Daniel Padro

Single-chain polyacrylic nanoparticles with multiple Gd(III) centres as potential MRI contrast agents

One of the approaches for obtaining magnetic resonance imaging (MRI) contrast agents with enhanced relaxivity is to construct macromolecular architectures with multiple Gd(III) centres. In some cases, simple grafting of Gd(III) chelates into polymeric backbones does not result in an increased relaxivity, due to high internal flexibility or a non-rigid attachment of the chelate to the macromolecule. Here we describe a new method for incorporating Gd(III) ions into a polymer in a rigid manner by complexing them into the cross-linker. Thus, acrylic copolymer 4 (Mw = 50 kDa) was forced to collapse intramolecularly by means of the bifunctional cross-linker 6 that had a diethylenetriaminepentaacetic acid (DTPA) group incorporated into it. This results in the formation of water-soluble polymeric nanoparticles (PNPs) 8, which were subsequently loaded with Gd(III) ions to give paramagnetic PNPs 9, with a mean particle size of 1.5 nm. The r1 relaxivity value for PNPs 9 on a per Gd basis was 6.78 mM−1s−1, which represents a 2-fold increase over Magnevist® and a 4-fold increase compared to its monometallic counterpart, the Gd-loaded cross-linker 7.

Magnetic Glyconanoparticles as a Versatile Platform for Selective Immunolabeling and Imaging of Cells

A versatile nanoplatform based on magnetic glyconanoparticles (glyco-ferrites) to attach well-oriented antibodies is described. An efficient ligand exchange process has been used to prepare water-soluble 6-nm-sized core-shell Fe(3)O(4)@Au nanoparticles bearing amphiphilic carbohydrates and aliphatic ethylene glycol chains ended in a carboxyl group. The covalent immobilization through the carboxyl group of an Fc receptor (protein G) enables successful well-oriented capture of immunoglobulins G onto the magnetic glyconanoparticle. A thorough characterization of structure and biofunctionality of the constructs is carried out by different techniques. The selective immunolabeling of cells by the antibody-magnetic glyconanoparticle conjugates is demonstrated by magnetic resonance imaging (MRI), as well as by fluorescence techniques.

Synthesis of single chain thermoresponsive polymer nanoparticles

Poly(N-isopropylacrylamide) (pNIPAM) is a thermoresponsive polymer of great interest in biomedical applications. In this work we combine RAFT polymerization and “click chemistry” for the synthesis of well defined single chain pNIPAM nanoparticles below 10 nm size with increasing degree of cross-linking. The method consists of the synthesis of NIPAM based terpolymers containing alkyne and azide functional groups by RAFT polymerization. Using this procedure terpolymers with polydispersity values below 1.2 were obtained. The terpolymers were collapsed by intramolecular “click” cycloaddition for the synthesis of the corresponding nanoparticles. Thermal phase transition behaviour has been studied for single-chain polymer nanoparticles (PNPs) and linear polymers. Temperature-dependent turbidimetry experiments showed a gradual decrease of transmittance for NPs compared to linear precursors, which presented a sharp decrease of transmittance. In the case of collapsed polymers, this effect was highly affected by the pH, exhibiting significant pH dependency in comparison to their linear precursors.

A solution NMR study of the interactions of oligomannosides and the anti-HIV-1 2G12 antibody reveals distinct binding modes for branched ligands

The structural and affinity details of the interactions of synthetic oligomannosides, linear (di-, tri-, and tetra-) and branched (penta- and hepta-), with the broadly neutralizing anti-HIV-1 antibody 2G12 (HIV=human immunodeficiency virus) have been investigated in solution by using ligand-based NMR techniques, specifically saturation transfer difference (STD) NMR spectroscopy and transferred NOE experiments. Linear oligomannosides show similar binding modes to the antibody, with the nonreducing terminal disaccharide Manα(1→2)Man (Man=mannose) making the closest protein/ligand contacts in the bound state. In contrast, the branched pentamannoside shows two alternate binding modes, involving both ligand arms (D2- and D3-like), a dual binding description of the molecular recognition of this ligand by 2G12 in solution that differs from the single binding mode deduced from X-ray studies. On the contrary, the antibody shows an unexpected selectivity for one arm (D1-like) of the other branched ligand (heptamannoside). This result explains the previously reported lack of affinity enhancement relative to that of the D1-like tetramannoside. Single-ligand STD NMR titration experiments revealed noticeable differences in binding affinities among the linear and branched ligands in solution, with the latter showing decreased affinity. Among the analyzed series of ligands, the strongest 2G12 binders were the linear tri- and tetramannosides because both show similar affinity for the antibody. These results demonstrate that NMR spectroscopic techniques can deliver abundant structural, dynamics, and affinity information for the characterization of oligomannose-2G12 binding in solution, thus complementing, and, as in the case of the pentamannoside, extending, the structural view from X-ray crystallography. This information is of key importance for the development of multivalent synthetic gp120 high-mannose glycoconjugate mimics in the context of vaccine development.

Water-soluble magnetic glyconanoparticles based on metal-doped ferrites coated with gold: Synthesis and characterization

Novel bimetallic superparamagnetic XFe2O4@Au (X = Fe, Mn and Co) nanocrystals of different sizes were synthesized in high-boiling point ether solvent in two steps. Reduction of gold salts on pre-made seeds in the presence of surfactants was performed resulting in the uniform coverage of the seeds. HAADF- and EDXS-STEM elemental mapping indicate the presence of two metals in the same nanocrystal. The apolar solvent-soluble nanoparticles were transferred into water using an amphiphilic linker and different neoglycoconjugates. The as prepared glyco-ferrites are superparamagnetic at room temperature and present comparable or higher transverse relaxivities (T2) than commercial contrast agents like Endorem, Resovist or Sinerem. The carbohydrate coating provides high stability under physiological conditions, low cytotoxicity and lack of immunogenicity. All glyco-ferrites prepared show comparable relaxation times in phantom imaging.

Specific labelling of cell populations in blood with targeted immuno-fluorescent/magnetic glyconanoparticles

Current performance of iron oxide nanoparticle-based contrast agents in clinical use is based on the unspecific accumulation of the probes in certain organs or tissues. Specific targeted biofunctional nanoparticles would significantly increase their potential as diagnostic and therapeutic tools in vivo. In this study, multimodal fluorescent/magnetic glyco-nanoparticles were synthesized from gold-coated magnetite (glyco-ferrites) and converted into specific probes by the covalent coupling of protein G and subsequent incubation with an IgG antibody. The immuno-magnetic-fluorescent nanoparticles were applied to the specific labelling of peripheral blood mononuclear cells (PBMCs) in a complex biological medium, as human blood. We have been able to label specifically PBMCs present in blood in a percentage as low as 0.10-0.17%. Red blood cells (RBCs) were also clearly labelled, even though the inherent T(2) contrast arising from the high iron content of these cells (coming mainly from haemoglobin). The labelling was further assessed at cellular level by fluorescence microscopy. In conclusion, we have developed new contrast agents able to label specifically a cell population under adverse biological conditions (low abundance, low intrinsic T(2), high protein content). These findings open the door to the application of these probes for the labelling and tracking of endogenous cell populations like metastatic cancer cells, or progenitor stem cells that exist in very low amount in vivo.

Sugar/gadolinium-loaded gold nanoparticles for labelling and imaging cells by magnetic resonance imaging

Targeted magnetic resonance imaging (MRI) probes for selective cell labelling and tracking are highly desired. We here present biocompatible sugar-coated paramagnetic Gd-based gold nanoparticles (Gd-GNPs) and test them as MRI T1 reporters in different cellular lines at a high magnetic field (11.7 T). With an average number of 20 Gd atoms per nanoparticle, Gd-GNPs show relaxivity values r1 ranging from 7 to 18 mM-1 s-1 at 1.41 T. The multivalent presentation of carbohydrates on the Gd-GNPs enhances the avidity of sugars for carbohydrate-binding receptors at the cell surface and increases the local concentration of the probes. A large reduction in longitudinal relaxation times T1 is achieved with both fixed cells and live cells. Differences in cellular labelling are obtained by changing the type of sugar on the gold surface, indicating that simple monosaccharides and disaccharides are able to modulate the cellular uptake. These results stress the benefits of using sugars to produce nanoparticles for cellular labelling. To the best of our knowledge this is the first report on labelling and imaging cells with Gd-based gold nanoparticles which use simple sugars as receptor reporters.

Targeted gold-coated iron oxide nanoparticles for CD163 detection in atherosclerosis by MRI

CD163 is a membrane receptor expressed by macrophage lineage. Studies performed in atherosclerosis have shown that CD163 expression is increased at inflammatory sites, pointing at the presence of intraplaque hemorrhagic sites or asymptomatic plaques. Hence, imaging of CD163 expressing macrophages is an interesting strategy in order to detect atherosclerotic plaques. We have prepared a targeted probe based on gold-coated iron oxide nanoparticles vectorized with an anti-CD163 antibody for the specific detection of CD163 by MRI. Firstly, the specificity of the targeted probe was validated in vitro by incubation of the probe with CD163(+) or (−) macrophages. The probe was able to selectively detect CD163(+) macrophages both in human and murine cells. Subsequently, the targeted probe was injected in 16 weeks old apoE deficient mice developing atherosclerotic lesions and the pararenal abdominal aorta was imaged by MRI. The accumulation of probe in the site of interest increased over time and the signal intensity decreased significantly 48 hours after the injection. Hence, we have developed a highly sensitive targeted probe capable of detecting CD163-expressing macrophages that could provide useful information about the state of the atheromatous lesions.

In vivo imaging of dopaminergic neurotransmission after transient focal ischemia in rats

The precise biologic mechanisms involved in functional recovery processes in response to stroke such as dopaminergic neurotransmission are still largely unknown. For this purpose, we performed in parallel in vivo magnetic resonance imaging and positron emission tomography (PET) with [18F]fluorodeoxyglucose ([18F]FDG) and [11C]raclopride at 1, 3, 7, 14, 21, and 28 days after middle cerebral artery occlusion in rats. In the ischemic territory, PET [18F]FDG showed a initial decrease in cerebral metabolism followed by a time-dependent recovery to quasi-normal values at day 14 after ischemia. The PET with [11C]raclopride, a ligand for dopamine D2 receptor, showed a sustained binding during the first week after ischemia that declined dramatically from day 14 to day 28. Interestingly, a slight increase in [11C]raclopride binding was observed at days 1 to 3 followed by the uppermost binding at day 7 in the contralateral territory. Likewise, in vitro autoradiography using [3H]raclopride confirmed these in vivo results. Finally, the neurologic test showed major neurologic impairment at day 1 followed by a recovery of the cerebral function at day 28 after cerebral ischemia. Taken together, these results might suggest that dopamine D2 receptor changes in the contralateral hemisphere could have a key role in functional recovery after cerebral ischemia.

[18F]fluorination of o-carborane via nucleophilic substitution: towards a versatile platform for the preparation of 18F-labelled BNCT drug candidates.

The mono-[(18)F]fluorination of o-carborane via nucleophilic substitution is reported. The new radiochemical transformation uses cyclotron produced [(18)F]F(-) and a carboranyl iodonium salt. Further derivatization of the (18)F-labelled carborane is achieved by formation of the C(c)-lithio salt and reaction with an aldehyde.