Fundamentals of chitosan-based hydrogels: elaboration and characterization techniques

CHAPTER

Fundamentals of chitosanbased hydrogels: elaboration and characterization techniques

3

Rejane Andrade Batista1, Caio Gomide Otoni2 and Paula J.P. Espitia3 1

Instituto Tecnolo´gico e de Pesquisas do Estado de Sergipe, Rua Campo do Brito, Aracaju, Brazil 2National Nanotechnology Laboratory for Agribusiness, Embrapa Instrumentac¸a˜o, Sa˜o Carlos, Brazil 3Nutrition and Dietetics School, Universidad del Atla´ntico, Atla´ntico, Colombia

3.1 INTRODUCTION Hydrogels can be defined as systems comprising of three-dimensional, physically or chemically bonded polymer networks entrapping water in intermolecular space (Ahmed, 2015). In other words, hydrogels may be referred to as hydrophilic gels or colloidal gels in which the dispersion medium is water (Ahmed et al., 2013). Depending on several factors (e.g., polymer nature, crosslinking nature, and density), hydrogels may be swollen with different amounts of water. The water sorption capacity of hydrogels results from their hydrophilicity, which in turn is provided mainly by capillary, osmotic, and hydration forces (Buwalda et al., 2014). Among the particularities of hydrogels two properties deserve special emphasis, namely: their remarkable ability of absorbing high amounts of liquids—more than 100% in relation to their dry weights—in a rapid fashion as well as their capacity to retain specific compounds without changing their structures in the swollen state when exposed to certain pressures (Bao et al., 2011; Feng et al., 2014; Mahdavinia et al., 2004). Fig. 3.1 illustrates a cellulose-based hydrogel in its original, swollen, and dried stages (Chang et al., 2010). Because of these peculiarities, hydrogels have become essential to numerous businesses as their application potential has been broadened for several areas, including agriculture, waste water treatment, water purification, tissue engineering, sensors, contact lenses, and drug release, among many others (Chang and Zhang, 2011; Feng et al., 2014). Thanks to their applicability potential, absorbent polymers and, particularly, hydrogels have been arousing growing interest from the scientific community. This reflects in a remarkable increase in the number of research and review articles since the 1980’s, as demonstrated in Fig. 3.2. Several classification criteria have been proposed in order to group hydrogels, as illustrated in Table 3.1 (Chang and Zhang, 2011; Zheng and Wang, 2015). Materials for Biomedical Engineering: Hydrogels and Polymer-based Scaffolds. DOI: https://doi.org/10.1016/B978-0-12-816901-8.00003-1 © 2019 Elsevier Inc. All rights reserved.

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FIGURE 3.1 Original (A), swollen (B), and dried (C) hydrogels comprised of cellulose powder and carboxymethylcellulose and crosslinked with epichlorohydrin. Adapted from Chang, C., Duan, B., Cai, J., Zhang, L., 2010. Superabsorbent hydrogels based on cellulose for smart swelling and controllable delivery. Eur. Polym. J. 46, 92
100 with the permission of Elsevier.

FIGURE 3.2 Number of scientific documents (research and review articles) published annually on superabsorbent polymers (black bars; topic: superabsorbent and polymer) and hydrogels (gray bars; topic: superabsorbent and hydrogel) retrieved from Web of Science Core Collection (as of January, 2017).

Hydrogels may be classified according to different properties or characteristics. The most used classifications rely on binding nature, that is, chemical or physical bonds. Physically bonded hydrogels are comprised of polymer chains ordered by intermolecular interactions (e.g., ionic or hydrogen bonds), whereas their chemically

3.1 Introduction

Table 3.1 Classification Criteria of Hydrogels and Main Features Considered Classification Criteria

Main Features Considered

Source

• • • • • • • • • • • • • • • • • • • • • • • • • • • •

Composition (monomers)

Crosslinking process Degradability Physical appearance

Electrical charge

Properties Chemically responsive

Biochemically responsive

Physically responsive

Natural Synthetic Hybrid Homopolymeric nature Copolymeric nature Multicomponent/interpenetrating network Physical Chemical Biodegradable Nonbiodegradable Matrix Film Microsphere (beads) Neutral/nonionic nature Ionic (anionic/cationic nature) Amphiphilic Conventional Smart pH Glucose Oxidant Antigens Enzymes Ligands Temperature Pressure Light Electric/magnetic field

Based on Ahmed, E.M., 2015. Hydrogel: preparation, characterization, and applications: a review. J. Adv. Res. 6, 105
121 and Ullah, F., Othman, M.B.H., Javed, F., Ahmad, Z., Akil, H.M., 2015. Classification, processing and application of hydrogels: a review. Mater. Sci. Eng. C 57, 414
433.

bound counterparts present chains connected by covalent bonds (Chang and Zhang, 2011). A combination of both is also possible (Buwalda et al., 2014). Another widely spread classification criterion takes into account the source of raw materials: natural, synthetic, or hybrid. The list of synthetic polymers used for hydrogel production is extensive and has been reviewed elsewhere (Kabiri et al., 2011; Ullah et al., 2015). Examples include polyacrylamide, poly(sodium acrylate), poly(acrylic acid), and polyvinylpyrrolidone, to mention a few.

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Hydrogels based on naturally occurring polymers, in turn, may be further sorted into two groups: polysaccharides or polypeptides (Zheng and Wang, 2015). The latter includes but is not limited to collagen, gelatin, soy, and fish proteins (Zohuriaan-Mehr et al., 2009), whereas the former encompasses starch (Chen et al., 2015), cellulose, and its derivatives (Bao et al., 2011; Chang and Zhang, 2011; Ma et al., 2015); sodium alginate (Wang and Wang, 2010), guar gum (Thombare et al., 2016), xanthan gum (Feng et al., 2014), and chitosan, and its derivatives, among others (Guilherme et al., 2015; Wang et al., 2013; Yu et al., 2010). Hydrogels made up of chitosan matrix, in particular, have been arousing rising interest because of the unique physicochemical, biological, and mechanical properties of this polymer. Concerning the biomedical industry, these materials present many advantages, such as biocompatibility, low toxicity, high bioactivity, multifunctionality, and biodegradability (Calo´ and Khutoryanskiy, 2015). These characteristics allow them to play their role in direct contact with the human body without any health damages. The aforementioned materials are capable of mimicking natural tissues, in addition to being adaptable and easy to handle as well as having lower cost than the polymers that are conventionally used for this purpose (Chang and Zhang, 2011; Feng et al., 2014). Chitosan-based hydrogels may be presented as fibers, films, gels, membranes, micro- or nanosized particles, and sponges without any functionality impairment (Bansal et al., 2011). When it comes to biomedical engineering, the feasible applications of hydrogels include, but are not limited to, contact lenses, drug release, artificial muscles, bone filling, and dermatology (Calo´ and Khutoryanskiy, 2015).

3.2 CHITOSAN NATURE AND MAIN PROPERTIES Chitosan is a carbohydrate polymer obtained from chitin through a deacetylation process. Chitin ranks second—preceded by cellulose only—amongst the most abundant biopolymers. It is of natural occurrence in marine crustaceans, mollusks, and insects—mainly as part of their exoskeleton—as well as in fungi, in which it plays a structural role (Alves and Mano, 2008). Large-scale chitin extraction from fungal mycelia is preferred over animal sources, chiefly due to the year-long (i.e., nonseasonal) availability of raw material and the completely controlled chitin production, which leads to a final product that features the standard physicochemical characteristics. Chitin extraction from fungi is also advantageous from a safety point of view due to the low allergenic risk, which otherwise would possibly be increased by crustacean components that might remain after the extraction and purification processes from marine sources (Croisier and Je´roˆme, 2013). In its native form, chitin is not suitable for several applications, mainly because of its chemical inertness and poor solubility (Croisier and Je´roˆme, 2013).

3.2 Chitosan Nature and Main Properties

FIGURE 3.3 Representation of chitosan extraction from chitin.

Thus, chitin must be subjected to a deacetylation process in a reaction with a concentrated alkali solution, which results in chitosan (Fig. 3.3). Chitin deacetylation is often carried out in the presence of sodium hydroxide or potassium hydroxide in addition to anhydrous hydrazine and hydrazine sulfate (Dash et al., 2011; Rinaudo, 2006). Unlike chitin, chitosan is highly reactive and is commercially available in powder, paste, fiber, or other forms (Agnihotri et al., 2004). Chemically, chitosan can be defined as a biopolymer with a linear arrangement of glucosamine and N-acetyl-glucosamine as structural and functional units linked by (1-4) glycosidic bonds. The chemical structure of chitosan is determined by the presence of glucosamine units, which affects the degree of deacetylation and, consequently, its reactivity. In this regard, chitosan solubility increases in aqueous-acid solutions when it presents a deacetylation degree higher than 50%, while chitosan biodegradability may be prevented when deacetylation degrees are higher than 69% (Alves and Mano, 2008; Berger et al., 2004). The main physicochemical characteristics of chitosan are presented in Table 3.2. Out of the major biological properties that make chitosan suitable for several applications, its antimicrobial, immunological, wound-healing activities, biodegradability, as well as its nonimmunogenic and noncarcinogenic characteristics may be highlighted (Dash et al., 2011). Regarding its antimicrobial properties, previous research has shown the biological activity of chitosan against Gramnegative and Gram-positive bacteria. One of the proposed mechanisms of action for chitosan antimicrobial activity is the interaction of this biopolymer with

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Table 3.2 Main Physicochemical Characteristics of Chitosan Physicochemical Characteristics Average molecular weight Deacetylation degree Ionic nature Solubility

3.8
20.0 kDa 60%
100% Cationic polysaccharide (at neutral or basic conditions) Insoluble in water, soluble in acid solutions

bacterial cell membranes due to the electrostatic interaction between chitosan— polycationic in nature—and anionic groups located on the bacterial surface. This interaction affects bacterial membrane permeability, resulting in its disruption and, consequently, leakage of intracellular content (Abdelgawad et al., 2014; Shahidi et al., 1999). The biodegradability of chitosan is a result of its biopolymeric nature, constituted of a polysaccharide and structured by means of glycosidic bonds (Croisier and Je´roˆme, 2013). In this regard, the main bonds of chitosan—that is, glucosamine-glucosamine, glucosamine-N-acetyl-glucosamine, and N-acetyl-glucosamine-N-acetyl-glucosamine—are targeted by enzymatic action, leading to chitosan degradation. Lysozyme and bacterial enzymes in the colon have been identified in vertebrates as responsible for chitosan biodegradation. Moreover, higher plants present chitinases, which show enzymatic activity on N-acetyl-glucosamine residues. This indicates a mechanism of self-protection against plant threats, such as microbes and insects, which might have chitin in their structure. On the other hand, the extent of chitosan biodegradation is determined by the deacetylation degree; in this context, high deacetylation degrees result in low biodegradability (Kean and Thanou, 2010). Additionally, chitosan has shown mucoadhesive properties, resulting mostly from its strong electrostatic interactions with the negative charges of sialic acid residues present on mucosal surfaces (Sinha et al., 2004). Therefore, the aforementioned biological properties make chitosan an interesting biopolymer with the potential to be used as a raw material for hydrogel preparation with innumerous biomedical and nonbiomedical applications.

3.3 FUNDAMENTALS OF CHITOSAN HYDROGELS Hydrogels are defined as tridimensional networks comprising of macromolecular compounds with the ability to retain water or biological fluids in their inner side (Berger et al., 2004). Regarding their structures, hydrogels are constituted by two phases: a liquid phase—mostly water or other biological fluids; and a solid phase—polymer chains that confer a gel-like consistency, allowing the structure to entrap water (Croisier and Je´roˆme, 2013). The liquid phase of hydrogels confers biocompatibility and allows for their widespread application in biomedicine,

3.3 Fundamentals of Chitosan Hydrogels

agriculture, food science, and nutrition, to mention a few. Generally, hydrogels are characterized by their particle size, which might range from the nanoscale to several centimeters. Also, they are expected to be highly flexible, allowing them to acquire the shape of the space they are contained in (Bhattarai et al., 2010). The main hydrogel component is a hydrophilic polymer, which can absorb different amounts of water depending on its hydrophilic nature (Bhattarai et al., 2010). As mentioned previously, hydrogels can be classified according to different criteria, such as the nature of the polymeric compound and the method of hydrogel constitution (network nature), among others (Berger et al., 2004). Besides its cationic nature, which allows for the formation of blends with a wide range of polysaccharides, chitosan is among the biopolymers that offer higher flexibility to hydrogel applications. This results from the ease of modification of its molecule through reactions of amino and hydroxyl groups, providing hydrogels with numerous possibilities of structural and morphological profiles as well as absorption potential. As a consequence, this broadens the range of the applications and functions of hydrogels. Such variations, however, are determined by the production techniques since crosslinking is the major cause of the structural, mechanical, and chemical properties of hydrogels, as highlighted in Table 3.3 (Barbucci et al., 2004; Deligkaris et al., 2010; Kalia et al., 2013; Omidian and Park, 2010). Moreover, some features of hydrogels are conditioned to changes in environmental factors, such as temperature, pH, and electric and magnetic properties. These are considered as nonconventional characteristics of chitosan-based hydrogels, which are mainly stimuli-responsive to the aforementioned environmental factors.

3.3.1 PHYSICAL HYDROGELS Hydrogels prepared by physical methods are constituted by noncovalent, reversible links or interactions between polymer chains, such as ionic, electrostatic and hydrophobic interactions, grafting, and entanglement, among others (Berger et al., 2004; Croisier and Je´roˆme, 2013). Factors that can affect hydrogel formation through physical methods include pH, polymer concentration, and temperature. Moreover, hydrogel integrity is determined by the number of interactions that occur between the reacting compounds; therefore, increasing interactions result in stiff hydrogels, while limited interactions result in a soft, weak hydrogel structure (Croisier and Je´roˆme, 2013). Hydrogels prepared by physical methods include polyelectrolyte complexed hydrogels, which are three-dimensional networks resulting from ionic interactions among their constituting polymers. In this regard, a three-dimensional network is formed when two polyelectrolytes with opposite charges react in solution. In the case of chitosan, electrostatic interactions take place between its amino group which features a cationic nature and the anionic group of the other compound present in the solution. This type of hydrogel is considered as an interesting alternative to hydrogels elaborated by chemical methods since no additional molecules

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Table 3.3 Crosslinking Methods Used to Produce Chitosan-Based Hydrogels Main Features

Crosslinking Methods Physical

Chemical

Radiation

Crosslinking agent

Absent

Absent

Interaction nature

Complex coacervation (Magnin et al., 2004) Freeze-thawing (Giannouli and Morris, 2003) H-bonding (Takigami et al., 2007) Heating/cooling a polymer solution (Funami et al., 2007) Ionic interaction (Zhao et al., 2009) Maturation (heat induced aggregation) (AlAssaf et al., 2009) Heat-induced aggregation Polymer solution heating/cooling Drying at high or low temperatures

Agents of different natures depending on the intended applications Covalent Radiation grafting (Cai et al., 2005) Chemical grafting (Spinelli et al., 2008)

Polymerization of the available functional groups Polymer-polymer crosslinking Reticulation triggered by a crosslinking agent Cosmetics

Producing free radicals in the polymer following exposure to high energy source, such as gamma ray, X-ray, or electron beam (Gulrez and Al-Assaf, 2011)

Applied techniques

Applications

Food industry Biomedical products

Aqueous state radiation (Zhao et al., 2003) Radiation in paste (Zhao et al., 2003) Solid state radiation (Kuang et al., 2008)

Biomedicine products Pharmaceutical industry

Based on Gulrez, S. K. H., & Al-Assaf, S. (2011). Hydrogels: methods of preparation, characterisation and applications. In: A. Carpi (Ed.), Progress in Molecular and Environmental Bioengineering-From Analysis and Modeling to Technology Applications (Vol. 1, pp. 117150). London, UK: IntechOpen.

(e.g., catalysts and initiators, among others) are needed for hydrogel formation. Therefore, there is no need for a purification step. Examples of natural polyelectrolytes with anionic nature include polysaccharides with carboxylic groups (COOH), such as xanthan gum, pectin, and alginate. Moreover, collagen, gelatin, keratin, albumin, and fibroin are proteins that have been previously studied for the development

3.4 Characterization Techniques

Table 3.4 Main Advantages and Disadvantages of Physical Hydrogels Advantages

Disadvantages

Additives (catalysts or initiators—mostly toxic) are not needed for hydrogel formation Biocompatibility—essential for biomedical applications Ability to shape the injured tissue as a template

Limited mechanical resistance Easy dissolution should environmental factors not be carefully controlled Limited control of hydrogel pore size

of polyelectrolyte complexed hydrogels (Berger et al., 2004). The major advantages and disadvantages of physical hydrogels are presented in Table 3.4.

3.3.2 CHEMICAL HYDROGELS Chemical hydrogels result from polymeric interactions through covalent bonding. These hydrogels are characterized by the need of chemical modification of chitosan structure and by their nature of having an irreversible structure. Linkages that might take place during the elaboration of chemical hydrogels include amide and ester bonding, as well as Schiff base, among others. During the elaboration process of chemical hydrogels, a crosslinker is required to allow the configuration of the tridimensional network. Most of the time these crosslinkers are small multifunctional molecules, such as tripolyphosphate, ethylene glycol, diglycidyl ether, and others, that react with chitosan and favor interaction with previously activated chitosan active functional groups (Croisier and Je´roˆme, 2013). However, one of the main characteristics of this kind of hydrogel is that a purification step is required after their elaboration, in order to avoid potential toxicity caused by covalent crosslinkers that can remain unreacted after the hydrogel elaboration process (Berger et al., 2004). The main factors that determine the structural characteristics of chemical hydrogels include the crosslinking density and the ratio of crosslinking molecules regarding the polymers used for hydrogel formation (Croisier and Je´roˆme, 2013; Dash et al., 2011).

3.4 CHARACTERIZATION TECHNIQUES Different techniques may be used to characterize the profile of hydrogels. Researchers are encouraged to choose certain ones depending on the desired hydrogel properties, as well as the requirements for specific practical applications. In biomedical engineering, for instance, rheological and cytotoxicological aspects as well as absorption and degradation potentials are of outmost importance.

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Overall, the main aspects that are considered in hydrogel characterization are morphology, swelling, and mechanical resistance. The relevant characterization techniques typically applied to chitosan-based hydrogels may be grouped into structural analysis or property measurements. Structural analyses mainly rely on microscopic and spectroscopic methods. On the other hand, the characterization of performance properties depends on the specific application of hydrogels (Table 3.5).

Table 3.5 Analytical Techniques Typically Used for Hydrogel Characterization Analysis

Technique

Analyzed Factors

References

Structural analyses

X-ray diffraction

• Identification of ions • Mapping and distribution of mineral chemical elements (compositional map) • Phase miscibility and compatibility in blends • Structure length and orientation; Modulation of chemical composition • Topographical analysis • Polymer chain conformation • Morphological, structural, and molecular characteristics of the matrix • Chemical quantification of different compounds (e.g., release of drugs, vitamins, and other active ingredients) • Identification of chemical compounds within the hydrogel matrix • Investigation of possible, important replacements in ingredients

Cullity and Stock (2001)

Scanning electron microscopy Transmission electron microscopy Atomic force microscopy

Ultraviolet
visible spectroscopy

Fourier-transform infrared spectroscopy

Zhou et al. (2008), Zhao et al. (2009) Kamel (2007)

Duran et al. (2006)

Zhou et al. (2008)

Zhao et al. (2009)

(Continued)

3.4 Characterization Techniques

Table 3.5 Analytical Techniques Typically Used for Hydrogel Characterization Continued Analysis

Technique

Analyzed Factors

References

Performance properties measurements

Release of active compounds

• Diffusion of active compounds • Diffusion-controlled mechanism • Relationship of active compound delivery and hydrogel pore size • Mechanical performance • Hydrogel structural integrity • Sol
gel transitions • Determination of hydrogel thermosensibility • Determination of the maximum amount of liquid (water or any biological fluid) retained inside the hydrogel structure • Quantification of hydrophilic degree of developed hydrogels

Bhattarai et al. (2010), Fan et al. (2015, 2016), Zhao et al. (2009), Zhou et al. (2008)

Mechanical resistance

Viscosity

Swelling index

Contact angle

3.4.1 STRUCTURAL ANALYSIS 3.4.1.1 Microstructural and spectroscopic analysis Direct imaging of chitosan hydrogels is important because it allows for the study of microstructural characteristics that might influence hydrogel structural integrity as well as in the loading and release of active compounds. In this regard, microscopic techniques, such as atomic force microscopy (AFM), transmission electron microscope (TEM), and scanning electron microscopy (SEM), allow for the direct imaging of chitosan hydrogels, the formation of new structures, matrix integrity, and porosity. AFM is one of the main tools for studying the surface of chitosan-based hydrogels. Studies done with AFM include the investigation of the threedimensional surface topography as well as the morphological, structural, and molecular properties of hydrogels on a nanoscale (Duran et al., 2006). These techniques allow for the characterization of phase morphology and distribution in blends and composites in addition to the conformation of polymer chains themselves. Such information supports the comprehension of a hydrogel profile.

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TEM allows for a detailed examination of hydrogel samples, including changes in the chemical composition, orientation, and aspect ratio (length-to-diameter or length-to-width ratio) of nanostructures; induction of electronic phase changes; and images based on material absorption. These data support the discussion of the influence of different treatments, as well as the optimization of hydrogel preparation conditions (Kamel, 2007; Williams and Carter, 1996). SEM is useful for determining the morphology of various hydrogel matrices. For SEM imaging, hydrogel samples are usually coated with a thin layer of gold under vacuum prior to sample exposure to electron beam. Zhou et al. (2008) used this technique to analyze thermosensitive chitosan hydrogels and observed that hydrogel morphology was dependent on chitosan characteristics and concentration. In this context, hydrogels prepared with 1% chitosan solution presented a loose and ramified configuration, while increased chitosan concentration resulted in a more compact microstructure. Also, chitosan characteristics, such as low molecular weight, resulted in a loose structure full of holes. Moreover, modifications to the degree of deacetylation resulted in more compact but irregular microstructures when increased from 75.4% to 85.5% (Fig. 3.4). Moreover, thermosensitive chitosan hydrogels combined with αβ-glycerophosphate were developed and characterized using an SEM technique (Zhao et al., 2009). To do so, samples were coated with gold under vacuum, and hydrogel surfaces and cross-sections were subjected to imaging analyses. On the other hand, when coupled with an energy dispersive system (EDS), SEM also determines qualitatively and semiquantitatively the elementary compositions of hydrogel samples by means of emitting X-rays. SEM may indicate miscibility of chitosan hydrogel as well as compatibility of polymer blends with the constituents of the hydrogel matrix. This technique has been previously used by Zhao et al. (2003), who studied the morphological structure, by SEM, as well as the elementary distribution pattern of blend hydrogels based on poly(vinyl alcohol) (PVA) and carboxymethylated chitosan (CM-chitosan) using EDS. In order to determine the pattern distribution of the CM-chitosan components on the surface of the blend hydrogels, developed hydrogels were immersed in a 5% (w/w) CuSO4 aqueous solution for 6 days at room temperature. After this time, the CuSO4-complexed amino groups in the developed hydrogels were determined using an EDS technique. As a result, it was confirmed that CM-chitosan was uniformly distributed in the developed blend hydrogels at low concentrations of CM-chitosan content (,8% w/w); this was probably because CM-chitosan is able to interact more effectively with PVA during hydrogel elaboration due to its higher hydrophilic nature compared to neat chitosan.

3.4.1.2 Ultraviolet
visible spectroscopy and Fourier-transform infrared spectroscopy Spectroscopy techniques are usually employed to determine chemical interactions among functional groups that constitute hydrogel structure. Also, Fourier-transform

3.4 Characterization Techniques

FIGURE 3.4 Scanning electron micrograph of chitosan and αβ-glycerophosphate CS-ab-GP gelation with different formulations (500 3 ). (A) Molecular weight (MW) 5 1360 kDa, degree of deacetylation (DD) 5 75.6%, chitosan concentration 5 1%; (B) MW 5 1360 kDa, DD 5 75.6%, chitosan concentration 5 2%; (C) MW 5 499 kDa, DD 5 75.4%, chitosan concentration 5 2%; and (D) MW 5 1340 kDa, DD 5 85.5%, chitosan concentration 5 2%. Zhou, H.Y., Chen, X.G., Kong, M., Liu, C.S., Cha, D.S., Kennedy, J.F., 2008. Effect of molecular weight and degree of chitosan deacetylation on the preparation and characteristics of chitosan thermosensitive hydrogel as a delivery system. Carbohydr. Polym. 73, 265
273 with permission.

infrared spectroscopy (FTIR) is often used to verify the development of polymerization reactions when hydrogels are formed. The FTIR technique has been used for the characterization of thermosensitive chitosan hydrogels by mixing 2 mg of hydrogel sample with 100 mg of KBr (Zhou et al., 2008). From the obtained results, researchers observed that hydrogel was successfully formed by the interaction of hydrogen bonding between C 5 O of chitosan and
OH of αβ-glycerophosphate and by the junction of N-H of chitosan and
OH of αβ-glycerophosphate. Li et al. (2017) proposed a novel method for preparing a chitosan-based hydrogel and corroborated, through FTIR, that the interaction between NH2 and Ag1 was the major factor leading to crosslinking and that the CTS-Ag1/NH3 hydrogel network was maintained by hydrogen bonds, leading to improved mechanical properties.

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3.4.2 PROPERTY MEASUREMENTS 3.4.2.1 Active compound release assessment The release for active compounds previously incorporated in hydrogels is determined by time and it could be controlled by diffusion, swelling ability of the hydrogel, or by chemical reactions, with the diffusion-controlled mechanism being the most common. However, many active compounds have sizes that are considerably smaller than the pore size of hydrogels, resulting in a burst delivery of these compounds and limiting their controlled release (Bhattarai et al., 2010).

3.4.2.2 Mechanical resistance One of the main characteristics of chitosan hydrogels is their adaptability to the environment where they are used. In this regard, in most cases chitosan hydrogels can mimic the body tissue allowing for their use as scaffolds for biomedical applications, as well as carriers for the delivery of active compounds (Croisier and Je´roˆme, 2013). Thus, chitosan hydrogels must present certain mechanical resistance associated to the maintenance of their structural integrity. For determining the mechanical resistance of chitosan hydrogels, samples are cut in a dumbbell shape. Parameters, such as tension (N) and elongation (mm), are determined using a universal testing machine or a texturometer, while the thickness of hydrogel samples is determined with a caliper (Fan et al., 2015). Finally, the tensile strength and percentage of elongation is determined according to these equations: Tensile strength ðMPaÞ 5 Elongation% 5

F ðW 3 HÞ

S 3 100 L

wherein F is the sample tension at break; S is the elongation at break; W is sample width; H is sample height; and L is sample length. Also, the mechanical resistance of chitosan hydrogel might be determined by puncture test with a texturometer (Gonc¸alves et al., 2017). In this test, a 12.5 mm Teflon probe is forced to puncture the hydrogel samples, allowing for the determination of the maximum force (Bloom index) required to alter a sample’s structural integrity.

3.4.2.3 Viscosity (sol
gel analysis) The measurement of hydrogel viscosity has been used to determine the occurrence of sol
gel transitions. This measurement is quite important when dealing with the development of chitosan thermosensitive hydrogels. Thermosensitive hydrogels are a kind of hydrogel that has the ability to transit from a solution into a gel with temperature modifications. In this regard, Zhou et al. (2008) have developed a thermosensitive hydrogel based on chitosan and αβ-glycerophosphate, which has the ability to transform from solution to gel at

3.4 Characterization Techniques

37 C. Considering viscosity, researchers stablished the sol phase as a physical state of the hydrogel with the main characteristic of flowing liquid, while the gel phase was stablished as a nonflowing physical state of the developed hydrogel. Generally, measurements to determine this property are done in a viscometer, and the hydrogel sample is placed in a water bath at target temperature for sol
gel transition (in this case 37 C 6 0.5 C).

3.4.2.4 Swelling index Swelling index is a quantitative parameter that allows for the determination of the amount of water or biological fluid that can be retained inside the tridimensional structural hydrogel network. Usually, the weight of the dry hydrogel is determined. Following this, the dry hydrogel is placed in distilled water or any other target biological fluid or simulant at room temperature for 48 hours. Also, assay temperatures might vary according to the specific characteristics of the tested hydrogel (e.g., whether it is thermosensible or not). Finally, the tested hydrogel is placed apart from the water, the excess liquid phase is removed from the hydrogel surface, and its final weight is determined (Fan et al., 2015). For swelling index determination this equation is often used: Sw 5

Ws 2 Wd Wd

wherein Sw represents the swelling index; Ws represents the weight of swollen hydrogel; and Wd represents the weight of dry hydrogel (Zhao et al., 2009). If desired the swelling index can be expressed as a percentage (Huber et al., 2017), as: %Sw 5 Sw 3 100

Argin et al. (2014) determined the swelling index of physically crosslinked xanthan-chitosan hydrogels in simulated gastrointestinal conditions. As a result, they observed that xanthan-chitosan hydrogels were sensible to simulated pH media. In this regard, the deionization of hydrogel functional groups affected the transport mechanism inside the tridimensional hydrogel network; thus, highly swellable hydrogels result in large amounts of free water in the hydrogel structure and, consequently, in increased solute release.

3.4.2.5 Contact angle Contact angle measurements allow for the quantification of the hydrophilicity of developed hydrogels. These measurements are usually done at room temperature with a contact angle goniometer, which releases a drop of distilled water onto the surface of the sample. Images of water drop on sample surface are recorded for a specific period of time for angle determination.

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3.4.2.6 Thermal analysis Thermal analysis is done to determine the thermal behavior of developed hydrogels. Hydrogel samples are tested in a thermogravimetric analysis system with temperatures ranging from room temperature to 600 C at a rate of 10 C/min, under an argon or nitrogen atmosphere (Klein et al., 2016; Konwar et al., 2015).

3.4.3 SPECIFIC PROPERTIES FOR BIOMEDICAL ENGINEERING APPLICATIONS 3.4.3.1 Degradability Hydrogels must be both biocompatible and biodegradable in order to be used for biomedical engineering applications (Hoffman, 2002; Shoichet, 2010). Investigating the in vitro degradability of these materials is hence indispensable. To do so, hydrogel samples are put in contact with a simulant medium and organic fluids. Lysozyme is one of the most applied enzymes for analyzing the degradation of chitosan derivatives because it mimics physiological conditions efficiently. Calculations consider sample weight loss throughout immersion in the simulant medium under stirring (80 rpm) at 37 C for approximately 28 days. Hydrogel samples are then removed from the simulant medium at pre-established intervals for weighing. The degradation pattern is plotted with the weight loss data, which is measured with this equation: Weight lossð%Þ 5

Wi 2 Wt 3 100 Wi

wherein Wi and Wt are hydrogel weight at the beginning of the measurements and after each time interval, respectively (Freier et al., 2005; Jin et al., 2009).

3.4.3.2 Cytotoxicity According to the International Standard Organization (ISO), an in vitro cytotoxicity assay is essential for any materials that are intended for direct contact with the human body. In this context, ISO 10993-5 (ISO 10993-5, 2009) described some standard methods for evaluating the toxicity of such materials. Most cytotoxicity tests involve the contact of a sample with a cell culture as well as the further investigation—through the addition of vital dyes as well as the inhibition of cell colonies, for instance—of possible cell disruptions (Nakayama et al., 1997).

3.5 POTENTIAL APPLICATIONS AND FUTURE TRENDS OF CHITOSAN HYDROGELS The development of chitosan hydrogel has wide potential applications in several fields of science and novel products, such as in drug delivery, tissue engineering (scaffolds), and wound-healing applications, among others (Alves and Mano, 2008;

References

Croisier and Je´roˆme, 2013; Dash et al., 2011). Chitosan has been shown as a promising compound for the partial or total substitution of synthetic materials for the elaboration of hydrogels. In this regard, the interest in this compound as a key material for hydrogel development has been notorious, with innumerous studies presenting its potential application in several fields, such as personal care products, agriculture, and pharmaceutics. The main properties of chitosan are its biodegradability, low toxicity, and rheological aspects, which are compatible with the current requirements of biomedical engineering. Thus, these features of chitosan have favored the development of an increasing number of studies toward a better understanding of the relationship between chitosan structure, its properties, and potential applications in new products. Moreover, studies regarding the interaction of chitosan with other biopolymers have been focused on the improvement of performance properties, such as mechanical resistance and swelling ability, aiming at efficient absorbing materials (Ng et al., 2016; Rodrı´guez-Va´zquez et al., 2015). Currently, the challenges regarding the development of hydrogels goes beyond crosslinking techniques and new formulations; these new challenges include further in vitro and in vivo tests, especially regarding drug release, genetic therapy, and others (Rani et al., 2010). Nevertheless, chitosan constitutes an excellent polymeric matrix for the development of hydrogels; some limitations still remain in its structure but they can be easily overcome by implementing new technologies (physical or chemical interactions) that allow for improving its functionality.

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