Lignin-based nanoparticles as both structural and active elements in self-assembling and self-healing multifunctional hydrogels for chronic wound management

Efficient wound healing is feasible when the dressing materials simultaneously target multiple factors causing wound chronicity, such as deleterious proteolytic and oxidative enzymes and bacterial infection. Herein, entirely bio-based multifunctional self-assembled hydrogels for wound healing were developed by simply mixing two biopolymers, thiolated hyaluronic acid (HA-SH) and silk fibroin (SF), with lignin-based nanoparticles (NPs) as both structural and functional elements. Sono-enzymatic lignin modification with natural phenolic compounds results in antibacterial and antioxidant phenolated lignin nanoparticles (PLN) capable of establishing multiple interactions with both polymers. These strong and dynamic polymer-NP interactions endow the hydrogels with self-healing and shear-thinning properties, and pH-responsive NP release is triggered at neutral to alkaline pH (7–9). Despite being a physically crosslinked hydrogel, the material was stable for at least 7 days, and its mechanical and functional properties can be tuned depending on the polymer and NP concentration. Furthermore, human skin cells in contact with the nanocomposite hydrogels for 7 days showed more than 93% viability, while the viability of clinically relevant Staphylococcus aureus and Pseudomonas aeruginosa was reduced by 99.7 and 99.0%, respectively. The hydrogels inhibited up to 52% of the activity of myeloperoxidase and matrix metalloproteinases, responsible for wound chronicity, and showed a strong antioxidant effect, which are crucial features promoting wound healing.Peer ReviewedPostprint (published version

Dual-function nanoparticles enzymatically conjugated with a custom-made polyurethane hydrogel for chronic wound treatment

Hydrogels are attractive drug delivery systems with the potential to protect their cargo and control its release. In particular, hydrogels based on synthetic polymers are gaining increasing interest by virtue of their controllable chemistry, ease of modification, and reproducibility. Moreover, the presence of specific side chains and pending functional groups in the polymer structure allows for the conjugation of drugs and other compounds resulting in improved control over drug release. Enzymes that catalyse reactions in a very specific way could also be used to control the conjugation of compounds to the polymeric chains to improve reproducibility and biocompatibility of the conjugation process. This contribution describes an innovative system for drug delivery comprising a bioartificial supramolecular hydrogel based on a customised polyurethane and α- cyclodextrins, and nanoparticles, for application in the treatment of chronic wounds. The system has the potential to reduce inflammation and eradicate infection by virtue of dual-function nanoparticles which incorporate cobalt as antimicrobial agent, and phenolated lignin as antioxidant. The nanoparticles are enzymatically conjugated to the hydrogel by means of the amine side groups exposed along the backbone of the ad-hoc synthesised polyurethane. The oxidase enzyme laccase is exploited to oxidize the phenol groups of lignin, to allow their interaction with the amines on the hydrogel. The effects of nanoparticles conjugation to the hydrogel are studied through gelification tests, stability tests, and rheology. Moreover, the release of nanoparticles from the hydrogel and their effects on patients’ wound fluids and against relevant bacterial strains are analysed in vitro

Influence of enzymatically hydrophobized hemp protein on morphology and mechanical properties of bio-based polyurethane and epoxy foams

Biomass fillers offer the possibility to modify the mechanical properties of foams, increasing their cost-effectiveness and reducing their carbon footprint. In this study, bio-based PU (soft, open cells for the automotive sector) and epoxy (EP, hard, closed cells for construction applications) composite foams were prepared by adding pristine and laccase-mediated lauryl gallate-hydrophobized hemp protein particles as filler (HP and HHP, respectively). The fillers were able to modify the density, the mechanical properties and the morphology of the PU and EP foams. The addition of HP filler increases the density of PU foams up to 100% and significantly increases the s values by 40% and Emod values. On the other hand, the inclusion of the HHP as filler in PU foams mostly results in reduced density, by almost 30%, and reduced s values in comparison with reference and HP-filled foams. Independently from filler concentration and type, the biomass increased the Emod values for all foams relative to the reference. In the case of the EP foams, the tests were only conducted for the foams filled with HHP due to the poor compatibility of HP with the EP matrix. HHP decreased the density, compressive strength and Emod values of the composites. For both foams, the fillers increased the size of the cells, while reducing the amount of open cells of PU foams and the amount of closed cells for EP foams. Finally, both types of foams filled with HHP reduced the moisture uptake by 80 and 45%, respectively, indicating the successful hydrophobization of the composites.Peer ReviewedPostprint (published version

Antibacterial properties and mechanisms of action of sonoenzymatically synthesized lignin-based nanoparticles

In recent years, lignin has drawn increasing attention for different applications due to its intrinsic antibacterial and antioxidant properties, coupled with biodegradability and biocompatibility. However, chemical modification or combination with metals is usually required to increase its antimicrobial functionality and produce biobased added-value materials for applications wherein bacterial growth should be avoided, such as biomedical and food industries. In this work, a sonoenzymatic approach for the simultaneous functionalization and nanotransformation of lignin to prepare metal-free antibacterial phenolated lignin nanoparticles (PheLigNPs) is developed. The grafting of tannic acid, a natural phenolic compound, onto lignin was achieved by an environmentally friendly approach using laccase oxidation upon the application of high-intensity ultrasound to rearrange lignin into NPs. PheLigNPs presented higher antibacterial activity than nonfunctionalized LigNPs and phenolated lignin in the bulk form, indicating the contribution of both the phenolic content and the nanosize to the antibacterial activity. Studies on the antibacterial mode of action showed that bacteria in contact with the functionalized NPs presented decreased metabolic activity and high levels of reactive oxygen species (ROS). Moreover, PheLigNPs demonstrated affinity to the bacterial surface and the ability to cause membrane destabilization. Antimicrobial resistance studies showed that the NPs did not induce resistance in pathogenic bacteria, unlike traditional antibiotics.Peer ReviewedPostprint (published version

Continuous sonochemical nanotransformation of lignin - process design and control

As the most abundant renewable aromatic polymer on the planet, lignin is gaining growing interest in replacing petroleum-based chemicals and products. However, only <5 % of industrial lignin waste is revalorized in its macromolecular form as additives, stabilizing agents or dispersant and surfactants. Herein, revalorization of this biomass was achieved by implementing an environmentally-friendly continuous sonochemical nano- transformation to obtain highly concentrated lignin nanoparticles (LigNPs) dispersions for added-value material applications. With the aim to further model and control a large-scale ultrasound-assisted lignin nano- transformation, a two-level factorial design of experiment (DoE) was implemented varying the ultrasound (US) amplitude, flow rate, and lignin concentration. Size and polydispersity measurements together with the UV–Vis spectra of lignin recorded at different time intervals of sonication allowed to monitor and understand the sonochemical process on a molecular level. The light scattering profile of sonicated lignin dispersions showed a significant particle size reduction in the first 20 min, followed by moderate particle size decrease below 700 nm until the end of the 2 h process. The response surface analysis (RSA) of the particle size data revealed that the lignin concentration and sonication time were the most important factors to achieve smaller NPs. From a mechanistic point of view, a strong impact of the particle–particle collisions due to sonication seems to be responsible for the decrease in particle size and homogenization of the particle distribution. Unexpectedly, a strong interaction between the flow rate and US amplitude on the particle size and nanotransformation efficiency was observed, yielding smaller LigNPs at high amplitude and low flow rate or vice versa. The data derived from the DoE were used to model and predict the size and polydispersity of the sonicated lignin. Furthermore, the use of the NPs spectral process trajectories calculated from the UV–Vis spectra showed similar RSA model as the dynamic light scattering (DLS) data and will potentially allow the in-line monitoring of the nanotransformation process.This research was funded by the European Union under the framework of the projects BIOMAT (H2020-953270) and rLightBioCom (HORIZON-101091691). G. F. acknowledges Universitat Politècnica de Catalunya and Banco Santander for his PhD grant (113 FPI-UPC 2018).Peer ReviewedPostprint (published version

Microbial cellulose from a komagataeibacter intermedius strain isolated from commercial wine vinegar

In this study a new bacterial cellulose (BC) producer isolated from commercial vinegar is identified as Komagataeibacter intermedius JF2 based on the examination of general taxonomical characteristics, 16S rDNA sequence analysis, and MALDI-TOF mass spectrometry. The cellulose produced is studied in terms of morphology by scanning electron microscopy, crystallinity by X-Ray diffraction, structure by Fourier transform infrared spectroscopy, and water absorption capacity. BC yield and characteristics of the cellulose produced by the new isolated JF2 are compared with those of the well-known and commonly-used BC producer Komagataeibacter xylinus. Yield of cellulose production was higher for JF2 than for K. xylinus grown on several culture media. JF2 exhibited maximum BC production (1.6 g/L) growing on HS medium supplemented with mannitol. The molecular structure of the produced cellulose was the same for both strains and it was in concordance with that of BC. The nanocellulose fibers produced by JF2 showed a higher degree of crystallinity and a more homogeneous size distribution than those produced by K. xylinus. The results suggested that Komagataeibacter intermedius JF2 could be a suitable candidate as a BC producer for biotechnological applications.Peer ReviewedPostprint (author's final draft

Laccase/TEMPO-mediated bacterial cellulose functionalization: production of paper-silver nanoparticles composite with antimicrobial activity

Bacterial cellulose (BC) was functionalized applying the Laccase/TEMPO oxidative treatment, leading to a five-fold increase of the concentration of carboxyl groups. Paper produced with this cellulose showed improved mechanical properties while maintaining barrier function against water and greases as compared to paper produced with non-oxidized BC. Also, the negative charge provided by the carboxyl groups on functionalized BC was used to generate silver nanoparticles (AgNPs), obtaining a BC paper and Ag composite. The presence of AgNPs in the composites was validated by SEM, EDS and ICP analysis, showing spherical, uniformly sized particles stabilized in the BC nanofibers matrix. Additionally, antimicrobial property of composites containing AgNPs was tested. The results showed the strong antimicrobial activity of the composites against Gram-positive and Gram-negative bacteria and fungi. The generation of Ag nanoparticles in a matrix that combine the physical characteristics of the BC nanofibers with the stiffness and the mechanical properties of paper produced composites that may have applicability in technological and biomedical uses

Laccase/TEMPO-mediated bacterial cellulose functionalization: production of paper-silver nanoparticles composite with antimicrobial activity

“This is a post-peer-review, pre-copyedit version of an article published in Cellulose. The final authenticated version is available online at: https://doi.org/10.1007/s10570-019-02678-5Bacterial cellulose (BC) was functionalized applying the Laccase/TEMPO oxidative treatment, leading to a five-fold increase of the concentration of carboxyl groups. Paper produced with this cellulose showed improved mechanical properties while maintaining barrier function against water and greases as compared to paper produced with non-oxidized BC. Also, the negative charge provided by the carboxyl groups on functionalized BC was used to generate silver nanoparticles (AgNPs), obtaining a BC paper and Ag composite. The presence of AgNPs in the composites was validated by SEM, EDS and ICP analysis, showing spherical, uniformly sized particles stabilized in the BC nanofibers matrix. Additionally, antimicrobial property of composites containing AgNPs was tested. The results showed the strong antimicrobial activity of the composites against Gram-positive and Gram-negative bacteria and fungi. The generation of Ag nanoparticles in a matrix that combine the physical characteristics of the BC nanofibers with the stiffness and the mechanical properties of paper produced composites that may have applicability in technological and biomedical usesPeer ReviewedPostprint (author's final draft

Antibacterial lignin-based nanoparticles and their use in composite materials

Lignin, one of the most abundant biopolymers on earth, has been traditionally considered a low-value by-product of the pulp and paper industries. This renewable raw material, besides being a source of valuable molecules for the chemical industry, also has antioxidant, UV-absorbing, and antibacterial properties in its macromolecular form. Moreover, lignin in the form of nanoparticles (LigNPs) presents advantages over bulk lignin, such as higher reactivity due to its larger surface-to-volume ratio. In view of the rapid surge of antimicrobial resistance (AMR), caused by the overuse of antibiotics, continuous development of novel antibacterial agents is needed. The use of LigNPs as antibacterial agents is a suitable alternative to conventional antibiotics for topical application or chemical disinfectants for surfaces and packaging. Besides, their multiple and unspecific targets in the bacterial cell may prevent the emergence of AMR. This review summarizes the latest developments in antibacterial nano-formulated lignin, both in dispersion and embedded in materials. The following roles of lignin in the formulation of antibacterial NPs have been analyzed: (i) an antibacterial active in nanoformulations, (ii) a reducing and capping agent for antimicrobial metals, and (iii) a carrier of other antibacterial agents. Finally, the review covers the inclusion of LigNPs in films, fibers, hydrogels, and foams, for obtaining antibacterial lignin-based nanocomposites for a variety of applications, including food packaging, wound healing, and medical coatings.Postprint (published version

The thumb domain is not essential for the catalytic action of HoLaMa DNA polymerase

A structural and kinetic characterization of a fragment of the HoLaMa DNA polymerase is presented here. In particular, a truncated form of HoLaMa, devoid of a consistent portion of the thumb domain, was isolated and purified. This HoLaMa fragment, denoted as Î\u94Nter-HoLaMa, is surprisingly competent in catalyzing DNA extension, albeit featuring a kcatone order of magnitude lower than the corresponding kinetic constant of its full-length counterpart. The conformational rearrangements, if any, of enzyme tryptophanes triggered by DNA binding or extension were assayed under pre-steady-state conditions. The fluorescence of HoLaMa tryptophanes was found to significantly change upon DNA binding and extension. On the contrary, no fluorescence changes of Î\u94Nter-HoLaMa tryptophanes were detected under the same conditions, suggesting that major conformational transitions are not required for DNA binding or extension by this truncated DNA polymerase