Assessment of Culture Systems to Produce Bacterial Cellulose with a Kombucha Consortium.

Bacterial cellulose (BC) is an emerging material for high-end applications due to its biocompatibility and physicochemical characteristics. However, the scale-up production of this material is still expensive, with the culture medium constituting one-third of the total cost. Herein, four different media (yeast nitrogen base, YNB; Murashige and Skoog, MSO; black tea; and NPK fertilizer solution) were compared while using sucrose as an additional carbon source. The yields of BC were best for YNB and fertilizer with 0.37 and 0.34 gBC/gC respectively. These two were then compared using glucose as a carbon source, with improvements in the production of 29% for the fertilizer, while only an 8% increase for YNB was seen; however, as the carbon concentration increased with a fixed N concentration, the yield was lower but the rate of production of BC increased. The obtained BC films were sanitized and showed low molecular weight and all the expected cellulose characteristic FT-IR bands while SEM showed nanofibers around 0.1 µm. Compared to traditional methods for lab-scale production, the use of the fertilizer and the consortium represent benefits compared to traditional lab-scale BC culture methods such as a competitive cost (two times lower) while posing resilience and tolerance to stress conditions given that it is produced by microbial communities and not with a single strain. Additionally, the low molecular weight of the films could be of interest for certain coating formulations.

Quartz Crystal Microbalance Application and In Silico Studies to Characterize the Interaction of Bovine Serum Albumin with Plasma Polymerized Pyrrole Surfaces: Implications for the Development of Biomaterials.

Plasma polymerized pyrrole/iodine (PPPy/I) microparticles and bovine serum albumin (BSA) protein have shown interesting results in experimental models for the treatment of traumatic spinal cord injury. By studying the interaction between BSA and PPPy/I by a quartz crystal microbalance (QCM) and docking, we obtained important results to elucidate possible cellular interactions and promote the use of these polymers as biomaterials. These measurements were also used to characterize the adsorption process using an equilibrium constant. In addition, atomic force microscopy (AFM) was used to obtain images of the QCM surface sensors before and after BSA adsorption. Furthermore, we carried out molecular dynamics simulations and molecular docking to characterize the molecular recognition between BSA and the previously reported PPPy/I structure. For this study, we used two combinatorial models that have not been tested. Thus, we could determine the electrostatic (ΔGele) and nonelectrostatic (ΔGnonelec) components of the free binding energy (ΔGb). We demonstrated that BSA is adsorbed on PPPy/I with an adsorption constant of K = 24.35 µ-1 indicating high affinity. This observation combined with molecular docking and binding free energy calculations showed that the interaction between BSA and both combinatorial models of the PPPy structure is spontaneous.

Electrostatic interactions control the adsorption of extracellular vesicles onto supported lipid bilayers.

Communication between cells located in different parts of an organism is often mediated by membrane-enveloped nanoparticles, such as extracellular vesicles (EVs). EV binding and cell uptake mechanisms depend on the heterogeneous composition of the EV membrane. From a colloidal perspective, the EV membrane interacts with other biological interfaces via both specific and non-specific interactions, where the latter include long-ranged electrostatic and van der Waals forces, and short-ranged repulsive "steric-hydration" forces. While electrostatic forces are generally exploited in most EV immobilization protocols, the roles played by various colloidal forces in controlling EV adsorption on surfaces have not yet been thoroughly addressed. In the present work, we study the adsorption of EVs onto supported lipid bilayers (SLBs) carrying different surface charge densities using a combination of quartz crystal microbalance with dissipation monitoring (QCM-D) and confocal laser scanning microscopy (CLSM). We demonstrate that EV adsorption onto lipid membranes can be controlled by varying the strength of electrostatic forces and we theoretically describe the observed phenomena within the framework of nonlinear Poisson-Boltzmann theory. Our modelling results confirm the experimental observations and highlight the crucial role played by attractive electrostatics in EV adsorption onto lipid membranes. They furthermore show that simplified theories developed for model lipid systems can be successfully applied to the study of their biological analogues and provide new fundamental insights into EV-membrane interactions with potential use in developing novel EV separation and immobilization strategies.

Sodium Alginate/Chitosan Scaffolds for Cardiac Tissue Engineering: The Influence of Its Three-Dimensional Material Preparation and the Use of Gold Nanoparticles.

Natural biopolymer scaffolds and conductive nanomaterials have been widely used in cardiac tissue engineering; however, there are still challenges in the scaffold fabrication, which include enhancing nutrient delivery, biocompatibility and properties that favor the growth, maturation and functionality of the generated tissue for therapeutic application. In the present work, different scaffolds prepared with sodium alginate and chitosan (alginate/chitosan) were fabricated with and without the addition of metal nanoparticles and how their fabrication affects cardiomyocyte growth was evaluated. The scaffolds (hydrogels) were dried by freeze drying using calcium gluconate as a crosslinking agent, and two types of metal nanoparticles were incorporated, gold (AuNp) and gold plus sodium alginate (AuNp+Alg). A physicochemical characterization of the scaffolds was carried out by swelling, degradation, permeability and infrared spectroscopy studies. The results show that the scaffolds obtained were highly porous (>90%) and hydrophilic, with swelling percentages of around 3000% and permeability of the order of 1 × 10−8 m2. In addition, the scaffolds proposed favored adhesion and spheroid formation, with cardiac markers expression such as tropomyosin, troponin I and cardiac myosin. The incorporation of AuNp+Alg increased cardiac protein expression and cell proliferation, thus demonstrating their potential use in cardiac tissue engineering.

Design and use of model membranes to study biomolecular interactions using complementary surface-sensitive techniques.

Cellular membranes are complex structures and simplified analogues in the form of model membranes or biomembranes are used as platforms to understand fundamental properties of the membrane itself as well as interactions with various biomolecules such as drugs, peptides and proteins. Model membranes at the air-liquid and solid-liquid interfaces can be studied using a range of complementary surface-sensitive techniques to give a detailed picture of both the structure and physicochemical properties of the membrane and its resulting interactions. In this review, we will present the main planar model membranes used in the field to date with a focus on monolayers at the air-liquid interface, supported lipid bilayers at the solid-liquid interface and advanced membrane models such as tethered and floating membranes. We will then briefly present the principles as well as the main type of information on molecular interactions at model membranes accessible using a Langmuir trough, quartz crystal microbalance with dissipation monitoring, ellipsometry, atomic force microscopy, Brewster angle microscopy, Infrared spectroscopy, and neutron and X-ray reflectometry. A consistent example for following biomolecular interactions at model membranes is used across many of the techniques in terms of the well-studied antimicrobial peptide Melittin. The overall objective is to establish an understanding of the information accessible from each technique, their respective advantages and limitations, and their complementarity.

Highly viscoelastic films at the water/air interface: α-Cyclodextrin with anionic surfactants.

This work showcases the remarkable viscoelasticity of films consisting of α-cyclodextrin (α-CD) and anionic surfactants (S) at the water/air interface, the magnitude of which has not been observed in similar systems. The anionic surfactants employed are sodium salts of a homologous series of n-alkylsulfates (n = 8-14) and of dodecylsulfonate. Our hypothesis was that the very high viscoelasticity can be systematically related to the bulk and interfacial properties of the system. Through resolution of the bulk distribution of species using isothermal titration calorimetry, the high dilatational modulus is related to (α-CD)2:S1 inclusion complexes in the bulk with respect to both the bulk composition and temperature. Direct interfacial characterization of α-CD and sodium dodecylsulfate films at 283.15 K using ellipsometry and neutron reflectometry reveals that the most viscoelastic films consist of a highly ordered monolayer of 2:1 complexes with a minimum amount of any other component. The orientation of the complexes in the films and their driving force for adsorption are discussed in the context of results from molecular dynamics simulations. These findings open up clear potential for the design of new functional materials or molecular sensors based on films with specific mechanical, electrical, thermal, chemical, optical or even magnetic properties.

Mechanical Properties of DPPC-POPE Mixed Langmuir Monolayers.

The mechanical properties of lipid monolayers and their responses to shear and compression stresses play an important role in processes such as breathing and eye blinking. We studied the mechanical properties of Langmuir monolayers of a model mixture, composed of an unsaturated lipid, 1-palmitoyl-2-oleoyl-sn-glycero-phosphoethanolamine (POPE), and a saturated lipid, 1,2-dipalmitoyl-sn-glycero-phosphocholine (DPPC). We performed isothermal compressions and sinusoidal shear deformations of these mixed monolayers. Also, the different phases were observed with Brewster angle microscopy. We found that the mechanical behavior is affected by the miscibility of both lipids. In the two-phase region, the compression elastic modulus increases with the amount of the LC phase but does not follow the predictions of a simple effective medium model. The discrepancies arise from the fact that, upon compression, the domains grow at a rate faster than the compression rate but not fast enough to reach thermodynamic equilibrium. Before reaching the LC phase, domain percolation is observed and compression and shear moduli become equal to those of the pure LC phase. Most of the monolayers behave as viscoelastic fluids at the frequencies investigated. A minimum in the compression modulus and shear viscosity was observed for mixtures close to equimolar composition, with the minimum being accompanied by a change in domain shapes.

Immobilization effects on the photocatalytic activity of CdS quantum Dots-Horseradish peroxidase hybrid nanomaterials.

The potential use of hybrid nanomaterials based on inorganic optically active nanoparticles known as quantum dots (QDs) and horseradish peroxidase (HRP) has been proposed by several authors as light-controllable nanocatalyzers, moreover, the immobilization within or over silica based supports represents an advantage over bulk-dispersed systems. However, the implications of the immobilization of such hybrid photoactivatable catalyzing systems have not been clarified with detail. Here, we present a thorough study of the functional photoactive efficiency and recycling of immobilized CdS QDs and HRP systems with different configurations, immobilized over silanized silica quartz crystal microbalance (QCM) sensors, allowing an accurate measure of the immobilized mass of each component and its correlation with the initial reaction rate of conversion of Amplex Red (AR) to resorufin. As well, the conversion efficiency is compared between the different systems and also to non-immobilized QD-HRP complexed systems.

Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

In this study, the interaction between the flavonoid pelargonidin and dairy proteins: ß-lactoglobulin (ß-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both ß-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins.

Complex Behavior of Aqueous α-Cyclodextrin Solutions. Interfacial Morphologies Resulting from Bulk Aggregation.

The spontaneous aggregation of α-cyclodextrin (α-CD) molecules in the bulk aqueous solution and the interactions of the resulting aggregates at the liquid/air interface have been studied at 283 K using a battery of techniques: transmission electron microscopy, dynamic light scattering, dynamic surface tensiometry, Brewster angle microscopy, neutron reflectometry, and ellipsometry. We show that α-CD molecules spontaneously form aggregates in the bulk that grow in size with time. These aggregates adsorb to the liquid/air interface with their size in the bulk determining the adsorption rate. The material that reaches the interface coalesces laterally to form two-dimensional domains on the micrometer scale with a layer thickness on the nanometer scale. These processes are affected by the ages of both the bulk and the interface. The interfacial layer formed is not in fast dynamic equilibrium with the subphase as the resulting morphology is locked in a kinetically trapped state. These results reveal a surprising complexity of the parallel physical processes taking place in the bulk and at the interface of what might have seemed initially like a simple system.

Surface adsorption and bulk aggregation of cyclodextrins by computational molecular dynamics simulations as a function of temperature: α-CD vs ß-CD.

The structural simplicity of native cyclodextrins (CDs) contrasts with their complex behavior in the bulk of aqueous solutions, mainly when they are combined with other cosolutes. Many scientific and industrial applications based on these molecules are supported only by empirical information. The lack of fundamental knowledge, which would allow one to rationally optimize many of these applications, is notable mainly at the solution/air interface. Basic information on phenomena such as the spontaneous adsorption of native CDs or on the structure of CD aggregates in the bulk solution is really scarce. In order to fill these gaps, a detailed computational study on the adsorption and aggregation of α- and ß-CDs as a function of temperature is presented here. Our simulations reproduce, at atomic resolution, the experimentally observed much higher ability of ß-CD to aggregate compared to that of α-CD at 298 K, as well as their dependence on temperature. The adsorption of both individual CDs and small CD aggregates (up to 20 molecules) to the solution/air interface is found to be negligible. 0.8 µs long trajectories of single CD molecules in aqueous solution reveal that the main differences in the behavior of both CDs are their flexibility, higher for ß-CD, and the occupancy of individual intramolecular hydrogen bonds that is significantly longer for the same cyclodextrin. The aggregation pattern of α- and ß-CDs is followed at the hundreds of ns time scale, allowing both the spontaneous self-assembly of cyclodextrins and their redistribution along the aggregates to be observed. This is the first attempt to study the adsorption and aggregation of native cyclodextrins by atomistic molecular dynamics simulations.

The cis-bis(decanoate)tin phthalocyanine/DPPC film at the air/water interface.

Films made of cis-bis-decanoate-tin(IV) phthalocyanine (PcSn10) and racemic dipalmitoylphosphatidylcholine (DPPC) are studied with compression isotherms and Brewster angle microscopy (BAM) at the air/water interface. Films enriched in PcSn10 present phase separation elliptical-shaped domains. These domains present optical anisotropy and molecular order. They are enriched in PcSn10, and the film outside these domains is enriched in DPPC, as shown in by high-angle annular dark-field transmission electron microscopy on Langmuir-Blodgett (LB) transferred films. Film collapse area and atomic force microscopy images of LB transferred films on mica indicate that the films are actually multilayers. A computational survey was performed to determine how the PcSn10 molecules prefer to self-assemble, in films basically made of PcSn10. The relative energetic stability for several dimeric assemblies was obtained, and a crystal model of the film was developed through packing and repeating the PcSn10 molecules, along the crystallographic directions of the unit cell. Our results contribute to understanding the strong interaction between PcSn10 and DPPC at the air/water interface, where even small quantities of DPPC (~1-2%) can modify the film in an important way.

DNA and cationic surfactant complexes at hydrophilic surfaces. An ellipsometry and surface force study.

The adsorption and formation of DNA and cationic surfactant complexes at the silica-aqueous interface have been studied by ellipsometry. The interaction between the DNA-surfactant complexes at the mica-aqueous interface has been determined by the interferometric surface force apparatus. Adsorption was as expected not observed on negatively charged hydrophilic surfaces for DNA and when DNA-cationic surfactant complexes were negatively charged. However, adsorption was observed when there is an excess of cationic surfactant, just below the point of phase separation. The adsorption process requires hours to reach steady state. The adsorbed layer thickness is large at low surface coverage but becomes more compact and thinner at high coverage. A long-range repulsive force was observed between adsorbed layers of DNA-cationic surfactant complexes, which was suggested to be of both electrostatic and steric origin. The forces were found to be dependent on the equilibration time and the experimental pathway.

Influence of DNA adsorption and DNA/cationic surfactant coadsorption on the interaction forces between hydrophobic surfaces.

The forces between hydrophobic surfaces with physisorbed DNA are markedly and irreversibly altered by exposure to DNA/cetyltrimethylammonium bromide (CTAB) mixtures. In this colloidal probe atomic force microscopy study of the interactions between a hydrophobic polystyrene particle and an octadecyltrimethylethoxysilane-modified mica surface in sodium bromide solutions, we measure distinct changes in colloidal forces depending on the existence and state of an adsorbed layer of DNA or CTAB-DNA complexes. For bare hydrophobic surfaces, a monotonically attractive approach curve and very large adhesion are observed. When DNA is adsorbed at low bulk concentrations, a long-range repulsive force dominates the approach, but on retraction some adhesion persists and DNA bridging is clearly observed. When the DNA solution is replaced with a CTAB-DNA mixture at relative low CTAB concentration, the length scale of the repulsive force decreases, the adhesion due to hydrophobic interactions greatly decreases, and bridging events disappear. Finally, when the surface is rinsed with NaBr solution, the length scale of the repulsive interaction increases modestly, and only a very tiny adhesion remains. These pronounced changes in the force behavior are consistent with CTAB-induced DNA compaction accompanied by increased DNA adsorption, both of which are partially irreversible.