pH-response of protein-polysaccharide multilayers adsorbed on a flat gold surface: A surface plasmon resonance study.

Polysaccharide-protein multilayers (PPMLs) consisting of bovine serum albumin (BSA) and chondroitin sulfate (CS) are assembled in acidic solution (pH 4.2) via layer-by-layer deposition method. The formation of PPMLs on gold surface and their responsiveness to pH change from 4.2 to 7 is investigated by Surface Plasmon Resonance Spectroscopy. The buildup of the multilayer at pH 4.2 exhibits non-linear growth while the formation of the first layers is strongly affected by the physicochemical properties of the gold surface. Neutral solution (pH 7) affects the interactions between the biopolymers and results in a partially disassemble (disintegration) of the multilayer film. On one hand, the single pair of layers, BSA-CS and the double pair of layers, (BSA-CS)2, assemblies are stable in neutral pH, a result that will be of interest for biomedical applications. On the other hand, multilayer films consisting of more than four layers that is (BSA-CS)2

Hydration effects on thermal transitions and molecular mobility in Xanthan gum polysaccharides.

In this work, the xanthan gum (XG) polysaccharide is studied over a wide range of temperatures and water fractions 0 ≤ hw ≤ 0.70 (on a wet basis) by employing differential scanning calorimetry (DSC) and broadband dielectric spectroscopy (BDS). The investigation reveals that the critical water fraction for ice formation is about 0.35. Glass transition temperature (Tg) was determined through calorimetry experiments for all the samples studied. Water acts as a strong plasticizer, i.e., decreasing Tg, for water fractions up to about 0.35. A secondary (local) relaxation process is recorded in both dry and hydrated samples, which is sensitive to the presence of water molecules. This fact indicates that this process originates due to the orientation of small polar groups of the side chain, or/and due to the local main chain dynamics. Two types of long-range charge transport processes were resolved. The first is related to the conductive paths being formed via bulk-like ice structures (at high hydration levels), whereas the second can be attributed to proton mobility via the hydrogen bond (HB) network of non-freezing water existing in XG. Interestingly, this process is exactly the same in all the hydrated samples with hw > 0.25. With respect to the sample with hw = 0.27, a Vogel-Tammann-Fulcher (VTF)-like polarization process has also been recorded which seems to be related to long-range charge mobility via interconnected water clusters. As far as we are aware, this is the first time that XG is studied in terms of glass transition and molecular mobility over a wide range of hydration levels combining DSC and BDS techniques.

Combining particle tracking microrheology and viscometry for the study of DNA aqueous solutions.

We use video particle tracking microrheology (VPTMR) in order to investigate the viscoelasticity of salmon DNA and correlate it to its steady-flow shear-thinning viscosity. Aqueous solutions of DNA are tested in a wide concentration range from the dilute to the semidilute unentangled concentration regime. The observed mean squared displacement shows power-law scaling with lag-time which is equivalent to power-law behavior of the complex modulus as a function of frequency that is, |G* (ω)| = S ∙ ω α . The relaxation exponent α changes abruptly with concentration in the semidilute regime from about 1 to about 0.5 which is the exponent predicted by the Rouse model. The quasi-property S follows the scaling of viscosity for uncharged polymers near θ-conditions in the semidilute regime that is, η ∼ c 1 / 3 ν eff - 1 with νeff = 0.50 - 0.51. The shear-thinning exponent observed by viscometry increases gradually towards the value of 0.5 which has been predicted for Rouse chains under flow. Our findings are in agreement with recent studies of DNA solutions where DNA is treated as a model polymer and addresses the low-molar mass regime of DNA viscoelasticity. This work demonstrates that the combination of passive particle tracking with viscometry can provide a complete picture on the viscoelasticity of DNA-based biopolymer materials.

Temperature-induced aggregation behavior in bovine pancreas trypsin solutions.

In this study, we investigate the effect of temperature treatment on Bovine Pancreas Trypsin (BPT) in aqueous solutions using dynamic, static and electrophoretic light scattering, fluorescence spectroscopy and circular dichroism. Static and dynamic light scattering at various solution conditions i.e. different salt content and pH, reveals that BPT aggregation is enhanced as temperature increases in a non-reversible manner. At acidic pH protein monomers are the dominant population over aggregates of globules, nevertheless the two populations co-exist at neutral and basic pH. The surface charge of the aggregates is intensified by aggregation and it is dominated by the negative residues of the protein at all pH conditions. Protein unfolding upon thermal treatment is probed by variation of the fluorescence spectrum which is caused by the exposure of tryptophan to the aqueous environment. The exposure of the hydrophobic interior of BPT upon heating may be considered as the reason of aggregation at the molecular level. Τhis study provides information that can be useful for utilizing thermal treatment protocols of BPT towards manufacturing protein-based nano formulated drugs.

Formation of complexes in aqueous solutions of amphiphilic triblock polyelectrolytes of different topologies and an oppositely charged protein.

The complexation of lysozyme with aggregates from two triblock amphiphilic polyelectrolytes of the same blocks but different topologies and block molar masses, namely PS-b-SCPI-b-PEO and SCPI-b-PS-b-PEO, is investigated by scattering and spectroscopy methods. Light scattering reveals that the interaction with lysozyme causes shrinkage of the self-assembled nanoparticles in the case of the hydrophobic-polyelectrolyte-hydrophilic sequence. In the polyelectrolyte-hydrophobic-hydrophilic sequence, the opposite trend is observed. Small angle neutron scattering confirms the existence of micellar and fractal aggregates and the complexation with lysozyme. The pH-dependence of the interactions and the stability of the hybrid protein/polymer nanoparticles upon salt addition are tested. The native conformation of the protein is found to be preserved during complexation. This study reveals that both micellar and fractal aggregates made of amphiphilic triblock polyelectrolytes are capable of loading with oppositely charged proteins in a controllable manner, tuned primarily by the structure of the triblock terpolymer.

Tuning the solution organization of cationic polymers through interactions with bovine serum albumin.

The interactions of bovine serum albumin (BSA) with aggregates of cationic polymers, i.e. quaternized poly(chloromethyl styrene) chains (QIm-PCMS), in aqueous solutions are investigated using small angle neutron scattering on length scales relevant to the size of BSA. The arrangement of the macromolecular chains within their aggregates is consistent with a blob description of overlapping chains that contain hydrophobic domains. The local conformations depend on the salt content as in typical linear polyelectrolytes. Although the hydrophobic content of the cationic polymers does not cause measurable local morphology differences, the interactions with BSA are enhanced in the case of the not fully quaternized polymer. The secondary structure of BSA is critically compromised by the interaction with the quaternized polymers as the signature of the alpha helix conformation is lost. The complexation with BSA and the resulting enhancement of interchain associations on higher length scales are verified using dynamic light scattering experiments. This study demonstrates the ability to tune the polyelectrolyte/protein interactions and polyelectrolyte chain-chain associations by modifying the hydrophobic content of the polyelectrolytes.

Micelles from HOOC-PnBA-b-PAA-C12H15 Diblock Amphiphilic Polyelectrolytes as Protein Nanocarriers.

We investigate the potential of self-assembled nanostructures of the PnBA-b-PAA amphiphilic diblock polyelectrolyte as candidates for protein nanocarriers. Three PnBA-b-PAA copolymers with different molecular weights and PnBA/PAA weight ratios are tested. The system with the most well-defined core-shell micellar structure is chosen for complexation with lysozyme. Its solutions are found to contain well-defined core-shell micelles that are stable upon increase in solution salt content to physiological levels. Upon mixing with lysozyme we find that the protein globules accumulate preferably at the outer parts of the hydrated corona of the micelles. Increasing the protein concentration, intermicellar aggregation is enhanced in a controllable way. At high salt content the number of proteins per micelle is lower compared with the low salt content, which points to an interaction of predominantly electrostatic nature. While light scattering is very sensitive to complexation, small-angle neutron scattering is able to distinguish between the contributions from individual micelles and aggregates. This work demonstrates the use of scattering techniques to characterize protein-polymer interactions in multiple hierarchical levels.

Thermoresponsive behavior of micellar aggregates from end-functionalized PnBA-b-PNIPAM-COOH block copolymers and their complexes with lysozyme.

The temperature response of micellar aggregates of poly(n-butyl acrylate)-b-poly(N-isopropylacrylamide)-carboxylic acid (PnBA-b-PNIPAM-COOH) end-functionalized diblock copolymers in aqueous solutions is investigated by small angle neutron scattering and light scattering techniques. The particular micellar aggregates present -COOH groups at their surface due to the molecular architecture of the block copolymer chains. Above the critical solution temperature micellar aggregation depends on the initial solution concentration, while at the highest polymer content intermicellar correlations are observed as a hard-sphere interaction intensity peak. Addition of lysozyme induces this morphological transition even at low concentrations. The scattering profiles are consistent with lysozyme accumulating in the vicinity of the micellar cores, a finding that is supported by measurements in lysozyme contrast matched solvent. Upon temperature increase negatively charged units are exposed to the surface of the aggregates during thermal transition which is a stabilizing force against the phase separating coil-to-globule transition of poly(N-isopropylacrylamide) (PNIPAM).

Complexation of lysozyme with adsorbed PtBS-b-SCPI block polyelectrolyte micelles on silver surface.

We present a study of the interaction of the positively charged model protein lysozyme with the negatively charged amphiphilic diblock polyelectrolyte micelles of poly(tert-butylstyrene-b-sodium (sulfamate/carboxylate)isoprene) (PtBS-b-SCPI) on the silver/water interface. The adsorption kinetics are monitored by surface plasmon resonance, and the surface morphology is probed by atomic force microscopy. The micellar adsorption is described by stretched-exponential kinetics, and the micellar layer morphology shows that the micelles do not lose their integrity upon adsorption. The complexation of lysozyme with the adsorbed micellar layers depends on the micelles arrangement and density in the underlying layer, and lysozyme follows the local morphology of the underlying roughness. When the micellar adsorbed amount is small, the layers show low capacity in protein complexation and low resistance in loading. When the micellar adsorbed amount is high, the situation is reversed. The adsorbed layers both with or without added protein are found to be irreversibly adsorbed on the Ag surface.

Thermally stabilized chondroitin sulfate-hemoglobin nanoparticles and their interaction with bioactive compounds.

The preparation of nanoparticles (NPs) based on hemoglobin (Hb) with a fully biocompatible methodology is presented. The spontaneous formation of electrostatic complexes of Hb with chondroitin sulfate (CS) at pH 4 in the polysaccharide/protein mass ratio regime where charge neutrality is met leads to spherical nanostructures with monomodal hydrodynamic radii distribution in the range of 50-100 nm. The integrity of the electrostatic complexes is disturbed at pH 7 as the net electric charge of Hb is very low. Treating the NPs at mildly elevated temperature stabilizes them against the pH increase taking advantage of Hb's ability of unfolding and self-associating upon thermal treatment. The NPs surface charge is pH-tunable and changes from positive to strongly negative upon pH increase to 7 proving the presence of negative surface patches of Hb and CS segments in their exterior. The α-helix content of Hb does not change significantly by thermal treatment. The NPs are found to bind the bioactive compounds curcumin and ß-carotene and are stable in solutions with high salt content. This investigation introduces a straightforward method to formulate Hb in NPs with possibilities in the nanodelivery of nutrients and drugs.

Advances in Small Angle Neutron Scattering on Polysaccharide Materials.

Polysaccharide materials and biomaterials gain the focus of intense research owing to their great versatility in chemical structures and modification possibilities, as well as their biocompatibility, degradability, and sustainability features. This review focuses on the recent advances in the application of SANS on polysaccharide systems covering a broad range of materials such as nanoparticulate assemblies, hydrogels, nanocomposites, and plant-originating nanostructured systems. It motivates the use of SANS in its full potential by demonstrating the features of contrast variation and contrast matching methods and by reporting the methodologies for data analysis and interpretation. As these soft matter systems may be organized in multiple length scales depending on the interactions and chemical bonds between their components, SANS offers exceptional and unique opportunities for advanced characterization and optimization of new nanostructured polysaccharide materials.

A sustainable bioprocess to produce bacterial cellulose (BC) using waste streams from wine distilleries and the biodiesel industry: evaluation of BC for adsorption of phenolic compounds, dyes and metals.

BACKGROUND: The main challenge for large-scale production of bacterial cellulose (BC) includes high production costs interlinked with raw materials, and low production rates. The valorization of renewable nutrient sources could improve the economic effectiveness of BC fermentation while their direct bioconversion into sustainable biopolymers addresses environmental pollution and/or resource depletion challenges. Herein a green bioprocess was developed to produce BC in high amounts with the rather unexplored bacterial strain Komagataeibacter rhaeticus, using waste streams such as wine distillery effluents (WDE) and biodiesel-derived glycerol. Also, BC was evaluated as a bio-adsorbent for phenolics, dyes and metals removal to enlarge its market diversification. RESULTS: BC production was significantly affected by the WDE mixing ratio (0-100%), glycerol concentration (20-45 g/L), type of glycerol and media-sterilization method. A maximum BC concentration of 9.0 g/L, with a productivity of 0.90 g/L/day and a water holding capacity of 60.1 g water/g dry BC, was achieved at 100% WDE and ≈30 g/L crude glycerol. BC samples showed typical cellulose vibration bands and average fiber diameters between 37.2 and 89.6 nm. The BC capacity to dephenolize WDE and adsorb phenolics during fermentation reached respectively, up to 50.7% and 26.96 mg gallic acid equivalents/g dry BC (in-situ process). The produced BC was also investigated for dye and metal removal. The highest removal of dye acid yellow 17 (54.3%) was recorded when 5% of BC was applied as the bio-adsorbent. Experiments performed in a multi-metal synthetic wastewater showed that BC could remove up to 96% of Zn and 97% of Cd. CONCLUSIONS: This work demonstrated a low-carbon approach to produce low-cost, green and biodegradable BC-based bio-adsorbents, without any chemical modification. Their potential in wastewater-treatment-applications was highlighted, promoting closed-loop systems within the circular economy era. This study may serve as an orientation for future research towards competitive or targeted adsorption technologies for wastewater treatment or resources recovery.

Physicochemical properties of electrostatically crosslinked carrageenan/chitosan hydrogels and carrageenan/chitosan/Laponite nanocomposite hydrogels.

In this work physical carrageenan/chitosan (Car/Chit) hydrogels are prepared by electrostatic complexation between the two oppositely charged polysaccharides. The hydrogels have storage moduli in the order of 5-10 kPa and swelling ratios in the order of 5000-6000 %. At conditions where both polysaccharides are highly charged (pH 5) the swelling ratios are lower than the ones at conditions of lower dissociation i.e., at pH 2 and 7 and the opposite trend is found for the storage modulus. Chit appears to act as a crosslinker for Car as increasing its concentration the swelling ratio decreases and the moduli increase. The hydrogels can incorporate the nanoclay Laponite (Lap) and form hybrid nanocomposites where the intercalation by the two biopolymers leads to exfoliation of the clay nanoplatelets in the presence of both Car and Chit. The composite hydrogels retain the mechanical properties of the Car/Chit hydrogels at the studied pH range (pH 2 to pH 7). This shows the prepared hydrogels can be potentially used as multifunctional biomaterials for drug delivery, tissue engineering and bone regeneration applications.

Protein-induced transformation of unilamellar to multilamellar vesicles triggered by a polysaccharide.

We report on the morphological transitions of didodecyldimethylammonium bromide (DDAB) cationic vesicles and hybrid DDAB/hyaluronic acid (HA) vesicles upon addition of BSA at pH 7 where BSA is overall negatively charged. Small angle neutron scattering (SANS) is used to extract the size distributions of the nanovesicles, the thickness of the DDAB bilayers and their lamellarity. Although the HA-decorated DDAB vesicles contain the negatively charged polysaccharide the interaction with BSA appears to be more intense in comparison to bare vesicles. Characteristic peaks in the SANS patterns indicate the presence of multilamellar interfaces while the formation of multilamellar vesicles induced by BSA depends on the amount of added HA. Consequently, higher lamellarities are observed at higher BSA contents. This work demonstrates a simple methodology to tune the encapsulation of globular proteins in vesicular nanoassemblies by affecting their lamellarity and has direct implications on the application of vesicles and liposomes in protein delivery.

Merging high doxorubicin loading with pronounced magnetic response and bio-repellent properties in hybrid drug nanocarriers.

Hybrid magnetic drug nanocarriers are prepared via a self-assembly process of poly(methacrylic acid)-graft-poly(ethyleneglycol methacrylate) (p(MAA-g-EGMA)) on growing iron oxide nanocrystallites. The nanocarriers successfully merge together bio-repellent properties, pronounced magnetic response, and high loading capacity for the potent anticancer drug doxorubicin (adriamicin), in a manner not observed before in such hybrid colloids. High magnetic responses are accomplished by engineering the size of the magnetic nanocrystallites (∼13.5 nm) following an aqueous single-ferrous precursor route, and through adjustment of the number of cores in each colloidal assembly. Complementing conventional magnetometry, the magnetic response of the nanocarriers is evaluated by magnetophoretic experiments providing insight into their internal organization and on their response to magnetic manipulation. The structural organization of the graft-copolymer, locked on the surface of the nanocrystallites, is further probed by small-angle neutron scattering on single-core colloids. Analysis showed that the MAA segments selectively populate the area around the magnetic nanocrystallites, while the poly(ethylene glycol)-grafted chains are arranged as protrusions, pointing towards the aqueous environment. These nanocarriers are screened at various pHs and in highly salted media by light scattering and electrokinetic measurements. According to the results, their stability is dramatically enhanced, as compared to uncoated nanocrystallites, owing to the presence of the external protective PEG canopy. The nanocarriers are also endowed with bio-repellent properties, as evidenced by stability assays using human blood plasma as the medium.

Current Advances of Polysaccharide-Based Nanogels and Microgels in Food and Biomedical Sciences.

Polysaccharides are natural polymers with hydrophilic, biocompatible and biodegradable characteristics and have many opportunities in the food and pharmaceutical sectors. This review focuses on the field of nano and microstructures whose internal structure is based on networked polysaccharide chains in 3D i.e., polysaccharide nanogels (NGs) and microgels (MGs). As it is observed the number of articles on NGs and MGs in peer reviewed scientific journals has been increasing over the last two decades. At the same time, the relative contribution of polysaccharides in this field is gaining place. This review focuses on the different applied methods for the fabrication of a variety of polysaccharide-based NGs and MGs and aims to highlight the recent advances on the subject and present their potentials and properties with regards to their integration in aspects of medicinal and food sciences. The presentation of the recent advances in the application of polysaccharide NGs and MGs is divided in materials with potential as emulsion stabilizers and materials with potential as carriers of bioactives. For applications in the medical sector the division is based on the fabrication processes and includes self-assembled, electrostatically complexed/ionically crosslinked and chemically crosslinked NGs and MGs. It is concluded that many advances are expected in the application of these polysaccharide-based materials and in particular as nutrient-loaded emulsion stabilizers, viscosity modifiers and co-assembled structures in combination with proteins.

Formation and physicochemical properties of glycogen phosphorylase in complex with a cationic polyelectrolyte.

The accumulation of rabbit muscle glycogen phosphorylase b (RMGPb) in electrostatic complexes with the cationic polyelectrolyte poly 2-(dimethylamino) ethyl methacrylate in its quenched form (QPDMAEMA) was studied in two buffer solutions. In the N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, large complexes of RMGPb-QPDMAEMA were formed which adopted smaller sizes as QPDMAEMA concentration increased. However, in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer, the hydrodynamic radius of the formed complexes gradually increased as the polymer concentration increased. Zeta potential measurements (ζp) showed that RMGPb significantly changed the ζp of the QPDMAEMA aggregates. Fluorescence studies showed that the interaction between RMGPb and QPDMAEAMA was enhanced as polymer concentration increased. Specifically, 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence indicated that in the BES buffer the aggregates became denser as more QPDMAEMA was added, while in the HEPES buffer the density of the formed structures decreased. RMGPb's secondary structure was examined by Attenuated Total Reflection - Fourier Transform Infrared (ATR-FTIR) and Circular Dichroism (CD) showing that QPDMAEMA interaction with RMGPb does not induce any changes to the secondary structure of the enzyme. These observations suggest that cationic polyelectrolytes may be utilized for the formulation of RMGPb in multifunctional nanostructures and be further exploited in innovative biotechnology applications and bioinspired materials development.

Preparation of trypsin-based nanoparticles, colloidal properties and ability to bind bioactive compounds.

Nanoparticles (NPs) based on the proteolytic enzyme trypsin (TRY) were prepared by a biocompatible methodology. TRY co-assembled with the anionic polysaccharide chondroitin sulfate (CS) in complexes with well-defined distributions of radii in the range of 100-200 nm by electrostatic complexation at acidic conditions. At pH 7 the complexes were unstable and lost their monomodal size distribution which is potentially related to TRY's weak positive net surface charge and a large negative charge patch that forms at neutral pH. Thermal treatment at conditions which were not expected to interfere with TRY's proteolytic activity was used to stabilize the complexes into NPs that resisted disintegration at pH 7 taking advantage of the ability of the TRY globules to thermally aggregate. The secondary conformation of TRY within the NPs was found fairly unperturbed even after thermal treatment which is crucial for its physiological function. The CS-TRY NPs could bind and encapsulate the bioactive substances curcumin (CUR) and ß-carotene (ß-C) owing to TRY's hydrophobic domains. The CS-TRY NPs may be considered as a platform for the immobilized active enzyme and multifunctional NPs for hydrophobic bioactive compounds.

Polysaccharide-Protein Multilayers Based on Chitosan-Fibrinogen Assemblies for Cardiac Cell Engineering.

The cell and tissue culture substrates play a pivotal role in the regulation of cell-matrix and cell-cell interactions. The surface properties of the materials control a wide variety of cell functions. Amongst various methods, layer-by-layer (LbL) assembly is a versatile surface coating technique for creating controllable bio-coatings. Here, polysaccharide/protein multilayers are proposed, which are fabricated by immersive LbL assembly and based on the chitosan/fibrinogen pair for improving the adhesion and spreading of cardiomyocytes. Two approaches in LbL assembly are employed for clarifying the effect of the bilayers order and their concentration on cardiomyocytes viability and morphology. Fourier transform infrared spectroscopy (FTIR) measurements show that the adsorption of the biopolymers is enhanced during the LbL deposition in a synergistic manner. Contact angle measurements indicate that the multilayers are alternating from less to more hydrophilic behavior depending on the biopolymer that is added last. Confocal microscopy with immunostained fibrinogen reveals that the amount of the protein is higher when the concentration of the immersion solution is increased, however, for low solution concentration it is speculated that interdigitation between the separate biopolymer layers takes place. This work motivates the use of fibrinogen in polysaccharide/protein multilayers for enhanced cytocompatibility in cardiac tissue engineering.

Development of biodegradable films using sunflower protein isolates and bacterial nanocellulose as innovative food packaging materials for fresh fruit preservation.

This study presents the valorization of side streams from the sunflower-based biodiesel industry for the production of bio-based and biodegradable food packaging following circular economy principles. Bacterial cellulose (BC) was produced via fermentation in 6 L static tray bioreactors using nutrient-rich supplements derived from the enzymatic hydrolysis of sunflower meal (SFM) combined with crude glycerol as carbon source. Novel biofilms were produced using either matrices of protein isolates extracted from sunflower meal (SFMPI) alone or SFMPI matrices reinforced with nanocellulose biofillers of commercial or bacterial origin. Acid hydrolysis was employed for ex-situ modification of BC to nanostructures (56 nm). The biofilms reinforced with bacterial nanocellulose structures (SFMPI-BNC) showed 64.5% higher tensile strength, 75.5% higher Young's modulus, 131.5% higher elongation at break, 32.5% lower water solubility and 14.1% lower water vapor permeability than the biofilms produced only with SFMPI. The biofilms were evaluated on fresh strawberries packaging showing that the SFMPI-BNC-based films lead to effective preservation at 10 °C considering microbial growth and physicochemical profile (weight loss, chemical characterization, color, firmness and respiration activity). The SFMPI-BNC-based films could be applied in fresh fruit packaging applications.