Strategies to Prepare Chitin and Chitosan-Based Bioactive Structures Aided by Deep Eutectic Solvents: A Review

Abstract

Chitin and chitosan, abundant biopolymers derived from the shells of crustaceans and the cell walls of fungi, have garnered considerable attention in pharmaceutical circles due to their biocompatibility, biodegradability, and versatile properties. Deep eutectic solvents (DESs), emerging green solvents composed of eutectic mixtures of hydrogen bond acceptors and donors, offer promising avenues for enhancing the solubility and functionality of chitin and chitosan in pharmaceutical formulations. This review delves into the potential of utilizing DESs as solvents for chitin and chitosan, highlighting their efficiency in dissolving these polymers, which facilitates the production of novel drug delivery systems, wound dressings, tissue engineering scaffolds, and antimicrobial agents. The distinctive physicochemical properties of DESs, including low toxicity, low volatility, and adaptable solvation power, enable the customization of chitin and chitosan-based materials to meet specific pharmaceutical requirements. Moreover, the environmentally friendly nature of DESs aligns with the growing demand for sustainable and eco-friendly processes in pharmaceutical manufacturing. This revision underscores recent advances illustrating the promising role of DESs in evolving the pharmaceutical applications of chitin and chitosan, laying the groundwork for the development of innovative drug delivery systems and biomedical materials with enhanced efficacy and safety profiles.

1. Introduction

Biopolymers, derived from renewable resources such as plants, animals, and microorganisms, offer a multitude of advantages over conventional petroleum-based polymers. Firstly, biopolymers are environmentally friendly, as they are biodegradable, reducing the burden of plastic pollution on ecosystems and/or marine life [1]; in fact, several studies have shown that biopolymers degrade faster in various environments compared to traditional plastics [2,3,4], thus paving the way for implementing a circular economy [5]. Additionally, the production of biopolymers typically requires lower energy consumption and emits fewer greenhouse gases, contributing to mitigating climate change [6]. Furthermore, biopolymers can be produced from sustainable feedstocks and/or industrial waste [7,8], reducing the reliance on finite fossil fuel resources and promoting agricultural diversification [9,10]. Another notable advantage is the potential for biopolymers to exhibit superior biocompatibility, making them suitable for biomedical applications such as drug delivery systems [11,12] and tissue engineering scaffolds [13,14]. Moreover, biopolymers offer versatility in terms of their properties and functionalities, with researchers exploring various modification techniques to tailor their mechanical [15,16], thermal [17,18], and barrier properties [19,20] to specific applications [21,22,23,24]. Overall, the adoption of biopolymers presents a promising pathway towards achieving sustainability goals while catering to diverse industrial and societal needs [25].

Among natural biopolymers, chitin stands out as the second most abundant natural polymer on Earth (only after cellulose, the principal component of plant cell walls and the most abundant source of renewable polysaccharides on Earth [26]). It can be found in a plethora of living beings [27], with some notable sources being crustacean shells [28,29,30,31,32], arthropods [33], mollusks [34], insects [35,36,37], and fungi [38]. Chemically, chitin is a linear polysaccharide composed by a repetition of subunits of 2-amino-2-deoxy-D-glucose (N-acetyl-glucosamine, GlcNAc, Figure 1a) connected by (β1→4) glycosidic bonds (Figure 1b,c). The presence of these (β1→4) glycosidic bonds generates a linear structure of chitin macromolecules, which permit an extended conformation resembling a regular zigzag pattern (Figure 1c). This configuration fosters robust intramolecular forces, notably hydrogen bonding [39]. These secondary interactions occur between the hydrogen atom of the hydroxyl group at carbon 3 and the oxygen atom within the cyclic structure of the preceding ring, as well as between the hydrogen atom of the hydroxyl group at carbon 6 and the oxygen atom of the acetylamino functional group of the monosaccharide preceding it, as shown in Figure 1d (blue color).

The resultant rigidity bolstered by these hydrogen bonds, along with the substantial bulk of the acetamide substituent, not only imparts inflexibility to the chains but also restricts rotation along the glycosidic bonds linking the monosaccharides, leading to highly stable chain conformations (Khoushab and Yamabhai, 2010). Chitin present in its primary natural reservoir, namely crustacean shells [28,29,30,31,32], adopts an antiparallel orientation (α-chitin), in which not only intramolecular but also intermolecular hydrogen bonding is present (Figure 1d, red color), resulting in an orthorhombic crystal structure [27,43]. Conversely, β-chitin (present in squid pens, clams, oyster shells, and bones of cuttlefish, [34]) features a parallel chain alignment, with no interchain bonding, giving rise to a monoclinic crystal symmetry [43] with higher solubilizing properties. Finally, γ-chitin, less abundant and only present in some insects, comprises a blend of both α- and β-chitin, with two parallel chains followed by an antiparallel chain [44]. Another point of interest related to chitin is related to its properties, as the polymer is biodegradable [45], biocompatible [46,47], non-toxic, and eatable [48]. Its availability, coupled with its inherent characteristics, have incited the biomedicine [49,50,51] and cosmetics industries [52] to employ chitin in various processes, while its potential use in sustainable agriculture is very promising [53,54,55].

However, it should be noted that it is difficult to work with chitin, as it is insoluble in aqueous acidic media due to its acetylated residues [45,56,57]. Thus, efforts have been made to use chitin to produce more soluble compounds, such as chitosan [58,59,60,61,62,63], a biopolymer generally obtained via the chemical [64] or biotechnological [65,66] deacetylation of chitin, which exhibits a unique, versatile, and variable structure [67,68,69,70,71,72,73]. Structurally, chitosan (Figure 2b) is a copolymer formed D-glucosamine units (GlcN, major component, Figure 2) and GlcNAc units [74] connected by linear β-(1,4)-links (Figure 2b).

Similar to chitin, chitosan also exhibits both parallel and antiparallel configurations of its chains (referred to as α, β, and γ) [27,75]. In contrast, conventional chitosan, derived from α-chitin deacetylation, demonstrates a hydrated polymorphic crystalline structure wherein chitosan macromolecules form elongated double helices within the crystal lattice, reinforced by hydrogen bonding between chitosan chains facilitated by water molecules [27]. In this sense, to be considered chitosan, a copolymer must contain at least 60% of GlcN units [76,77]. The degree of deacetylation (DDA), representing the proportion of GlcN units relative to GlcNAc, significantly influences its physicochemical properties; in fact, higher DDA values result in increased protonation of amino groups, rendering chitosan more soluble and positively charged in acidic conditions [60,74]. This property is pivotal for its interaction with negatively charged surfaces and molecules, enabling applications such as drug delivery [11,12,78,79,80,81,82], wound healing [83,84,85,86], and antimicrobial coatings [87,88,89,90]. Additionally, the molecular weight of chitosan, determined by its polymerization degree, impacts its viscosity, solubility, and biodegradability [91,92]. Chitosan’s structure also facilitates the formation of polyelectrolyte complexes with oppositely charged polymers, leading to the development of advanced materials with tailored properties. Understanding the intricacies of chitosan’s structure is paramount for optimizing its performance in diverse applications in pharma [60,93,94], biomedicine [95,96], environmentally friendly agricultural formulation [53], or even wastewater treatment [97].

Although chitosan’s solubility in acidic media is higher compared to chitin, above pH 6.5, it is not soluble in water [98,99]. Moreover, chitin and chitosan, like other biopolymers, face challenges related to their low or null solubility in solvents different from water [100], which may impede their broad applicability. One of the primary issues stems from both the inherent hydrophilic nature of most biopolymers and their complex hierarchical structures (Figure 1d), including intra- and intermolecular hydrogen bonds and crystalline regions, which makes them insoluble or poorly soluble in common organic solvents [56]. This limitation restricts their processability and hinders their potential applications in industries where solvent-based processing methods are prevalent. Attempts to enhance the solubility of chitin/chitosan and other biopolymers often involve chemical modifications, such as derivatization or grafting of functional groups [101], to disrupt these structural arrangements and introduce solubilizing moieties; specifically for chitin/chitosan modification, many examples can be found in the literature [63,95,102,103,104,105]. However, such modifications can alter the biopolymers’ properties and biodegradability, posing challenges in maintaining their eco-friendly characteristics.

Thus, ongoing research efforts are focused on developing innovative strategies to improve the solubility of biopolymers while preserving their sustainability and biocompatibility for broader industrial applications. Emerging alternatives involving what are called green solvents [106] or neoteric solvents [107] such as Room-Temperature Ionic Liquids (RTILs), bio-derived solvents, or deep eutectic solvents (DESs) are gaining attention for biomass pretreatment, extraction, and processing of natural polymers. Among them, the use of DESs is probably the most promising alternative; according to the more accurate definition of DESs, they are eutectic solvents (eutectic-type systems that are liquid at a given desired temperature where at least one of its components would, otherwise, be a solid) whose components (hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs), Lewis or Brønsted acids and bases) present enthalpic-driven negative deviations from thermodynamic ideality [108]. DESs have been proven to be very useful not only for biopolymer processing [109,110,111,112] but also for many other uses [113,114,115,116], including the preparation of bioactive compounds trough catalytic procedures [117,118,119,120], due to their several notable advantages, including biodegradability, low toxicity, and easy, cost-effective, and solvent-free preparation from readily available natural sources [108]. In this sense, and to highlight this last aspect, the term NADESs (natural deep eutectic solvents) was coined some time ago [121,122]. Though the concept of NADESs holds allure and may find relevance in plant biology or biochemistry, it has been overexploited within the domain of novel green solvents, being generally accepted as a paradigm of the inherent sustainability of these solvents [114,123,124,125,126,127]. Yet, the sustainability and natural origin of the precursors most widely used to prepare DESs are questionable, as recently emphasized by Abranches and Coutinho [108]; for instance, choline, although it could be sourced from biochemical pathways and living organisms, is produced on a large scale by hydrogen chloride reacting with trimethylamine and ethylene oxide. Similarly, urea is mass-produced (for fertilizer and chemical industry raw materials) through ammonia–carbon dioxide reactions. Furthermore, some other ideal properties of (NA)DESs, such as biodegradability [128,129], lack of toxicity [130,131], reuse capability [132], easy product recovery and disposal after reaction [133], or inexpensiveness [134], cannot be taken for granted, but rather must be proved.

In this review, we present recent advancements in and insights into chitin/chitosan’s biomolecular behavior within DES-containing systems, aiming to produce bioactive molecules. As the first step, we will focus on the utility of DESs in the extraction of chitin from natural sources; secondly, the use of biocatalysis in DESs for the generation of chitin/chitosan-based materials will be commented on. Finally, the generation of bioactive materials will be presented.

2. DES-Assisted Chitin Extraction

The extraction of chitin and chitosan from their natural sources requires several independent processes, such as demineralization, deproteinization, and deacetylation [74,77,135]. Traditionally, the long-established methods to carry out these processes are chemical, using acid treatments with hydrochloric acid for demineralization [136] and basic treatments with sodium hydroxide at high temperature for deproteinization and deacetylation [64,137]. Conventional chemical processes have several disadvantages; most of these are associated with possible environmental contamination derived from the production of corrosive waste [138,139]. Another issue is related to the high energy consumption required to heat the sample in the deproteinization and deacetylation processes, as well as the concomitant CO₂ release (estimated at about 0.9 kg CO₂ per kg chitin [61]). Therefore, these disadvantages contrast with one of the most relevant aspects of the use of natural biopolymers, which is their contribution to the reduction of the environmental impact [140].

Due to this, one of the primary options for extracting chitin and chitosan, owing to their previously described characteristics, is the utilization of DESs. A recent review by Khajavian et al. covers some well-known processes involving DESs [141]; in any case, the earliest literature documenting the capability of DESs to (partially) dissolve chitin dates back to 2013, as reported by Sharma et al. [142]. These authors tested mixtures of different HBAs (choline halides or betaine) and used (thio)urea as HBD to solubilize α-chitin. In the best of the scenarios (ChCl/thiourea 1:2, 6 h heating at 100 °C), a moderate 9% w/w solubility was reported. Nevertheless, this seminal paper paved the way for further studies [57,143,144]. Nowadays, it is broadly accepted that the partial solubilization of chitin in DESs does not directly depend on the pH or pKa values of DESs, but rather on the crystallinity index and/or degree of deacetylation of chitin, and it is caused by the DES-mediated disruption of the strong hydrogen-bonding networks within chitin’s structure, facilitating a fractional dissolution and subsequent regeneration of interactions with DESs components [144].

Derived from the above-mentioned partial solubilization of chitin in DESs, currently, there are numerous studies focusing on the use of DESs in the extraction of chitin from various sources, some of which are summarized in

Table 1. Some examples of DES-assisted extraction protocols.

Source	DES	Molar Ratio	Solid/Liquid Ratio	Temp. Reaction a	Yield (%)	Protein (%)	Ash (%)	Ref.
Lobster shells	ChCl/malonic acid	1:2	7% (w/w)	50	20.63	2.02	0.3	[145]
	ChCl/malonic acid	1:2	1:10 (w/w)	50	22.21	1.81	0.21	[146]
	ChCl/malic acid	1:1		50	23.06	3.11	0.31
	ChCl/lactic acid	1:2		50	23.31	3.25	0.35
	ChCl/levulinic acid	1:2		50	20.23	2.62	0.30
Shrimp shells	ChCl/malonic acid	1:2	1:25 (w/v)	80	23.86	1.53	0.74	[147]
	ChCl/malic acid	1:2		80	25.00	3.59	1.44
	ChCl/lactic acid	1:2		80	29.20	3.01	0.48
	ChCl/citric acid	1:2		80	25.18	8.37	1.18
	ChCl/lactic acid	1:2.5	1:20	150	-	0.61	0.064	[148]
	N-methylurea, N-methylacetamide/acetic acid	1:1:3	1:30 (w/w)	-- b		5.71	3.26	[149]
Squid pens	K2CO3/glycerol	1:10	5% w/w	80	32.00	-	-	[150]
	Bet/urea	1:1	1:25	120	31.7	-	-	[151]
	K2CO3/glycerol	1:5	1:25	120	31.5	-	-
Insect	ChCl/lactic acid	1:2	1:10	50	23.33	8.50	0.25	[152]
				80	16.40	5.00	0.25
	ChCl/urea	1:2		50	26.02	4.00	3.25
				80	22.82	7.50	1.45
	Bet/lactic acid	1:2		50	25.70	10.00	2.45
				80	14.27	11.50	0.50
	Bet/urea	1:1		50	26.71	12.50	0.90
				80	12.01	3.75	1.25
Mushroom	ChCl/acetic acid	1:2	1:10	75	-	94.40	-	[153]
				95	-	82.80	-
				55 c	-	90.00	-
				-- d	-	61.30	-
				-- e	-	25.00	-
	ChCl/lactic acid	1:1		-- e	-	35.00	-
	ChCl/glycerol	1:2		-- e	-	45.00	-

^a Reaction time of 2 h. ^b All values are expressed as percentages (%). ^c Ultrasonic bath for 2 h. ^d Microwave-assisted extraction for 3 min. ^e Microwave-assisted extraction for 9 min.

. In all these research endeavors, subsequent to extraction, the authors analyze the end product; however, the variability observed in terms of the reported parameters is considerable. Some researchers solely assess the yield obtained relative to the weight of the initial powder, without taking into account the specific content of proteins or minerals removed from the sample in question. Conversely, others gauge the mineral and protein content extracted from the sample in the DES solution or the final product, presenting it as a percentage by weight of the sample. Nevertheless, unlike the former approaches, certain studies present these protein or mineral results as a percentage relative to the quantity removed via some conventional chemical method. Consequently, it would be highly advantageous to standardize or establish uniform criteria for reporting results to facilitate comprehension and subsequent comparison of the studies.

In a paper published in 2017, Zhu et al. extracted chitin from lobster shells using four different DESs [145]. These authors reported that, while the use of different mixtures of choline chloride (ChCl) with thiourea, urea, or glycerol had no significant effects on demineralization or deproteinization, a one-pot procedure using a ChCl/malonic acid mixture allowed them to reach deproteinization and demineralization values comparable to those obtained by a two-step chemical method (HCl for demineralization and NaOH for deproteinization). In 2018, Hong et al. [146] confirmed that, using different DESs composed of ChCl and four organic acids (malonic, malic, lactic, or levulinic acid), the best results were obtained with malonic acid for the demineralization and deproteinization of lobster shells. It is also important to note that, depending on the temperature and on the acid used in chitin extraction, the molecular weight of the extracted chitin was significatively different; for instance, chitin extracted using malonic acid at 50 °C and 100 °C yielded molecular weights of 312 KDa and 199 KDa, respectively, whereas extraction using malic acid at 100 °C resulted in a molecular weight of 91 KDa [146]. When concentrating on an alternative source, such as shrimp shells, Saravana et al. found that the most efficient NADES was again ChCl/malonic acid [147]. These researchers observed that combinations of ChCl with urea, thiourea, oxalic acid, glycerol, or 1,6-hexanediol yielded chitin samples with ash and protein content exceeding 40% and 15%, respectively. Although these studies have shown promising results, they are not free of other issues, as the use of malonic acid can lead to water pollution, and NADESs consisting of malic acid and choline chloride may undergo pyrolysis at 150 °C. Furthermore, there has been insufficient discussion on the entire process of chitin preparation using NADESs. In this regard, Sun et al. [148] introduced an environmentally friendly approach using choline chloride/lactic acid for chitin extraction from shrimp shells. Additionally, they conducted a systematic investigation into the reaction mechanisms involved in demineralization and deproteinization. They opted for a molar ratio of 1:2.5, reasoning that part of the lactic acid would initially react with CaCO₃, while the remaining lactic acid and choline chloride would form DESs to aid in protein removal. Furthermore, they chose a temperature of 150 °C for the reaction because, at this temperature, there is enhanced thermal movement of molecules, which increases the likelihood of collisions between DES and target molecules [154]. Finally, He et al. [149] recently reported an innovative ternary DES comprising N-methylurea, N-methylacetamide, and acetic acid for chitin extraction. To achieve this, shrimp shells were combined with the DES in various proportions and exposed to either microwave heating for varying durations or magnetic stirring at room temperature. The highest levels of demineralization and deproteinization were attained following an 11 min microwave treatment using a shell/DES ratio of 1:30.

Squid pen composition usually has 25–50% of β-chitin, 50–75% of protein, and <5% of minerals [155]. Due to the higher protein content and lower amounts of minerals, respectively, of β-chitin from squid compared to α-chitin from shrimp or crab shells, the acidic DESs are probably not efficient enough solvents to treat this source. In this sense, although Lv et al. did not mention the residual protein and mineral content in chitin, they proved that, using an alkaline DES (K₂CO₃-Glycerol), they were capable to remove 64.53% and 1.07% of proteins and minerals, respectively [150]. McReynolds et al. [151] also investigated the extraction of chitin from squid pens using acidic, neutral, and alkaline DESs. They conducted a comparison with yields obtained through conventional chemical methods and observed significantly higher yields with acidic and neutral DESs, attributed to their limited demineralization and deproteinization capabilities. However, the efficiency observed with alkaline DESs at temperatures of 100 or 120 °C was very similar to that of the chemical extraction method.

For Zhou et al. [152], the best decalcification of chitin from insects (skimmed black soldier fly (SBSF, Hermetia illucens) prepupae) was achieved with ChCl/lactic acid and betaine/urea at 80 °C. Interestingly, they also showed that the higher the operating temperature, the easier the decalcification. Regarding deproteinization, they observed the best efficiency of the betaine/urea treatment at 80 °C. Although none of the DESs used in that study achieved a deproteinization like that achieved by the chemical method, it is worth noting that the difference was insignificant (3.75% with betaine/urea vs. 2.5% with the chemical method). Chitin extracted not only from insects [152] but also from mushrooms [153] had a high deacetylation degree (DDA), higher than 60%; therefore, it can surely be considered chitosan [74]. To reach similar compositions, a second deacetylation step must be carried out to obtain chitosan from lobster, shrimp, or squid pen chitin. Once again, this deacetylation is traditionally carried out by chemical treatments with NaOH via homogeneous or heterogeneous processes [99]. However, as was mentioned, these processes have many disadvantages and can be substituted by DESs.

Unlike chitin extraction, there are fewer deacetylation studies on obtaining chitosan. An example is the study of Vicente et al., who were the first to report on deacetylation with DESs requiring only mild reaction conditions [156]. To this end, chitin was mixed with several DESs (acidic, neutral, and alkaline) in a ratio of 1:50 (w/v). DESs were tested for their ability to promote chitin deacetylation and the results were compared with the initial DDA of chitin. They observed how ChCl/malic acid showed the best result, yielding a DDA of 40% after 24 h of reaction at 120 °C. However, this degree of deacetylation is too low for the obtained product to be considered chitosan. Recently, in 2024, Sun et al. [157] proposed the use of a NADES composed of betaine and glycerol (1:2.5 molar ratio) in the deacetylation reaction of chitin to facilitate the subsequent attack of the N-acetyl groups with 25 wt% NaOH at 100 °C for 12 h. Conventionally, concentrations of 50% NaOH are employed in chemical methods to obtain chitosan, but with the mixture of betaine/glycerol and 25% NaOH, chitosan with 83.77% deacetylation was obtained. Similar work was previously conducted by Vicente et al. in 2020 [143], but they used 30% NaOH and the DDA achieved was lower (70–80%).

3. Biocatalysis for Chitin/Chitosan Modifications

Biocatalysis, the use of biological catalysts for the generation of many different valuable products, has proven to be a very useful tool, not only in the lab [158,159,160] but also at an industrial scale [161,162,163], undoubtedly fostered by the inherent sustainability associated with biocatalysis [164,165,166]. Additionally, the well-reported capability of enzymes to work in media different from water [167,168,169], and particularly in DESs [119,170,171,172,173], has expanded their applicability, up to the point that some authors are pointing towards a golden age for biocatalysis [174].

In this sense, biocatalysis is also a valuable tool for processing chitinaceous substrates [103,175,176,177]. For instance, chitin deacetylases (EC 3.5.1.41, [178,179,180,181]) are enzymes capable of catalyzing the hydrolysis of GlcNAc subunits to convert them into GlcN, while chitooligosacharide deacetylases (EC 3.5.1.105) can perform the same deacetylation on chitosan and chitooligosacharides following different endo- or exo-mechanisms [181]. These enzymes would therefore be the biotechnological option for converting chitin into chitosan, as the first step in a valorization cycle [61] leading to a chitin-based biorefinery [28].

Regarding chitin and chitosan depolymerization, these biopolymers can be specifically recognized by different glycosidase-type hydrolases present in nature, leading to different oligomeric structures, as depicted in Figure 3. Thus, chitinases [182,183] are enzymes which act on chitin following two types of mechanisms; on the one hand, endo-chitinases (EC 3.2.1.14) are enzymes which hydrolyze internal glycosidic bonds between GlcNAc subunits, producing various fragments with different sizes, ranging from dimers to polymers. Conversely, exo-chitinases (also named β-N-acetyl-glucosaminidases, EC 3.2.21.52) selectively hydrolyze chitin starting from either the reducing or non-reducing end, generating GlcNAc and, to lesser extent, dimeric (DP = 2) GlcNAc units. Chitosanases (EC 3.2.1.132 [184,185,186]) cleave bonds in chitosan following an endo path (Figure 3b); according to their specificity, they are divided into several types: type I (cutting GlcNAc-GlcN and GlcN-GlcN bonds), type II (only GlcN-GlcN), type III (hydrolysing GlcN-GlcN and GlcN-GlcNAc), and type IV (cleaving all bonds except GlcNAc-GlcNAc). Another type of enzymes acting on chitosan are exo-β-D-glucosaminidases (EC 3.2.1.165), which hydrolyze chitosan from its non-reducing end [187].

Besides hydrolysis by chitinases and chitosanases, chitin and chitosan can be nonspecifically degraded with other nonspecific glycosyl hydrolases, such as endoglucanases, cellobiohydrolases, endoxylanases, endomannases, lisozyme, α- and β-amylases, or hyalurodinases [175,176]. Remarkably, other hydrolases, such as proteases or lipases, are also able to degrade chitosan to render chitooligosacharides (COs) with different molecular weights. These hydrolases show a hydrolytic endo-mode of action, with optimal pH at slightly acidic values (around 5 [175]).

Now, it should be noted that achieving high hydrolytic efficiency proves challenging when employing enzymes. Particularly, chitin has a highly ordered crystalline structure that limits the accessibility of enzymes. Therefore, research efforts have been made to improve the efficacy of enzymatic processing of chitin and chitosan. One strategy would involve the synergistic utilization of physical treatments, such as microwave [188], ultrasonication [189], or mechanochemistry [190,191,192,193], to help in the enzymatic degradation of chitin by promoting its partial solubilization. Another option would be the use of strong organic solvents which have been reported to dissolve chitin [57], such as such as LiCl/dimethylacetamide (DMAc) [194], CaCl2/MeOH [195], or hexafluoroisopropanol (HFIP, [196]). These solvents were previously used in the solubilization of chitin before enzymatic hydrolysis, but they were not viable for enzyme stability [197].

In this context, as DESs are perfectly compatible with enzymes [119,171,198], the synergic combination of these solvents for promoting the solubilization of chitinaceous biomaterials and enzymatic catalysis would constitute a smart approach. Nevertheless, although the positive effect of DESs on the chitin deacetylation has been reported and explained by molecular modeling [156,157] (see Table 1), to the best of our knowledge, no examples have been reported for the combined use of enzymes and DESs, neither for chitin deacetylation nor for chitin or chitosan depolymerization.

Yet, the use of DESs and enzymes has been reported for the quaternization of chitosan. This process involves the introduction of a quaternary ammonium moiety onto or adjacent to the chitosan backbone through reactions with primary amino and hydroxyl groups under various experimental conditions [199,200]. By incorporating a quaternary moiety into chitosan, the resultant polymer gains permanent cationic charges on its backbone, leading to enhancements in properties like water solubility, antimicrobial activity, mucoadhesiveness, and permeability [201,202]. This modification enables multiple applications, primarily in the biomedical and pharmaceutical fields [203,204]. Traditional forms of quaternized chitosan, such as N,N,N-trimethyl chitosan (TMC) and N-[(2-hydroxy-3-trimethyl ammonium) propyl] chitosan (HTCC), have been widely used, alongside newer variants incorporating pyridinium or phosphonium salts [199]. A significant challenge of this chemical modification lies in its low selectivity, resulting in a mixture of O- and N-methylated products, as well as the non-desired breaking of the chitosan polymer due to the stringent conditions required (organic solvents such as N-methyl-2-pyrrolidone (NMP), dimethyl sulfate (DMS), or dimethylformamide (DMF); iodomethane as a methylating agent and NaOH as a base), consequently reducing the molecular weight of the final product. Thus, two sequential publications by Dandekar, Jain, and coworkers [205,206] reported the use of lipases from Burkholderia cepacia or Candida rugosa for the transformation of chitosan into the desired methylated products in DES/water mixtures using dimethyl carbonate (DMC) as a sustainable methylating agent (without the need for a base additive) and at lower process temperatures compared to the chemical synthesis (Figure 4).

The conventional method for synthesizing N-methylated chitosan typically involves the use of organic solvents under alkaline conditions, with methyl iodide serving as the methylating agent. However, this approach lacks selectivity in N-methylation and often leads to significant polymer scission. Thus, Bangde et al. [205] evaluated two types of DESs, ChCl/urea 1:2 and ChCl/glycerol 1:2, either alone or in combination with other solvents (water and/or DMF) for chitosan methylation, showing selective N-methylation of products when using ChCl/urea 1:2, while those ChCl/glycerol 1:2 showed some O-methylation, with a significant reduction in polymer scission compared to traditional methods. When adding lipase, the enzyme from Burkhorlderia cepacia in ChCl/urea 1:2 furnished mainly N,N-dimethylchitosan (around 36%) and some O-methylation, while immobilized Candida antartica lipase failed in this regard. Conversely, Mahajan et al. [206] reported the use of ternary DESs composed of ChCl/urea/glycerol, showing that the 1:2:1 ternary mixture allowed for a high degree of N,N,N-trimethylation when using lipase from B. cepacia, while lipase from Candida rugosa led mainly to the O-methylated product.

4. DESs for the Generation of Chitinaceous Bioactive Materials

As already pointed out in the Introduction, and because of its unique structure, chitosan exhibits plenty of intrinsic therapeutical properties [60,62,63,77,91,207,208], summarized in

Table 2. Therapeutical properties of chitosan.

Property/Activity	References
Antidiabetic	[209,210,211,212]
Anti-inflammatory	[213,214,215,216]
Antifungal	[217,218,219,220,221,222]
Antimicrobial	[90,223,224]
Antioxidant	[90,223,225]
Antitumoral	[226,227,228,229,230]
Antiviral	[231,232,233,234,235]
Hemostatic	[84,236,237,238]
Hepatoprotective	[239,240,241,242,243]
Mucoadhesive	[244,245,246]
Neuroprotective	[247,248,249]
Skin regeneration/wound healing	[84,250,251,252]
Stem cell proliferation	[253,254,255,256,257]

(containing some updated references).

The medicinal relevance of chitosan is even higher if we consider that, aside from its inherent therapeutic applications per se, its ability to be processed into various forms, such as films, hydrogels, nanoparticles, and 3D scaffolds, increases their applicability to an even higher extent [96,208,258]. In this sense, the use of DESs is also highly valuable. Some recent examples are shown in

Table 3. DES-assisted preparation of chitosan biomaterials.

Application	DES	Biomaterial	Ref.
Reducing bacterial biofilms	ChCl/urea	Porous monoliths and films	[259]
Antibacterial wound dressing formulation	ChCl/glucose	Sodium hyaluronate, dopamine, chitosan, and aloe vera	[260]
Antibacterial wound dressing formulation	ChCl/lactic acid	Chitosan biofilm	[261]
Antibacterial wound dressing formulation	Glycerol/lactic acid	Chitosan/PVA skeleton-type 3D networks	[262]
Drug delivery (curcumin)	ChCl/glycerol	Chitosan/alginate hydrogen beads	[263]
Reducing atherosclerosis	ChCl/urea	Chitin–glucan complexes	[264]
Anticoagulant and antiviral properties	Sulfamic acid/urea	Sulfated chitosan	[265]
Alternative to traditional fluorophores and metal-based catalysts	ChCl/urea	Luminescent nitrogen-doped carbon dots from chitin	[266]
Catalyst for aerobic oxidation of β-isophorone	ChCl/urea	Metallophthalocyanines on chitosan	[267]
Food packaging	ChCl/several HBDs	Chitosan films	[268]
	ChCl/glycerol	Chitosan films	[269]
	ChCl/lactic acid	Chitosan films	[270]
	ChCl/lactic acid	Chitosan/lignin films	[271]
	Betaine/lactic acid	Chitosan/lignin nanoparticle films	[272]
	Thymol/octanoic acid	Chitosan/gelatin films	[273]
Self-healing biomaterials	ChCl/citric acid	Self-healing chitosan films	[274]
Biocomposites	ChCl/thiourea	Chitosan nanofibers	[275]
Biocomposites	ChCl/urea	cellulose nanowhiskers (CNW)/chitosan nanocomposite	[276]
Non-porous (dense) membranes for pervaporation	L-proline/sulfolane	Chitosan crosslinked with glutaraldehyde	[277]
Dye absorption	ChCl/urea	Chitosan beads	[278]
	ChCl/lactate	Chitosan/lignin	[279]

.

Focusing on therapeutic applications, Delgado-Rangel et al. [259] reported the use of ChCl/urea for producing 3D chitosan materials targeting V. cholerae biofilm. They developed an environmentally friendly method for creating porous monoliths and films, showcasing DES-assisted phase separation processes’ versatility. The production involved a three-step process of evaporation-induced phase separation, blending 2% chitosan with acetic acid and the DES. The resulting plasticized film structure was achieved after evaporating the acidic aqueous solvent. Opting for an equal-weight ratio of chitosan/DES yielded films with the most suitable porous structure. The thermal stability of chitosan was influenced by the presence of residual DES, impacting V. cholerae growth in the chitosan films. In another example, Wang et al. [260] reported a fully natural hydrogel formulation containing sodium hyaluronate, dopamine, aloe vera, chitosan, and a DES formed by ChCl/glucose as an eco-friendly, biodegradable wound dressing, showing excellent cytocompatibility with NIH-3T3 fibroblast cells and antibacterial efficacy against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. After characterizing the gel using different methodologies, these authors concluded that this hydrogel displayed not only remarkable cytocompatibility and antibacterial properties, but also facilitated skin tissue regeneration, promoting effective wound healing in mouse skin within 12 days post-surgery. Similarly, Sun et al. [261] have reported how a DES formed by ChCl/lactic acid allowed for the preparation of chitosan biofilms for antibacterial wound dressing, very active against Staphylococcus aureus and Escherichia coli, easily degradable in both soil and water, and also showing UV barrier properties.

Sun et al. [263] have recently reported the generation of a biocompatible chitosan-based supramolecular 3D aerogel, also intended for wound healing, formed by chitosan, polyvinyl alcohol, and a DES (glycerol/lactic acid) joined by hydrogen bonding, which was also very active against S. aureus and E. coli and was very active in promoting wound healing.

Chitosan-based nanoparticles and hydrogels have emerged as effective drug delivery systems due to their high loading capacity, sustained release kinetics, and targeted delivery [82,252,280]. Chitosan nanoparticles can encapsulate both hydrophilic and hydrophobic drugs, protecting them from enzymatic degradation and facilitating their transport across biological barriers. Additionally, the mucoadhesive properties of chitosan enhance the residence time of drug-loaded nanoparticles at mucosal surfaces, thereby improving drug absorption and bioavailability. Because of their unique properties, DESs are also especially suited for drug delivery [281]. As an example of this synergy, Silva et al. [263] reported a system wherein curcumin dissolved in ChCl/glycerol was encapsulated within beads formed via ionotropic gelation with chitosan and alginate. The beads were manufactured using an extrusion-dripping technique. The primary objective of the investigation was to develop hydrogel beads loaded with curcumin, exhibiting enhanced solubility and stability throughout transit along the gastrointestinal tract, therefore becoming a sustainable and promising alternative to surmount solubility challenges and the necessity for eliminating organic solvents.

Chitin present in mushrooms is bonded to β-glucan, forming complexes (CGCs, chitin–glucan complexes). Kim et al. [264] utilized ChCl/urea to pretreat white button mushrooms for the formation of CGCs (which have shown to present beneficial effect for the treatment of atherosclerosis [282]) under mild and environmentally friendly conditions. These author prepared CGCs with different compositions, varying the percentage of chitin or chitosan, in all cases resulting in different materials compared to those obtained from the extraction from shrimp chitin that displayed a distinctive morphology which was favorable for subsequent functionalization.

Chitosan’s limited water solubility restricts its utility, but introducing a sulfate group enhances solubility and biodegradability; thus, sulfated chitosan gains biological activity akin to heparin and heparin sulfate [283,284], exhibiting anticoagulant and antiviral properties, serving as substitutes for heparin or heparin sulfate in biomedicine [285]. In this area, Kazachenko et al. [265] reported the sulfation process of chitosan using sulfamic acid, both in 1,4-dioxane with urea and in a DES formed by sulfamic acid/urea. The optimal reaction conditions were fairly similar in terms of temperature (89.2 °C vs. 88.0 °C) and time (2.9 vs. 2.1 h). FTIR was used to confirm the introduction of a sulfate group into the chitosan structure, while XRD analysis revealed amorphization during the sulfation process of chitosan. Particle size distributions on the films of sulfated chitosan, obtained in both 1,4-dioxane and the sulfamic acid/urea deep eutectic solvent, were also found to be very similar or identical (around 160 nm). GPC data confirmed the main difference, as sulfated chitosan obtained using 1,4-dioxane had a molecular weight (Mw) of approximately 36.6 kDa, whereas sulfated chitosan obtained with the DES had a slightly lower molecular weight, around 27.2 kDa.

Chitosan presents a promising option for food packaging because of its biodegradability, antimicrobial activity, and barrier properties against gases and liquids [286]. Its film-forming ability allows for the creation of biodegradable and edible coatings, extending the shelf life of perishable foods by reducing microbial growth and moisture loss. Additionally, chitosan’s compatibility with food ingredients and its safety for human consumption make it a suitable material for eco-friendly and sustainable food packaging solutions, contributing to reducing plastic waste and environmental impacts [287]. Thus, it is not surprising to find plenty of uses of DESs as auxiliaries/additives to improve chitosan-based food packaging biomaterials, as shown in Table 3. In this sense, it is worth mentioning the synergic combination of chitosan (derived from chitin, the world’s most abundant renewable resource and a by-product of the fishing industry [27]) and lignin (derived from cellulose, the most abundant biopolymer on Earth [26]) for the sustainable production of biomaterials useful for food packaging [271,272]. This synergy is well known, as both chitin and lignin as natural ingredients show interesting applications in the substitution of usual synthetic chemicals for the production of innovative/healthy products [288]; in addition, chitin and lignin are used in production of phenolic monomers via catalytic depolymerization processes [289,290].

Self-healing materials, prized for their ability to repair damage autonomously, find extensive application in coating design, energy storage, corrosion protection, and tissue engineering [291]. Chitosan-derived polymers have demonstrated inherent self-healing properties due to reversible interactions leading to a dynamic physical or chemical crosslinked 3D network established either by noncovalent interactions (hydrogen bonding, electrostatic and hydrophobic interactions) or dynamic covalent bonds in chitosan-modified materials [292]. Moreover, the natural propensity of DES components to form hydrogen bonds offers a favorable characteristic for crafting self-healing ion gels. For instance, Smirnov et al. [274] prepared plasticized chitosan films with a high content (67–82 wt%) of ChCl/citric acid DES. The film, containing 67 wt% of NADES, displayed a 56% recovery of elongation at break and 72% of initial strength post-rupture, alongside self-healing capabilities. This was credited to the dynamic network of hydrogen bonds between the polymer and DES components, as confirmed by FTIR analysis. The film’s highly hydrophilic nature, coupled with internal osmotic pressure, could enable swift reversible vapor-induced movement, mimicking certain plant movements.

Up to this point, the preponderant role of chitosan for generating different biocomposites has been clearly proved [293]. DESs have been also used for facilitating the generation of chitosan-based nanofibers [275] or nanowhiskers [276]. As an additional proof of the synergic combination of DESs and chitosan, different biomaterials useful for dye absorption [278,279] or for pervaporation membranes [277] have been described.

5. Conclusions

Based on the data outlined in this review, our aim has been to document, albeit not exhaustively, the significant advantages of utilizing deep eutectic solvents (DESs) in the realm of chitin/chitosan extraction and modification. We have sought to underscore the inherent sustainability linked to the combined use of biocompatible, biodegradable, and versatile chitinaceous biomaterials in conjunction with DESs, environmentally friendly solvents comprising distinctive blends of hydrogen bond acceptors and donors with specific physicochemical properties, including low toxicity, minimal volatility, and adaptable solvation capabilities. This synergy facilitates the development of pioneering drug delivery systems, wound dressings, tissue engineering scaffolds, and antimicrobial agents. We firmly believe that we are witnessing a kind of golden era in the application of DESs for the production of therapeutic biomaterials derived from biopolymers in general, and chitin/chitosan in particular, anticipating a plethora of new and exciting developments in this field over the coming years.