aSchool of Textile and Garment, Anhui Polytechnic University, Wuhu 241000, China
bState Key Laboratory of New Textile Materials and Advanced Processing Technologies & Key Laboratory of Textile Fiber and Products, Ministry of Education & Hubei International Scientific and Technological Cooperation Base of Intelligent Textile Materials and Application, School of Textile Science and Engineering, Wuhan Textile University, Wuhan 430200, China
cDepartment of Poultry Science, Auburn University, Auburn, AL 36849, USA
The purpose of this research was to develop a chitosan sulfobetaine (CS-SNCC) film via the solution-casting method as a biodegradable antibacterial material for biomedical applications. Chitosan and monochloro-triazine sulfobetaine were used as the raw materials for CS-SNCC preparation, and Fourier-transform infrared (FTIR), ultraviolet-visible (UV-Vis), energy-dispersive X-ray (EDX), and X-ray photoelectron spectroscopy (XPS) spectra were used to characterize and analyze the structure of the synthesized CS-SNCC. Furthermore, the swelling property, thermal stability, biodegradability, cytocompatibility, and antibacterial properties of the CS-SNCC film were comprehensively investigated and compared with those of the chitosan film. The results for the film’s enzymatic biodegradation behavior show that the CS-SNCC film undergoes a weight loss of 45.54% after 21 days of incubation. In addition, the CS-SNCC film effectively resists bacterial adhesion, prevents the formation of bacteria biofilms, and exhibits high antibacterial activity, with inactivation rates of 93.43% for Escherichia coli and 91.00% for Staphylococcus aureus. Moreover, the CS-SNCC film shows good cellular activity and cytocompatibility according to the cytotoxicity results. Therefore, the prepared biodegradable, cytocompatible, antibacterial, and biofilm-controlling CS-SNCC film has potential for biomedical applications.
Maoli Yin, Yingfeng Wang, Xuehong Ren, Tung-Shi Huang.
Development of a Biodegradable, Cytocompatible, Antibacterial, and Biofilm-Controlling Chitosan Sulfobetaine Derivative Film as a Biological Material.
Engineering, 2024, 35(4): 99-107 DOI:10.1016/j.eng.2023.06.020
Every year, patients suffer from a wide range of skin wounds due to mechanical trauma, surgical procedures, burns, or ulcers, or simply from daily activities. Such wounds are prone to causing bacterial infection, which results in exudate formation and inflammation, hindering wound repair [1], [2]. Hence, researchers have developed numerous biomedical materials to protect wounds, inhibit bacterial invasion, and promote healing, such as nanofibers, sponges, hydrogels, films, and coatings [3], [4], [5], [6], [7], [8]. Polymer film materials have been attracting a great deal of attention due to their simple manufacturing process, uniform and transparent structure that permits the observation of wound changes, and close adherence to the wound [6], [9]. Furthermore, biomaterial films based on natural biopolymers have gained importance because of their biocompatible and biodegradable properties and the hydrophilic nature of these molecules. Because such biomaterial films are easily removed from the wound, their use can avoid secondary damage; they also moisten the wound environment and facilitate wound recovery [9], [10], [11].
Chitosan, a biodegradable polycationic polysaccharide, is composed of glucosamine and randomly distributed N-acetylglucosamine monomers, connected by β-1,4-glycosidic bonds [3]. In the past few years, chitosan has come into focus as a potential biomaterial, as it possesses inherent antibacterial properties, good film-forming abilities, biocompatibility, non-antigenicity, multiple biological activities, and analgesic and hemostatic activities [12], [13]. In particular, chitosan-based antibacterial bioactive film materials have been widely studied. However, chitosan’s moderate antibacterial activity may limit its applications. Therefore, many researchers have aimed to improve the antibacterial properties of chitosan through modification, grafting, and compositing [14], resulting in derivatives such as chitosan quaternary ammonium salt [15], alkyl and aromatic chitosan [16], amino acid and antimicrobial peptide-grafted chitosan [17], and chitosan/inorganic nanomaterials [18], [19].
Zwitterionic compounds, a family of biomaterials that contain both cations and anions on the same macromolecular segment, have been reported to be efficient wound-dressing materials [20], [21]. Moreover, these materials possess good biodegradability, biocompatibility, hydrophilicity, and antibacterial activity; effectively prevent non-specific protein adsorption pollution; and have great potential for improving the adhesion resistance of biomaterials [22], [23], [24]. More specifically, sulfobetaine, which contains both an anionic sulfonate group and a cationic trimethyl ammonium group, is one of the most attractive zwitterionic compounds for biomaterials, owing to its easy synthesis and purification and its biomimetic ability [23]. The addition of sulfobetaine could significantly improve chitosan’s antibacterial properties [22]; however, research on biomaterial films based on chitosan sulfobetaine derivatives has rarely been reported.
In this work, a novel triazine zwitterionic sulfobetaine (SNCC; Fig. 1(a)) was synthesized and used to modify chitosan; the prepared chitosan sulfobetaine (CS-SNCC) film was then tested to evaluate its use for biomaterial applications (Fig. 1(b)). The synthesized CS-SNCC derivative was systematically studied and analyzed through Fourier-transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, energy-dispersive X-ray (EDX) spectroscopy, and X-ray photoelectron spectroscopy (XPS). The crystalline structure of the CS-SNCC was studied and a thermal analysis was performed. The swelling, biodegradability, cytocompatibility, antibacterial, and biofilm-controlling properties of the CS-SNCC film were also investigated, and the film’s applicability for the field of wound dressing was assessed.
2. Materials and methods
2.1. Materials
Chitosan (viscosity average molecular weight Mv = 50 kDa, 90% deacetylation) was provided by Zhejiang Aoxing Biochemical Co., Ltd. (China). N,N-Dimethylethanolamine, 1,3-propanesultone, and 2,4,6-trichloro-1,3,5-triazine were provided by J&K Scientific Ltd. (China). Sodium carbonate, glacial acetic acid, and acetone were provided by Shanghai Sinopharm Chemical Reagent Co., Ltd. (China). Chemical reagents were used according to the quality purchased without additional purification. Lysozyme (from chicken egg white) was obtained from Aladdin Reagent (Shanghai) Co., Ltd. (China). The Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) O157:H7 used were American Type Culture Collection (ATCC) 6538 and ATCC 43895, respectively (American Type Culture Collection, USA). Green fluorescent protein (GFP)-expressed NIH-3T3 fibroblast cells were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China.)
2.2. Synthesis of SNCC
The SNCC was synthesized via two processes: ① a substitution reaction of s-triazine with N,N-dimethylethanolamine; and ② an epoxy ring-opening between 1,3-propanesultone and monochloro triazine di-tertiary amine. Fig. 1(a) provides a schematic of the SNCC synthesis. In a typical procedure, 3.6 g of N,N-dimethylethanolamine was dissolved in 50 mL of acetone, and then dropped into 100 mL of acetone solution containing 3.64 g of 2,4,6-trichloro-1,3,5-trizine. The solution was reacted for 2 h in an ice bath, and the pH was adjusted to 7-8 by adding 20 wt% Na2CO3. Next, the solution was heated to 40 °C and the reaction was allowed to continue for 4 h. Then, 1,3-propanesultone (4.48 g) was dropped into the reaction system, which was heated to 60 °C for 5 h. A large amount of white precipitate was obtained, which was then filtered, washed, and purified thoroughly with diethyl ether. The product (SNCC) was dried at 50 °C for 24 h in a vacuum drying oven. Finally, the resulting SNCC was obtained with a yield of 92.16%.
2.3. Synthesis of CS-SNCC
CS-SNCC was synthesized by grafting SNCC onto chitosan amino groups (Fig. 1(a)). Chitosan (1.61 g, 0.01 mol) was completely dissolved in a 2 vol% aqueous solution of glacial acetic acid. Next, 4.12 g of SNCC was slowly added under mechanical stirring at 90 °C for 24 h. After the reaction, most of the solvent was removed using a rotary evaporator, and the solution was precipitated in a large volume of acetone. The sediment was collected through filtration and rinsed thoroughly with ethanol and acetone. The product (CS-SNCC) was then dried at 45 °C for 24 h.
2.4. The degree of substitution of chitosan by SNCC
The contents of carbon (C), oxygen (O), nitrogen (N), and sulfur (S) in the chitosan and CS-SNCC were determined using EDX spectroscopy (EDAX Octane EDS-30, AMETEK Inc., USA). The mass percentages of C, O, N, and S in the chitosan and CS-SNCC samples were determined by means of elemental analysis. According to the mass ratio of N, S, and C, the degrees of deacetylation of chitosan (WN/C) and the substitution of sulfobetaine (WS/C) on CS-SNCC were calculated with the following equations (Eqs. (1), (2), respectively) [15]:
where and respectively represents the proportion of constitutional units of chitosan and chitin in the chitosan and CS-SNCC; represents the proportion of constitutional units of chitosan sulfobetaine in the CS-SNCC; , , , , , and are the number of moles of nitrogen, sulfur, and carbon per mole of corresponding constitutional unit; , , and are the relative molar mass of N, S, and C, respectively; and and are the elemental mass ratios of N, S, and C of the measured products.
2.5. Preparation of the CS-SNCC film
The synthesized CS-SNCC was completely dissolved in 1 vol% aqueous solution of glacial acetic acid at ambient temperature for 4 h. Then, 100 mL of the solution was coated onto a Teflon mold, and the sample was allowed to naturally dry overnight to form a film. The prepared film was soaked in 20 g∙L−1 NaOH solution at ambient temperature for 1 h, and then immersed in deionized water. The obtained film was further dried and kept at a constant temperature and humidity for further study.
2.6. Instruments and characterizations
FTIR spectroscopy of the SNCC, chitosan, and CS-SNCC was carried out from 4000 to 500 cm−1 by means of a Nicolet Nexus 470 spectrophotometer (Thermo Fisher Scientific, USA). The UV-Vis spectra of the SNCC, chitosan, and CS-SNCC were obtained using a UNICO UV-2802S (Shimadzu Corporation, Kyoto, Japan) at test wavelengths from 200 to 800 nm. An Avance III 400 MHz digital NMR spectrometer (Bruker AXS GmbH, Germany) was used to detect the 1H NMR and 13C NMR spectra of the SNCC. The chitosan and modified chitosan underwent XPS on an Escalab 250 Xi (Thermo Fisher Scientific). X-ray diffraction (XRD) spectroscopy was used to characterize the crystalline texture of the chitosan and modified chitosan for the range of 5°-60° by means of a D8 Advance X-ray diffractometer (Bruker AXS GmbH). Thermogravimetric analysis (TGA)-derivative thermogravimetry (DTG) was performed under a nitrogen atmosphere by heating about 5 mg of film from 50 to 600 °C at a speed of 10 °C∙min−1 on a Q500 TGA instrument (TA Instruments, USA).
2.7. Swelling and enzymatic degradation behavior
The swelling kinetics of the chitosan film (referred to herein as the “CS film”) and the CS-SNCC film were observed by soaking dehydrated films in phosphate-buffered saline (PBS) solution over a period of 24 h at room temperature. After each predetermined time period, the film was taken out from the solution, and the water on the surface of the film was sucked out with filter paper and then weighed. The degree of swelling (SD) of each film was calculated using the following formula:
where Wd is the weight of film before immersion and Ws is the weight of the swollen film at the predetermined time period.
The enzymatic biodegradation of the CS film and CS-SNCC film was assessed using the proportion of the residual weight at 37 °C in lysozyme/PBS solution (1 mg∙mL−1, 15 mL) over a period of 20 days. CS and CS-SNCC films in PBS solution (15 mL) were used as the control group. The weights of the CS and CS-SNCC films were measured before testing (W0). After each impregnation period, three pieces of CS and CS-SNCC films were extracted from the impregnation solution and dried until the weight (Wt) remained constant. To evaluate the degree of degradation, the film’s relative weight (FW) was calculated using the following formula:
2.8. Antibacterial effectiveness
S. aureus and E. coli were used to evaluate the biocidal effectiveness of the CS and CS-SNCC films by means of a modified version of American Association of Textile Chemists and Colorists (AATCC) 100-2004 test method [3]. In short, 25 μL of bacterial suspension was placed in the middle of a film (2.54 cm × 2.54 cm); then, an identical piece of film was placed on top of the inoculated film. A sterile weight was added on top of the films to ensure that the bacteria were in full contact with the films. After each predetermined period of time, the samples were transferred into a sterile centrifuge tube with 5.0 mL of PBS solution and were vortexed for about 2 min. After that, the bacterial suspension was serially diluted ten-fold with PBS and inoculated on trypticase soy agar (TSA) plates. The plates were then incubated at 37 °C for 24 h, and the number of bacterial colonies was counted to calculate the biocidal effectiveness.
2.9. Anti-adhesion and biofilm-controlling behavior
The ability of the CS-SNCC film to resist bacteria and prevent biofilm formation was evaluated via the colony-counting method and through scanning electron microscope (SEM) assessment [25]. The model bacteria E. coli and S. aureus were cultured, harvested, and resuspended in PBS solution to obtain a bacterial suspension of about 107 colony-forming units (CFU)∙mL−1. The CS and CS-SNCC films (∼1 cm × 1 cm) were soaked in 10 mL of bacterial suspension and incubated at 37 °C in a shaking chamber at 60 r∙min−1 for 30 min. Next, the films were removed from the bacterial suspensions and gently washed three times with 10 mL of PBS solution in order to remove any loosely attached bacteria. After that, the films were soaked in 10 mL of fresh nutrient broth and cultivated at 37 °C for 24 h. The extracted films were gently washed with non-flowing sterile PBS solution for the next steps. For the colony-counting method, some of the films described above were immersed in 10 mL of PBS solution and eddied for 2 min to disperse the bacteria attached on the films into the PBS solution. The bacterial suspension was then serially diluted ten-fold with PBS and plated on TSA plates for the bacteria enumeration. For the SEM assessment, some of the prepared films were immobilized with 2.5 wt% glutaraldehyde/PBS solution at 4 °C for 24 h. After fixation, the films were subjected to gradient dehydration by means of a battery of alcohol/PBS solution (25%, 50%, 75%, 85%, 95%, and 100%) for 30 min. Then the fixed films were vacuum dried for 24 h before SEM observation.
2.10. Cytocompatibility assay
An MTT assay was used to investigate the cytocompatibility and cell proliferation of the films, and NIH-3T3 fibroblast cells were directly exposed to the test films [26]. The films were cut into 8 mm disks and sterilized with ultraviolet (UV) radiation for 30 min. After that, the films were immersed in culture media for 24 h in a sterile environment. An NIH-3T3 fibroblast cell suspension was added into 48-well plates and incubated at 37 °C for 24 h with 5% CO2. Next, the film disks were placed into the wells to provide contact with cells and were incubated for 24 and 48 h, respectively. The film disks were then removed, and MTT assays were performed to determine cell viability. The results were determined by the percentage of the absorbance value of each component relative to the control. NIH-3T3 fibroblast cells seeded on wells without films were taken as a comparison group. The cell morphology and proliferation were observed using an inverted fluorescence microscope (Motic AE31, Motic Microscopes, China). The cell viability (CV) of was calculated by
where As and Ac are the absorbance of sample and control group, respectively.
3. Results and discussion
3.1. Structural determination via 1H NMR, 13C NMR, UV-Vis, and FTIR
CS-SNCC was prepared by grafting monochloro-triazine disulfonic acid betaine onto chitosan. 1H NMR and 13C NMR were used to confirm the synthesis of SNCC. Figs. 2(a) and (b) show the 1H NMR and 13C NMR spectra of the SNCC in D2O. The chemical shift at 3.14 parts per million (ppm) (12H) is ascribed to the hydrogen on the quaternary ammonium [3]. The peaks at 4.01 ppm (4H), 3.48-3.53 ppm (8H), 2.94 ppm (4H), and 2.21 ppm (2H) are attributed to the hydrogens of the zwitterionic species at the H1, H2 and H4, H5, and H6 positions, respectively [22], [27]. Moreover, the chemical shifts of the carbon atoms in the monochloro-triazine disulfonic acid betaine were at 159.21 ppm (C1), 151.68 ppm (C2), 63.60 ppm (C3), 63.42 ppm (C4), 51.40 ppm (C5), 55.33 ppm (C6), 17.95 and 47.15 ppm (C6), respectively [27], as shown in Fig. 2(b). The analysis of the NMR (1H and 13C) results indicates that the monochloro-triazine disulfonic acid betaine has been successfully synthesized.
The UV-Vis spectra of chitosan, SNCC, and CS-SNCC are shown in Fig. 2(c). There is a distinct characteristic absorption peak at 228 nm in the spectra of SNCC and CS-SNCC, which is attributed to the triazine ring structure [28]. However, the spectra of chitosan were not found. The peak at 228 nm for CS-SNCC indicates that the triazine compound has been grafted onto the chitosan. The FTIR spectra of chitosan, SNCC, and CS-SNCC are shown in Fig. 2(d). The peaks at 1172 and 1040 cm−1 in the SNCC FTIR spectra are respectively attributed to the asymmetric and symmetric stretching vibrations of the SO3− group [29]. The peak of the symmetric stretching vibration of the C-S bond is located at 600 cm−1 [22]. Moreover, there is an obvious characteristic peak at 1470 cm−1, which is attributed to the methyl groups of the trimethyl quaternary ammonium [15]. The FTIR spectrum of CS-SNCC shows several new peaks at 1470, 1172, and 600 cm−1, which correspond to the functional group peaks of the SO3− group and the quaternary ammonium group, respectively. Furthermore, the triazine ring’s peaks at 764 to 793 cm−1 are shown in the FTIR spectra of CS-SNCC [30]. These new absorption peaks in the FTIR spectrum of CS-SNCC reveal that SNCC has been successfully grafted onto the chitosan.
3.2. EDX and XPS spectra of CS and CS-SNCC
To study the elemental content on the surfaces of the CS and CS-SNCC films and the degree of CS-SNCC substitution, the mass percentages of C, O, N, and S for the two samples were determined by means of EDX, as shown in Figs. 3(a) and (b). According to Eq. (1), the degree of deacetylation of chitosan was 92.2%, which is very close to the value of the production specification of chitosan. Furthermore, a new sulfur absorption peak in the EDX spectrum of CS-SNCC corresponds to the element sulfur in the zwitterionic sulfobetaine. The degree of substitution of SNCC in CS-SNCC is 15.55%, based on Eq. (2).
The binding energies of the surface chemical bonds of chitosan and CS-SNCC were analyzed via XPS to confirm the introduction of SNCC into the chitosan. The results, which are presented in Figs. 3(c)-(f), contain core-level patterns of the N 1s and S 2p. Two peaks can be seen on the N 1s spectrum of chitosan (Fig. 3(d)) at the binding energies of 398.98 and 399.98 eV, which are ascribed to the primary amine -NH2 (deacetylated units) and acetamido group -NHCO- (acetylated units), respectively [31], [32]. Theoretically, no S 2p peak exists in the XPS spectrum of chitosan (Fig. 3(c)). However, the XPS spectra showed significant differences after SNCC was introduced into the chitosan chains. Most importantly, two obvious peaks can be seen on the N 1s core-level pattern of CS-SNCC at the binding energies of 400.82 and 402.12 eV, which are attributed to the C-N=C bond on the homotriazine ring and the quaternary ammonium groups (-N+(CH3)-) of SNCC, respectively [21], [32]. Furthermore, characteristic signals located at 167.11 and 168.32 eV can be seen on the S 2p spectra, which are assigned to the S 2p3/2 and S 2p1/2 of the sulfonate group (SO3−). Based on the above characterizations, a new monochloro-triazine disulfonic acid betaine has been successfully synthesized and introduced into chitosan.
3.3. Crystalline structure and thermal analysis
XRD studies were used to characterize the crystal structure changes of CS-SNCC and chitosan, and the results are presented in Fig. 4(a). The chitosan shows obvious peaks at 19.80°, 15.25°, and 10.67°, which are attributed to the characteristic peaks of the chitosan macromolecular backbone aligned by intermolecular interactions [33]. The intensity of the peak at 2θ of 19.80° for CS-SNCC decreased after the introduction of SNCC, indicating that the chitosan’s crystal structure was destroyed; this may be ascribed to the breakage of intermolecular and intramolecular hydrogen bonds after the introduction of SNCC, and to the fact that CS-SNCC has a looser structure than chitosan [22].
The dehydration and decomposition of the prepared films were analyzed by means of TGA. The TGA-DTG curves of chitosan and CS-SNCC show two steps of thermogravimetric loss (Fig. 4(b)). The first step is attributed to the evaporation of absorbed water and intermolecularly bound water, while the thermal degradation of chitosan corresponds to the second step of heat loss [33], [34]. The first step of thermogravimetric loss is approximately 5% at 50-120 °C, while the second step is approximately 44% at 235-380 °C. The first step of the thermogravimetric loss of CS-SNCC is very similar to that of chitosan, albeit with a slightly lower starting temperature and a broader range of thermal decomposition. This phenomenon mainly arises from the fact that the introduction of SNCC destroys the interaction force between the chitosan macromolecules and the crystallization structure is damaged, forming more amorphous regions, which is consistent with the results from XRD [22], [33].
3.4. Swelling and enzymatic biodegradation behavior
Water absorption and swelling properties are essential features for biomedical materials. The PBS absorption efficiencies of the CS and CS-SNCC films are shown in Fig. 4(c). All the films exhibited a rapid water-absorption capability and reached swelling equilibrium within 2 h. Moreover, the films showed a high swelling ability (over 100% swelling capability), since chitosan has many hydrophilic groups, including hydroxyl and amino groups, which promote water absorption. The CS-SNCC film exhibited a quicker water absorption rate and a higher equilibrium swelling degree than the CS film, which is mainly attributed to the presence of the hydrophilic sulfonate group (SO3−) improving the hydrophilicity and wettability of the film, as well as accelerating the water penetration and swelling of the film [24].
The biodegradability of the CS-SNCC film is very important if it is to be used as a biomaterial. To evaluate the biodegradability, the weight loss of CS-SNCC was tested in lysozyme/PBS solution at 37 °C over 20 days. Lysozyme is a glycoside hydrolase that naturally exists in typical wound exudation fluid with inflammation; it can hydrolyze the β-1,4-glycosidic bonds in polysaccharides [35], [36]. As shown in Fig. 4(d), lysozyme accelerated the biodegradation of both the CS and CS-SNCC films. The CS-SNCC film degraded faster than the CS film in both the lysozyme/PBS solution and the PBS solution alone. After incubating for 21 days, the CS and CS-SNCC films respectively exhibited a 19.88% and 23.13% weight loss in PBS solution and a weight loss of 41.59% and 45.54% in 1 mg∙mL−1 lysozyme solution. According to research on the biodegradation of chitosan derivatives, lysozyme can scissor the chitosan polymer backbone by cleaving the β-1,4-glucosidic bonds [37]. The observed accelerated degradation of the CS-SNCC film is due to the introduction of zwitterionic sulfobetaine groups into the chitosan molecular chain, which destroys the crystal structure of CS-SNCC, giving the lysozyme additional opportunities to rapidly access the β-1,4-glucosidic bonds for specific cleaving degradation. The better swelling and biodegradability of the CS-SNCC film provide further evidence that the chitosan derivative CS-SNCC has potential for biomedical applications.
3.5. Anti-adhesion and biofilm-controlling function
Zwitterionic surfaces have been reported to possess effective anti-adhesion properties against bacteria and to prevent biofilm formation due to their anionic and cationic groups with equal amounts but opposite electric charges [29], [38]. To evaluate this effect in the CS-SNCC film, SEM images were obtained, as shown in Fig. 5(a). The images show the distribution of two types of bacteria on the surface of the CS and CS-SNCC films after 24 h of incubation. As shown in Fig. 5(a), numerous bacteria cells adhere to the surface of the CS film, forming microcolonies and biofilms; in contrast, only a few scattered bacteria are found and no biofilms have formed on the CS-SNCC film for both S. aureus and E. coli. These SEM images intuitively indicate that the chitosan zwitterionic sulfobetaine film has anti-adhesion properties against bacteria and a biofilm-controlling function. To further quantitatively analyze the film’s anti-adhesion effect against bacteria, the bacteria on the CS and CS-SNCC films were evaluated using the colony-counting method. Fig. 5(b) shows the quantitative data of the recoverable bacteria on the surface of the CS and CS-SNCC films. The CS-SNCC surface had a low bacterial population (S. aureus: 2.95 × 106 CFU∙cm−2; E. coli: 1.15 × 106 CFU∙cm−2) compared with the surface of the CS film (S. aureus: 2.25 × 107 CFU∙cm−2; E. coli: 1.98 × 107 CFU∙cm−2); thus, bacterial adhesion on the CS-SNCC film was reduced by approximately 86.89% for S. aureus and 94.19% for E. coli adhesion. All these results strongly suggest that the chitosan zwitterionic sulfobetaine film is effectively resistant to bacterial adhesion and prevents the formation of bacterial biofilms.
3.6. Antibacterial efficacies
The antibacterial efficacies of the CS and CS-SNCC films against S. aureus and E. coli (approximately 106 CFU per swatch) were studied, and the results are shown in Fig. 6. The CS film showed a certain degree of antibacterial activity, as 51.83% of the E. coli and 87.77% of the S. aureus were killed after 60 min of exposure, due to the inherent antibacterial properties of chitosan. Chitosan amino groups easily protonate to gain a positive charge, which can combine with the negatively charged molecule on the bacterial cell wall to destroy the physical-chemical properties of the cell wall, thus leading to bacterial inactivation [39], [40]. The CS-SNCC film inactivated 93.43% of the E. coli and 91.00% of the S. aureus after 60 min of contact, exhibiting a more effective killing ability for both E. coli and S. aureus in comparison with the CS film. This finding is similar to those of previous studies on the antibacterial properties of chitosan betaine sulfonate derivatives [22], [25]. Therefore, the CS-SNCC film possesses antibacterial, anti-adhesion, and biofilm-controlling properties, as shown in Fig. 1(b).
3.7. Cytocompatibility
Cytocompatibility is a major concern for the application of antibacterial films as biomedical materials. The cytotoxicity of the CS and CS-SNCC films was evaluated by incubating NIH-3T3 fibroblast cells with the films via classic MTT assays; the results are shown in Fig. 7. After cultivation for 24 and 48 h, the metabolic activities of the cells incubated with the CS films were not obviously affected, and the cell viabilities were about 98.21% and 96.83%, respectively. After the SNCC was grafted onto the chitosan, the cell survival rates on the CS-SNCC film were over 90% after cultivation for 24 and 48 h. The lack of cytotoxicity of CS-SNCC indicates that it has potential for application as a biomedical material. These results are consistent with previous studies reporting that sulfobetaine shows no toxicity and no skin irritation [22], [27].
Fluorescent images of NIH-3T3 fibroblast cells incubated with the films were used to analyze the cell morphology, proliferation, and metabolic activity [23]. As shown in Fig. 7, after cultivation for 24 and 48 h, most of the NIH-3T3 fibroblasts were green and showed spindle shapes; thus, they were not significantly different from the control group. The films cultured for 48 h presented significantly higher cell numbers than those cultured for 24 h, indicating the films’ good cellular cytocompatibility.
4. Conclusions
In this study, a novel biodegradable, cytocompatible, antibacterial, and biofilm-controlling chitosan derivative film was produced for application as a biomedical material. A new chitosan derivative denoted as CS-SNCC was synthesized by grafting SNCC onto chitosan, and the chemical structure of CS-SNCC was verified by means of FTIR, UV-Vis, EDX, and XPS spectra. The crystalline structures and thermostability results indicated that the introduction of SNCC in chitosan reduced the crystallinity and thermal stability of chitosan to a certain extent. The swelling and biodegradation behavior showed that the CS-SNCC film possesses excellent water absorption and degradability, with the degradation of the CS-SNCC film being accelerated by lysozyme. Furthermore, the cytocompatibility study revealed that the CS-SNCC film has no cytotoxicity. In addition, testing the film’s resistance to bacteria adhesion indicated that the CS-SNCC film can reduce around 86.89% of S. aureus and 94.19% of E. coli adhesion on its surface. The CS-SNCC film also exhibits a good bactericidal performance and can kill 91.00% and 93.43% of S. aureus and E. coli, respectively, within 60 min of contact. As a result, the synthesized CS-SNCC film, with its good cytocompatibility, enzymatic biodegradability, antibacterial, and biofilm-controlling properties, has excellent potential for application as a biomedical material.
Acknowledgments
This work was supported by research funding from the Starting Research Fund from the Anhui Polytechnic University (2021YQQ040), the Natural Science Foundation of Anhui Province, China (2008085QE255), and the Hubei Provincial Central Leading Local Science and Technology Development Special Fund (2022BGE253).
Compliance with ethics guidelines
Maoli Yin, Yingfeng Wang, Xuehong Ren, and Tung-Shi Huang declare that they have no conflict of interest or financial conflicts to disclose.
X.Zhao, Y.P.Liang, Y.Huang, J.H.He, Y.Han, B.L.Guo. Physical double-network hydrogel adhesives with rapid shape adaptability, fast self-healing, antioxidant and NIR/pH stimulus-responsiveness for multidrug-resistant bacterial infection and removable wound dressing. Adv Funct Mater, 30 (17) (2020), p. 1910748.
[2]
M.L.Yin, S.S.Wan, X.H.Ren, C.C.Chu. Development of inherently antibacterial, biodegradable, and biologically active chitosan/pseudo-protein hybrid hydrogels as biofunctional wound dressings. ACS Appl Mater Interfaces, 13 (12) (2021), pp. 14688-14699.
[3]
M.L.Yin, Y.F.Wang, Y.Zhang, X.H.Ren, Y.Y.Qiu, T.S.Huang. Novel quaternarized N-halamine chitosan and polyvinyl alcohol nanofibrous membranes as hemostatic materials with excellent antibacterial properties. Carbohydr Polym, 232 (2020), p. 115823.
[4]
M.Li, J.Aveyard, K.G.Doherty, R.C.Deller, R.L.Williams, K.N.Kolegraff, et al. Antimicrobial nitric oxide-releasing electrospun dressings for wound healing applications. ACS Mater Au, 2 (2) (2022), pp. 190-203.
[5]
Y.P.Liang, J.H.He, B.L.Guo. Functional hydrogels as wound dressing to enhance wound healing. ACS Nano, 15 (8) (2021), pp. 12687-12722.
[6]
L.Li, X.Cao, X.Shen, S.T.Yu, F.Su, S.W.Liu, et al. Alkyl chitosan film—high strength, functional biomaterials. J Biomed Mater Res Part A, 105 (11) (2017), pp. 3034-3041.
[7]
S.M.Zhang, L.Li, X.H.Ren, T.S.Huang. N-Halamine modified multiporous bacterial cellulose with enhanced antibacterial and hemostatic properties. Int J Biol Macromol, 161 (2020), pp. 1070-1078.
[8]
B.Demir, R.M.Broughton, M.Y.Qiao, T.H.Huang, S.D.Worley. N-Halamine biocidal materials with superior antimicrobial efficacies for wound dressings. Molecules, 22 (10) (2017), p. 1582.
[9]
M.G.A.Vieira, M.A. daSilva, L.O. dosSantos, M.M.Beppu. Natural-based plasticizers and biopolymer films: a review. Eur Polym J, 47 (3) (2011), pp. 254-263.
[10]
S.Patel, S.Srivastava, M.R.Singh, D.Singh. Preparation and optimization of chitosan-gelatin films for sustained delivery of lupeol for wound healing. Int J Biol Macromol, 107 (2018), pp. 1888-1897.
[11]
C.Liu, H.R.Shan, X.X.Chen, Y.Si, X.Yin, J.Y.Yu, et al. Novel inorganic-based N-halamine nanofibrous membranes as highly effective antibacterial agent for water disinfection. ACS Appl Mater Interfaces, 10 (51) (2018), pp. 44209-44215.
[12]
J.H.Li, S.L.Zhuang. Antibacterial activity of chitosan and its derivatives and their interaction mechanism with bacteria: current state and perspectives. Eur Polym J, 138 (2020), p. 109984.
[13]
P.S.Bakshi, D.Selvakumar, K.Kadirvelu, N.S.Kumar. Chitosan as an environment friendly biomaterial—a review on recent modifications and applications. Int J Biol Macromol, 150 (2020), pp. 1072-1083.
[14]
P.Sahariah, M.Masson. Antimicrobial chitosan and chitosan derivatives: a review of the structure-activity relationship. Biomacromolecules, 18 (11) (2017), pp. 3846-3868.
[15]
M.L.Yin, X.H.Lin, T.Ren, Z.G.Li, X.H.Ren, T.S.Huang. Cytocompatible quaternized carboxymethyl chitosan/poly(vinyl alcohol) blend film loaded copper for antibacterial application. Int J Biol Macromol, 120 (Pt A) (2018), pp. 992-998.
[16]
P.Sahariah, B.E.Benediktssdottir, M.A.Hjalmarsdottir, O.E.Sigurjonsson, K.K.Sorensen, M.B.Thygesen, et al. Impact of chain length on antibacterial activity and hemocompatibility of quaternary N-alkyl and N,N-dialkyl chitosan derivatives. Biomacromolecules, 16 (5) (2015), pp. 1449-1460.
[17]
V.Patrulea, B.H.Gan, K.Perron, X.Cai, P.Abdel-Sayed, E.Sublet, et al. Synergistic effects of antimicrobial peptide dendrimer-chitosan polymer conjugates against Pseudomonas aeruginosa. Carbohydr Polym, 280 (2022), p. 119025.
[18]
Y.Z.Ma, K.Gao, H.H.Yu, W.X.Liu, Y.K.Qin, R.G.Xing, et al. C-coordinated O-carboxymethyl chitosan Cu(II) complex exerts antifungal activity by disrupting the cell membrane integrity of Phytophthora capsici Leonian. Carbohydr Polym, 261 (2021), p. 117821.
[19]
D.D.Li, X.Gao, X.C.Huang, P.L.Liu, W.Xiong, S.T.Wu, et al. Preparation of organic-inorganic chitosan@silver/sepiolite composites with high synergistic antibacterial activity and stability. Carbohydr Polym, 249 (2020), p. 116858.
[20]
H.C.He, Z.C.Xiao, Y.J.Zhou, A.Q.Chen, X.Xuan, Y.Y.Li, et al. Zwitterionic poly(sulfobetaine methacrylate) hydrogels with optimal mechanical properties for improving wound healing in vivo. J Mater Chem B, 7 (10) (2019), pp. 1697-1707.
[21]
H.T.Lin, A.Venault, Y.Chang. Zwitterionized chitosan based soft membranes for diabetic wound healing. J Membr Sci, 591 (2019), p. 117319.
[22]
Y.X.Chen, J.N.Li, Q.Q.Li, Y.Y.Shen, Z.C.Ge, W.W.Zhang, et al. Enhanced water-solubility, antibacterial activity and biocompatibility upon introducing sulfobetaine and quaternary ammonium to chitosan. Carbohydr Polym, 143 (2016), pp. 246-253.
[23]
X.H.Chu, M.Zhang, N.L.Zhou, F.Wu, B.H.Sun, J.Shen. Synthesis and characterization of a novel antibacterial material containing poly(sulfobetaine) using reverse atom transfer radical polymerization. RSC Adv, 8 (58) (2018), pp. 33000-33009.
[24]
X.Yang, D.Sha, H.Jiang, K.Shi, J.D.Xu, C.Yu, et al. Preparation of antibacterial poly(sulfobetaine methacrylate) grafted on poly(vinyl alcohol)-formaldehyde sponges and their properties. J Appl Polym Sci, 136 (6) (2019), p. 47047.
[25]
R.Wang, K.G.Neoh, E.T.Kang. Integration of antifouling and bactericidal moieties for optimizing the efficacy of antibacterial coatings. J Colloid Interface Sci, 438 (2015), pp. 138-148.
[26]
X.Zhao, B.L.Guo, H.Wu, Y.P.Liang, P.X.Ma. Injectable antibacterial conductive nanocomposite cryogels with rapid shape recovery for noncompressible hemorrhage and wound healing. Nat Commun, 9 (1) (2018), p. 2784.
[27]
L.He, C.Gao, S.Li, C.T.W.Chung, J.H.Xin. Non-leaching and durable antibacterial textiles finished with reactive zwitterionic sulfobetaine. J Ind Eng Chem, 46 (2017), pp. 373-378.
[28]
C.Chen, S.G.Yang, Y.P.Guo, C.Sun, C.G.Gu, B.Xu. Photolytic destruction of endocrine disruptor atrazine in aqueous solution under UV irradiation: products and pathways. J Hazard Mater, 172 (2-3) (2009), pp. 675-684.
[29]
S.Chen, L.Yuan, Q.Li, J.Li, X.Zhu, Y.Jiang, et al. Durable antibacterial and nonfouling cotton textiles with enhanced comfort via zwitterionic sulfopropylbetaine coating. Small, 12 (26) (2016), pp. 3516-3521.
[30]
Y.J.Chen, W.Wang, Y.Qiu, L.S.Li, L.J.Qian, F.Xin. Terminal group effects of phosphazene-triazine bi-group flame retardant additives in flame retardant polylactic acid composites. Polym Degrad Stabil, 140 (2017), pp. 166-175.
[31]
D.W.Yuan, K.Cadien, Q.Liu, H.B.Zeng. Adsorption characteristics and mechanisms of O-carboxymethyl chitosan on chalcopyrite and molybdenite. J Colloid Interface Sci, 552 (2019), pp. 659-670.
[32]
R.L.Tang, Y.Zhang, Y.Zhang, Z.M.Yu. Synthesis and characterization of chitosan based dye containing quaternary ammonium group. Carbohydr Polym, 139 (2016), pp. 191-196.
[33]
M.L.Yin, X.L.Chen, R.Li, D.Huang, X.Y.Fan, X.H.Ren, et al. Preparation and characterization of antimicrobial PVA hybrid films with N-halamine modified chitosan nanospheres. J Appl Polym Sci, 133 (46) (2016), p. 44204.
M.Nieto-Suárez, M.A.López-Quintela, M.Lazzari. Preparation and characterization of crosslinked chitosan/gelatin scaffolds by ice segregation induced self-assembly. Carbohydr Polym, 141 (2016), pp. 175-183.
[36]
S.Ganguly, P.P.Maity, S.Mondal, P.Das, P.Bhawal, S.Dhara, et al. Polysaccharide and poly(methacrylic acid) based biodegradable elastomeric biocompatible semi-IPN hydrogel for controlled drug delivery. Mater Sci Eng C, 92 (2018), pp. 34-51.
[37]
M.Y.He, A.Potuck, Y.Zhang, C.C.Chu. Arginine-based polyester amide/polysaccharide hydrogels and their biological response. Acta Biomater, 10 (6) (2014), pp. 2482-2494.
[38]
Y.Ma, J.Y.Li, Y.Si, K.Huang, N.Nitin, G.Sun. Rechargeable antibacterial N-halamine films with antifouling function for food packaging applications. ACS Appl Mater Interfaces, 11 (19) (2019), pp. 17814-17822.
[39]
A.M.Heimbuck, T.R.Priddy-Arrington, M.L.Padgett, C.B.Llamas, H.H.Barnett, B.A.Bunnell, et al. Development of responsive chitosan-genipin hydrogels for the treatment of wounds. ACS Appl Bio Mater, 2 (7) (2019), pp. 2879-2888.
[40]
W.Y.Li, B.X.Wang, M.H.Zhang, Z.T.Wu, J.X.Wei, Y.Jiang, et al. All-natural injectable hydrogel with self-healing and antibacterial properties for wound dressing. Cellulose, 27 (5) (2020), pp. 2637-2650.
PDF
(2075KB)
