Rafael T. M. de Rosales

Vascular Smooth Muscle Cell Calcification Is Mediated by Regulated Exosome Secretion

RATIONALE Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. OBJECTIVE The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. METHODS AND RESULTS Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. CONCLUSIONS This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.

99mTc-Bisphosphonate-Iron Oxide Nanoparticle Conjugates for Dual-Modality Biomedical Imaging

The combination of radionuclide-based imaging modalities such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) with magnetic resonance imaging (MRI) is likely to become the next generation of clinical scanners. Hence, there is a growing interest in the development of SPECT- and PET-MRI agents. To this end, we report a new class of dual-modality imaging agents based on the conjugation of radiolabeled bisphosphonates (BP) directly to the surface of superparamagnetic iron oxide (SPIO) nanoparticles. We demonstrate the high potential of BP-iron oxide conjugation using (⁹⁹m)Tc-dipicolylamine(DPA)-alendronate, a BP-SPECT agent, and Endorem/Feridex, a liver MRI contrast agent based on SPIO. The labeling of SPIOs with (⁹⁹m)Tc-DPA-alendronate can be performed in one step at room temperature if the SPIO is not coated with an organic polymer. Heating is needed if the nanoparticles are coated, as long as the coating is weakly bound as in the case of dextran in Endorem. The size of the radiolabeled Endorem (⁹⁹m)Tc-DPA-ale-Endorem) was characterized by TEM (5 nm, Fe₃O₄ core) and DLS (106 ± 60 nm, Fe₃O₄ core + dextran). EDX, Dittmer-Lester, and radiolabeling studies demonstrate that the BP is bound to the nanoparticles and that it binds to the Fe₃O₄ cores of Endorem, and not its dextran coating. The bimodal imaging capabilities and excellent stability of these nanoparticles were confirmed using MRI and nanoSPECT-CT imaging, showing that (⁹⁹m)Tc and Endorem co-localize in the liver and spleen In Vivo, as expected for particles of the composition and size of (⁹⁹m)Tc-DPA-ale-Endorem. To the best of our knowledge, this is the first example of radiolabeling SPIOs with BP conjugates and the first example of radiolabeling SPIO nanoparticles directly onto the surface of the iron oxide core, and not its coating. This work lays down the basis for a new generation of SPECT/PET-MR imaging agents in which the BP group could be used to attach functionality to provide targeting, stealth/stability, and radionuclides to Fe₃O₄ nanoparticles using very simple methodology readily amenable to GMP.

Bisphosphonate-anchored PEGylation and radiolabeling of superparamagnetic iron oxide: long-circulating nanoparticles for in vivo multimodal (T1 MRI-SPECT) imaging

The efficient delivery of nanomaterials to specific targets for in vivo biomedical imaging is hindered by rapid sequestration by the reticuloendothelial system (RES) and consequent short circulation times. To overcome these two problems, we have prepared a new stealth PEG polymer conjugate containing a terminal 1,1-bisphosphonate (BP) group for strong and stable binding to the surface of ultrasmall-superparamagnetic oxide nanomaterials (USPIOs). This polymer, PEG(5)-BP, can be used to exchange the hydrophobic surfactants commonly used in the synthesis of USPIOs very efficiently and at room temperature using a simple method in 1 h. The resulting nanoparticles, PEG(5)-BP-USPIOs are stable in water or saline for at least 7 months and display a near-zero ζ-potential at neutral pH. The longitudinal (r1) and transverse (r2) relaxivities were measured at a clinically relevant magnetic field (3 T), revealing a high r1 of 9.5 mM–1 s–1 and low r2/r1 ratio of 2.97, making these USPIOs attractive as T1-weighted MRI contrast agents at high magnetic fields. The strong T1-effect was demonstrated in vivo, revealing that PEG(5)-BP-USPIOs remain in the bloodstream and enhance its signal 6-fold, allowing the visualization of blood vessels and vascular organs with high spatial definition. Furthermore, the optimal relaxivity properties allow us to inject a dose 4 times lower than with other USPIOs. PEG(5)-BP-USPIOs can also be labeled using a radiolabeled-BP for visualization with single photon emission computed tomography (SPECT), and thus affording dual-modality contrast. The SPECT studies confirmed low RES uptake and long blood circulation times (t1/2 = 2.97 h). These results demonstrate the potential of PEG(5)-BP-USPIOs for the development of targeted multimodal imaging agents for molecular imaging.

An artificial di-iron oxo-protein with phenol oxidase activity.

Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site.

Synthesis of 64CuII–Bis(dithiocarbamatebisphosphonate) and Its Conjugation with Superparamagnetic Iron Oxide Nanoparticles: In Vivo Evaluation as Dual-Modality PET–MRI Agent

The synergistic combination of positron emission tomography (PET) and magnetic resonance imaging (MRI) is likely to become the next generation of dual-modality scanners in medical imaging. These instruments will provide us with accurate diagnoses thanks to the sensitive and quantifiable signal of PET and the high soft-tissue resolution of MRI. Furthermore, patients will receive less radiation dose and spend less time in the procedure relative to current dual-modality scanners (e.g. PET–computed tomography (CT)). As a consequence, there has been increasing interest recently in the development of dual-modality PET–MRI agents.[1]

188Re(CO)3-dipicolylamine-alendronate: a new bisphosphonate conjugate for the radiotherapy of bone metastases

The palliation of pain due to bone metastases using targeted compounds containing beta-emitters such as rhenium-188 ((188)Re) is an accepted and effective form of treatment. Here, we describe the efficient synthesis and preclinical evaluation of (188)Re(CO)(3)-dipicolylamine(DPA)-alendronate, a novel bifunctional bisphosphonate for the palliative treatment of bone metastases. (188)Re(CO)(3)-DPA-alendronate can be easily synthesized with high specific activities and yields (18.8 GBq/mg, radiochemical yield > or =96%) in two steps using kit-based methodology, and in contrast with the clinically approved bisphosphonate (186/188)Re-HEDP, it forms inert, single species that have been well-characterized. In vivo imaging and biodistribution studies demonstrate that (188)Re(CO)(3)-DPA-alendronate is superior to (188)Re-HEDP in targeting and accumulating in areas of high metabolic bone activity while having low soft-tissue uptake. In addition to these studies, a simple and convenient new method for purifying its precursor, fac-[(188)Re(CO)(3)(H(2)O)(3)](+), is described.

Magnetically Decorated Multiwalled Carbon Nanotubes as Dual MRI and SPECT Contrast Agents

Carbon nanotubes (CNTs) have been proposed as one of the most promising nanomaterials to be used in biomedicine for their applications in drug/gene delivery as well as biomedical imaging. The present study developed radio-labeled iron oxide decorated multi-walled CNTs (MWNT) as dual magnetic resonance (MR) and single photon emission computed tomography (SPECT) imaging agents. Hybrids containing different amounts of iron oxide were synthesized by in situ generation. Physicochemical characterisations revealed the presence of superparamagnetic iron oxide nanoparticles (SPION) granted the magnetic properties of the hybrids. Further comprehensive examinations including high resolution transmission electron microscopy (HRTEM), fast Fourier transform simulations (FFT), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) assured the conformation of prepared SPION as γ-Fe2O3. High r2 relaxivities were obtained in both phantom and in vivo MRI compared to the clinically approved SPION Endorem®. The hybrids were successfully radio-labeled with technetium-99m through a functionalized bisphosphonate and enabled SPECT/CT imaging and γ-scintigraphy to quantitatively analyze the biodistribution in mice. No abnormality was found by histological examination and the presence of SPION and MWNT were identified by Perls stain and Neutral Red stain, respectively. TEM images of liver and spleen tissues showed the co-localization of SPION and MWNT within the same intracellular vesicles, indicating the in vivo stability of the hybrids after intravenous injection. The results demonstrated the capability of the present SPION-MWNT hybrids as dual MRI and SPECT contrast agents for in vivo use.

Aluminium hydroxide stabilised MnFe2O4 and Fe3O4 nanoparticles as dual-modality contrasts agent for MRI and PET imaging

Magnetic nanoparticles (NPs) MnFe2O4 and Fe3O4 were stabilised by depositing an Al(OH)3 layer via a hydrolysis process. The particles displayed excellent colloidal stability in water and a high affinity to [18F]-fluoride and bisphosphonate groups. A high radiolabeling efficiency, 97% for 18F-fluoride and 100% for 64Cu-bisphosphonate conjugate, was achieved by simply incubating NPs with radioactivity solution at room temperature for 5 min. The properties of particles were strongly dependant on the thickness and hardness of the Al(OH)3 layer which could in turn be controlled by the hydrolysis method. The application of these Al(OH)3 coated magnetic NPs in molecular imaging has been further explored. The results demonstrated that these NPs are potential candidates as dual modal probes for MR and PET. In vivo PET imaging showed a slow release of 18F from NPs, but no sign of efflux of 64Cu.

Efficient site-specific radiolabeling of a modified C2A domain of synaptotagmin I with [99mTc(CO)3]+: a new radiopharmaceutical for imaging cell death.

We describe the design and synthesis of a new Tc-99m labeled bioconjugate for cell-death imaging, based on C2A, the phosphatidylserine (PS)-binding domain of rat synaptotagmin I. Since several lysine residues in this protein are critical for PS binding, we engineered a new protein, C2AcH, to include the C-terminal sequence CKLAAALEHHHHHH, incorporating a free cysteine (for site-specific covalent modification) and a hexahistidine tag (for site-specific radiolabeling with [99mTc(CO)3(OH2)3]+). We also engineered a second derivative, C2Ac, in which the C-terminal sequence included only the C-terminal cysteine. These proteins were characterized by electrospray mass spectrometry, SDS/PAGE, and size exclusion chromatography and radiolabeled with [99mTc(CO)3(OH2)3]+. Conjugates of the proteins with the rhenium analogue [Re(CO)3(OH2)3]+ were also synthesized. Site-specific labeling was confirmed by performing a tryptic digest of rhenium tricarbonyl-labeled C2AcH, and only peptides containing the His-tag contained the [Re(CO)3]+. The labeled proteins were tested for binding to red blood cells (RBC) with exposed PS in a calcium dependent manner. Labeling 100 microg of C2AcH with [99mTc(CO)3(OH2)3]+ at 37 degrees C for 30 min gave a radiochemical yield of > 96%. However, C2AcH that had first been conjugated with fluorescein maleimide or iodoacetamide via the Cys residue gave only 50% and 83% radiochemical yield, respectively, after incubation for 30 min at 37 degrees C. Serum stability results indicated that >95% of radiolabeled C2AcH remained stable for at least 18 h at 37 degrees C. Site-specifically labeled C2AcH exhibited calcium-dependent binding to the PS on the RBC, whereas a nonspecifically modified derivative, C2AcH-B, in which lysines had been modified with benzyloxycarbonyloxy, did not. We conclude that (i) the combination of Cys and a His-tag greatly enhances the rate and efficiency of labeling with [99mTc(CO)3(OH2)3]+ compared to either the His-tag or the Cys alone, and this sequence deserves further evaluation as a radiolabeling tag; (ii) non-site-specific modification of C2A via lysine residues impairs target binding affinity; (iii) 99mTc-C2AcH has excellent radiolabeling, stability and PS binding characteristics and warrants in vivo evaluation as a cell-death imaging agent.

Iron(II) Complexes with Tetradentate Bis(aminophenolate) Ligands: Synthesis and Characterization, Solution Behavior, and Reactivity with O2

Tetradentate bis(aminophenolate) ligands H(2)salan(X) and H(2)bapen(X) (where X refers to the para-phenolate substituent = H, Me, F, Cl) react with [Fe{N(SiMe(3))(2)}(2)] to form iron(II) complexes, which in the presence of suitable donor ligands L (L = pyridine or THF) can be isolated as the complexes [Fe(salan(X))(L)(2)] and [Fe(bapen(X))(L)(2)]. In the absence of donor ligands, either mononuclear complexes, for example, [Fe(salan(tBu,tBu))], or dinuclear complexes of the type [Fe(salan(X))](2) are obtained. The dynamic coordination behavior in solution of the complexes [Fe(salan(F))(L)(2)] and [Fe(bapen(F))(L)(2)] has been investigated by VT (1)H and (19)F NMR spectroscopy, which has revealed equilibria between isomers with different ligand coordination topologies cis-α, cis-β and trans. Exposure of the iron(II) salan(X) complexes to O(2) results in the formation of oxo-bridged iron(III) complexes of the type [{Fe(salan(X))}(2)(μ-O)] or [{Fe(salan(X))(L)}(2)(μ-O)]. The lack of catalytic activity of the iron(II) salan and bapen complexes in the oxidation of cyclohexane with H(2)O(2) as the oxidant is attributed to the rapid formation of stable and catalytically inactive oxo-bridged iron(III) complexes.