Thiolated Chitosans: A Multi-talented Class of Polymers for Various Applications

Various properties of chitosan can be customized by thiolation for very specific needs in a wide range of application areas. Since the discovery of thiolated chitosans, many studies have proven their advantageous characteristics, such as adhesion to biological surfaces, adjustable cross-linking and swelling behavior, controllable drug release, permeation as well as cellular uptake enhancement, inhibition of efflux pumps and enzymes, complexation of metal ions, antioxidative properties, and radical scavenging activity. Simultaneously, these polymers remain biodegradable without increased toxicity. Within this Review, an overview about the different possibilities to covalently attach sulfhydryl ligands to the polymeric backbone of chitosan is given, and the resulting versatile physiochemical properties are discussed in detail. Furthermore, the broad spectrum of applications for thiolated chitosans in science and industry, ranging from their most advanced use in pharmaceutical and medical science over wastewater treatment to the impregnation of textiles, is addressed.


INTRODUCTION
Derived from chitin via deacetylation, chitosan displays a cationic charge, differentiating it from other polymers and making it a unique polysaccharide with properties like biocompatibility, biodegradability, and antimicrobial activity. Various derivatives as well as forms of chitosan have been developed to further adjust the properties of this polymer for different applications. 1−10 Among these derivatives, thiolated chitosans, obtained by the covalent attachment of different −SH group-bearing ligands mainly to the primary amino but also to the hydroxyl groups of the polymer, might be the most auspicious ones. As these kind of chitosans were shown to exhibit substantially improved properties over the unmodified polymer, the variety of thiolated chitosans and the number of sound applications has strongly increased since their discovery in 1998. 11−13 Having the great potential of these polymers in mind, and taking into consideration all the opportunities offered by more and more new types of thiolated chitosans exhibiting additional functions, we are certain that they will further alter the landscape of biomaterial sciences and engineering.
This Review should encourage and motivate scientists in academia and industry to move in or intensify their activities in this promising research field. It provides an overview of the various thiolated chitosans and the chemistry behind them. The basic characteristics of unmodified chitosan and its thiolation are described, followed by the properties gained by the addition of immobilized thiol groups. Furthermore, applications of thiolated chitosans in science and industry ranging from their pharmaceutical and biomedical use over cosmetics and wastewater treatment to the impregnation of textiles are discussed.

CHITOSAN: ITS CHEMISTRY AND THIOLATION
Chitosan, as displayed in Figure 1, is a copolymer consisting of N-acetyl-D-glucosamine and D-glucosamine connected by linear β-(1→4) glycosidic bonds. 14 It is obtained via partial deacetylation of chitin, a polysaccharide that is a main part of the exoskeleton of fungi, insects, and crustaceans and, after cellulose, the most abundant natural polymer. 15 The acetyl groups are cleaved off either enzymatically by chitin deacetylase or in alkaline conditions using concentrated sodium hydroxide, resulting in different degrees of deacetylation (DD). Together with the molecular weight of chitosan, the DD determines the physicochemical characteristics, such as solubility, of chitosan. As the resulting amino groups show a pK a value of 6.5, chitosan is protonated in acidic surroundings and therefore displays water solubility as well as a cationic charge. 16 In general, the syntheses of thiolated chitosans can be divided into methods affecting the hydroxy moieties, the amino groups, or both. Subsequently, these methods can be further categorized according to the way thiol groups are introduced, either by direct substitution or by attaching a −SH-bearing ligand. The thiol groups of these ligands directly exist as such or need to be first disclosed via additional chemical treatment. Furthermore, the resulting −SH moieties can be S-protected/ preactivated via disulfide formation with another thiol-bearing ligand. Thereby, the free thiol groups are protected from oxidation and show an increased reactivity over a broader pH range. 17 Attaching an already S-protected/preactivated ligand is another way to synthesize these derivatives. As an example, the synthesis of S-protected/preactivated chitosan thioglycolic acid is depicted in Figure 1. In a first step, thioglycolic acid is attached to the amino group of pristine chitosan via carbodiimide-mediated amidation, and afterward, the resulting free −SH moieties are S-protected/preactivated by a disulfide exchange reaction with 6-mercaptonicotinamide or 6,6′dithionicotinamide.
Since the synthesis of the first thiolated chitosan, 12 numerous derivatives with various ligands, illustrated in Table 1, have been synthesized. For the analysis of the amount of immobilized −SH groups of these thiolated chitosans, Ellman's reagent optionally with previous reduction of disulfide bonds with borohydride or iodometric titration can be used. 18 Phadungcharoen et al., for instance, have conducted the Ellman's assay via smartphone, providing an easy, fast, and reliable analysis of thiolated polymers without access to sophisticated equipment. 19

PROPERTIES OF THIOLATED CHITOSANS
Due to the covalent attachment of thiol groups to chitosan, various properties of this polysaccharide, as outlined in Figure  2, can be gained. These properties are discussed in detail within this section.
3.1. Adhesion to Biological Surfaces. Adhesion to substructures of biological surfaces like mucins or keratins is a key parameter of thiolated chitosans, as for instance in drug delivery a prolonged residence time on mucosal membranes is essential for local therapy or for non-invasive systemic delivery. 121 To understand the underlying mechanism of mucoadhesion, the composition of the mucus has to be considered. It mainly consists of water, inorganic salts, lipids, and mucin glycoproteins, whereby its composition as well as thickness varies at different application sites. 15 As mucin glycoproteins bear negatively charged sialic and sulfonic acid moieties, mucoadhesive properties of pristine chitosan mainly result from ionic interactions of its positively charged amino groups with these sialic and sulfonic acids. 15,122 Besides these ionic interactions also van der Waals, hydrogen, and hydrophobic bonds take part in the mucoadhesion of unmodified chitosan. 123,124 By introducing thiol-bearing ligands to chitosan, covalent bonds are added to the so far mentioned interactions with mucosal surfaces due to the formation of disulfides with    125,126 This covalent bond formation is also responsible for the adhesion of thiolated chitosans to keratinous surfaces, as keratins display a high cysteine content of 7−20%. 127 Thus, thiolated chitosan exhibits an enhanced mucosal as well as keratinous adhesion compared to the unmodified polymer, whereby a lower pH of the surrounding medium intensifies this effect due to a reduced reactivity of the thiol groups, resulting in a lower extent of oxidation prior to surface contact. 42,89 Therefore, preactivation of the attached −SH groups can further increase adhesiveness Where applicable limited to three references; for further references please contact the corresponding author.
Biomacromolecules pubs.acs.org/Biomac Review of thiolated chitosans, as these preactivated forms display on the one hand an improved reactivity over a wide pH range and are, on the other hand, less prone to oxidization. 17,89,125 Samprasit et al., for example, showed the enhanced mucoadhesive properties of thiolated chitosans by an in vivo study with human volunteers using cysteine thiolated nanofiber mats. As illustrated in Figure 3, an over 3-fold prolonged adhesion time to buccal mucosa compared to unmodified nanofiber mats was observed, in good accordance with in vitro data acquired also within this study on porcine buccal mucosa. 128 3.2. Cross-Linking. Thiolated chitosans display enhanced in situ gelling properties compared to pristine chitosan, caused by cross-linking due to the formation of intra-as well as interchain disulfide bridges via oxidation. 129−131 This crosslinking process can be accelerated by the addition of different  Biomacromolecules pubs.acs.org/Biomac Review oxidizing agents such as hydrogen peroxide, leading, for example, to a dynamic viscosity increase for chitosan− thioglycolic acid of up to 16 500-fold within 20 min. 132 Furthermore, Hintzen et al. showed an impact of the thiolated chitosan's molecular weight on in situ gelation. A lower molecular weight resulted in a distinct increase in viscosity due to a higher flexibility as well as mobility of polymer chains, leading to a higher degree of cross-linking. 133 Moreover, as with an increasing hydroxide ion concentration more negatively charged thiolate anions are available, the tendency for disulfide bond formation increases. Therefore, thiolated chitosans like chitosan−thioglycolic acid do not form disulfides at pH < 5, and so they are not useful as in situ gelling systems for drug delivery at applications sites with acidic pH.
Based on these considerations, Millotti et al. synthesized a thiolated chitosan, namely chitosan−mercaptonicotinic acid, with pH-independent cross-linking properties. This thiolated chitosan bears two tautomeric forms ( Figure 4) and, thus, can also react in a protonated state, as one of its forms displays a nucleophile (CS) as well as proton donor (N−H) within its structure, enabling the formation of disulfides. 134 In addition to the mentioned gelling process via oxidation, thiol−ene reactions between thiolated chitosans and alkenes can be utilized to generate in situ gelling systems. 135 The gelling characteristics of such systems can be altered by the concentration of available thiol groups on chitosan and by the type of reaction partner. 72,136,137 Apart from in situ gelation, cross-linking is also used to improve the mechanical properties of chitosan. For example, Wu et al. increased the physical strength of a biodegradable and biocompatible hydrogel composed of an N-isopropylacrylamide−chitosan copolymer via coupling of N-acetylcysteine. The compressive modulus, as highlighted in Figure 5, of a thiolated chitosan copolymer gel was over 9-fold improved compared to that of the unmodified copolymer gel. 138 Furthermore, Miles et al. investigated the mechanical properties of chitosan films in order to construct a robust material. A 4-fold higher tensile strength, 6-fold higher breaking strain, and  . Average compressive modulus of hydrogels composed of N-isopropylacrylamide and chitosan or chitosan−N-acetylcysteine calculated from the slope of the stress−strain curve in a range of 10−20% strain (toe region). NC = N-isopropylacrylamide-g-chitosan copolymer; TNC = thiol-modified N-isopropylacrylamide-g-chitosan with different amounts of free thiol groups (TNC50: 101.84 ± 18.35 μmol/g, TNC100: 141.91 ± 27.15 μmol/g, TNC200: 299.39 ± 8.11 μmol/g). Significantly higher moduli were observed for TNC100 as well as TNC200 compared to NC and TNC50 (p < 0.05). Indicated values are means (n = 3) ± SD. Reprinted with permission from ref 138. Copyright 2018 Elsevier. Biomacromolecules pubs.acs.org/Biomac Review 14-fold increased average toughness were observed for chitosan−N-acetylcysteine with a 20% degree of substitution compared to the unmodified polymer. Additionally, the resilience of the prepared thiolated polymer films, characterizing the ability to deform without energy dissipation, was significantly enhanced. In contrast, the crystallinity of the films decreased due to the attachment of thiol groups. Usually, a higher crystallinity results in stronger mechanical properties. Cross-linking within the polymer was compensating this behavior, leading to a material with lower crystallinity and, at the same time, enhanced mechanical properties. 139 3.3. Swelling Behavior. Introduced −SH groups have an impact on the swelling characteristics of chitosan, as these groups display a lower hydrophilicity compared to hydroxyl and amino groups and can additionally cross-link the polymer chains via disulfide bridges. 25,90 Thus, the swelling of thiolated chitosans is in many cases slower and less pronounced than that of unmodified chitosan. A slow and stable swelling process resulting in robust and firm networks is desired for applications such as wound healing, where high amounts of exudate can occur over an extended time period. 44,137 Apart from −SH groups, the type of attached ligand has also a substantial impact on the swelling behavior of the polymer. Medeiros et al., for example, observed a 20% and 30% minor swelling capacity of chitosan−mercaptoundecanoic acid in comparison to chitosan−cysteine and unmodified chitosan, respectively, caused by the hydrophobicity of the attached acyl chain. 25 For a thiolated chitosan with a hydrophilic ligand like chitosan−homocysteine thiolactone, however, an up to 5-fold increased swelling ratio was determined in comparison to unmodified chitosan. 102 Furthermore, preactivating thiolated chitosans with hydrophobic ligands such as 6-mercaptonicotinamide can result in a decrease in swelling capacity, as a 40% lower weight gain of S-preactivated chitosan−thioglycolic acid compared to chitosan−thioglycolic acid without preactivation was observed. 17 Accordingly, the swelling capacity of a thiolated chitosan can be adjusted to match the intended area of application by using an appropriate ligand and degree of S-preactivation.
3.4. Controlled Release. A sustained release of an active pharmaceutical ingredient (API) can be essential for drug delivery, as drug concentrations remaining within the therapeutic window for an extended period of time can reduce side effects and increase treatment efficacy. Moreover, patient compliance and convenience can be improved with controlled drug delivery systems. 140 The release of APIs is influenced by the cross-linking density of a hydrogel, as thereby, the swelling behavior of the hydrogel is altered, resulting in a minor drug diffusion. 89,141 For instance, Moreno et al. obtained differently swelling gels based on increasing amounts of 6-mercaptonicotinic acid and cysteine attached to chitosan and could modulate the release of ranibizumab and aflibercept with these gels over 7 days. As the release profile and retention of these two drugs out of the hydrogels were comparable to those of fluorescein isothiocyanate−dextran, with an average molecular weight of 40 kDa, physical entrapment in the gel systems was identified as the main cause for sustained drug release. 44 For formulations like liposomes, micro-or nanoparticles composed of or coated with thiolated chitosans, various studies have shown a sustained release of encapsulated drugs resulting from disulfide bridge formation within and between the polymer chains controlling API liberation. 75 66 Furthermore, polymer tablets based on thiolated chitosans display longer disintegration times in comparison to tablets comprising just unmodified chitosan due to a higher mechanical stability caused by formed disulfide bridges. Consequently, drug release can be sustained. Millotti et al., for instance, showed that 25% of insulin is released from chitosan−6-mercaptonicotinic acid tablets within 180 min, whereas at this time point already 80% of the APIs are released from unmodified chitosan tablets. 43 Additionally, it is possible to delay API release by utilizing non-covalent drug−thiol ligand interactions. Due to these interactions, a significantly different liberation of metronidazole was observed out of tablets consisting of chitosan− N-acetylcysteine entirely S-protected with 6-mercaptonicotinamide with 320 μmol ligand/gram polymer, in comparison to tablets composed of the thiolated chitosan with 160 μmol of ligand/gram polymer. 96 Another way to achieve a sustained release is based on binding drugs covalently to carrier systems via disulfide bridges with the thiol ligands. Via this mechanism, Chen et al. as well as Liu et al. achieved a controlled release for the BMP2-derived peptide P24. In both studies the peptide was coupled to chitosan−4-thiobutylamidine, which was confirmed via FT-IR 147 or by X-ray photoelectron spectroscopy. 148 As shown in Figure 6, the hydrogel prepared by Liu et al. displayed a zeroorder release of the peptide over 16 days. 148 Chen et al., however, extended this time frame to 90 days with their hydrogel formulation and observed an enhanced performance regarding the repair of bone defects in vivo in rats. 147 3.5. Enhancement of Permeation. A major reason for the ability of thiolated chitosans to enhance the permeation of incorporated APIs is based on the opening of tight junctions by Biomacromolecules pubs.acs.org/Biomac Review interacting with thiol groups of cysteine-bearing membrane receptors and enzymes. 92,149 In detail, different mechanisms are claimed to contribute to this tight junction opening. On the one hand, thiolated chitosan could inhibit protein tyrosine phosphatase dephosphorylating tyrosine subunits on occludin, thus opening tight junctions. 149 On the other hand, as membrane receptors such as epidermal growth factor and insulin-like growth factor contain high amounts of cysteine, thiolated chitosans could interact with these and cause the activation of the intracellular protein tyrosine kinase Src due to phosphorylation. As a result, claudin-4 proteins are disrupted, leading to tight junction opening. 92 The structure and pK a of attached ligands have substantial impacts on the permeationenhancing effect, whereby for 6-mercaptonicotinic acid the most pronounced effect was observed. 150 Responsible for this superior effect are likely the two tautomeric forms of 6mercaptonicotinic acid, leading to more reactive thiol groups under physiological pH conditions, as described in section 3.2. 134 Other attached thiol ligands can be oxidized, especially at pH values ≥5, causing an impaired permeation enhancing effect. As a result, S-protected forms display a more pronounced permeation enhancement due to their higher pH stability. 151,152 Moreover, in the case of thiolated chitosans, the remaining cationic amino groups can electrostatically interact with anionic carboxyl groups of transmembrane receptors like integrin αvβ3, thereby inducing claudin-4 down-regulation and opening of tight junctions. 92 Additionally, a prolonged mucosal residence time provided by the mucoadhesive properties of thiolated chitosans, as well as their inhibiting effect on API degrading enzymes as described in section 3.8, may contribute to the permeation enhancing effect. Another advantage of thiolated chitosans is their high molecular weight, avoiding their absorption and consequently preventing systemic toxicity and prolonging their permeation enhancing effect. 153 Fur-thermore, as the permeation enhancing mechanism of thiolated chitosans differs from those of other permeation enhancers, a combination can lead to an additive effect. 154 The permeation enhancement of thiolated chitosan for different APIs could be observed in various in vivo studies. 43 143 3.6. Enhancement of Cellular Uptake. Thiolated chitosans increase cellular uptake by different mechanisms. 162 The first mechanism is based on reactions of thiol functions present on the surface of the polymeric carrier with exofacial thiols of transmembrane proteins such as glycosylphosphatidylinositol-linked proteins or proteins non-covalently bound to the plasma membrane, leading to a more efficient absorptive endocytosis. As shown in Figure 8, three different types of these reactions can be assumed. First, an exofacial thiol group can react with a disulfide bond within the cargo-bearing vector, leading to the formation of a mixed disulfide bond. Second, a disulfide exchange reaction between a −SH group of the carrier and a disulfide bond within a cell surface protein may take place. Finally, the third possibility involves the formation of an intermolecular disulfide bond between a thiol group of the carrier and one of a cell surface protein. In each case, the resulting complex is internalized, and the cargo is released after internal cleavage of the previously formed disulfide bond. 163 This cleavage mainly takes place within the reducing environment of the cytosol where there is a higher glutathione content compared the extracellular space. Thus, thiolated carrier systems can be designed for targeted intracellular release which is, for instance, important for gene therapy. 164  Consequently, the cellular uptake decreases when the thiol groups of a thiolated chitosan carrier are oxidized to a large extent, as only the reaction of an exofacial thiol group with a disulfide bond of the carrier can take place. Additionally, overstabilization of the carrier surface due to the oxidization process can further decrease the uptake. 166 The second mechanism contributing to enhanced cellular uptake is based on the stability of thiolated chitosan vectors during incubation with nucleases. Within the uptake process, the cargo needs to be protected from enzymatic degradation inside and outside the cells, for instance, in the small-intestinal fluid. On the one hand, the steric access of the nucleases is hampered to thiolated chitosan carriers with a cross-linked surface. On the other hand, thiolated chitosans form complexes with divalent cations (see section 3.9) necessary for the activity of nucleases. 167 Third, opening of tight junctions increases the total cell surface area available for interacting with carrier systems. This opening of tight junctions may also enable the access of carriers to the basolateral surface of cells, from which uptake may occur. Moreover, as mentioned for the ability of thiolated chitosan to enhance the permeation, the increased mucoadhesion of thiolated carriers may contribute to the cellular uptake efficiency.
He et al., for instance, observed a 74% reduced serum TNFα production after oral administration of siRNA encapsulated in trimethyl chitosan−cysteine nanoparticles to mice, whereas the naked administered TNF-α siRNA showed no effect. 35 3.7. Inhibition of Efflux Pumps. As multi-drug-resistant proteins like P-gp and MRP1, being responsible for a reduced bioavailability of various APIs and resistance of tumors to chemotherapy as well as of bacteria to antibiotics, exhibit cysteine subunits within their transmembrane-channel-forming structures, thiolated chitosans can form disulfide bonds with these efflux pumps and thereby inhibit the activity of these transporters. 56,168−170 Sakloetsakun et al. confirmed this mechanism by analyzing the influence of the amount of thiol groups attached to chitosan on P-gp inhibition. The most pronounced effect was found for the highest concentration of −SH groups. 150 Moreover, by attaching pyridyl substructures to thiolated chitosans and thereby forming S-protected derivatives with a higher reactivity of thiol groups, a more distinct P-gp inhibition could be achieved. 152 Huo et al. utilized this efflux pump Biomacromolecules pubs.acs.org/Biomac Review inhibiting effect by first confirming a P-gp inhibition of Nmercaptoacetyl-N′-octyl-O,N″-glycol chitosan using the P-gp substrate Rhodamine-123. Afterward, this thiolated polymer was used to prepare micelles loaded with the P-gp substrate paclitaxel, an API for cancer treatment. With this carrier, a 3.8fold as well as 1.4-fold increased bioavailability of paclitaxel, as illustrated in Figure 9, in rats compared to the market product Taxol and the non-sulfhydrylated micelles, respectively, was achieved. 80 3.8. Inhibition of Enzymes. Thiolated chitosans have also shown to impede various enzymes. Again, disulfide bridge formation between the thiol groups of chitosan and cysteine substructures of enzymes seems to be responsible for this effect. The ability of thiolated chitosans to chelate divalent cations (see section 3.9, representing essential cofactors for most enzymes to maintain their activity, further contributes to this effect. Accordingly, chitosan−thioglycolic acid could be identified as an inhibitor of different enzymes, namely the drug-metabolizing CYP3A4 and CYP2A6, which together participate in the metabolism of over 60% of administered APIs, and of trypanothione reductase, a vital enzyme for parasitic protozoa such as leishmania and trypanosomes. 171,172 Moreover, for chitosan−4-thiobutylamidine, an inhibitory activity was found against myeloperoxidase and metalloproteinases, enzymes important for wound healing, whereas overexpression of these enzymes can interfere with the healing process and lead to chronic wounds. 99, 173 3.9. Complexation of Metal Ions. Thiolated chitosans have the ability to form complexes with different metal ions, especially divalent metal ions, due to their −SH groups. As depicted in Figure 10, for example, nanoparticles made from N-(2-hydroxyl)propyl-3-trimethylammonium chitosan chloride−cysteine showed an improved removal of Hg 2+ , Cu 2+ , Zn 2+ , Cd 2+ , as well as Pb 2+ from aqueous solutions compared to non-thiolated nanoparticles. 32 Cd 2+ was also more efficiently adsorbed from dithiocarbamate−chitosan beads in comparison to beads prepared with the unmodified polymer, and nanoparticles coated with chitosan−4-thiobutylamidine showed stronger Ca 2+ binding compared to unmodified polymer-coated nanoparticles. 174 Furthermore, chitosan−thioglycolic acid could be identified as an effective Ni 2+ adsorbent, as it displays a 40% higher binding capacity than the unmodified polymer. 175 Additionally, a thiolated chitosan prepared by microwave-assisted substitution of the amine/hydroxyl groups with thiol groups was able to bind 85.4% of As 3+ as well as 87.0% of As 5+ within sorption studies and was utilized as a Hg 2+ sorbent. 118,120 The described complex formation with metal ions contributes to the ability of thiolated chitosans to enhance the permeation of different substances and to inhibit various enzymes.
3.10. Antioxidative and Radical Scavenging Activity. The antioxidative and radical scavenging activity of thiols results from their ability to form disulfides according to the following equation: Due to this equilibrium reaction, thiols can take part in many biological processes and are able, via the emerging electrons and protons, to inactivate toxic radicals, leading, for  Biomacromolecules pubs.acs.org/Biomac Review example, to insufficient wound healing. 176,177 In the case of chitosan, even the non-thiolated polymer displays an antioxidative effect caused by the available amino groups. A higher degree of deacetylation consequently results in an increased antioxidative activity, whereby this effect can be strongly improved due to thiolation. 99,119,178,179 Chauhan et al. showed a radical scavenging activity of up to 82% for chitosan modified by thioglycolic acid at the amino as well as hydroxyl groups, compared to an activity of about 20% for the unmodified polymer, whereby the antioxidative activity increased with the amount of attached −SH groups. 119 This correlation was also observed by Stefanov et al., as shown in Figure 11, utilizing chitosan−4-thiobutylamidine hydrogels for application on chronic wounds. As these hydrogels were prepared using the antioxidative active chicoric acid as a crosslinker, higher applied concentrations of this acid contributed to the antioxidative activity of resulting gels due to an increased amount of released chicoric acid. 99 3.11. Safety Profile of Thiolated Chitosans. Despite the fact that unmodified chitosan is a biocompatible polymer, each thiolated chitosan has to be regarded as a novel compound with specific properties and with its own safety profile. 180 As highlighted in Table 2, various studies have shown the biocompatibility of different thiolated chitosans. Among them, in vivo studies and in particular clinical investigations in humans are of most importance to determine the safety profile of thiolated chitosans. For instance, Schmidl et al. observed no serious adverse events within a controlled randomized double-blind study including 38 patients for chitosan−N-acetylcysteine administered as eye drops. 181 These results were confirmed by Lorenz et al. via an investigation with 102 patients and within further clinical trials. 182−186 Moreover, nanofiber mats based on chitosan− cysteine were proven to be non-toxic when applied in the human oral cavity for dental caries prevention. 128 To prevent long-term toxic effects when thiolated chitosans are used, for instance, in drug delivery or tissue engineering, biodegradability has to be ensured. In general, as chitosan itself is a polysaccharide exhibiting cleavable glycosidic bonds, different enzymes such as lysozyme, cellulose, and pectinase are able to degrade the polymeric chain to non-toxic oligosaccharides. 180,187 A decelerated degradation, however, can be of interest, depending on the application of the thiolated chitosan. For example, in tissue engineering, the formulation, on the one hand, has to display stability to substitute damaged or missing tissue for a sufficiently long time period to promote cell growth and, on the other hand, has to be biodegradable to enable its replacement by the cured tissue, requiring no further surgical intervention to change or remove the applied system. 71,180 In the case of thiolated chitosan, the extent and rate of degradation can be adjusted by the attachment of a ligand, whereby a more hydrophobic ligand seems to increase the affinity for lysozyme, cellulase, and pectinase, resulting in a fast and more pronounced degradation in comparison to that of chitosan itself. In contrast, when hydrophilic ligands like glutathione are used, a slower degradation compared to the unmodified polymer is achieved. 187 As enzymes like lysozyme used for biodegradability studies exhibit disulfide bridges within their structure, being essential to preserve their active center, thiolated chitosans can react with these substructures, leading to conformational alterations and consequently an inactivation of such enzymes. Moreover, cross-linking of thiolated chitosans via oxidation results in a lower degradation due to an impeded access of enzymes to cleavable sites. 99, 106,187 4. APPLICATIONS OF THIOLATED CHITOSANS As described in section 3, thiolated chitosans exhibit versatile properties enabling their use in different application areas. These applications are summarized in Table 3 as well as Table  4 and discussed in detail in the following.
4.1. Use as Therapeutic Agents. Topical therapy with biopolymers such as carboxymethylcellulose or hyaluronic acids is commonly used to treat dry eye syndrome (DES) symptoms. Due to short resident times of these lubricants, frequent instillation is necessary. As thiolated chitosans display pronounced adhesion to biological surfaces (see section 3.1), chitosan−N-acetylcysteine (C-NAC) was evaluated within various clinical studies for the treatment of DES within the past decade. 181−185 Within these studies, substantial improvements in symptoms of DES were achieved. A controlled randomized double-blind study, for instance, demonstrated a significant increase in tear film thickness, lasting for 24 h after one single instillation, and corneal damage could be reduced in >60% of patients. 181 As a result, C-NAC-based eye drops have been available in European pharmacies since 2019 under the trade name Lacrimera.
However, as several authors of the aforementioned study stated a conflict of interest (the study was sponsored by Lacrimera's producer Croma-Pharma GmbH), the results must be treated with some reservation. Therefore, the effect of C-NAC on DES was independently evaluated by Messina and     181 but also suggested that the indications for the use of Lacrimera may be extended to patients suffering from other ocular surface pathology associated with dry eyes. Diagnosis and severity of dry eye disease pre and post treatment evaluated within this study showing the effectiveness of C-NAC-based eye drops are listed Table 5. 195 The efficacy of Lacrimera in the treatment of corneal epithelial defects was also observed by Fischak et al. in an in vivo study in rabbits, as applying Lacrimera two times Biomacromolecules pubs.acs.org/Biomac Review daily resulted in a significantly faster corneal wound healing compared to placebo. These outcomes further underlined the aforementioned recommendation to extend the application area of Lacrimera. 194 In addition to eye drops based on C-NAC, the corneal wound healing capacity of nanoparticles composed of chitosan−cysteine was evaluated by Zahir-Jouzdani et al. in vivo. After 21 days, no visual difference in corneal appearance was found between mice with induced corneal damage treated with chitosan−cysteine nanoparticles and animals without inflicted corneal wounds. As these results were confirmed by histological investigations, further products for ocular treatment containing thiolated chitosans will enter the global market within the next years. 197 4.2. Drug Delivery Systems. The application of thiolated chitosans within drug delivery systems has already been described in detail within numerous reviews. 1,2,248−250 Furthermore, a summary of dosage forms, APIs, and routes of administration, and results of in vivo studies, are listed in Table 3 and Table 6, respectively.
In the following, results of a study conducted with docetaxel are described in detail to illustrate the advantages of thiolated chitosans for drug delivery. For this API, increased intestinal retention through thiol-mediated mucoadhesion, enhancement in paracellular transport through the opening of tight junctions, protection from P-gp recognition, P-gp-independent transcytosis across the intestinal endothelium, and inhibition of Pgp efflux were identified using thiolated chitosan delivery  Figure 12, achieved a 10.6-fold higher oral bioavailability of docetaxel administered to rats via nanoparticles coated with chitosan−glutathione in comparison to the market formulation. These results exemplify the potential of thiolated chitosans for drug delivery. 63 4.3. Theranostics: Photodynamic and Photothermal Therapy. Photodynamic and photothermal therapy are known as therapies involving electromagnetic energy as the trigger and either a photosensitizer, leading to the formation of reactive oxygen species (ROS), or a metal, converting electromagnetic energy into heat (via the surface plasmon resonance phenomena). In both ways, ROS or heat is used to selectively kill cells. Gold nanorods (GNRs), synthesized via seedmediated growth assisted by cetyltrimethylammonium bromide (CTAB), represent the most studied nanoparticular system for these therapies. Due to a high unspecific cytotoxicity, the use of CTAB-linked GNR is limited.
Almada et al. evaluated the cytotoxicity and photothermal efficiency of GNRs prepared with a chitosan−3-mercaptopropionic acid conjugate. Thereby, the substitution of CTAB with thiolated chitosan significantly increased the viability of MDA-MB-231 cells from 40% to 100%. However, to overcome a nearly neutral zeta potential at physiological pH, GNRs based on thiolated chitosan were coated with an additional polymer to prevent aggregation. GNRs formed with thiolated chitosan and coated with either poly(vinyl alcohol) or alginate achieved an equivalent photothermal efficiency compared to CTAB-GNR, as illustrated in Figure 13. 237 Similar results were obtained by Iqbal et al., who showed the photothermal and photodynamic activity of cobalt-dotted zinc oxide particles coated with thiolated chitosan in sunlight. Thereby, an antibacterial activity against methicillin-resistant Staphylococcus aureus of 3−5% in the dark and 100% after activation in sunlight for 15 min was observed. Moreover, nanoparticles coated with thiolated chitosan displayed a significantly enhanced antimicrobial activity upon photoinactivation of methicillin-resistant S. aureus compared to uncoated nanoparticles. 62 This synergistic effect of ROS production and antimicrobial activity was also found for iron-doped nanocerias (nanoparticles formed with cerium) that were utilized to eradicate antibiotic-resistant bacteria prevalent in hospital wastewater in sunlight. 168 However, a major drawback of photodynamic therapy is toxicity due to a broad distribution of the utilized reagent in the body. Therefore, chitosan glutathione was used to develop multifunctional photosensitive selenium nanoparticles with the potential to target activated macrophages via CD44 as well as FR-β receptors and convert H 2 O 2 to singlet oxygen ( 1 O 2 ) to specifically eradicate inflammatory macrophages. To generate such a carrier, the −SH groups of the thiolated chitosan were utilized to couple the photosensitizer Rose Bengal via amide bond formation, and catalase was conjugated by disulfide bond formation. After internalization, the intracellular reduction of disulfide bonds between catalase and the nanoparticles triggered the release of the enzyme, causing the degradation of H 2 O 2 to O 2 . The resulting O 2 was converted to 1 O 2 by Rose Bengal, leading to a cytotoxic effect on proinflammatoryactivated macrophages. In addition, quenching of intracellular H 2 O 2 allowed for better fluorescence imaging, as well as inhibiting inflammation-associated nitric oxide production. In conclusion, more specific imaging and stronger photodynamic therapy effects for detecting and killing activated macrophages make this system highly attractive for theranostic applications. 65 Additionally, Bharathiraja et al. successfully demonstrated the applicability of chitosan thioglycolic acid in the field of theranostics in vivo. Tumors, induced by injecting MDA-MB-231 cells into mice, were relapsed and undetectable after 20 days following the administration of nanocomposites composed of thiolated chitosan, palladium nanoparticles, and RGD peptide (for MDA-MB-231 targeted delivery) and laser irradiation. 236 Thus, the lower cytotoxicity of thiolated chitosan while maintaining sufficient photothermal efficiency and the redox-responsive release of S−S coupled enzymes make thiolated chitosans highly interesting for theranostics research and development.
4.4. Tissue Engineering. In addition to transplantation, tissue engineering provides another way to treat tissue and organ loss or damage. Thereby, cells from the patient, from another genetically non-identical individual, or from animal species are seeded to a biocompatible 3D matrix combined with the possibility to incorporate bioactive molecules within this scaffold to improve cellular function and desired tissue formation.
The polymer matrix should exhibit hydration characteristics similar to those of the tissue and an interconnected microstructure to promote the transport of nutrients as well as oxygen, and should enable an easy modification of the chemical ligand for cell attachment, while maintaining its mechanical structure until the tissue has been completely formed. 138,251 Among others, chitosan-based hydrogels exhibit such properties; however, they also show several common shortcomings, such as easy breakability, low strength, and poor elasticity. 242,252,253 In order to overcome these issues, Wu et al. attached N-acetylcysteine to an N-isopropylacrylamide− chitosan-based hydrogel and observed improved mechanical  156 rabbits nanoparticles ocular curcumin delivery For TC-coated nanoparticles, the significantly highest ocular retention was observed by fluorescence imaging, and a 29.9-fold increased AUC was obtained compared to that with curcumin eye drops. Uncoated nanoparticles displayed a 6.0-fold higher AUC, and for CS-coated nanoparticles a 12.3-fold increased AUC was detected.

67
nasal delivery of selegiline Animals treated with a system based on TC showed a significantly reduced immobility time, increased sucrose water intake, and higher locomotor activity compared to the group receiving a formulation with unmodified polymer.

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Biomacromolecules pubs.acs.org/Biomac Review properties, such as an over 9-fold improvement of the compressive modulus and a correlation between storage modulus and cross-linking density, as demonstrated in Figure  14. Simultaneously, the hydrogel facilitated adequate cell proliferation of mesenchymal stem cells and fibroblasts as well as osteoblasts. 138 Liu et al. utilized a similar method to establish a stable dual network gel formulation by coupling chitosan−N-acetylcysteine to silk fibroin. The introduced thiolated chitosan enhanced the strength and stiffness of the gel, whereas the silk fibroin component primarily contributed to its elasticity.
The resulting scaffold facilitated a three-dimensional growth of chondrocytes, making the composite highly interesting for cartilage repair. 242 Moreover, Chen et al. demonstrated the applicability of thiolated chitosan for bone tissue engineering in vivo. The osteoinductive protein P24 was coupled via disulfide bond formation on a biomimetic scaffold based on thiolated chitosan and hydroxyapatite. Compared to the control (scaffold without P24), a significantly higher ectopic osteogenesis level in rat dorsal muscle pockets as well as superior performance in the reconstruction of calvarial bone defects were observed for the P24-loaded scaffold. 147 4.5. Wound Treatment. Treatment of chronic wounds requires intensive medical intervention at huge healthcare costs, and the incidence of such wounds increases as more people get older and suffer from diabetes. 173 Different strategies have been developed to make the wound healing process faster and less painful.
For instance, the wound healing properties of chitosan−Nacetylcysteine on corneal tissue were assessed in rabbits with monocular epithelial debridement. Time for wound healing was significantly reduced in the thiolated chitosan group compared to the placebo group treated with phosphate buffered saline. The authors of the study stated that the underlying mechanism is not entirely clear but may be at least partially related to well-known effects of unmodified chitosan on wound healing. 194 In vitro studies conducted on fibroblasts revealed no statistically significant difference in migration and viability of cells treated with thiolated chitosan and its parent polymer. 88 In a similar approach, Zahir-Jouzdani et al. demonstrated in vitro that the thiolation of chitosan through cysteine conjugation had no effect on the anti-fibrotic and antiangiogenic properties compared to treatment with the polymer. However, thiolated chitosan was preferred for the following in vivo studies in mice, due to the increased mucoadhesion to the cornea compared to pristine chitosan. For assessing the wound healing capacity of chitosan−cysteine, corneal wounds were induced by applying NaOH to the right eye of the animals. After 21 days, the corneal appearance of the group treated with thiolated chitosan was similar to that of healthy animals, and only minor inflammation was observed. The treatment could also successfully prevent hyphema, a usually painful collection of blood inside the anterior chamber of the eye that can cause permanent vision problems if left untreated. 197 Moreover, sustained antibacterial activity against infection is of great importance for wound healing. Therefore, Trp-rich peptide (PSI) was coupled via disulfide formation to chitosan cysteine to provide a sustained in vitro release of this polypeptide with a broad-spectrum antimicrobial activity over 20 days. However, no significant difference in wound closure was observed in vivo in mice, as hydrogels loaded with PSI based on thiolated chitosan as well as pristine chitosan had    Figure 15. The wound healing capacity and wound closure rate of a thiolated chitosan alginate zinc oxide nanoparticles bandage were more enhanced compared to those of the reference groups treated with either the unmodified chitosan alginate bandage with incorporated zinc oxide nanoparticles or a marketed product. The authors stated that enhanced mucoadhesion and retention of the bandage ensured proper dosing and enhanced antimicrobial activity, and resulted in an increased healing capacity and wound closure. 71 Nevertheless, inhibitory activity of sulfhydryl groups against the chronic wound enzymes observed in vitro and ex vivo might also be responsible for improved wound closure. 99,173 4.6. Coating Material. Coating with thiolated chitosan to alter surface characteristics is a promising approach to inhibit bacterial adhesion and biofilm formation on materials prone to fouling. For instance, carboxymethyl chitosan−4-thiobutylamidine was coupled via Michael addition on maleimidocontaining tannic acid anchored on stainless steel. Thereby, the surface coated with thiolated chitosan exhibited a 70% reduced protein adsorption and 91% decreased adhesion of Escherichia coli compared to stainless steel modified with maleimidocontaining tannic acid. 255 The reduced antifouling can be attributed to the increased hydrophilicity of the surface, since protein adsorption and bacterial adhesion correlate with the hydrophobicity of the surface. 256,257 However, no conclusion based on these results could be made on the anti-adhesive effect of the thiol moiety.
In another study conducted by Costa et al., a bacterial antiadhesive coating was developed to avoid Staphylococcus aureus adhesion and biofilm formation. Biological studies confirmed chitosan−N-acetylcysteine to be a promising material, as it promoted a ∼95% decrease in bacterial adhesion compared to unmodified chitosan coating (Figure 16a). The authors stated that the reduction of free available amino groups of chitosan as well as the increase of surface hydrophilicity might decrease bacterial adhesion directly or indirectly through the decrease of protein adsorption. This theory was underlined by higher adherence of bacteria on a gold surface and a gold surface coated with pristine chitosan in the presence of protein from human plasma (Figure 16b). Moreover, thiolation of chitosan promoted a 5.5-and 2.2-fold reduction of protein adsorption from human plasma compared to gold and pristine chitosan, respectively (Figure 16c). Anti-adherence properties of thiolated chitosan were further proven through the quantification of the total biofilm mass, as this parameter was efficiently reduced using this thiolated chitosan for surface modification (Figure 16d). As declared by the authors, it was unclear if the exposed thiol groups had any direct contribution to preventing specific adhesion (e.g., degrading relevant disulfide bridges of bacterial adhesins), or if the overall mechanism was purely based on non-specific anti-adhesive  Biomacromolecules pubs.acs.org/Biomac Review effects. 245 Therefore, protein as well as bacterial adhesion studies performed with coatings of chitosan with different amounts of free thiol groups might be helpful to assess the influence of the thiol moiety. Furthermore, pegylated chitosan−4-thiobutylamidine was investigated within the development of a polymer film intended for use as a food-packaging material. The thiolmodified film exhibited antibacterial activity, which was absent for native chitosan, and displayed higher compatibility with polyethylene, as a more transparent film was obtained. 101 4.7. Textile Industry. From yarn formation to a finished evening dress, industrial textile manufacturing includes several processing steps that end in a final finishing operation, where biocidal finishing agents are used to provide antimicrobial characteristics to shield the end commodities from microbial degradation. 258 For such biocidal finishing agents, not only the antimicrobial efficiencies but also environmental, health, and safety aspects must be considered. Its biocompatibility and biodegradability make chitosan a highly promising agent for this application. However, its weak adhesion to fibers results in a gradual leaching from the textile with repetitive washing.    was higher compared to most of the reported materials. Adsorption kinetic studies indicated that the adsorption was mainly promoted by a chemical process. Furthermore, chemisorption, ion exchange, surface complexation, physical adsorption, electrostatic interaction, and precipitation improved the adsorption capacity of the composite for the tested metal ions. Additionally, a sufficient regeneration performance was observed. In order to enforce the adhesion of chitosan, several methods were developed using cross-linking agents, combinations of physical treatments with UV or plasma sources, as well as biological treatments like enzymatic methods. 259 More recently, Zhang et al. investigated a novel approach, illustrated in Figure 17, for grafting of thiolated chitosan onto wool via disulfide bond formation between the thiol-bearing side chain N-acetylcysteine and sulfhydryl groups of the wool proteins (keratins).
Besides the improvement of several characteristics important for textile manufacturing (shrink-resistance, dyeing ability), bacteriostatic rates up to 58.32% and 63.96% were achieved for disulfide-linked chitosan and adsorbed unmodified chitosan, respectively. Thiolation of chitosan decreased the number of free amino groups, causing a slight weakening of the antibacterial properties. However, considering that the greatest disadvantage of chitosan as a biocidal is its weak adhesion, 260 disulfide cross-linking seems to be a novel, highly promising approach for the use of thiolated chitosans within the textile industry. 52 4.8. Water Treatment. Pollution of drinking water is an issue that has been increasing over the past few years. Due to their physiochemical, mechanical, and biological properties, thiolated chitosans offer a great potential for applications within water treatment by complexing and adsorbing inorganic pollutants. 116−118 However, a comparison of different studies is delicate, since experimental conditions are not always the same (temperature, pH, concentration of metal as well as adsorbent, incubation time, and interfering ions/metal). Nevertheless, the outcomes summarized in Table 7 underline the potential of thiolated chitosans as adsorbents in wastewater treatment.
Moreover, the complexation capability of chitosan-SH was also utilized for colorimetric determination of Hg 2+ . The developed sensor showed a quick response, good selectivity, and high sensitivity, with a detection limit of 0.465 ppb by the naked eye, below the upper limit of Hg 2+ in drinking water of 6 ppb according to the WHO. 120 Besides inorganic pollutants, thiolated chitosans are also under investigation for treatment of organic pollutants within wastewater. Recently, an Fe 3 O 4 − thiolated chitosan hybrid composite was synthesized for laccase immobilization to decolorize the textile dyes Reactive Blue 171 and Acid Blue 74. The enzyme was covalently attached via disulfide bond formation onto the composite and retained its high decolorization activity during repeated use. 261 In a different approach, Carvalho et al. evaluated 3D sponges based on a chitosan−mercaptoundecanoic acid conjugate as adsorbent. Their study revealed a high adsorption capacity of 388 mg/g and a removal efficiency greater than 90% over a broad range of concentration from 20 to 1200 mg/L for the model organic pollutant Methyl Orange. 50 Furthermore, thiolated chitosans were applied within the removal of antimicrobial contaminations. Khan et al. developed irondoped nanoceria (nanoparticles consisting of cerium) coated with folate-grafted chitosan−thioglycolic acid. Metal-doped nanoceria are generally used due to their ability to generate ROS by photocatalytic activity. In their study, they effectively demonstrated a total eradication of all bacteria present in hospital wastewater by applying nanoparticles in a concentration of 100 μg/mL. 168 Overall, these studies underline the potential of thiolated chitosans for application in water treatment by removing organic as well as inorganic pollutants, immobilizing enzymes utilized to degrade specific substances, and for developing composites to eliminate antimicrobial contaminations.
4.9. Cosmetics. The application of cosmetics based on thiolated chitosans for hair, eyelashes, and eyebrows treatments (styling gels, repairing agents, coloring agents, detergents, and coating), nail polishes, make-up, and antiperspirants is highly interesting, due to the thiol-bearing proteins of hair, nails, and skin. 262 Thus, thiolated chitosans exhibit excellent adhesion via covalent disulfide bond formation with cysteine subunits of these surface proteins, stabilizing the product after application without smearing and dissolution. 263 As a result, several patents regarding the use of thiolated chitosans as cosmetic ingredients were filed. 262−264 However, the European Commission database for information on cosmetic substances and ingredients (CosIng) has currently listed only one thiolated chitosan applied as a filmforming, skin-conditioning, and skin-protecting agent within cosmetic products, namely chitosan−glutathione. According to a patent filed for this thiolated chitosan, it displays a 40% higher binding capacity for Ni 2+ ions in comparison to unmodified chitosan. 175 As heavy metals and in particular Ni 2+ from, for instance, jewelry are common causes of contact dermatitis, chitosan−glutathione, so-called Chitatione, is utilized in the cosmetic lines Nidiesque and Nuev to coat the skin with a film and thereby prevent the permeation of metals ions, inhibiting allergy symptoms, as illustrated in Figure 18. 265,266 5. FUTURE PERSPECTIVES The great future potential of thiolated chitosans lies in the broad diversity of sulfhydryl ligands that can be covalently attached to the polymeric backbone. Due to this diversity, specific properties can be adjusted on demand, and additional functions can be introduced. As thiol/disulfide exchange reactions and the formation of disulfides are directed by opposite charges in their surroundings attracting each other and bringing the reactive sulfur species close to each other, in particular charged ligands will likely be used to a higher extent in the years to come. Such ligands will allow the formation of more targeted disulfide bonds between thiol or disulfide substructures of interest. Another category of sulfhydryl ligands Biomacromolecules pubs.acs.org/Biomac Review that will definitely shape the future landscape of thiolated chitosans are less reactive S-protected thiols. So far, thiol groups on chitosan were S-protected by the formation of disulfide bonds with mercaptopyridine analogues, as illustrated in Figure 1. Due to the electron-withdrawing effect of the πsystem of the pyridine, the disulfide bond is highly reactive toward free thiols. In contrast, thiol groups on chitosan, being S-protected by the formation of disulfide bonds with sulfhydryl ligands that do not exhibit such an electron-withdrawing effect like cysteine or N-acetylcysteine, are less reactive. Very recently, Netsomboon et al. demonstrated that, when thiol groups are S-protected with N-acetylcysteine instead of with a highly reactive mercaptopyridine analogue, even higher mucoadhesive properties can be achieved. This effect was explained by a more pronounced interpenetration of the less reactive thiolated chitosan into deeper regions of the mucus gel layer, which is more tightly anchored there than highly reactive S-protected thiolated chitosans reacting already with just loose mucus on the surface of the gel layer. 267 Furthermore, having the booming thiol−ene cross-linking of biopolymers in mind, 268 we expect the development of numerous potential hydrogels containing thiolated chitosans that are cross-linked by this type of reaction.
On the application side, further product developments will follow, as the success of the first products containing thiolated chitosans on the global market sends out a strong positive signal. For example, Lacrimera eye drops showed impressive improvements in late-stage clinical trials in treatment of dry eye syndrome. Accordingly, the same thiolated chitosan will likely show similar potential for the treatment of dry mouth and vaginal syndromes. In the case of drug delivery, thiolated chitosans are already in clinical trials. Other areas of applications, such as the use of thiolated chitosans in the textile industry, are comparatively young, but there is no doubt that sound products based on thiolated chitosans will also emerge from there.