Chemically Enhanced Immunogenicity of Bacteria by Supramolecular Functionalization with an Adjuvant.

Many pathogens blunt immune responses because they lack immunogenic structural features, which typically results in disease. Here, we show evidence suggesting that pathogen immunogenicity can be chemically enhanced. Using supramolecular host-guest chemistry, we complexed onto the surface of a poorly immunogenic bacterium (Staphylococcus aureus) a TLR7 agonist-based adjuvant. "Adjuvanted" bacteria were readily recognized by macrophages and induced a more pro-inflammatory immunophenotype. Future applications of this concept could yield treatment modalities that bolster the immune system's response to pathogenic microbes.

Nerve Targeting via Myelin Protein Zero and the Impact of Dimerization on Binding Affinity.

BACKGROUND: Surgically induced nerve damage is a common but debilitating side effect. By developing tracers that specifically target the most abundant protein in peripheral myelin, namely myelin protein zero (P0), we intend to support fluorescence-guided nerve-sparing surgery. To that end, we aimed to develop a dimeric tracer that shows a superior affinity for P0. METHODS: Following truncation of homotypic P0 protein-based peptide sequences and fluorescence labeling, the lead compound Cy5-P0101-125 was selected. Using a bifunctional fluorescent dye, the dimeric Cy5-(P0101-125)2 was created. Assessment of the performance of the mono- and bi-labeled compounds was based on (photo)physical evaluation. This was followed by in vitro assessment in P0 expressing Schwannoma cell cultures by means of fluorescence confocal imaging (specificity, location of binding) and flow cytometry (binding affinity; KD). RESULTS: Dimerization resulted in a 1.5-fold increase in affinity compared to the mono-labeled counterpart (70.3 +/- 10.0 nM vs. 104.9 +/- 16.7 nM; p = 0.003) which resulted in a 4-fold increase in staining efficiency in P0 expressing Schwannoma cells. Presence of two targeting vectors also improves a pharmacokinetics of labeled compounds by lowering serum binding and optical stability by preventing dye stacking. CONCLUSIONS: Dimerization of the nerve-targeting peptide P0101-125 proves a valid strategy to improve P0 targeting.

Au3+-Induced gel network formation of proteins.

The formation of protein gel networks in aqueous systems is a result of protein intermolecular interactions after an energy input, like heating. In this research, we report that a redox reaction between Au3+ ions and proteins can also lead to the formation of a protein gel network. Amino acids, like cysteine and tyrosine, get oxidized and form covalent bonds with neighboring protein molecules, while Au3+ ions get reduced to Au+ and Au0, nucleate and form gold nanoparticles. The protein gel network formation occurs within 2 h at room temperature and can be tuned by varying Au3+/protein ratio and accelerated by increasing the incubation temperature. The proposed Au3+-induced gel network formation was applied to different proteins, like egg yolk high-density lipoprotein, bovine serum albumin and whey protein. This research opens new insights for the investigation of the metal-protein interactions and may aid in the design of novel hybrid-soft nanocomposite materials.

Dendrimicelles with pH-controlled aggregation number of core-dendrimers and stability.

We present a simple way to build up well-controlled coacervate-core dendrimicelles by assembly of anionic PAMAM dendrimers with a cationic-neutral diblock copolymer. Upon increasing pH, the formation of micellar structures shows constant size but the number of dendrimer molecules incorporated in one micelle decreases, following the charge stoichiometry formation rules; concomitantly, the salt stability increases. This study shows the straightforward tuning of macromolecular core-units and related micelle properties.

Response of metal-coordination-based polyelectrolyte complex micelles to added ligands and metals.

Polyelectrolyte complex based micelles have attracted significant attention due to their potential regarding bio-applications. Although the morphology and functions have been studied extensively, dynamic properties, particularly component exchange with other surrounding molecules, have remained elusive to date. Here, we show how micelles based on metal-ligand coordination complex coacervate-core micelles (M-C3Ms) respond to addition of extra ligand and metal ions. The micelles are prepared from a polycationic-neutral diblock copolymer and an anionic coordination polyelectrolyte, which is obtained by coordination between metal ions (lanthanides Ln3+ and Zn2+) and a bis-ligand (LEO) containing two dipicolinic acid (DPA) groups connected by a tetra-ethylene oxide spacer (4EO). Our findings show that the bis-ligand LEO is essential for the growth of coordination polymers and consequently the formation of micelles, leading to equilibrium structures with the same micellar composition and structure independent of the order of mixing. In other words, adding single DPA has no effect on the formed M-C3Ms. As for metal exchange, we find that added Zn2+ can replace some of the Ln3+ from Ln-C3Ms, leading to a hybrid coordination structure with both Ln3+ and Zn2+. We find that component exchange occurs in these coordination polyelectrolyte micelles, but it is more favorable in the direction of replacing the weak binding components with strong ones. Hence, the designed M-C3Ms based on the strong binding components, such as Ln-C3Ms, shall be relatively stable in biological surroundings, paving the way for the application of such particles as bio-imaging probes.

COvalent monolayer patterns in Microfluidics by PLasma etching Open Technology - COMPLOT.

Plasma microcontact patterning (PµCP) and replica molding were combined to make PDMS/glass microfluidic devices with ß-cyclodextrin (ß-CD) patterns attached covalently on the glass surface inside microchannels. The supramolecular reactivity, reusability and association constant of ß-CD with Cy5-Ad2 was tested by analyzing signal-to-noise ratios of patterns vs. spacing with fluorescence microscopy.

Characterizing the structure of aerobic granular sludge using ultra-high field magnetic resonance.

Despite aerobic granular sludge wastewater treatment plants operating around the world, our understanding of internal granule structure and its relation to treatment efficiency remains limited. This can be attributed in part to the drawbacks of time-consuming, labor-intensive, and invasive microscopy protocols which effectively restrict samples sizes and may introduce artefacts. Time-domain nuclear magnetic resonance (NMR) allows non-invasive measurements which describe internal structural features of opaque, complex materials like biofilms. NMR was used to image aerobic granules collected from five full-scale wastewater treatment plants in the Netherlands and United States, as well as laboratory granules and control beads. T1 and T2 relaxation-weighted images reveal heterogeneous structures that include high- and low-density biofilm regions, water-like voids, and solid-like inclusions. Channels larger than approximately 50 µm and connected to the bulk fluid were not visible. Both cluster and ring-like structures were observed with each granule source having a characteristic structural type. These structures, and their NMR relaxation behavior, were stable over several months of storage. These observations reveal the complex structures within aerobic granules from a range of sources and highlight the need for non-invasive characterization methods like NMR to be applied in the ongoing effort to correlate structure and function.

Regulation of Plasmodium sporozoite motility by formulation components.

BACKGROUND: The protective efficacy of the most promising malaria whole-parasite based vaccine candidates critically depends on the parasite's potential to migrate in the human host. Key components of the parasite motility machinery (e.g. adhesive proteins, actin/myosin-based motor, geometrical properties) have been identified, however the regulation of this machinery is an unknown process. METHODS: In vitro microscopic live imaging of parasites in different formulations was performed and analysed, with the quantitative analysis software SMOOTIn vitro, their motility; their adherence capacity, movement pattern and velocity during forward locomotion. RESULTS: SMOOTIn vitro enabled the detailed analysis of the regulation of the motility machinery of Plasmodium berghei in response to specific (macro)molecules in the formulation. Albumin acted as an essential supplement to induce parasite attachment and movement. Glucose, salts and other whole serum components further increased the attachment rate and regulated the velocity of the movement. CONCLUSIONS: Based on the findings can be concluded that a complex interplay of albumin, glucose and certain salts and amino acids regulates parasite motility. Insights in parasite motility regulation by supplements in solution potentially provide a way to optimize the whole-parasite malaria vaccine formulation.

Sorting of Molecular Building Blocks from Solution to Surface.

We demonstrate that molecular gradients on an organic monolayer is formed by preferential binding of ruthenium complexes from solutions also containing equimolar amounts of isostructural osmium complexes. The monolayer consists of a nanometer-thick assembly of 1,3,5-tris(4-pyridylethenyl)benzene (TPEB) covalently attached to a silicon or metal-oxide surface. The molecular gradient of ruthenium and osmium complexes is orthogonal to the surface plane. This gradient propagates throughout the molecular assembly with thicknesses over 30 nm. Using other monolayers consisting of closely related organic molecules or metal complexes results in the formation of molecular assemblies having an homogeneous and equimolar distribution of ruthenium and osmium complexes. Spectroscopic and computational studies revealed that the geometry of the complexes and the electronic properties of their ligands are nearly identical. These subtle differences cause the isostructural osmium and ruthenium complexes to pack differently on modified surfaces as also demonstrated in crystals grown from solution. The different packing behavior, combined with the organic monolayer significantly contributes to the observed differences in chemical composition on the surface.

Illumination of Nanoliter-NMR Spectroscopy Chips for Real-Time Photochemical Reaction Monitoring.

We report the use of a small-volume nuclear-magnetic-resonance (NMR)-spectroscopy device with integrated fiber-optics for the real-time detection of UV-vis-light-assisted chemical reactions. An optical fiber is used to guide the light from LEDs or a laser diode positioned safely outside the magnet toward the 25 nL detection volume and placed right above the microfluidic channel, irradiating the transparent back of the NMR chip. The setup presented here overcomes the limitations of conventional NMR systems for in situ UV-vis illumination, with the microchannel permitting efficient light penetration even in highly concentrated solutions, requiring lower-power light intensities, and enabling high photon flux. The efficacy of the setup is illustrated with two model reactions activated at different wavelengths.

Cyclodextrin-based complex coacervate core micelles with tuneable supramolecular host-guest, metal-to-ligand and charge interactions.

Micelles have been recognized as versatile platforms for different biomedical applications, from bioimaging to drug delivery. Complex coacervate core micelles present great advantages compared to traditional micelles, however controlling the number of charges per core-unit and the stability is still a challenge. We here present cyclodextrin-based complex coacervate core micelles where the charge per core-unit can be straightforwardly tuned by cyclodextrin host-guest interactions. By varying the ratio between two adamantane guest molecules, 1-adamantanecarboxylic acid and 1,3-adamantanediacetic acid, the charge of the monomeric core-units can be finely tuned from 6- to 9-. By adding an adamantane bislinker, monomeric core-units can be combined together in dimeric and polymeric structures, increasing the micelles' stability. The orthogonal supramolecular host-guest and coordination-chemistry allows for well-controlled cyclodextrin-based complex coacervate core micelles that offer a versatile platform for designing future, e.g., responsive systems.

Supramolecular Virus-Like Nanorods by Coassembly of a Triblock Polypeptide and Reversible Coordination Polymers.

We investigate a new case of a self-assembly-stimulated self-assembly in which a triblock polypeptide is combined with a anionic coordination polymer of a dipicolinic acid bis-ligand, and d- or f- block metal ions like ZnII or EuIII . The polypeptide not only has a silk-like domain that can fold and stack, but also a C-terminal cationic sequence by which it can interact with the supramolecular (coordination) polyanion. In the presence of all three ingredients (polypeptide, bis-ligand, and metal ions), we observe the initiation and slow growth of well-defined metal-containing nanorods of up to 150 nm in length, proving that self-assembly of the polypeptide is triggered by the self-assembly of the coordination polyelectrolyte and vice versa. The particles, which have a striking resemblance to rod-like viruses, are stable up to 1.2 m NaCl, and can be made fluorescent when lanthanides like EuIII are used, showing the potential to exploit functional properties and applications of virus-like supramolecular structures.

Revealing and tuning the core, structure, properties and function of polymer micelles with lanthanide-coordination complexes.

Controlling self-assembly processes is of great interest in various fields where multifunctional and tunable materials are designed. We here present the versatility of lanthanide-complex-based micelles (Ln-C3Ms) with tunable coordination structures and corresponding functions (e.g. luminescence and magnetic relaxation enhancement). Micelles are prepared by charge-driven self-assembly of a polycationic-neutral diblock copolymer and anionic coordination complexes formed by Ln(III) ions and the bis-ligand L2EO4, which contains two dipicolinic acid (DPA) ligand groups (L) connected by a tetra-ethylene oxide spacer (EO4). By varying the DPA/Ln ratio, micelles are obtained with similar size but with different stability, different aggregation numbers and different oligomeric and polymeric lanthanide(III) coordination structures in the core. Electron microscopy, light scattering, luminescence spectroscopy and magnetic resonance relaxation experiments provide an unprecedented detailed insight into the core structures of such micelles. Concomitantly, the self-assembly is controlled such that tunable luminescence or magnetic relaxation with Eu-C3Ms, respectively, Gd-C3Ms is achieved, showing potential for applications, e.g. as contrast agents in (pre)clinical imaging. Considering the various lanthanide(III) ions have unique electron configurations with specific physical chemical properties, yet very similar coordination chemistry, the generality of the current coordination-structure based micellar design shows great promise for development of new materials such as, e.g., hypermodal agents.

Determination of Kinetic Parameters within a Single Nonisothermal On-Flow Experiment by Nanoliter NMR Spectroscopy.

Conventional methods to determine the kinetic parameters for a certain reaction require multiple, separate isothermal experiments, resulting in time- and material-consuming processes. Here, an approach to determine the kinetic information within a single nonisothermal on-flow experiment is presented, consuming less than 10 µmol of reagents and having a total measuring time of typically 10 min. This approach makes use of a microfluidic NMR chip hyphenated to a continuous-flow microreactor and is based on the capabilities of the NMR chip to analyze subnanomole quantities of material in the 25 nL detection volume. Importantly, useful data are acquired from the microreactor platform in specific isothermal and nonisothermal frames. A model fitting the experimental data enables rapid determination of kinetic parameters, as demonstrated for a library of isoxazole and pyrazole derivatives.

Lanthanide-Dipicolinic Acid Coordination Driven Micelles with Enhanced Stability and Tunable Function.

Lanthanide-incorporated polymer micelles have been prepared driven by the lanthanide-dipicolinic acid (Ln-DPA) coordination. The terdentate DPA ligand is grafted to the PVP block of a diblock copolymer poly(4-vinylpyridine)-b-poly(ethylene oxide) (P4VP48-b-PEO193). Upon addition of Eu(III) ions to a solution of the DPA16-g-P4VP48-b-PEO193 block copolymer, intermolecular cross-links form and the ligand-carrying blocks assemble, leading to the formation of micelles, stabilized by the hydrophilic PEO blocks. The DPA exhibits a dual function in this study. First, the chelate group strongly coordinates to Eu(III) in a three to one ratio, and leads to high stability of the formed micelles, as proven by light scattering and luminescence spectroscopy. Second, DPA acts as an antenna that transfers energy to the Eu(III) ion and dramatically enhances the luminescence emission. The Eu(III) emission is moreover most sensitive for local environment and allows to shine light on the internal structure of this class of self-assembled 36 nm size soft nanoparticles. With the same strategy gadolinium(III) can be incorporated providing micelles which show enhanced magnetic relaxation rates. Micelles capping a mixture of Eu(III) and Gd(III) show both enhanced luminescence emission and magnetic relaxation rates, and the functions can be tuned by regulating the mixing ratio of Eu(III) and Gd(III), showing great potential for developing multimodal imaging agents for diagnostic as well as therapeutic applications.

MMP-2/9-Specific Activatable Lifetime Imaging Agent.

Optical (molecular) imaging can benefit from a combination of the high signal-to-background ratio of activatable fluorescence imaging with the high specificity of luminescence lifetime imaging. To allow for this combination, both imaging techniques were integrated in a single imaging agent, a so-called activatable lifetime imaging agent. Important in the design of this imaging agent is the use of two luminophores that are tethered by a specific peptide with a hairpin-motive that ensured close proximity of the two while also having a specific amino acid sequence available for enzymatic cleavage by tumor-related MMP-2/9. Ir(ppy)3 and Cy5 were used because in close proximity the emission intensities of both luminophores were quenched and the influence of Cy5 shortens the Ir(ppy)3 luminescence lifetime from 98 ns to 30 ns. Upon cleavage in vitro, both effects are undone, yielding an increase in Ir(ppy)3 and Cy5 luminescence and a restoration of Ir(ppy)3 luminescence lifetime to 94 ns. As a reference for the luminescence activation, a similar imaging agent with the more common Cy3-Cy5 fluorophore pair was used. Our findings underline that the combination of enzymatic signal activation with lifetime imaging is possible and that it provides a promising method in the design of future disease specific imaging agents.

Controlling the number of dendrimers in dendrimicelle nanoconjugates from 1 to more than 100.

Herein, we present a facile strategy to controllably build up dendrimicelles by self-assembly of anionic PAMAM dendrimers with cationic-neutral diblock copolymers. We present a systematic study incorporating a full decade (0-9) of dendrimer generations, tracing the gradual variation from aggregates (G0 and G1) to self-assembled micelles (G2-G8), and an unidendrimer micelle structure (G9) by different scattering techniques (light and X-ray). The formed micelles (G2-G9) are spherical in shape with a hydrodynamic radius of about 25 nm. Interestingly, the micellar size, structure and number of incorporated block copolymers are independent of the dendrimer generation (for G2 to G9), while the aggregation number of the dendrimers decreases from 108 to 1, and the stability of the micelles increases upon an increase in the dendrimer generation. Moreover, the micelles with lower generation dendrimers transform from spherical into worm-like structures upon an increase in the positive charge fraction (excess polymers) or ionic strength, while micelles with higher generation dendrimers do not show such a transition. This differential behavior is in-line with a change from a flexible configuration into rigid globular nanoparticles with increasing dendrimer generation. The reported systematic investigation of dendrimicelles comprising a full decade of dendrimer generations provides the basis for versatile strategies focused on building up new (multi)functional materials, e.g. by packing multiple types of dendrimers with different functional groups or encapsulated cargos controllably within one micelle.

Nonlinear amplification of a supramolecular complex at a multivalent interface.

Competition with a monovalent cyclodextrin host (blue cones) in solution drives the multivalent binding of a Eu(3+) complex and a sensitizer molecule to cyclodextrin monolayers through a nonlinear self-assembly process. Adamantyl groups (light-blue spheres) are attached to the EDTA ligand (black) and the antenna molecule (orange), which has a carboxylate group for coordination to the Eu(3+) ion (yellow or red in free or complexed form, respectively).

Multinuclear 1D and 2D NMR with 19F-Photo-CIDNP hyperpolarization in a microfluidic chip with untuned microcoil.

Nuclear Magnetic Resonance (NMR) spectroscopy is a most powerful molecular characterization and quantification technique, yet two major persistent factors limit its more wide-spread applications: poor sensitivity, and intricate complex and expensive hardware required for sophisticated experiments. Here we show NMR with a single planar-spiral microcoil in an untuned circuit with hyperpolarization option and capability to execute complex experiments addressing simultaneously up to three different nuclides. A microfluidic NMR-chip in which the 25 nL detection volume can be efficiently illuminated with laser-diode light enhances the sensitivity by orders of magnitude via photochemically induced dynamic nuclear polarization (photo-CIDNP), allowing rapid detection of samples in the lower picomole range (normalized limit of detection at 600 MHz, nLODf,600, of 0.01 nmol Hz1/2). The chip is equipped with a single planar microcoil operating in an untuned circuit that allows different Larmor frequencies to be addressed simultaneously, permitting advanced hetero-, di- and trinuclear, 1D and 2D NMR experiments. Here we show NMR chips with photo-CIDNP and broadband capabilities addressing two of the major limiting factors of NMR, by enhancing sensitivity as well as reducing cost and hardware complexity; the performance is compared to state-of-the-art instruments.

Chemical Composition, Biomolecular Analysis, and Nuclear Magnetic Resonance Spectroscopic Fingerprinting of Posidonia oceanica and Ascophyllum nodosum Extracts.

A detailed analysis of the elemental and molecular composition of Posidonia oceanica (PO) and Ascophyllum nodosum (AN) is presented. In particular, an in-depth study of the molecular identification via NMR spectroscopy of aqueous and organic extracts of PO and AN was carried out, exploiting 2D COSY and pseudo-2D DOSY data to aid in the assignment of peaks in complex 1D proton NMR spectra. Many metabolites were identified, such as carbohydrates, amino acids, organic acids, fatty acids, and polyphenols, with NMR complementing the characterization of the two species by standard elemental analysis, HPLC analysis, and colorimetric testing. For PO, different parts of the live plant (roots, rhizomes, and leaves) were analysed, as well as the residues of the dead plant which typically deposit along the coasts. The combination of the various studies made it possible to recognize bioactive compounds naturally present in the two plant species and, in particular, in the PO residues, opening the door for their possible recycling and use in, for example, fertilizer. Furthermore, NMR is proven to be a powerful tool for the metabolomic study of plant species as it allows for the direct identification of specific biomarkers as well as providing a molecular fingerprint of the plant variety.