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.

Magnetic Shape-Memory Heuslers Turn to Bio: Cytocompatibility of Ni-Mn-Ga Films and Biomedical Perspective.

Magnetic shape-memory (MSM) Heuslers have attracted great attention in recent years for both caloric and magnetomechanical applications. Thanks to their multifunctional properties, they are also promising for a vast variety of biomedical applications. However, this topic has been rarely investigated so far. In this communication, we present the first report on the absence of cytotoxicity of MSM Heuslers in Ni-Mn-Ga epitaxial thin films and the perspective toward bioapplications. Qualitative and quantitative biological characterizations reveal that Ni-Mn-Ga films can promote the adhesion and proliferation of human fibroblasts without eliciting any cytotoxic effect. Additionally, our findings show that the morphology, composition, microstructure, phase transformation, and magnetic characteristics of the films are well preserved after the biological treatments, making the material a promising candidate for further investigations.

Electroconductive scaffolds based on gelatin and PEDOT:PSS for cardiac regeneration.

Electroconductive biomaterials have been emerged to support the recovery of the degenerated electrically conductive tissues, especially the cardiac ones after myocardial infarction. This work describes the development of electroconductive scaffolds for cardiac tissue regeneration by using a biocompatible and conductive polymer - i.e. poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) - combined with a biomimetic polymer network of gelatin. Our approach involves the use of dehydrothermal (DHT) treatment in vacuum conditions to fabricate suitably stable scaffolds without using any additional crosslinking agent. The resulting scaffolds mimic the Young modulus - an essential mechanical performance - of native cardiac tissue and are endowed with a well-interconnected porosity coupled with a good swelling ability and stability in physiological conditions. Additionally, the presence of PEDOT:PSS is able to enhance the electroconductivity of resulting materials. All the scaffolds are non-cytotoxic towards H9C2 cardiomyoblasts and the presence of PEDOT:PSS enhances cell adhesion - especially at early timeframes, an essential condition for a successful outcome after the implantation - proliferation, and spreading on scaffolds. Considering the permissive interaction of scaffolds with cardiomyoblasts, the present biomimetic and electroconductive scaffolds display potential applications as implantable biomaterials for regeneration of electroconductive tissues, especially cardiac tissue, and as a promising 3D tissue model for in vitro biomolecules screening.

Electroconductive and injectable hydrogels based on gelatin and PEDOT:PSS for a minimally invasive approach in nervous tissue regeneration.

This work describes the development of electroconductive hydrogels as injectable matrices for neural tissue regeneration by exploiting a biocompatible conductive polymer - poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) - combined with a biomimetic polymer network made of gelatin. Our approach involved also genipin - a natural cross-linking agent - to promote gelation of gelatin networks embedding PEDOT:PSS. The achieved results suggest that physical-chemical properties of the resulting hydrogels, like impedance, gelation time, mechanical properties, swelling and degradation in physiological conditions, can be finely tuned by the amount of PEDOT:PSS and genipin used in the formulation. Furthermore, the presence of PEDOT:PSS (i) enhances the electrical conductivity, (ii) improves the shear modulus of the resulting hydrogels though (iii) partially impairing their resistance to shear deformation, (iv) reduces gelation time and (v) reduces their swelling ability in physiological medium. Additionally, the resulting electroconductive hydrogels demonstrate enhanced adhesion and growth of primary rat cortical astrocytes. Given the permissive interaction of hydrogels with primary astrocytes, the presented biomimetic, electroconductive and injectable hydrogels display potential applications as minimally invasive systems for neurological therapies and damaged brain tissue repair.

Controlled Liposome Delivery from Chitosan-Based Thermosensitive Hydrogel for Regenerative Medicine.

This work describes the development of an injectable nanocomposite system based on a chitosan thermosensitive hydrogel combined with liposomes for regenerative medicine applications. Liposomes with good physicochemical properties are prepared and embedded within the chitosan network. The resulting nanocomposite hydrogel is able to provide a controlled release of the content from liposomes, which are able to interact with cells and be internalized. The cellular uptake is enhanced by the presence of a chitosan coating, and cells incubated with liposomes embedded within thermosensitive hydrogels displayed a higher cell uptake compared to cells incubated with liposomes alone. Furthermore, the gelation temperature of the system resulted to be equal to 32.6 °C; thus, the system can be easily injected in the target site to form a hydrogel at physiological temperature. Given the peculiar performance of the selected systems, the resulting thermosensitive hydrogels are a versatile platform and display potential applications as controlled delivery systems of liposomes for tissue regeneration.

Strain Hardening in Highly Acetylated Chitosan Gels.

Strain hardening has recently emerged as a near-universal response of biological tissues to mechanical stimulation as well as a powerful regulator of cell fate. Understanding the mechanistic basis for this nonlinear elasticity is crucial for developing bioinspired materials that mimic extracellular matrix mechanics. Here, we show that covalent networks built from highly acetylated chitosans exhibit strain hardening at physiological pH and osmolarity. While varying the chitosan physical-chemical composition and network connectivity, we provide evidence that temporary nodes arising from the entangling of chains between stable cross-links are at the root of nonlinear elasticity. The contour length (Lc) of the said chains revealed that the larger the chain length between the cross-links, the greater is the entanglement over disentanglement upon network stretching. To this end, we calculated that the minimum number of Khun's segments in Lc that contributes to the onset of strain hardening is 15. Furthermore, we identified a relationship between critical strain marking nonlinear elasticity and the network connectivity, being similar to that found for the cytoskeletal collagen matrix, indicating the potential use of semiflexible (neutral pH-soluble) chitosans in assembling extracellular matrix mimics.

Temporary/Permanent Dual Cross-Link Gels Formed of a Bioactive Lactose-Modified Chitosan.

Mounting evidences have recognized that dual cross-link and double-network gels can promisingly recapitulate the complex living tissue architecture and overcome mechanical limitations of conventional scaffolds used hitherto in regenerative medicine. Here, dual cross-link gels formed of a bioactive lactose-modified chitosan reticulated via both temporary (boric acid-based) and permanent (genipin-based) cross-linkers are reported. While boric acid rapidly binds to lactitol flanking diols increasing the overall viscosity, a slow temperature-driven genipin binding process takes place allowing for network strengthening. Combination of frequency and stress sweep experiments in the linear stress-strain region shows that ultimate gel strength, toughness, and viscoelasticity depend on polymer-to-genipin molar ratio. Notably, herewith it is demonstrated that linear stretching correlates with strain energy dissipation through boric acid binding/unbinding dynamics. Strain-hardening effect in the nonlinear regime, along with good biocompatibility in vitro, points at an interesting role of present system as biological extracellular matrix substitute.

On the Formation and Stability of Chitosan/Hyaluronan-Based Complex Coacervates.

This contribution is aimed at extending our previous findings on the formation and stability of chitosan/hyaluronan-based complex coacervates. Colloids are herewith formed by harnessing electrostatic interactions between the two polyelectrolytes. The presence of tiny amounts of the multivalent anion tripolyphosphate (TPP) in the protocol synthesis serves as an adjuvant "point-like" cross-linker for chitosan. Hydrochloride chitosans at different viscosity average molar mass, , in the range 10,000-400,000 g/mol, and fraction of acetylated units, FA, (0.16, 0.46 and 0.63) were selected to fabricate a large library of formulations. Concepts such as coacervate size, surface charge and homogeneity in relation to chitosan variables are herein disclosed. The stability of coacervates in Phosphate Buffered Saline (PBS) was verified by means of scattering techniques, i.e., Dynamic Light Scattering (DLS) and Small-Angle X-ray Scattering (SAXS). The conclusions from this set of experiments are the following: (i) a subtle equilibrium between chitosan FA and does exist in ensuring colloidal stability; (ii) once diluted in PBS, osmotic swelling-driven forces trigger the enlargement of the polymeric mesh with an ensuing increase of coacervate size and porosity.

N-isopropyl chitosan. A pH- and thermo-responsive polysaccharide for gel formation.

The present contribution deals with the synthesis and characterization of N-isopropyl chitosan in which the introduction of hydrophobic groups leads to an increased flexibility of the polysaccharide backbone. The isopropyl groups extend the solubility of the modified-chitosan samples and render the modified chitosan a pH- and thermo-sensitive system for hydrogel formation. Indeed, upon varying the pH of the system and/or its temperature within a range compatible with biological applications, a non-reversible sol-gel transition occurs, as determined through extended rheological analyses. The modified chitosan samples show a very good biocompatibility as determined through preliminary viability and cell growth experiments.

pH-Assisted Gelation of Lactose-Modified Chitosan.

We report on a controlled process allowing for the gelation of a diol-rich chitosan-derivative named CTL (lactose-modified chitosan) in the presence of boric acid as the cross-linker. A two-step approach is described, namely (i) the mixing of CTL and boric acid at pH = 5, a condition where the inorganic component is mildly reactive; (ii) the addition of sodium bicarbonate (NaHCO3) as a trigger, allowing for the gradual and slow pH increase. The goal was to convert gradually the almost inert neutral boric acid into the much more reactive borate anion, the latter promoting the formation of borate esters with CTL diols. Gelling kinetics as well as mechanical behavior at small and large deformations was investigated by rheometry. CTL-boric acid gels behaved essentially as transient networks, hence continuously assembling and dissociating in a highly dynamic fashion. The present gelling mechanism preserves the strain-hardening behavior in the nonlinear region of stress-strain response, corroborating the already suggested potential applications of such gels as mimics of biological soft tissues.

Chitosan Acetylation Degree Influences the Physical Properties of Polysaccharide Nanoparticles: Implication for the Innate Immune Cells Response.

The aim of the present contribution is twofold as it reports (i) on the role played by chitosan acetylation degree for the stability of nanoparticles (NPs) formed with hyaluronan and (ii) on the effect of the interaction of such NPs with immune cells. Chitosans with similar viscosity-average molecular weight, [Formula: see text], (i.e., 200 000) and different fractions of acetylated units ( FA) together with low-molecular-weight hyaluronan were chosen for developing a select library of formulations via electrostatic complex coacervation. The resulting NPs were analyzed in terms of size, polydispersity, surface charge, and stability in physiological-mimicked media by dynamic light scattering. Only medium acetylated chitosan ( FA = 0.16) guaranteed the stability of NPs. To explore the effect of NPs interaction with immune cells, the release of proinflammatory cytokines and the reactive oxygen species production by human macrophages and neutrophils, respectively, were evaluated. Strikingly, a structure-function relationship emerged, showing that NPs made of chitosans with FA = 0.02, 0.25, 0.46, and 0.63 manifested a proinflammatory activity, linked to the instability of the system. Conversely, NPs made of chitosan with FA = 0.16 neither modified the functional response of macrophages nor that of neutrophils. Of note, such NPs were found to possess additional properties potentially advantageous in applications such as delivery of therapeutics to target inflamed sites: (i) they are devoid of cytotoxic effects, (ii) they avoid engulfment during the early stage of interaction with macrophages, and (iii) they are muco-adhesive, thereby providing for site-specificity and long-residence effects.

Nucleation, reorganization and disassembly of an active network from lactose-modified chitosan mimicking biological matrices.

Developing synthetic materials able to mimic micro- and macrorheological properties of natural networks opens up to novel applications and concepts in materials science. The present contribution describes an active network based on a semi-synthetic polymer, a lactitol-bearing chitosan derivative (Chitlac), and a transient inorganic cross-linker, boric acid. Due to the many and diverse anchoring points for boric acid on the flanking groups of Chitlac, the cross-links constantly break and reform in a highly dynamic fashion. The consequence is a network with unusual non-equilibrium and mechanical properties closely resembling the rheological behavior of natural three-dimensional arrangements and of cytoskeleton. Concepts like network nucleation, reorganization and disassembly are declined in terms of amount of the cross-linker, which acts as a putative motor for remodeling of the network upon application of energy. The out-of-equilibrium and non-linear behavior render the semi-synthetic system of great interest for tissue engineering and for developing in-vitro mimics of natural active matrices.

Biomimetic, Multiresponsive, and Self-Healing Lactose-Modified Chitosan (CTL)-Based Gels Formed via Competitor-Assisted Mechanism.

The present paper describes an original method to form under physiological conditions homogeneous lactose-modified chitosan (CTL) gels avoiding syneresis. Specifically, combination of boric acid-i.e., the cross-linker-and mannitol-i.e., a polyol competitor for boron binding-were exploited to reduce the very fast kinetics of CTL/boron self-assembly. Resulting gels were homogeneous as proved by scattering analyses. An in-depth rheological characterization was undertaken to identify the correct mannitol-to-boron ratio at which gels showed homogeneity without weakening. Stress sweep and frequency sweep tests were performed to investigate the viscoelastic properties of these dynamic networks, highlighting a marked strain-hardening behavior, which is pivotal in native tissues. Notably, herein we report for the first time that CTL-boric acid gels are multiresponsive systems, whose mechanics can be tailored by different stimuli such as the presence of small molecules like glucose. Moreover, we demonstrate that these networks spontaneously self-heal after breakage. The biocompatibility of such gels was studied under 2D and 3D conditions toward three different cell models, namely, pig primary chondrocytes, human Dental Pulp Stem Cells (hDPSCs), and mouse fibroblasts. Giving the peculiar mechanical performance of selected systems and considering the well-known bioactivity of the chitosan derivative, CTL-boric acid networks are promising candidates as multiresponsive gels to be used in the field of tissue engineering, especially for articular cartilage regeneration.

Lactose-Modified Chitosan Gold(III)-PEGylated Complex-Bioconjugates: From Synthesis to Interaction with Targeted Galectin-1 Protein.

Galectins (Gal) are a family of glycan-binding proteins characterized by their affinity for ß-galactosides. Galectin-1 (Gal-1), a dimeric lectin with two galactoside-binding sites, regulates cancer progression and immune responses. Coordination chemistry has been engaged to develop versatile multivalent neoglycoconjugates for binding Gal-1. In this study we report a fast and original method to synthesize hybrid gold nanoparticles in which a hydrochloride lactose-modified chitosan, named CTL, is mixed with dicarboxylic acid-terminated polyethylene glycol (PEG), leading to shell-like hybrid polymer-sugar-metal nanoparticles (CTL-PEG-AuNPs). The aim of this paper is to preliminarily study the interaction of the CTL-PEG-AuNPs with a target protein, namely, Gal-1, under specific conditions. The molecular interaction has been measured by Transmission Electron Microscopy (TEM), UV-vis, and Raman Spectroscopy on a large range of Gal-1 concentrations (from 0 to 10-12 M). We observed that the interaction was strongly dependent on the Gal-1 concentration at the surface of the gold nanoparticles.

Concepts for Developing Physical Gels of Chitosan and of Chitosan Derivatives.

Chitosan macro- and micro/nano-gels have gained increasing attention in recent years, especially in the biomedical field, given the well-documented low toxicity, degradability, and non-immunogenicity of this unique biopolymer. In this review we aim at recapitulating the recent gelling concepts for developing chitosan-based physical gels. Specifically, we describe how nowadays it is relatively simple to prepare networks endowed with different sizes and shapes simply by exploiting physical interactions, namely (i) hydrophobic effects and hydrogen bonds-mostly governed by chitosan chemical composition-and (ii) electrostatic interactions, mainly ensured by physical/chemical chitosan features, such as the degree of acetylation and molecular weight, and external parameters, such as pH and ionic strength. Particular emphasis is dedicated to potential applications of this set of materials, especially in tissue engineering and drug delivery sectors. Lastly, we report on chitosan derivatives and their ability to form gels. Additionally, we discuss the recent findings on a lactose-modified chitosan named Chitlac, which has proved to form attractive gels both at the macro- and at the nano-scale.

Boric Acid Induced Transient Cross-Links in Lactose-Modified Chitosan (Chitlac).

The present paper explores the effect of boric acid on Chitlac, a lactose-modified chitosan which had previously shown interesting biological and physical-chemical features. The herewith-reported experimental evidences demonstrated that boric acid binds to Chitlac, producing conformational and association effects on the chitosan derivative. The thermodynamics of boric acid binding to Chitlac was explored by means of 11B NMR, circular dichroism (CD), and UV-vis spectroscopy, while macromolecular effects were investigated by means of viscometry and dynamic light scattering (DLS). The experimental results revealed a chain-chain association when limited amounts of boric acid were added to Chitlac. However, upon exceeding a critical boric acid limit dependent on the polysaccharide concentration, the soluble aggregates disentangle. The rheological behavior of Chitlac upon treatment with boric acid was explored showing a dilatant behavior in conditions of steady flow. An uncommonly high dependence in the scaling law between the zero-shear viscosity and the concentration of Chitlac was found, i.e., η0 ∝ CCTL5.8, pointing to interesting potential implications of the present system in biomaterials development.

Highly monodisperse colloidal coacervates based on a bioactive lactose-modified chitosan: From synthesis to characterization.

The present contribution aims at describing the fabrication of coacervates in the nano-size range starting from a 1-deoxylactit-1-yl chitosan (in this manuscript termed as CTL60) and the multivalent anion tripolyphosphate (TPP). Colloidal coacervates have been obtained for precise values of the molar ratio of TPP to CTL60 repeating unit. Coacervation is ensured only at pH 4.5 and not at 7.4, thus demonstrating the key role of electrostatic interactions in the stabilization of the coacervates. At a variance with chitosan, CTL60 favors the formation of highly homogeneous coacervates with very low values of the polydispersity index (PDI). Moreover, CTL60 coacervates can be freeze-dried without any cryoprotectant, they can host a model molecule and are stable up to three weeks at 4°C. Conversely, such coacervates dissolve upon increasing pH and ionic strength. By considering the bioactive polycation CTL60, the present system can be suggested as a first step in the development of innovative biologically-active nano-carriers to be used as drug delivery systems.