Trametes versicolor laccase activity modulated by the interaction with gold nanoparticles.

In this study, the impact of gold nanoparticles (AuNPs) on the structure and activity of laccase from Trametes versicolor (Lc) was described. Fluorescence experiments revealed that AuNPs efficiently quench Lc's tryptophan fluorescence by a static and dynamic process. By using differential scanning microcalorimetry and circular dichroism spectroscopy, it was determined how the concentration of nanoparticles and the composition of the medium affected the secondary structure of Lc. The data revealed that upon binding with AuNPs, conformational changes take place mainly in presence of high amounts of nanoparticles. The complex kinetic analysis unveiled the Lc activity enhancement at low concentrations of AuNPs as opposed to the concentrated regime, where it can be reduced by up to 55%. The Michaelis-Menten tests highlighted that the activity of the biocatalyst is closely related to the concentration of AuNPs, while the Selwyn analysis demonstrated that even in a concentrated regime of Lc it is not deactivated regardless of the amount of AuNPs added. The thermal parameters improved by twofold in the presence of low AuNPs concentration, whereas the activation energy increased with AuNPs content, implying that not all collisions are beneficial to the enzyme structure. The effect of AuNPs on the decomposition of a recalcitrant dye (naphthol green B, NG) by Lc was also evaluated, and the Michaelis-Menten model revealed that only the high AuNPs content influenced negatively the Lc activity. The isothermal titration calorimetry revealed that hydrogen bonds are the main intermolecular forces between Lc and AuNPs, while electrostatic interactions are responsible for NG adsorption to AuNPs. The results of the docking analysis show the binding of NG near the copper T1 site of Lc with hydrogen bonds, electrostatic and hydrophobic interactions. The findings of this work provide important knowledge for laccase-based bio-nanoconjugates and their use in the field of environmental remediation.

Green Synthesis of Hydrogel-Based Adsorbent Material for the Effective Removal of Diclofenac Sodium from Wastewater.

The removal of pharmaceutical contaminants from wastewater has gained considerable attention in recent years, particularly in the advancements of hydrogel-based adsorbents as a green solution for their ease of use, ease of modification, biodegradability, non-toxicity, environmental friendliness, and cost-effectiveness. This study focuses on the design of an efficient adsorbent hydrogel based on 1% chitosan, 40% polyethylene glycol 4000 (PEG4000), and 4% xanthan gum (referred to as CPX) for the removal of diclofenac sodium (DCF) from water. The interaction between positively charged chitosan and negatively charged xanthan gum and PEG4000 leads to strengthening of the hydrogel structure. The obtained CPX hydrogel, prepared by a green, simple, easy, low-cost, and ecological method, has a higher viscosity due to the three-dimensional polymer network and mechanical stability. The physical, chemical, rheological, and pharmacotechnical parameters of the synthesized hydrogel were determined. Swelling analysis demonstrated that the new synthetized hydrogel is not pH-dependent. The obtained adsorbent hydrogel reached the adsorption capacity (172.41 mg/g) at the highest adsorbent amount (200 mg) after 350 min. In addition, the adsorption kinetics were calculated using a pseudo first-order model and Langmuir and Freundlich isotherm parameters. The results demonstrate that CPX hydrogel can be used as an efficient option to remove DCF as a pharmaceutical contaminant from wastewater.

Formation of Alginate/Chitosan Interpenetrated Networks Revealed by EPR Spectroscopy.

This study analyzes the physico-chemical properties of interpenetrated polymer networks (IPNs) and semi-IPN resulting from cross-linking chitosan with glutaraldehyde and alginate with Ca2+ cations, as a function of the order in which the cross-linking agents are added to the polymer mixture. Three physico-chemical methods were used to assess the differences between systems: rheology, IR spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy. While rheology and IR spectroscopy are commonly used to characterize gel materials, EPR spectroscopy is rarely used, but has the advantage of providing local information about the dynamics of a system. The rheological parameters, which describe the global behavior of the samples, show that semi-IPN systems have a weaker gel behavior and the order of introducing the cross-linker in the polymer systems plays a role. The IR spectra of samples resulting by adding only Ca2+ or Ca2+ as the first cross-linker are similar to that of the alginate gel, while the spectra of samples in which glutaraldehyde is firstly added resemble the chitosan gel spectrum. Using spin-labeled alginate and spin-labeled chitosan, we monitored the changes occurring in the dynamic of the spin labels due to the formation of IPN and semi-IPN. The results show that the order of adding the cross-linking agents influences the dynamic of the IPN network, and that the formation of the alginate network determines the characteristics of the entire IPN system. The EPR data were correlated with the rheological parameters and IR spectra of the analyzed samples.

Antibacterial Aloe vera Based Biocompatible Hydrogel for Use in Dermatological Applications.

The present research aims to describe a new methodology to obtain biocompatible hydrogels based on Aloe vera used for wound healing applications. The properties of two hydrogels (differing in Aloe vera concentration, AV5 and AV10) prepared by an all-green synthesis method from raw, natural, renewable and bioavailable materials such as salicylic acid, allantoin and xanthan gum were investigated. The morphology of the Aloe vera based hydrogel biomaterials was studied by SEM analysis. The rheological properties of the hydrogels, as well as their cell viability, biocompatibility and cytotoxicity, were determined. The antibacterial activity of Aloe vera based hydrogels was evaluated both on Gram-positive, Staphylococcus aureus and on Gram-negative, Pseudomonas aeruginosa strains. The obtained novel green Aloe vera based hydrogels showed good antibacterial properties. In vitro scratch assay demonstrated the capacity of both AV5 and AV10 hydrogels to accelerate cell proliferation and migration and induce closure of a wounded area. A corroboration of all morphological, rheological, cytocompatibility and cell viability results indicates that this Aloe vera based hydrogel may be suitable for wound healing applications.

ROS-Induced DNA-Damage and Autophagy in Oral Squamous Cell Carcinoma by Usnea barbata Oil Extract-An In Vitro Study.

Oxidative stress is associated with aging, cancers, and numerous metabolic and chronic disorders, and phenolic compounds are well known for their health-promoting role due to their free-radical scavenging activity. These phytochemicals could also exhibit pro-oxidant effects. Due to its bioactive phenolic secondary metabolites, Usnea barbata (L.) Weber ex. F.H. Wigg (U. barbata) displays anticancer and antioxidant activities and has been used as a phytomedicine for thousands of years. The present work aims to analyze the properties of U. barbata extract in canola oil (UBO). The UBO cytotoxicity on oral squamous cell carcinoma (OSCC) CLS-354 cell line and blood cell cultures was explored through complex flow cytometry analyses regarding apoptosis, reactive oxygen species (ROS) levels, the enzymatic activity of caspase 3/7, cell cycle, nuclear shrinkage (NS), autophagy (A), and synthesis of deoxyribonucleic acid (DNA). All these studies were concomitantly performed on canola oil (CNO) to evidence the interaction of lichen metabolites with the constituents of this green solvent used for extraction. The obtained data evidenced that UBO inhibited CLS-354 oral cancer cell proliferation through ROS generation (316.67 × 104), determining higher levels of nuclear shrinkage (40.12%), cell cycle arrest in G0/G1 (92.51%; G0 is the differentiation phase, while during G1 phase occurs preparation for cell division), DNA fragmentation (2.97%), and autophagy (62.98%) than in blood cells. At a substantially higher ROS level in blood cells (5250.00 × 104), the processes that lead to cell death-NS (30.05%), cell cycle arrest in G0/G1 (86.30%), DNA fragmentation (0.72%), and autophagy (39.37%)-are considerably lower than in CLS-354 oral cancer cells. Our work reveals the ROS-mediated anticancer potential of UBO through DNA damage and autophagy. Moreover, the present study suggests that UBO pharmacological potential could result from the synergism between lichen secondary metabolites and canola oil phytoconstituents.

New Amorphous Hydrogels with Proliferative Properties as Potential Tools in Wound Healing.

The study and discovery of bioactive compounds and new formulations as potential tools for promoting the repair of dermoepidermal tissue in wound healing is of continuing interest. We have developed a new formulation of amorphous hydrogel based on sodium alginate (NaAlg); type I collagen, isolated by the authors from silver carp tails (COL); glycerol (Gli); Aloe vera gel powder (AV); and silver nanoparticles obtained by green synthesis with aqueous Cinnamomum verum extract (AgNPs@CIN) and vitamin C, respectively. The gel texture of the amorphous hydrogels was achieved by the addition of Aloe vera, demonstrated by a rheological analysis. The evaluations of the cytotoxicity and cell proliferation capacity of the experimental amorphous hydrogels were performed against human foreskin fibroblast Hs27 cells (CRL-1634-ATCC). The developed gel formulations did not show a cytotoxic effect. The hydrogel variant containing AgNPs@CIN in a concentration of 8 µg Ag/gel formulation and hydrogel variant with vitamin C had proliferative activity. In addition, the antibacterial activity of the hydrogels was evaluated against S. aureus ATCC 6538, Ps. aeruginosa ATCC 27853, and E. coli ATCC 25922. The results demonstrated that the gel variant based on AgNPs@CIN in a concentration of 95 µg Ag/gel formulation and the hydrogel based on vitamin C show antibacterial activity. Therefore, the developed hydrogels with AgNPs@CIN and vitamin C could be promising alternatives in wound healing.

Green synthesis of bioinspired chitosan-ZnO-based polysaccharide gums hydrogels with propolis extract as novel functional natural biomaterials.

A facile, green synthesis methodology to obtain zinc oxide nanoparticles using three polysaccharide gums (Acacia gum, Guar gum and Xanthan gum) of biological origin was developed. Subsequently, biosynthesized zinc oxide nanoparticles were incorporated into a sustainable chitosan hydrogel matrix functionalized with propolis extract. This study has revealed that the selected polysaccharides as chelates represents a suitable approach to synthesize ZnO nanoparticles of particular interest with controlled morphology. The formation of ZnO nanoparticles using polysaccharide gums was confirmed by FTIR, XRD, UV-Vis spectroscopy, thermal analysis, SEM, Raman and photoluminescence spectroscopies. The rheological behaviour of obtained hydrogels was evaluated. The AFM studies demonstrate that all synthesized chitosan incorporated ZnO composites hydrogels functionalized with propolis extract exhibit corrugated topographies. The present study highlights the possible incorporation of various guest molecules into hydrogel matrix due to its tuneable morphologies. The obtained hydrogel composites were cytocompatible in L929 fibroblast cell culture, in a range of concentrations between 50 and 1000 µg/mL, as assessed by MTT, LDH and Live/Dead double staining assays. By enhancing the biological properties, these novel green hydrogels show attractive superior performance in a wide concentration range to develop future in vivo suitable natural platforms as effective delivery systems of pharmacologic agents for biomedical applications.

Multifunctional Composite Coatings Based on Photoactive Metal-Oxide Nanopowders (MgO/TiO2) in Hydrophobic Polymer Matrix for Stone Heritage Conservation.

Multifunctional composite coatings composed of metal oxide nanoparticles dispersed in polymer matrices are an advanced solution to solve the problem of stone heritage deterioration. Their innovative design is meant to be stable, durable, transparent, easy to apply and remove, non-toxic, hydrophobic, and permeable. Coating formulations for the protection of buildings and monuments have been intensively researched lately. Such formulations are based on multifunctional composite coatings incorporating metal oxides. The present work aims to combine the hydrophobic properties of sodium polyacrylate (NaPAC16) with the antimicrobial effectiveness, with promising antimicrobial results even in the absence of light, and good compatibility of MgO (a safe to use, low cost and environmentally friendly material) and TiO2 (with antibacterial and antifungal properties), in order to develop coatings for stone materials protection. MgO (pure phase periclase) and TiO2 (pure phase anatase) nanopowders were prepared through sol-gel method, specifically routes. Aqueous dispersions of hydrophobically modified polymer (NaPAC16, polyacrylic acid sodium salt) and MgO/TiO2 nanopowders were deposited through layer-by-layer dip coating technique on glass slides and through immersion on stone fragments closely resembling the mosaic stone from the fourth century AD Roman Mosaic Edifice, from Constanta, Romania. The oxide nanopowders were characterized by: Thermal analysis (TG/DTA), scanning electron microscopy (SEM), X-ray diffraction (XRD), BET specific surface area and porosity, and UV-Vis spectroscopy for band gap determination. An aqueous dispersion of modified polyacrylate polymer and oxide nanopowders was deposited on different substrates (glass slides, red bricks, gypsum mortars). Film hydrophobicity was verified by contact angle measurements. The colour parameters were evaluated. Photocatalytic and antimicrobial activity of the powders and composite coatings were tested.

Polymorphism of chitosan-based networks stabilized by phytate investigated by molecular dynamics simulations.

Chitosan can associate in the presence of polyphosphates into insoluble hydrogels capable of drug encapsulation and safe and efficient release. On the one hand, chitosan hydrogels were synthesized using the phytate anion as a crosslinking agent and were characterized by employing dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). On the other hand, an effective chitosan-phytate model with atomistic details was created to examine the underlying physical crosslinking pattern, and the structure and dynamics of the chitosan-phytate complex were systematically investigated by using molecular dynamics (MD) simulations. To harbor the crosslinker potential for obtaining chitosan-based hydrogels, the impact of the phytate concentration and the functional groups of the chitosan on the reticulation process was addressed. The phytate association was determined by the phosphates' capacity for H-bonding to the amine and hydroxyl groups belonging to two consecutive glucosidic units. The physical crosslinking pattern was determined by the number of chitosan chains bound by one phytate anion and the phytate orientation relative to the glucopyranose neighbors. Cross-linking of two up to six chitosan chains mediated by a phytate anion represented favorable states, and the number distribution of cross-linked chains depended on the phytate concentration. The circular distribution of the cross-linkable phosphates regulated the nearly isotropic orientation of the chitosan chains and phytate at the junction, and the variety of topological crosslinking demonstrated the phytate ion's potential for developing chitosan-based hydrogels with improved structural attributes.

Enhancement of laccase immobilization onto wet chitosan microspheres using an iterative protocol and its potential to remove micropollutants.

This study was focused on creating a new and effective immobilization method for Trametes versicolor laccase (Lc) by using chitosan (CS) microspheres activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. The activation of the support alternated with immobilization of the enzyme, in repetitive procedures, led to obtaining three different products. Also, the physicochemical properties of the new products were investigated and compared with those of free laccase. The discoloration and reusability properties of the immobilized Lc were evaluated using indigo carmine (IC) as a model micropollutant. The ESEM and FT-IR methods demonstrated that the Lc was successfully immobilized. The relative reaction rate and the total amount of immobilized Lc were tripled using the iterative protocol as proved by specific and Bradford assays. The maximum amount of immobilized Lc was 8.4 mg Lc/g CS corresponding to the third immobilization procedure. Compared to the free Lc, the operational stability of the immobilized Lc was significantly improved, presenting a maximum activity plateau over a pH range of 3-5 and a temperature range of 25-50 °C. The thermal inactivation study at 55 °C proved that the immobilized enzyme is three times more stable than the free Lc. The isoconversional and Michaelis-Menten methods showed that the immobilization did not affect the enzyme catalytic properties. After 32 days of storage, the residual activities are 85% for the immobilized laccase and 40% for the free one. In similar conditions, the free and immobilized Lc (2.12 x 10-6 M) completely decolorized IC (7.15 x 10-5 M) within 14 min. The immobilized Lc activity remained almost constant (80%) during 10 reusability cycles. All these results highlight the substantial advantages of the new immobilization protocol and demonstrate that immobilized Lc can be used as a promising micropollutant removal from real wastewater.

Interaction of piroxicam with bovine serum albumin investigated by spectroscopic, calorimetric and computational molecular methods.

The binding of drugs to serum proteins is governed by weak non-covalent forces. In this study, the nature and magnitude of the interactions between piroxicam (PRX) and bovine serum albumin (BSA) was assessed using spectroscopic, calorimetric and computational molecular methods. The fluorescence data revealed an atypical behavior during PRX and BSA interaction. The quenching process of tryptophan (Trp) by PRX is a dual one (approximately equal static and dynamic quenched components). The FRET results indicate that a non-radiative transfer of energy occurred. The association constant and the number of binding sites indicate moderate PRX and BSA binding. The competitive binding study indicates that PRX is bound to site I from the hydrophobic pocket of subdomain IIA of BSA. The synchronous spectra showed that the microenvironment around the BSA fluorophores and protein conformation do not change considerably. The Trp lifetimes revealed that PRX mainly quenches the fluorescence of Trp-213 situated in the hydrophobic domain. The CD and DSC investigation show that addition of PRX stabilizes the protein structure. ITC results revealed that BSA-PRX binding involves a combination of electrostatic, hydrophobic and hydrogen interactions. The analysis of the computational data is consistent with the experimental results. This thorough investigation of the PRX-BSA binding may provide support for other studies concerning moderate affinity drugs with serum protein.Communicated by Ramaswamy H. Sarma.