**2.1 Chitin esters in dressing materials**

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

proliferation of fibroblasts and influences vascularization [6–11].

which can be classified into the group of amino-polysaccharides.

Despite the very good biological properties of chitin, its practical use is moderate, which is related to its low solubility causing difficulties in its processing. Therefore, chitin is used as a raw material to obtain new derivatives with better performance parameters. In terms of practical use, the most important chitin derivatives are its esters and chitosan, which is a product of chitin deacetylation,

interactions with patients' bodies.

treatment at elevated temperature during sterilization, after exposure to X-ray, detergents and aseptic. Polysaccharide biopolymers, like most polymeric materials, degrade after some time of use, so it is also important that their decomposition products do not cause inflammation, allergic or immune reactions or any other

Chitin is a polysaccharide composed of N-acetylglucosamine residues linked by β-1,4-glycosidic bonds. Chitin is the main component of the fungal walls and the shells of arthropods (crustaceans, insects, and arachnids), but is also present in sponges, corals, and mollusks. However, for laboratory and industrial purposes, it is obtained mainly from marine invertebrates such as: crabs, shrimps, lobsters and krill. The properties of chitin depend on its origin. Chitin used in the production of medical devices must come from certified, controlled fisheries and must be properly purified. The methods of isolating chitin from natural sources are strictly dependent on the choice of the organism from which it is isolated. This polysaccharide is similar in structure to cellulose. It differs in the presence of an acetyl amide group (-NHCOCH3) in place of one of the hydroxyl groups (**Figure 1**). The presence of this group means that there are much stronger intermolecular hydrogen bonds in chitin, which results in its greater mechanical strength compared to cellulose [1, 2]. Depending on the origin source, chitin can occur in three amorphous forms: α, β and γ [2, 3]. The most widespread is α chitin found in fungi, shells of crustaceans and krill, and the skeletons of insects. The β form, which can mainly be isolated from squids, is much less common. The differences in the crystal structure of both amorphous forms of chitin affect their processing capabilities. The ordered crystal structure of chitin limits its solubility in commonly used solvents, and thus, reduces its use in industry. α-Chitin is moderately soluble in aqueous thiourea solution, aqueous alkaline urea solution, 5% LiCl/DMAC, some ionic liquids, hexafluoroacetone, hexafluoro-2-propanol, methanesulfonic acid [4, 5]. The form of β-chitin, on the other hand, swells in water (forms a suspension) and is soluble in formic acid. Chitin has no cytotoxic effect *in vitro*, is physiologically inert, biodegradable, has antibacterial properties and is characterized by a high affinity to proteins. During its biodegradation in the wound environment, its oligomers and units are released. Most often, it is used in the form of gel, membranes, fibers, polymer films or is a component of polymer blends. Chitin activates macrophages, stimulates the

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**Figure 1.**

*Structural resemblance of cellulose, chitin and chitosan.*

The esterification of chitin hydroxyl groups allows to increase the utility potential of the polysaccharide by introducing various substituents, and thus, influencing the physical, chemical and biological properties of materials. The best known are chitin esters, in which the hydroxyl groups are esterified with one type of acylating reagent (presence of the same ester groups on both hydroxyl groups of chitin). Acetylated chitin derivatives (CH3CO- substituent) are prepared with acetic anhydride in the presence of an acid catalyst. However, the physicochemical properties conditioning the processing of chitin acetate turned out to be unsatisfactory [12]. The use of a mixture of orthophosphoric acid and trifluoroacetic acid anhydride as a catalyst allowed to obtain a variety of chitin esters derived from: butyric acid, cyclopropanecarboxylic acid, cyclobutanecarboxylic acid, cyclopentanecarboxylic acid, cyclohexanecarboxylic acid and substituted benzoic acids. In the case of chitin butyrate, the process efficiency (DS (degree of substitution) included in the range 1.9–2.38) was dependent on the excess of butyric acid anhydride use [13–15]. Di-butyrylchitin (chitin di-butyrate, DBC) is an example of a chitin derivative soluble in typical organic solvents [16]. DBC is obtained by chitin esterification with butyric anhydride. Typically, it is a two-stage process. In the first step, chitin is purified from calcium salts with 2 M hydrochloric acid. The next stage is the process of proper esterification of purified chitin. The substrates of the reaction, apart from chitin, are butyric anhydride and the catalyst, which is most often chloric (VII) acid. The reaction is carried out in a heterogeneous system by adding powdered chitin in appropriate proportions to the reaction mixture consisting of butyric anhydride and chloric (VII) acid. The classical esterification process requires the use of substrates in a molar ratio of acid anhydride to N-acetylaminoglucose unit of 10: 1. It is also crucial to carry out the reaction at a temperature of 20°C. Increasing the reaction temperature to 40°C causes a rapid lowering of the molecular weight of the modified polymer. The catalyst concentration has a direct influence on the efficiency of the esterification reaction. The yield of the reaction is the higher when more concentrated chloric (VII) acid is used. However, it should be remembered that the use of too much concentrated chloric (VII) acid results in the macromolecule degradation. The esterification process is completed by adding diethyl ether to the reaction mixture. The isolated product is then heated with water to remove residual chloric (VII) acid. The product obtained in this way is treated for 24 hours with acetone, in which only di-butyrylchitin is dissolved. Then, the solution is concentrated to 5–6%. After the desired concentration is reached, the solution is poured into deionized water to precipitate the polymer, then the product is dried to obtain solid di-butyryl chitin. The above-described process of chitin esterification allows the conversion of free hydroxyl groups on the C3 and C6 carbon of the chitin into ester groups (CH3(CH2)2CO- substituent). Di-butyrylchitin is composed of dibutyl-N-acetyl-amino-glucose units linked by 1,4-β-glycosidic bonds. The polymer is stabilized by hydrogen bonds between the polymer chains. Hydrogen bonds are formed with the participation of the hydrogen atom from the acetylamino group and the oxygen atom from the ester group. This kind of intermolecular interaction determines its good mechanical properties [12–15]. Di-butyrylchitin does not dissolve and does not swell in water, but it dissolves in many organic solvents such as: acetone, methanol, ethanol, tetrahydrofuran (THF), dimethylformamide (DMF), chloroform, methylene chloride and others. Di-butyryl chitin is not easily degraded, it is resistant to γ-radiation (possibility of radiation sterilization), while enzymatic degradation under the influence of lysozyme and CE econase occurs at a

slow rate, which causes a slight change in molecular weight. Di-butyrylchitin with a molecular weight above 100000 Da has film-forming and fiber-forming properties [1, 2, 12–15]. Thus, obtaining DBC with the desired molecular weights directly determines its further processing capabilities (in particular electrospinning and leaching). The most important biological parameters of di-butyrylchitin are: prolongation of blood clotting time and good wettability. The use of DBC dressings has a positive effect on the granulation process (increasing the level of glycosaminoglycans in the wound), collagen cross-linking (generating more durable tissue), accelerating the wound healing process to form a healthy epidermis without scarring and protecting the wound against excessive moisture loss (optimal moist environment) [1, 2, 6–11]. In the course of treatment, the dressing slowly bio-degrades and resorbs until it disappears completely, which eliminates the painful act of changing it. The spontaneous, anti-pain effect of the dressing was also noted. DBC does not show cytotoxicity or irritation, it is a biocompatible polymer [9]. Di-butyrylchitin fibers are obtained by two methods: wet and dry-wet. The choice of the method used determines the structure of the obtained fibers. The fibers obtained in the wet-spinning process are less regular in shape, with a greater surface development than in the case of dry-wet spinning. DBC fibers produced by wet spinning are used as a raw material for the production of nonwovens. The technique of producing nonwovens from DBC depends on cutting the fibers into 6 cm long sections, from which the fleece is produced using a mechanical method on carding machines, and then the fibers in the fleece are joined by needling and calendaring [16, 17].

The dry-wet method of forming fibers from DBC hinges on preparing a spinning solution with a concentration of 15 to 25% in ethanol, heating it to 60°C and squeezing it through a spinning die. Then, the incompletely solidified fiber is introduced into a water bath, where it is completely solidified. The fiber is then wound onto drums, stretched, and dried. A microporous DBC fiber with a linear mass of 1.7 to 5.6 g is obtained, depending on the concentration of the spinning solution used. The fibers obtained by the dry method have an elongated and curved cross-sectional shape, similar to a croissant. The degree of crystallinity of the fibers determined in X-ray examinations is similar in both methods and amounts to approx. 19%, and the transverse dimension of the crystallites approx. 23 Å. It is also easy to obtain chitin materials (the so-called regenerated chitin) from these materials without damaging their macro-structure after a mild alkaline treatment. Fibers made of regenerated chitin and di-butyrylchitin do not induce cytotoxic, haemolytic or irritating effects and cause minimal local tissue reaction after implantation [17–19]. Di-butyrylchitin and regenerated chitin fibers can be used to obtain dry dressing materials, as well as materials for other biomedical purposes. DBC-based woven dressings are biodegradable within the wound and do not need to be replaced during use [16, 17]. Chitin di-pentanoate (chitin divalerate, Di-O-Valeryl-Chitin, DVCH) is also used for the production of commercially available dressings, where chitin is esterified with two valeryl groups (CH3(CH2)3CO- substituent) at the 3 and 6 positions of N-acetylglucosamine units. The high degree of DVCH esterification was achieved by using a large excess of valeric anhydride used both as acylating agent and reaction medium, and perchloric acid as catalyst. It turned out to be optimal to conduct the reaction where the molar ratio of valeric acid anhydride to chitin was 7:1, which also facilitated thorough mixing of the components during the reaction and temperature control. The performance of the reaction under conditions of high homogeneity of the solution has a great influence on the quality of the product. Insufficient mixing of the solution during the acylation step led to a local temperature rise, uneven chitin acylation and ultimately resulted in products with varying degrees of esterification and higher polydispersity. Additionally, at elevated temperature it was observed reduction of the molecular

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*Modulating the Physicochemical Properties of Chitin and Chitosan as a Method of Obtaining…*

weight of the biopolymer as a result of the acidic degradation of chitin that occurs in parallel with the acylation reaction in the presence of a strong acid. To obtain products with a high degree of esterification, 0.5 M perchloric acid was used (deacetylation of the N-acetyl group was not observed). The separation of the raw product from the reaction mixture takes place during the neutralization of the valeric acid excess with a 2.5% NaHCO3 solution. The use of sodium bicarbonate as a weak base prevents deacetylation of the N-acetyl group. Depending on the reaction time and temperature, products with different parameters are obtained. The lower temperature leads to a product with a higher molecular weight. A longer reaction time increases the yield of the reaction, but is associated with a reduction in molecular weight due to acidic degradation of the polymer. The DVCH polydispersity index ranged from 1.47 to 2.06, suggesting a low molecular weight distribution. Due to the good solubility of DVCH in organic solvents such as acetone or ethanol, it is possible to prepare thin polymer layers by casting or porous structures by salt leaching. The DVCH shows a semi-ductile behavior and breaks when it exceeds the yield point. The stretching properties of DVCH films depend on the molecular weight. The modulus, yield stress, tensile stress as well as strain at break increase continuously with increasing DVCH molecular weight. The increase in the modulus with molecular weight results in higher mechanical strength of DVCH films. The elongation at break, although slightly increases with increasing molecular weight, remains low, not exceeding 4%. As a consequence, the higher DVCH molecular weight is, it behaves like a stiff plastic that can withstand relatively high stresses but does not withstand high elongation before breakage. Using the salt leach method, it is also possible to develop porous materials from DVCH. The structure of porous DVCH-based materials consists of a unified network of interconnected channels. This structure is characterized by a high content of open pores of various sizes. Two pore populations can be distinguished: large with a size in the range 150–780 μm (average pore size approx. 435 μm ± 168 μm) and small with a size in the range of 4–22 μm (average pore size 7.7 μm ± 3.3 μm [16–19]. Chitin divalerate is a technologically friendly biopolymer. The good solubility of DVCH in organic solvents (ethanol, DMAC, DMSO, acetone) due to the presence of hydrophobic valeryl groups in C-3 and C-6 positions enables its easy processing of its particles. The DVCH maintains the film-forming ability of chitin, so it can easily be used in the production of threads, films, foams and scaffolds, as well as non-woven fabrics. Biological data show that DVCH is not cytotoxic to fibroblasts and does not cause irritation or allergy to the skin of animals [20]. For the synthesis of chitin dihexanoate (DHCH) it is also possible to use appropriate acid anhydrides and methanesulfonic acid as a catalyst. In order to increase the homogeneity of the solution and better control the temperature in the process, by analogy to the synthesis of the valerate ester, an excess of acid anhydride and methanesulfonic acid are used, the mixture being the reagents and the reaction medium. Optimal methanesulfonic acid to chitin molar ratios are 16:1 and 10:1 for chitin di-hexanoate and chitin di-butyrate, respectively. This approach will result in a high degree of substitution of hydroxyl groups, equal to almost 2, and a low polydispersity. Moreover, under optimal conditions, no hydrolysis of the N-acetyl bond was

observed. Good chitin solubility in methanesulfonic acid, even at low temperatures, allows the esterification process to be carried out under milder conditions. The key parameter is the intensity of agitation of the reaction suspension. Insufficient heat transfer due to poor mixing of the solution, similar to the synthesis of chitin di-pentanoate, leads to a lower degree of esterification, high polydispersity of the final product and a reduction in molecular weight. The neutralization process is carried out with a 4% sodium bicarbonate solution. The synthesis of DBC at a low temperature and short reaction time (temperature 0°C and 8°C) is ineffective due

*DOI: http://dx.doi.org/10.5772/intechopen.95815*

## *Modulating the Physicochemical Properties of Chitin and Chitosan as a Method of Obtaining… DOI: http://dx.doi.org/10.5772/intechopen.95815*

weight of the biopolymer as a result of the acidic degradation of chitin that occurs in parallel with the acylation reaction in the presence of a strong acid. To obtain products with a high degree of esterification, 0.5 M perchloric acid was used (deacetylation of the N-acetyl group was not observed). The separation of the raw product from the reaction mixture takes place during the neutralization of the valeric acid excess with a 2.5% NaHCO3 solution. The use of sodium bicarbonate as a weak base prevents deacetylation of the N-acetyl group. Depending on the reaction time and temperature, products with different parameters are obtained. The lower temperature leads to a product with a higher molecular weight. A longer reaction time increases the yield of the reaction, but is associated with a reduction in molecular weight due to acidic degradation of the polymer. The DVCH polydispersity index ranged from 1.47 to 2.06, suggesting a low molecular weight distribution. Due to the good solubility of DVCH in organic solvents such as acetone or ethanol, it is possible to prepare thin polymer layers by casting or porous structures by salt leaching. The DVCH shows a semi-ductile behavior and breaks when it exceeds the yield point. The stretching properties of DVCH films depend on the molecular weight. The modulus, yield stress, tensile stress as well as strain at break increase continuously with increasing DVCH molecular weight. The increase in the modulus with molecular weight results in higher mechanical strength of DVCH films. The elongation at break, although slightly increases with increasing molecular weight, remains low, not exceeding 4%. As a consequence, the higher DVCH molecular weight is, it behaves like a stiff plastic that can withstand relatively high stresses but does not withstand high elongation before breakage. Using the salt leach method, it is also possible to develop porous materials from DVCH. The structure of porous DVCH-based materials consists of a unified network of interconnected channels. This structure is characterized by a high content of open pores of various sizes. Two pore populations can be distinguished: large with a size in the range 150–780 μm (average pore size approx. 435 μm ± 168 μm) and small with a size in the range of 4–22 μm (average pore size 7.7 μm ± 3.3 μm [16–19]. Chitin divalerate is a technologically friendly biopolymer. The good solubility of DVCH in organic solvents (ethanol, DMAC, DMSO, acetone) due to the presence of hydrophobic valeryl groups in C-3 and C-6 positions enables its easy processing of its particles. The DVCH maintains the film-forming ability of chitin, so it can easily be used in the production of threads, films, foams and scaffolds, as well as non-woven fabrics. Biological data show that DVCH is not cytotoxic to fibroblasts and does not cause irritation or allergy to the skin of animals [20]. For the synthesis of chitin dihexanoate (DHCH) it is also possible to use appropriate acid anhydrides and methanesulfonic acid as a catalyst. In order to increase the homogeneity of the solution and better control the temperature in the process, by analogy to the synthesis of the valerate ester, an excess of acid anhydride and methanesulfonic acid are used, the mixture being the reagents and the reaction medium. Optimal methanesulfonic acid to chitin molar ratios are 16:1 and 10:1 for chitin di-hexanoate and chitin di-butyrate, respectively. This approach will result in a high degree of substitution of hydroxyl groups, equal to almost 2, and a low polydispersity. Moreover, under optimal conditions, no hydrolysis of the N-acetyl bond was observed. Good chitin solubility in methanesulfonic acid, even at low temperatures, allows the esterification process to be carried out under milder conditions. The key parameter is the intensity of agitation of the reaction suspension. Insufficient heat transfer due to poor mixing of the solution, similar to the synthesis of chitin di-pentanoate, leads to a lower degree of esterification, high polydispersity of the final product and a reduction in molecular weight. The neutralization process is carried out with a 4% sodium bicarbonate solution. The synthesis of DBC at a low temperature and short reaction time (temperature 0°C and 8°C) is ineffective due

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

slow rate, which causes a slight change in molecular weight. Di-butyrylchitin with a molecular weight above 100000 Da has film-forming and fiber-forming properties [1, 2, 12–15]. Thus, obtaining DBC with the desired molecular weights directly determines its further processing capabilities (in particular electrospinning and leaching). The most important biological parameters of di-butyrylchitin are: prolongation of blood clotting time and good wettability. The use of DBC dressings has a positive effect on the granulation process (increasing the level of glycosaminoglycans in the wound), collagen cross-linking (generating more durable tissue), accelerating the wound healing process to form a healthy epidermis without scarring and protecting the wound against excessive moisture loss (optimal moist environment) [1, 2, 6–11]. In the course of treatment, the dressing slowly bio-degrades and resorbs until it disappears completely, which eliminates the painful act of changing it. The spontaneous, anti-pain effect of the dressing was also noted. DBC does not show cytotoxicity or irritation, it is a biocompatible polymer [9]. Di-butyrylchitin fibers are obtained by two methods: wet and dry-wet. The choice of the method used determines the structure of the obtained fibers. The fibers obtained in the wet-spinning process are less regular in shape, with a greater surface development than in the case of dry-wet spinning. DBC fibers produced by wet spinning are used as a raw material for the production of nonwovens. The technique of producing nonwovens from DBC depends on cutting the fibers into 6 cm long sections, from which the fleece is produced using a mechanical method on carding machines, and

then the fibers in the fleece are joined by needling and calendaring [16, 17].

The dry-wet method of forming fibers from DBC hinges on preparing a spinning solution with a concentration of 15 to 25% in ethanol, heating it to 60°C and squeezing it through a spinning die. Then, the incompletely solidified fiber is introduced into a water bath, where it is completely solidified. The fiber is then wound onto drums, stretched, and dried. A microporous DBC fiber with a linear mass of 1.7 to 5.6 g is obtained, depending on the concentration of the spinning solution used. The fibers obtained by the dry method have an elongated and curved cross-sectional shape, similar to a croissant. The degree of crystallinity of the fibers determined in X-ray examinations is similar in both methods and amounts to approx. 19%, and the transverse dimension of the crystallites approx. 23 Å. It is also easy to obtain chitin materials (the so-called regenerated chitin) from these materials without damaging their macro-structure after a mild alkaline treatment. Fibers made of regenerated chitin and di-butyrylchitin do not induce cytotoxic, haemolytic or irritating effects and cause minimal local tissue reaction after implantation [17–19]. Di-butyrylchitin and regenerated chitin fibers can be used to obtain dry dressing materials, as well as materials for other biomedical purposes. DBC-based woven dressings are biodegradable within the wound and do not need to be replaced during use [16, 17]. Chitin di-pentanoate (chitin divalerate, Di-O-Valeryl-Chitin, DVCH) is also used for the production of commercially available dressings, where chitin is esterified with two valeryl groups (CH3(CH2)3CO- substituent) at the 3 and 6 positions of N-acetylglucosamine units. The high degree of DVCH esterification was achieved by using a large excess of valeric anhydride used both as acylating agent and reaction medium, and perchloric acid as catalyst. It turned out to be optimal to conduct the reaction where the molar ratio of valeric acid anhydride to chitin was 7:1, which also facilitated thorough mixing of the components during the reaction and temperature control. The performance of the reaction under conditions of high homogeneity of the solution has a great influence on the quality of the product. Insufficient mixing of the solution during the acylation step led to a local temperature rise, uneven chitin acylation and ultimately resulted in products with varying degrees of esterification and higher polydispersity. Additionally, at elevated temperature it was observed reduction of the molecular

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to the low reaction yields and possibly incomplete esterification of the chitin hydroxyl groups, resulting in the formation of a significant amount of insoluble gel fractions when dissolved in acetone prior to precipitation with water. For DHCH, it is preferable to use low synthesis temperatures (0°C and 8°C). The yield of DHCH synthesis was relatively high (above 70%), with the highest efficiency observed at 21°C (84 to 95%). Unfortunately, carrying out the synthesis of DHCH at 21°C resulted in a low molecular weight product. A trend analogous to that of chitin di-pentanoate was observed, indicating that the longer the reaction time, the higher the reaction performance and the lower the molecular weight of the obtained biopolymers. Although in DHCH the hydroxyl groups of chitin are substituted with longer alkyl chains than in DVCH or DBC, it has been found that DHCH retains good solubility in solvents such as ethanol, acetone, dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide, N,N-dimethylacetamide and ethyl acetate and no solubility in water. Good solubility, filmogenic and fiber-forming properties of DHCH give greater possibilities of its processing (film casting, washing method, electrospinning method) compared to chitin alone. The mechanical properties of DHCH and DBC in the form of thin solid layers poured from solution were investigated in relation to their molecular weights. DHCH and DBC showed semicontinuous properties and cracked rapidly upon exceeding the plasticity point, similar to that observed for DVCH. The elongation at break was small and did not exceed 4%. For both biopolymers, their tensile properties correlate with the molecular weight. Parameters such as modulus of elasticity, stress at yield, as well as stress and strain at break, were found to increase with increasing DHCH and DBC molecular weight. Comparing the mechanical properties of DHCH, DBC and DVCH, it can be concluded that Young's modulus decreases with increasing chain length of the acyl group of chitin diesters (a similar relationship is observed for chitin monoesters, where only one hydroxyl group is acylated). Due to the good solubility of hydrophobic chitin diesters in organic solvents and their insolubility in water, it is possible to obtain porous structures based on DHCH and DBC by using the salt leaching method. The prepared porous materials are characterized by a united network of interconnected channels and a high degree of open porosity with pores of various sizes (pore size in the range 78–421 μm, average pore size 253 μm ± 93 μm) [21–24]. Due to its physic-chemical properties, DHCH can successfully replace or support materials based on di-butyrylchitin (e.g. in the form of mixtures of both biopolymers) and thus it can be used as a material for medical and pharmaceutical applications, especially in tissue engineering scaffolds and in healing wounds. The described procedure of chitin esterification to obtain products of high purity. Moreover, this method is universal (the possibility of preparation various chitin diesters) and is easy to produce and is not time-consuming [21]. Another method of chemical modification of chitin is esterification leading to carboxymethylchitin (CMCht, CM-chitin) [22, 23] or dicarboxymethylchitin using monochloroacetic or mono-chloropropionic acid followed by substitution of halogen with a hydroxyl group. This modification leads to the loss of the supramolecular structure of chitin and the formation of water-soluble derivatives [24].
