**1. Introduction**

Supramolecular chemistry relies on non-covalent interactions, like hydrogen bonds, hydrophobic and Van der Waals forces, and metal–ligand coordination. These interactions control many self-assembly processes, such as biological processes, and can be used to design innovative products for biomedical applications. Polysaccharides, due to their promising biomedical and biological applications, such as tissue engineering, biosensor and wound healing, are a particularly interesting class of molecules: intra- and inter-chain interactions, together with ion pairs, originate primary, secondary, tertiary, and quaternary structures, leading to supramolecular architectures [1,2]. Such architectures are suitable for a variety of applications, ranging from drug delivery to enhance the bioavailability of poorly soluble drugs [3]. Hydrophobized polysaccharides were synthetized to produce supramolecular structures in water, and their interactions with soluble proteins or other molecular assemblies, such as monolayers, black lipid membranes, liposomes and oil-inwater emulsion were studied [4].

Among the extensive number of polysaccharides available as candidates for biopolymer engineering, glycosaminoglycans (GAGs) are highly promising candidates [5]. GAGs

**Citation:** Ladiè, R.; Cosentino, C.; Tagliaro, I.; Antonini, C.; Bianchini, G.; Bertini, S. Supramolecular Structuring of Hyaluronan-Lactose-Modified Chitosan Matrix: Towards High-Performance Biopolymers with Excellent Biodegradation. *Biomolecules* **2021**, *11*, 389. https:// doi.org/10.3390/biom11030389

Academic Editor: Dragana Nikitovic

Received: 28 January 2021 Accepted: 26 February 2021 Published: 5 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

are linear polysaccharides, which consist of repeating disaccharide units, usually include a uronic acid component (such as glucuronic acid), and a hexosamine component (such as N-acetyl-D-glucosamine).

In particular, hyaluronic acid (HA) is a linear polysaccharide with a poly-repeating disaccharide structure [(1→3)- β-D-GlcNAc-(1→4)- β-D-GlcA], which can be found ubiquitously in all vertebrate tissues extracellular matrix (ECM) [6,7]. Glucuronic acid residues contain a carboxyl group, which confers a negative charge on HA. Despite its relatively simple chemical composition, HA is involved in several biological functions, such as morphogenesis, tissue remodelling, inflammation, and tumours development and metastasis; in addition, HA contributes directly to the maintenance of tissue homeostasis and biomechanics [7]. Biocompatibility, biodegradability, high viscoelasticity, and immunoneutrality make HA an attractive polymer for biomedical and pharmaceutical applications. Frequently, HA acts as a space filler, applied to treat joint diseases such as in osteoarthritis and in eye surgery as replacement fluid, for drug delivery, and tissue engineering applications. HA is availed in viscosupplementation therapy, with the therapeutic goal to restore the viscoelasticity of synovial fluid. Indeed, HA is continuously secreted in the articular cavity and is one of the most common components of synovial liquid, giving rise to its characteristic viscoelastic properties. These rheological properties are essential for the lubrication and shock protection of healthy joints, allowing the protection of cartilage and soft tissues. In the event of traumatic and degenerative diseases, the amount of HA is lowered and synovial fluid experiences a viscosity drop, impairing joint functionality, and causing pain [8–10]. For applications, unmodified HA and chemically modified or cross-linked HA are used [11,12]; the derivatization of HA increases its mechanical properties and stability, which allows it to be used as a biomaterial [13–15].

Chitosan is also a linear polysaccharide, derived from deacetylation of chitin, the structural component of fungal cell walls, and exoskeleton of arthropods. It is composed of β-1→4 glucosamine units, with some residual interspersed N-acetyl-glucosamine residues; it is soluble in acidic solution, with a positive charge density, dependent on pH and percentage of deacetylation. Chitosan is well-known for its numerous and interesting biological properties—it is biocompatible, bioresorbable, and bioactive. Availability, safety for medical use, and biodegradability make chitosan very interesting for tissue engineering and biomaterials products. Chitosan can be further improved from the bioactive features and the physical−chemical behaviour. In particular, Chitlac (Ch) is a compound obtained by the modification of chitosan with lactitol moieties [16], in which the oligosaccharide pendant groups alter its solubility at physiological pH. The physico-chemical and biological properties of Ch are already reported in the literature [17].

Both HA and Ch are hydrolyzed by enzymes; linear HA can be easily degraded by enzymes, such as bovine testicular hyaluronidase, an endo-β-N-D-acetylhexosaminidase that hydrolyzes HA at the β (1→4)-N-acetylglucosaminide bonds [18]. Hyaluronidases simultaneously display both hydrolytic and transglycosylation activities—the optimal conditions for the hydrolysis of HA by hyaluronidase are pH 4.0 and the presence of NaCl, whereas for transglycosylation they are pH 7.0 and the absence of NaCl [19]. In the case of Ch, there is no specific enzyme; nonetheless, previous studies report that degradation can be performed by lysozyme, which is, for example, present at concentrations ranging from 4 to 13 mg/L in serum and in tears (450 to 1230 mg/L) [20].

A polyelectrolyte complex is formed when polymers with opposite charges are combined in solution. A strong complex is obtained if the anions and cations in the polymers contain strong acids and bases, or if the polyions attain their fully ionized forms and vice versa. The polyelectrolyte complexes are recently gaining attention as supramolecular carriers for controlled release of drugs and proteins [21], which is widely used in many applications such as membranes, medical prosthetics, environmental sensors, and protein separation systems [22,23]. These complexes prepared from natural polymers, such as polysaccharides, have the additional advantage of being non-toxic and bioabsorbable [24]. For example, biomaterials, constituted of a Chondroitin sulphate-Chitosan complex, have

interesting biological properties, such as wound-healing acceleration and cellular assistance for skin and cartilage recovery [25]. Some applications of Chitosan and HA complexes include ophthalmic surgery, arthritis treatment, scaffolds for wound healing, tissue engineering, and the use as a component in implant materials. In particular, different authors showed that the potential of Chitosan/HA complexes coacervates in the biomaterials field [26–31]. A recent study about self-supporting multi-layered film containing a Chitosan and HA polyelectrolyte complex, showed high selectivity during the separation of water from the ethanol–water mixtures for membrane technology applications [32].

Moreover, recent studies highlighted promising results for biological and medical applications of the HA/Ch complex, such as in the treatment of osteoarthritis [33,34]; in vivo osteoarthritis treatments with a viscosupplementation containing Ch and HA showed a decrease in morphological and histolopathological cartilage damage and synovial membrane inflammation, in comparison to the treatment performed with HA alone [33]. A combination of Ch and HA-attenuated macrophage-induced inflammation, inhibited metalloproteinases expression, and exhibited anti-oxidative effects, providing interesting insights into the biological effects of mixture of these polysaccharides for the development of osteoarthritis treatments [34]. However, the literature does not provide exhaustive chemical-physical characterization of HA/Ch for a better understanding of the observed biological performances.

As such, in this work we investigated the properties of the polyelectrolyte complex to understand the interaction between HA and Ch. Specifically, the molecular weight of the supramolecular product through size-exclusion chromatography with a triple detector array (HP-SEC-TDA) was determined. The viscosity property and the molecular mobility was elucidated through dynamic viscosity and NMR diffusion ordered spectroscopy (DOSY) experiments, respectively. Finally, the stability toward enzymatic actions was evaluated.
