Juan Gallo

CXCR4-Targeted and MMP-Responsive Iron Oxide Nanoparticles for Enhanced Magnetic Resonance Imaging†

MRI offers high spatial resolution with excellent tissue penetration but it has limited sensitivity and the commonly administered contrast agents lack specificity. In this study, two sets of iron oxide nanoparticles (IONPs) were synthesized that were designed to selectively undergo copper-free click conjugation upon sensing of matrix metalloproteinase (MMP) enzymes, thereby leading to a self-assembled superparamagnetic nanocluster network with T2 signal enhancement properties. For this purpose, IONPs with bioorthogonal azide and alkyne surfaces masked by polyethylene glycol (PEG) layers tethered to CXCR4-targeted peptide ligands were synthesized and characterized. The IONPs were tested in vitro and T2 signal enhancements of around 160 % were measured when the IONPs were incubated with cells expressing MMP2/9 and CXCR4. Simultaneous systemic administration of the bioorthogonal IONPs in tumor-bearing mice demonstrated the signal-enhancing ability of these ‘smart’ self-assembling nanomaterials.

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.

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.

Lanthanide(III) Complexes of Rhodamine–DO3A Conjugates as Agents for Dual-Modal Imaging

Two novel dual-modal MRI/optical probes based on a rhodamine–DO3A conjugate have been prepared. The bis(aqua)gadolinium(III) complex Gd.L1 and mono(aqua)gadolinium(III) complex Gd.L2 behave as dual-modal imaging probes (r1 = 8.5 and 3.8 mM–1 s–1 for Gd.L1 and Gd.L2, respectively; λex = 560 nm and λem = 580 nm for both complexes). The rhodamine fragment is pH-sensitive, and upon lowering of the pH, an increase in fluorescence intensity is observed as the spirolactam ring opens to give the highly fluorescent form of the molecule. The ligands are bimodal when coordinated to Tb(III) ions, inducing fluorescence from both the lanthanide center and the rhodamine fluorophore, on two independent time frames. Confocal imaging experiments were carried out to establish the localization of Gd.L2 in HEK293 cells and primary mouse islet cells (∼70% insulin-containing β cells). Colocalization with MitoTracker Green demonstrated Gd.L2’s ability to distinguish between tumor and healthy cells, with compartmentalization believed to be in the mitochondria. Gd.L2 was also evaluated as an MRI probe for imaging of tumors in BALB/c nude mice bearing M21 xenografts. A 36.5% decrease in T1 within the tumor was observed 30 min post injection, showing that Gd.L2 is preferentially up taken in the tumor. Gd.L2 is the first small-molecule MR/fluorescent dual-modal imaging agent to display an off–on pH switch upon its preferential uptake within the more acidic microenvironment of tumor cells.

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.

Live Imaging of Mouse Endogenous Neural Progenitors Migrating in Response to an Induced Tumor

Adult neurogenesis is restricted to specific brain regions. Although involved in the continuous supply of interneurons for the olfactory function, the role of neural precursors in brain damage-repair remains an open question. Aiming to in vivo identify endogenous neural precursor cells migrating towards a brain damage site, the monoclonal antibody Nilo2 recognizing cell surface antigens on neuroblasts, was coupled to magnetic glyconanoparticles (mGNPs). The Nilo2-mGNP complexes allowed, by magnetic resonance imaging in living animals, the in vivo identification of endogenous neural precursors at their niche, as well as their migration to a lesion site (induced brain tumor), which was fast (within hours) and orderly. Interestingly, the rapid migration of neuroblasts towards a damage site is a characteristic that might be exploited to precisely localize early damage events in neurodegenerative diseases. In addition, it might facilitate the study of regenerative mechanisms through the activation of endogenous neural cell precursors. A similar approach, combining magnetic glyconanoparticles linked to appropriate antibodies could be applied to flag other small cell subpopulations within the organism, track their migration, localize stem cell niches, cancer stem cells or even track metastatic cells.

Uptake and intracellular fate of fluorescent-magnetic glyco-nanoparticles.

Iron oxide based nanoparticles are finding their way as leading actors in nanotechnology applications to medicine. Magnetite nanoparticles are currently being used in clinics for the detection of hepatic tumors based on their unspecific accumulation in liver. More and more works are being published on potential applications of magnetic nanoparticles in diagnostics and therapy. But the interaction between magnetic nanoparticles and human cells at the subcellular level is only now beginning to be studied and more basic research is needed in this field. This work studies the interaction between carbohydrate functionalized gold-coated magnetite nanoparticles and C33 tumoural human cells as the first step towards the in vivo application of these nanoparticles. The uptake of this magnetic material follows a similar trend to that described for other nanoparticles. The intracellular fate of these nanoparticles once internalized has been unveiled, and an automatic methodology for the analysis of co-localization data has also been tested and compared to a more classic approach. These results can help in the optimization of the design of magnetic nanoparticles depending on their final application.

PET imaging with multimodal upconversion nanoparticles

A series of new upconversion nanoparticles have been functionalised with tumour-targeting molecules and metal chelates, prepared following standard peptidic and thiol chemistry. The targeting strategy has been delivered via the αvβ3 integrin, which is a heterodimeric cell surface receptor that is up-regulated in a variety of cancers, such as melanoma and breast cancer. The well-known DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) motif allows coordination to the radionuclide (68)Ga. Radiolabelling experiments were optimised under relatively mild conditions, and are rare amongst nanoparticulate materials. In vivo application of these probes in mouse tumour models revealed their potential as specific cancer contrast agents for PET imaging.

RGD-targeted MnO nanoparticles as T1 contrast agents for cancer imaging – the effect of PEG length in vivo

As magnetic resonance imaging (MRI) contrast agents, T1 Gd3+ chelates are generally the preferred option for radiologists over T2 iron oxide nanoparticles. The main reason for the popularity of T1 agents is the easier interpretation of T1-weighted MR images. However, the chemical versatility of nanoparticulate platforms makes them ideal candidates for the next generation of targeted MRI contrast agents. In this context, we present herein the design and preparation of a nanoparticulate contrast agent based on MnO, which presents T1 contrast enhancement properties as well as nanoparticle formulation. Functionalization of MnO nanoparticles with the extensively studied RGD peptide was used to target tumours over-expressing the αvβ3 integrin. PEG (polyethylene glycol) molecules were used to increase the blood half-life of the nanoparticles in vivo, and the effect of different PEG lengths on the final contrast on MR images was investigated.

A step-heating procedure for the synthesis of high-quality FePt nanostars

A step-heating procedure was used to synthesize high quality FePt nanostars by confined decomposition of Fe(acac)2 and reduction of Pt(acac)2.