Chitosan-Based Antibacterial Polyurethane Materials

Chitosan-Based Antibacterial Polyurethane Materials

Introduction

Infectious diseases have been one of humanity's most significant challenges for centuries. Contagious diseases caused by pathogenic microorganisms are becoming an increasingly pressing issue in public health with each passing day. Contagious diseases typically spread through direct transmission of bacteria, viruses or other microbes from one person to another. Infection can spread through contact with a contaminated surface or when an infected person coughs or sneezes. Hospitals are efficient breeding grounds for resistant pathogens. In this context, resistant pathogens can cause hospital-acquired infections that are more difficult to treat compared to other pathogenic infections. By rendering common materials used in public transport, hospitals, schools and similar areas antibacterial in nature, it is possible to slow down and prevent the spread of microorganisms. Interest in antibacterial materials is growing with each passing day. Similarly, in the polyurethane sector, there has been growing interest in antibacterial materials, particularly in shoe insoles, integral systems such as armrests, steering wheels and gear shifters that we touch many times in daily life, and surface coating materials. Due to their unique properties such as high biocompatibility, non-toxicity, biodegradability, low allergenicity and low cytotoxicity, studies have been conducted on the use of chitosan-based materials. Within the scope of this article, chitosan-based cationic polymers were synthesized and then incorporated into polyurethane-based materials. (Scheme 1) The surface biocidal efficacy of the material was tested against E. coli and S. aureus bacteria. [caption id="attachment_158316" align="aligncenter"] Scheme 1. Chitosan-based antibacterial polyurethane coating[/caption]

2. Natural Polymers and Chitosan

Polymers are widely used due to their diverse functionality, light weight, low cost and chemical stability. Plastic production continues to increase year after year worldwide, and non-biodegradable, petroleum-based polymeric materials present a major environmental problem. In this context, there has been an increase in research on recycling technologies, bio-based and/or biodegradable raw materials. Polymeric materials used in our daily lives have a wide range of applications including food packaging, surface coating materials, insulation materials, and textile and healthcare industries, and their use is increasing with each passing day. However, these materials are not antibacterial and must be given biocidal properties to prevent bacterial infection. Bio-based polymers, such as natural polymers from natural monomers, polymers obtained from microbial fermentation, and plant oil-based polymers, have gained popularity in recent years as an alternative to crude oil sources. Biopolymers are polymers obtained from biological sources and soluble in nature. Among natural polymers, polysaccharides are the most abundant natural polymers in the world. Starch, cellulose and chitin are abundantly found in nature, biodegradable and renewable natural polymers. Chitin, the second most abundant biopolymer after cellulose, is found in various living organisms such as shrimp, crab, insects and turtles, as well as in the cell walls of fungi, in the internal structures of invertebrates and in the exoskeleton of arthropods. [1-3] Synthetically, it can also be prepared through a non-biosynthetic route of a chitobiose oxazoline derivative via chitinase-catalyzed polymerization.[4-6] Chitin is the most frequently encountered polysaccharide after cellulose on Earth. Chitin is a natural polymer composed of monomers called N-acetylglucosamine (NAG). (Figure 1) The polymer consists of interconnected chains of NAG monomers and crystallizes to varying degrees. [caption id="attachment_158317" align="aligncenter"] Figure 1. Chemical structure of chitin and deacetylated chitin (chitosan)[/caption] Chitosan, one of the most important derivatives of chitin, is a polycationic biopolymer obtained through partial or complete deacetylation of chitin in an alkaline environment (removal of the acetyl functional group from an organic compound).[7,8] Chitosan, which can be obtained in large quantities from many natural sources containing chitin such as fungi, crayfish, shrimp and crab exoskeletons, has properties such as lack of toxic characteristics for organisms, easy biodegradability and biocompatibility.[9,10] For this reason, chitosan is used in many industrial fields including food, medical, pharmaceutical, cosmetic, agriculture, wastewater treatment and textiles.[11] In addition to having antiviral, antibacterial and antifungal properties, chitosan is an effective agent in disease control and reducing their spread by promoting plants' defense systems. Furthermore, it has begun to be used in agricultural improvements due to its ability to chelate metal ions in its environment (water, soil, etc.) and its ability to prevent the absorption of toxic metals in plants.[12,13] Particularly, there is growing interest in antibacterial materials. However, chitosan applications as a biocide are only used in acidic environments due to its low solubility under neutral and basic conditions.[14] For this reason, chemical modifications are needed to increase the solubility of chitosan. Quaternization on amine groups (increasing cationic charge density and hydrophobic properties) significantly enhances biocidal efficacy. The main factors affecting chitosan's biocidal efficacy [15] are: (i) microbial species related to microorganisms; (ii) chitosan's positive charge density, molecular weight, concentration, hydrophilic/hydrophobic structural balance and chelation capacity; (iii) physical state, i.e., chitosan in water-soluble and solid form; (iv) environmental factors, namely ionic strength, pH, temperature and reaction time in the medium. There are numerous studies on chitosan-based antimicrobial coatings. For example, the surface of polylactic acid (PLA) films was produced with chitosan-based surfaces by dip coating.[16] The antimicrobial activity of functionalized surfaces was examined against S. aureus and P. aeruginosa. It was found that biocidal activity was 96% and the activity depended on the molecular weight of chitosan. Antimicrobial and antiviral nanocomposites containing PLA, chitosan and silver nanoparticles were synthesized using a thermochemical method. PLA-Ag-chitosan polymer nanocomposites showed strong antimicrobial activity particularly against S. aureus and E. coli strains.[17] Furthermore, antiviral activity was demonstrated against Influenza A virus, herpes simplex virus type 1 and adenovirus serotype 2. A polyurethane nanocomposite film was synthesized by adding modified chitosan and graphene oxide within the scope of polyurethane coating.[18] As a result of antibacterial efficacy and MTT (toxicity) analyses, the nanocomposite obtained showed good biocompatibility and high efficacy against S. aureus and E. coli bacteria. In another study, a chitosan coating on tomatoes successfully delayed fruit spoilage; it also preserved the fruit's texture and firmness compared to uncoated fruit; consequently, it contributed to extending shelf life at room temperature.[19] In another study, polyurethanes transesterified with triethanolamine (TEA) and pentaerythritol (PE) were synthesized based on castor oil and containing 1% chitosan. The surface of the chitosan-based polyurethane material was found to prevent bacteria from adhering to the surface.[20] Transesterification of the polyol helped modify mechanical properties, making them more suitable for cardiovascular applications without altering biological properties such as biocompatibility and hemocompatibility. Polyurethanes synthesized by the addition of castor oil and 1% chitosan do not support cytotoxicity, hemolysis or platelet adhesion when in contact with cells, making these materials highly suitable for use in cardiovascular devices.[20] Furthermore, in another study conducted, polysaccharide-based biosorbents can be used for the development of edible and safe biosorbents that would reduce excess cholesterol and bile salts in the gastrointestinal passage. Following analysis of polysaccharide-based biosorbents developed to adsorb cholesterol and bile salts from the gastrointestinal passage and common modification methods for these adsorbents, polysaccharides including β-cyclodextrin, pectin, chitin/chitosan, dietary fiber extract and cellulose were recommended for adsorbing cholesterol and bile salts in the gastrointestinal passage as biosorbents.[21] Within the scope of this article, research was conducted on the use of chitosan, which belongs to the natural polymers class, in polyurethane foam formulation and the examination of its antibacterial effect.

3. Method

In our project work, the objective was to develop chitosan-based polyurethane materials that could be used in industry, healthcare and textiles. Within the scope of the project, biopolymers consisting of a total of 9 different modified chitosans were synthesized with 3 different chitosans of low molecular weight, medium molecular weight and high molecular weight using 3 different methods. Figure 2 shows modified biopolymers (ZK-1; ZK-2 and ZK-PEG series) resulting from 3 different methods. Functional chitosans contain cationic methyl salts, cationic decane salts and polyethylene glycol (PEG) substituted groups. [caption id="attachment_158318" align="aligncenter"] Figure 2. Chitosan molecules synthesized within the scope of the study[/caption]

3.1. Synthesis of the ZK-1 Series

In this study, each of the three different molecular weight chitosans (ZK-1La, ZK-1Ma, ZK-1Ha) was methylated and converted into quaternary chitosan polymers.

Sample Formulation Study within the Scope of ZK-1 Series Synthesis

Into a glass balloon containing 1 gram of chitosan, 50 mL NMP and 15 mL of 1.5 molar NaOH solution were placed. The mixture was refluxed for 30 minutes at 50°C in an oil bath under N₂ gas. To prepare 1.5 M NaOH, 60 grams NaOH was added to a 1000 mL round-bottom flask. Subsequently, 1.08 g sodium iodide and 4.9 mL methyl iodide were added to the glass balloon. The mixture was refluxed for 24 hours (1 day) in an oil bath at 50°C under N₂ gas. The orange-colored material obtained from the reflux had a viscous structure and unsuitable pore size, making filtration with a Gooch erlenmeyer flask impossible. Acetone was added dropwise to a 500 mL beaker and the mixture was kept at -18°C for 15 minutes. Yellow precipitation was observed in the material. Subsequently, after washing again with 100 mL acetone and 100 mL THF, the material was dried in a vacuum oven. This synthesis was applied for each molecular weight chitosan.[22]

3.2. Synthesis of the ZK-2 Series

This series represents isobutyraldehyde-methyl quaternization of chitosan. In this study, each of the three different molecular weight chitosans (ZK-2Hb, ZK-2Mb, ZK-2Lb) are polymers formed as a result of reaction with isobutyraldehyde. Sample formulation study within the scope of ZK-2 series synthesis. To 1 gram of chitosan in a glass balloon, 0.6 mL isobutyraldehyde and 1% acetic acid solution were added. The mixture was stirred at pH 3 for 1 hour under N₂ gas using a magnetic stirrer with a fish bone stirrer. Subsequently, 0.35 grams of sodium borohydride was added to the mixture to bring pH to 4.5, and stirring was continued for one and a half hours under N₂ gas. Then, a freshly prepared 1M NaOH solution was slowly added to the balloon until pH reached 10, and pH was continuously monitored at each pH adjustment stage. The material brought to desired properties was separated into falcons. Each falcon was centrifuged with distilled water until pH reached 7. The samples were then placed in a lyophilizer and the modified chitosan was obtained as a dry white material. For quaternization with methyl iodide, 0.51 grams of the isobutyraldehyde-functional chitosan obtained after lyophilization, 25 mL NMP and 7.5 mL of freshly prepared 1.5 M NaOH were added to a glass balloon. The mixture was dissolved at 60°C for 30 minutes in an oil bath under N₂ gas. Subsequently, 0.54 grams of sodium iodide and 2.5 mL methyl iodide were added to the reaction medium and the reaction continued for 24 hours in an oil bath under N₂ gas. After the reaction vessel was brought to room temperature, 400 mL acetone was added dropwise and then stored at -18°C. The cream-colored product was partially dried with N₂ gas and then dried in a vacuum oven for 3 hours before being stored at +4°C. This synthesis was applied for each molecular weight chitosan.[22]

3.3. Synthesis of the ZK-PEG Series

In this series, PEG is added to quaternary chitosan structures. In this study, each of the three different molecular weight quaternary chitosans (ZK-2HbPEGG, ZK-2MbPEGG, ZK-2LbPEGG) are chitosan polymers formed by the attachment of PEG structures. Sample formulation study within the scope of ZK-PEG series synthesis. The solubility in distilled water of chitosan that had undergone isobutyraldehyde-methyl quaternization (resulting from method 2) was examined. Once solubility was confirmed, 1 gram of chitosan, 30 mL methanol were placed in a glass balloon and stirred in a magnetic stirrer at 25°C under N₂ gas until complete dissolution was achieved over 1 hour. In a separate beaker, 5 mL methanol and 0.1 grams PEG monoacrylate were mixed, added dropwise to the glass balloon at 25°C under N₂ gas and left to mix for 2 days. The methanol solvent in the balloon was then partially removed using a rotary evaporator, and the product in concentrated methanol was precipitated in 400 mL cold acetone. A white material was obtained after filtration and vacuum oven drying. This synthesis was applied for each molecular weight isobutyraldehyde-methyl quaternized chitosan.

3.4. Structural Characterization

During the experiments, FTIR and NMR analyses were performed at each stage and the experiments were continued according to the results of these analyses. As an example, Figure 3 shows FTIR analysis results of modified chitosans obtained by adding PEG to quaternary chitosan structures. After modified chitosan obtained by adding PEG to quaternary chitosan structures was obtained, FTIR was taken. In Figure 3, for PEG quaternized chitosan, the strong peak at 1050 cm⁻¹ in the C-O peak confirms this. The intense peak at 2879 due to the aliphatic C-H structure indicates the presence of alkyl groups attached to chitosan. For the vinyl group and C=O peaks of the PEG acrylate structure, peaks were observed at 841 cm⁻¹ and approximately 1660 cm⁻¹, respectively. However, following Michael addition, the characteristic peaks of PEG acrylate were found to have disappeared. In Figure 3, characteristic ester carbonyl stretching at 1720 cm⁻¹ was also observed after PEG was attached to chitosan. Thus, the reaction was confirmed to have proceeded successfully. [caption id="attachment_158319" align="aligncenter"] Figure 3. FTIR of modified chitosans obtained as a result of Method 3 (Addition of PEG to quaternary chitosan structures) (I) high molecular weight chitosan, (II) medium molecular weight chitosan, (III) PEG, (IV) low molecular weight chitosan[/caption] Table 1 shows the solubility of modified chitosans in common solvents. It is observed that samples do not dissolve particularly in low polarity solvents. While chitosan only dissolves in acidic water, once it becomes cationic, it dissolves in solvents such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and methanol. [caption id="attachment_158320" align="aligncenter"] DCM: Dichloromethane; DMF: Dimethylformamide; DMSO: Dimethyl sulfoxide[/caption] Based on the solubility data, modified chitosans obtained using Method 1 were selected with distilled water as solvent and modified chitosans obtained using Methods 2 and 3 were selected with methanol as the coating solution solvent. A total of 6 glass panes × 9 samples = 54 glass coatings were applied using the spin coating apparatus on clean glass. The glass coatings were sent to the microbiology laboratory for bacterial testing. The glass coating method was used as a reference method, and the efficacy value obtained was used with high polymer (functional chitosan) in the subsequent polyurethane foam application. Bacterial tests were examined as % killing (10⁶ cfu/mL). For testing, 2 bacteria were selected, E. coli (gram-negative) and S. aureus (gram-positive). The bacterial test results on glass coatings are shown in Table 2. Functional chitosan-coated surfaces were found to have higher efficacy against S. aureus. This is attributed to differences arising from the cell walls of both bacteria. Lower biocidal efficacy against S. aureus was observed in E. coli due to the presence of a double phospholipid layer and lipopolysaccharide layer in the E. coli cell wall. Following bacterial testing on glass surfaces, the polymer resulting from isobutyraldehyde-methyl quaternization of chitosan was found to have the best bacteria-killing effect. In this project, to slow the spread of microorganisms and protect against infectious diseases, polyurethane foams were coated using the spray method with chitosans of three different molecular weights. In this context, a total of 4×4=16 homogeneous, sterile foam cubes were used for coating ZK-2Hb, ZK-2Mb, ZK-2Lb and Blank (unfilled). 0.2 grams of each modified chitosan was taken and mixed with 6 mL distilled water to obtain 4% solutions. 0.2 mL of solution was sprayed onto each foam. The spraying was repeated 4 times at 5-minute intervals. As a result, 16 foam cubes were dried in an oven at 60°C for 1 hour after which they were packaged and sent for bacterial testing. [caption id="attachment_158322" align="aligncenter"] Figure 4. Polyurethane foams with chitosan derivatives on the surface[/caption] Following foam coating testing shown in Figure 4, it was found that the high molecular weight chitosan resulting from isobutyraldehyde-methyl quaternization, which gave the best results against S. aureus, i.e., the best bacteria-killing polymer, was the one that was most effective. Blank (unfilled) polyurethane foam has 0% killing effect. The test results are shown in Figure 5. [caption id="attachment_158323" align="aligncenter"] Figure 5. Modified Chitosan Test Results According to Bacterial Killing Percentages[/caption]

Conclusion

Chitin and chitosan are amino polysaccharides with multidimensional properties and are natural polymers. Polyurethanes, on the other hand, are materials frequently used in our daily lives. Within the scope of this project, studies were conducted to render polyurethane foam materials antibacterial starting from natural raw material chitosan. Cationic chitosans with different functional groups were added to the surface of polyurethane foam materials using the spray method. As a result, high efficacy particularly against S. aureus was achieved. It is anticipated that the products obtained within the scope of the project can be used to render materials used particularly in hospital environments antibacterial. Research continues on the incorporation of chitosan into polyurethane formulation and increasing its efficacy against E. coli.

Acknowledgments

We thank Flokser Kimya San. ve Tic. A.Ş. for polyurethane foam formulation. We thank TÜBİTAK for supporting our project work (project application no: 1139B412101110).   References [1] Rinaudo, M. (2008). Main properties and current applications of some polysaccharides as biomaterials. Polymer international, 57(3), 397-430. [2] Vincent, J. F., & Wegst, U. G. (2004). Design and mechanical properties of insect cuticle. Arthropod structure & development, 33(3), 187-199. [3] Raabe, D., Al-Sawalmih, A., Yi, S. B., & Fabritius, H. (2007). Preferred crystallographic texture of α-chitin as a microscopic and macroscopic design principle of the exoskeleton of the lobster Homarus americanus. Acta Biomaterialia, 3(6), 882-895. [4] Kobayashi, S., Kiyosada, T., & Shoda, S. I. (1996). Synthesis of artificial chitin: irreversible catalytic behavior of a glycosyl hydrolase through a transition state analogue substrate. Journal of the American Chemical Society, 118(51), 13113-13114. [5] Kobayashi, S., Makino, A., Tachibana, N., & Ohmae, M. (2006). Chitinase-Catalyzed Synthesis of a Chitin-Xylan Hybrid Polymer: A Novel Water-Soluble-(1→ 4) Polysaccharide Having an N-Acetylglucosamine-Xylose Repeating Unit. Macromolecular rapid communications, 27(10), 781-786. [6] Kadokawa, J. I. (2011). Precision polysaccharide synthesis catalyzed by enzymes. Chemical reviews, 111(7), 4308-4345. [7] Kumar, M. N. R. (2000). A review of chitin and chitosan applications. Reactive and functional polymers, 46(1), 1-27. [8] Pandey, P., Verma, M. K., & De, N. (2018). Chitosan in agricultural context–a review. Bulletin of Environment, Pharmacology and Life Sciences, 7(4), 87-96. [9] Dutta, P. K., Ravikumar, M. N. V., & Dutta, J. (2002). Chitin and chitosan for versatile applications. Journal of Macromolecular Science, Part C: Polymer Reviews, 42(3), 307-354. [10] Malerba, M., & Cerana, R. (2019). Recent applications of chitin- and chitosan-based polymers in plants. Polymers, 11(5), 839. [11] Demir, A., & Seventekin, N. (2009). Kitin, kitosan ve genel kullanım alanları. Tekstil Teknolojileri Elektronik Dergisi, 3(2), 92-103. [12] Vasconcelos, M. W. (2014). Chitosan and chitooligosaccharide utilization in phytoremediation and biofortification programs: current knowledge and future perspectives. Frontiers in plant science, 5. [13] Malerba, M., & Cerana, R. (2016). Chitosan effects on plant systems. International journal of molecular sciences, 17(7), 996. [14] Martins, A. F., Facchi, S. P., Follmann, H. D., Pereira, A. G., Rubira, A. F., & Muniz, E. C. (2014). Antimicrobial activity of chitosan derivatives containing N-quaternized moieties in its backbone: a review. International Journal of Molecular Sciences, 15(11), 20800-20832. [15] Kong, M., Chen, X. G., Xing, K., & Park, H. J. (2010). Antimicrobial properties of chitosan and mode of action: a state of the art review. International journal of food microbiology, 144(1), 51-63. [16] Lima, M., Teixeira-Santos, R., Gomes, L. C., Faria, S. I., Valcarcel, J., Vázquez, J. A., ... & Mergulhão, F. J. (2021). Development of chitosan-based surfaces to prevent single- and dual-species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa. Molecules, 26(14), 4378. [17] Demchenko, V., Rybalchenko, N., Zahorodnia, S., Naumenko, K., Riabov, S., Kobylinskyi, S., ... & Kowalczuk, M. (2022). Preparation, characterization, and antimicrobial and antiviral properties of silver-containing nanocomposites based on polylactic acid–chitosan. ACS Applied Bio Materials, 5(6), 2576-2585. [18] Najafabadi, S. A. A., Mohammadi, A., & Kharazi, A. Z. (2020). Polyurethane nanocomposite impregnated with chitosan-modified graphene oxide as a potential antibacterial wound dressing. Materials Science and Engineering: C, 115, 110899. [19] Meenu, B., Raman, M., Sreelakshmi, P. U., & Mathew, P. T. (2023). Effect of mushroom chitosan coating on the quality and storability of tomato (Solanum lycopersicum L.). Journal of Postharvest Technology, 11(1), 133-144. [20] Morales-González, M., Navas-Gómez, K., Diaz, L. E., Gómez-Tejedor, J. A., & Valero, M. F. (2023). Incorporation of Chitosan in Polyurethanes Based on Modified Castor Oil for Cardiovascular Applications. Polymers, 15(18), 3733. [21] Chen, L., He, X., Pu, Y., Cao, J., & Jiang, W. (2023). Polysaccharide-based biosorbents for cholesterol and bile salts in gastric-intestinal passage: Advances and future trends. Comprehensive Reviews in Food Science and Food Safety. [22] Najafabadi, S. A. A., Mohammadi, A., & Kharazi, A. Z. (2020). Polyurethane nanocomposite impregnated with chitosan-modified graphene oxide as a potential antibacterial wound dressing. Materials Science and Engineering: C, 115, 110899.   Prof. Dr. Tarık Eren Chemistry Department Yıldız Teknik Üniversitesi Zeynep Küçükersen Master's Student Yıldız Teknik Üniversitesi L. Yusuf Yivlik Research and Development Manager Flokser Kimya Prof. Dr. Melda Altıkatoğlu Professor Chemistry Department Yıldız Teknik Üniversitesi