Melanin Biopolymers: Tailoring Chemical Complexity for Materials Design.

Melanins, a group of dark insoluble pigments found widespread in nature, have become the focus of growing interest in materials science for various biomedical and technological applications, including opto-bioelectronics, nanomedicine and mussel-inspired surface coating. Recent progress in the understanding of melanin optical, paramagnetic redox, and conductivity properties, including photoconductivity, would point to a revision of the traditional concept of structural disorder in terms of more sophisticated and interrelated levels of chemical complexity which however have never been defined and codified. Herein, we bring to focus the various levels of structural disorder that emerged from spectral and chemical signatures over the past decade. A revised approach to structure-property relationships in terms of intermolecular interactions is also provided that may pave the way towards the rational design of next-generation melanin-based functional materials.

Spontaneous wrinkle emergence in nascent eumelanin thin films.

Self-patterning processes originated by physical stimuli have been extensively documented in thin films, whereas spontaneous wrinkling phenomena due to chemical transformation processes are, to the best of our knowledge, unprecedented. Herein we report a case of spontaneous polymerization-driven surface nano-patterning (∼500 nm) that develops in smooth thin solid films of 5,6-dihydroxyindole (DHI), a major precursor of eumelanin polymers, over a time scale of 30 to 60 days in air at room temperature. The phenomenon can be observed only above a critical film thickness of ∼250 nm and it is affected by exposure to ammonia vapors causing acceleration of the oxidation process. The thickness-dependent onset of wrinkling can be attributed to non-homogeneous rates of oxidation through the film causing slow swelling/expansion of the inner layers followed by fast stiffening and cross-linking in the outer layer exposed to higher oxygen levels.

Surface-Functionalization of Nanostructured Cellulose Aerogels by Solid State Eumelanin Coating.

Bioinspired aerogel functionalization by surface modification and coating is in high demand for biomedical and technological applications. In this paper, we report an expedient three-step entry to all-natural surface-functionalized nanostructured aerogels based on (a) TEMPO/NaClO promoted synthesis of cellulose nanofibers (TOCNF); (b) freeze-drying for aerogel preparation; and (c) surface coating with a eumelanin thin film by ammonia-induced solid state polymerization (AISSP) of 5,6-dihydroxyindole (DHI) or 5,6-dihydroxyindole-2-carboxylic acid (DHICA) previously deposited from an organic solution. Scanning electron microscopy showed uniform deposition of the dark eumelanin coating on the template surface without affecting porosity, whereas solid state (13)C NMR and electron paramagnetic resonance (EPR) spectroscopy confirmed the eumelanin-type character of the coatings. DHI melanin coating was found to confer to TOCNF templates a potent antioxidant activity, as tested by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays as well as strong dye adsorption capacity, as tested on methylene blue. The unprecedented combination of nanostructured cellulose and eumelanin thin films disclosed herein implements an original all-natural multifunctional aerogel biomaterial realized via an innovative coating methodology.

Superior photoprotective motifs and mechanisms in eumelanins uncovered.

Human pigmentation is a complex phenomenon commonly believed to serve a photoprotective function through the generation and strategic localization of black insoluble eumelanin biopolymers in sun exposed areas of the body. Despite compelling biomedical relevance to skin cancer and melanoma, eumelanin photoprotection is still an enigma: What makes this pigment so efficient in dissipating the excess energy brought by harmful UV-light as heat? Why has Nature selected 5,6-dihydroxyindole-2-carboxylic acid (DHICA) as the major building block of the pigment instead of the decarboxylated derivative (DHI)? By using pico- and femtosecond fluorescence spectroscopy we demonstrate herein that the excited state deactivation in DHICA oligomers is 3 orders of magnitude faster compared to DHI oligomers. This drastic effect is attributed to their specific structural patterns enabling multiple pathways of intra- and interunit proton transfer. The discovery that DHICA-based scaffolds specifically confer uniquely robust photoprotective properties to natural eumelanins settles a fundamental gap in the biology of human pigmentation and opens the doorway to attractive advances and applications.

Cyan Hydrogen Process: A New Route for Simultaneous Hydrogen Production and Carbon Valorization.

Hydrogen is expected to largely contribute to the near-future circular economy. Today, most hydrogen is still produced from fossil fuels or renewable pathways with low efficiency and high cost. Herein, a proof of concept for a novel hydrogen production process is proposed, here named cyan hydrogen, inspired by a combination of the green and blue processes, due to the key role played by water and the low carbon content in the gas phase, respectively. The developed novel process, recently patented and demonstrated at the lab scale, is based on successive steps in which ethanol (5.0 mL) and water (10.0 mL) are alternately fed, with a fixed initial amount of sodium metaborate (2.0 g), in a batch reactor at 300 °C. Preliminary results showed the simultaneous production of a 95% v/v hydrogen stream, a polymeric byproduct with a repetitive carbon pattern -CH2-CH2-, and a liquid phase rich in oxygenated chemicals at temperatures lower than conventional hydrogen production processes.

Designing bioinspired multifunctional nanoplatforms to support wound healing and skin regeneration: Mg-hydroxyapatite meets melanins.

Melanin is a multifunctional biological pigment that recently emerged as endowed with anti-inflammatory, antioxidant, and antimicrobial properties and with high potentialities in skin protection and regenerative medicine. Here, a biomimetic magnesium-doped nano-hydroxyapatite (MgHA) was synthesized and decorated with melanin molecules starting from two different monomeric precursors, i.e. 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and dopamine (DA), demonstrating to be able to polymerize on the surface of MgHA nanostructures, thus leading to a melanin coating. This functionalization was realized by a simple and green preparation method requiring mild conditions in an aqueous medium and room temperature. Complementary spectroscopy and electron imaging analyses were carried out to define the effective formation of a stable coating, the percentage of the organic compounds, and the structural properties of resulting melanin-coated nanostructures, which showed good antioxidant activity. The in vitro interaction with a cell model, i.e. mouse fibroblasts, was investigated. The excellent biocompatibility of all bioinspired nanostructures was confirmed from a suitable cell proliferation. Finally, the enhanced biological performances of the nanostructures coated with melanin from DHICA were confirmed by scratch assays. Jointly our findings indicated that low crystalline MgHA and melanin pigments can be efficiently combined, and the resulting nanostructures are promising candidates as multifunctional platforms for a more efficient approach for skin regeneration and protection.

Biosustainable Hybrid Nanoplatforms as Photoacoustic Agents.

The development of biosafe theranostic nanoplatforms has attracted great attention due to their multifunctional behavior, reduced potential toxicity, and improved long-term safety. When considering photoacoustic contrast agents and photothermal conversion tools, melanin and constructs like melanin are highly appealing due to their ability to absorb optical energy and convert it into heat. Following a sustainable approach, in this study, silver-melanin like-silica nanoplatforms are synthesized exploiting different bio-available and inexpensive phenolic acids as potential melanogenic precursors and exploring their role in tuning the final systems architecture. The UV-Vis combined with X-Ray Diffraction investigation proves metallic silver formation, while Transmission Electron Microscopy analysis reveals that different morphologies can be obtained by properly selecting the phenolic precursors. By looking at the characterization results, a tentative formation mechanism is proposed to explain how phenolic precursors' redox behavior may affect the nanoplatforms' structure. The antibacterial activity experiments showed that all synthesized systems have a strong inhibitory effect on Escherichia coli, even at low concentrations. Furthermore, very sensitive Photoacoustic Imaging capabilities and significant photothermal behavior under laser irradiation are exhibited. Finally, a marked influence of phenol nature on the final system architecture is revealed resulting in a significant effect on both biological and photoacoustic features of the obtained systems. These melanin-based hybrid systems exhibit excellent potential as triggerable nanoplatforms for various biomedical applications.

Free radical coupling of o-semiquinones uncovered.

As a rule, o-semiquinones decay through disproportionation leading to equimolar amounts of catechol and o-quinone products. However, the o-semiquinone 1S generated by pulse radiolysis oxidation of the eumelanin precursor 5,6-dihydroxyindole (1) decays with second-order kinetics to generate broad visible chromophores that are incompatible with the predicted absorption of 5,6-indolequinone (1Q). Using an integrated chemical, pulse radiolytic and computational approach as well as deuterium labeling, we show herein that 1S and related 5,6-dihydroxyindole semiquinones decay mainly by a free radical coupling mechanism. This conclusion was supported by the inverse kinetic isotope effect observed with deuterated 1S, the identification of unprecedented dihydrobiindole products by one-electron oxidation of 1, the good matching of simulated absorption profiles of free radical coupling products of 1S with experimental spectra, and a detailed computational analysis of the kinetics and thermodynamics of the disproportionation equilibrium and free radical coupling of 1S versus 1-1Q coupling. These results disclose, to the best of our knowledge, the first example of free radical dimerization of o-semiquinones outcompeting the classic disproportionation-driven catechol-quinone coupling and suggest that this hitherto unrecognized process may be of broader relevance than previously believed.

Neuroglobin modification by reactive quinone species.

The physiological functions of neuroglobin (Ngb), the heme protein of the globin family expressed in the nervous tissue, have not yet been clarified. Besides O2 storage and homeostasis, Ngb is thought to play a role in neuroprotection as a scavenger of toxic reactive species generated in vivo under conditions of oxidative stress. Herein, the interaction of Ngb with the quinones generated by oxidation of catecholamines (dopamine, norepinephrine) and catechol estrogens (2-hydroxyestradiol and 4-hydroxyestradiol), which have been implicated in neurodegenerative pathologies like Parkinson's and Alzheimer's diseases, has been investigated. The cytotoxicity of quinones has been ascribed to the derivatization of amino acid residues (mainly cysteine) in proteins through the formation of covalent bonds with the aromatic rings. Combined studies of tandem mass spectrometry and protein unfolding indicate the presence of quinone-promoted modifications in all of the Ngb derivatives analyzed (i.e., obtained employing either catecholamines or catechol estrogens as the source of the reactive species). Among protein residues, the highest reactivity of cysteines (Cys46, Cys55, and Cys120 in human Ngb) toward quinone species has been confirmed, and the dependence of the extent of protein modification on the method employed for catechol oxidation has been observed. When the oxidation reaction proceeds by one-electron steps, the involvement of semiquinone reactivity has been observed. The whole analysis of the data of Ngb modification suggests that the catecholamine-oxidation products can extensively modify proteins (likely by catecholamine oligomers, the compounds initially formed during the transformation of catecholamine to melanin). The modification mediated by catechol estrogens is less pronounced but strongly affects the interactions with the solvent as well as the protein stability.

Enhanced Photoacoustic Response by Synergistic Ag-Melanin Interplay at the Core of Ternary Biocompatible Hybrid Silica-Based Nanoparticles.

Photoacoustics (PA) is gaining increasing credit among biomolecular imaging methodologies by virtue of its poor invasiveness, deep penetration, high spatial resolution, and excellent endogenous contrast, without the use of any ionizing radiation. Recently, we disclosed the excellent PA response of a self-structured biocompatible nanoprobe, consisting of ternary hybrid nanoparticles with a silver core and a melanin component embedded into a silica matrix. Although preliminary evidence suggested a crucial role of the Ag sonophore and the melanin-containing nanoenvironment, whether and in what manner the PA response is controlled and affected by the self-structured hybrid nanosystems remained unclear. Because of their potential as multifunctional platforms for biomedical applications, a detailed investigation of the metal-polymer-matrix interplay underlying the PA response was undertaken to understand the physical and chemical factors determining the enhanced response and to optimize the architecture, composition, and performance of the nanoparticles for efficient imaging applications. Herein, we provide the evidence for a strong synergistic interaction between eumelanin and Ag which suggests an important role in the in situ-generated metal-organic interface. In particular, we show that a strict ratio between melanin and silver precursors and an accurate choice of metal nanoparticle dimension and the kind of metal are essential for achieving strong enhancements of the PA response. Systematic variation of the metal/melanin component is thus shown to offer the means of tuning the stability and intensity of the photoacoustic response for various biomedical and theranostic applications.

Nanoparticle Coatings on Glass Surfaces to Prevent Pseudomonas fluorescens AR 11 Biofilm Formation.

Microbial colonization of surfaces is a sanitary and industrial issue for many applications, leading to product contamination and human infections. When microorganisms closely interact with a surface, they start to produce an exo-polysaccaridic matrix to adhere to and protect themselves from adverse environmental conditions. This type of structure is called a biofilm. The aim of our work is to investigate novel technologies able to prevent biofilm formation by surface coatings. We coated glass surfaces with melanin-ZnO2, melanin-TiO2, and TiO2 hybrid nanoparticles. The functionalization was performed using cold plasma to activate glass-substrate-coated surfaces, that were characterized by performing water and soybean oil wetting tests. A quantitative characterization of the antibiofilm properties was done using Pseudomonas fluorescens AR 11 as a model organism. Biofilm morphologies were observed using confocal laser scanning microscopy and image analysis techniques were used to obtain quantitative morphological parameters. The results highlight the efficacy of the proposed surface coating to prevent biofilm formation. Melanin-TiO2 proved to be the most efficient among the particles investigated. Our results can be a valuable support for future implementation of the technique proposed here in an extended range of applications that may include further testing on other strains and other support materials.

High density polyethylene (HDPE) biodegradation by the fungus Cladosporium halotolerans.

Polyethylene (PE) is high molecular weight synthetic polymer, very hydrofobic and hardly biodegradable. To increase polyethylene bio-degradability it is very important to find microorganisms that improve the PE hydrophilic level and/or reduce the length of its polymeric chain by oxidation. In this study, we isolated Cladosporium halotolerans, a fungal species, from the gastric system of Galleria mellonella larvae. Here, we show that C. halotolerans grows in the presence of PE polymer, it is able to interact with plastic material through its hyphae and secretes enzymes involved in PE degradation.

Eumelanin decorated poly(lactic acid) electrospun substrates as a new strategy for spinal cord injury treatment.

Spinal cord injury (SCI) is characterized by neuroinflammatory processes that are marked by an uncontrolled activation of microglia, which directly damages neurons. Natural and synthetic melanins represent an effective tool to treat neuroinflammation because they possess immunomodulatory properties. Here, the main objective was to evaluate the effect of eumelanin-coated poly(lactic acid) (EU@PLA) aligned microfibers on in vitro model of neuroinflammation related to spinal cord injury in terms of inflammatory mediators' modulation. Aligned fibers were chosen to provide physical cues to guide axonal growth in a specific direction thus restoring the synaptic connection. Eumelanin decorated PLA electrospun substrates were produced combining electrospinning, spin coating and solid-state polymerization processes (oxidative coupling under oxygen atmosphere). Biological response in terms of antioxidant and anti-inflammatory activity was analyzed on an in vitro model of neuroinflammation [microglial cells stimulated with lipopolysaccharide (LPS)]. Cell morphology and EU@PLA mechanism of action, in terms of toll-like receptor-4 (TLR-4) involvement were assessed. The results show that EU@PLA fibers were able to decrease reactive oxygen species, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) expression >50 % compared to PLA + LPS and interleukin 6 (IL-6) secretion about 20 %. Finally, the mechanism of action of EU@PLA in microglia was found to be dependent on the TLR-4 signaling. Protein expression analysis revealed a decreased in TLR-4 production induced by LPS stimulation in presence of EU@PLA. Overall, our results show that EU@PLA represents an innovative and effective strategy for the control of inflammatory response in central nervous system.

Bioactive Composite Methacrylated Gellan Gum for 3D-Printed Bone Tissue-Engineered Scaffolds.

Gellan gum (GG) was chemically modified with methacrylic moieties to produce a photocrosslinkable biomaterial ink, hereinafter called methacrylated GG (GGMA), with improved physico-chemical properties, mechanical behavior and stability under physiological conditions. Afterwards, GGMA was functionalized by incorporating two different bioactive compounds, a naturally derived eumelanin extracted from the black soldier fly (BSF-Eumel), or hydroxyapatite nanoparticles (HAp), synthesized by the sol-gel method. Different ink formulations based on GGMA (2 and 4% (w/v)), BSF-Eumel, at a selected concentration (0.3125 mg/mL), or HAp (10 and 30% wHAp/wGGMA) were developed and processed by three-dimensional (3D) printing. All the functionalized GGMA-based ink formulations allowed obtaining 3D-printed GGMA-based scaffolds with a well-organized structure. For both bioactive signals, the scaffolds with the highest GGMA concentration (4% (w/v)) and the highest percentage of infill (45%) showed the best performances in terms of morphological and mechanical properties. Indeed, these scaffolds showed a good structural integrity over 28 days. Given the presence of negatively charged groups along the eumelanin backbone, scaffolds consisting of GGMA/BSF-Eumel demonstrated a higher stability. From a mechanical point of view, GGMA/BSF-Eumel scaffolds exhibited values of storage modulus similar to those of GGMA ones, while the inclusion of HAp at 30% (wHAp/wGGMA) led to a storage modulus of 32.5 kPa, 3.5-fold greater than neat GGMA. In vitro studies proved the capability of the bioactivated 3D-printed scaffolds to support 7F2 osteoblast cell growth and differentiation. BSF-Eumel and HAp triggered a different time-dependent physiological response in the osteoblasts. Specifically, while the ink with BSF-Eumel acted as a stimulus towards cell proliferation, reaching the highest value at 14 days, a higher expression of alkaline phosphatase activity was detected for scaffolds consisting of GGMA and HAp. The overall findings demonstrated the possible use of these biomaterial inks for 3D-printed bone tissue-engineered scaffolds.

Towards nanostructured red-ox active bio-interfaces: Bioinspired antibacterial hybrid melanin-CeO2 nanoparticles for radical homeostasis.

Redox-active nano-biointerfaces are gaining weight in the field of regenerative medicine since they can act as enzymes in regulating physiological processes and enabling cell homeostasis, as well as the defense against pathogen aggression. In particular, cerium oxide nanoparticles (CeO2 NPs) stand as intriguing enzyme-mimicking nanoplatforms, owing to the reversible Ce+3/Ce+4 surface oxidation state. Moreover, surface functionalization leads to higher catalytic activity and selectivity, as well as more tunable enzyme-mimicking performances. Conjugation with melanin is an adequate strategy to boost and enrich CeO2 NPs biological features, because of melanin redox properties accounting for intrinsic antioxidant, antimicrobial and anti-inflammatory power. Herein, hybrid Melanin/CeO2 nanostructures were designed by simply coating the metal-oxide nanoparticles with melanin chains, obtained in-situ through ligand-to-metal charge transfer mechanism, according to a bioinspired approach. Obtained hybrid nanostructures underwent detailed physico-chemical characterization. Morphological and textural features were investigated through TEM, XRD and N2 physisorption. The nature of nanoparticle-melanin interaction was analyzed through FTIR, UV-vis and EPR spectroscopy. Melanin-coated hybrid nanostructures exhibited a relevant antioxidant activity, confirmed by a powerful quenching effect for DPPH radical, reaching 81 % inhibition at 33 µg/mL. A promising anti-inflammatory efficacy of the melanin-coated hybrid nanostructures was validated through a significant inhibition of BSA denaturation after 3 h. Meanwhile, the enzyme-mimicking activity was corroborated by a prolonged peroxidase activity after 8 h at 100 µg/mL and a relevant catalase-like action, by halving the H2O2 level in 30 min at 50 µg/mL. Antimicrobial assays attested that conjugation with melanin dramatically boosted CeO2 biocide activity against both Gram (-) and Gram (+) strains. Cytocompatibility tests demonstrated that the melanin coating not only enhanced the CeO2 nanostructures biomimicry, resulting in improved cell viability for human dermal fibroblast cells (HDFs), but mostly they proved that Melanin-CeO2 NPs were able to control the oxidative stress, modulating the production of nitrite and reactive oxygen species (ROS) levels in HDFs, under physiological conditions. Such remarkable outcomes make hybrid melanin-CeO2 nanozymes, promising redox-active interfaces for regenerative medicine.

Eumelanin buildup on the nanoscale: aggregate growth/assembly and visible absorption development in biomimetic 5,6-dihydroxyindole polymerization.

Establishing structure-property relationships in the black insoluble eumelanins, the key determinants of human pigmentation and skin photoprotective system, is a considerable conceptual and experimental challenge in the current drive for elucidation of the biological roles of these biopolymers and their application as advanced materials for organoelectronics. Herein, we report a new breakthrough toward this goal by the first detailed investigation on the nanoscale level of the oxidative polymerization of 5,6-dihydroxyindole (DHI), a model process of eumelanin synthesis. On the basis of a combined use of spectrophotometry, dynamic light scattering (DLS), and small-angle neutron scattering (SANS) investigations, it was possible to unveil the dynamics of the aggregation process before precipitation, the key relationships with visible light absorption and the shape of fundamental aggregates. The results indicated a polymerization mechanism of the type: Polymer(n) + DHI(x) = Polymer(n+x), where DHI(x) indicates monomer, dimer, or low oligomers (x ≤ 5). During polymerization, visible absorption increases rapidly, reaching a plateau. Particle growth proceeds slowly, with formation of 2-D structures ~55 nm thick, until precipitation occurs, that is, when large aggregates with a maximum hydrodynamic radius (R(h)) of ~1200 nm are formed. Notably, markedly smaller R(h) values, up to ~110 nm, were determined in the presence of poly(vinyl alcohol) (PVA) that was shown to be an efficient aggregation-preventing agent for polymerizing DHI ensuring water solubilization. Finally, it is shown that DHI monomer can be efficiently and partially irreversibly depleted from aqueous solutions by the addition of eumelanin suspensions. This behavior is suggested to reflect oxidant-independent competing pathways of polymer synthesis and buildup via monomer conversion on the active aggregate surface contributing to particle growth. Besides filling crucial gaps in DHI polymerization, these results support the attractive hypothesis that eumelanins may behave as a peculiar example of living biopolymers. The potential of PVA as a powerful tool for solution chemistry-based investigations of eumelanin supramolecular organization and for technological manipulation purposes is underscored.

Eumelanin pigment precursor 2-carboxy-5,6-dihydroxyindole and 2-amino-6-methylbenzothiazole chromophore integration towards melanin inspired chemoresponsive materials: the case of the Zn2+ ion.

The 2-amino-6-methylbenzothiazole chromophore is introduced at the carboxyl group of the melanin precursor 2-carboxy-5,6-dihydroxyindole achieving a novel dihydroxyindole derivative with metal chelation properties not depending on the catechol moiety. In view of potential exploitation in charge storage systems, systematic investigation of the interaction of the new amide derivative with metal ions is carried out, in comparison with that of the parent 2-carboxy-5,6-dihydroxyindole, and the stoichiometry of the zinc-amide complex is determined.

The interplay of chemical structure, physical properties, and structural design as a tool to modulate the properties of melanins within mesopores.

The design of modern devices that can fulfil the requirements for sustainability and renewable energy applications calls for both new materials and a better understanding of the mixing of existing materials. Among those, surely organic-inorganic hybrids are gaining increasing attention due to the wide possibility to tailor their properties by accurate structural design and materials choice. In this work, we'll describe the tight interplay between porous Si and two melanic polymers permeating the pores. Melanins are a class of biopolymers, known to cause pigmentation in many living species, that shows very interesting potential applications in a wide variety of fields. Given the complexity of the polymerization process beyond the formation and structure, the full understanding of the melanins' properties remains a challenging task. In this study, the use of a melanin/porous Si hybrid as a tool to characterize the polymer's properties within mesopores gives new insights into the conduction mechanisms of melanins. We demonstrate the dramatic effect induced on these mechanisms in a confined environment by the presence of a thick interface. In previous studies, we already showed that the interactions at the interface between porous Si and eumelanin play a key role in determining the final properties of composite materials. Here, thanks to a careful monitoring of the photoconductivity properties of porous Si filled with melanins obtained by ammonia-induced solid-state polymerization (AISSP) of 5,6-dihydroxyindole (DHI) or 1,8-dihydroxynaphthalene (DHN), we investigate the effect of wet, dry, and vacuum cycles of storage from the freshly prepared samples to months-old samples. A computational study on the mobility of water molecules within a melanin polymer is also presented to complete the understanding of the experimental data. Our results demonstrate that: (a) the hydration-dependent behavior of melanins is recovered in large pores (≈ 60 nm diameter) while is almost absent in thinner pores (≈ 20 nm diameter); (b) DHN-melanin materials can generate higher photocurrents and proved to be stable for several weeks and more sensitive to the wet/dry variations.

Eumelanin from the Black Soldier Fly as Sustainable Biomaterial: Characterisation and Functional Benefits in Tissue-Engineered Composite Scaffolds.

An optimized extraction protocol for eumelanins from black soldier flies (BSF-Eumel) allows an in-depth study of natural eumelanin pigments, which are a valuable tool for the design and fabrication of sustainable scaffolds. Here, water-soluble BSF-Eumel sub-micrometer colloidal particles were used as bioactive signals for developing a composite biomaterial ink for scaffold preparation. For this purpose, BSF-Eumel was characterized both chemically and morphologically; moreover, biological studies were carried out to investigate the dose-dependent cell viability and its influence on human mesenchymal stem cells (hMSCs), with the aim of validating suitable protocols and to find an optimal working concentration for eumelanin-based scaffold preparation. As proof of concept, 3D printed scaffolds based on methacrylated hyaluronic acid (MEHA) and BSF-Eumel were successfully produced. The scaffolds with and without BSF-Eumel were characterized in terms of their physico-chemical, mechanical and biological behaviours. The results showed that MEHA/BSF-Eumel scaffolds had similar storage modulus values to MEHA scaffolds. In terms of swelling ratio and stability, these scaffolds were able to retain their structure without significant changes over 21 days. Biological investigations demonstrated the ability of the bioactivated scaffolds to support the adhesion, proliferation and osteogenic differentiation of human mesenchymal stem cells.

Tuning the specificity of the recombinant multicomponent toluene o-xylene monooxygenase from Pseudomonas sp. strain OX1 for the biosynthesis of tyrosol from 2-phenylethanol.

Biocatalysis is today a standard technology for the industrial production of several chemicals, and the number of biotransformation processes running on a commercial scale is constantly increasing. Among biocatalysts, bacterial multicomponent monooxygenases (BMMs), a diverse group of nonheme diiron enzymes that activate dioxygen, are of primary interest due to their ability to catalyze a variety of complex oxidations, including reactions of mono- and dihydroxylation of phenolic compounds. In recent years, both directed evolution and rational design have been successfully used to identify the molecular determinants responsible for BMM regioselectivity and to improve their activity toward natural and nonnatural substrates. Toluene o-xylene monooxygenase (ToMO) is a BMM isolated from Pseudomonas sp. strain OX1 which hydroxylates a wide spectrum of aromatic compounds. In this work we investigate the use of recombinant ToMO for the biosynthesis in recombinant cells of Escherichia coli strain JM109 of 4-hydroxyphenylethanol (tyrosol), an antioxidant present in olive oil, from 2-phenylethanol, a cheap and commercially available substrate. We initially found that wild-type ToMO is unable to convert 2-phenylethanol to tyrosol. This was explained by using a computational model which analyzed the interactions between ToMO active-site residues and the substrate. We found that residue F176 is the major steric hindrance for the correct positioning of the reaction intermediate leading to tyrosol production into the active site of the enzyme. Several mutants were designed and prepared, and we found that the combination of different mutations at position F176 with mutation E103G allows ToMO to convert up to 50% of 2-phenylethanol into tyrosol in 2 h.