Surface Modi cation of Film Chitosan Materials With Aldehydes for Wettability and Biodegradation Control

Chitosan is one of prospective polymer for use in regenerative medicine which has unique properties such as biocompatibility, biodegradability, antimicrobial, antiinflammatory, and antitumor potency. In this article, we study the peculiarities of the surface modification of chitosan films with carbonyl-containing compounds, which differed both in molecular characteristics and in their hydrophilic and hydrophobic properties. The potential for controlling the biodegradation of the resulting materials has been established, which can be used in the creation of wound dressings. Both the destruction time and lyophilic properties of the surface depend on the length of the modifier's hydrocarbon radical. The contact angle and water absorption of obtained film materials correlate with hydrophobicity, which estimated by the calculated value of the hydrophilic–lipophilic balance (HLB). The


INTRODUCTION
Since the early 2000s, there has been an increasing interest in biodegradable polymers and materials based on them in regenerative medicine [1]. Unlike biologically inert implants, these biomaterials are designed for controlled degradation and promote the growth of new tissues or support the growth of the body's own cells [2][3][4][5][6][7]. In addition to biodegradability, materials for tissue engineering should have biocompatibility, i.e., they should be incorporated into the body without causing adverse clinical implications [4,8], and biological potency, such as antimicrobial, anti-inflammatory, and antitumor potency [9,10].
One of these polymers is chitosan, with chitosan-based materials being able to degrade not only in the environment [11,12] but also enzymatically in the human body [13]. It is biocompatible, biologically potent [14] and biodegradable [15], which make this polymer promising for use in regenerative medicine [16] for healing burns and wounds without scarring [17,18].
Owing to the presence of functional hydroxyl, carboxyl, carbonyl, hemiacetal, and amine groups, polysaccharides are hydrophilic polymers that swell to a limited extent in water. Thus, materials of this kind lose their shape in a humid environment, including in the human body [1,19]. It is known that the hydrophilicity of chitosan accelerates its biodegradability in the presence of moisture due to the active reproduction of microorganisms in such an environment and the appearance of enzymes and ions in water that affect the degradation kinetics [20]. Moreover, imparting hydrophobicity with contact angles on the order of 120° and higher makes it impossible for such materials to absorb wound exudates and therefore lead to rejection by the body. Thus, it is necessary to regulate the hydrophilic-hydrophobic properties of such materials to control the destruction time.
Aldehydes are effective modifying agents for chitosan [21][22][23][24][25][26] that retain a wide range of its biological potency. Interaction with aldehydes leads to the formation of Schiff bases and to a change in the wettability of chitosan-based materials [27]. There are two approaches for the chemical modification of chitosan with low molecular weight [28] and polymeric [29] modifiers. The first one involves chitosan dissolution in acetic or lactic acid in the presence of ethanol at moderate temperatures for a long time [30]. The disadvantage of this method is the formation of partially cross-linked products due to intermacromolecular interactions, which impede film formation. The simplest and most promising method is the surface treatment of chitosan-based materials (films, fibers, sponges, etc.) with alcohol and aqueous solutions of aldehydes at room temperature [31][32][33].
Research in this area is mainly devoted to the study of the interaction of chitosan dissolved in acetic acid with individual carbonyl-containing modifying agents. The nature effect of the aldehyde on the polysaccharide properties is practically not covered, and the study of lyophilic properties of the resulting materials, which affect their destruction time, is beyond the scope of this article.
The difficulty of comparative analysis of these publications lies in the fact that in their works the authors used chitosan samples that differed in nature, molecular weight, molecular weight distribution, degree of deacetylation, degree of crystallinity, etc. The aforementioned factors affect the deformation and strength characteristics of the resulting films and fibers and the reactivity of chitosan.
This study used chitosan from the same batch; in addition, an extended set of aldehydes [34] were used, which differed both in molecular characteristics and in their hydrophilic and hydrophobic properties (soluble and slightly soluble in water or in organic solvents). This enabled us to conduct a comparative assessment of the structural organization and the physical, mechanical, and lyophilic properties of the chitosan films upon surface modification with aldehydes of different chemical composition.
This study aimed to investigate the peculiarities of modification of chitosan films with aldehydes for the creation of materials that have controlled wettability, biodegradability, and biocompatibility with human tissues.

Preparation of a chitosan films
The preparation of solutions and the formation of chitosan-based materials with subsequent reduction from the salt to the basic form were carried out according to methods described earlier [34,35]. The dried films had a thickness of 30-40 µm and were used for further studies of structural, physical, mechanical, hydrophilic, and hydrophobic properties and for determining their biocompatibility with human skin cells.

Modification of chitosan films with aldehydes
The film sample was placed separately in a bath with 40 mL of aldehyde solutions of various concentrations in water or alcohol. The film was cured for 25-30 min in 0.005-0.05% aqueous solutions of acrolein and 0.5-5% alcohol solutions of the remaining aldehydes, which corresponded to the optimal modification time. To remove unreacted aldehyde from the film surface, the samples were washed with water or alcohol and then subjected to Soxhlet extraction with methyl alcohol for 10 h. The washed samples were dried at room temperature until a constant weight.
The degree of grafting was determined gravimetrically as the ratio of the mass of the grafted aldehyde to the mass of the initial modified chitosan-based material, and the grafting efficiency was determined as the ratio of the mass of the grafted modifier to the mass of aldehyde in the modification solution.
The resulting Schiff bases were reduced by film surface treatment with excess (20 mL of a 2% solution) sodium borohydride in water for 24 h at room temperature [36]. The reduced samples were washed in containers with water until neutral reaction of the medium and dried at 40°C until reaching a constant weight.

Methods
Structural studies were carried out by IR spectroscopy using a SPECORD M-82 (Germany) and with an attenuated total reflection (ATR) accessory using an InfraLUM FT-08 (Russia) apparatus. Before spectral measurements, the samples were cured at room temperature in a desiccator with a relative humidity of 66% (over sodium nitrate). The IR absorption spectra were interpreted using literature data according to assignments of frequencies of functional groups in the spectra of analogous compounds.
The strength characteristics of the resulting films were determined at a tensile speed of 10 mm/min using a Zwicki Line "5kN zwicki" (Germany) tensile testing machine according to GOST 14236-81. Samples with a size of 2 × 20 mm were used for testing. Before testing, the samples were conditioned for at least 16 h at a temperature of 23±2°C and a relative humidity of 66% (over sodium nitrate) according to GOST 12423-66.
The hydrophilic and hydrophobic properties of the initial and modified chitosan films were studied by determining the amount of moisture sorbed by the film samples from the change in the sample mass in desiccators with a moisture content of 0% (over phosphorus pentoxide) and 98% (over a saturated solution of potassium sulfate). In addition, the contact angle of the surface was determined using a DataPhysics OCA 15EC apparatus (Germany) [37]. The measurements were carried out by applying drops of distilled water with a volume of 5-7 μL on the surface of the test material at room temperature with the contact angle of a sessile drop calculated according to the Young-Laplace method.
The HLB (hydrophilic-lipophilic balance) value was calculated using the Davis method (formula (1)) [38]: where m is the number of hydrophilic groups in the molecule; HLB i is the number for the i-th hydrophilic group; and n is the number of lipophilic groups in the molecule.
Biodegradability was studied using chitosan films in the form of 20 × (3-4)mm samples that were used as a substrate. The test samples were subjected to in vitro soil degradation. The soil was "activated" at a temperature of 20±5°C for 30 days. During this period, the soil was stirred on a daily basis, and the moisture content (should be 30±5%) was determined once a week. For this purpose, the soil was moistened with distilled water every 48 h. The pH of the aqueous extract in the soil was determined before testing. The soil was considered fit for testing at The cytotoxicity of the chitosan-based matrices was determined according to a method described earlier [34,40]. A toxicity measure for the materials is the cytotoxic index (CI), which was calculated using Formula (2). Cultured plastic was used as a comparison object: where a is the reference optical density (supernatant was cultured plastic without the addition of the preparation), and b is the test optical density (supernatant with the addition of the preparation).
range of 0 to 50% of the reference.

DISCUSSION
Aqueous solutions of acetic or formic acids, which form the corresponding salts with chitosan, were used as a solvent to obtain chitosan films, sponges, and fibers. Dry chitosan films are homogeneous, transparent, colorless, and non-brittle materials in which the ultimate tensile stress and elongation are 60-70 MPa and 7-10%, respectively [41,42].   (Table 1). Because the use of hydrophobic agents on smooth surfaces enables contact angles of no more than 120 degrees [43], it is necessary to study the effect of the structure of aldehydes on the morphology and texture of the surface of chitosanbased films. It was found that surface modification with formic (FA) and acetic (AA) aldehydes slightly affects the hydrophobicity of the films, while reducing the strength characteristics, which would be expected in accordance with Table 1.
The structures of the resulting compounds were confirmed by IR spectroscopy  In addition, the spectra of the chitosan films were obtained using the attenuated total reflection (ATR) method ( Figure 3). In the wavelength range of 1500 to 1700 cm −1 , there was also an increase in the absorption intensity at 1652 cm −1 (for modified films), the intensity of which changed upon interaction with aldehydes. However, there was a decrease in the peak intensity at 1581 cm −1 (amino group), which indicated a decrease in the number of amino groups, indicating the formation of imino groups. We previously showed that the optimal concentration of aldehyde in the modification solution was 0.0125 wt.% for Ac [34]. Table 2 shows that the greatest degree of grafting was achieved by using propionaldehyde as a modifier, with the modification efficiency decreasing with an increase in the concentration of aldehydes, which was due to the filling of available amino groups on the material surface of the film.   anisotropy of properties in different directions of the film materials, which was due to the reorientation of macromolecules during molding and drying as well as due to the fact that treatment modified only the surface and near-surface layers of the films without penetration of the modifier into the bulk of the samples.
The effects of surface modification of chitosan on the strength and lyophilic characteristics of the formed materials are described in Table 3. The strengths of the modified chitosan films increase in the following order of modifiers: SA < PA < IVA < Ac. Relative elongation increases in the following order of modifiers: Ac < SA < IVA < PA.
Based on the structure of the alkyl radical in the aldehyde, it was expected that modification with isovaleraldehyde would impart the highest hydrophobicity to the chitosan films. However, it turned out that the Schiff bases based on chitosan and acrolein are superior to the used modifiers in these terms (the contact angle was ≈110 degrees). This can be explained by the presence of -N=C-C=C-    Figure 7).
Previously [34], we presented results for the initial chitosan films and chitosan films modified with acrolein. Of note, the films modified with PA, SA, IVA demonstrated faster cell proliferation than films that were modified with Ac.
The toxicity measure for materials is the cytotoxic index (CI). The cytotoxic effect on human skin cells was estimated by counting viable cells in the chamber before and after their incubation with the preparation.
Calculation of the cytotoxic index requires determination of the optical density of the test object, which was cultured plastic. In this study, this was 0.298 after 5 days of cultivation. Afterwards, the indices shown in Figure 7 were determined using formula (2). Optical density showed the ability to grow new cells, and their toxicity was within the safe range (less than 50% of reference). However, the greatest effect was observed after 5 days of cell incubation. Among the films tested, those modified with SA and IVA demonstrated the best results, with their cytotoxic indices being below the index of the initial chitosan.
Schiff bases formed on the surface of chitosan materials containing imino groups are unstable in an alkaline medium at moderate temperatures; therefore, their further use requires additional reduction by treatment with sodium borohydride, which follows the scheme shown in Figure 8   The method for calculating the HLB value for N-alkylchitosans and iminochitosans yielded similar values with the exception of films modified with acrolein, which was also confirmed by measuring the lyophilic properties of the reduced Schiff bases presented in Table 2. The decrease in the hydrophobicity of the samples treated with acrolein and borohydride indicated a partial reduction of vinyl groups on the surface.

CONCLUSION
The peculiarities of the surface modification of chitosan films with aldehydes have been studied for the first time, and the dependence of the hydrophilic and hydrophobic properties of the surface of chitosan films on the structure of the hydrocarbon radical of the modifier has been determined. The hydrophobic properties increase upon modification of chitosan films with the corresponding aldehydes with the formation of Schiff bases on their surface in the following order: formic aldehyde < acetaldehyde < propionic aldehyde < salicylic aldehyde < isovaleraldehyde < acrolein.
The potential for use of the surface modification of chitosan-based film materials with low molecular weight aldehydes for the control of wettability, biodegradation time, and physical and mechanical properties has been revealed.
Modification of chitosan films with acrolein yielded the largest contact angles up to 110° and the lowest moisture absorption of approximately 10%, while the initial chitosan was characterized by a contact angle of 65±5° and moisture absorption of 40%.
The complete soil destruction of the initial chitosan film materials required 75 to 80 days, while the films modified with aldehydes were destroyed by 60-70% (wt.) during the same time period, depending on the chemical structure of the modifier.
The cytotoxicity studies of modified chitosan films showed that the resulting materials are non-toxic (cytotoxic index <50 %) for human skin cell cultures, which increases their potential use for external wound healing. IR spectra of the initial chitosan lm (1) and chitosan lm treated with 0.0125% Ac solution (2), 2.5% PA solution (3), 2.5% SA solution (4), and 2.5% IVA solution (5) Comparison of the contact angles of modi ed chitosan lms with HLB data of hydrocarbon substituents of aldehydes Figure 6 Biodegradability of the initial chitosan lms (1) and chitosan lms modi ed with salicylic aldehyde (2) and acrolein (3) Figure 7 Changes in proliferation of human dermal broblasts on unmodi ed and modi ed chitosan lms Figure 8