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sodium alginate composite films with enhanced antibacterial property
Carbohydrate Polymers 132 (2015) 351–358
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Development of silver sulfadiazine loaded bacterial cellulose/sodium alginate composite films with enhanced antibacterial property Wei Shao a,∗ , Hui Liu a , Xiufeng Liu b , Shuxia Wang a , Jimin Wu a , Rui Zhang a , Huihua Min c , Min Huang a a
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 c Advanced Analysis and Testing Center, Nanjing Forestry University, Nanjing 210037, PR China b
a r t i c l e
i n f o
Article history: Received 5 April 2015 Received in revised form 21 June 2015 Accepted 22 June 2015 Available online 25 June 2015 Keywords: Bacterial cellulose Silver sulfadiazine Sodium alginate Antibacterial activity
a b s t r a c t Sodium alginate (SA) and bacterial cellulose (BC) are widely used in many applications such as scaffolds and wound dressings due to its biocompatibility. Silver sulfadiazine (AgSD) is a topical antibacterial agents used as a topical cream on burns. In the study, novel BC/SA–AgSD composites were prepared and characterized by SEM, FTIR and TG analyses. These results indicate AgSD successfully impregnated into BC/SA matrix. The swelling behaviors in different pH were studied and the results showed pH-responsive swelling behaviors. The antibacterial performances of BC/SA–AgSD composites were evaluated with Escherichia coli, Staphylococcus aureus and Candida albicans. Moreover, the cytotoxicity of BC/SA–AgSD composites was performed on HEK 293 cells. The experimental results showed BC/SA–AgSD composites have excellent antibacterial activities and good biocompatibility, thus confirming its utility as potential wound dressings. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Development of novel wound dressing has attracted more and more attentions in recent years. Silver is known to be a powerful antibacterial agent with effective broad-spectrum against Gram-positive and Gram-negative microorganisms (Barud et al., 2011; Jung, Kim, Kim, & Jin, 2009). Silver-based materials with antimicrobial properties were studied by many researchers (Maneerung, Tokura, & Rujiravanit, 2008; Maria et al., 2010). Bacterial cellulose-silver nanocomposites were successfully prepared and they exhibited excellent antibacterial activity (Barud et al., 2008; Maria et al., 2009; Shao et al., 2015). Silver compounds such as silver sulfadiazine (AgSD), are used widespreadly in burn and wound treatment for its broad activity spectrum and wound healing promotion (Aguzzi et al., 2014). In particular, AgSD is considered to be the first choice for treatment in skin chronic lesions and burns (Atiyeh, Costagliola, Hayek, & Dibo, 2007). However, there may be some adverse effects of AgSD in clinical studies, such as cytotoxicity and allergic reactions, which lead to retard wound healing
∗ Corresponding author. Tel.: +86 25 85427024; fax: +86 25 85418873. E-mail address: [email protected] (W. Shao). http://dx.doi.org/10.1016/j.carbpol.2015.06.057 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
processes (Dellera et al., 2014; Sandri et al., 2014). Therefore, an alternative strategy is required to improve drug efficacy. Sodium alginate (SA), a linear unbranched copolymer of 1,4linked -d-mannuronate (M) and ␣-d-guluronate (G), is isolated from marine algae (Seo et al., 2012). It is widely used in many applications such as scaffolds and wound dressings due to its biocompatibility, biodegradability under normal physiological conditions and capacity for bioresorption of the constituent ´ Boˇzanic, ´ materials (Becker, Kipke, & Brandon, 2001; Trandafilovic, ´ 2012). However, the rigid ´ ´ Luyt, & Djokovic, Dimitrijevic-Brankovi c, and fragile nature of the gelatinous SA may also be unfavorable in processing into non-spherical forms such as films and filaments via the gel state (Shalumon et al., 2011). A method to overcome this drawback is to blend SA with a compatible polysaccharide biopolymer. Bacterial cellulose (BC) is another biopolymer of great potentials, which features a distinctive three-dimensional structure consisting of an ultrafine network of cellulose nanofibers (Czaja, Young, Kawecki, & Brown, 2007). This unique micromorphology enables it to have great water holding capacity, good conformability, high porosity, high crystallinity, excellent mechanical strength and large surface area, which determines its potential application as an excellent wound dressing material (Jonas & Farah, 1998; Yang, Xie, Hong, Cao, & Yang, 2012). A novel composite matrix which
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gained the beneficial properties of both BC and SA was developed. Previous studies have shown that SA and BC blends are biocompatible, the blend leading to an increase in the thermomechanical stability (Shi, Zheng, Wang, Lin, & Fan, 2014). However, both BC and SA are lack of antibacterial property, resulting in failing to provide a barrier against wound infection, which limits the possibilities of application in wound dressing areas. To gain the beneficial properties of AgSD, BC and SA, a novel porous composites based AgSD loaded a blend of BC and SA as an efficient wound healing dressing was developed in this study. In our study, the ratio of BC:SA was chosen to be 4:1 since the tensile strength and elongation at break of BC/SA composite with this ratio were the higher than other ratios (3:2, 2:3, 1:4 and pure SA) (Phisalaphong, Suwanmajo, & Tammarate, 2008). Furthermore, the structure of BC/SA composites with alginate less than 30% was more stable after 2 h of immersion in PBS (Chiaoprakobkij, Sanchavanakit, Subbalekha, Pavasant, & Phisalaphong, 2011). BC/SA–AgSD composites were characterized by Scanning Electron Microscope (SEM), Fourier transform infrared spectra (FTIR) and thermogravimetric analyses (TG). The swelling behaviors of BC/SA–AgSD composites at different pH values were studied. The antibacterial activities of the obtained BC/SA–AgSD composites were investigated by Gram-negative Escherichia coli (E. coli) ATCC 25922, Gram-positive Staphylococcus aureus (S. aureus) ATCC 6538 and yeast Candida albicans (C. albicans) CMCC(F) 98001, respectively.
2.3. Characterization A JSM-7600F Scanning Electron Microscope (SEM) operating at an accelerating voltage of 10–15 kV was used to investigate the surface morphologies of BC and BC-Ag nanocomposites. The samples were coated with a thin layer of gold under high vacuum conditions (20 mA, 100 s). Fourier transform infrared (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 . Thermogravimetric analysis (TG) was carried out by using a TA Instruments model Q5000 TGA. The samples were heated from 20 to 800 ◦ C with a heating rate of 10 ◦ C/min under nitrogen atmosphere. 2.4. Porosity calculation The bulk density of the composite (f ) was calculated with Eq. (1): f =
W0 V0
(1)
2. Materials and methods
where W0 is the weight of the composite and V0 is the volume of the composite, which was measured with the modified method of Lin et al. (2014). Briefly, the samples were infiltrated with 99% ethanol in a 25-mL beaker under −0.08 MPa for 5 min in a vacuum oven. Subsequently, the tested sample was weighed in a 5-mL test tube and recorded as W1 and weighed again after ethanol was filled in the tube and recorded as W2 . V0 was calculated from Eq. (2):
2.1. BC preparation
V0 = 5 −
BC was prepared in a static culture medium by A. xylinum GIM1.327, which was purchased from BNBio Tech Co., Ltd., China. The method of preparing BC was well-established and described in literature (Ge et al., 2011). Briefly, in a static culture system enriched with polysaccharides, bacterial strain was incubated and was able to produce a thin layer of BC in the interface of liquid/air (Shi et al., 2012). 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 films were further washed thoroughly with de-ionized water until pH became neutral.
where ethanol is the density of ethanol and is 0.79 g/cm3 at room temperature. The density of the solid BC/SA material (BS ) was calculated with Eq. (3): BS =
To prepare BC/SA–AgSD composite, the preparation procedure is separated into three stages. Firstly, 20 g obtained wet BC membranes were cut into small pieces and crushed by high speed homogenizer at 15,000 rpm for 30 min to form BC fiber slurry. Secondly, SA was dissolved in distilled water to achieve 2.0% (w/v) at room temperature to form a gel-like solution. To prepare BC/SA hybrid composite, BC slurry and SA solution, were mixed with the weight ratios of wet BC and SA solution at 4:1, to obtain homogenous BC/SA dispersions (marked as BS0 ). Thirdly, AgSD was added into the BC/SA dispersions and mixed for 30 min. The weight ratio of AgSD to BC/SA was controlled to be 0.008 wt%, 0.016 wt%, 0.024 wt%, 0.06 wt% and 0.1 wt% (marked as BS1 , BS2 , BS3 , BS4 and BS5 , respectively). Then the mixture was treated by ultrasonication for degassing at supersonic power of 500 W for 3 min under ice-water bath. 0.25 mL mixture was placed in a 48-well plate and cross-linked by an aqueous solution of 5% CaCl2 for 3 h and rinsed by de-ionized water to remove the excess cross-linking agents. The homogeneous dispersions were freeze-dried at −40 ◦ C for 10 h.
ethanol
1 [f /BC + (1 − f )/SA ]
(2)
(3)
where f is the weight fraction of BC in the BC/SA composite which is 80% in our study; BC and SA refer to the densities of BC and 2 wt% SA and were calculated to be 1.01 and 1.013 g/cm3 . The density of the solid BC/SA–AgSD composite (s ) was calculated with Eq. (4): s =
2.2. Production of BC/SA–AgSD composites
(W − W ) 2 1
1 [(1 − AgSD )/BS + AgSD /AgSD ]
(4)
where AgSD is the AgSD weight fraction in the BC/SA–AgSD composite; AgSD refer to the densities of AgSD and were 1.496 g/cm3 . The porosity of the composites was calculated from Eq. (5): Porosity =
1−
f s
× 100%
(5)
2.5. Swelling behavior assays The swelling behaviors of the composites under different pH values were determined through a gravimetric method (Li et al., 2011). Initially, the tested films were cut into round pieces in diameter of 10 mm and their dry weights (W0 ) were accurately measured. The dry samples were immersed in PBS solution with pH 7.4, and the solutions with pH 2.5 and 11.5 that prepared by dilution of 1 M NaOH and 1 M HCl at room temperature. After 15 h immersion to obtain equilibrium swelling, the swollen membranes were withdrawn. The wet weight of the swollen membranes (W1 ) was measured after the removal of excess surface water by gently blotting with a filter paper. All testing was proceeded in triplicate; the
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Fig. 1. SEM images of BC/SA and BC/SA–AgSD composites: BS0 (A), BS1 (B), BS2 (C), BS3 (D), BS4 (E), BS5 (F); BS0 (G) and BS5 (H) with lower magnification.
equilibrium swelling ratio of the composites was calculated with Eq. (6):
3. Results and discussion 3.1. Surface morphology
swelling ratio =
(W1 − W0 ) W0
(6)
2.6. Antibacterial and antifungal activities The antibacterial activities of BC/SA–AgSD composites were investigated against E. coli ATCC 25922, S. aureus ATCC 6538 and C. albicans CMCC(F) 98001 by disk diffusion method. BC/SA–AgSD composites and BC/SA composite were cut into round shapes with 10 mm diameter and sterilized by ultraviolet lamp for 60 min. Lawns of test bacteria (about 1 × 105 CFU/mL) were prepared on TSA. The sterilized samples were then carefully placed upon the lawns and BC/SA composite was used as control. The plates were placed in a 37 ◦ C incubator for 24 h. Then inhibitory action of tested samples on the growth of the bacteria was determined by measuring diameter of inhibition zone.
2.7. Cytotoxicity tests The HEK 293 cell line was cultured in RPMI medium supplemented with 10% FBS, 100 g/mL penicillin and 100 g/mL streptomycin. The cells were then incubated for 3 days in a humidified 5% CO2 -containing balanced-air incubator at 37 ◦ C. The cytotoxicity was measured using the MTT assay method. 200 L of HEK 293 cells, at a density of 1 × 105 , were placed in each well of a 48-well plate. Then the cells were incubated over night at 37 ◦ C in a humidified 5% CO2 -containing atmosphere. After that, media was discarded. BC/SA–AgSD composites with same size (5 mm × 5 mm) were placed slightly on the top of cells 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 media containing sample was changed with fresh media and 20 L of dimethyl thiazolyl diphenyl (MTT) was added and the incubation continued for 6 h. Medium was removed, and 200 L 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.
Surface morphologies of BC/SA and BC/SA–AgSD composites were investigated (Fig. 1). Fig. 1A shows the morphology of BC/SA composite and Fig. 1G is BC/SA composite with lower magnification, which exhibited a highly porous structure with interconnected pores with size in the range of 100–300 m throughout the hybrid composites (Fig. 1A), which is in accordance with other reports (Chiaoprakobkij et al., 2011; Shi et al., 2014). In the case of BC/SA–AgSD composites, a denser network structure with clearly dispersed AgSD particles in the BC/SA matrix is illustrated in the composites (Fig. 1B–F). AgSD particles were displayed as needle shaped crystals which can be easily found in the composites. With the increase of AgSD particles in the composites, the surfaces are becoming smoother and more compact (Fig. 1B–D). However, in respect to BC/SA–AgSD composites with higher AgSD contents (Fig. 1E and F), large AgSD particles aggregates were observed in the matrix. This behavior can be attributed to the fact that high AgSD loadings leads to the AgSD particles overlap each other within the BC/SA matrix. Fig. 1H is the morphology of BS5 with lower magnification, which exhibited a lower porous structure compared to BS0 (Fig. 1G). Meanwhile, with the supplementation of AgSD, the calculated porosity decreased from 90.02% for BS0 to 69.9% for BS5 (Table 1) and led to a much more compact structure in the composite, which is in accordance with SEM results. 3.2. FTIR spectroscopy FTIR analysis was carried out to evaluate the interaction between BC/SA and AgSD. Fig. 2 displays the FTIR spectra of BC/SA and BC/SA–AgSD composites with different loadings of AgSD. In the case of BC/SA (curve a), the FTIR spectrum was typical and the dominating signal is at 3200–3500 cm−1 , corresponding to OH Table 1 Porosity of BC/SA–AgSD composites. Sample
s (g/cm3 )
f /s
BS BS1 BS2 BS3 BS4 BS5
1.01006 1.01009 1.01011 1.01014 1.01026 1.01039
0.10 0.17 0.18 0.23 0.28 0.30
Porosity (%) ± ± ± ± ± ±
0.01 0.02 0.01 0.02 0.01 0.00
90.02 83.23 81.64 77.28 72.31 69.90
± ± ± ± ± ±
1.18 1.83 1.15 1.55 0.64 0.35
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Fig. 2. FTIR analysis of BC/SA and BC/SA–AgSD composites: BS (a), BS1 (b), BS2 (c), BS3 (d), BS4 (e), BS5 (f), AgSD (g).
stretching from both BC and SA (Feng, Zhang, Shen, Yoshino, & Feng, 2012; Wan et al., 2007). In the C O stretching vibration region, the peaks at 1163, and 1061 cm−1 correspond to the C O asymmetric bridge stretching and the C O C pyranose ring skeletal vibration of BC, respectively (Park, Cheng, Choi, Kim, & Hyun, 2013). The absorption bands at 1633 cm−1 and 1419 cm−1 , which correspond to asymmetric COO stretching vibration and symmetric COO stretching vibration of SA, respectively (Chiew et al., 2014). The infrared spectrum of pure AgSD exhibited four characteristic bands, which was shown in Fig. 2 (curve g). The first band associated with vibrational stretching of its phenyl structure conjugated to the NH2 group was observed at 1552 cm−1 (Zepon, Petronilho, Soldi, Salmoria, & Kanis, 2014). The second band appeared at 1233 cm−1 was assigned to the asymmetrical stretching of the S O bonding. The third and fourth bands presented at 3344 and 3391 cm−1 were assigned as NH2 stretching bands. For BC/SA–AgSD composites (curves b–f in Fig. 2), the peak intensities of AgSD increased with the increase of AgSD loadings in the composites. And the samples with higher loadings of AgSD showed a more significant shoulder at 3391 cm−1 due to the free N H (Fajardo et al., 2013). Moreover, hydrogen bonding formed between amine group from AgSD and carbonyl and hydroxyl groups from BC/SA, which could promote NH2 stretching bands to shift to minor wavenumber. Similar observations were previously reported in cornstarch/cellulose acetate/AgSD blend membrane (Zepon et al., 2014). 3.3. Thermal properties The thermal degradation behavior of BC/SA–AgSD composites was studied in the range of 25–800 ◦ C under a nitrogen atmosphere. The introduction of AgSD has an influence on the thermal stability of the BC/SA composite, which can be found in Fig. 3. TGA curves exhibit the same trend and three significant weight loss stages below 800 ◦ C are observed in the thermal
Fig. 3. TG profiles of BC/SA and BC/SA–AgSD composites: BS (a), BS1 (b), BS2 (c), BS3 (d), BS4 (e), BS5 (f).
degradation curves. For BC/SA composite (curve a), the total weight loss was 61.88%. The initial weight loss occurred around 100 ◦ C, which is attributed to the evaporation of absorbed moisture. Physically adsorbed and hydrogen bond linked water molecules were lost at this first stage (Pavlidou, 2008). The second weight loss occurred in the range of 220–270 ◦ C involving the complex process of degradation of polysaccharides of SA (Huq et al., 2012; Liu, Dai, Shi, Xiong, & Zhou, 2015). The third rapid weight loss appears in the range of 270–350 ◦ C which can be assigned to the thermal degradation and decomposition of BC, which involves the formation of levoglucosan, transglycosylation and free radical reaction, followed by generation of C, CO, CO2 , H2 O and combustible volatiles (Qin et al., 2014; Yan, Chen, Wang, Wang, & Jiang, 2008). In the case of BC/SA–AgSD composites, the third weight loss stages shifted to lower temperature with the increase of AgSD loadings. This is due to the decomposition temperature of AgSD is around 320 ◦ C (Zepon et al., 2014). However, the residues increased with the increasing AgSD loadings, e.g. the residue were highly enhanced from 38.12% for BC to 49.76% of BS5 , which was related to the existence of AgSD particles. Therefore, AgSD particles are demonstrated successfully to be incorporated into BC/SA composites.
3.4. Swelling behaviors in vitro Swelling behavior and structural stability of composites play an important role in their practical use in wound dressing applications. The swelling behaviors of BC/SA–AgSD composites under different pH values were investigated and the results were shown in Fig. 4. The structural stability of the freeze-dried composites was investigated by 15 h immersion in different pH values and the morphologies of BS5 were displayed in Fig. 5. Fig. 4 showed the dynamic swelling profiles of the composites with different AgSD loadings in the solutions with pH 2.5 (a), 7.4 (b) and 11.5 (c). The swelling ratio was the lowest in pH 2.5 with less than 20 times of its dry weight. In neutral and alkaline environment, the swelling ratio was considerably higher. As evidenced from Fig. 4b, the swelling ratio reached to about 23 times as the pH value increased to 7. At pH 11.5, the maximum weight of BC/SA–AgSD hybrid composites was approximately 28 times of its dry weight. The swelling ratio result showed that the alkaline condition led to swelling more, while the acidic condition lowered the swelling degree. The reason for this is because SA in the composites has many carboxylic groups, which was protonated and converted to COOH groups, and the hydrogen bonding can be formed among OH and COOH groups in low pH value, which is responsible for the small swelling ratio. An increase of the swelling behaviors occurred when the alginate converted to sodium salt in higher pH condition. Hydrogen bands were break when alginic acid was deprotonated in higher pH condition. The electrostatic repulsion generated and resulted in the increase of swelling ratio (Shi et al., 2014). Moreover, there is an increasing tendency of swelling ratio with further AgSD loading. This could be due to the fact that AgSD is negative charge which promotes the electrostatic repulsion between polymer molecules, resulting in higher swelling ratio. Fig. 5 exhibited the morphologies of BS5 after 15 h swelling in different pH values. The structure of BS5 could remain stable in acidic and neutral conditions without any damage of its morphology. However, BS5 was slightly corrupted due to cross-linked calcium ions could form Ca(OH)2 in alkaline condition, which lead to erosion and disintegration of the composite. The in vitro study confirms the swelling ability of BC/SA–AgSD composites to be excellent and stable, facilitating adsorption of wound exudates with better potential application in a wide range of clinical settings (Chiaoprakobkij et al., 2011).
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Fig. 4. Swelling behaviors of BC/SA–AgSD composites in different pH values: pH 2.5 (A), pH 7.4 (B) and pH 11.5 (C).
3.5. Antibacterial and antifungal activities Three strains including Gram-negative E. coli ATCC 25922, Gram-positive S. aureus ATCC 6538 and Yeast C. albicans CMCC(F) 98001 were selected for antibacterial tested because they are usually associated with the infections during wound healing procedure (Fajardo et al., 2013; Jo, Jung, Choi, & Lee, 2012). The antibacterial activities of BC/SA and BC/SA–AgSD composites were investigated by disc diffusion method. The prepared composites were placed on a lawn of tested bacteria in TSA, respectively. The antibacterial activity is measured by the clear zone of inhibition around the samples after 24 h incubation and the images are shown in Fig. 6. As expected, no inhibition zones were observed for BC/SA as control (a), implying that BC/SA composite do not have any antibacterial properties against the tested three stains. The average diameters of inhibition zones of
prepared BC/SA–AgSD composites calculated from the disc diffusion method are listed in Fig. 7. With increasing AgSD loadings in the composite, the inhibition zones increase. The antibacterial activity is due to the presence of AgSD in the BC/SA matrix, which allows the release of sulfadiazine and silver ions, being effective for controlling bacterial growth (Mi et al., 2002). The silver ions provide the primary antibacterial activity against a range of bacteria and fungi, while sulfadiazine has bacteriostatic properties (Laura et al., 2013). BC/SA–AgSD composites showed a better antibacterial activity against S. aureus than E. coli. The differences observed in the diameter of zone of inhibition may be due to the difference in the susceptibility of different bacteria to the prepared BC/SA–AgSD composites. This phenomenon was probably due to structural differences in the outer membrane of bacteria. S. aureus, is a Gram-positive bacterium which possess single peptidoglycan layer structure, allowing sulfadiazine to easily penetrate
Fig. 5. Morphologies of BS5 after 15 h swelling in different pH values: pH 2.5 (A and D), pH 7.4 (B and E) and pH 11.5 (C and F).
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Fig. 6. Optical images of inhibition zones of BC/SA and BC/SA–AgSD composites: E. coli (A), S. aureus (B) and C. albicans (C) (In all plates, a–f indicate BS0 , BS1 , BS2 , BS3 , BS4 and BS5 ).
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4. Conclusion
Fig. 7. Average diameters of inhibition zones of BC/SA and BC/SA–AgSD composites, includes disk diameter of 10 mm.
their cell walls to interfere in its metabolic pathway. E. coli, as a Gram-negative bacterium, has a thick layer of lipopolysaccharide outer membrane covering the cell wall, which lead more resistant to hydrophobic substance (Zhang, Liu, Wang, Jiang, & Quek, 2016). The results reported in this study are consistent with other works on bactericidal action of AgSD (Fajardo et al., 2013). Furthermore, BC/SA–AgSD composites also exhibited good antifungal behavior against C. albicans. The present study clearly indicates that BC/SA–AgSD composites show excellent antibacterial activities against Gram negative organism, Gram positive organism and yeast. Combining all beneficial qualities, make the prepared BC/SA–AgSD composites good antibacterial wound dressing materials as well as in other biomedical applications.
3.6. Cytotoxicity Cytotoxicity studies were performed to investigate the effect of AgSD in the BC/SA matrix on proliferation of HEK 293 cell line. It is important to determine the effective AgSD concentration in vitro. The effect of BC/SA, without AgSD, was evaluated in vitro to ensure that AgSD did not have an independent toxicity effect. The cell viability of HEK 293 cells was evaluated by MTT assay. The MTT results were illustrated in Fig. 8 as relative viability of the cells by comparison with the control well containing only the cells. All the materials showed negligible toxicity although the cell viability slightly decreased with increasing AgSD loadings. The results showed that AgSD does not inhibit the proliferation of HEK 293 cells significantly, even at a high loading since HEK 293 cells do not seem to be affected obviously (less than 20%) from their incubation with BC/SA–AgSD composites. Therefore, based on these results, the prepared BC/SA–AgSD composites showed accepted cytotoxicity.
Fig. 8. Cell viability percentage treated with BC/SA–AgSD composites for 24 h.
In this study, BC/SA–AgSD composites were prepared by introducing AgSD particles into BC/SA matrix. SEM, FTIR and TG analysis confirmed the existence of AgSD particles in the BC/SA matrix. Swelling behaviors under different pH conditions were tested and the results displayed pH-responsive swelling behaviors. The swelling ratio of BC/SA–AgSD composites increased with increasing pH value. Antibacterial and cytotoxicity tests revealed that BC/SA–AgSD composites displayed excellent antibacterial performances on E. coli, S. aureus and C. albicans and good biocompatibility, which confirms BC/SA–AgSD composites utility as potential wound dressings. Acknowledgements The work was financially supported by the National Natural Science Foundation of China (51401109), the Major Program of the Natural Science Foundation of Jiangsu Higher Education of China (14KJB430018), the High-level Talent Project of Nanjing Forestry University (GXL201301) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The authors would like to thank the Advanced Analysis & Testing Center of Nanjing Forestry University. References Aguzzi, C., Sandri, G., Bonferoni, C., Cerezo, P., Rossi, S., Ferrari, F., et al. (2014). Solid state characterisation of silver sulfadiazine loaded on montmorillonite/chitosan nanocomposite for wound healing. Colloids and Surfaces B: Biointerfaces, 113, 152–157. Atiyeh, B. S., Costagliola, M., Hayek, S. N., & Dibo, S. A. (2007). Effect of silver on burn wound infection control and healing: Review of the literature. Burns, 33, 139–148. Becker, T. A., Kipke, D. R., & Brandon, T. (2001). Calcium alginate gel: A biocompatible and mechanically stable polymer for endovascular embolization. Journal of Biomedical Materials Research, 54, 76–86. Barud, H. S., Barrios, C., Regiani, T., Marques, R. F. C., Verelst, M., Dexpert-Ghys, J., et al. (2008). Self-supported silver nanoparticles containing bacterial cellulose membranes. Materials Science and Engineering C, 28, 515–518. Barud, H. S., Regiani, T., Marques, R. F. C., Lustri, W. R., Messaddeq, Y., & Ribeiro, S. J. L. (2011). Antimicrobial bacterial cellulose-silver nanoparticles composite membranes. Journal of Nanomaterials, http://dx.doi.org/10.1155/2011/721631 Chiaoprakobkij, N., Sanchavanakit, N., Subbalekha, K., Pavasant, P., & Phisalaphong, M. (2011). Characterization and biocompatibility of bacterial cellulose/alginate composite sponges with human keratinocytes and gingival fibroblasts. Carbohydrate Polymers, 85, 548–553. Chiew, C. S. C., Poh, P. E., Pasbakhsh, P., Tey, B. T., Yeoh, H. K., & Chan, E. S. (2014). Physicochemical characterization of halloysite/alginate bionanocomposite hydrogel. Applied Clay Science, 101, 444–454. Czaja, W. K., Young, D. J., Kawecki, M., & Brown, R. M. (2007). The future prospects of microbial cellulose in biomedical applications. Biomacromolecules, 8, 1–12. Dellera, E., Bonferoni, M. C., Sandri, G., Rossi, S., Ferrari, F., Fante, C. D., et al. (2014). Development of chitosan oleate ionic micelles loaded with silver sulfadiazine to be associated with platelet lysate for application in wound healing. European Journal of Pharmaceutics and Biopharmaceutics, 88, 643–650. Fajardo, A. R., Lopes, L. C., Caleare, A. O., Britta, E. A., Nakamura, C. V., Rubira, A. F., et al. (2013). Silver sulfadiazine loaded chitosan/chondroitin sulfate films for a potential wound dressing application. Materials Science and Engineering C, 33, 588–595. 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, 644–649. Ge, H. J., Du, S. K., Lin, D. H., Zhang, J. N., Xiang, J. L., & Li, Z. X. (2011). Gluconacetobacter hansenii subsp. nov., a high-yield bacterial cellulose producing strain induced by high hydrostatic pressure. Applied Biochemistry and Biotechnology, 165, 1519–1531. Huq, T., Salmieri, S., Khan, A., Khan, R. A., Tien, C. L., Riedl, B., et al. (2012). Nanocrystalline cellulose (NCC) reinforced alginate based biodegradable nanocomposite film. Carbohydrate Polymers, 90, 1757–1763. Jonas, R., & Farah, L. H. (1998). Production and application of microbial cellulose. Polymer Degradation and Stability, 59, 101–106. Jo, E. R., Jung, P. M., Choi, J., & Lee, J. W. (2012). Radiation sensitivity of bacteria and virus in porcine xenoskin for dressing agent. Radiation Physics and Chemistry, 81, 1259–1262. Jung, R., Kim, Y., Kim, H. S., & Jin, H. J. (2009). Antimicrobial properties of hydrated cellulose membranes with silver nanoparticles. Journal of Biomaterials Science, Polymer Edition, 20, 311–324.
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Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Development of silver sulfadiazine loaded bacterial cellulose/sodium alginate composite films with enhanced antibacterial property Wei Shao a,∗ , Hui Liu a , Xiufeng Liu b , Shuxia Wang a , Jimin Wu a , Rui Zhang a , Huihua Min c , Min Huang a a
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 c Advanced Analysis and Testing Center, Nanjing Forestry University, Nanjing 210037, PR China b
a r t i c l e
i n f o
Article history: Received 5 April 2015 Received in revised form 21 June 2015 Accepted 22 June 2015 Available online 25 June 2015 Keywords: Bacterial cellulose Silver sulfadiazine Sodium alginate Antibacterial activity
a b s t r a c t Sodium alginate (SA) and bacterial cellulose (BC) are widely used in many applications such as scaffolds and wound dressings due to its biocompatibility. Silver sulfadiazine (AgSD) is a topical antibacterial agents used as a topical cream on burns. In the study, novel BC/SA–AgSD composites were prepared and characterized by SEM, FTIR and TG analyses. These results indicate AgSD successfully impregnated into BC/SA matrix. The swelling behaviors in different pH were studied and the results showed pH-responsive swelling behaviors. The antibacterial performances of BC/SA–AgSD composites were evaluated with Escherichia coli, Staphylococcus aureus and Candida albicans. Moreover, the cytotoxicity of BC/SA–AgSD composites was performed on HEK 293 cells. The experimental results showed BC/SA–AgSD composites have excellent antibacterial activities and good biocompatibility, thus confirming its utility as potential wound dressings. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Development of novel wound dressing has attracted more and more attentions in recent years. Silver is known to be a powerful antibacterial agent with effective broad-spectrum against Gram-positive and Gram-negative microorganisms (Barud et al., 2011; Jung, Kim, Kim, & Jin, 2009). Silver-based materials with antimicrobial properties were studied by many researchers (Maneerung, Tokura, & Rujiravanit, 2008; Maria et al., 2010). Bacterial cellulose-silver nanocomposites were successfully prepared and they exhibited excellent antibacterial activity (Barud et al., 2008; Maria et al., 2009; Shao et al., 2015). Silver compounds such as silver sulfadiazine (AgSD), are used widespreadly in burn and wound treatment for its broad activity spectrum and wound healing promotion (Aguzzi et al., 2014). In particular, AgSD is considered to be the first choice for treatment in skin chronic lesions and burns (Atiyeh, Costagliola, Hayek, & Dibo, 2007). However, there may be some adverse effects of AgSD in clinical studies, such as cytotoxicity and allergic reactions, which lead to retard wound healing
∗ Corresponding author. Tel.: +86 25 85427024; fax: +86 25 85418873. E-mail address: [email protected] (W. Shao). http://dx.doi.org/10.1016/j.carbpol.2015.06.057 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
processes (Dellera et al., 2014; Sandri et al., 2014). Therefore, an alternative strategy is required to improve drug efficacy. Sodium alginate (SA), a linear unbranched copolymer of 1,4linked -d-mannuronate (M) and ␣-d-guluronate (G), is isolated from marine algae (Seo et al., 2012). It is widely used in many applications such as scaffolds and wound dressings due to its biocompatibility, biodegradability under normal physiological conditions and capacity for bioresorption of the constituent ´ Boˇzanic, ´ materials (Becker, Kipke, & Brandon, 2001; Trandafilovic, ´ 2012). However, the rigid ´ ´ Luyt, & Djokovic, Dimitrijevic-Brankovi c, and fragile nature of the gelatinous SA may also be unfavorable in processing into non-spherical forms such as films and filaments via the gel state (Shalumon et al., 2011). A method to overcome this drawback is to blend SA with a compatible polysaccharide biopolymer. Bacterial cellulose (BC) is another biopolymer of great potentials, which features a distinctive three-dimensional structure consisting of an ultrafine network of cellulose nanofibers (Czaja, Young, Kawecki, & Brown, 2007). This unique micromorphology enables it to have great water holding capacity, good conformability, high porosity, high crystallinity, excellent mechanical strength and large surface area, which determines its potential application as an excellent wound dressing material (Jonas & Farah, 1998; Yang, Xie, Hong, Cao, & Yang, 2012). A novel composite matrix which
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gained the beneficial properties of both BC and SA was developed. Previous studies have shown that SA and BC blends are biocompatible, the blend leading to an increase in the thermomechanical stability (Shi, Zheng, Wang, Lin, & Fan, 2014). However, both BC and SA are lack of antibacterial property, resulting in failing to provide a barrier against wound infection, which limits the possibilities of application in wound dressing areas. To gain the beneficial properties of AgSD, BC and SA, a novel porous composites based AgSD loaded a blend of BC and SA as an efficient wound healing dressing was developed in this study. In our study, the ratio of BC:SA was chosen to be 4:1 since the tensile strength and elongation at break of BC/SA composite with this ratio were the higher than other ratios (3:2, 2:3, 1:4 and pure SA) (Phisalaphong, Suwanmajo, & Tammarate, 2008). Furthermore, the structure of BC/SA composites with alginate less than 30% was more stable after 2 h of immersion in PBS (Chiaoprakobkij, Sanchavanakit, Subbalekha, Pavasant, & Phisalaphong, 2011). BC/SA–AgSD composites were characterized by Scanning Electron Microscope (SEM), Fourier transform infrared spectra (FTIR) and thermogravimetric analyses (TG). The swelling behaviors of BC/SA–AgSD composites at different pH values were studied. The antibacterial activities of the obtained BC/SA–AgSD composites were investigated by Gram-negative Escherichia coli (E. coli) ATCC 25922, Gram-positive Staphylococcus aureus (S. aureus) ATCC 6538 and yeast Candida albicans (C. albicans) CMCC(F) 98001, respectively.
2.3. Characterization A JSM-7600F Scanning Electron Microscope (SEM) operating at an accelerating voltage of 10–15 kV was used to investigate the surface morphologies of BC and BC-Ag nanocomposites. The samples were coated with a thin layer of gold under high vacuum conditions (20 mA, 100 s). Fourier transform infrared (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 . Thermogravimetric analysis (TG) was carried out by using a TA Instruments model Q5000 TGA. The samples were heated from 20 to 800 ◦ C with a heating rate of 10 ◦ C/min under nitrogen atmosphere. 2.4. Porosity calculation The bulk density of the composite (f ) was calculated with Eq. (1): f =
W0 V0
(1)
2. Materials and methods
where W0 is the weight of the composite and V0 is the volume of the composite, which was measured with the modified method of Lin et al. (2014). Briefly, the samples were infiltrated with 99% ethanol in a 25-mL beaker under −0.08 MPa for 5 min in a vacuum oven. Subsequently, the tested sample was weighed in a 5-mL test tube and recorded as W1 and weighed again after ethanol was filled in the tube and recorded as W2 . V0 was calculated from Eq. (2):
2.1. BC preparation
V0 = 5 −
BC was prepared in a static culture medium by A. xylinum GIM1.327, which was purchased from BNBio Tech Co., Ltd., China. The method of preparing BC was well-established and described in literature (Ge et al., 2011). Briefly, in a static culture system enriched with polysaccharides, bacterial strain was incubated and was able to produce a thin layer of BC in the interface of liquid/air (Shi et al., 2012). 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 films were further washed thoroughly with de-ionized water until pH became neutral.
where ethanol is the density of ethanol and is 0.79 g/cm3 at room temperature. The density of the solid BC/SA material (BS ) was calculated with Eq. (3): BS =
To prepare BC/SA–AgSD composite, the preparation procedure is separated into three stages. Firstly, 20 g obtained wet BC membranes were cut into small pieces and crushed by high speed homogenizer at 15,000 rpm for 30 min to form BC fiber slurry. Secondly, SA was dissolved in distilled water to achieve 2.0% (w/v) at room temperature to form a gel-like solution. To prepare BC/SA hybrid composite, BC slurry and SA solution, were mixed with the weight ratios of wet BC and SA solution at 4:1, to obtain homogenous BC/SA dispersions (marked as BS0 ). Thirdly, AgSD was added into the BC/SA dispersions and mixed for 30 min. The weight ratio of AgSD to BC/SA was controlled to be 0.008 wt%, 0.016 wt%, 0.024 wt%, 0.06 wt% and 0.1 wt% (marked as BS1 , BS2 , BS3 , BS4 and BS5 , respectively). Then the mixture was treated by ultrasonication for degassing at supersonic power of 500 W for 3 min under ice-water bath. 0.25 mL mixture was placed in a 48-well plate and cross-linked by an aqueous solution of 5% CaCl2 for 3 h and rinsed by de-ionized water to remove the excess cross-linking agents. The homogeneous dispersions were freeze-dried at −40 ◦ C for 10 h.
ethanol
1 [f /BC + (1 − f )/SA ]
(2)
(3)
where f is the weight fraction of BC in the BC/SA composite which is 80% in our study; BC and SA refer to the densities of BC and 2 wt% SA and were calculated to be 1.01 and 1.013 g/cm3 . The density of the solid BC/SA–AgSD composite (s ) was calculated with Eq. (4): s =
2.2. Production of BC/SA–AgSD composites
(W − W ) 2 1
1 [(1 − AgSD )/BS + AgSD /AgSD ]
(4)
where AgSD is the AgSD weight fraction in the BC/SA–AgSD composite; AgSD refer to the densities of AgSD and were 1.496 g/cm3 . The porosity of the composites was calculated from Eq. (5): Porosity =
1−
f s
× 100%
(5)
2.5. Swelling behavior assays The swelling behaviors of the composites under different pH values were determined through a gravimetric method (Li et al., 2011). Initially, the tested films were cut into round pieces in diameter of 10 mm and their dry weights (W0 ) were accurately measured. The dry samples were immersed in PBS solution with pH 7.4, and the solutions with pH 2.5 and 11.5 that prepared by dilution of 1 M NaOH and 1 M HCl at room temperature. After 15 h immersion to obtain equilibrium swelling, the swollen membranes were withdrawn. The wet weight of the swollen membranes (W1 ) was measured after the removal of excess surface water by gently blotting with a filter paper. All testing was proceeded in triplicate; the
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Fig. 1. SEM images of BC/SA and BC/SA–AgSD composites: BS0 (A), BS1 (B), BS2 (C), BS3 (D), BS4 (E), BS5 (F); BS0 (G) and BS5 (H) with lower magnification.
equilibrium swelling ratio of the composites was calculated with Eq. (6):
3. Results and discussion 3.1. Surface morphology
swelling ratio =
(W1 − W0 ) W0
(6)
2.6. Antibacterial and antifungal activities The antibacterial activities of BC/SA–AgSD composites were investigated against E. coli ATCC 25922, S. aureus ATCC 6538 and C. albicans CMCC(F) 98001 by disk diffusion method. BC/SA–AgSD composites and BC/SA composite were cut into round shapes with 10 mm diameter and sterilized by ultraviolet lamp for 60 min. Lawns of test bacteria (about 1 × 105 CFU/mL) were prepared on TSA. The sterilized samples were then carefully placed upon the lawns and BC/SA composite was used as control. The plates were placed in a 37 ◦ C incubator for 24 h. Then inhibitory action of tested samples on the growth of the bacteria was determined by measuring diameter of inhibition zone.
2.7. Cytotoxicity tests The HEK 293 cell line was cultured in RPMI medium supplemented with 10% FBS, 100 g/mL penicillin and 100 g/mL streptomycin. The cells were then incubated for 3 days in a humidified 5% CO2 -containing balanced-air incubator at 37 ◦ C. The cytotoxicity was measured using the MTT assay method. 200 L of HEK 293 cells, at a density of 1 × 105 , were placed in each well of a 48-well plate. Then the cells were incubated over night at 37 ◦ C in a humidified 5% CO2 -containing atmosphere. After that, media was discarded. BC/SA–AgSD composites with same size (5 mm × 5 mm) were placed slightly on the top of cells 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 media containing sample was changed with fresh media and 20 L of dimethyl thiazolyl diphenyl (MTT) was added and the incubation continued for 6 h. Medium was removed, and 200 L 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.
Surface morphologies of BC/SA and BC/SA–AgSD composites were investigated (Fig. 1). Fig. 1A shows the morphology of BC/SA composite and Fig. 1G is BC/SA composite with lower magnification, which exhibited a highly porous structure with interconnected pores with size in the range of 100–300 m throughout the hybrid composites (Fig. 1A), which is in accordance with other reports (Chiaoprakobkij et al., 2011; Shi et al., 2014). In the case of BC/SA–AgSD composites, a denser network structure with clearly dispersed AgSD particles in the BC/SA matrix is illustrated in the composites (Fig. 1B–F). AgSD particles were displayed as needle shaped crystals which can be easily found in the composites. With the increase of AgSD particles in the composites, the surfaces are becoming smoother and more compact (Fig. 1B–D). However, in respect to BC/SA–AgSD composites with higher AgSD contents (Fig. 1E and F), large AgSD particles aggregates were observed in the matrix. This behavior can be attributed to the fact that high AgSD loadings leads to the AgSD particles overlap each other within the BC/SA matrix. Fig. 1H is the morphology of BS5 with lower magnification, which exhibited a lower porous structure compared to BS0 (Fig. 1G). Meanwhile, with the supplementation of AgSD, the calculated porosity decreased from 90.02% for BS0 to 69.9% for BS5 (Table 1) and led to a much more compact structure in the composite, which is in accordance with SEM results. 3.2. FTIR spectroscopy FTIR analysis was carried out to evaluate the interaction between BC/SA and AgSD. Fig. 2 displays the FTIR spectra of BC/SA and BC/SA–AgSD composites with different loadings of AgSD. In the case of BC/SA (curve a), the FTIR spectrum was typical and the dominating signal is at 3200–3500 cm−1 , corresponding to OH Table 1 Porosity of BC/SA–AgSD composites. Sample
s (g/cm3 )
f /s
BS BS1 BS2 BS3 BS4 BS5
1.01006 1.01009 1.01011 1.01014 1.01026 1.01039
0.10 0.17 0.18 0.23 0.28 0.30
Porosity (%) ± ± ± ± ± ±
0.01 0.02 0.01 0.02 0.01 0.00
90.02 83.23 81.64 77.28 72.31 69.90
± ± ± ± ± ±
1.18 1.83 1.15 1.55 0.64 0.35
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Fig. 2. FTIR analysis of BC/SA and BC/SA–AgSD composites: BS (a), BS1 (b), BS2 (c), BS3 (d), BS4 (e), BS5 (f), AgSD (g).
stretching from both BC and SA (Feng, Zhang, Shen, Yoshino, & Feng, 2012; Wan et al., 2007). In the C O stretching vibration region, the peaks at 1163, and 1061 cm−1 correspond to the C O asymmetric bridge stretching and the C O C pyranose ring skeletal vibration of BC, respectively (Park, Cheng, Choi, Kim, & Hyun, 2013). The absorption bands at 1633 cm−1 and 1419 cm−1 , which correspond to asymmetric COO stretching vibration and symmetric COO stretching vibration of SA, respectively (Chiew et al., 2014). The infrared spectrum of pure AgSD exhibited four characteristic bands, which was shown in Fig. 2 (curve g). The first band associated with vibrational stretching of its phenyl structure conjugated to the NH2 group was observed at 1552 cm−1 (Zepon, Petronilho, Soldi, Salmoria, & Kanis, 2014). The second band appeared at 1233 cm−1 was assigned to the asymmetrical stretching of the S O bonding. The third and fourth bands presented at 3344 and 3391 cm−1 were assigned as NH2 stretching bands. For BC/SA–AgSD composites (curves b–f in Fig. 2), the peak intensities of AgSD increased with the increase of AgSD loadings in the composites. And the samples with higher loadings of AgSD showed a more significant shoulder at 3391 cm−1 due to the free N H (Fajardo et al., 2013). Moreover, hydrogen bonding formed between amine group from AgSD and carbonyl and hydroxyl groups from BC/SA, which could promote NH2 stretching bands to shift to minor wavenumber. Similar observations were previously reported in cornstarch/cellulose acetate/AgSD blend membrane (Zepon et al., 2014). 3.3. Thermal properties The thermal degradation behavior of BC/SA–AgSD composites was studied in the range of 25–800 ◦ C under a nitrogen atmosphere. The introduction of AgSD has an influence on the thermal stability of the BC/SA composite, which can be found in Fig. 3. TGA curves exhibit the same trend and three significant weight loss stages below 800 ◦ C are observed in the thermal
Fig. 3. TG profiles of BC/SA and BC/SA–AgSD composites: BS (a), BS1 (b), BS2 (c), BS3 (d), BS4 (e), BS5 (f).
degradation curves. For BC/SA composite (curve a), the total weight loss was 61.88%. The initial weight loss occurred around 100 ◦ C, which is attributed to the evaporation of absorbed moisture. Physically adsorbed and hydrogen bond linked water molecules were lost at this first stage (Pavlidou, 2008). The second weight loss occurred in the range of 220–270 ◦ C involving the complex process of degradation of polysaccharides of SA (Huq et al., 2012; Liu, Dai, Shi, Xiong, & Zhou, 2015). The third rapid weight loss appears in the range of 270–350 ◦ C which can be assigned to the thermal degradation and decomposition of BC, which involves the formation of levoglucosan, transglycosylation and free radical reaction, followed by generation of C, CO, CO2 , H2 O and combustible volatiles (Qin et al., 2014; Yan, Chen, Wang, Wang, & Jiang, 2008). In the case of BC/SA–AgSD composites, the third weight loss stages shifted to lower temperature with the increase of AgSD loadings. This is due to the decomposition temperature of AgSD is around 320 ◦ C (Zepon et al., 2014). However, the residues increased with the increasing AgSD loadings, e.g. the residue were highly enhanced from 38.12% for BC to 49.76% of BS5 , which was related to the existence of AgSD particles. Therefore, AgSD particles are demonstrated successfully to be incorporated into BC/SA composites.
3.4. Swelling behaviors in vitro Swelling behavior and structural stability of composites play an important role in their practical use in wound dressing applications. The swelling behaviors of BC/SA–AgSD composites under different pH values were investigated and the results were shown in Fig. 4. The structural stability of the freeze-dried composites was investigated by 15 h immersion in different pH values and the morphologies of BS5 were displayed in Fig. 5. Fig. 4 showed the dynamic swelling profiles of the composites with different AgSD loadings in the solutions with pH 2.5 (a), 7.4 (b) and 11.5 (c). The swelling ratio was the lowest in pH 2.5 with less than 20 times of its dry weight. In neutral and alkaline environment, the swelling ratio was considerably higher. As evidenced from Fig. 4b, the swelling ratio reached to about 23 times as the pH value increased to 7. At pH 11.5, the maximum weight of BC/SA–AgSD hybrid composites was approximately 28 times of its dry weight. The swelling ratio result showed that the alkaline condition led to swelling more, while the acidic condition lowered the swelling degree. The reason for this is because SA in the composites has many carboxylic groups, which was protonated and converted to COOH groups, and the hydrogen bonding can be formed among OH and COOH groups in low pH value, which is responsible for the small swelling ratio. An increase of the swelling behaviors occurred when the alginate converted to sodium salt in higher pH condition. Hydrogen bands were break when alginic acid was deprotonated in higher pH condition. The electrostatic repulsion generated and resulted in the increase of swelling ratio (Shi et al., 2014). Moreover, there is an increasing tendency of swelling ratio with further AgSD loading. This could be due to the fact that AgSD is negative charge which promotes the electrostatic repulsion between polymer molecules, resulting in higher swelling ratio. Fig. 5 exhibited the morphologies of BS5 after 15 h swelling in different pH values. The structure of BS5 could remain stable in acidic and neutral conditions without any damage of its morphology. However, BS5 was slightly corrupted due to cross-linked calcium ions could form Ca(OH)2 in alkaline condition, which lead to erosion and disintegration of the composite. The in vitro study confirms the swelling ability of BC/SA–AgSD composites to be excellent and stable, facilitating adsorption of wound exudates with better potential application in a wide range of clinical settings (Chiaoprakobkij et al., 2011).
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Fig. 4. Swelling behaviors of BC/SA–AgSD composites in different pH values: pH 2.5 (A), pH 7.4 (B) and pH 11.5 (C).
3.5. Antibacterial and antifungal activities Three strains including Gram-negative E. coli ATCC 25922, Gram-positive S. aureus ATCC 6538 and Yeast C. albicans CMCC(F) 98001 were selected for antibacterial tested because they are usually associated with the infections during wound healing procedure (Fajardo et al., 2013; Jo, Jung, Choi, & Lee, 2012). The antibacterial activities of BC/SA and BC/SA–AgSD composites were investigated by disc diffusion method. The prepared composites were placed on a lawn of tested bacteria in TSA, respectively. The antibacterial activity is measured by the clear zone of inhibition around the samples after 24 h incubation and the images are shown in Fig. 6. As expected, no inhibition zones were observed for BC/SA as control (a), implying that BC/SA composite do not have any antibacterial properties against the tested three stains. The average diameters of inhibition zones of
prepared BC/SA–AgSD composites calculated from the disc diffusion method are listed in Fig. 7. With increasing AgSD loadings in the composite, the inhibition zones increase. The antibacterial activity is due to the presence of AgSD in the BC/SA matrix, which allows the release of sulfadiazine and silver ions, being effective for controlling bacterial growth (Mi et al., 2002). The silver ions provide the primary antibacterial activity against a range of bacteria and fungi, while sulfadiazine has bacteriostatic properties (Laura et al., 2013). BC/SA–AgSD composites showed a better antibacterial activity against S. aureus than E. coli. The differences observed in the diameter of zone of inhibition may be due to the difference in the susceptibility of different bacteria to the prepared BC/SA–AgSD composites. This phenomenon was probably due to structural differences in the outer membrane of bacteria. S. aureus, is a Gram-positive bacterium which possess single peptidoglycan layer structure, allowing sulfadiazine to easily penetrate
Fig. 5. Morphologies of BS5 after 15 h swelling in different pH values: pH 2.5 (A and D), pH 7.4 (B and E) and pH 11.5 (C and F).
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Fig. 6. Optical images of inhibition zones of BC/SA and BC/SA–AgSD composites: E. coli (A), S. aureus (B) and C. albicans (C) (In all plates, a–f indicate BS0 , BS1 , BS2 , BS3 , BS4 and BS5 ).
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4. Conclusion
Fig. 7. Average diameters of inhibition zones of BC/SA and BC/SA–AgSD composites, includes disk diameter of 10 mm.
their cell walls to interfere in its metabolic pathway. E. coli, as a Gram-negative bacterium, has a thick layer of lipopolysaccharide outer membrane covering the cell wall, which lead more resistant to hydrophobic substance (Zhang, Liu, Wang, Jiang, & Quek, 2016). The results reported in this study are consistent with other works on bactericidal action of AgSD (Fajardo et al., 2013). Furthermore, BC/SA–AgSD composites also exhibited good antifungal behavior against C. albicans. The present study clearly indicates that BC/SA–AgSD composites show excellent antibacterial activities against Gram negative organism, Gram positive organism and yeast. Combining all beneficial qualities, make the prepared BC/SA–AgSD composites good antibacterial wound dressing materials as well as in other biomedical applications.
3.6. Cytotoxicity Cytotoxicity studies were performed to investigate the effect of AgSD in the BC/SA matrix on proliferation of HEK 293 cell line. It is important to determine the effective AgSD concentration in vitro. The effect of BC/SA, without AgSD, was evaluated in vitro to ensure that AgSD did not have an independent toxicity effect. The cell viability of HEK 293 cells was evaluated by MTT assay. The MTT results were illustrated in Fig. 8 as relative viability of the cells by comparison with the control well containing only the cells. All the materials showed negligible toxicity although the cell viability slightly decreased with increasing AgSD loadings. The results showed that AgSD does not inhibit the proliferation of HEK 293 cells significantly, even at a high loading since HEK 293 cells do not seem to be affected obviously (less than 20%) from their incubation with BC/SA–AgSD composites. Therefore, based on these results, the prepared BC/SA–AgSD composites showed accepted cytotoxicity.
Fig. 8. Cell viability percentage treated with BC/SA–AgSD composites for 24 h.
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