3D printable self-healing hyaluronic acid/chitosan polycomplex hydrogels with drug release capability

https://doi.org/10.1016/j.ijbiomac.2021.08.022Get rights and content

Abstract

Multifunctional printable biomaterials are at the base of advanced biomedical applications. Chitosan (CHI) and hyaluronic acid (HA) allow the development of polycomplex hydrogels with tailorable properties, including self-healing and controlled drug release. This work correlates and optimizes the mucoadhesive, swelling, biodegradation, mechanical and rheological properties of HA/CHI polycomplex hydrogels with synthesis parameters such as polysaccharide content and complexation time, according to the interaction forces established between both polyelectrolytes. Related to these dynamic forces, the self-healing ability of the hydrogels was investigated together with the potential of the HA/CHI polycomplex hydrogels for 3D printing. Finally, their capability to modulate and promote controlled release of a variety of drugs (anionic and anti-inflammatory sodium diclofenac and the neutral antibiotic rifampicin) was demonstrated. Thus, the reported tunable properties, self-repair ability, printability and drug release properties, demonstrate the suitability of HA/CHI hydrogels for advanced biomedical applications.

Introduction

Natural hydrogels formed by a simple mixture of biopolymers and free of chemical additives are biomaterials with a large range of applications in fields like cosmetic, food industry, biomedicine and regenerative medicine, among others. Hydrogels are formed by the crosslinking of polymer chains leading to three-dimensional (3D) structures capable of retaining large amounts of water without dissolving [1]. Further, they can respond to external stimulus such as pH, temperature or light [2]. Chemical or physical hydrogels are distinguished according to the kind of crosslinking, the former being referred to hydrogels with covalent bonds between polymer chains and the latter, to polymeric chains bounded by non-covalent interactions, such as hydrogen bonds, hydrophobic or electrostatic interactions. Hydrogels formed by electrostatic interactions between polyelectrolytes are known as polyelectrolyte complex (PEC) hydrogels and are originated by the association of polymers with a large number of ionisable groups by electrostatic interactions between the chains [3]. The formation of PECs depends on the molecular weight, mixing ratio and concentration of the polymers and pH, among other factors [4], which sometimes can lead to materials with poor biocompatibility. It is important to highlight the pH-dependence of polyelectrolyte complexes: while weak polyions dissociate in a specific range of pH, strong polyions are dissociated in all pH values.

Polyelectrolyte complex hydrogels have a wide range of applications in the biomedical field [5], such as wound dressing [6] or drug delivery [7]. In general, the applicability of PEC hydrogels in medicine is enhanced by the self-healing capability, because it augments materials lifetime and reduces replacement costs. Self-healing is the term used to refer to materials able to repair themselves after being injured [8]. This ability is the result of the restoration of broken bonds or interactions, which may be because of the release of a self-healing agent (extrinsic) or the reversible nature of covalent or non-covalent bonds. Electrostatic interactions have gained attention as non-covalent reversible bonds for self-healing behaviour due to their simplicity that can be exploited to increase biocompatibility and minimize side reactions concerns [9]. For example, hydrogen bonding promotes self-healing ability of poly (vinyl acetate) hydrogels [10], as well as electrostatic interactions between poly(acrylic acid)/poly(ethylenimine) [9].

The unique properties of hydrogels for mimicking natural tissues, such as their soft and highly hydrated nature, have positioned them as essential constituents for personalized tissue engineered devices, scaffolds for regenerative medicine, or the development of customized implants [11], promoting also the increasing interest on hydrogels made with 3D printing technology [12], [13]. Hydrogels are ideal candidates for the development of bioinks for 3D printing mediated by extrusion-, laser- or inkjet-based methods. Cell compatible hydrogels must present some features in order to act as bioinks: apart from biocompatibility, adequate viscosity, appropriate degradation kinetics, non-toxic degradation products and similar morphology to living tissue, mechanical properties, such as shear-thinning, also have to be considered, since this capacity can protect cells from mechanical damage when printing [14]. Hydrogels that show shear-thinning behaviour act like low-viscosity liquids when extruding, but after shear stress removal, recover the original stiffness [15]. In order to allow this behaviour in hydrogels for 3D printing, crosslinked hydrogels with strong physical interactions, such as electrostatic interactions, are commonly studied. In addition, the formation of covalent bonds is generally not compatible with in situ encapsulations of cells or proteins due to the presence of coupling agents [16]. Thus, physical hydrogels with autonomous healing property are highly preferred for the development of new bioinks, being exposed to different mechanical solicitation during cells growth, migration and proliferation [17].

Typically, printable hydrogels as components of bioinks formulations are made of natural polymers, biopolymers or a natural-synthetic mixture [18]. One of the most exploited natural polymers is alginate, due to the versatility that it offers regarding to its simple crosslinking mechanisms by ionic interactions between –COO groups and Ca2+ cations, its low cost, and biocompatibility. As an example, Markstedt et al. [19] reported an alginate/nanocelullose based ink that as a consequence of its shear thinning behaviour enabled printing of 2D grid-like and 3D complex structures. However, since alginate hydrogels show low cell adhesion and undergo rapid dissolution, alginate is not an ideal candidate for cell-laden printing. However, its combination with functional biomaterials, such as gelatine has shown promising results, for example, for bone tissue engineering applications in combination with cellulose nanocrystals [20]. Thus, despite significant progress in the 3D printing of hydrogels formed by a single polymer, nowadays, the limited variety of printable hydrogel systems limits the expected progress of this field [21].

Regarding other promising biopolymer candidates for 3D printing purposes, it is worth to highlight chitosan due to its physico- chemical and cell adhesion properties [22]. Chitosan is a linear and natural polysaccharide derived from the partial deacetylation of chitin, found in crustaceans' exoskeleton. It is a weak polycation with pKa 6.5, formed by two randomly ordered units, N-acetyl-2-amino-2-deoxy-d-glucose and 2-amino-2-deoxy-d-glucose. It is biodegradable, biocompatible, non-toxic and, due to its cationic nature, it shows antibacterial activity causing the disruption of bacteria cell membranes, which are negatively charged [23]. In addition, it is also known for its mucoadhesive properties, linked to the hydroxyl and amine groups of chitosan, which can interact with sialic acid groups of mucus glycoproteins forming hydrogen bonds and electrostatic interactions [24].

The combination of the polycationic chitosan with weak polyanionic polysaccharides such as alginate [25], carboxymethylcellulose [26] or hyaluronic acid [27] results in polyelectrolyte complex (PEC) hydrogels. According to the external pH and the pKa values of both polysaccharides, electrostatic interactions between carboxylate groups (-COO) of the polyanions, and protonated amine groups (-NH3+) of chitosan, or H-bonding between no ionized groups, are established (Fig. 1), leading to the formation of the PEC network.

Hyaluronic acid (HA) is a linear and high molecular weight glycosaminoglycan composed of a repeating disaccharide molecules, β-(1,4)-D-glucuronic acid and β-(1,3)-N-acetil-D-glucosamine. It is a weak polyanion, with pKa 2.9, found mainly in the extracellular and pericellular matrix, though it has also been detected intracellularly [28]. Its main characteristics are biodegradability, biocompatibility, non-toxicity and viscoelasticity; moreover, it is one of the most hydrophilic molecules known, and accordingly, it is widely used as moisturiser. All these features enhance HA derived materials biocompatibility and wound dressing ability [29]. HA is also known to play an essential role during the regulation of cell motility and adhesion as a consequence of its ability to interact with the cluster of differentiation 44 (CD44) [30].

Taking this into consideration, this work proposes the synthesis of biocompatible, biodegradable and potentially mucoadhesive polyelectrolyte complex hydrogels based on both natural polysaccharides: hyaluronic acid and chitosan. In fact, the direct mixture of pure chitosan and hyaluronic acid has been poorly exploited in the literature for hydrogels synthesis. Kim et al. [31] explored the influence of CHI/HA ratio on the swelling of CHI/HA PEC films, that shown a non-Fickian diffusion. Lately, Shi et al. [32] following a similar experimental procedure, i.e., casting of slightly turbid solutions of CHI/HA mixtures in highly acidic medium, prepared CHI/HA PEC hydrogels with strong tensile strain and strain rate dependence. Conversely, Vignesh et al. [33] prepared CHI/HA PEC hydrogels by addition of HA solution to a previously prepared chitosan hydrogel gelled by basification with NaOH solution. Deferoxamine loaded PLGA nanoparticles (DFO NPs) were also incorporated in the obtained PEC hydrogels for enhancing angiogenesis. More recently, CHI/HA PEC hydrogels have been prepared by direct mixture of high molecular weight HA and CHI solutions adjusting the pH to an intermediate value between the pKa of both polysaccharides, this is, 2.9 < pKa < 6.5 [34]. PECs were also obtained in the form of nanometric polyelectrolyte multilayers (PEMs) onto poly(ethylene terephthalate) surfaces, both PECs systems showing a rapid self- healing behaviour. In the light of these promising properties, herein is reported the novel correlation between the synthesis conditions of HA/CHI PEC hydrogels (polysaccharide content in solutions and complexation time) and the mechanical, mucoadhesive, swelling and rheological properties of the polycomplex hydrogels. As a result, self-healing capacity and drug delivery ability for ionic and non-ionic drugs, are also expected and herein demonstrated for HA/CHI PEC hydrogels. In addition, this work aims to explore for the first time, the printability of the plain combination of HA/CHI PEC hydrogels as extruded bioinks to their potential application as 3D printed biodegradable scaffolds and soft implants with self-healing and drug release capability.

Section snippets

Materials

Hyaluronic acid (Contipro, high molecular weight, 2.1 × 106 ± 1.01 × 105 g/mol (PDI = 1.003)) and chitosan from crab shells (Sigma Aldrich, highly viscous, 1.2 × 106 ± 153.9 g/mol (PDI = 1.037), deacetylation degree of 71% determined by 1H NMR) were used for the formation of polyelectrolyte complex hydrogels. The average molecular weights were measured by gel permeation chromatography equipped with refractive index (RID) and light scattering (LS15 and LS90) detectors (HPLC Agilent Technologies,

Synthesis and physico-chemical characterization of PEC hydrogels

The electrostatic interactions between two natural polysaccharides results on the formation of biocompatible and biodegradable polyelectrolyte complex (PEC) hydrogels. Electrostatic interactions between the carboxylate group (-COO) of hyaluronic acid and protonated amine (-NH3+) of chitosan were favoured by adjusting the pH at an intermediate value between hyaluronic acid and chitosan pKa value, 2.9 and 6.5, respectively, in which ionization of both polymers is maximum (Fig. 1a). PECs were

Conclusions

Polyelectrolyte complex hydrogels based on two natural polysaccharides, hyaluronic acid and chitosan, have been developed with self-healing, drug-release and printability capabilities. The hydrogels have been optimized in terms of synthesis concentrations (2%) and complexation time (24 h). For this, mucoadhesiveness, rheological and mechanical properties were evaluated, showing a not significant effect of the polysaccharide content and a great influence of the complexation time. In addition,

CRediT authorship contribution statement

Conceptualization: L.P.A., U. S., J.L.V and S.L.M; Data curation: S.M.F. and N.B.; Formal analysis S.M.F., N. B. and L.P.A.; Funding acquisition: S.L.M.; Investigation S.M.F., N.B. and L.P.A.; Supervision J.L.V.; Writing - review & editing: S.M.F, N.B. L.P.A, S.L.M.

Acknowledgments

The authors acknowledge funding by Spanish State Research Agency (AEI) and the European Regional Development Fund (ERFD) through the project PID2019-106099RB-C43/AEI/10.13039/501100011033, as well as, from the Basque Government Industry Department under the ELKARTEK program. The authors thank Dra. Cristina Eguizabal for giving them access to the group of cell therapy, stem cells and tissues linked to the Basque Center for Transfusion and Human Tissues at the Galdakao hospital. Technical and

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