An Overview of Current Knowledge on the Properties, Synthesis and Applications of Quaternary Chitosan Derivatives

Chitosan is a polycationic polymer (pKa = 6.2–6.8) obtained by partial chitin N-deacetylation under alkaline conditions, thus consisting of a linear mucopolysaccharide formed by β -(1 → 4)-2-amino-2-deoxy- d -glucose and β -(1 → 4)-2-acetamide-2-deoxy- d -glucose copolymers. This cationic character is the basis of most of its applications as an antimicrobial compound in several fields, including food, agriculture, biomedicine and textiles [ 1 ]. As a result that it presents itself as a biodegradable [ 2 ] and biocompatible [ 3 ] material, chitosan has been arousing the interest of several research groups due to its physicochemical properties and unique bioactivity [ 4 , 5 , 6 , 7 ]. However, chitosan has low solubility in aqueous solutions and organic solvents, in addition to having a short useful life, thus limiting its applicability [ 8 ]. One of the methodologies used to solve this limitation is the quaternization of chitosan. Chitosan quaternization consists of replacing the chitosan amino terminals with quaternary terminals or by inserting functional cationic groups [ 9 , 10 ]. This quaternization improves the solubility of chitosan, in addition to enhancing its antimicrobial action [ 11 ]. The introduction of permanent positive charges in the chitosan chain can be achieved by preparing a quaternary ammonium chitosan salt, covalently adding a substituent containing a quaternary ammonium group or quaternizing the amino group of the original polymer [ 12 ]. This permanent introduction of positive charges in the polymer, regardless of the pH of the aqueous medium, provides solubility in water as well as better antimicrobial activity [ 13 , 14 ]. This improvement in antimicrobial activity may be related to the fact that the positive charges in chitosan are captured by cells, inhibiting the transition of DNA and the synthesis of RNA and proteins, or even, the disorganization and denaturation of the proteins of the microorganism’s membrane caused by the interaction between positive chitosan groups and the negative charges of the microorganism’s membrane and therefore causing cell death [ 15 , 16 , 17 ]. The most studied and simple form of quaternary ammonium chitosan derivative is N,N,N-trimethyl chitosan (TMC) [ 18 ]. This derivative is obtained by reaction with methyl iodide, sodium iodide or sodium hydroxide [ 19 ]. The insertion of portions of quaternary ammonium outside the chitosan structure by an alkylation reaction is the second most common way to quaternize chitosan, forming the compound N-[(2-hydroxy-3-trimethyl ammonium) propyl] chitosan (HTCC) and it is commonly synthesized from a reaction between chitosan and gycidyltrimethyl ammonium chloride [ 20 , 21 , 22 ]. Recently, new quaternized chitosan derivatives with pyridinium salts and phosphonium salts have been gaining interest from several research groups due to the antimicrobial properties presented by these quaternary salts in addition to providing better solubility to chitosan [ 23 , 24 , 25 , 26 , 27 ]. These derivatives, based on the literature, have been shown to be quite efficient in microbial activities when compared to chitosan [ 28 , 29 , 30 , 31 , 32 ], in addition to being able to be applied in areas such as cosmetic agents [ 33 ], pharmaceuticals and biomedical [ 34 , 35 , 36 , 37 ], food industry [ 38 , 39 ], antimicrobial agent [ 24 , 40 , 41 ] and wastewater treatment [ 42 , 43 ]. This review focuses on presenting the main quaternized compounds of chitosan, from the most used, such as TMC and HTCC, to those that have aroused a greater interest of researchers, such as the quaternized chitosan derivatives with pyridinium or phosphonium salts. We addressed them in terms of their structure, properties, synthesis routes and applications. In addition, other less explored compounds are also presented, involving the main findings and future perspectives in the field of quaternized chitosans.

Until 2016, there were four main methods for preparing TMC, according to Wu et al. [ 69 ] and Kulkarni et al. [ 70 ]. These methods are described below, followed by alternative methods and approaches that were able to successfully produce TMC in the last four years. A scheme with a summary of the most common methodologies employed is displayed in Figure 2 . 2.2.1. Conventional Methods The simplest way of obtaining TMC is by a one-step reaction of chitosan with methyl iodide (CH 3 I) under strong alkaline conditions using N-methyl-2-pyrrolidone (NMP) as a solvent and sodium iodide (NaI) as a catalyst [ 48 ]. Curti et al. [ 12 ] verified that an excess of NaOH resulted in higher trimethylation, even reaction yield was lower and O-methylation was favored. As a result of this, water solubility decreased as the alkaline agent and CH 3 I excess was increased. The average degrees of quaternization obtained in this study ranged from 10% to 45%. By using methyl iodide as methylating agent, N,N,N-trimethyl chitosan iodide is obtained. As a result of its toxicity and, therefore, unsuitability of application in several fields, it is normally converted to TMC chloride, either by reacting with NaCl [ 48 , 71 ] or hydrochloric acid (HCl) [ 52 ]. In this context, Zhang et al. [ 72 ] prepared four TMC salts (citrate, acetylsalicylate, ascorbate and gallate) by dissolving iodide precursor in solutions of sodium salts with the four desired couteranions. As a result, the gallate and ascorbate derivatives presented better antioxidant activity than chitosan and TMC iodide. Another method, initially developed by Muzzarelli and Tanfani [ 47 ], involved the synthesis of N,N-dimethyl chitosan (DMC) by reacting chitosan with formaldehyde in acidic media and following addition of sodium borohydride. Only then was CH 3 I added to the system to produce TMC. The obtained compound was water insoluble, even with a DQ as high as 60%. A modification of this method was later executed by Verheul et al. [ 51 ]. In the study, instead of sodium borohydride, DMC was synthesized using a formic acid–formaldehyde mixture. Trimethylation was then carried out with an excess of methyl iodide. Through this method, the authors were able to obtain O-methyl free water soluble TMC with no chain scission. They also stated that TMCs with DQs as high as 75% could be obtained by varying reaction time. A variation of this method was attempted by some authors [ 19 , 65 ]. In these approaches, a pre-alkylation of chitosan was carried out by used different aldehydes than formaldehyde in order to obtain a Schiff-base intermediate. Later on, quaternization was carried out traditionally by using CH 3 I. By previously introducing methyl groups on the nitrogen atom, the formation of quaternary ammonium salts is facilitated along with the hindering of O-methylation. However, because of the use of different and more complex aldehydes in the pre-alkylation step, other quaternized chitosan derivatives, such as N-N-Propyl-N,N-dimethyl chitosan and N-Furfuryl-N,N-dimethyl chitosan, can be obtained along with TMC [ 19 ]. In spite of the efficiency obtained by using CH 3 I as a methylation agent, it is an expensive, volatile and toxic reagent. In addition, halide ions are difficult to separate from solution. Thus, De Britto et al. [ 60 ] synthesized TMC with the use of dimethyl sulfate (DMS). Compared to methyl iodide methods, this approach results in a less expensive, less toxic and more simple method, as DMS can act both as an agent and a solvent for the reaction, avoiding the use of NMP. In addition, TMC chloride was directly obtained as NaCl is used in the medium. In this study, the calculated DQs ranged from 15.8 to 52.5% and were found to be time and temperature dependent, with high temperatures favoring O-methylation over N-methylation. Due to the fact that methylation agents are not selective and O-methylation occurs in almost all of the methods cited above, even if chitosan’s amino groups are much more reactive than C-3 and C-6 hydroxyl groups, it is a good strategy to protect these less reactive groups through O-silylation. First, chitosan is reacted with methanesulfonic acid (CH 3 SO 3 H) to obtain chitosan metasylate, followed by the insertion of tert-butyldimethylsilyl (TBDMS) groups, producing 3,6-di-O-tert-butyldimethylsilyl-chitosan. This compound displays excellent solubility in a number of common organic solvents [ 73 ]. Once the hydroxyl groups are protected and O-methylation can no longer occur, the methylation of this chitosan derivative is executed by using CH 3 I. According to Benediktsdóttir et al. [ 74 ], such procedure leads to a product with 100% trimethylation, composed of N,N,N-Trimethyl-di-TBDMS chitosan. Finally, the hydroxyl groups can be deprotected through the use of a tetrabutylammonium fluoride (TBAF) solution in NMP. The authors also reported, for the first time, a full N,N,N-trimethyl chitosan without the presence of O-methylated or N-mono o

TMC has been applied to several fields in the form of nanoparticles, films, blends, emulsions, etc. Biomedicine is, by far, the field that have been the most studied for trimethyl chitosan and its derivatives. In addition, some research has been done about TMC in environmental and food applications. In this context, some reviews have been written. Kulkarni et al. [ 70 ] wrote a review paper focused on applications of TMC in the form of nanoparticles and some other specific applications have been tracked by Mourya and Inamdar [ 77 ], Wu et al. [ 69 ] and Zhao et al. [ 78 ]. Therefore, this review will focus more on recent applications. It is important to be aware that “TMC” almost always refers to TMC iodide, but it is hardly ever specified by authors in their publications and thus the term “iodide” will be not used throughout this section. Whenever another counter-anion, such as chloride, replaces iodide, it will be explicitly stated below. Chitosan and its derivatives have been widely studied as adsorption enhancers, especially of peptide and protein drugs. This is due to the opening of tight junctions in between epithelial cells, which facilitates the paracellular diffusion across mucosal epithelia [ 79 ]. Kotzé et al. [ 80 ] demonstrated that TMC was able to increase transport of the hydrophilic compounds [ 14 C]-mannitol, a fluorescein isothiocyanate-labeled dextran and the peptide drug buserelin across Caco-2 cell monolayers. Since then, several studies have reported TMC’s efficiency as a permeation enhancer. Thanou et al. [ 58 ] evaluated the effect of the degree of quaternization of trimethyl chitosan on the absorption enhancement. The authors showed that high charge density is necessary for TMC to significantly improve the paracellular permeability, which means that the transport enhancement across intestinal epithelia is improved with higher DQs. He et al. [ 81 ] reported improved transdermal permeation of testosterone by using TMC and also verified a stronger enhancement with high DQs. Trimethyl chitosan displayed penetration enhancement towards intestinal [ 82 ], nasal [ 83 ], corneal [ 84 ] and bronchial epithelia, [ 85 ] and buccal mucosa [ 54 ]. As a result of TMC’s excellent mucoadhesive and absorption-enhancing properties, it shows potential to be used, especially, in the delivery of oral drugs. It can increase protein bioavailability in gastrointestinal environments and permeability in the intestinal barrier, in addition to its low toxicity and high susceptibility to biodegradation. In the last few years, trimethyl chitosan and its derivatives have been successfully applied in systems aiming oral delivery of insulin [ 86 , 87 ], natural antioxidants [ 88 ], flavonols [ 89 ], anticancer drugs, such as paclitaxel [ 90 ] and gemcitabine [ 91 ], antifungal drugs [ 92 ] and antiviral drugs [ 28 ]. In addition to oral administration, researchers have developed systems composed of TMC to deliver drugs through nasal [ 93 ], pulmonary [ 94 ] and intravenous [ 90 ]. Drug delivery can also be done through ocular administration. In this sense, efforts have been made in order to develop new drug carriers that are able to increase ocular absorption and improve drug bioavailability. Asasutjarit et al. [ 36 ] developed formulations of TMC NPs loaded with diclofenac sodium (DC) for ophthalmic use. Eye irritation tests revealed that this material is safe for use. Additionally, in vivo ophthalmic absorption studies conducted with rabbits showed that bioavailability of DC is increased and the system could be used for treatment of ocular inflammation with lower frequency of administration than that of common DC eye drops. Similarly, Li et al. [ 95 ] prepared TMC-coated lipid nanoparticles of baicalein (TMC-BAI-LNPs). In vitro and in vivo studies indicated that the material had no ocular irritation and resulted in prolongation of drug retention time in tears and improvement of its ocular bioavailability. Cationic chitosan is able to form a polyelectrolyte complex with anionic DNA and thus it is applied to gene delivery systems. Additionally, polymeric vehicles seem more convenient than other gene delivery approaches because of efficiency and safety issues. Rahmani et al. [ 96 ] investigated DNA transfection efficiency of three chitosan derivatives, one of them being a TMC derivative (thiolated trimethyl chitosan). The authors reported that the three compounds are efficient vehicles for gene delivery and that specifically for SKOV-3 and MCF-7 cells, TMC exhibited the highest transfection efficiency of DNA nanocomplexes. According to this study, the thiol group can enhance the transfection process, even though cell type is the determining factor in a delivery system. Baghaei et al. [ 97 ] evaluated polyelectrolyte complexes of TMC and some polyanions (hyaluronan, alginate and dextran sulfate) in their gene delivery efficiency to MCF7 cells through an experimental design. In vitro studies showed non-toxicity and hi

The most common and most widely studied method for obtaining HTCC is by the reaction between chitosan and GTMAC, according to the synthesis route shown in Figure 4 a. In general, this reaction is more favored under acidic conditions, which makes the epoxy group more reactive and easier to open. Furthermore, under acidic conditions the epoxy group reacts more favorably to NH 2 groups of chitosan, while under basic conditions, the reaction by the OH groups of chitosan is more favored [ 22 ]. Most of the studies that used the aforementioned methodology either performed it in an aqueous medium or used acetic acid as a reaction catalyst. Therefore, chitosan is dispersed in deionized water (and acetic acid, when applicable). GTMAC aqueous solution is added in different amounts to obtain different DQs. In general, the acid-catalyzed reaction is conducted over a 10–18 h period, at temperatures ranging from 50–80 °C [ 22 , 35 , 117 , 120 , 138 ]. In cases where the catalyst is not used, a longer time (up to 24 h) or a higher temperature (85 °C) may be necessary to achieve expected results [ 21 , 29 , 130 , 139 ]. As reported by Seong et al. [ 117 ], who studied the influence of the time and temperature parameters on the DQ, the DQ increases with increasing temperature and reaction time, up to an optimum, in their case 80 °C and 18 h, with little variation in DQ values above these conditions. It is also possible to find more specific cases of the reaction of chitosan and GTMAC. Lu et al. [ 140 ], Wu et al. [ 141 ] and Zhou et al. [ 125 ] carried out the reaction by dissolving chitosan in isopropanol, with a reaction time in the range of 7–10 h, at a temperature of 80 °C. Nam et al. [ 142 ] used Zn(BF 4 ) 2 as a catalyst for the reaction, with elevated time (20 h) and temperature (100 °C) conditions, and Ruihua et al. [ 143 ] dispersed chitosan in deionized water and perchloric acid, the reaction occurring at 80 °C for 8 h. As noted, HTCC synthesis is normally carried out in a heterogeneous reaction medium and involves several types of volatile organic reagents or other aggressive chemical reagents. To overcome these issues, Yang et al. [ 118 ] proposed the employment of an ionic liquid of 1-allyl-3-methylimidazole chloride (AmimCl) as a green and homogeneous reaction medium. Thus, the chitosan was dispersed in AmimCl until complete dissolution. GTMAC was added and maintained in reaction for 8 h at 80 °C. The second most used route for the production of HTCC is through the reaction between chitosan and 3-chloro-2-hydroxy-propyl trimethyl ammonium chloride (CTA), a lower cost reagent than the usual GTMAC [ 9 ]. CTA is used as an etherification reagent due to its characteristic of generating an epoxide in alkaline conditions, which is similar to GTMAC [ 144 ]. In general, chitosan is dispersed in isopropanol or 2-propanol, over which NaOH solution is added and kept under stirring for a time that can vary from 2–5 h. After this step, aqueous CTA solution is added and the reaction can be conducted under conditions ranging from 6–10 h and 40–60 °C [ 145 , 146 , 147 ]. Ali and Singh [ 148 ], in turn, dispersed the chitosan in water, requiring reaction conditions of 33 °C and 18 h, a significantly longer time than that performed by authors who used other solvents. Tan et al. [ 149 ] dispersed chitosan in an alkaline aqueous solution of LiOH/KOH/urea/H 2 O, which is considered an environment-friendly solution. Later, they added CTA solution dropwise to the chitosan solution, the reaction taking 12 h to complete. In addition to HTCC synthesis, some studies report the synthesis of other compounds similar to HTCC, such as O-HTCC (O-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride), characterized by the insertion of quaternary terminals in the hydroxyl group of chitosan. This reaction also occurs through the contact of chitosan with GTMAC, though under conditions where the NH 2 functional groups are protected [ 150 ]. The procedure for obtaining the O-HTCC usually proceeds through three steps: the first to protect the NH 2 functional group, the second to insert the quaternary terminal into the hydroxyl group, and the third to release the NH 2 functional group. In general, chitosan is dispersed in acetic acid, to which benzaldehyde or any benzoyl hydride is added to protect the NH 2 group, producing N-benzylidene chitosan. After neutralization with NaOH, solution of GTMAC dispersed in isopropyl alcohol is added and maintained in reaction for 16 h at 70 °C. The product generated in this step is added to an ethanolic HCl solution, obtaining crude O-HTCC, which must be purified in suitable solvents [ 150 , 151 , 152 ]. Figure 4 b presents the synthesis route aforementioned [ 153 ]. Additionally through the contact of chitosan with GTMAC, it is possible to obtain a third product, N,O-HTCC (N,O-[(2-hydroxyl-3-trimethyl-ammonium) propyl] chitosan chloride), obtained from an additional quaternization of HTCC through hydroxyl gr

Pyridinium salts constitute a class of unsaturated heterocyclic molecules ( Figure 5 ) and the main route of obtaining them is through SN 2 type reactions between pyridine and alkali halides [ 174 , 175 , 176 ]. Pyridine derivatives were introduced into the polymer backbone to improve polymer properties including solubility, physicochemical and biological properties [ 63 ]. Several studies have proven that pyridinium salts have a wide range of applications, including antiviral, antibacterial and antifungal activity [ 177 , 178 ]. According to Sowmiah et al. [ 179 ], pyridinium salts are known to inhibit the growth of various microorganisms, such as bacteria, viruses and fungi. This inhibition occurs through the cationic groups present in the pyridinium that are attracted by negatively charged microorganisms, resulting in disorganization and denaturation of proteins in the microorganism membrane and, thus, causing cell death [ 15 ]. The first study on the incorporation of pyridinium salts in chitosan was reported in 2010 by Li et al. [ 180 ]. The authors presented three new quaternary chitosan derivatives, called PACS, CHPACS and BHPACS, synthesized from the reaction between chloroacetyl chitosan (CACS) and pyridine, as shown in Figure 6 a, and tested the antifungal activity of both against four pathogenic fungi, namely, Cladosporium cucumerinum , Monilinia fructicola , Colletotrichum lagenarium and Fusarium oxysporum . The CHPACS and BHPACS derivatives were synthesized from a solution of 5-chlorosalicylaldehyde or 5-bromosalicylaldehyde and the product of this reaction was added together with CACS. The authors observed that the derivatives CHPACS and BHPACS had an inhibitory effect against pathogenic fungi superior to that presented by the other derivatives and the chitosan itself, presenting an effect of 100% in concentrations of 500 μ g/mL and 1000 μ g/mL which happened by the presence of groups 5-chloro-2-hydroxybenzylideneamino and 5-bromo-2-hydroxybenzylideneamino in the structure of the derivatives. This fact was also reported by Guo et al. [ 181 ] in their work, where they noticed an increase in the inhibitory effect of the chitosan derivative when the group 5-chloro-2-hydroxybenzylideneamino was inserted in the chitosan structure. Tan et al. [ 23 ] developed a cationic derivative of chitosan containing N-methyl-1,2,3-triazolium and N-methyl-pyridinium via efficient cuprous-catalyzed azide-alkali cycloaddition reaction (CuACC). The derivative was synthesized in three stages: initially, the cationic derivative of propargyl chitosan (a) was synthesized from the reaction of chitosan with propargyl bromide; then, derivative (a) was dissolved in a DMSO solution and 3-azidopyridine, triethylamine and cuprous iodide were added, producing the cationic derivative containing 1,2,3-triazole and pyridine (b); finally, derivative (c), containing 1,2,3-triazolium and pyridinium was synthesized from N-methylation, reacting derivative (b) with iodomethane. Figure 6 b shows a summary of the synthesis of compound (c). The authors tested the antifungal potential of the derivative with three phytopathogenic fungi, C. lagenarium , W. fusarium and F. oxysporum . The derivative containing 1,2,3-trizolium and pyridinium in its structure showed a superior antifungal action when compared to the other derivatives, presenting a maximum inhibition rate of 98.44% for C. lagenarium , followed by 79.16% and 67.56% for W. fusarium and F. oxysporum , respectively, at 1.0 mg/mL, showing a potential antifungal agent. In addition, it has better solubility in water, especially at alkaline pH. Jia et al. [ 24 ] introduced, by nucleophilic substitution, portions of pyridine to obtain N-(1-carboxbutyl-4-pyridinium) chloride chitosan. The synthesis process ( Figure 6 c) of this new derivative took place through an initial treatment of chitosan with 4-chlorobutyl chloride for the production of N-chlorobutyl chitosan, which then reacted with pyridine, finally forming a chloride of quaternary ammonium, N-(1-carboxybutyl-4-pyridinium) chitosan. The antifungal activity of this derivative was tested with B. cinerea and F. fulva , where the antifungal index of pyridine chitosan was 75%, while that of chitosan was only 58.9%, in addition, serious morphological changes of B. cinerea when treated with pyridine chitosan, where it caused the damage and deformation of the structure of fungal hyphae and, subsequently, the inhibition of the growth of the strain. A similar fact was observed by [ 182 ], where the positively charged portion of the cationic molecule directly interfered with the fungal cell surface, changing the permeability of the plasma membrane and thus inhibiting the growth of fungi. A recent study, developed by Omidi and Kakanejadifard [ 25 ], made modifications of chitosan and chitosan nanoparticles by long-chain pyridinium compounds via imine binding. In this work, the chitosan reaction was carried out in an acidic aqueous solution using

As the quaternization of chitosan has already demonstrated the possibility of obtaining materials with improved properties, many studies have expanded this modification using a number of reagents and obtaining a variety of quaternized chitosans, in addition to the most common ones aforementioned. Some of the options for obtaining different quaternized derivatives involve modifying the most common quaternized chitosans, such as TMC and HTCC, or using their precursors to obtain other quaternizing agents. Xu et al. [ 20 ] developed compounds of N,O-di-quaternary ammonium chitosan with different degrees of O-substitution, by the reaction of TMC with CTA. All derivatives showed better bacterial activity than chitosan and N,N,N-trimethyl-O-(2-hydroxy-3-trimethylammonium propyl) chitosan, obtained after the O-quaternization step, demonstrated stronger activity than that of TMC, with an increase proportional to the degree of O-substitution. Qi et al. [ 190 ] synthesized the quaternary ammonium salt 3-chloro-2-hydroxypropyl dimethyl dehydroabietyl ammonium chloride (CHPDMDHA), from dehydroabietylamine, with a similar structure to that of CTA. CHPDMDHA, when reacting with low molecular weight chitosan (LWCS), generates a grafted polymeric cationic surfactant, called LWCS-g-CHPDMDHA. The properties of the surfactant were directly influenced by DQ: the critical micellar concentration (CMC), above which aggregates (or micelles) of surfactant begin to form, decreased with increasing DQ, as well as the surface tension in CMC. According to Holappa et al. [ 191 ], the traditional route of obtaining TMC does not allow achieving structurally uniform polymers, since obtaining TMC does not occur without also methylating hydroxyl fractions of chitosan. To compensate, the authors suggested the synthesis of another quaternized derivative of chitosan, the chitosan N-betainates. Through subsequent steps of protection and deprotection of the amino and hydroxyl groups, it is obtained a full N-substituted chitosan, with a quaternary betaine moiety. The authors found a low antibacterial activity in neutral conditions and an increase in this activity with decreased DQ in acidic conditions. This means that the location of the positive charge in relation to the chitosan backbone affects antimicrobial activity. The same research group reported in Holappa et al. [ 192 ] obtaining another quaternized derivative of chitosan, in order to eliminate the problems of obtaining TMC: the mono- and di-quaternary piperazine derivatives. In Korjamo et al. [ 193 ], the research group evaluated the effect of these novel chitosan derivatives (N-betainates and N-piperazines) on the paracellular transport of mannitol and its cytotoxicity. The authors found an increase in paracellular transport in compounds with lower DQs and, in addition, N-betainates proved to be less toxic than N-piperazines, although both were found to have low toxicity. Once again, the authors proved that the permanent positive charge of the betaine and piperazine groups does not resemble the positive charge in the free amino group in terms of biological activity. Thus, the substitutes must be present at their minimum value until the pH-independent solubility is reached, in order to exercise its maximum activity. Zambito et al. [ 194 ] and Zambito et al. [ 195 ] reported that the property of enhanced transmucosal absorption of drugs in common quaternized chitosans, such as TMC, is affected by molecular weight, DQ and structural features. Thus, the authors proposed novel quaternized chitosans instead of the traditional TMC to improve the absorption of intraocular drugs, generating the so-called N,O-[N,N-diethylaminomethyl (diethyldimethylene ammonium)n] methyl chitosans. The obtained derivatives enhanced the penetration of hydrophilic and hydrophobic molecules through the porcine buccal epithelium, with a remarkably stronger effect than that of TMC. Drug penetration through the paracellular route has been improved by all derivatives, while the transcellular route has been enhanced by derivatives with high DQ. Regarding the ex vivo and in vivo permeability through the cornea of a rabbit, all derivatives improved the permeability, with better results than the TMC and more effective for the highest DQ. With the purpose of obtaining a quaternary chitosan derivative different from the traditional N-substituted ones, Cao et al. [ 196 ] synthesized N,N-dimethyl-O-quaternary ammonium chitosan using GTMAC as a quaternizing agent. The generated product had better moisture absorption and retention capacity than chitosan, in addition to stronger antibacterial activity. Li et al. [ 197 ] also used GTMAC to produce a novel O-quaternary ammonium N-acyl thiourea chitosan (OQCATUCS), containing double antimicrobial groups: the quaternary ammonium moiety and the thiourea group. An initial N-protection step was performed for the grafting of the quaternary ammonium group into the hydroxyl group, followed by