Bis(phenylethylamide) derivatives of Gd-DTPA as potential receptor-specific MRI contrast agents

peer reviewedDTPA-bis(amide) derivatives bearing phenyl, phenol or catechol groups that mimic side chains of naturally occurring amino acids, such as phenylatanine, tyrosine or dopamine, were synthesized and characterized by elemental analysis, electrospray mass spectrometry, NMR spectroscopy and IR spectroscopy. The gadolinium(III) complexes of the ligands DTPA-bis(tyramide) [DTPA-(TA)(2)], DTPA-bis(3-hydroxytyramide) [DTPA-(HTA)(2)] and DTPA-bis(phenylalanine ethyl ester) [DTPA-(PAE)(2)], were prepared and then studied in vitro by O-17 NMR spectroscopy and by nuclear magnetic relaxation dispersion (NMRD) measurements. The residence time of the coordinated water in gadolinium(III) complexes was obtained from O-17 NMR relaxometric T-2 measurements. At 310 K, the following tau(M) values were obtained: Gd-DTPA(TA)(2) 582 ns, Gd-DTPA-(HTA)(2) 372 ns and Gd-DTPA-(PAE)(2) 809 ns. As shown by the analysis of the proton NMRD profiles, the larger proton relaxivities of the gadolinium(III) complexes at 310 K relative to that of the parent Gd-DTPA complex are mainly because of the increase in the rotational correlation time

Development of a robust protocol for vulnerable plaque characterization by using two peptide-functionalized USPIO derivaties

Purpose: Rupture of atherosclerotic plaque is the primary cause of sudden cardiac death mainly in the industrialized countries. The term 'plaque rupture' defines a structural defect in the fibrous cap that separates a necrotic core from the lumen resulting in its exposure to the blood via a gap in the cap (Schwartz SM et al, Arterioscler Thromb Vasc Biol, 2007, 27, 705). In most patients, acute ischemic events are caused by the disruption of type IV and Va lipid-rich lesions, which are often not angiographically visible (Frank H, Am Heart J, 2001, 141, S45). ApoE-KO mouse model of atherosclerosis is characterized, in certain circumstances, by human-like plaque rupture events (Johnson JL & Jackson CL, Atherosclerosis, 2001, 154, 399). VCAM-1 and apoptotic cell-targeted peptides identified and validated during our previous work (Burtea C et al, J Med Chem, 2009, 52, 4725; Burtea C et al, Mol Pharm, 2009, 6, 1903) were now conjugated to USPIO (USPIO-R832 for VCAM-1 targeting; USPIO-R826 for apoptosis targeting) and assessed by MRI on ApoE-KO mice. The results were then correlated with several biomarkers of plaque vulnerability which were evaluated by immunohistochemistry. Materials and Methods: Female ApoE-KO mice injected with 100 µmol Fe/kg were imaged on a 4.7 T Bruker MRI at the level of abdominal aorta with RARE (TR/TE = 3000/20 ms, spatial resolution = 90 µm) and FLASH (TR/TE = 175/1.88 ms, flip angle = 90°, spatial resolution = 172 µm) imaging protocols. After MRI investigations, aortic samples were examined by histochemistry for the binding of contrast agent (Perl's staining protocol), the presence of collagen and thrombus (Masson's trichrome staining), of angiogenic blood vessels (VCAM-1 and PECAM-1 staining), apoptotic cells (caspase-3), macrophages (Mac 1), cholesterol (Sudan IV), and smooth muscle cells (?-actin staining). The MR images and histological pictures were then analyzed with ImageJ software. Results: Both USPIO-R832 and USPIO-R826 produced a maximum negative contrast 30 min after administration (Fig. 1), being constant until the end of MRI studies (90 min). The plaque surface was measured on images and it was correlated to the level of plaque enhancement and to the histological observations. USPIO-R826 has mainly enhanced lipid-rich plaques, while this parameter did not seem to influence the binding of USPIO-R832 which enhanced fibrous plaques as well. Conclusion: Our VCAM-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 min after administration

Development and validation of a peptide-vectorized superparamagnetic imaging probe designed for detection of inflammation in atherosclerotic plaque

Introduction Atherosclerosis develops in response to vascular injury and involves inflammation and vessel remodeling. VCAM-1 is an important biomarker of various inflammatory disorders having a particular relevance for the pathological process of atherosclerotic disease. Molecularly targeted imaging probes have gained great popularity during the last decade in the context of theranostic strategies. The imaging of VCAM-1 expression has thus been approached with antibodies or peptides conjugated to nuclear, magnetooptical, or ultrasound probes. However, the size of most atherosclerotic lesions is below the spatial resolution of nuclear or ultrasound imaging systems. MRI is the only clinical imaging technique able to attain a spatial resolution in the order of micrometers. Nevertheless, the low sensitivity achieved with magnetic probes constitutes a real challenge for molecular targeting by MRI. Owing to their large NMR efficacy, iron oxide nanoparticles may represent an attractive probe alternative [1, 2]. During our previous work [3], we have identified and validated a VCAM-1-targeted cyclic heptapeptide which was able to detect specifically this adhesion molecule both in a mouse a model of T cell mediated hepatitis, and in atherosclerotic plaque of ApoE-KO mice. In the present work, this peptide was conjugated to USPIO (USPIO-R832), and VCAM-1 binding was first confirmed on HUVEC stimulated with TNF-alpha. Subsequently, USPIO-R832 was evaluated by MRI at 4.7T on ApoE-KO mice, by using T2 and T2*-weighted imaging sequences. The ability to bind to atherosclerotic plaque of this molecular imaging probe was furthermore corroborated by histochemistry. The control imaging probe was represented by USPIO vectorized by a non-specific peptide (USPIO-NSP). Materials and methods The peptides were conjugated to USPIO as previously described [4]. The size of functionalized USPIO was of ~30 nm, while their r2 at 60 MHz and 37°C was of 86 s-1 mM-1 for USPIO-R832 and of 90 s-1 mM-1 for USPIO-NSP. Female C57Bl ApoEtm1unc mice received a Western diet (0.21% cholesterol) for 3 months prior to the MRI studies. The contrast agents were assessed blindly on 6 mice each at a dose of 100 µmol Fe/kg. Images were acquired (4.7 T Bruker imaging system, Bruker, Ettlingen, Germany) at the level of abdominal aorta with a T2-weighted RARE sequence (TR/TE = 3000/20 ms, RARE factor = 4, NEX = 4, matrix = 256x256, FOV = 2.3 cm, slice thickness 1 mm, 20 axial slices, spatial resolution = 90 µm) and a T2*-weighted FLASH imaging protocol (TR/TE = 175/1.88 ms, flip angle = 90°, NEX = 8, matrix = 128x128, FOV = 2.5 cm, slice thickness 1.25 mm, 15 axial slices, spatial resolution = 172 µm). A 3D-TOF sequence (TR/TE = 10/2 ms, flip angle = 20°, NEX = 2, FOV = 4x2x4 cm, matrix = 256x128x64, slice thickness = 1 mm, 60 axial slices, spatial resolution = 156x156x625 µm) was used with the aim to confirm the anatomical location of the aorta in the image slice. SI values for each time point were measured within ROIs drawn manually by using the ImageJ image analysis software in the arterial wall of the abdominal aorta. The standard deviation (SD) of noise was also measured in a region situated out of the animal's image. SI enhancement (%SNR) was then calculated. VCAM-1 expression in atherosclerotic aorta was confirmed by immunohistochemistry, while contrast agents were stained on aortic samples by Perl's Prussian blue staining protocol. Results The maximum negative contrast produced by USPIO-R832 on RARE images occurred between ~30 min and 94 min post-injection, probably depending on the level of plaque neovascularization, which influences the diffusion of the contrast agent. This could also be a sign of plaque vulnerability. The plaque surface was measured on images and it was correlated to the level of the plaque enhancement and to the histological observations. In the case of FLASH images, the maximum negative contrast occurred between ~40 min and 80 min post-injection. With the exception of one mouse, the negative contrast was almost absent in the case of USPIO-NSP both on RARE and FLASH images. The histochemistry studies confirmed the MRI results and have shown an extensive VCAM-1 expression, as well as the presence of capillary-like structures that could be of angiogenic nature. Conclusions Our peptide-vectorized, VCAM-1-targeted, superparamagnetic imaging probe seems to be a highly promising tool for atherosclerosis imaging, by considering its ability to attain its target in lower doses and as fast as 30 min after administration. This represents an important progress in comparison with previously developed superparamagnetic agents designed for the same purpose. The lower immunogenic potential and the cost-effectiveness when compared with antibody-conjugated contrast agents represent supplementary arguments for a possible implementation in the clinical practice. Bibliography 1. Kelly KA, Allport JR, Tsurkas A, Shinde-Patil VR, Josephson L, Weissleder R, Circ Res, 96, 2005, 327-336. 2. McAteer MA, Schneider JE, Ali ZA, Warrick N, Bursill CA, von zur Muhlen C, Greaves DR, Neubauer S, Channon KM, Choudhury RP, Arterioscler Thromb Vasc Biol, 28, 2008, 77-83. 3. Burtea C, Laurent S, Port M, Lancelot E, Ballet S, Rousseaux O, Toubeau G, Vander Elst L, Corot C, Muller RN, J Med Chem, 52, 2009, 4725-4742. 4. Port M, Corot C, Raynal I, Rousseaux O, US Patent 2004/0253181 A1

Crucial Ignored Parameters on Nanotoxicology: The Importance of Toxicity Assay Modifications and “Cell Vision”

Until now, the results of nanotoxicology research have shown that the interactions between nanoparticles (NPs) and cells are remarkably complex. In order to get a deep understanding of the NP-cell interactions, scientists have focused on the physicochemical effects. However, there are still considerable debates about the regulation of nanomaterials and the reported results are usually in contradictions. Here, we are going to introduce the potential key reasons for these conflicts. In this case, modification of conventional in vitro toxicity assays, is one of the crucial ignored matter in nanotoxicological sciences. More specifically, the conventional methods neglect important factors such as the sedimentation of NPs and absorption of proteins and other essential biomolecules onto the surface of NPs. Another ignored matter in nanotoxicological sciences is the effect of cell “vision” (i.e., cell type). In order to show the effects of these ignored subjects, we probed the effect of superparamagnetic iron oxide NPs (SPIONs), with various surface chemistries, on various cell lines. We found thatthe modification of conventional toxicity assays and the consideration of the “cell vision” concept are crucial matters to obtain reliable, and reproducible nanotoxicology data. These new concepts offer a suitable way to obtain a deep understanding on the cell-NP interactions. In addition, by consideration of these ignored factors, the conflict of future toxicological reports would be significantly decreased

Relaxivities of paramagnetic liposomes: on the importance of the chain type and the length of the amphiphilic complex

peer reviewedNuclear magnetic relaxation dispersion (NMRD) profiles of unilamellar DPPC liposomes incorporating Gd-DTPA-bisamides with alkyl chains of 12 to 18 C atoms in their external and internal layers were recorded in order to study the influence that the chain length and structure of Gd-bisamides incorporated in the liposomal membrane have on their proton relaxivity. The NMRD profiles recorded at 310 K show that the relaxivity reaches a minimum value when the carbon chain lengths of the phospholipid and of the Gd complex match and is at a maximum in the presence of a carbon-carbon double bond. For these DPPC paramagnetic liposomes, the longer the aliphatic chains of the complex, the larger will be its immobilization in the membrane. In addition, the presence of an unsaturated carbon-carbon bond in the alkyl chain of the Gd complex induces an increase of its mobility and of its water exchange rate with, as a result, a much greater efficiency as an MRI contrast agent

Paramagnetic liposomes: Inner versus outer membrane relaxivity of DPPC liposomes incorporating lipophilic gadolinium complexes

peer reviewedProton relaxometric properties of unilamellar DPPC liposomes embedding an amphiphilic paramagnetic chelate (Gd-DTPA-BC(14)A) in both layers of the phospholipid membrane or only in the external one are compared. The results show that the membrane's water permeability is able to quench the effect of the paramagnetic complexes located in the internal layer of DPPC liposomes, leading thus to an apparent lower global relaxivity

Description of the cell lines used in MTT and XTT studies (DMEM: Dulbecco's modified Eagle's medium; Ham's: Nutrient Mixture F-10; FBS: fetal bovine serum; RPMI-1640 (Roswell Park Memorial Institute)).

<p>Description of the cell lines used in MTT and XTT studies (DMEM: Dulbecco's modified Eagle's medium; Ham's: Nutrient Mixture F-10; FBS: fetal bovine serum; RPMI-1640 (Roswell Park Memorial Institute)).</p

Induced lysosomes and ROS level in various cell lines upon interaction with (a) PEG- and (b) APTES-coated SPIONs.

<p>Induced lysosomes and ROS level in various cell lines upon interaction with (a) PEG- and (b) APTES-coated SPIONs.</p

Bis(phenylethylamide) derivatives of Gd-DTPA as potential receptor-specific MRI contrast agents

DTPA-bis(amide) derivatives bearing phenyl, phenol or catechol groups that mimic side chains of naturally occurring amino acids, such as phenylatanine, tyrosine or dopamine, were synthesized and characterized by elemental analysis, electrospray mass spectrometry, NMR spectroscopy and IR spectroscopy. The gadolinium(III) complexes of the ligands DTPA-bis(tyramide) [DTPA-(TA)(2)], DTPA-bis(3-hydroxytyramide) [DTPA-(HTA)(2)] and DTPA-bis(phenylalanine ethyl ester) [DTPA-(PAE)(2)], were prepared and then studied in vitro by O-17 NMR spectroscopy and by nuclear magnetic relaxation dispersion (NMRD) measurements. The residence time of the coordinated water in gadolinium(III) complexes was obtained from O-17 NMR relaxometric T-2 measurements. At 310 K, the following tau(M) values were obtained: Gd-DTPA(TA)(2) 582 ns, Gd-DTPA-(HTA)(2) 372 ns and Gd-DTPA-(PAE)(2) 809 ns. As shown by the analysis of the proton NMRD profiles, the larger proton relaxivities of the gadolinium(III) complexes at 310 K relative to that of the parent Gd-DTPA complex are mainly because of the increase in the rotational correlation time.status: publishe