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Biocompatible cellulose-based superabsorbent hydrogels with antimicrobial activity
Carbohydrate Polymers 137 (2016) 59–64
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Biocompatible cellulose-based superabsorbent hydrogels with antimicrobial activity Na Peng a,b , Yanfeng Wang a , Qifa Ye a,∗ , Lei Liang b , Yuxing An b , Qiwei Li b , Chunyu Chang b,c,∗∗ a
Zhongnan Hospital of Wuhan University, Institute of Hepatobiliary Diseases of Wuhan University, Wuhan 430071, China Guangzhou Sugarcane Industry Research Institute, Guangzhou 510316, China c College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China b
a r t i c l e
i n f o
Article history: Received 15 September 2015 Received in revised form 6 October 2015 Accepted 15 October 2015 Available online 19 October 2015 Keywords: Cellulose hydrogel Superabsorbent property Biocompatible Antimicrobial activity
a b s t r a c t Current superabsorbent hydrogels commercially applied in the disposable diapers have disadvantages such as weak mechanical strength, poor biocompatibility, and lack of antimicrobial activity, which may induce skin allergy of body. To overcome these hassles, we have developed novel cellulose based hydrogels via simple chemical cross-linking of quaternized cellulose (QC) and native cellulose in NaOH/urea aqueous solution. The prepared hydrogel showed superabsorbent property, high mechanical strength, good biocompatibility, and excellent antimicrobial efficacy against Saccharomyces cerevisiae. The presence of QC in the hydrogel networks not only improved their swelling ratio via electrostatic repulsion of quaternary ammonium groups, but also endowed their antimicrobial activity by attraction of sections of anionic microbial membrane into internal pores of poly cationic hydrogel leading to the disruption of microbial membrane. Moreover, the swelling properties, mechanical strength, and antibacterial activity of hydrogels strongly depended on the contents of quaternary ammonium groups in hydrogel networks. The obtained data encouraged the use of these hydrogels for hygienic application such as disposable diapers. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Superabsorbent hydrogels are lightly crosslinked polyelectrolyte polymer networks with unique absorption capacity of up to several hundred times of their dried weight (Chang, Duan, Cai, & Zhang, 2010). They have been widely used in many fields such as drug delivery systems, tissue engineering, immobilization of protein and cells, agriculture and horticulture, and sanitary products (Sharma, Dua, & Malik, 2014; Zhang, Wang, & Wang, 2007; Zohuriaan-Mehr, Omidian, Doroudiani, & Kabiri, 2010; Chang & Zhang, 2011; Gawande & Mungray, 2015). Superabsorbent hydrogels were industrially developed in Japan and USA as early as 1980s for hygienic application, such as baby diapers and feminine napkins, which originated from the product of hydrolysis of starch-g-polyacrylonitrile in 1970 (Kabiri, Omidian, ZohuriaanMehr, & Doroudiani, 2011). The global demand for superabsorbent
∗ Corresponding author. ∗∗ Corresponding author at: Wuhan University, College of Chemistry and Molecular Sciences, Wuhan, China. Tel.: +86 2787219274. E-mail addresses: yqf [email protected] (Q. Ye), [email protected] (C. Chang). http://dx.doi.org/10.1016/j.carbpol.2015.10.057 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
hydrogels is increasing and reaches 1.9 million metric tons in 2015 (Zhang et al., 2014). However, lack of biocompatibility and biodegradability is the major hassle for the present superabsorbent materials used worldwide as they are made from petroleum based monomers like acrylic acid and acrylamide. The remedy for this problem is to design the hydrogels by using natural polymers which usually would be biocompatible and biodegradable, but they have inferior mechanical properties compared to petroleum based polymers (Spagnol et al., 2012). The techniques to improve the mechanical properties of hydrogels include increasing the cross-linking density and incorporation of inorganic materials or suitable polymers (Huang et al., 2007; Gong, Katsuyama, Kurokawa, & Osada, 2003; Haraguchi & Li, 2005). However, the former resulted in the decrease of swelling ratio of hydrogels while the latter need add new components to hydrogel networks. So these approaches were inapplicable to develop superabsorbent hydrogels with good mechanical strength. In our previous works, we found that cellulose with stiff molecular chains could act as support ingredient in the hydrogel networks and superabsorbent hydrogels can be obtained by introducing other hydrophilic polymers into the networks (Chang, He, Zhou, & Zhang, 2011; Chang, Duan, & Zhang, 2009). Therefore, it may be a
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N. Peng et al. / Carbohydrate Polymers 137 (2016) 59–64
Table 1 Conditions for the preparation of QC/cellulose hydrogels. Code
Gel9-2 Gel9-4 Gel9-6 Gel25-2 Gel25-4 Gel25-6
QC
Cellulose
Weight ratio
Swelling ratio
Mw (×10−4 )
DS
Mw (×10−4 )
QC/cellulose
g/g
9.4 9.4 9.4 25.6 25.6 25.6
0.23 0.42 0.61 0.28 0.44 0.69
9.4 9.4 9.4 9.4 9.4 9.4
9:1 9:1 9:1 9:1 9:1 9:1
206.6 337.3 433.9 247.1 607.5 983.9
relatively simple, cost-effective, and green approach way to fabricate cellulose based superabsorbent hydrogels. Traditional diapers are breeding grounds for harmful bacteria after absorbing urine, which seriously threaten human health. To avoid the growth of bacteria and provide a healthy environment for baby skin during usage of a diaper, the antimicrobial activity of products is essential and new technique must be developed to suit the needs. Among the various antimicrobial agents, metallic nanoparticles have shown broad spectrum antibacterial activity (Cui et al., 2012; Loo et al., 2015), but both metallic nanoparticle and their syntheses may cause environmental toxicity or biological hazards (Kittler, Greulich, Diendorf, Koller, & Epple, 2010; Cheng, Betts, Kelly, Schaller, & Heinze, 2013; Sharma, Yngard, & Lin, 2009). Fortunately, modification of cellulose fibers with bactericidal species, such as quaternary ammonium, -cyclodextrin with ciprofloxacin, siloxane sulfopropylbetaine, and N-halamine siloxanes, has become a facile way to endow them with antibacterial performance (Roy, Knapp, Guthrie, & Perrier, 2008; Dong et al., 2014; Ren et al., 2008; Chen et al., 2011). These results demonstrated that the effective modification of cellulose fiber can enhance the antibacterial performance of cellulose materials. Almost all studies confined only to modify the surface of cellulose fibers, but the technique to covalently incorporate bactericidal species into cellulose hydrogel networks have not been reported. Therefore, we attempt to introduce quaternary ammonium groups into hydrogel networks by solution blending of cellulose solution and quaternized cellulose solution, aiming to develop novel hydrogel materials with good mechanical strength, superabsorbent property, biocompatibility and antimicrobial activity. This paper reports the preparation of cellulose based hydrogels through chemical cross-linking cellulose and quaternized cellulose in NaOH/urea aqueous solution, besides the evaluation of superabsorbent properties, mechanical properties, biocompatibility, and antimicrobial activities of hydrogels, as well as the influence of chemical structure of quaternized cellulose on the properties of hydrogels. 2. Experimental 2.1. Materials Two kinds of cellulose samples (cotton linter pulps) were supplied by Hubei Chemical Fiber Co. Ltd. Their weight-average molecular weights (Mw ) were determined by static laser light scattering (DAWN DSP, Wyatt Technology Co.) to be 9.4 × 104 and 2.56 × 104 . 3-Chloro-2-hydroxypropyltrimethylammonium chloride was purchased from Guofeng Fine Chemical Co. Ltd. (Shandong, China). Epichlorohydrin (ECH), sodium hydroxide (NaOH), and urea were from Chemical Agents, Ltd. Co. (Shanghai, China) without further purification. The Saccharomyces cerevisiae was obtained from European S. cerevisiae Archive for Functional Analysis (Frankfurt, Germany). And other biochemical reagents were purchased from Thermo Fisher Oxoid (Shanghai, China).
2.2. Fabrication of cellulose based hydrogels Quaternized celluloses (QC) were synthesized by quaternization of cellulose with 3-chloro-2-hydroxypropyltrimethylammonium chloride in NaOH/urea aqueous solutions according to our previous method (Chang et al., 2011). Their degree of substitutions (DS) were determined by an elemental analyzer (CHN-O-Rapid, Hanau, Germany) (see Table 1). For the preparation of hydrogels, cellulose was dissolved in 7 wt% NaOH/12 wt% urea aqueous solutions at low temperature, while QC was also dissolved in 7 wt% NaOH/12 wt% urea aqueous solutions at room temperature. Then, QC and cellulose solution were mixed with a weight ratio of 9:1 to obtain a 3 wt% polymer concentration. 1 mL ECH as crosslinker was added to 10 g QC/cellulose mixed solution under stirring, and reacted at 60 ◦ C for 2 h. Finally, the hydrogels were taken out and immersed in ultrapure water to remove the residual NaOH, urea and un-reacted ECH to get pure samples. Hydrogels were coded as Gel9-2, Gel9-4, Gel9-6, Gel25-2, Gel25-4, and Gel25-6, according to the molecular weight and DS of QC (Table 1). 2.3. Characterization The dried samples were analyzed in KBr discs by FTIR (Perkin Elmer Spectrum One, Wellesley, MA, USA) in the region of 400–4000 cm−1 . The mechanical properties of the hydrogels were measured on a universal testing machine (CMT6503, Shenzhen SANS Test Machine Co. Ltd., Shenzhen, China). Swelling ratios of the hydrogels in the ultrapure water at 37 ◦ C were measured by the gravimetric method (Chang et al., 2011). The swelling ratio was calculated as Swelling ratio =
Ws Wd
(1)
where Ws is the weight of the swollen gel after equilibrium at 37 ◦ C, and Wd is the weight of the gel in the dry state. 2.4. Cell viability assay Hydrogels samples were sterilized in an autoclave at 150 ◦ C for 15 min before the cells were cultured. L02 cells (5 × 104 cells per well) were seeded on the surface of the sterilized hydrogel matrices in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) with 10% FBS, and incubated at 37 ◦ C. After incubation at 37 ◦ C for 48 h, the medium was removed. Fresh medium (1 mL) and MTT (60 L, 5 mg/mL) were added to each well, followed by 4 h of incubation at 37 ◦ C. Subsequently, the supernatant was carefully removed, and 1 mL DMSO was added to each well. The absorbance of the solution was measured with microplate reader (Bio-Rad 550) at 570 nm to determine the Optical Density (OD) value. The cell viability was evaluated by MTT assay using the cells cultured on the cell culture plate as control and calculated as follows:
Cell viability =
ODgel ODcontrol
× 100%
(2)
N. Peng et al. / Carbohydrate Polymers 137 (2016) 59–64
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Fig. 1. Proposed mechanism for cross-linking reaction of cellulose and quaternized cellulose (QC) in alkali aqueous solution with ECH.
Fig. 2. Photographs of QC/cellulose hydrogels after swelling equilibrium in distilled water.
where ODgel is obtained for the cells cultured on gels. 2.5. Antimicrobial activity evaluation Shaking flask method was applied to detect the antimicrobial performance of QC/cellulose hydrogels. S. cerevisiae N85 was inoculated in yeast extract peptone dextrose (YPD) medium (containing 10 g bacto yeast extract, 20 g bacto peptone, and 20 g dextrose per 1 L) and cultured at 30 ◦ C for 24 h in a rotary shaker at 200 rev/min. The strain was harvested by centrifugation and diluted 20 times (106 –107 CFU/mL). The samples Gel9-2, Gel9-4, Gel9-6, Gel25-2, Gel25-4, and Gel25-6 were cut into disk with the weight of 0.02 g and then put into flasks with 50 mL bacteria suspension, respectively. The flasks were held in a shaking table at 30 ◦ C for 24 h, and then about 3 mL of suspension was taken out for OD600 nm measurement. The results were recorded in an UV spectrophotometer (UV-6100PCS, Shanghai, China). Three parallel samples were adopted for each experimental group to ensure the correction of experimental results, and all the conditions were kept sterile. A control culture without sample was also treated in the similar way.
Table 1). The molecular weight of cellulose was 9.4 × 104 and the QC/cellulose weight ratio was fixed to be 9:1. The appearance of QC/cellulose hydrogels are shown in Fig. 2, where the hydrogels were swollen and transparent. The size of hydrogels after equilibrium swelling which prepared in the same models increased from Gel25-2 to Gel 25-6, with the increase of DS of QC in hydrogel networks from 0.28 to 0.69. Fig. 3 shows the FTIR spectra of the cellulose hydrogel, QC, and QC/cellulose hydrogel. The broad band at about 3350 cm−1 was assigned to O H stretching vibration, which can be found in all samples. The sharp peak around 2900 cm−1 in the spectra of samples included the antisymmetric and symmetric stretching
3. Results and discussion 3.1. Appearance and structure of QC/cellulose hydrogels Fig. 1 shows the proposed mechanism for cross-linking reaction of quaternized cellulose (QC) and cellulose in alkali aqueous solution. Epichlorohydrin was used as a difunctional cross-linker which could react with the hydroxyl groups of QC and cellulose. Six hydrogel samples were synthesized and named as Gel9-2, Gel9-4, Gel9-6, Gel25-2, Gel25-4, and Gel25-6, respectively, according to the molecular weight (Mw = 9.4 × 104 and 2.56 × 105 ) and degree of substitution (from 0.23 to 0.69) of QC used in this work (see
Fig. 3. FTIR spectra of the cellulose hydrogel (Gel0), QC (QC25-6), and QC/cellulose hydrogels (Gel25-6).
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Fig. 6. The cytotoxicity tests of QC/cellulose hydrogels. Fig. 4. Equilibrium swelling ratio of QC/cellulose hydrogels in distilled water at room temperature.
vibration of CH2 groups. The most striking difference between cellulose hydrogel and other samples was the peak at 1482 cm−1 , which could not be found in the spectrum of cellulose hydrogel corresponding to the methyl groups of ammonium (Song, Sun, Zhang, Zhou, & Zhang, 2008). Moreover, the presence of another additional absorption at 1413 cm−1 was assignable to the C N stretching vibration in spectrum of QC (Pal, Mal, & Singh, 2005). These results indicated that a cationic moiety has been incorporated onto the backbone of cellulose, and quaternary ammonium salt groups existed in QC/cellulose hydrogel networks. 3.2. Swelling and mechanical properties of QC/cellulose hydrogels The swelling ratio of QC/cellulose hydrogels are listed in Table 1, ranging from 206.6 g/g to 983.9 g/g, which showed superabsorbent properties and far exceeded that of cellulose hydrogels (Chang, Zhang, Zhou, Zhang, & Kennedy, 2010). This phenomena could be explained by the presence of positive charges (cationic quaternary ammonium) in the hydrogel networks, where the electrostatic repulsion of positive charges expanded the hydrogel networks and improved the swelling ratio of samples. To investigate the roles of DS and molecular weight of QC in the swelling properties of hydrogel, the swelling ratio of QC/cellulose hydrogels are shown in Fig. 4. It can be found that the samples containing QC with higher molecular weight exhibited higher swelling ratio than those containing lower molecular weight QC with similar DS values. This result indicated that high molecular weight QC could combine
with more water in hydrogel networks, leading to higher swelling ratio of hydrogel samples. On the other hand, the swelling ratio of hydrogels increased with the increasing of DS of QC. The higher DS of QC raised the numbers of positive charges in the hydrogel networks, resulting in the expansion of the networks by the electrostatic repulsion of cationic quaternary ammonium in the hydrogels, leading to the improvement of their swelling ratio. For the mechanical properties of samples, compression tests are conducted in this section. The QC/cellulose hydrogels are elastic and their compressive stress–strain curves are shown in Fig. 5. For Gel9 series, when the DS of QC increased from 0.23 to 0.61, the compressive stress decreased from 28.7 kPa to 11.2 kPa. While the similar tendency could also be found in Gel25 series, that is, when the DS of QC increased from 0.28 to 0.69 for Gel25 series, the compressive stress decreased from 30.6 kPa to 2.11 kPa. These results showed that the DS of QC played an important role in the compressive stress of hydrogel. However, the mechanical properties of samples did not depend on molecular weight of QC in the hydrogels, which was different with traditional cellulose hydrogels (Cai & Zhang, 2006). It could be explained by the superabsorbent properties of hydrogels, which could hold a lot of water several hundred times of their own weight, resulting in the independence of QC molecular weight. 3.3. Biocompatibility and antimicrobial activity of QC/cellulose hydrogels To explore the cytocompatibility of QC/cellulose hydrogels, the normal human hepatic L02 cells was used as model cell lines. From the results of L02 cells culture experiment, we found that L02 cells
Fig. 5. Compressive stress–strain curves of QC/cellulose hydrogels.
N. Peng et al. / Carbohydrate Polymers 137 (2016) 59–64
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Fig. 7. Antibacterial activity of QC/cellulose hydrogel samples against S. cerevisiae: Pictures of S. cerevisiae growth on different samples (left) and OD600 nm value of S. cerevisiae after a 24 h culture.
could adhere to the surface of hydrogels and their spreading and proliferation on the surface were also realized. After 48 h incubation, the cells seed on the QC/cellulose hydrogels exhibited normal morphology compared to the control. Fig. 6 shows the results of cytotoxicity tests on the hydrogels. It can be seen that the cell viability values on the all hydrogel samples were greater than 80%, indicating good biocompatibility to L02 cells. The cytotoxicity of QC has been reported which gradually increased with the increasing of its DS value due to the presence of a higher amount of cationic cellulose damaging the cellular membranes (Song et al., 2008). Therefore, the lower cytotoxicity of QC/cellulose hydrogels observed in this work confirmed that the combination of QC and cellulose in the hydrogel networks could improve the cytocompatibility of samples, leading to better compatibility of materials. The antibacterial property of QC/cellulose hydrogel was tested in a 24 h growth inhibition assay using S. cerevisiae. Cellulose hydrogel was also evaluated in parallel as control. One hydrogel disk was placed in flask and submerged in yeast extract peptone dextrose medium inoculated with S. cerevisiae. Fig. 7 shows the pictures of S. cerevisiae growth on hydrogel samples with diluted culture medium. After 24 h incubation at 30 ◦ C, the colony forming unit (CFU) of S. cerevisiae cultured on Gel9-4, Gel9-6, and Gel256 declined obviously compared to that cultured on control. The OD600 nm of the supernatant of each samples was also measured and recorded as an indication for bacteria population density (Fig. 7, right). All hydrogels showed S. cerevisiae growth inhibition compared to the control. The survival rate of S. cerevisiae on QC/cellulose hydrogels were 88%, 33.3%, 33.6%, 87.1%, 66.9%, and 19.9% for Gel9-2, Gel9-4, Gel9-6, Gel25-2, Gel25-4, and Gel25-6, respectively. Gel25-6 exhibited the best antibacterial activity among all hydrogel samples, due to the presence of QC with the highest DS in the hydrogel networks. Therefore, the mechanism of antimicrobial activity of Gel25-6 sample could be explained by that attraction of sections of anionic microbial membrane into internal pores of poly cationic hydrogel led to the disruption of microbial membrane and the death of microbial (Li et al., 2011). 4. Conclusions We demonstrated a paradigm to fabricate novel hydrogels with antimicrobial activity using chemical crosslinking of natural polymer and its cationic derivative. The transparent hydrogels exhibited superabsorbent property, good mechanical performance, and biocompatibility. In the hydrogel networks, quaternized cellulose not only played an important role in the improvement of the swelling ratio of samples by the electrostatic repulsion of quaternary ammonium groups, but also endowed them with antimicrobial activity through the interaction between anionic microbial membrane and poly cationic hydrogel resulting in the death of microbial. The
swelling ratio and antimicrobial activity of QC/cellulose hydrogels could be effectively tuned by the DS of QC. Therefore, utilization of this method to fabricate hydrogels could potentially facilitate the development of materials for hygienic application. Acknowledgments This work was supported by Pearl River S&T Nova Program of Guangzhou (201506010101), Guangzhou science and technology project (201510010221), Hubei Province Science Foundation for Youths (2015CFB499), and the National Natural Science Foundation of China (21304021). References Cai, J., & Zhang, L. (2006). Unique gelation behavior of cellulose in NaOH/urea aqueous solution. Biomacromolecules, 7(1), 183–189. Chang, C., Duan, B., Cai, J., & Zhang, L. (2010). Superabsorbent hydrogels based on cellulose for smart swelling and controllable delivery. European Polymer Journal, 46(1), 92–100. Chang, C., Duan, B., & Zhang, L. (2009). Fabrication and characterization of novel macroporous cellulose–alginate hydrogels. Polymer, 50(23), 5467–5473. Chang, C., He, M., Zhou, J., & Zhang, L. (2011). Swelling behaviors of pH- and salt-responsive hydrogels from cellulose. Macromolecules, 44(6), 1642–1648. Chang, C., & Zhang, L. (2011). Cellulose-based hydrogels: Present status and application. Carbohydrate Polymers, 84(1), 40–53. Chang, C., Zhang, L., Zhou, J., Zhang, L., & Kennedy, J. F. (2010). Structure and properties of hydrogels prepared from cellulose in NaOH/urea aqueous solution. Carbohydrate Polymers, 82(1), 122–127. Chen, S., Chen, S., Jiang, S., Xiong, M., Luo, J., Tang, J., et al. (2011). Environmentally friendly antibacterial cotton textiles finished with siloxane sulfoproylbetaine. ACS Applied Materials & Interfaces, 3(4), 1154–1162. Cheng, F., Betts, J. W., Kelly, S. M., Schaller, J., & Heinze, T. (2013). Synthesis and antibacterial effects of aqueous colloidal solution of silver nanoparticles using aminocellulose as a combined reducing and capping reagent. Green Chemistry, 15(4), 989–998. Cui, J., Hu, C., Yang, Y., Wu, Y., Yang, L., Wang, Y., et al. (2012). Facile fabrication of carbonaceous nanospheres loaded with silver nanoparticles as antibacterial materials. Journal of Materials Chemistry, 22(16), 8121–8126. Dong, C., Ye, Y., Qian, L., Zhao, G., He, B., & Xiao, H. (2014). Antibacterial modification of cellulose fibers by grafting -cyclodextrin and inclusion with ciprofloxacin. Cellulose, 21(3), 1921–1932. Gawande, N., & Mungray, A. A. (2015). Superaborbent polymer hydrogels for protein enrichment. Separation and Purification Technology, 150, 86–94. Gong, J. P., Katsuyama, Y., Kurokawa, T., & Osada, Y. (2003). Double-network hydrogels with extremely high mechanical strength. Advanced Materials, 15(14), 1155–1158. Haraguchi, K., & Li, H. J. (2005). Control of the coil-to-globule transition and ultrahigh mechanical properties of PNIPA in nanocomposite hydrogels. Angewandte Chemie International Edition, 44(40), 6500–6504. Huang, T., Xu, H., Jiao, K., Zhu, L., Brown, H., & Wang, H. (2007). A novel hydrogel with high mechanical strength: A macromolecular microsphere composite hydrogel. Advanced Materials, 19(12), 1622–1626. Kabiri, K., Omidian, H., Zohuriaan-Mehr, M. J., & Doroudiani, S. (2011). Superabsorbent hydrogel composites and nanocomposites: A review. Polymer Composite, 32(2), 277–289. Kittler, S., Greulich, C., Diendorf, J., Koller, M., & Epple, M. (2010). Toxicity of silver nanoparticles increases during storage because of slow dissolution under release of silver ions. Chemistry of Materials, 22(16), 4548–4554.
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Song, Y., Sun, Y., Zhang, X., Zhou, J., & Zhang, L. (2008). Homogeneous rogelquaternization of cellulose in NaOH/urea aqueous solution as gene carriers. Biomacromolecules, 9(9), 2259–2264. Spagnol, C., Rogdrigues, F. H. A., Pereira, A. G. B. P., Fajardo, A. R., Rubira, A. F., & Muniz, E. C. (2012). Superabsorbent hydrogel nanocomposites based on starch-g-poly(sodium acrylate) matrix filled with cellulose nanowhiskers. Cellulose, 19(4), 1225–1237. Zhang, J., Wang, Q., & Wang, A. (2007). Synthesis and characterization of chitosan-g-poly(acrylic acid)/attapulgite superabsorbent composites. Carbohydrate Polymer, 68(2), 367–374. Zhang, M., Cheng, Z., Zhao, T., Liu, M., Hu, M., & Li, J. (2014). Synthesis, characterization, and swelling behaviors of salt-sensitive maize bran-poly(acrylic acid) superabsorbent hydrogel. Journal of Agriculture and Food Chemistry, 62(35), 8867–8874. Zohuriaan-Mehr, M. J., Omidian, H., Doroudiani, S., & Kabiri, K. (2010). Advances in non-hygienic applications of superabsorbent hydrogel materials. Journal of Material Science, 45(21), 5711–5735.
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Biocompatible cellulose-based superabsorbent hydrogels with antimicrobial activity Na Peng a,b , Yanfeng Wang a , Qifa Ye a,∗ , Lei Liang b , Yuxing An b , Qiwei Li b , Chunyu Chang b,c,∗∗ a
Zhongnan Hospital of Wuhan University, Institute of Hepatobiliary Diseases of Wuhan University, Wuhan 430071, China Guangzhou Sugarcane Industry Research Institute, Guangzhou 510316, China c College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China b
a r t i c l e
i n f o
Article history: Received 15 September 2015 Received in revised form 6 October 2015 Accepted 15 October 2015 Available online 19 October 2015 Keywords: Cellulose hydrogel Superabsorbent property Biocompatible Antimicrobial activity
a b s t r a c t Current superabsorbent hydrogels commercially applied in the disposable diapers have disadvantages such as weak mechanical strength, poor biocompatibility, and lack of antimicrobial activity, which may induce skin allergy of body. To overcome these hassles, we have developed novel cellulose based hydrogels via simple chemical cross-linking of quaternized cellulose (QC) and native cellulose in NaOH/urea aqueous solution. The prepared hydrogel showed superabsorbent property, high mechanical strength, good biocompatibility, and excellent antimicrobial efficacy against Saccharomyces cerevisiae. The presence of QC in the hydrogel networks not only improved their swelling ratio via electrostatic repulsion of quaternary ammonium groups, but also endowed their antimicrobial activity by attraction of sections of anionic microbial membrane into internal pores of poly cationic hydrogel leading to the disruption of microbial membrane. Moreover, the swelling properties, mechanical strength, and antibacterial activity of hydrogels strongly depended on the contents of quaternary ammonium groups in hydrogel networks. The obtained data encouraged the use of these hydrogels for hygienic application such as disposable diapers. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Superabsorbent hydrogels are lightly crosslinked polyelectrolyte polymer networks with unique absorption capacity of up to several hundred times of their dried weight (Chang, Duan, Cai, & Zhang, 2010). They have been widely used in many fields such as drug delivery systems, tissue engineering, immobilization of protein and cells, agriculture and horticulture, and sanitary products (Sharma, Dua, & Malik, 2014; Zhang, Wang, & Wang, 2007; Zohuriaan-Mehr, Omidian, Doroudiani, & Kabiri, 2010; Chang & Zhang, 2011; Gawande & Mungray, 2015). Superabsorbent hydrogels were industrially developed in Japan and USA as early as 1980s for hygienic application, such as baby diapers and feminine napkins, which originated from the product of hydrolysis of starch-g-polyacrylonitrile in 1970 (Kabiri, Omidian, ZohuriaanMehr, & Doroudiani, 2011). The global demand for superabsorbent
∗ Corresponding author. ∗∗ Corresponding author at: Wuhan University, College of Chemistry and Molecular Sciences, Wuhan, China. Tel.: +86 2787219274. E-mail addresses: yqf [email protected] (Q. Ye), [email protected] (C. Chang). http://dx.doi.org/10.1016/j.carbpol.2015.10.057 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
hydrogels is increasing and reaches 1.9 million metric tons in 2015 (Zhang et al., 2014). However, lack of biocompatibility and biodegradability is the major hassle for the present superabsorbent materials used worldwide as they are made from petroleum based monomers like acrylic acid and acrylamide. The remedy for this problem is to design the hydrogels by using natural polymers which usually would be biocompatible and biodegradable, but they have inferior mechanical properties compared to petroleum based polymers (Spagnol et al., 2012). The techniques to improve the mechanical properties of hydrogels include increasing the cross-linking density and incorporation of inorganic materials or suitable polymers (Huang et al., 2007; Gong, Katsuyama, Kurokawa, & Osada, 2003; Haraguchi & Li, 2005). However, the former resulted in the decrease of swelling ratio of hydrogels while the latter need add new components to hydrogel networks. So these approaches were inapplicable to develop superabsorbent hydrogels with good mechanical strength. In our previous works, we found that cellulose with stiff molecular chains could act as support ingredient in the hydrogel networks and superabsorbent hydrogels can be obtained by introducing other hydrophilic polymers into the networks (Chang, He, Zhou, & Zhang, 2011; Chang, Duan, & Zhang, 2009). Therefore, it may be a
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Table 1 Conditions for the preparation of QC/cellulose hydrogels. Code
Gel9-2 Gel9-4 Gel9-6 Gel25-2 Gel25-4 Gel25-6
QC
Cellulose
Weight ratio
Swelling ratio
Mw (×10−4 )
DS
Mw (×10−4 )
QC/cellulose
g/g
9.4 9.4 9.4 25.6 25.6 25.6
0.23 0.42 0.61 0.28 0.44 0.69
9.4 9.4 9.4 9.4 9.4 9.4
9:1 9:1 9:1 9:1 9:1 9:1
206.6 337.3 433.9 247.1 607.5 983.9
relatively simple, cost-effective, and green approach way to fabricate cellulose based superabsorbent hydrogels. Traditional diapers are breeding grounds for harmful bacteria after absorbing urine, which seriously threaten human health. To avoid the growth of bacteria and provide a healthy environment for baby skin during usage of a diaper, the antimicrobial activity of products is essential and new technique must be developed to suit the needs. Among the various antimicrobial agents, metallic nanoparticles have shown broad spectrum antibacterial activity (Cui et al., 2012; Loo et al., 2015), but both metallic nanoparticle and their syntheses may cause environmental toxicity or biological hazards (Kittler, Greulich, Diendorf, Koller, & Epple, 2010; Cheng, Betts, Kelly, Schaller, & Heinze, 2013; Sharma, Yngard, & Lin, 2009). Fortunately, modification of cellulose fibers with bactericidal species, such as quaternary ammonium, -cyclodextrin with ciprofloxacin, siloxane sulfopropylbetaine, and N-halamine siloxanes, has become a facile way to endow them with antibacterial performance (Roy, Knapp, Guthrie, & Perrier, 2008; Dong et al., 2014; Ren et al., 2008; Chen et al., 2011). These results demonstrated that the effective modification of cellulose fiber can enhance the antibacterial performance of cellulose materials. Almost all studies confined only to modify the surface of cellulose fibers, but the technique to covalently incorporate bactericidal species into cellulose hydrogel networks have not been reported. Therefore, we attempt to introduce quaternary ammonium groups into hydrogel networks by solution blending of cellulose solution and quaternized cellulose solution, aiming to develop novel hydrogel materials with good mechanical strength, superabsorbent property, biocompatibility and antimicrobial activity. This paper reports the preparation of cellulose based hydrogels through chemical cross-linking cellulose and quaternized cellulose in NaOH/urea aqueous solution, besides the evaluation of superabsorbent properties, mechanical properties, biocompatibility, and antimicrobial activities of hydrogels, as well as the influence of chemical structure of quaternized cellulose on the properties of hydrogels. 2. Experimental 2.1. Materials Two kinds of cellulose samples (cotton linter pulps) were supplied by Hubei Chemical Fiber Co. Ltd. Their weight-average molecular weights (Mw ) were determined by static laser light scattering (DAWN DSP, Wyatt Technology Co.) to be 9.4 × 104 and 2.56 × 104 . 3-Chloro-2-hydroxypropyltrimethylammonium chloride was purchased from Guofeng Fine Chemical Co. Ltd. (Shandong, China). Epichlorohydrin (ECH), sodium hydroxide (NaOH), and urea were from Chemical Agents, Ltd. Co. (Shanghai, China) without further purification. The Saccharomyces cerevisiae was obtained from European S. cerevisiae Archive for Functional Analysis (Frankfurt, Germany). And other biochemical reagents were purchased from Thermo Fisher Oxoid (Shanghai, China).
2.2. Fabrication of cellulose based hydrogels Quaternized celluloses (QC) were synthesized by quaternization of cellulose with 3-chloro-2-hydroxypropyltrimethylammonium chloride in NaOH/urea aqueous solutions according to our previous method (Chang et al., 2011). Their degree of substitutions (DS) were determined by an elemental analyzer (CHN-O-Rapid, Hanau, Germany) (see Table 1). For the preparation of hydrogels, cellulose was dissolved in 7 wt% NaOH/12 wt% urea aqueous solutions at low temperature, while QC was also dissolved in 7 wt% NaOH/12 wt% urea aqueous solutions at room temperature. Then, QC and cellulose solution were mixed with a weight ratio of 9:1 to obtain a 3 wt% polymer concentration. 1 mL ECH as crosslinker was added to 10 g QC/cellulose mixed solution under stirring, and reacted at 60 ◦ C for 2 h. Finally, the hydrogels were taken out and immersed in ultrapure water to remove the residual NaOH, urea and un-reacted ECH to get pure samples. Hydrogels were coded as Gel9-2, Gel9-4, Gel9-6, Gel25-2, Gel25-4, and Gel25-6, according to the molecular weight and DS of QC (Table 1). 2.3. Characterization The dried samples were analyzed in KBr discs by FTIR (Perkin Elmer Spectrum One, Wellesley, MA, USA) in the region of 400–4000 cm−1 . The mechanical properties of the hydrogels were measured on a universal testing machine (CMT6503, Shenzhen SANS Test Machine Co. Ltd., Shenzhen, China). Swelling ratios of the hydrogels in the ultrapure water at 37 ◦ C were measured by the gravimetric method (Chang et al., 2011). The swelling ratio was calculated as Swelling ratio =
Ws Wd
(1)
where Ws is the weight of the swollen gel after equilibrium at 37 ◦ C, and Wd is the weight of the gel in the dry state. 2.4. Cell viability assay Hydrogels samples were sterilized in an autoclave at 150 ◦ C for 15 min before the cells were cultured. L02 cells (5 × 104 cells per well) were seeded on the surface of the sterilized hydrogel matrices in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) with 10% FBS, and incubated at 37 ◦ C. After incubation at 37 ◦ C for 48 h, the medium was removed. Fresh medium (1 mL) and MTT (60 L, 5 mg/mL) were added to each well, followed by 4 h of incubation at 37 ◦ C. Subsequently, the supernatant was carefully removed, and 1 mL DMSO was added to each well. The absorbance of the solution was measured with microplate reader (Bio-Rad 550) at 570 nm to determine the Optical Density (OD) value. The cell viability was evaluated by MTT assay using the cells cultured on the cell culture plate as control and calculated as follows:
Cell viability =
ODgel ODcontrol
× 100%
(2)
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Fig. 1. Proposed mechanism for cross-linking reaction of cellulose and quaternized cellulose (QC) in alkali aqueous solution with ECH.
Fig. 2. Photographs of QC/cellulose hydrogels after swelling equilibrium in distilled water.
where ODgel is obtained for the cells cultured on gels. 2.5. Antimicrobial activity evaluation Shaking flask method was applied to detect the antimicrobial performance of QC/cellulose hydrogels. S. cerevisiae N85 was inoculated in yeast extract peptone dextrose (YPD) medium (containing 10 g bacto yeast extract, 20 g bacto peptone, and 20 g dextrose per 1 L) and cultured at 30 ◦ C for 24 h in a rotary shaker at 200 rev/min. The strain was harvested by centrifugation and diluted 20 times (106 –107 CFU/mL). The samples Gel9-2, Gel9-4, Gel9-6, Gel25-2, Gel25-4, and Gel25-6 were cut into disk with the weight of 0.02 g and then put into flasks with 50 mL bacteria suspension, respectively. The flasks were held in a shaking table at 30 ◦ C for 24 h, and then about 3 mL of suspension was taken out for OD600 nm measurement. The results were recorded in an UV spectrophotometer (UV-6100PCS, Shanghai, China). Three parallel samples were adopted for each experimental group to ensure the correction of experimental results, and all the conditions were kept sterile. A control culture without sample was also treated in the similar way.
Table 1). The molecular weight of cellulose was 9.4 × 104 and the QC/cellulose weight ratio was fixed to be 9:1. The appearance of QC/cellulose hydrogels are shown in Fig. 2, where the hydrogels were swollen and transparent. The size of hydrogels after equilibrium swelling which prepared in the same models increased from Gel25-2 to Gel 25-6, with the increase of DS of QC in hydrogel networks from 0.28 to 0.69. Fig. 3 shows the FTIR spectra of the cellulose hydrogel, QC, and QC/cellulose hydrogel. The broad band at about 3350 cm−1 was assigned to O H stretching vibration, which can be found in all samples. The sharp peak around 2900 cm−1 in the spectra of samples included the antisymmetric and symmetric stretching
3. Results and discussion 3.1. Appearance and structure of QC/cellulose hydrogels Fig. 1 shows the proposed mechanism for cross-linking reaction of quaternized cellulose (QC) and cellulose in alkali aqueous solution. Epichlorohydrin was used as a difunctional cross-linker which could react with the hydroxyl groups of QC and cellulose. Six hydrogel samples were synthesized and named as Gel9-2, Gel9-4, Gel9-6, Gel25-2, Gel25-4, and Gel25-6, respectively, according to the molecular weight (Mw = 9.4 × 104 and 2.56 × 105 ) and degree of substitution (from 0.23 to 0.69) of QC used in this work (see
Fig. 3. FTIR spectra of the cellulose hydrogel (Gel0), QC (QC25-6), and QC/cellulose hydrogels (Gel25-6).
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Fig. 6. The cytotoxicity tests of QC/cellulose hydrogels. Fig. 4. Equilibrium swelling ratio of QC/cellulose hydrogels in distilled water at room temperature.
vibration of CH2 groups. The most striking difference between cellulose hydrogel and other samples was the peak at 1482 cm−1 , which could not be found in the spectrum of cellulose hydrogel corresponding to the methyl groups of ammonium (Song, Sun, Zhang, Zhou, & Zhang, 2008). Moreover, the presence of another additional absorption at 1413 cm−1 was assignable to the C N stretching vibration in spectrum of QC (Pal, Mal, & Singh, 2005). These results indicated that a cationic moiety has been incorporated onto the backbone of cellulose, and quaternary ammonium salt groups existed in QC/cellulose hydrogel networks. 3.2. Swelling and mechanical properties of QC/cellulose hydrogels The swelling ratio of QC/cellulose hydrogels are listed in Table 1, ranging from 206.6 g/g to 983.9 g/g, which showed superabsorbent properties and far exceeded that of cellulose hydrogels (Chang, Zhang, Zhou, Zhang, & Kennedy, 2010). This phenomena could be explained by the presence of positive charges (cationic quaternary ammonium) in the hydrogel networks, where the electrostatic repulsion of positive charges expanded the hydrogel networks and improved the swelling ratio of samples. To investigate the roles of DS and molecular weight of QC in the swelling properties of hydrogel, the swelling ratio of QC/cellulose hydrogels are shown in Fig. 4. It can be found that the samples containing QC with higher molecular weight exhibited higher swelling ratio than those containing lower molecular weight QC with similar DS values. This result indicated that high molecular weight QC could combine
with more water in hydrogel networks, leading to higher swelling ratio of hydrogel samples. On the other hand, the swelling ratio of hydrogels increased with the increasing of DS of QC. The higher DS of QC raised the numbers of positive charges in the hydrogel networks, resulting in the expansion of the networks by the electrostatic repulsion of cationic quaternary ammonium in the hydrogels, leading to the improvement of their swelling ratio. For the mechanical properties of samples, compression tests are conducted in this section. The QC/cellulose hydrogels are elastic and their compressive stress–strain curves are shown in Fig. 5. For Gel9 series, when the DS of QC increased from 0.23 to 0.61, the compressive stress decreased from 28.7 kPa to 11.2 kPa. While the similar tendency could also be found in Gel25 series, that is, when the DS of QC increased from 0.28 to 0.69 for Gel25 series, the compressive stress decreased from 30.6 kPa to 2.11 kPa. These results showed that the DS of QC played an important role in the compressive stress of hydrogel. However, the mechanical properties of samples did not depend on molecular weight of QC in the hydrogels, which was different with traditional cellulose hydrogels (Cai & Zhang, 2006). It could be explained by the superabsorbent properties of hydrogels, which could hold a lot of water several hundred times of their own weight, resulting in the independence of QC molecular weight. 3.3. Biocompatibility and antimicrobial activity of QC/cellulose hydrogels To explore the cytocompatibility of QC/cellulose hydrogels, the normal human hepatic L02 cells was used as model cell lines. From the results of L02 cells culture experiment, we found that L02 cells
Fig. 5. Compressive stress–strain curves of QC/cellulose hydrogels.
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Fig. 7. Antibacterial activity of QC/cellulose hydrogel samples against S. cerevisiae: Pictures of S. cerevisiae growth on different samples (left) and OD600 nm value of S. cerevisiae after a 24 h culture.
could adhere to the surface of hydrogels and their spreading and proliferation on the surface were also realized. After 48 h incubation, the cells seed on the QC/cellulose hydrogels exhibited normal morphology compared to the control. Fig. 6 shows the results of cytotoxicity tests on the hydrogels. It can be seen that the cell viability values on the all hydrogel samples were greater than 80%, indicating good biocompatibility to L02 cells. The cytotoxicity of QC has been reported which gradually increased with the increasing of its DS value due to the presence of a higher amount of cationic cellulose damaging the cellular membranes (Song et al., 2008). Therefore, the lower cytotoxicity of QC/cellulose hydrogels observed in this work confirmed that the combination of QC and cellulose in the hydrogel networks could improve the cytocompatibility of samples, leading to better compatibility of materials. The antibacterial property of QC/cellulose hydrogel was tested in a 24 h growth inhibition assay using S. cerevisiae. Cellulose hydrogel was also evaluated in parallel as control. One hydrogel disk was placed in flask and submerged in yeast extract peptone dextrose medium inoculated with S. cerevisiae. Fig. 7 shows the pictures of S. cerevisiae growth on hydrogel samples with diluted culture medium. After 24 h incubation at 30 ◦ C, the colony forming unit (CFU) of S. cerevisiae cultured on Gel9-4, Gel9-6, and Gel256 declined obviously compared to that cultured on control. The OD600 nm of the supernatant of each samples was also measured and recorded as an indication for bacteria population density (Fig. 7, right). All hydrogels showed S. cerevisiae growth inhibition compared to the control. The survival rate of S. cerevisiae on QC/cellulose hydrogels were 88%, 33.3%, 33.6%, 87.1%, 66.9%, and 19.9% for Gel9-2, Gel9-4, Gel9-6, Gel25-2, Gel25-4, and Gel25-6, respectively. Gel25-6 exhibited the best antibacterial activity among all hydrogel samples, due to the presence of QC with the highest DS in the hydrogel networks. Therefore, the mechanism of antimicrobial activity of Gel25-6 sample could be explained by that attraction of sections of anionic microbial membrane into internal pores of poly cationic hydrogel led to the disruption of microbial membrane and the death of microbial (Li et al., 2011). 4. Conclusions We demonstrated a paradigm to fabricate novel hydrogels with antimicrobial activity using chemical crosslinking of natural polymer and its cationic derivative. The transparent hydrogels exhibited superabsorbent property, good mechanical performance, and biocompatibility. In the hydrogel networks, quaternized cellulose not only played an important role in the improvement of the swelling ratio of samples by the electrostatic repulsion of quaternary ammonium groups, but also endowed them with antimicrobial activity through the interaction between anionic microbial membrane and poly cationic hydrogel resulting in the death of microbial. The
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