No abstract

Metal nanoclusters (NCs) are considered ideal nanomaterials for biological applications owing to their strong photoluminescence (PL), excellent photostability, and good biocompatibility. This study presents a simple and versatile strategy to design proteins, via incorporation of a di‐histidine cluster coordination site, for the sustainable synthesis and stabilization of metal NCs with different metal composition. The resulting protein‐stabilized metal NCs (Prot‐NCs) of gold, silver, and copper are highly photoluminescent and photostable, have a long shelf life, and are stable under physiological conditions. The biocompatibility of the clusters was demonstrated in cell cultures in which Prot‐NCs showed efficient cell internalization without affecting cell viability or losing luminescence. Moreover, the approach is translatable to other proteins to obtain Prot‐NCs for various biomedical applications such as cell imaging or labeling.

Multidimensional kinetic analysis of immobilized enzymes is essential to understand the enzyme functionality at the interface with solid materials. However, spatiotemporal kinetic characterization of heterogeneous biocatalysts on a microscopic level and under operando conditions has been rarely approached. As a case study, we selected self-sufficient heterogeneous biocatalysts where His-tagged cofactor-dependent enzymes (dehydrogenases, transaminases, and oxidases) are co-immobilized with their corresponding phosphorylated cofactors [nicotinamide adenine dinucleotide phosphate (NAD(P)H), pyridoxal phosphate (PLP), and flavin adenine dinucleotide (FAD)] on porous agarose microbeads coated with cationic polymers. These self-sufficient systems do not require the addition of exogenous cofactors to function, thus avoiding the extensive use of expensive cofactors. To comprehend the microscopic kinetics and thermodynamics of self-sufficient systems, we performed fluorescence recovery after photobleaching measurements, time-lapse fluorescence microscopy, and image analytics at both single-particle and intraparticle levels. These studies reveal a thermodynamic equilibrium that rules out the reversible interactions between the adsorbed phosphorylated cofactors and the polycations within the pores of the carriers, enabling the confined cofactors to access the active sites of the immobilized enzymes. Furthermore, this work unveils the relationship between the apparent Michaelis–Menten kinetic parameters and the enzyme density in the confined space, eliciting a negative effect of molecular crowding on the performance of some enzymes. Finally, we demonstrate that the intraparticle apparent enzyme kinetics are significantly affected by the enzyme spatial organization. Hence, multiscale characterization of immobilized enzymes serves as an instrumental tool to better understand the in operando functionality of enzymes within confined spaces.

The zeta-potential of PSS/PAH and PSS/PDADMAC coated silica particles was studied in the presence of ClO4(-) and H2PO4(-) salts. In the presence of ClO4(-), layer-by-layer (LbL) coated silica particles with PDADMAC as the top layer show a reversal in the surface charge with increasing salt concentration but remain positive in phosphate solutions. LbL particles with PAH as the top layer become, however, negative in the presence of H2PO4(-) but retain their positive charge in the presence of ClO4(-). Charge reversal was explained by specific interaction of ClO4(-) ions with the quaternary amine groups and of H2PO4(-) with the primary amines through hydrogen bonding. Atomic force microscopy (AFM) and quartz crystal microbalance with dissipation (QCM-D) were employed to study the corresponding layer stability on planar surfaces.

Remyelination is a key aspect in multiple sclerosis pathology and a special effort is being made to promote it. However, there is still no available treatment to regenerate myelin and several strategies are being scrutinized. Myelination is naturally performed by oligodendrocytes and microRNAs have been postulated as a promising tool to induce oligodendrocyte precursor cell differentiation and therefore remyelination. Herein, DSPC liposomes and PLGA nanoparticles were studied for miR-219a-5p encapsulation, release and remyelination promotion. In parallel, they were compared with biologically engineered extracellular vesicles overexpressing miR-219a-5p. Interestingly, extracellular vesicles showed the highest oligodendrocyte precursor cell differentiation levels and were more effective than liposomes and polymeric nanoparticles crossing the blood–brain barrier. Finally, extracellular vesicles were able to improve EAE animal model clinical evolution. Our results indicate that the use of extracellular vesicles as miR-219a-5p delivery system can be a feasible and promising strategy to induce remyelination in multiple sclerosis patients.

Herein we describe the results of a combined theoretical and spectroscopic investigation into the design of a simple molecular system intended to act as a memory storage bank. The main operating principle revolves around the two-electron reduction of an aryl disulfide bond. Addition of the first electron leads to elongation of the S-S bond but it breaks only if there is accompanying protonation. Adding a second electron causes S-S bond cleavage, with or without protonation. The structural changes have been assessed by way of quantum chemical calculations and molecular dynamics simulations. Electrochemical studies show that the two-electron reduced product can be re-oxidised at mildly anodic potentials and the cycle can be repeated many times. Both theory and experiment point towards pronounced potential inversion whereby the second reduction potential lies at a significantly more positive potential than that for the first step. Computer simulations of the cyclic voltammograms give rise to numerical values for the reduction potentials that are in quite good agreement with the computed values and also allow determination of the electrochemical rate constants and transfer coefficients. Accurate simulation of the experimental data can be realised only if one proton accompanies the second reduction step. The possibility to design an effective molecular-scale memory device around this system is discussed briefly.

In this communication, polyanionic poly(potassium 3‐sulfopropyl methacrylate) (PSPM) brushes were switched from hydrophilic to hydrophobic by exchange of the counter cations. First, poly(potassium 3‐sulfopropyl methacrylate) brushes were grown by means of atom transfer radical polymerization (ATRP) from thiol monolayers of initiating ω‐mercaptoundecyl bromoisobutyrate and mixed monolayers of thiol initiator and 1‐undecanothiol (blank thiol) attached to gold surfaces. The kinetics of the polymerization reaction were followed by means of the quartz microbalance technique with dissipation (QCM‐D). The collapse of PSPM brushes in the presence of cationic surfactants like quaternary ammonium salts (tetraethylammonium bromide, hexadecyltrimethylammonium bromide) and imidazolium salts (1‐dodecyl‐3‐methylimidazolium bromide, 1H,1H,2H,2H‐perfluoro‐1‐decyl‐3‐methylimidazolium bromide) was shown by QCM‐D. Water contact angle measurements proved that the wettability of the surface could be tuned reversibly from hydrophilic values (<30 °) to hydrophobic ones (>85 °).magnified image

Fabrication of protein-based biomaterials is an arduous and time-consuming procedure with multiple steps. In this work, we describe a portable toolkit that integrates both cell-free protein synthesis (CFPS) and protein immobilization in one pot just by mixing DNA, solid materials, and a CFPS system. We have constructed a modular set of plasmids that fuse the N-terminus of superfolded green fluorescent protein (sGFP) with different peptide tags (poly(6X)Cys, poly(6X)His, and poly(6X)Lys), which drive the immobilization of the protein on the tailored material (agarose beads with different functionalities, gold nanorods, and silica nanoparticles). This system also enables the incorporation of azide-based amino acids into the nascent protein for its selective immobilization through copper-free click reactions. Finally, this technology has been expanded to the synthesis and immobilization of enzymes and antibody-binding proteins for the fabrication of functional biomaterials. This synthetic biological platform has emerged as a versatile tool for on-demand fabrication of therapeutic, diagnostic, and sensing biomaterials.