Hyaluronic acid/lactose-modified chitosan electrospun wound dressings – Crosslinking and stability criticalities

Abstract

Polysaccharide electrospun wound dressings should be an effective strategy in the field of wound care, as they combine an extracellular matrix-like structure with excellent biomimicry. However, their high hydrophilicity and large surface area cause a rapid dissolution in aqueous environments, compromising their clinical employment. In the present paper, electrospun membranes prepared using hyaluronic acid, a bioactive lactose-modified chitosan (CTL), and polyethylene oxide have been crosslinked using glutaraldehyde, genipin, EDC/NHS or thermal treatments, obtaining very poor results in terms of membrane stability. Therefore, carbonyldiimidazole (CDI) and methacrylic anhydride were investigated in an innovative way, where CDI proved to be the best compromise between nanofiber water resistance, architecture maintenance and degradability. Indeed, the swelling and degradation behavior as well as the water vapor permeability of these matrices were tested, revealing the effectiveness of the electrospun products in absorbing large amount of liquid while maintaining the balance between water retention and gas permeability.

Introduction

Non-healing or chronic wounds are characterized by a dysregulated healing path, where the normal timeline of coagulation and hemostasis, inflammation, proliferation, and remodeling stalls in the inflammation phase, causing fibrosis, tissue loss, and insurgence of infections (Andrabi et al., 2021; Hauck et al., 2021; Yang, Zhao, et al., 2021; Zhang et al., 2021). Surgical debridement and negative pressure are common strategies for the cleaning and preparation of the wound bed, but a wound dressing (passive or active) must be then applied to protect the wound site (Khandelwal et al., 2021). There is no single definition of the characteristics that an ideal wound dressing should have, but some key factors clearly influence the goodness of the medical device; these include, for example, biocompatibility, antimicrobial and anti-scarring potential, water adsorption capacity along with gas permeability, adaptability to the wound shape, mimicking of extracellular matrix (ECM) structure and mechanical properties, and cost-effectiveness (Fu et al., 2021; Kraskouski et al., 2021; Liu et al., 2011). In this context, electrospinning has received much attention as a simple and effective technique to produce biomimetic nanofibrous wound dressings, with a large surface area and a highly interconnected porous structure, that mimics ECM architecture and favors gaseous exchanges, drainage of excess fluid, and hemostasis Li, Wang, et al., 2021; Tonda-Turo et al., 2018). This moisturizing ability is then responsible for the anti-scarring potential of electrospun mats, as they accelerate wound repair and closure avoiding scar insurgence (Ekambaram & Dharmalingam, 2020).

The use of FDA-approved synthetic polymers as bulk materials for electrospun wound dressings has been largely exploited in recent years due to their good biocompatibility and mechanical strength as well as their good degradation profile or thermal stability. However, they are usually hydrophobic and lack intrinsic bioactive properties and biological cues directly recognized by cells (Kai et al., 2014; Wang, Song, et al., 2021; Zhou et al., 2021). Attention was then drawn to natural polymers, namely proteins (such as collagen, fibrinogen, fibroin) and polysaccharides (such as alginate, chitosan, hyaluronic acid, cellulose, dextran, to name a few). In addition to their excellent biocompatibility and ability to mimic ECM composition, which is essentially fibrous structural proteins (mainly collagen and elastin) and polysaccharides (such as hyaluronan and dermatan sulfate), natural polymers are hydrophilic and provide an optimal regenerative substrate due to their recognition signals for cells, even if they do not confer proper mechanical strength to the final product (Dodero, Schlatter, et al., 2021; Izadyari Aghmiuni et al., 2021; Memic et al., 2019). Among them, hyaluronic acid and chitosan are widely employed in the synthesis of wound dressing devices (Suo et al., 2021; Wu & Li, 2021; Xia et al., 2020; Yang, Xie, et al., 2021; Zhong et al., 2020). Hyaluronic acid is a high molecular weight non-sulfated glycosaminoglycan given by a linear repetition of (β 1 → 4)-glucuronic acid and (β 1 → 3)-N-acetyl-d-glucosamine. The ECM of skin contains up to 50% of the total amount of hyaluronic acid in the body, where it acts as lubricant preventing skin dehydration. Furthermore, hyaluronic acid can be recognized by cells through CD44 receptor, promoting cell adhesion, proliferation, and differentiation (Alven & Aderibigbe, 2021; Makvandi et al., 2019; Mauro et al., 2021; Tokudome et al., 2018). All these properties, together with its antioxidant and anti-inflammatory activity, make it an ideal candidate for the preparation of wound dressings (Chen et al., 2018; Corrêa et al., 2020). Chitosan, on the other hand, is a high molecular weight polysaccharide that is widely used as a biocompatible, biodegradable, hemostatic, and antimicrobial polymer. It is obtained from the deacetylation of chitin and is structured in a linear repeat of (β 1 → 4)-linked glucosamine and N-acetylglucosamine residues (Chen, Lin, et al., 2021). Thanks to the presence of numerous available amino groups, highly deacetylated chitosan can be exploited for the insertion of specific ligands, such as oligosaccharides (Donati, Haung, et al., 2007). Among them, CTL (1-deoxylactic-1-γ-l-chitosan also known as Chitlac) is a hydrophilic lactose-modified chitosan obtained by reductive amination with the lactose aldehydic group. This gives CTL several chemical-physical advantages over chitosan, including higher solubility at pH values closer to neutrality (Cok et al., 2018; Donati, Borgogna, et al., 2007), allowing the employment of non-toxic solvents, as water, ensuring the biocompatibility of the final product. CTL even possesses bioactive properties; for example, it induces the aggregation of articular chondrocytes by stimulating the production of glycosaminoglycans and type-II collagen and interacting with Galectin-1 (Marcon et al., 2005) or it promotes the differentiation of multipotent stem cells (namely, human dental pulp stem cells) into an osteoblast phenotype (Porrelli, Gruppuso, et al., 2021).

Despite all the advantages, electrospinning of natural polymers has some difficulties, due to the high viscosity, surface tension, and conductivity of the solutions, so that the addition of synthetic polymers and/or surfactants to the polysaccharide solutions is necessary (Bazmandeh et al., 2020; Rošic et al., 2012). This includes polyethylene oxide (PEO), a hydrophilic, water-soluble, inert synthetic polymer often used to increase polymer chain entanglement in the solution and refine polysaccharides electrospinnability (Darbasizadeh et al., 2019; Zhao et al., 2016). On the other hand, the addition of surfactants, as Tween® 20, reduces the surface tension of the solution and improves its conductivity, resulting in thinner and bead-free fibers (Kriegel et al., 2009; Liu et al., 2011).

However, the most important and critical issue related to the electrospinning of natural polymers, and especially polysaccharides, is represented by their high solubility and thus almost immediate dissolution in aqueous environment, which requires an additional and properly selected crosslinking step (Baker et al., 2016; Campiglio et al., 2019; Zheng, Yang, et al., 2021). Over the years, numerous crosslinking strategies have been employed, ranging from physical to chemical to enzymatic methods (Gruppuso et al., 2021). Physical methods include irradiation (γ-irradiation, UV irradiation, high-energy electron beam irradiation) or heat treatment. Chemical crosslinking, on the other hand, involves the formation of covalent bonds between the functional units of the polymer chains. This is the case of glutaraldehyde, genipin, or EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide), to name the most commonly used methods. The enzymatic approach exploits enzymes (as transglutaminase or oxidoreductases) to catalyze specific chemical reactions (Dodero, Scarfi, et al., 2021; Grabska-Zielińska et al., 2021; Koosha et al., 2019).

The aim of this work is to present a novel electrospun wound dressing based on hyaluronic acid and CTL, paying special attention to all critical aspects related to the chemical and structural stabilization of the final matrix. Despite the huge variety of chitosan-based wound dressing materials, to the best of the author's knowledge, this is the first time that CTL-based electrospun wound dressings are produced, with numerous possible advantages, firstly related to the use of water as solvent and to the multiplicity of bioactive properties exhibited by CTL. Moreover, various crosslinking methods are here reported, by comparing strategies documented in literature with innovative ones. The results show that all the exploited traditional approaches do not stabilize the mats or maintain their fibrous structure, mechanical strength, and integrity. Hyaluronic acid/CTL mats were also compared with electrospun matrices based on poly(−ε-caprolactone) (PCL), non-electrospun polysaccharide membranes, and the commercial product Chitoderm® to highlight the advantages of using electrospun matrices compared to non electrospun ones, where the use of bioactive polysaccharides enables to switch from biologically inert to bioactive medical devices. The hypotheses on which this work is based are: i) that it will be possible to electrospun water-soluble CTL, ii) that it will be possible, given the chemical structure of hyaluronic acid and CTL, to stabilize the electrospun membranes produced with these polymers availing of novel crosslinking methods, and iii) that the polysaccharide-based electrospun membranes here produced will present swelling and degradation behaviors, together with vapor permeability, exploitable for wound dressing applications.