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Novel bioactive surface functionalization of bacterial cellulose membrane
Carbohydrate Polymers 178 (2017) 270–276
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Research paper
Novel bioactive surface functionalization of bacterial cellulose membrane a,⁎
a
a
a
a
Wei Shao , Jimin Wu , Hui Liu , Shan Ye , Lei Jiang , Xiufeng Liu a b
b,⁎
MARK
College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, PR China State Key Laboratory of Natural Medicines, Department of Biotechnology of TCM, China Pharmaceutical University, Nanjing 210009, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Bacterial cellulose Aminoalkylsilane group Antibacterial activity Biocompatibility
Bacterial cellulose (BC) membrane is a promising biopolymer which can be used for tissue implants, wound healing, and drug delivery due to its unique properties, such as high crystallinity, high mechanical strength, ultrafine fiber network structure, good water holding capacity and biocompatibility. However, BC does not intrinsically present antibacterial properties. In the present study, functionalized BC membranes were prepared. FTIR, SEM and XPS were used to characterize the chemical composition and surface morphology. Static water contact angles were measured to investigate surface wettability. Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans were used to evaluate the antibacterial properties of membranes. HEK293 cell lines were applied to assess the biocompatibility of membrane surfaces by MTT assay and their morphologies were observed by Confocal Microscopy. Interestingly, the resultant functionalized BC membranes exhibiting excellent antibacterial property and good biocompatibility demonstrated great utility and potential as biomaterial materials.
1. Introduction Bacterial cellulose (BC) membrane is a polysaccharide produced mainly by the acetic acid bacterium Gluconacetobacter xylinus (formerly Acetobacter xylinus) (Li, Schulz, Ackley, & Fenske, 2010). BC has a unique micromorphology with its distinctive three-dimensional structure consisting of an ultrafine network of cellulose nanofibers (Czaja, Young, Kawecki, & Brown, 2007). BC displays many unique properties, such as high porosity, high crystallinity, excellent mechanical strength, large surface area, great water holding capacity and biocompatibility (Karuppuswamy, Reddy Venugopal, Navaneethan, Luwang Laiva, & Ramakrishna, 2015; Kenawy et al., 2002; Yang, Xie, Hong, Cao, & Yang, 2012). Based on its special structure and its excellent properties, BC has various innovative applications such as oil absorbents (Hoque, Konai, Sequeira, Samaddar, & Haldar, 2016), fuel cells (Lin, Liang, Chen, & Lai, 2013) and catalyst industry (Chen et al., 2015). Beside these areas, BC has been recently explored in the biomedical applications such as controlled drug delivery, scaffolds for cartilage tissue engineering, skin repair and wound dressings (Fu et al., 2012; Kirdponpattara, Khamkeaw, Sanchavanakit, Pavasant, & Phisalaphong, 2016; Numata, Mazzarino, & Borsali, 2015). However, BC is not able to prevent bacterial growth at all, resulting in failing to provide a barrier against infection, which limits the possibilities of applications in biomedical applications. Bacterial adhesion is the first step in the development of such infections. When bacteria attach to a biomedical
⁎
material, a biofilm forms after a multistep process. It is very difficult to treat clinically because the bacteria on the interior of the biofilm are protected from phagocytosis and antibiotics (Shao & Zhao, 2010). Thereby, an alternative strategy is required to control infections. Since bacterial adhesion to biomaterial surfaces is the primary step in the process of infections, modifications to biomaterial surfaces are considered to be the first choice in order to diminish infections by inhibiting initial bacterial adhesion (Katsikogianni & Missirlis, 2004). Antibacterial biopolymer have been obtained by incorporating active agents (Ringot, Sol, Granet, & Krausz, 2009), such as quaternary ammonium salts (Poverenov et al., 2013), N-halamines (Li et al., 2014), guanidine polymers (Kukharenko et al., 2014), antibiotics (Bajpai, Pathak, & Soni, 2015), molecularly engineered peptides (Fan et al., 2014) or compounds releasing bactericidal moieties such as metal ions (Rathore, Sharma, Pathania, & Gupta, 2014; Shao, Liu, Liu, Sun, 2015a,2015; Wan & Li, 2015). However, the resultant materials possess some limitations in their applications because of the easy loss of antibacterial properties in non-covalent systems, the release of environmentally hazardous leached agents and emergence of antibacterial resistance (Mbakidi et al., 2013). To overcome these major drawbacks, chemical modification of the biopolymer structure is necessary. Chemical grafting of bioactive moieties onto the surface is an attractive strategy to form novel surfaces with permanent biocide activity. Recent studies on surface functionalization of cellulosic substrates via covalent link have been reported. The grafting of acrylamide onto the cellulose
Corresponding authors. E-mail addresses: [email protected] (W. Shao), [email protected] (X. Liu).
http://dx.doi.org/10.1016/j.carbpol.2017.09.045 Received 30 June 2017; Received in revised form 11 September 2017; Accepted 13 September 2017 Available online 14 September 2017 0144-8617/ © 2017 Elsevier Ltd. All rights reserved.
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(Thermo Fisher, USA) using Al Ka radiation (1486.6 eV, 150 W). The base pressure was less than 10−8 Torr. Static water contact angles were measured using the sessile drop method with a JC2000D contact angle analyzer (Powereach, China). Five contact angle measurements were made on each sample.
surface followed by entrapment of silver nanoparticles results in development of a novel antibacterial biomaterial which can be used as an antibacterial packaging material (Tankhiwale & Bajpai, 2009). The poly (2-(dimethylamino)ethyl methacrylate)-grafted cellulose fibers were prepared by reversible addition-fragmentation chain transfer polymerization and displayed particularly high antibacterial activity (Roy, Knapp, Guthrie, & Perrier, 2008). Aminoalkyl moieties have potent antibacterial activity and were reported to be effective against bacteria. In addition, cationic aminoalkyl groups exhibit low toxicity and good environmental stability (Baâzaoui et al., 2015; Zobel et al., 1999). In this study, the aminoalkylsilane-grafted BC (A-g-BC) membranes were fabricated by chemical grafting method. The formed A-g-BC membranes were characterized using Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscope (SEM) and X-ray Photoelectron Spectroscopy (XPS). The wettability was evaluated by measuring static water contact angles. Antibacterial activities of A-g-BC membranes were investigated by plate count method against Gramnegative Escherichia coli (E. coli) ATCC 25922, Gram-positive Staphylococcus aureus (S. aureus) ATCC 6538, Bacillus subtilis (B. subtilis) ATCC 9372 and fungus Candida albicans (C. albicans) CMCC(F) 98001, respectively. In addition, the biocompatibility of A-g-BC membranes was further investigated.
2.4. Antibacterial activity The antibacterial activities of A-g-BC membranes were investigated by plate count method against E. coli ATCC 25922 and S. aureus ATCC 6538, B. subtilis ATCC 9372 and C. albicans CMCC(F) 98001. A-g-BC membranes and BC (the control) were cut into round shapes with 10 mm diameter and sterilized by ultraviolet lamp for 60 min. The sterilized samples were put into the tank containing 60 mL bacterial suspension with the concentration of about 1 × 107 CFU/mL and incubated at 37 °C for 1 h under a gentle stirring at 20 rpm. Each sample was taken from the suspension using sterile forceps and was gently dipped into sterile de-ionized water to remove loosely bound bacteria. Then the bacteria adhered to each sample was removed to sterile glass beakers containing 10 mL sterile de-ionized water by ultrasonication for 5 min. 100 μL of the sonication suspension and 10−1, 10−2 and 10−3 dilutions were plated out on TSA plates and incubated overnight at 37 °C. The colonies were counted on the following day. The total number of bacteria in 100 μL of bacterial suspension was obtained for each concentration and hence the total number of bacteria as colony-forming units (CFU)/cm2 attached to the tested sample as CFU was obtained. The experiments were carried out in triplicate to confirm reproducibility, then the average values and error bars were calculated. Antibacterial ratio was calculated with the following formula:
2. Experimental 2.1. BC preparation BC was prepared in a static culture medium by Acetobacter xylinum GIM1.327, which was purchased from BNBio Tech Co., Ltd, China. Briefly, in a static culture system enriched with polysaccharides, bacterial strain was incubated at 30 °C for 5 days and was able to produce a thin layer of BC in the interface of liquid/air (Kirdponpattara et al., 2016). This layer was washed by de-ionized water and then boiled in a 0.1 M NaOH solution at 80 °C for 60 min to eliminate impurities such as medium components and attached cells. BC membrane was further washed with de-ionized water until pH became neutral.
Ratio = (N0–N1)/N0 × 100%
where N0 is the number of attached bacteria on BC and N1 is the number of attached bacteria on A-g-BC membranes. 2.5. Cytotoxicity tests Human embryonic kidney 293 cells (HEK293; ATCC) were cultured in RPMI medium supplemented with 10% FBS, 100 μg/mL penicillin and 100 μg/mL streptomycin. The cytotoxicity was measured using the MTT assay method. The cytotoxicity was measured using the MTT assay. 200 μL of HEK293 cells, at a density of 1 × 105, were placed in each well of a 24-well plate. Then the cells were incubated over night at 37 °C in a humidified 5% CO2-containing atmosphere. A-g-BC membranes with same size (5 mm × 5 mm) were placed slightly in the transwell chambers and then fresh media was added. Wells containing only the cells were used as control. The cells were treated for another 24 h. Then the transwell chambers with samples were removed. The media in plate was changed with fresh media and 20 μL of dimethyl thiazolyldiphenyl (MTT) was added and the incubation continued for 6 h. Medium was removed, and 200 μL DMSO was added to each well to dissolve the formazan. The absorbance was measured with a test wavelength of 570 nm and a reference wavelength of 630 nm. Empty wells (DMSO alone) were used as blanks. The relative cell viability was measured by comparison with the control well containing only the cells. On the other hand, HEK293 cells were plated on the confocal culture dish. As cells reached to 30% confluence in all groups then the cells were treated with A-g-BC membranes for another 24 h. Cells were fixed and stained with FITC-Phalloidin and DAPI. The morphologies were visualized by Confocal Microscopy (Leica DM2500, Germany).
2.2. Production of A-g-BC membranes A-g-BC membranes were prepared via alkoxysilane polycondensation using (3-aminopropyl)triethoxysilane (APTES, Sinopharm Chemical Reagent Co. Ltd.). In brief, BC membranes were cut into 25 mm × 25 mm pieces and immersed in a previously prepared solution of 2, 4, 6, 8 and 10 wt.% APTES in ethanol (5 mL) and the mixture was gently stirred at 20 rpm at 25 °C for 4 h. The modified BC membranes were washed with ethanol to remove the unreacted APTES and other impurities. Then they were freeze-dried at −40 °C for 24 h. The final Ag-BC membrane films were named as BC2, BC4, BC6, BC8 and BC10, respectively. The grafting yield (GY) of grafted BC membrane was determined gravimetrically using Eq. (1). The experiments were carried out in five times replicate.
Grafting Yield =
(m1 − m 0) × 100% m0
(2)
(1)
where m0 and m1 represent the weight of dried BC membrane (mg) and dried grafted BC membrane (mg), respectively. 2.3. Characterization A JSM-7600F SEM operating at an accelerating voltage of 10–15 kV was used to investigate the surface morphologies of BC and A-g-BC membranes. The samples were coated with a thin layer of gold under high vacuum conditions (20 mA, 100 s). FTIR spectra were recorded on a Spectrum Two Spectrometer (Perkin Elmer, USA) with the wavenumber range of 4000–400 cm−1 at a resolution of 4 cm−1. XPS measurements were carried out with Thermo Escalab 250Xi instrument
3. Results and discussion 3.1. Functionalization mechanism Synthesis mechanism of the chemical grafting of aminoalkylsilane 271
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Scheme 1. Synthesis mechanism of aminoalkylsilane groups grafted BC membrane.
Table 1 The grafting yields, zeta potential and antibacterial ratios of A-g-BC membranes. Sample
BC BC2 BC4 BC6 BC8 BC10
Grafting Yield (%)
0 0.12 0.38 0.72 2.16 3.05
± 0.01 ± 0.01 ± 0.03 ± 0.02 ± 0.04
Zeta potential (mV)
−15.37 ± 0.15 2.69 ± 0.10 15.83 ± 0.87 22.83 ± 1.88 44.63 ± 1.83 47 ± 3.02
Anti-bacterial Ratio (%) E.coli
S.aureus
0 46 ± 4.1 82.7 ± 1.6 95.5 ± 0.5 98.3 ± 0.4 100
0 74.3 92.1 97.8 98.3 99.4
± ± ± ± ±
B.subtilis
0.2 0.5 0.3 0.1 0.1
0 96.3 98.6 99.3 99.7 99.9
± ± ± ± ±
C.albicans
0.6 0.2 0.1 0.1 0.1
0 61.9 75.6 93.2 98.1 98.7
± ± ± ± ±
2.2 1.0 1.2 0.1 0.1
peak intensities at 2864 cm−1 associated with the CH2 vibrations of the propyl moiety of the silane moiety become more evident with increasing grafting yield of A-g-BC. The typical vibrations of SieOeC and SieOeSi bridges appear at around 1150 and 1135 cm−1, are hardly seen in the curves since their peaks should be masked by the large and intense CeOeC vibration bands of BC (Fernandes et al., 2013; Meng et al., 2015).
groups onto BC membrane was shown in Scheme 1. It involves three steps: (i) APTES hydrolyzed into its corresponding silanol; (ii) the adsorption of silanol onto BC matrix through hydrogen bonding; (iii) siloxane bridges (SieOeSi) grafted onto BC membrane surface through SieOeC bonds via chemical condensation method. Therefore, A-g-BC membranes were prepared via forming a polysiloxane network form on the BC membrane surface (Fernandes et al., 2013; Meng et al., 2015). The grafting yields are listed in Table 1 and the result shows that the grafting yield increased from 0.12 to 3.05% with increasing the aminoalkylsilane moieties in the system.
3.3. Surface morphology The morphologies of prepared BC and BC10 membranes were analyzed using SEM (Fig. 1). Fig. 1a and c show the surface and crosssection morphologies of BC, respectively. BC exhibited a nanoporous three-dimensional web-like structure (Kenawy et al., 2002) with a random arrangement of ribbon-shaped nanofibrils without any preferential orientation, resulting in a large surface area and high porosity. In the case of BC10 membrane, a thick and dense morphology was observed because of the coverage with aminoalkylsilane groups (Fig. 1b). This morphology is identical with FTIR spectrum result that Si-O-Si bridges form throughout the BC matrix. It is worthy to note that the cross-section morphology of BC10 became to a lamellar structure with highly ordered fibers after being grafted (Fig. 1d). These phenomenon could be explained due to the increase of repulsive forces between fibers with the fact that the absolute value of zeta potential of BC increases from 15.37 mV to 47 mV. The detailed zeta potentials of BC and A-g-BC are listed in Table 1. The EDS element maps of BC10 membrane were displayed in Fig. 1e and f, which showed that the Si and N
3.2. FTIR measurements FTIR analysis was performed to compare BC membranes before and after grafting aminoalkylsilane groups (Fig. S1). In the case of BC (curve a), the FTIR spectrum was typical and the dominating signal is at 3200–3500 cm−1, corresponding to the intramolecular hydrogen bond for 3O⋯HeO5 and the hydroxyl group (Feng, Zhang, Shen, Yoshino, & Feng, 2012; Kumar, Rao, Kwon, Lee, & Han, 2017; Shao et al., 2015a, 2015b). In the CeO stretching vibration region, the adsorption between 1170 and 1150 cm−1 corresponds to the CeOeC pyranose ring skeletal vibration (Meng et al., 2015). For A-g-BC membranes (curves b–f), two new bands at 1560 cm−1 and 780 cm−1 appear that are attributed to the bending of primary amino groups (NH2) and stretching vibration of Si-O-Si, respectively (Kumar, Rao, & Han, 2017), which comes from APTES, thus verifying the successful grafting of aminoalkylsilane groups onto the BC membrane. In addition, the 272
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Fig. 1. SEM surface images of BC (a) and BC10 (b) membranes, cross-section images of BC (c) and BC10 (d) membranes, Si element map (e) and N element map (f) of BC10 membranes.
elements from aminoalkylsilane groups were distributed uniformly. Herein, the aminoalkylsilane groups were successfully grafted onto BC membranes. 3.4. XPS analysis The presence of amine and silane functionalities of BC membrane, which could represent covalent successfully, was also confirmed by XPS analysis. In Fig. 2, the survey spectra clearly indicate there were two major peaks at 285.7 eV and 531.7 eV correspond to C 1s and O 1s adsorptions in the BC membrane. For A-g-BC (BC10) membrane, a new peak at 398.8 eV in the N 1s region was observed (Yu, Ge, Atewologun, López, & Stiff-Roberts, 2014). Moreover, two new peaks appeared at 101.8 eV and 152.8 eV corresponding to the binding energies of Si 2s
Fig. 2. XPS survey scans of BC and A-g-BC (BC10) membranes, and high-resolution XPS spectra of N 1s and Si 2p regions of BC10 membrane.
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and 2p (Kim, Park, & Lee, 2011). The corresponding composition of BC and BC10 membranes is listed in Table S1. It can be seen that carbon content increases for BC10 membrane which is due to the existence of aminoalkylsilane group. Curve fitting of the C 1s spectra of BC and BC10 membrane is shown in Figs. S2a and b. The deconvolution of the C 1s levels of BC membrane produce three peaks at 284.4, 286, and 288.2 eV, which correspond to C1 (CeC, CeH), C2 (CeO), and C3 (OeCeO), respectively (Figs. S2 a) (Tang et al., 2016; Vega-Figueroa et al., 2017). For BC10 membrane, the relative intensity of the C1 peak increased and C1 peak decreased (Figs. S2 b). The enhanced C1 and weakened C2 signals are due to the successful grafting aminoalkylsilane groups. High-resolution XPS spectra of BC10 membrane confirming the grafted aminoalkylsilane groups via N and Si are also shown in Fig. 2. The successful grafting of APTES onto BC was confirmed by the N 1s spectrum showing a peak which is attributed to nitrogen from NH2 terminal groups on APTES at 399.2 eV (Xue, Zhou, Zhao, Lu, & Han, 2011; Zhuang et al., 2017). The appearance of Si 2p spectrum at about 102.3 eV in BC10 membrane was assigned to Si of APTES (Manakhov, Čechal, Michlíček, & Shtansky, 2017), thereby confirming the successful grafting of aminoalkylsilane group onto BC membrane. The same result was achieved with FTIR analysis. 3.5. Surface wettability The surface wettability behavior of A-g-BC membranes was investigated by measuring static water contact angles. The obtained contact angles and the water drop profiles are shown in Fig. 3. As it is clearly showed, the grafting yield plays an important role in the hydrophobicity of BC membranes. BC is a very hydrophilic material with the contact angle of water 48.3° over its surface. With the increase of grafting yield of the BC membranes, there is an increase of the contact angle due to the covalent linkage with hydrophobic aminoalkylsilane groups. The contact angles of water over A-g-BC membranes are in the range of 59.3–86.4°. These data prove that the hydrophobicity of BC membrane is considerably increased via grafting of APTES. It was reported that the increase of hydrophobicity is advantageous to the enhanced antibacterial performance (Hoque, Konai, Sequeira, Samaddar, & Haldar, 2016). 3.6. Antibacterial and antifungal activities The antibacterial and antifungal activities of BC and A-g-BC membranes were investigated. Fig. 4 shows the numbers of bacteria colonies attached to the A-g-BC membrane and to pristine BC at 37 °C after a contact time of 1 h. The A-g-BC membranes performed much better than BC in reducing bacterial attachment, and it was found that bacterial adhesion decreased with increasing graft yield of the BC membrane. The calculated antibacterial ratios are listed in Table 1. BC10 membranes could reduce E. coli attachment by 100%, S. aureus attachment by 99.4% and B. subtilis attachment by 99.9% at 1 h
Fig. 4. Number of bacteria adhesion onto A-g-BC membranes: (A) E. coli, (B) S. aureus, (C) B. subtilis and (D) C. albicans.
compared with pristine BC, respectively. In general, these results indicate that A-g-BC membranes have excellent antibacterial activities against Gram negative E. coli, Gram positive S. aureus and S. subtilis. Moreover, great antifungal property against C. albicans of A-g-BC membranes has been established.
Fig. 3. Static water contact angles of BC and A-g-BC membranes.
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Clearly, the strong antibacterial activity is due to the introduction of aminoalkylsilane groups into BC matrix. A-g-BC membranes become polycationic after functionalized with aminoalkylsilane groups (Table S2). Thus, polycationic A-g-BC membranes with extremely high charge density can easily adsorb onto the negatively charged bacterial cell membrane by electrostatic interaction effect, thus, leading to the disruption of the bacterial cell membrane. In addition, the grafted hydrophobic aminoalkylsilane groups can affect the mode of surface interaction between functionalized BC and the cytoplasmic membrane of the bacteria though binding to the cytoplasmic membrane. Thus, the disruption of membrane and subsequent leakage of K+ ions and other cytoplasmic constituents can lead to the death of bacteria (Meng et al., 2015; Tashiro, 2001). Combining all beneficial qualities, the prepared A-g-BC membranes possess broad spectrum of antibacterial property, making them especially promising biomaterials which can be used in various biomedical applications.
3.7. Cytotoxicity of A-g-BC membranes Cellular growth and proliferation on surfaces are a symbol of cytocompatibility for materials which can be used to assess the potential of materials for application in tissue engineering (Shao et al., 2015a, 2015b). Cytotoxicity studies were performed to investigate the effect of grafting yield of the BC membrane on proliferation of HEK293 cell lines. The effect of pristine BC was evaluated in vitro to ensure that the grafted BC did not have an independent toxicity effect. The cell viability of HEK293 cells was evaluated by MTT assay. The cell cytotoxicity imparted by A-g-BC membranes being placed slightly in the transwell chambers was studied. HEK293 cells were treated for 24 h and then the transwell chambers with samples were removed. The MTT results were illustrated in Fig. 5 as relative viability of the cells by comparison with the control well containing only the cells. All the materials showed negligible toxicity. No obvious reduced cell viability following their incubation with A-g-BC membranes was shown. The results show that grafted BC did not inhibit the growth of HEK293 cells, even at a high concentration because HEK293 cells do not seem to be affected from their incubation with A-g-BC membranes. The relative cell viability is slightly decreased with grafting yield increasing of the BC matrix. However, it is still in the considerable range that no obvious cell cytotoxicity was detected. The possible reason for the slightly lower cell viability could be due to the morphology differences between pristine BC and functionalized BC, which is shown in Fig. 1. As it is shown that BC has a 3D porous network, therefore it is ideal for harboring cell growth. In the case of A-g-BC membranes, the introduction of aminoalkylsilane groups onto the BC nanofibrills reduces the membranes porosity, which leads to the lower cell viability. To determine whether the morphology of HEK293 cells was affected by A-g-BC membranes, the cells were stained with FITC-Phalloidin (green) and DAPI (blue), and then observed using Confocal Microscopy
Fig. 6. Cell morphologies treated with A-g-BC membranes.
(Fig. 6). The morphologies of HEK293 cells treated with A-g-BC membranes were similar to the blank one. Thus, the prepared A-g-BC membranes display no effect on the morphology of HEK293 cells. These results show that A-g-BC membranes are promising candidates for biomedical applications. 4. Conclusion In summary, antibacterial and biocompatible A-g-BC membranes were fabricated via grafting aminoalkylsilane groups. A-g-BC membrane displayed a thick and dense network structure compared to nanoporous three-dimensional network structure of BC. The hydrophobicity of BC membrane increases by grafting aminoalkylsilane groups. A-g-BC membranes display effective antibacterial and antifungal activities against E. coli, S. aureus, S. subtilis and C. albicans, which could be used as promising antibacterial materials in combating challenging pathogenic infection. Furthermore, A-g-BC membranes possess excellent biocompatibility. Therefore, the developed A-g-BC membranes have great potential applications in biomedical applications. Acknowledgements The work was financially supported by the Natural Science Foundation of Jiangsu Province (BK20161528), Postgraduate
Fig. 5. Cell viability percentages treated with A-g-BC membranes for 24 h.
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Research & Practice Innovation Program of Jiangsu Province (KYZZ16_0320, KYCX17_0847) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.2017.09.045. References Baâzaoui, M., Béjaoui, I., Kalfat, R., Amdouni, N., Hbaieb, S., & Chevalier, Y. (2015). Preparation and characterization of nanoparticles made from amphiphilic mono and per-aminoalkyl-â-cyclodextrins. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 484, 365–376. Bajpai, S. K., Pathak, V., & Soni, B. (2015). Minocycline-loaded cellulose nano whiskers/ poly(sodium acrylate) composite hydrogel films as wound dressing. International Journal of Biological Macromolecules, 79, 76–85. Chen, M., Kang, H., Gong, Y., Guo, J., Zhang, H., & Liu, R. (2015). Bacterial cellulose supported gold nanoparticles with excellent catalytic properties. ACS Applied Materials & Interfaces, 7(39), 21717–21726. Czaja, W. K., Young, D. J., Kawecki, M., & Brown, J. R. M. (2007). The future prospects of microbial cellulose in biomedical applications. Biomacromolecules, 8(1), 1–12. Fan, L., Peng, M., Zhou, X., Wu, H., Hu, J., Xie, W., & Liu, S. (2014). Modification of carboxymethyl cellulose grafted with collagen peptide and its antioxidant activity. Carbohydrate Polymers, 112, 32–38. Feng, Y., Zhang, X., Shen, Y., Yoshino, K., & Feng, W. (2012). A mechanically strong, flexible and conductive film based on bacterial cellulose/graphene nanocomposite. Carbohydrate Polymers, 87(1), 644–649. Fernandes, S. C., Sadocco, P., Alonso-Varona, A., Palomares, T., Eceiza, A., Silvestre, A. J., ... Freire, C. S. (2013). Bioinspired antimicrobial and biocompatible bacterial cellulose membranes obtained by surface functionalization with aminoalkyl groups. ACS Applied Materials & Interfaces, 5(8), 3290–3297. Fu, L., Zhang, Y., Li, C., Wu, Z., Zhuo, Q., Huang, X., ... Yang, G. (2012). Skin tissue repair materials from bacterial cellulose by a multilayer fermentation method. Journal Of Materials Chemistry, 22(24), 12349–12357. Hoque, J., Konai, M. M., Sequeira, S. S., Samaddar, S., & Haldar, J. (2016). Antibacterial and antibiofilm activity of cationic small molecules with spatial positioning of hydrophobicity: An in vitro and in vivo evaluation. Journal of Medicinal Chemistry, 59(23), 10750–10762. Karuppuswamy, P., Reddy Venugopal, J., Navaneethan, B., Luwang Laiva, A., & Ramakrishna, S. (2015). Polycaprolactone nanofibers for the controlled release of tetracycline hydrochloride. Materials Letters, 141, 180–186. Katsikogianni, M., & Missirlis, Y. F. (2004). Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating bacteria-material interactions. European Cells & Materials, 8, 37–57. Kenawy, E.-R., Bowlin, G. L., Mansfield, K., Layman, J., Simpson, D. G., Sanders, E. H., & Wnek, G. E. (2002). Release of tetracycline hydrochloride from electrospun poly (ethylene-co-vinylacetate), poly(lactic acid), and a blend. Journal of Controlled Release, 81, 57–64. Kim, S. S., Park, J. E., & Lee, J. (2011). Properties and antimicrobial efficacy of cellulose fiber coated with silver nanoparticles and 3-mercaptopropyltrimethoxysilane (3MPTMS). Journal of Applied Polymer Science, 119(4), 2261–2267. Kirdponpattara, S., Khamkeaw, A., Sanchavanakit, N., Pavasant, P., & Phisalaphong, M. (2016). Structural modification and characterization of bacterial cellulose-alginate composites caffolds for tissue engineering. Carbohydrate Polymers, 132, 146–155. Kukharenko, O., Bardeau, J.-F., Zaets, I., Ovcharenko, L., Tarasyuk, O., Porhyn, S., ... Kozyrovska, N. (2014). Promising low cost antimicrobial composite material based on bacterial cellulose and polyhexamethylene guanidine hydrochloride. European Polymer Journal, 60, 247–254. Kumar, A., Rao, K. M., & Han, S. S. (2017). Synthesis of mechanically stiff and bioactive hybrid hydrogels for bone tissue engineering applications. Chemical Engineering Journal, 317, 119–131. Kumar, A., Rao, K. M., Kwon, S. E., Lee, Y. N., & Han, S. S. (2017). Xanthan gum/bioactive silica glass hybrid scaffolds reinforced with cellulose nanocrystals: Morphological, mechanical and in vitro cytocompatibility study. Materials Letters, 193, 274–278. Li, Z., Schulz, L., Ackley, C., & Fenske, N. (2010). Adsorption of tetracycline on kaolinite with pH-dependent surface charges. Journal of Colloid and Interface Science, 351(1),
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Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Research paper
Novel bioactive surface functionalization of bacterial cellulose membrane a,⁎
a
a
a
a
Wei Shao , Jimin Wu , Hui Liu , Shan Ye , Lei Jiang , Xiufeng Liu a b
b,⁎
MARK
College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, PR China State Key Laboratory of Natural Medicines, Department of Biotechnology of TCM, China Pharmaceutical University, Nanjing 210009, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Bacterial cellulose Aminoalkylsilane group Antibacterial activity Biocompatibility
Bacterial cellulose (BC) membrane is a promising biopolymer which can be used for tissue implants, wound healing, and drug delivery due to its unique properties, such as high crystallinity, high mechanical strength, ultrafine fiber network structure, good water holding capacity and biocompatibility. However, BC does not intrinsically present antibacterial properties. In the present study, functionalized BC membranes were prepared. FTIR, SEM and XPS were used to characterize the chemical composition and surface morphology. Static water contact angles were measured to investigate surface wettability. Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans were used to evaluate the antibacterial properties of membranes. HEK293 cell lines were applied to assess the biocompatibility of membrane surfaces by MTT assay and their morphologies were observed by Confocal Microscopy. Interestingly, the resultant functionalized BC membranes exhibiting excellent antibacterial property and good biocompatibility demonstrated great utility and potential as biomaterial materials.
1. Introduction Bacterial cellulose (BC) membrane is a polysaccharide produced mainly by the acetic acid bacterium Gluconacetobacter xylinus (formerly Acetobacter xylinus) (Li, Schulz, Ackley, & Fenske, 2010). BC has a unique micromorphology with its distinctive three-dimensional structure consisting of an ultrafine network of cellulose nanofibers (Czaja, Young, Kawecki, & Brown, 2007). BC displays many unique properties, such as high porosity, high crystallinity, excellent mechanical strength, large surface area, great water holding capacity and biocompatibility (Karuppuswamy, Reddy Venugopal, Navaneethan, Luwang Laiva, & Ramakrishna, 2015; Kenawy et al., 2002; Yang, Xie, Hong, Cao, & Yang, 2012). Based on its special structure and its excellent properties, BC has various innovative applications such as oil absorbents (Hoque, Konai, Sequeira, Samaddar, & Haldar, 2016), fuel cells (Lin, Liang, Chen, & Lai, 2013) and catalyst industry (Chen et al., 2015). Beside these areas, BC has been recently explored in the biomedical applications such as controlled drug delivery, scaffolds for cartilage tissue engineering, skin repair and wound dressings (Fu et al., 2012; Kirdponpattara, Khamkeaw, Sanchavanakit, Pavasant, & Phisalaphong, 2016; Numata, Mazzarino, & Borsali, 2015). However, BC is not able to prevent bacterial growth at all, resulting in failing to provide a barrier against infection, which limits the possibilities of applications in biomedical applications. Bacterial adhesion is the first step in the development of such infections. When bacteria attach to a biomedical
⁎
material, a biofilm forms after a multistep process. It is very difficult to treat clinically because the bacteria on the interior of the biofilm are protected from phagocytosis and antibiotics (Shao & Zhao, 2010). Thereby, an alternative strategy is required to control infections. Since bacterial adhesion to biomaterial surfaces is the primary step in the process of infections, modifications to biomaterial surfaces are considered to be the first choice in order to diminish infections by inhibiting initial bacterial adhesion (Katsikogianni & Missirlis, 2004). Antibacterial biopolymer have been obtained by incorporating active agents (Ringot, Sol, Granet, & Krausz, 2009), such as quaternary ammonium salts (Poverenov et al., 2013), N-halamines (Li et al., 2014), guanidine polymers (Kukharenko et al., 2014), antibiotics (Bajpai, Pathak, & Soni, 2015), molecularly engineered peptides (Fan et al., 2014) or compounds releasing bactericidal moieties such as metal ions (Rathore, Sharma, Pathania, & Gupta, 2014; Shao, Liu, Liu, Sun, 2015a,2015; Wan & Li, 2015). However, the resultant materials possess some limitations in their applications because of the easy loss of antibacterial properties in non-covalent systems, the release of environmentally hazardous leached agents and emergence of antibacterial resistance (Mbakidi et al., 2013). To overcome these major drawbacks, chemical modification of the biopolymer structure is necessary. Chemical grafting of bioactive moieties onto the surface is an attractive strategy to form novel surfaces with permanent biocide activity. Recent studies on surface functionalization of cellulosic substrates via covalent link have been reported. The grafting of acrylamide onto the cellulose
Corresponding authors. E-mail addresses: [email protected] (W. Shao), [email protected] (X. Liu).
http://dx.doi.org/10.1016/j.carbpol.2017.09.045 Received 30 June 2017; Received in revised form 11 September 2017; Accepted 13 September 2017 Available online 14 September 2017 0144-8617/ © 2017 Elsevier Ltd. All rights reserved.
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(Thermo Fisher, USA) using Al Ka radiation (1486.6 eV, 150 W). The base pressure was less than 10−8 Torr. Static water contact angles were measured using the sessile drop method with a JC2000D contact angle analyzer (Powereach, China). Five contact angle measurements were made on each sample.
surface followed by entrapment of silver nanoparticles results in development of a novel antibacterial biomaterial which can be used as an antibacterial packaging material (Tankhiwale & Bajpai, 2009). The poly (2-(dimethylamino)ethyl methacrylate)-grafted cellulose fibers were prepared by reversible addition-fragmentation chain transfer polymerization and displayed particularly high antibacterial activity (Roy, Knapp, Guthrie, & Perrier, 2008). Aminoalkyl moieties have potent antibacterial activity and were reported to be effective against bacteria. In addition, cationic aminoalkyl groups exhibit low toxicity and good environmental stability (Baâzaoui et al., 2015; Zobel et al., 1999). In this study, the aminoalkylsilane-grafted BC (A-g-BC) membranes were fabricated by chemical grafting method. The formed A-g-BC membranes were characterized using Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscope (SEM) and X-ray Photoelectron Spectroscopy (XPS). The wettability was evaluated by measuring static water contact angles. Antibacterial activities of A-g-BC membranes were investigated by plate count method against Gramnegative Escherichia coli (E. coli) ATCC 25922, Gram-positive Staphylococcus aureus (S. aureus) ATCC 6538, Bacillus subtilis (B. subtilis) ATCC 9372 and fungus Candida albicans (C. albicans) CMCC(F) 98001, respectively. In addition, the biocompatibility of A-g-BC membranes was further investigated.
2.4. Antibacterial activity The antibacterial activities of A-g-BC membranes were investigated by plate count method against E. coli ATCC 25922 and S. aureus ATCC 6538, B. subtilis ATCC 9372 and C. albicans CMCC(F) 98001. A-g-BC membranes and BC (the control) were cut into round shapes with 10 mm diameter and sterilized by ultraviolet lamp for 60 min. The sterilized samples were put into the tank containing 60 mL bacterial suspension with the concentration of about 1 × 107 CFU/mL and incubated at 37 °C for 1 h under a gentle stirring at 20 rpm. Each sample was taken from the suspension using sterile forceps and was gently dipped into sterile de-ionized water to remove loosely bound bacteria. Then the bacteria adhered to each sample was removed to sterile glass beakers containing 10 mL sterile de-ionized water by ultrasonication for 5 min. 100 μL of the sonication suspension and 10−1, 10−2 and 10−3 dilutions were plated out on TSA plates and incubated overnight at 37 °C. The colonies were counted on the following day. The total number of bacteria in 100 μL of bacterial suspension was obtained for each concentration and hence the total number of bacteria as colony-forming units (CFU)/cm2 attached to the tested sample as CFU was obtained. The experiments were carried out in triplicate to confirm reproducibility, then the average values and error bars were calculated. Antibacterial ratio was calculated with the following formula:
2. Experimental 2.1. BC preparation BC was prepared in a static culture medium by Acetobacter xylinum GIM1.327, which was purchased from BNBio Tech Co., Ltd, China. Briefly, in a static culture system enriched with polysaccharides, bacterial strain was incubated at 30 °C for 5 days and was able to produce a thin layer of BC in the interface of liquid/air (Kirdponpattara et al., 2016). This layer was washed by de-ionized water and then boiled in a 0.1 M NaOH solution at 80 °C for 60 min to eliminate impurities such as medium components and attached cells. BC membrane was further washed with de-ionized water until pH became neutral.
Ratio = (N0–N1)/N0 × 100%
where N0 is the number of attached bacteria on BC and N1 is the number of attached bacteria on A-g-BC membranes. 2.5. Cytotoxicity tests Human embryonic kidney 293 cells (HEK293; ATCC) were cultured in RPMI medium supplemented with 10% FBS, 100 μg/mL penicillin and 100 μg/mL streptomycin. The cytotoxicity was measured using the MTT assay method. The cytotoxicity was measured using the MTT assay. 200 μL of HEK293 cells, at a density of 1 × 105, were placed in each well of a 24-well plate. Then the cells were incubated over night at 37 °C in a humidified 5% CO2-containing atmosphere. A-g-BC membranes with same size (5 mm × 5 mm) were placed slightly in the transwell chambers and then fresh media was added. Wells containing only the cells were used as control. The cells were treated for another 24 h. Then the transwell chambers with samples were removed. The media in plate was changed with fresh media and 20 μL of dimethyl thiazolyldiphenyl (MTT) was added and the incubation continued for 6 h. Medium was removed, and 200 μL DMSO was added to each well to dissolve the formazan. The absorbance was measured with a test wavelength of 570 nm and a reference wavelength of 630 nm. Empty wells (DMSO alone) were used as blanks. The relative cell viability was measured by comparison with the control well containing only the cells. On the other hand, HEK293 cells were plated on the confocal culture dish. As cells reached to 30% confluence in all groups then the cells were treated with A-g-BC membranes for another 24 h. Cells were fixed and stained with FITC-Phalloidin and DAPI. The morphologies were visualized by Confocal Microscopy (Leica DM2500, Germany).
2.2. Production of A-g-BC membranes A-g-BC membranes were prepared via alkoxysilane polycondensation using (3-aminopropyl)triethoxysilane (APTES, Sinopharm Chemical Reagent Co. Ltd.). In brief, BC membranes were cut into 25 mm × 25 mm pieces and immersed in a previously prepared solution of 2, 4, 6, 8 and 10 wt.% APTES in ethanol (5 mL) and the mixture was gently stirred at 20 rpm at 25 °C for 4 h. The modified BC membranes were washed with ethanol to remove the unreacted APTES and other impurities. Then they were freeze-dried at −40 °C for 24 h. The final Ag-BC membrane films were named as BC2, BC4, BC6, BC8 and BC10, respectively. The grafting yield (GY) of grafted BC membrane was determined gravimetrically using Eq. (1). The experiments were carried out in five times replicate.
Grafting Yield =
(m1 − m 0) × 100% m0
(2)
(1)
where m0 and m1 represent the weight of dried BC membrane (mg) and dried grafted BC membrane (mg), respectively. 2.3. Characterization A JSM-7600F SEM operating at an accelerating voltage of 10–15 kV was used to investigate the surface morphologies of BC and A-g-BC membranes. The samples were coated with a thin layer of gold under high vacuum conditions (20 mA, 100 s). FTIR spectra were recorded on a Spectrum Two Spectrometer (Perkin Elmer, USA) with the wavenumber range of 4000–400 cm−1 at a resolution of 4 cm−1. XPS measurements were carried out with Thermo Escalab 250Xi instrument
3. Results and discussion 3.1. Functionalization mechanism Synthesis mechanism of the chemical grafting of aminoalkylsilane 271
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Scheme 1. Synthesis mechanism of aminoalkylsilane groups grafted BC membrane.
Table 1 The grafting yields, zeta potential and antibacterial ratios of A-g-BC membranes. Sample
BC BC2 BC4 BC6 BC8 BC10
Grafting Yield (%)
0 0.12 0.38 0.72 2.16 3.05
± 0.01 ± 0.01 ± 0.03 ± 0.02 ± 0.04
Zeta potential (mV)
−15.37 ± 0.15 2.69 ± 0.10 15.83 ± 0.87 22.83 ± 1.88 44.63 ± 1.83 47 ± 3.02
Anti-bacterial Ratio (%) E.coli
S.aureus
0 46 ± 4.1 82.7 ± 1.6 95.5 ± 0.5 98.3 ± 0.4 100
0 74.3 92.1 97.8 98.3 99.4
± ± ± ± ±
B.subtilis
0.2 0.5 0.3 0.1 0.1
0 96.3 98.6 99.3 99.7 99.9
± ± ± ± ±
C.albicans
0.6 0.2 0.1 0.1 0.1
0 61.9 75.6 93.2 98.1 98.7
± ± ± ± ±
2.2 1.0 1.2 0.1 0.1
peak intensities at 2864 cm−1 associated with the CH2 vibrations of the propyl moiety of the silane moiety become more evident with increasing grafting yield of A-g-BC. The typical vibrations of SieOeC and SieOeSi bridges appear at around 1150 and 1135 cm−1, are hardly seen in the curves since their peaks should be masked by the large and intense CeOeC vibration bands of BC (Fernandes et al., 2013; Meng et al., 2015).
groups onto BC membrane was shown in Scheme 1. It involves three steps: (i) APTES hydrolyzed into its corresponding silanol; (ii) the adsorption of silanol onto BC matrix through hydrogen bonding; (iii) siloxane bridges (SieOeSi) grafted onto BC membrane surface through SieOeC bonds via chemical condensation method. Therefore, A-g-BC membranes were prepared via forming a polysiloxane network form on the BC membrane surface (Fernandes et al., 2013; Meng et al., 2015). The grafting yields are listed in Table 1 and the result shows that the grafting yield increased from 0.12 to 3.05% with increasing the aminoalkylsilane moieties in the system.
3.3. Surface morphology The morphologies of prepared BC and BC10 membranes were analyzed using SEM (Fig. 1). Fig. 1a and c show the surface and crosssection morphologies of BC, respectively. BC exhibited a nanoporous three-dimensional web-like structure (Kenawy et al., 2002) with a random arrangement of ribbon-shaped nanofibrils without any preferential orientation, resulting in a large surface area and high porosity. In the case of BC10 membrane, a thick and dense morphology was observed because of the coverage with aminoalkylsilane groups (Fig. 1b). This morphology is identical with FTIR spectrum result that Si-O-Si bridges form throughout the BC matrix. It is worthy to note that the cross-section morphology of BC10 became to a lamellar structure with highly ordered fibers after being grafted (Fig. 1d). These phenomenon could be explained due to the increase of repulsive forces between fibers with the fact that the absolute value of zeta potential of BC increases from 15.37 mV to 47 mV. The detailed zeta potentials of BC and A-g-BC are listed in Table 1. The EDS element maps of BC10 membrane were displayed in Fig. 1e and f, which showed that the Si and N
3.2. FTIR measurements FTIR analysis was performed to compare BC membranes before and after grafting aminoalkylsilane groups (Fig. S1). In the case of BC (curve a), the FTIR spectrum was typical and the dominating signal is at 3200–3500 cm−1, corresponding to the intramolecular hydrogen bond for 3O⋯HeO5 and the hydroxyl group (Feng, Zhang, Shen, Yoshino, & Feng, 2012; Kumar, Rao, Kwon, Lee, & Han, 2017; Shao et al., 2015a, 2015b). In the CeO stretching vibration region, the adsorption between 1170 and 1150 cm−1 corresponds to the CeOeC pyranose ring skeletal vibration (Meng et al., 2015). For A-g-BC membranes (curves b–f), two new bands at 1560 cm−1 and 780 cm−1 appear that are attributed to the bending of primary amino groups (NH2) and stretching vibration of Si-O-Si, respectively (Kumar, Rao, & Han, 2017), which comes from APTES, thus verifying the successful grafting of aminoalkylsilane groups onto the BC membrane. In addition, the 272
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Fig. 1. SEM surface images of BC (a) and BC10 (b) membranes, cross-section images of BC (c) and BC10 (d) membranes, Si element map (e) and N element map (f) of BC10 membranes.
elements from aminoalkylsilane groups were distributed uniformly. Herein, the aminoalkylsilane groups were successfully grafted onto BC membranes. 3.4. XPS analysis The presence of amine and silane functionalities of BC membrane, which could represent covalent successfully, was also confirmed by XPS analysis. In Fig. 2, the survey spectra clearly indicate there were two major peaks at 285.7 eV and 531.7 eV correspond to C 1s and O 1s adsorptions in the BC membrane. For A-g-BC (BC10) membrane, a new peak at 398.8 eV in the N 1s region was observed (Yu, Ge, Atewologun, López, & Stiff-Roberts, 2014). Moreover, two new peaks appeared at 101.8 eV and 152.8 eV corresponding to the binding energies of Si 2s
Fig. 2. XPS survey scans of BC and A-g-BC (BC10) membranes, and high-resolution XPS spectra of N 1s and Si 2p regions of BC10 membrane.
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and 2p (Kim, Park, & Lee, 2011). The corresponding composition of BC and BC10 membranes is listed in Table S1. It can be seen that carbon content increases for BC10 membrane which is due to the existence of aminoalkylsilane group. Curve fitting of the C 1s spectra of BC and BC10 membrane is shown in Figs. S2a and b. The deconvolution of the C 1s levels of BC membrane produce three peaks at 284.4, 286, and 288.2 eV, which correspond to C1 (CeC, CeH), C2 (CeO), and C3 (OeCeO), respectively (Figs. S2 a) (Tang et al., 2016; Vega-Figueroa et al., 2017). For BC10 membrane, the relative intensity of the C1 peak increased and C1 peak decreased (Figs. S2 b). The enhanced C1 and weakened C2 signals are due to the successful grafting aminoalkylsilane groups. High-resolution XPS spectra of BC10 membrane confirming the grafted aminoalkylsilane groups via N and Si are also shown in Fig. 2. The successful grafting of APTES onto BC was confirmed by the N 1s spectrum showing a peak which is attributed to nitrogen from NH2 terminal groups on APTES at 399.2 eV (Xue, Zhou, Zhao, Lu, & Han, 2011; Zhuang et al., 2017). The appearance of Si 2p spectrum at about 102.3 eV in BC10 membrane was assigned to Si of APTES (Manakhov, Čechal, Michlíček, & Shtansky, 2017), thereby confirming the successful grafting of aminoalkylsilane group onto BC membrane. The same result was achieved with FTIR analysis. 3.5. Surface wettability The surface wettability behavior of A-g-BC membranes was investigated by measuring static water contact angles. The obtained contact angles and the water drop profiles are shown in Fig. 3. As it is clearly showed, the grafting yield plays an important role in the hydrophobicity of BC membranes. BC is a very hydrophilic material with the contact angle of water 48.3° over its surface. With the increase of grafting yield of the BC membranes, there is an increase of the contact angle due to the covalent linkage with hydrophobic aminoalkylsilane groups. The contact angles of water over A-g-BC membranes are in the range of 59.3–86.4°. These data prove that the hydrophobicity of BC membrane is considerably increased via grafting of APTES. It was reported that the increase of hydrophobicity is advantageous to the enhanced antibacterial performance (Hoque, Konai, Sequeira, Samaddar, & Haldar, 2016). 3.6. Antibacterial and antifungal activities The antibacterial and antifungal activities of BC and A-g-BC membranes were investigated. Fig. 4 shows the numbers of bacteria colonies attached to the A-g-BC membrane and to pristine BC at 37 °C after a contact time of 1 h. The A-g-BC membranes performed much better than BC in reducing bacterial attachment, and it was found that bacterial adhesion decreased with increasing graft yield of the BC membrane. The calculated antibacterial ratios are listed in Table 1. BC10 membranes could reduce E. coli attachment by 100%, S. aureus attachment by 99.4% and B. subtilis attachment by 99.9% at 1 h
Fig. 4. Number of bacteria adhesion onto A-g-BC membranes: (A) E. coli, (B) S. aureus, (C) B. subtilis and (D) C. albicans.
compared with pristine BC, respectively. In general, these results indicate that A-g-BC membranes have excellent antibacterial activities against Gram negative E. coli, Gram positive S. aureus and S. subtilis. Moreover, great antifungal property against C. albicans of A-g-BC membranes has been established.
Fig. 3. Static water contact angles of BC and A-g-BC membranes.
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Clearly, the strong antibacterial activity is due to the introduction of aminoalkylsilane groups into BC matrix. A-g-BC membranes become polycationic after functionalized with aminoalkylsilane groups (Table S2). Thus, polycationic A-g-BC membranes with extremely high charge density can easily adsorb onto the negatively charged bacterial cell membrane by electrostatic interaction effect, thus, leading to the disruption of the bacterial cell membrane. In addition, the grafted hydrophobic aminoalkylsilane groups can affect the mode of surface interaction between functionalized BC and the cytoplasmic membrane of the bacteria though binding to the cytoplasmic membrane. Thus, the disruption of membrane and subsequent leakage of K+ ions and other cytoplasmic constituents can lead to the death of bacteria (Meng et al., 2015; Tashiro, 2001). Combining all beneficial qualities, the prepared A-g-BC membranes possess broad spectrum of antibacterial property, making them especially promising biomaterials which can be used in various biomedical applications.
3.7. Cytotoxicity of A-g-BC membranes Cellular growth and proliferation on surfaces are a symbol of cytocompatibility for materials which can be used to assess the potential of materials for application in tissue engineering (Shao et al., 2015a, 2015b). Cytotoxicity studies were performed to investigate the effect of grafting yield of the BC membrane on proliferation of HEK293 cell lines. The effect of pristine BC was evaluated in vitro to ensure that the grafted BC did not have an independent toxicity effect. The cell viability of HEK293 cells was evaluated by MTT assay. The cell cytotoxicity imparted by A-g-BC membranes being placed slightly in the transwell chambers was studied. HEK293 cells were treated for 24 h and then the transwell chambers with samples were removed. The MTT results were illustrated in Fig. 5 as relative viability of the cells by comparison with the control well containing only the cells. All the materials showed negligible toxicity. No obvious reduced cell viability following their incubation with A-g-BC membranes was shown. The results show that grafted BC did not inhibit the growth of HEK293 cells, even at a high concentration because HEK293 cells do not seem to be affected from their incubation with A-g-BC membranes. The relative cell viability is slightly decreased with grafting yield increasing of the BC matrix. However, it is still in the considerable range that no obvious cell cytotoxicity was detected. The possible reason for the slightly lower cell viability could be due to the morphology differences between pristine BC and functionalized BC, which is shown in Fig. 1. As it is shown that BC has a 3D porous network, therefore it is ideal for harboring cell growth. In the case of A-g-BC membranes, the introduction of aminoalkylsilane groups onto the BC nanofibrills reduces the membranes porosity, which leads to the lower cell viability. To determine whether the morphology of HEK293 cells was affected by A-g-BC membranes, the cells were stained with FITC-Phalloidin (green) and DAPI (blue), and then observed using Confocal Microscopy
Fig. 6. Cell morphologies treated with A-g-BC membranes.
(Fig. 6). The morphologies of HEK293 cells treated with A-g-BC membranes were similar to the blank one. Thus, the prepared A-g-BC membranes display no effect on the morphology of HEK293 cells. These results show that A-g-BC membranes are promising candidates for biomedical applications. 4. Conclusion In summary, antibacterial and biocompatible A-g-BC membranes were fabricated via grafting aminoalkylsilane groups. A-g-BC membrane displayed a thick and dense network structure compared to nanoporous three-dimensional network structure of BC. The hydrophobicity of BC membrane increases by grafting aminoalkylsilane groups. A-g-BC membranes display effective antibacterial and antifungal activities against E. coli, S. aureus, S. subtilis and C. albicans, which could be used as promising antibacterial materials in combating challenging pathogenic infection. Furthermore, A-g-BC membranes possess excellent biocompatibility. Therefore, the developed A-g-BC membranes have great potential applications in biomedical applications. Acknowledgements The work was financially supported by the Natural Science Foundation of Jiangsu Province (BK20161528), Postgraduate
Fig. 5. Cell viability percentages treated with A-g-BC membranes for 24 h.
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