Development of acid-free chitosan films in food coating applications: Provolone cheese as a case study.

Chitosan has been extensively explored in food coatings. Still, its practical application is largely hampered by its conventional wet processing in acetic acid, whose residuals negatively impact food quality and safety. Here, we propose a new method to formulate chitosan coatings for food applications by avoiding organic acid processing and validate them on a cheese model. The procedure entails modifying a previously reported process based on HCl chitosan treatment and neutralising the resulting gel. The obtained chitosan is solubilised in water using carbonic acid that forms in situ by dissolving carbon dioxide gas. The reversibility of water carbonation allows for easy removal of carbonic acid residues, resulting in acid-free chitosan films and coatings. The performance of the coating was tested against state-of-the-art chitosan-based and polymeric coatings. We preliminarily characterised the films' properties (water stability, barrier, and optical properties). Then, we assessed the performance of the coating on Provolone cheese as a food model (mass transfer and texture profiles over 14 days). The work demonstrated the advantage of the proposed approach in solving some main issues of food quality and safety, paving the way for an effective application of chitosan in future food contact applications.

Editorial on the Special Issue: "Advances in Xerogels: From Design to Applications".

Xerogels are solid materials derived from gels which consist of interconnected particles or polymers dispersed in a liquid [...].

Towards a More Realistic In Vitro Meat: The Cross Talk between Adipose and Muscle Cells.

According to statistics and future predictions, meat consumption will increase in the coming years. Considering both the environmental impact of intensive livestock farming and the importance of protecting animal welfare, the necessity of finding alternative strategies to satisfy the growing meat demand is compelling. Biotechnologies are responding to this demand by developing new strategies for producing meat in vitro. The manufacturing of cultured meat has faced criticism concerning, above all, the practical issues of culturing together different cell types typical of meat that are partly responsible for meat's organoleptic characteristics. Indeed, the existence of a cross talk between adipose and muscle cells has critical effects on the outcome of the co-culture, leading to a general inhibition of myogenesis in favor of adipogenic differentiation. This review aims to clarify the main mechanisms and the key molecules involved in this cross talk and provide an overview of the most recent and successful meat culture 3D strategies for overcoming this challenge, focusing on the approaches based on farm-animal-derived cells.

Eco-Friendly Engineered Nanomaterials Coupled with Filtering Fine-Mesh Net as a Promising Tool to Remediate Contaminated Freshwater Sludges: An Ecotoxicity Investigation.

The use of eco-friendly engineered nanomaterials represents a recent solution for an effective and safe treatment of contaminated dredging sludge. In this study, an eco-designed engineered material based on cross-linked nanocellulose (CNS) was applied for the first time to decontaminate a real matrix from heavy metals (namely Zn, Ni, Cu, and Fe) and other undesired elements (mainly Ba and As) in a lab-scale study, with the aim to design a safe solution for the remediation of contaminated matrices. Contaminated freshwater sludge was treated with CNS coupled with a filtering fine-mesh net, and the obtained waters were tested for acute and sublethal toxicity. In order to check the safety of the proposed treatment system, toxicity tests were conducted by exposing the bacterium Aliivibrio fischeri and the crustacean Heterocypris incongruens, while subtoxicity biomarkers such as lysosomal membrane stability, genetic, and chromosomal damage assessment were performed on the freshwater bivalve Dreissena polymorpha. Dredging sludge was found to be genotoxic, and such genotoxicity was mitigated by the combined use of CNS and a filtering fine-mesh net. Chemical analyses confirmed the results by highlighting the abetment of target contaminants, indicating the present model as a promising tool in freshwater sludge nanoremediation.

Smart Methylcellulose Hydrogels for pH-Triggered Delivery of Silver Nanoparticles.

Infection is a severe complication in chronic wounds, often leading to morbidity or mortality. Current treatments rely on dressings, which frequently contain silver as a broad-spectrum antibacterial agent, although improper dosing can result in severe side effects. This work proposes a novel methylcellulose (MC)-based hydrogel designed for the topical release of silver nanoparticles (AgNPs) via an intelligent mechanism activated by the pH variations in infected wounds. A preliminary optimization of the physicochemical and rheological properties of MC hydrogels allowed defining the optimal processing conditions in terms of crosslinker (citric acid) concentration, crosslinking time, and temperature. MC/AgNPs nanocomposite hydrogels were obtained via an in situ synthesis process, exploiting MC both as a capping and reducing agent. AgNPs with a 12.2 ± 2.8 nm diameter were obtained. MC hydrogels showed a dependence of the swelling and degradation behavior on both pH and temperature and a noteworthy pH-triggered release of AgNPs (release ~10 times higher at pH 12 than pH 4). 1H-NMR analysis revealed the role of alkaline hydrolysis of the ester bonds (i.e., crosslinks) in governing the pH-responsive behavior. Overall, MC/AgNPs hydrogels represent an innovative platform for the pH-triggered release of AgNPs in an alkaline milieu.

3D Bioprinting of Pectin-Cellulose Nanofibers Multicomponent Bioinks.

Pectin has found extensive interest in biomedical applications, including wound dressing, drug delivery, and cancer targeting. However, the low viscosity of pectin solutions hinders their applications in 3D bioprinting. Here, we developed multicomponent bioinks prepared by combining pectin with TEMPO-oxidized cellulose nanofibers (TOCNFs) to optimize the inks' printability while ensuring stability of the printed hydrogels and simultaneously print viable cell-laden inks. First, we screened several combinations of pectin (1%, 1.5%, 2%, and 2.5% w/v) and TOCNFs (0%, 0.5%, 1%, and 1.5% w/v) by testing their rheological properties and printability. Addition of TOCNFs allowed increasing the inks' viscosity while maintaining shear thinning rheological response, and it allowed us to identify the optimal pectin concentration (2.5% w/v). We then selected the optimal TOCNFs concentration (1% w/v) by evaluating the viability of cells embedded in the ink and eventually optimized the writing speed to be used to print accurate 3D grid structures. Bioinks were prepared by embedding L929 fibroblast cells in the ink printed by optimized printing parameters. The printed scaffolds were stable in a physiological-like environment and characterized by an elastic modulus of E = 1.8 ± 0.2 kPa. Cells loaded in the ink and printed were viable (cell viability >80%) and their metabolic activity increased in time during the in vitro culture, showing the potential use of the developed bioinks for biofabrication and tissue engineering applications.

Cellular Responses Induced by Zinc in Zebra Mussel Haemocytes. Loss of DNA Integrity as a Cellular Mechanism to Evaluate the Suitability of Nanocellulose-Based Materials in Nanoremediation.

Zinc environmental levels are increasing due to human activities, posing a threat to ecosystems and human health. Therefore, new tools able to remediate Zn contamination in freshwater are highly recommended. Specimens of Dreissena polymorpha (zebra mussel) were exposed for 48 h and 7 days to a wide range of ZnCl2 nominal concentrations (1-10-50-100 mg/L), including those environmentally relevant. Cellulose-based nanosponges (CNS) were also tested to assess their safety and suitability for Zn removal from freshwater. Zebra mussels were exposed to 50 mg/L ZnCl2 alone or incubated with 1.25 g/L of CNS (2 h) and then removed by filtration. The effect of Zn decontamination induced by CNS has been verified by the acute toxicity bioassay Microtox®. DNA primary damage was investigated by the Comet assay; micronuclei frequency and nuclear morphological alterations were assessed by Cytome assay in mussels' haemocytes. The results confirmed the genotoxic effect of ZnCl2 in zebra mussel haemocytes at 48 h and 7-day exposure time. Zinc concentrations were measured in CNS, suggesting that cellulose-based nanosponges were able to remove Zn(II) by reducing its levels in exposure waters and soft tissues of D. polymorpha in agreement with the observed restoration of genetic damage exerted by zinc exposure alone.

Bioactive Hydrogels: Design and Characterization of Cellulose-Derived Injectable Composites.

Cellulose represents a low cost, abundant, and renewable polysaccharide with great versatility; it has a hierarchical structure composed of nanofibers with high aspect ratio (3-4 nm wide, hundreds of µm long). TEMPO-mediated oxidation represents one of the most diffused methods to obtain cellulose nanofibers (CNFs): It is possible to obtain physically crosslinked hydrogels by means of divalent cation addition. The presence of inorganic components, such as calcium phosphates (CaP), can improve not only their mechanical properties but also the bioactivity of the gels. The aim of this work is to design and characterize a TEMPO-oxidized cellulose nanofibers (TOCNFs) injectable hydrogel embedded with inorganic particles, CaP and CaP-GO, for bone tissue regeneration. Inorganic particles act as physical crosslinkers, as proven by rheological characterization, which reported an increase in mechanical properties. The average load value registered in injection tests was in the range of 1.5-4.4 N, far below 30 N, considered a reasonable injection force upper limit. Samples were stable for up to 28 days and both CaP and CaP-GO accelerate mineralization as suggested by SEM and XRD analysis. No cytotoxic effects were shown on SAOS-2 cells cultured with eluates. This work demonstrated that the physicochemical properties of TOCNFs-based dispersions could be enhanced and modulated through the addition of the inorganic phases, maintaining the injectability and bioactivity of the hydrogels.

Dispersions of Zirconia Nanoparticles Close to the Phase Boundary of Surfactant-Free Ternary Mixtures.

The achievement of a homogeneous dispersion of nanoparticles is of paramount importance in supporting their technological application. In wet processing, stable dispersions were largely obtained via surfactant or surface functionalization: although effective, the use of dispersant can alter, or even impair, the functional properties of the resulting nanostructured systems. Herein, we report a novel integrated modeling and experimental approach to obtain stable ZrO2 nanoparticle (NP) dispersions at native dimensions (about 5 nm) in homogeneous ternary mixtures of solvents (i.e., water, ethanol, and 1,2-dichlorobenzene) without any further surface functionalization. A miscibility ternary diagram was computed exploiting the universal quasi-chemical functional-group activity coefficient (UNIFAC) model, which was then experimentally validated. Dynamic light scattering (DLS) on these mixtures highlights that nanometric structures, resembling nanoemulsion droplets, form close to the mixture two-phase boundary, with a size that depends on the ternary mixture composition. ZrO2-NPs were then synthesized following a classic sol-gel approach and characterized by XRD and Raman spectroscopy. ZrO2-NPs were dispersed in HCl and mixed with different mixtures of ethanol and 1,2-dichlorobenzene (DCB), obtaining homogeneous and stable dispersions. These dispersions were then studied by means of DLS as a function of DCB concentration, observing that the nanoparticles can be dispersed at their native dimensions when the mass fraction of DCB was lower than 60%, whereas the increase of the hydrophobic solvent leads to the NPs' agglomeration and sedimentation. The proposed approach not only offers specific guidelines for the design of ZrO2-NPs dispersions in a ternary solvent mixture but can also be extended to other complex solvent mixtures in order to achieve stable dispersions of nanoparticles with no functionalization.

2D Correlation Spectroscopy (2DCoS) Analysis of Temperature-Dependent FTIR-ATR Spectra in Branched Polyethyleneimine/TEMPO-Oxidized Cellulose Nano-Fiber Xerogels.

Fourier transform infrared spectroscopy in attenuated total reflectance geometry (FTIR-ATR), combined with a 2D correlation analysis, was here employed to investigate temperature-induced spectral changes occurring in a particular type of novel cellulosic-based nano-material prepared using 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) oxidized and ultra-sonicated cellulose nano-fibers (TOUS-CNFs) as three-dimensional scaffolds, and branched polyethyleneimine (bPEI) as cross-linking agent. The aim was to highlight the complex sequential events involving the different functional groups of the polymeric network, as well as to gain insight into the interplay between the amount of bPEI and the resulting sponge-like material, upon increasing temperature. In this framework, synchronous and asynchronous 2D spectra were computed and analyzed in three wavenumber regions (900-1200 cm-1, 1500-1700 cm-1 and 2680-3780 cm-1), where specific vibrational modes of the cellulosic structure fall, and over a T-range between 250 K and 340 K. A step-by-step evolution of the different arrangements of the polymer functional groups was proposed, with particular regard to how the cooperativity degree of inter- and intramolecular hydrogen bonds (HBs) changes upon heating. Information acquired can be useful, in principle, in order to develop a next-generation, T-sensitive novel material to be used for water remediation applications or for drug-delivery nano-vectors.

Synthesis and Application of Cellulose-Polyethyleneimine Composites and Nanocomposites: A Concise Review.

Cellulose/polyethyleneimine composites have increasingly attracted the attention of scientific community, devoted to the design and development of new synthetic strategies and materials for different application fields. In this review, after introducing the main characteristics of the two polymeric components, we provide in the second section a critical overview on the main protocols for the synthesis of these composites, considering both the several cellulose sources and forms, and the different cross-linkers and cross-linking procedures developed for this purpose, outlining advantages and limits for the reported approaches. The last section analyses the principal results obtained in different application fields. A wide discussion is dedicated to the principal use of cellulose/polyethyleneimine composites as sorbents for water remediation from heavy metal ions and organic contaminants. Subsequently, we introduce the literature describing the use of these composites, functionalized appropriately, where necessary, as drug delivery systems, sensors, and heterogeneous catalysts for organic reactions. Finally, after a brief description of other random applications, we furnish a personal analysis of actual limits and potentialities for these systems.

Suitability of a Cellulose-Based Nanomaterial for the Remediation of Heavy Metal Contaminated Freshwaters: A Case-Study Showing the Recovery of Cadmium Induced DNA Integrity Loss, Cell Proliferation Increase, Nuclear Morphology and Chromosomal Alterations on Dreissena polymorpha.

The contamination of freshwaters by heavy metals represents a great problem, posing a threat for human and environmental health. Cadmium is classified as carcinogen to humans and its mechanism of carcinogenicity includes genotoxic events. In this study a recently developed eco-friendly cellulose-based nanosponge (CNS) was investigated as a candidate in freshwater nano-remediation process. For this purpose, CdCl2 (0.05 mg L-1) contaminated artificial freshwater (AFW) was treated with CNS (1.25 g L-1 for 2 h), and cellular responses were analyzed before and after CNS treatment in Dreissena polymorpha hemocytes. A control group (AFW) and a negative control group (CNS in AFW) were also tested. DNA primary damage was evaluated by Comet assay while chromosomal damage and cell proliferation were assessed by Cytome assay. AFW exposed to CNS did not cause any genotoxic effect in zebra mussel hemocytes. Moreover, DNA damage and cell proliferation induced by Cd(II) turned down to control level after 2 days when CNS were used. A reduction of Cd(II)-induced micronuclei and nuclear abnormalities was also observed. CNS was thus found to be a safe and effective candidate in cadmium remediation process being efficient in metal sequestering, restoring cellular damage exerted by Cd(II) exposure, without altering cellular physiological activity.

Silver Nanoparticles for Water Pollution Monitoring and Treatments: Ecosafety Challenge and Cellulose-Based Hybrids Solution.

Silver nanoparticles (AgNPs) are widely used as engineered nanomaterials (ENMs) in many advanced nanotechnologies, due to their versatile, easy and cheap preparations combined with peculiar chemical-physical properties. Their increased production and integration in environmental applications including water treatment raise concerns for their impact on humans and the environment. An eco-design strategy that makes it possible to combine the best material performances with no risk for the natural ecosystems and living beings has been recently proposed. This review envisages potential hybrid solutions of AgNPs for water pollution monitoring and remediation to satisfy their successful, environmentally safe (ecosafe) application. Being extremely efficient in pollutants sensing and degradation, their ecosafe application can be achieved in combination with polymeric-based materials, especially with cellulose, by following an eco-design approach. In fact, (AgNPs)-cellulose hybrids have the double advantage of being easily produced using recycled material, with low costs and possible reuse, and of being ecosafe, if properly designed. An updated view of the use and prospects of these advanced hybrids AgNP-based materials is provided, which will surely speed their environmental application with consequent significant economic and environmental impact.

Effect-Based Approach to Assess Nanostructured Cellulose Sponge Removal Efficacy of Zinc Ions from Seawater to Prevent Ecological Risks.

To encourage the applicability of nano-adsorbent materials for heavy metal ion removal from seawater and limit any potential side effects for marine organisms, an ecotoxicological evaluation based on a biological effect-based approach is presented. ZnCl2 (10 mg L-1) contaminated artificial seawater (ASW) was treated with newly developed eco-friendly cellulose-based nanosponges (CNS) (1.25 g L-1 for 2 h), and the cellular and tissue responses of marine mussel Mytilus galloprovincialis were measured before and after CNS treatment. A control group (ASW only) and a negative control group (CNS in ASW) were also tested. Methods: A significant recovery of Zn-induced damages in circulating immune and gill cells and mantle edges was observed in mussels exposed after CNS treatment. Genetic and chromosomal damages reversed to control levels in mussels' gill cells (DNA integrity level, nuclear abnormalities and apoptotic cells) and hemocytes (micronuclei), in which a recovery of lysosomal membrane stability (LMS) was also observed. Damage to syphons, loss of cilia by mantle edge epithelial cells and an increase in mucous cells in ZnCl2-exposed mussels were absent in specimens after CNS treatment, in which the mantle histology resembled that of the controls. No effects were observed in mussels exposed to CNS alone. As further proof of CNS' ability to remove Zn(II) from ASW, a significant reduction of >90% of Zn levels in ASW after CNS treatment was observed (from 6.006 to 0.510 mg L-1). Ecotoxicological evaluation confirmed the ability of CNS to remove Zn from ASW by showing a full recovery of Zn-induced toxicological responses to the levels of mussels exposed to ASW only (controls). An effect-based approach was thus proven to be useful in order to further support the environmentally safe (ecosafety) application of CNS for heavy metal removal from seawater.

TEMPO-Nanocellulose/Ca2+ Hydrogels: Ibuprofen Drug Diffusion and In Vitro Cytocompatibility.

Stable hydrogels with tunable rheological properties were prepared by adding Ca2+ ions to aqueous dispersions of 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)-oxidized and ultra-sonicated cellulose nanofibers (TOUS-CNFs). The gelation occurred by interaction among polyvalent cations and the carboxylic units introduced on TOUS-CNFs during the oxidation process. Both dynamic viscosity values and pseudoplastic rheological behaviour increased by increasing the Ca2+ concentration, confirming the cross-linking action of the bivalent cation. The hydrogels were proved to be suitable controlled release systems by measuring the diffusion coefficient of a drug model (ibuprofen, IB) by high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. IB was used both as free molecule and as a 1:1 pre-formed complex with ß-cyclodextrin (IB/ß-CD), showing in this latter case a lower diffusion coefficient. Finally, the cytocompatibility of the TOUS-CNFs/Ca2+ hydrogels was demonstrated in vitro by indirect and direct tests conducted on a L929 murine fibroblast cell line, achieving a percentage number of viable cells after 7 days higher than 70%.

Naked-Eye Heterogeneous Sensing of Fluoride Ions by Co-Polymeric Nanosponge Systems Comprising Aromatic-Imide-Functionalized Nanocellulose and Branched Polyethyleneimine.

Heterogeneous colorimetric sensors for fluoride ions were obtained by cross-linking TEMPO-oxidized cellulose nanofibers (TOCNF) with chemically modified branched polyethyleneimine 25 kDa (bPEI). Functionalization of bPEI primary amino groups with aromatic anhydrides led to the formation of the corresponding mono- and bis-imides on the grafted polymers (f-bPEI). A microwave-assisted procedure allowed the optimization of the synthetic protocol by reducing reaction time from 17 h to 30 minutes. Hydrogels obtained by mixing different ratios of TOCNF, bPEI and f-bPEI were lyophilized and thermally treated at about 100 °C to promote the formation of amide bonds between the amino groups of poly-cationic polymers and the carboxylic groups of cellulose nanofibers. This approach generated a series of cellulose nanosponges S1-S3 which were characterized by FT-IR and by solid state 13 C CPMAS NMR. These sponge materials can act as colorimetric sensors for the selective naked-eye recognition of fluoride ions over chloride, phosphate and acetate ions at concentrations of up to 0.05 M in DMSO. Moreover, when the sponges were functionalized with perylene tetracarboxylic diimide, successful naked-eye detection was achieved with only 0.02 % w/w of chromophore units per gram of material.

Environmentally Sustainable and Ecosafe Polysaccharide-Based Materials for Water Nano-Treatment: An Eco-Design Study.

Nanoremediation, which is the use of nanoparticles and nanomaterials for environmental remediation, is widely explored and proposed for preservation of ecosystems that suffer from the increase in human population, pollution, and urbanization. We herein report a critical analysis of nanotechnologies for water remediation by assessing their sustainability in terms of efficient removal of pollutants, appropriate methods for monitoring their effectiveness, and protocols for the evaluation of any potential environmental risks. Our purpose is to furnish fruitful guidelines for sustainable water management, able to promote nanoremediation also at European level. In this context, we describe new nanostructured polysaccharide-based materials obtained from renewable resources as alternative efficient and ecosafe solutions for water nano-treatment. We also provide eco-design indications to improve the sustainability of the production of these materials, based on life-cycle assessment methodology.

TEMPO-mediated oxidation of polysaccharides: An ongoing story.

The oxidation of natural polysaccharides by TEMPO has become by now an "old chemical reaction" which led to numerous studies mainly conducted on cellulose. This regioselective oxidation of primary alcohol groups of neutral polysaccharides has generated a new class of polyuronides not identified before in nature, even if the discovery of enzymes promoting an analogous oxidation has been more recently reported. Around the same time, the scientific community discovered the surprising biological and techno-functional properties of these anionic macromolecules with a high potential of application in numerous industrial fields. The objective of this review is to establish the state of the art of TEMPO chemistry applied to polysaccharide oxidation, its history, the resulting products, their applications and the associated modifying enzymes.

Mechanical and Drug Release Properties of Sponges from Cross-linked Cellulose Nanofibers.

All-organic porous sponges were obtained throughout the direct and solvent-free (oven 105 °C, time>6 h) crosslinking of TEMPO-oxidized cellulose nanofibers (TOCNF) with 25 kDa branched polyethyleneimine (bPEI) in the presence of different amounts of citric acid (CA) as co-crosslinker. The chemical and mechanical stability of these materials was provided by the formation of amide bonds between the carboxylic moieties of TOCNF and CA with the primary amines of bPEI. The mechanical properties were investigated under static and dynamic loads with both dry and wet samples. The materials had the interesting capability to recover their shape with reduced losses in mechanical resistance, while their Young's modulus progressively increased with the content of CA. In work toward developing possible applications of bPEI-TOCNF sponges in drug delivery, amoxicillin (AM) and ibuprofen (IB) were considered as model drugs. All materials showed very good performance in adsorbing both AM and IB (ca. 200 mg g-1 ) from methanol solution. In particular, an increased adsorption of IB was observed in parallel to the increase of citrate moieties in the samples. Moreover, samples crosslinked in presence of CA showed slower kinetic release in aqueous environments than materials obtained without CA.

The diketopiperazine-fused tetrahydro-ß-carboline scaffold as a model peptidomimetic with an unusual α-turn secondary structure.

Aiming at restricting the conformational freedom of tryptophan-containing peptide ligands, we designed a THBC (tetrahydro-ß-carboline)-DKP (diketopiperazine)-based peptidomimetic scaffold capable of arranging in an unusual α-turn conformation. The synthesis is based on a diastereoselective Pictet-Spengler condensation to give the THBC core, followed by an intramolecular lactamization to complete the tetracyclic THBC-DKP fused ring system. The presence of conformers bearing the intramolecular thirteen-membered hydrogen bond that characterizes the α-turn structure is confirmed by (1)H NMR conformational studies. To the best of our knowledge, this scaffold represents one of the rare examples of a designed constrained α-turn mimic.