic Imaging Probe

The vascular cell adhesion molecule-1 (VCAM-1) has distinct roles in inflammatory cell recruitment to the damaged vessel wall. In the present work, a cyclic heptapeptide phage displayed library was screened in vitro during four rounds of biopanning. On the basis of Kd and IC50 values, a peptide (encoded as R832) was selected for in vitro and in vivo validation. After conjugation to Gd-DOTA, VCAM-1 imaging was assessed by MRI on a model of T cell mediated hepatitis, induced in mice by concanavalin A. On histological samples, the location of biotinylated R832 (R832-Bt) around liver veins in hepatitis resembles the pattern of MRI enhancement. Gd-DOTA-R832 was then assessed on ApoE(-/-) mice and produced an important signal enhancement of the aortic wall, while R832-Bt interacted with morphologic structures comparable to those marked by anti-VCAM-1 antibody. In conclusion, the in vitro and in vivo evaluation of peptide R832 suggests a specific interaction with the targeted biomolecule. Its conjugation to imaging reporters could assist the diagnosis of inflammatory diseases.

Hard corona composition and cellular toxicities of the graphene sheets

Graphene nanomaterials are recognized as one of the most promising nanomaterials because of their unique and highly attractive physicochemical properties (e.g., thermal conductivity, superlative mechanical strength, and ultrahigh surface-to-volume ratios). It is well established that when nanomaterials interact with biological medium, biomolecules and in particular proteins attach to their surfaces, which form a complex between surface of nanoparticles and proteins called corona. Thus, the interaction of the biological system with the nanomaterials depends on the composition of the protein layer, rather than the surface characteristics of the nanomaterials itself. Although there is a significant increase of interest in the application of graphene in medical science, there has been a little attention to the nanotoxicological aspects of these newly developed materials. For this reason, we aimed to investigate whether the effect of the interactions between graphene-sheets with various human plasma concentrations (i.e. both in vitro (cells/tissues) and in vivo simulating states) is toxic. The results showed that by increasing the human plasma concentration, the affinity of proteins with low molecular weights to graphene-sheets surface is significantly increased. Fluorescence microscopy of Hela and Panc-1 cell lines showed a reduction of nuclei number and an increase of reactive oxygen species (ROS) production respectively after a longer incubation of graphene-sheets with plasma proteins. ROS production was higher in Panc-1 cell line, when used as protein source for graphene-sheets than HeLa cell line.

Polyglycerol-grafted superparamagnetic iron oxide nanoparticles: highly efficient MRI contrast agent for liver and kidney imaging and potential scaffold for cellular and molecular imaging.

Polyglycerol as a water-soluble and biocompatible hyperbranched polymer was covalently grafted on the surface of superparamagnetic iron oxide nanoparticles. With this aim, superparamagnetic magnetite nanoparticles were prepared by coprecipitation in aqueous media, then the surface of nanoparticles was modified to introduce the reactive groups on the surface of nanoparticles. After that, polyglycerol was grafted on the surface of nanoparticles by ring-opening anionic polymerization of glycidol using n-bulyllithium as initiator. The magnetometry, relaxometry and phantom MRI experiments of this highly stable ferrofluid showed its high potential as a negative MRI contrast agent. Calculated r(1) and r(2) relaxivities at different magnetic fields were higher than the values reported for commercially available iron oxide contrast agents. The in vivo MRI studies showed that, after intravenous injection into mice, the particles produced a strong negative contrast in liver and kidneys, which persisted for 80 min (in liver) to 110 min (in kidneys). The negative contrast of the liver and kidneys weakened over the time, suggesting that polyglycerol coating renders the nanoparticles stealth and possibly optimal for renal excretion.

Potential amyloid plaque-specific peptides for the diagnosis of Alzheimer's disease.

Amyloid plaques (AP) represent one of the main molecular hallmarks of Alzheimers disease (AD). In order to develop new AP-specific contrast agents for AD molecular imaging, the phage display technology was used to identify peptides specific to amyloid-beta (A beta(42)). A random disulfide constrained heptapeptide phage display library was screened against A beta(42). After biopanning, 72 phage clones were isolated and their binding affinity to A beta(42) was evaluated by enzyme-linked immunosorbent assay (ELISA). The final library was enriched in two peptide sequences. The K(d) of candidate phage clones for binding to A beta(42) are in the picomolar range. The binding affinity for A beta(42) of two selected peptides was confirmed by ELISA, and the specific interaction with AP was validated by immunohistochemistry on brain sections. The preliminary MRI in vivo study, which was performed with a peptide functionalized contrast agent on AD transgenic mouse, showed encouraging results. To conclude, low molecular weight peptides presenting a specific affinity for A beta(42) were identified by phage display. As specific carriers, they have a real potential for molecular imaging of AD thanks to AP binding.