Biocompatible Peptide-Coated Ultrasmall Superparamagnetic Iron Oxide Nanoparticles for In Vivo Contrast-Enhanced Magnetic Resonance Imaging.

The biocompatibility and performance of reagents for in vivo contrast-enhanced magnetic resonance imaging (MRI) are essential for their translation to the clinic. The quality of the surface coating of nanoparticle-based MRI contrast agents, such as ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs), is critical to ensure high colloidal stability in biological environments, improved magnetic performance, and dispersion in circulatory fluids and tissues. Herein, we report the design of a library of 21 peptides and ligands and identify highly stable self-assembled monolayers on the USPIONs' surface. A total of 86 different peptide-coated USPIONs are prepared and selected using several stringent criteria, such as stability against electrolyte-induced aggregation in physiological conditions, prevention of nonspecific binding to cells, and absence of cellular toxicity and contrast-enhanced in vivo MRI. The bisphosphorylated peptide 2PG-S*VVVT-PEG4-ol provides the highest biocompatibility and performance for USPIONs, with no detectable toxicity or adhesion to live cells. The 2PG-S*VVVT-PEG4-ol-coated USPIONs show enhanced magnetic resonance properties, r1 (2.4 mM-1·s-1) and r2 (217.8 mM-1·s-1) relaxivities, and greater r2/ r1 relaxivity ratios (>90) when compared to those of commercially available MRI contrast agents. Furthermore, we demonstrate the utility of 2PG-S*VVVT-PEG4-ol-coated USPIONs as a T2 contrast agent for in vivo MRI applications. High contrast enhancement of the liver is achieved as well as detection of liver tumors, with significant improvement of the contrast-to-noise ratio of tumor-to-liver contrast. It is envisaged that the reported peptide-coated USPIONs have the potential to allow for the specific targeting of tumors and hence early detection of cancer by MRI.

Characterizing Self-Assembled Monolayers on Gold Nanoparticles.

A key aspect of nanoscience is to control the assembly of complex materials from a "bottom-up" approach. The self-assembly and self-organization of small ligands at the surface of nanoparticles represent a possible starting route for the preparation of (bio)nanomaterials with precise (bio)physical and (bio)chemical properties. However, surface characterization and elucidation of the structure-properties relationship, essential to envisioning such control, remain challenging and are often poorly investigated. This Topical Review aims to discuss different levels of surface characterization, giving an overview of the experimental and computational approaches that are used to provide insights into the self-assembled monolayer with molecular details. The methods and strategies discussed focus on the characterization of self-assembled monolayers at the gold nanoparticle surface, but most of them could also be applied to other types of nanoparticles.

Computational and Experimental Investigation of the Structure of Peptide Monolayers on Gold Nanoparticles.

The self-assembly and self-organization of small molecules on the surface of nanoparticles constitute a potential route toward the preparation of advanced proteinlike nanosystems. However, their structural characterization, critical to the design of bionanomaterials with well-defined biophysical and biochemical properties, remains highly challenging. Here, a computational model for peptide-capped gold nanoparticles (GNPs) is developed using experimentally characterized Cys-Ala-Leu-Asn-Asn (CALNN)- and Cys-Phe-Gly-Ala-Ile-Leu-Ser-Ser (CFGAILSS)-capped GNPs as a benchmark. The structure of CALNN and CFGAILSS monolayers is investigated using both structural biology techniques and molecular dynamics simulations. The calculations reproduce the experimentally observed dependence of the monolayer secondary structure on the peptide capping density and on the nanoparticle size, thus giving us confidence in the model. Furthermore, the computational results reveal a number of new features of peptide-capped monolayers, including the importance of sulfur movement for the formation of secondary structure motifs, the presence of water close to the gold surface even in tightly packed peptide monolayers, and the existence of extended 2D parallel ß-sheet domains in CFGAILSS monolayers. The model developed here provides a predictive tool that may assist in the design of further bionanomaterials.

Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding.

Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages' pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages' core and low non-specific binding to the cages' outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage's core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently available approaches and provides a new route to design tailored and well-controlled hybrid nanoparticles.

Photothermally responsive gold nanoparticle conjugated polymer-grafted porous hollow silica nanocapsules.

Polymer-grafted porous hollow silica nanoparticles prepared by reversible addition-fragmentation chain transfer polymerisation have an upper critical solution temperature of 45 °C. Conjugation of 5 nm gold nanoparticles onto polymer-grafted porous hollow silica nanoparticles enables remarkable specific photothermally-induced controlled release of encapsulated Rhodamine B by laser-stimulation at physiological temperature.

Targeting Cell Membrane Lipid Rafts by Stoichiometric Functionalization of Gold Nanoparticles With a Sphingolipid-Binding Domain Peptide.

A non-membrane protein-based nanoparticle agent for the tracking of lipid rafts on live cells is produced by stoichiometric functionalization of gold nanoparticles with a previously characterized sphingolipid- and cell membrane microdomain-binding domain peptide (SBD). The SBD peptide is inserted in a self-assembled monolayer of peptidol and alkane thiol ethylene glycol, on gold nanoparticles surface. The stoichiometric functionalization of nanoparticles with the SBD peptide, essential for single molecule tracking, is achieved by means of non-affinity nanoparticle purification. The SBD-nanoparticles have remarkable long-term resistance to electrolyte-induced aggregation and ligand-exchange and have no detectable non-specific binding to live cells. Binding and diffusion of SBD-nanoparticles bound to the membrane of live cells is measured by real-time photothermal microscopy and shows the dynamics of sphingolipid-enriched microdomains on cells membrane, with evidence for clustering, splitting, and diffusion over time of the SBD-nanoparticle labeled membrane domains. The monofunctionalized SBD-nanoparticle is a promising targeting agent for the tracking of lipid rafts independently of their protein composition and the labelling requires no prior modification of the cells. This approach has potential for further functionalization of the particles to manipulate the organization of, or targeting to microdomains that control signaling events and thereby lead to novel diagnostics and therapeutics.

Designing non-native iron-binding site on a protein cage for biological synthesis of nanoparticles.

In biomineralization processes, a supramolecular organic structure is often used as a template for inorganic nanomaterial synthesis. The E2 protein cage derived from Geobacillus stearothermophilus pyruvate dehydrogenase and formed by the self-assembly of 60 subunits, has been functionalized with non-native iron-mineralization capability by incorporating two types of iron-binding peptides. The non-native peptides introduced at the interior surface do not affect the self-assembly of E2 protein subunits. In contrast to the wild-type, the engineered E2 protein cages can serve as size- and shape-constrained reactors for the synthesis of iron nanoparticles. Electrostatic interactions between anionic amino acids and cationic iron molecules drive the formation of iron oxide nanoparticles within the engineered E2 protein cages. The work expands the investigations on nanomaterial biosynthesis using engineered host-guest encapsulation properties of protein cages.

Ferritin-templated synthesis and self-assembly of Pt nanoparticles on a monolithic porous graphene network for electrocatalysis in fuel cells.

The monolithic three-dimensional (3D) graphene network is used as the support for Pt nanoparticles (NPs) to fabricate an advanced 3D graphene-based electrocatalyst. Distinct from previous strategies, the monodispersed Pt NPs with ultrafine particle size (∼3 nm) are synthesized using ferritin protein nanocages as the template and subsequently self-assembled on the 3D graphene by leveraging on the hydrophobic interaction between the ferritin and the graphene. Following the self-assembly, the ferritins are removed, resulting in a stable Pt NP/3D graphene composite. The composite exhibits much enhanced electrocatalytic activity for methanol oxidation as compared with both Pt NP/chemically reduced graphene oxide (Pt/r-GO) and state-of-the-art Pt/C catalyst. The observed electrocatalytic activity also shows marked improvement over Pt/3D graphene prepared by pulse electrodeposition of Pt. This study demonstrates that protein nanocage templating and assembly are promising strategies for the fabrication of functional composites in catalysis and fuel cell applications.

Chemical cross-linkers for protein structure studies by mass spectrometry.

The cross-linking approach combined with MS for protein structure determination is one of the most striking examples of multidisciplinary success. Indeed, it has become clear that the bottleneck of the method was the detection and the identification of low-abundance cross-linked peptides in complex mixtures. Sample treatment or chromatography separation partially addresses these issues. However, the main problem comes from over-represented unmodified peptides, which do not yield any structural information. A real breakthrough was provided by high mass accuracy measurement, because of the outstanding technical developments in MS. This improvement greatly simplified the identification of cross-linked peptides, reducing the possible combinations matching with an observed m/z value. In addition, the huge amount of data collected has to be processed with dedicated software whose role is to propose distance constraints or ideally a structural model of the protein. In addition to instrumentation and algorithms efficiency, significant efforts have been made to design new cross-linkers matching all the requirements in terms of reactivity and selectivity but also displaying probes or reactive systems facilitating the isolation, the detection of cross-links, or the interpretation of MS data. These chemical features are reviewed and commented on in the light of the more recent strategies.

Solid-Phase Cross-Linking (SPCL): a new tool for protein structure studies.

A wide range of chemical reagents are available to study the protein-protein interactions or protein structures. After reaction with such chemicals, covalently modified proteins are digested, resulting in shorter peptides that are analyzed by mass spectrometry (MS). Used especially when NMR of X-ray data are lacking, this methodology requires the identification of modified species carrying relevant information, among the unmodified peptides. To overcome the drawbacks of existing methods, we propose a more direct strategy relying on the synthesis of solid-supported cleavable monofunctional reagents and cross-linkers that react with proteins and that selectively release, after protein digestion and washings, the modified peptide fragments ready for MS analysis. Using this Solid-Phase Cross-Linking (SPCL) strategy, only modified sequences are analyzed and consistent data can be easily obtained since the signals of interest are not masked or suppressed by over-represented unmodified materials.

A new generation of cross-linkers for selective detection by MALDI MS.

We designed a new cross-linker bearing a CHCA moiety. The use of the CHCA-tagged cross-linker JMV 3378 in conjunction with a neutral MALDI matrix alpha-cyano-4-hydroxycinnamic methyl ester enabled specific signal enhancement in MALDI-TOF MS of cross-link containing peptides. Discrimination between modified and non-modified peptides can be achieved by comparison of two spectra, one using CHCA and the other using the alpha-cyano-4-hydroxycinnamic methyl ester matrix. The methodology was validated using cytochrome c and apo-myoglobine as model proteins.

MSX-3D: a tool to validate 3D protein models using mass spectrometry.

MOTIVATION: The technique of chemical cross-linking followed by mass spectrometry has proven to bring valuable information about the protein structure and interactions between proteic subunits. It is an effective and efficient way to experimentally investigate some aspects of a protein structure when NMR and X-ray crystallography data are lacking. RESULTS: We introduce MSX-3D, a tool specifically geared to validate protein models using mass spectrometry. In addition to classical peptides identifications, it allows an interactive 3D visualization of the distance constraints derived from a cross-linking experiment. AVAILABILITY: Freely available at http://proteomics-pbil.ibcp.fr