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.

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.

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.