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ovalbumin films layer-by-layer self-assembled polyacrylonitrile nanofibrous mats and their antibacterial activity
Colloids and Surfaces B: Biointerfaces 108 (2013) 322–328
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Silver ions/ovalbumin films layer-by-layer self-assembled polyacrylonitrile nanofibrous mats and their antibacterial activity Rukun Song a,b,1 , Jinjiao Yan a,b,c,1 , Shasha Xu a,b , Yuntao Wang a,b , Ting Ye a,b , Jing Chang d , Hongbing Deng a,b,∗ , Bin Li a,b,∗∗ a
Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, Wuhan 430070, China College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China c Asia Biomaterials (Wuhan) Co., Ltd, Wuhan, China d Acrylic Development Centre, Daqing Petrochemical Co., Ltd, Daqing, China b
a r t i c l e
i n f o
Article history: Received 30 November 2012 Received in revised form 17 February 2013 Accepted 3 March 2013 Available online 19 March 2013 Keywords: Layer-by-layer Ovalbumin Silver ions Nanofibrous mats Cytotoxicity Antibacterial activity
a b s t r a c t The CN groups of polyacrylonitrile (PAN) can strongly adsorb silver ions. The possibility of using this attraction as a layer-by-layer (LBL) self-assembly driving force was investigated. Firstly, the surface of the PAN nanofibrous mats was modified by silver ions to make sure it was positively charged. Then oppositely charged ovalbumin (OVA) and silver ions in aqueous media were alternatively deposited onto the surface of the obtained composite mats by layer-by-layer self-assembly technique. The morphology of the LBL films coating mats was observed by field emission scanning electron microscope (FE-SEM). The deposition of silver ions and OVA was confirmed by X-ray photoelectron spectroscopy (XPS) and wide-angle X-ray diffraction (XRD). The thermal degradation properties were investigated by thermo-gravimetric analysis (TGA). Besides these, the cytotoxicity and antibacterial activity of the prepared mats were studied via flow cytometry (FCM) and inhibition zone test, respectively. The results showed that the composite mats after LBL self-assembly processing exhibited improved thermal stability, slightly decreased cytotoxicity, and excellent antibacterial activity against Escherichia coil and Staphylococcus aureus. © 2013 Elsevier B.V. All rights reserved.
1. Introduction As one of the classic synthetic fiber-forming polymers, polyacrylonitrile (PAN) has been widely used in areas of industry, agriculture, construction and biomedicine. PAN fibers could be produced through not only the conventional “wet” and “dry” spinning process [1], but also the electrospun methods [2–6]. The diameter of the nanoscale PAN fibers obtained by electrospinning is well distributed at the range of 100 to 500 nm, depending on the preparation conditions[2,7]. Super-hydrophobic, well aligned, and high strength PAN nanofibers could be prepared through simple modifications [3–5,8]. PAN ultrafiltration membranes have been widely used as dialyzers, however, the biocompatibility of the PAN membrane is still insufficient [9,10]. Polymer surfaces modification techniques could
∗ Corresponding author at: Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, Wuhan 430070, China. Tel.: +86 2787282111; fax: +86 2787282111. ∗∗ Corresponding author at: Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, Wuhan 430070, China. Tel.: +86 2763730040; fax: +86 2787282966. E-mail addresses: [email protected] (H. Deng), [email protected] (B. Li). 1 These authors contributed equally to this work. 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.03.008
be introduced to improve the properties of PAN membrane. There are many options, such as physical blending, wet chemical oxidation, plasma treatment, UV irradiation, classical organic chemistry, and attachment of polymer chains [11,12]. Besides these, electrostatic layer-by-layer (LBL) self-assembly technique, a powerful and effective method to form multilayer ultra-thin films, which was introduced by Decher and Hong [13], is widely adopted on the modification of nanofibrous mats obtained via electrospinning [14,15]. Initially, LBL film assembly is based on adsorption of oppositely charged polyions and proteins from their solutions in alternate steps. The main driving force of classic LBL film assembly is considered to be electrostatic interaction between oppositely charged species [16]. Afterwards, driving forces such as hydrogen bonding and hydrophobic attraction have been introduced in this technique [17]. The active nitrile groups presented in acrylonitrile copolymers allow additional functional groups to be introduced by special polymer reactions. Cathionite, anionite, and ampholyte, used as mental chelating fibers or optical sensors, could be obtained via modifying of PAN fibers [18,19]. In our previous study, it was found that the unmodified PAN fibers could absorb many kinds of mental ions, including silver ions. This phenomenon, which was also confirmed by some researches [20,21], inspired us to investigate the possibility of using the attractive interaction as a LBL assembly driving force to modify electrospun ultrafine PAN fibers.
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Silver ions with excellent antimicrobial effects have been widely used in biomedical fields [22]. Many researches have combined silver nanoparticles with electrospun technique [23,24]. Because of their high surface area-to-volume ratio, electrospun antimicrobial ultrafine fibers would exhibit much stronger antimicrobial activity than the conventional wound dressing materials, tissue scaffolds, and antimicrobial filters products [25,26]. The positively charged ovalbumin (OVA) was selected to self-assemble with silver ions for a few reasons. It is a classic protein applied in electrospinning as well as in self-assembly processing [27–29]. What is more, it has the high biocompatibility to be used as biomedical materials [30] and carriers for controlled release of active compounds [31]. The cytotoxicity of PAN and silver ions was expected to reduce by the adding of OVA. In this study, a novel PAN electrospun nanofibrous mat coated by Ag/OVA LBL films was prepared and characterized. Both the cytotoxicity and the antibacterial activity of the mats were investigated. It was expected that the attractive interactions between the CN groups of PAN and the silver ions could be applied in LBL self-assembly processing as a novel driving force. 2. Experimental details 2.1. Materials The starting materials included ovalbumin (OVA, A5253, SigmaAldrich Co., USA) and polyacrylonitrile (PAN, Spectrum Co., Shanghai, China). Silver nitrate (SN), sodium chloride and N, N-dimethyl formamide (DMF) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All other chemicals were of analytical grade and used as received. All aqueous solutions were prepared using purified water with a resistance of 18.2 M cm. 2.2. Preparation of PAN electrospun nanofibers 8% PAN solution was prepared by adding PAN powder into DMF with gentle magnetic stirring for 5 h at room temperature. PAN nanofibrous mats were fabricated by electrospinning technique. Briefly, the PAN solution was fed into a plastic syringe, which was driven by a syringe pump (LSP02-1B, Baoding Long Precision Pump Co., Ltd., China). The positive electrode of a high voltage power supply (DW-P303-1ACD8, Tianjin Dongwen Co., China) was clamped to a metal needle tip with the inner diameter of 7 mm, and the cylindrical collector covered by aluminum foil was grounded. The applied voltage was 15 kV, the flow rate was 1.0 ml/h, and the distance between the collector and the spinneret was 16 cm. The ambient temperature and relative humidity were maintained at 25 ◦ C and 45%, respectively. The prepared fibrous mats were dried at 30 ◦ C in vacuum for further use. 2.3. Silver ions and OVA LBL modification PAN nanofibrous mats Silver nitrate and OVA were dissolved in distilled water to obtain 1 mg/ml solutions, respectively. The LBL films were assembled on the surface of PAN nanofibers mats by alternating adsorption of silver nitrate and OVA using the method described previously [14,17]. Firstly, PAN mats were immersed in the silver nitrate solution for 20 min followed by 3 min of rising in three distilled water baths. Then the mats were immersed in OVA solution for 20 min followed by the same washing steps. The absorption and rinsing steps were repeated until the desired number of deposition layers was obtained. Herein, (Ag/OVA)n was referred to label the LBL structure films, where n was the number of the Ag/OVA bilayers. The outermost layer was OVA when n was 5 and 10, and Ag when n equaled
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to 5.5 and 10.5. All resultant samples were dried at 25 ◦ C in vacuum for 24 h prior to the subsequent characterizations. 2.4. Characterization The morphology of samples was measured by field emission scanning electron microscopy (FE-SEM, JSM-6700F, Jeol Ltd., Japan). The diameters of the fibers were measured using an image analyzer (Adobe Photoshop 5.0). The surface elemental composition of samples was identified by X-ray photoelectron spectroscopy (XPS, VG Multilab 2000, Thermo Electron Co., USA). The wide-angle X-ray diffraction (XRD) was performed using D/Max-IIIA (Rigaku, Japan). The scanning rate and scope of 2 were 10◦ /min and 6–60◦ , respectively. Thermo-gravimetric analysis (TGA, 2000, Preiser Co., USA) was introduced to investigate the thermal degradation of the prepared mats at a heating rate of 10 ◦ C/min in nitrogen from 40 to 600 ◦ C. 2.5. Annexin V-PI double staining assay for apoptosis detection of C2C12 cells C2C12 cells were selected for the cellular compatibility experiments. The prepared mats were cut into the size that could fit in the cell culture plates, sterilized by UV light for 30 min, washed three times with phosphate buffered saline (PBS) solutions, and then transferred to the culture plate. After 24 h incubation on the prepared mats in the culture plate at 37 ◦ C under 5% CO2 , a total number of 1 × 106 cells per sample was washed with PBS three times to remove the unattached cells. Afterwards, the cells was digested with trypsin, and adjusted to 5 × 105 /ml with PBS. Annexin V-FITC and PI were added to each sample. Samples were mixed gently and incubated at room temperature in the dark for 5 min, and then analyzed by flow cytometry (FACSCalibur, Becton Dickinson) without delay. Green fluorescence of Annexin V was collected under 488 nm and 530 nm, and red fluorescence of PI under 575 nm. Data were stored as list mode files, and further converted with FACSConvert software into Cellquest v 3.1 software formats for analysis. 2.6. Antibacterial activities of nanofibrous mats against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) The inhibition zone test was used to study the bacterial inhibition activity of nanofibrous mats [32]. Gram-negative E. coil and gram-positive S. aureus were selected as representative microorganism and cultivated in culture medium in an incubator. Unmodified PAN mats were used as negative controls. Filter paper which was soaked in 5 mg/ml SN solution for 20 min and then dried at 25 ◦ C was used as positive control. The testing mats were cut into round disks with a diameter of 6 mm, sterilized under an ultraviolet radiation lamp for 30 min. One hundred macro-liters of 5.0–10.0 × 105 cfu/ml E. coil or 5.0–10.0 × 105 cfu/ml S. aureus bacteria levitation liquid was placed onto pre-autoclave sterilized meat-peptone broth and coated uniformly, respectively. Then the prepared mats were tiled on the surface of meat-peptone broth to cling to the bacteria levitation liquid. After incubated at 37 ◦ C in an air-bathing thermostat shaker with a rotating speed of 120r/min for 24 h, the bacterial inhibition zones were measured by a micrometer with a tolerance of 1 mm. Each sample was repeated three times. 3. Results and discussion Schematic diagram of the formation of the Ag/OVA LBL filmcoating PAN mats was shown in Scheme 1. PAN nanofibrous mats were obtained via electrospun processing. It has been reported that there were strong attractive interactions between silver ions and the CN groups of PAN [20]. The PAN nanofibrous mats were
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Scheme 1. Schematic diagram illustrating the fabrication process of the LBL film-coating PAN mats.
dipped into prepared silver nitrate solutions, so that the silver ions were absorbed by the CN groups of the PAN chains. Then the PAN nanofibrous mats were positively charged. Afterwards, the PAN nanofibrous mats were immersed in OVA solutions. Subsequently, the negatively charged OVA molecules were immobilized to the silver ions by electrostatic force. This process was repeated until desired film layers were assembled onto the mats. The influences of the coated layer number and the composition of the outmost layer on the morphology of the LBL film-coating nanofibrous mats were investigated by FE-SEM. The morphology and diameter distribution of the PAN mats were shown in Fig. 1. The pure PAN mats were composed of loosely packed cylindrical fibers with a uniformly distributed diameter of 208.53 ± 31.50 nm (Fig. 1a and a ). Not only the diameter but also the morphology of all the mats changed evidently after the LBL films deposition. However, they all maintained the nanofibrous 3D structure, which was a typical feature of the electrospun nanofibrous mats. The diameter enlarged gradually but not linearly with the increase of the LBL coated layers, and distributed evenly. The alignment of the pure PAN nanofibers was out of order, looking like a delicate, well-distributed fishing net. After the LBL films coating, the fibers tended to align with each other and some parallel bundles of fibers could be observed among the mats. This phenomenon has been observed in other LBL film-coating nanofibers mats as well [15], which was attributed to the LBL coating processing. The adjacent fibers adsorbed each other in the LBL solutions, and assembled together after the evaporation of the solvent. As the number of LBL layers increased, more bundles, bigger junctions and thicker deposition were observed. The sheet-like deposition in Fig. 1c and c as well as the grainy ones were caused by the imbalanced depositing. It could be concluded that the morphology of LBL film-coating mats was affected by the LBL processing greatly.
XPS scans were performed to verify the surface compositions of the prepared mats. Fig. 2 shows the XPS data of PAN mats and (Ag/OVA)10.5 LBL sample, respectively. The carbon and nitrogen peaks could be observed in PAN mats (Fig. 2a) with a C: N ratio of 79.5:15.96. After LBL film-coating, sliver and sulphur peaks were found in the XPS spectrum (Fig. 2b–d). Moreover, the C:N:O ratio changed, with an obvious increase of O content. The results verified that Ag and OVA were assembled onto the PAN mats successfully. The C1s, N1s and O1s photoemissions spectra were not shifted by the LBL modification, implying that no other interactions were involved besides the adsorption behavior of PAN for Ag and the electrostatic attraction between silver ion and OVA molecules. According to previous literatures [24,25,33], the peaks at the binding energy of 367.5 eV, 367.8 eV and 368.2 eV represent AgO, Ag2 O and Ag, respectively. The shift of the binding energies of AgNO3 was attributed to the photoreduction and oxidation of silver ions. Fig. 3 presents the XRD patterns of pure PAN and LBL filmcoating mats. The narrow crystalline peak centers at about 17◦ and 30◦ , and a broad non-crystalline peak (20–30◦ ) could be attributed to the pure PAN polymer phase [6,34,35]. After electrospinning, both peaks were weakened. It was shown that the pure PAN product was poorly crystallized, and the amorphous phase OVA has no peak in the XRD pattern. The peak at 35◦ (Fig. 3b and c) could be attributed to silver oxide, while the peak at 47◦ was a feature of silver [33,36]. The results mentioned above illustrated that Ag has been deposited on the surface of PAN mats. The thermal stability of PAN mats and LBL films coating mats was evaluated by TGA analysis (Fig. 4). The curves could be divided into three zones [37]: water lose, the decomposition of biopolymers and the carbonization of the degraded products to ash. From the initial temperature to about 410 ◦ C (water lose and the decomposition zone), the mass loss of LBL structured mats were slightly
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Fig. 1. FE-SEM images of (a) nanofibrous PAN mats, and LBL structured mats coated with: (b) (Ag/OVA)5 , (c) (Ag/OVA)5.5 , (d) (Ag/OVA)10 , and (e) (Ag/OVA)10.5 . Images (a –e ) showed high magnification images of a–e, respectively. Images (a –e ) showed the fiber size distributions of a–e, respectively.
different from that of the pure PAN mats. Within this temperature range, it could be observed that the mass loss of the (Ag/OVA)5 and (Ag/OVA)10 coated mats (the outmost layer of which were OVA) was slightly higher than that of the PAN mats at the same temperature, while the mass loss of the (Ag/OVA)5.5 and (Ag/OVA)10.5 coated mats (the outmost layer of which were Ag) were slightly lower. This implied that on the one hand the OVA on the outmost layer would weaken the thermal stability of the LBL film-coating mats. On the other hand, the thermal instability would enhance when the outmost layer was the inorganic Ag. When the temperature was higher than 410 ◦ C, the rate of weight loss increased with the increase of the coating layer number, and so was the Tmax (the temperature where the rate of weight loss reached a maximum) of all LBL
structured mats. This phenomenon stated that (Ag/OVA) filmcoating mats exhibited better thermal stability, no matter what component the outmost layer was. The cytotoxicity of the LBL film-coating mats was investigated by flow cytometry. The data in Table 1 was expressed as a percentage of the corresponding viable and non-viable cells. The percentage of non-viable cells is represented by the sum of UR and LR, containing both the early phase apoptosis and the necrotic cells. PAN mats showed mildly cytotoxicity on C2C12 cells. The cytotoxicity of the LBL film-coating mats decreased as the layers increased, however, ascended again over 10 layers. The (Ag/OVA)5.5 showed the minimal toxicity, while there was no significant difference between that of the PAN and (Ag/OVA)10.5 mats. No connection
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Fig. 2. XPS peak fitting curves of: (a) PAN mats, (b) (Ag/OVA)10.5 coated PAN mats, and core-level spectra of (c) Ag 3d and (d) S 2p narrow scans of (Ag/OVA)10.5 coated PAN mats. Table 1 FITC-Annexin/PI flow cytometry of C2C12 cells cultured on PAN nanofibrous mats and LBL structured nanofibrous mats coated with: (Ag/OVA)5 , (Ag/OVA)5.5 , (Ag/OVA)10 , and (Ag/OVA)10.5. LL represents vital cells, negative for both FITC-Annexin V binding and PI uptake; UL: damaged cells, FITC-Annexin V-negative and PI-positive; LR: apoptotic cells, FITC-Annexin V-positive and PI-negative, demonstrating cytoplasmic membrane integrity; UR refers to the non-viable, necrotic cells, positive for FITC-Annexin V binding and for PI uptake. The experiment was repeated three times. Group
% of total UR
PAN (Ag/OVA)5 (Ag/OVA)5.5 (Ag/OVA)10 (Ag/OVA)10.5
5.67 3.81 4.03 3.91 5.87
LR ± ± ± ± ±
1.00 1.45 1.98 2.00 2.01
18.16 7.48 5.77 13.77 17.71
UL ± ± ± ± ±
4.23 3.12 2.14 4.51 5.10
was observed between the component of the outmost layer and the change of cytotoxicity. It could be induced that a small amount of assembled Ag and OVA possessed favorable biocompatibility. At first, the cytotoxicity of the LBL film-coating mats reduced because of the adding of OVA, but then enhanced as the silver ions increased. The results of antimicrobial activities of the LBL film-coating PAN nanofibrous mats against E. coil and S. aureus were shown
Fig. 3. XRD patterns of (a) pure PAN powders, and LBL structured nanofibrous mats coated with: (b) (Ag/OVA)10 and (c) (Ag/OVA)10.5 .
11.45 10.59 10.64 11.03 12.55
LL ± ± ± ± ±
5.12 3.98 2.11 4.42 5.14
64.72 78.12 79.56 71.29 63.87
UR + LR ± ± ± ± ±
9.14 6.14 4.96 5.44 6.41
23.83 11.29 9.8 17.68 23.58
± ± ± ± ±
5.12 6.46 3.12 4.22 8.42
in Fig. 5. It could be observed that the pure PAN nanofibrous mats had no antibacterial effects, since the diameter of the inhibition zone was 6 mm. Nanofibrous mats coated with Ag and OVA exhibited obvious antibacterial activity against E. coil and S.
Fig. 4. TGA thermograms of (a) PAN nanofibrous mats and LBL structured nanofibrous mats coated with: (b) (Ag/OVA)5 , (c) (Ag/OVA)5.5 , (d) (Ag/OVA)10 , and (e) (Ag/OVA)10.5 .
R. Song et al. / Colloids and Surfaces B: Biointerfaces 108 (2013) 322–328
Fig. 5. Antimicrobial activities against E. coli and S. aureus of (a) fibrous PAN mats, (b) AgNO3 filter paper, and LBL structured mats coated with: (c) (Ag/OVA)5 , (d) (Ag/OVA)5.5 , (e) (Ag/OVA)10 , and (f) (Ag/OVA)10.5. The experiment was repeated three times.
aureus. The inhibition ability against S. aureus was slightly better compared with that against E. coil. This result was identical with previous reports [38]. Compared with the positive controls, the antibacterial effects of the Ag/OVA coated mats were even better. It could be attributed to the immobilization and protection effect of the nanofibrous mats to the LBL coated composition. It could be speculated that the LBL film-coating mats with Ag on the outmost layer {(Ag/OVA)5.5 and (Ag/OVA)10.5 } would exhibit better antibacterial effect than the ones whose outmost layer was OVA {(Ag/OVA)5 and (Ag/OVA)10 }. However, the antibacterial effect did not increase with the increase of Ag coated times. This pattern was similar to that of the cellular compatibility of the mats. 4. Conclusions Ag/OVA films coated PAN nanofibrous mats were successfully fabricated by electrospinning and LBL self-assembly technique. Both the strong attractive interactions between silver ions and the CN groups of PAN and the electrostatic attractions between Ag and OVA were applied in the LBL self-assembly processing. The XPS and XRD results evidenced that Ag and OVA were deposited onto the surface of PAN templates The addition of Ag/OVA resulted in larger fiber diameter and better thermal stability which could broaden the applications in food packaging, catalysis, sensors, textiles, tissue engineering and antimicrobial wound dressing. Additionally, flow cytometry assay suggested that the number of coating bilayers was a critical point affecting the cytotoxicity of the fibrous mats. The toxicity of the nanofibrous mats had been improved after LBL modification. The addition of silver ions onto the nanofibrous mats led to excellent antibacterial activity against E. coil and S. aureus. Acknowledgment Financial support from contract grant sponsors: The National Natural Science Foundation of China (Grant Nos. 31071607 and 31101365). References [1] B. Geller, Status and prospects for development of polyacrylonitrile fibre production. A review, Fibre Chem. 34 (2002) 151–161. [2] Z.M. Huang, Y.Z. Zhang, M. Kotaki, S. Ramakrishna, A review on polymer nanofibers by electrospinning and their applications in nanocomposites, Compos. Sci. Technol. 63 (2003) 2223–2253.
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Silver ions/ovalbumin films layer-by-layer self-assembled polyacrylonitrile nanofibrous mats and their antibacterial activity Rukun Song a,b,1 , Jinjiao Yan a,b,c,1 , Shasha Xu a,b , Yuntao Wang a,b , Ting Ye a,b , Jing Chang d , Hongbing Deng a,b,∗ , Bin Li a,b,∗∗ a
Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, Wuhan 430070, China College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China c Asia Biomaterials (Wuhan) Co., Ltd, Wuhan, China d Acrylic Development Centre, Daqing Petrochemical Co., Ltd, Daqing, China b
a r t i c l e
i n f o
Article history: Received 30 November 2012 Received in revised form 17 February 2013 Accepted 3 March 2013 Available online 19 March 2013 Keywords: Layer-by-layer Ovalbumin Silver ions Nanofibrous mats Cytotoxicity Antibacterial activity
a b s t r a c t The CN groups of polyacrylonitrile (PAN) can strongly adsorb silver ions. The possibility of using this attraction as a layer-by-layer (LBL) self-assembly driving force was investigated. Firstly, the surface of the PAN nanofibrous mats was modified by silver ions to make sure it was positively charged. Then oppositely charged ovalbumin (OVA) and silver ions in aqueous media were alternatively deposited onto the surface of the obtained composite mats by layer-by-layer self-assembly technique. The morphology of the LBL films coating mats was observed by field emission scanning electron microscope (FE-SEM). The deposition of silver ions and OVA was confirmed by X-ray photoelectron spectroscopy (XPS) and wide-angle X-ray diffraction (XRD). The thermal degradation properties were investigated by thermo-gravimetric analysis (TGA). Besides these, the cytotoxicity and antibacterial activity of the prepared mats were studied via flow cytometry (FCM) and inhibition zone test, respectively. The results showed that the composite mats after LBL self-assembly processing exhibited improved thermal stability, slightly decreased cytotoxicity, and excellent antibacterial activity against Escherichia coil and Staphylococcus aureus. © 2013 Elsevier B.V. All rights reserved.
1. Introduction As one of the classic synthetic fiber-forming polymers, polyacrylonitrile (PAN) has been widely used in areas of industry, agriculture, construction and biomedicine. PAN fibers could be produced through not only the conventional “wet” and “dry” spinning process [1], but also the electrospun methods [2–6]. The diameter of the nanoscale PAN fibers obtained by electrospinning is well distributed at the range of 100 to 500 nm, depending on the preparation conditions[2,7]. Super-hydrophobic, well aligned, and high strength PAN nanofibers could be prepared through simple modifications [3–5,8]. PAN ultrafiltration membranes have been widely used as dialyzers, however, the biocompatibility of the PAN membrane is still insufficient [9,10]. Polymer surfaces modification techniques could
∗ Corresponding author at: Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, Wuhan 430070, China. Tel.: +86 2787282111; fax: +86 2787282111. ∗∗ Corresponding author at: Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, Wuhan 430070, China. Tel.: +86 2763730040; fax: +86 2787282966. E-mail addresses: [email protected] (H. Deng), [email protected] (B. Li). 1 These authors contributed equally to this work. 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.03.008
be introduced to improve the properties of PAN membrane. There are many options, such as physical blending, wet chemical oxidation, plasma treatment, UV irradiation, classical organic chemistry, and attachment of polymer chains [11,12]. Besides these, electrostatic layer-by-layer (LBL) self-assembly technique, a powerful and effective method to form multilayer ultra-thin films, which was introduced by Decher and Hong [13], is widely adopted on the modification of nanofibrous mats obtained via electrospinning [14,15]. Initially, LBL film assembly is based on adsorption of oppositely charged polyions and proteins from their solutions in alternate steps. The main driving force of classic LBL film assembly is considered to be electrostatic interaction between oppositely charged species [16]. Afterwards, driving forces such as hydrogen bonding and hydrophobic attraction have been introduced in this technique [17]. The active nitrile groups presented in acrylonitrile copolymers allow additional functional groups to be introduced by special polymer reactions. Cathionite, anionite, and ampholyte, used as mental chelating fibers or optical sensors, could be obtained via modifying of PAN fibers [18,19]. In our previous study, it was found that the unmodified PAN fibers could absorb many kinds of mental ions, including silver ions. This phenomenon, which was also confirmed by some researches [20,21], inspired us to investigate the possibility of using the attractive interaction as a LBL assembly driving force to modify electrospun ultrafine PAN fibers.
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Silver ions with excellent antimicrobial effects have been widely used in biomedical fields [22]. Many researches have combined silver nanoparticles with electrospun technique [23,24]. Because of their high surface area-to-volume ratio, electrospun antimicrobial ultrafine fibers would exhibit much stronger antimicrobial activity than the conventional wound dressing materials, tissue scaffolds, and antimicrobial filters products [25,26]. The positively charged ovalbumin (OVA) was selected to self-assemble with silver ions for a few reasons. It is a classic protein applied in electrospinning as well as in self-assembly processing [27–29]. What is more, it has the high biocompatibility to be used as biomedical materials [30] and carriers for controlled release of active compounds [31]. The cytotoxicity of PAN and silver ions was expected to reduce by the adding of OVA. In this study, a novel PAN electrospun nanofibrous mat coated by Ag/OVA LBL films was prepared and characterized. Both the cytotoxicity and the antibacterial activity of the mats were investigated. It was expected that the attractive interactions between the CN groups of PAN and the silver ions could be applied in LBL self-assembly processing as a novel driving force. 2. Experimental details 2.1. Materials The starting materials included ovalbumin (OVA, A5253, SigmaAldrich Co., USA) and polyacrylonitrile (PAN, Spectrum Co., Shanghai, China). Silver nitrate (SN), sodium chloride and N, N-dimethyl formamide (DMF) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All other chemicals were of analytical grade and used as received. All aqueous solutions were prepared using purified water with a resistance of 18.2 M cm. 2.2. Preparation of PAN electrospun nanofibers 8% PAN solution was prepared by adding PAN powder into DMF with gentle magnetic stirring for 5 h at room temperature. PAN nanofibrous mats were fabricated by electrospinning technique. Briefly, the PAN solution was fed into a plastic syringe, which was driven by a syringe pump (LSP02-1B, Baoding Long Precision Pump Co., Ltd., China). The positive electrode of a high voltage power supply (DW-P303-1ACD8, Tianjin Dongwen Co., China) was clamped to a metal needle tip with the inner diameter of 7 mm, and the cylindrical collector covered by aluminum foil was grounded. The applied voltage was 15 kV, the flow rate was 1.0 ml/h, and the distance between the collector and the spinneret was 16 cm. The ambient temperature and relative humidity were maintained at 25 ◦ C and 45%, respectively. The prepared fibrous mats were dried at 30 ◦ C in vacuum for further use. 2.3. Silver ions and OVA LBL modification PAN nanofibrous mats Silver nitrate and OVA were dissolved in distilled water to obtain 1 mg/ml solutions, respectively. The LBL films were assembled on the surface of PAN nanofibers mats by alternating adsorption of silver nitrate and OVA using the method described previously [14,17]. Firstly, PAN mats were immersed in the silver nitrate solution for 20 min followed by 3 min of rising in three distilled water baths. Then the mats were immersed in OVA solution for 20 min followed by the same washing steps. The absorption and rinsing steps were repeated until the desired number of deposition layers was obtained. Herein, (Ag/OVA)n was referred to label the LBL structure films, where n was the number of the Ag/OVA bilayers. The outermost layer was OVA when n was 5 and 10, and Ag when n equaled
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to 5.5 and 10.5. All resultant samples were dried at 25 ◦ C in vacuum for 24 h prior to the subsequent characterizations. 2.4. Characterization The morphology of samples was measured by field emission scanning electron microscopy (FE-SEM, JSM-6700F, Jeol Ltd., Japan). The diameters of the fibers were measured using an image analyzer (Adobe Photoshop 5.0). The surface elemental composition of samples was identified by X-ray photoelectron spectroscopy (XPS, VG Multilab 2000, Thermo Electron Co., USA). The wide-angle X-ray diffraction (XRD) was performed using D/Max-IIIA (Rigaku, Japan). The scanning rate and scope of 2 were 10◦ /min and 6–60◦ , respectively. Thermo-gravimetric analysis (TGA, 2000, Preiser Co., USA) was introduced to investigate the thermal degradation of the prepared mats at a heating rate of 10 ◦ C/min in nitrogen from 40 to 600 ◦ C. 2.5. Annexin V-PI double staining assay for apoptosis detection of C2C12 cells C2C12 cells were selected for the cellular compatibility experiments. The prepared mats were cut into the size that could fit in the cell culture plates, sterilized by UV light for 30 min, washed three times with phosphate buffered saline (PBS) solutions, and then transferred to the culture plate. After 24 h incubation on the prepared mats in the culture plate at 37 ◦ C under 5% CO2 , a total number of 1 × 106 cells per sample was washed with PBS three times to remove the unattached cells. Afterwards, the cells was digested with trypsin, and adjusted to 5 × 105 /ml with PBS. Annexin V-FITC and PI were added to each sample. Samples were mixed gently and incubated at room temperature in the dark for 5 min, and then analyzed by flow cytometry (FACSCalibur, Becton Dickinson) without delay. Green fluorescence of Annexin V was collected under 488 nm and 530 nm, and red fluorescence of PI under 575 nm. Data were stored as list mode files, and further converted with FACSConvert software into Cellquest v 3.1 software formats for analysis. 2.6. Antibacterial activities of nanofibrous mats against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) The inhibition zone test was used to study the bacterial inhibition activity of nanofibrous mats [32]. Gram-negative E. coil and gram-positive S. aureus were selected as representative microorganism and cultivated in culture medium in an incubator. Unmodified PAN mats were used as negative controls. Filter paper which was soaked in 5 mg/ml SN solution for 20 min and then dried at 25 ◦ C was used as positive control. The testing mats were cut into round disks with a diameter of 6 mm, sterilized under an ultraviolet radiation lamp for 30 min. One hundred macro-liters of 5.0–10.0 × 105 cfu/ml E. coil or 5.0–10.0 × 105 cfu/ml S. aureus bacteria levitation liquid was placed onto pre-autoclave sterilized meat-peptone broth and coated uniformly, respectively. Then the prepared mats were tiled on the surface of meat-peptone broth to cling to the bacteria levitation liquid. After incubated at 37 ◦ C in an air-bathing thermostat shaker with a rotating speed of 120r/min for 24 h, the bacterial inhibition zones were measured by a micrometer with a tolerance of 1 mm. Each sample was repeated three times. 3. Results and discussion Schematic diagram of the formation of the Ag/OVA LBL filmcoating PAN mats was shown in Scheme 1. PAN nanofibrous mats were obtained via electrospun processing. It has been reported that there were strong attractive interactions between silver ions and the CN groups of PAN [20]. The PAN nanofibrous mats were
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Scheme 1. Schematic diagram illustrating the fabrication process of the LBL film-coating PAN mats.
dipped into prepared silver nitrate solutions, so that the silver ions were absorbed by the CN groups of the PAN chains. Then the PAN nanofibrous mats were positively charged. Afterwards, the PAN nanofibrous mats were immersed in OVA solutions. Subsequently, the negatively charged OVA molecules were immobilized to the silver ions by electrostatic force. This process was repeated until desired film layers were assembled onto the mats. The influences of the coated layer number and the composition of the outmost layer on the morphology of the LBL film-coating nanofibrous mats were investigated by FE-SEM. The morphology and diameter distribution of the PAN mats were shown in Fig. 1. The pure PAN mats were composed of loosely packed cylindrical fibers with a uniformly distributed diameter of 208.53 ± 31.50 nm (Fig. 1a and a ). Not only the diameter but also the morphology of all the mats changed evidently after the LBL films deposition. However, they all maintained the nanofibrous 3D structure, which was a typical feature of the electrospun nanofibrous mats. The diameter enlarged gradually but not linearly with the increase of the LBL coated layers, and distributed evenly. The alignment of the pure PAN nanofibers was out of order, looking like a delicate, well-distributed fishing net. After the LBL films coating, the fibers tended to align with each other and some parallel bundles of fibers could be observed among the mats. This phenomenon has been observed in other LBL film-coating nanofibers mats as well [15], which was attributed to the LBL coating processing. The adjacent fibers adsorbed each other in the LBL solutions, and assembled together after the evaporation of the solvent. As the number of LBL layers increased, more bundles, bigger junctions and thicker deposition were observed. The sheet-like deposition in Fig. 1c and c as well as the grainy ones were caused by the imbalanced depositing. It could be concluded that the morphology of LBL film-coating mats was affected by the LBL processing greatly.
XPS scans were performed to verify the surface compositions of the prepared mats. Fig. 2 shows the XPS data of PAN mats and (Ag/OVA)10.5 LBL sample, respectively. The carbon and nitrogen peaks could be observed in PAN mats (Fig. 2a) with a C: N ratio of 79.5:15.96. After LBL film-coating, sliver and sulphur peaks were found in the XPS spectrum (Fig. 2b–d). Moreover, the C:N:O ratio changed, with an obvious increase of O content. The results verified that Ag and OVA were assembled onto the PAN mats successfully. The C1s, N1s and O1s photoemissions spectra were not shifted by the LBL modification, implying that no other interactions were involved besides the adsorption behavior of PAN for Ag and the electrostatic attraction between silver ion and OVA molecules. According to previous literatures [24,25,33], the peaks at the binding energy of 367.5 eV, 367.8 eV and 368.2 eV represent AgO, Ag2 O and Ag, respectively. The shift of the binding energies of AgNO3 was attributed to the photoreduction and oxidation of silver ions. Fig. 3 presents the XRD patterns of pure PAN and LBL filmcoating mats. The narrow crystalline peak centers at about 17◦ and 30◦ , and a broad non-crystalline peak (20–30◦ ) could be attributed to the pure PAN polymer phase [6,34,35]. After electrospinning, both peaks were weakened. It was shown that the pure PAN product was poorly crystallized, and the amorphous phase OVA has no peak in the XRD pattern. The peak at 35◦ (Fig. 3b and c) could be attributed to silver oxide, while the peak at 47◦ was a feature of silver [33,36]. The results mentioned above illustrated that Ag has been deposited on the surface of PAN mats. The thermal stability of PAN mats and LBL films coating mats was evaluated by TGA analysis (Fig. 4). The curves could be divided into three zones [37]: water lose, the decomposition of biopolymers and the carbonization of the degraded products to ash. From the initial temperature to about 410 ◦ C (water lose and the decomposition zone), the mass loss of LBL structured mats were slightly
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Fig. 1. FE-SEM images of (a) nanofibrous PAN mats, and LBL structured mats coated with: (b) (Ag/OVA)5 , (c) (Ag/OVA)5.5 , (d) (Ag/OVA)10 , and (e) (Ag/OVA)10.5 . Images (a –e ) showed high magnification images of a–e, respectively. Images (a –e ) showed the fiber size distributions of a–e, respectively.
different from that of the pure PAN mats. Within this temperature range, it could be observed that the mass loss of the (Ag/OVA)5 and (Ag/OVA)10 coated mats (the outmost layer of which were OVA) was slightly higher than that of the PAN mats at the same temperature, while the mass loss of the (Ag/OVA)5.5 and (Ag/OVA)10.5 coated mats (the outmost layer of which were Ag) were slightly lower. This implied that on the one hand the OVA on the outmost layer would weaken the thermal stability of the LBL film-coating mats. On the other hand, the thermal instability would enhance when the outmost layer was the inorganic Ag. When the temperature was higher than 410 ◦ C, the rate of weight loss increased with the increase of the coating layer number, and so was the Tmax (the temperature where the rate of weight loss reached a maximum) of all LBL
structured mats. This phenomenon stated that (Ag/OVA) filmcoating mats exhibited better thermal stability, no matter what component the outmost layer was. The cytotoxicity of the LBL film-coating mats was investigated by flow cytometry. The data in Table 1 was expressed as a percentage of the corresponding viable and non-viable cells. The percentage of non-viable cells is represented by the sum of UR and LR, containing both the early phase apoptosis and the necrotic cells. PAN mats showed mildly cytotoxicity on C2C12 cells. The cytotoxicity of the LBL film-coating mats decreased as the layers increased, however, ascended again over 10 layers. The (Ag/OVA)5.5 showed the minimal toxicity, while there was no significant difference between that of the PAN and (Ag/OVA)10.5 mats. No connection
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Fig. 2. XPS peak fitting curves of: (a) PAN mats, (b) (Ag/OVA)10.5 coated PAN mats, and core-level spectra of (c) Ag 3d and (d) S 2p narrow scans of (Ag/OVA)10.5 coated PAN mats. Table 1 FITC-Annexin/PI flow cytometry of C2C12 cells cultured on PAN nanofibrous mats and LBL structured nanofibrous mats coated with: (Ag/OVA)5 , (Ag/OVA)5.5 , (Ag/OVA)10 , and (Ag/OVA)10.5. LL represents vital cells, negative for both FITC-Annexin V binding and PI uptake; UL: damaged cells, FITC-Annexin V-negative and PI-positive; LR: apoptotic cells, FITC-Annexin V-positive and PI-negative, demonstrating cytoplasmic membrane integrity; UR refers to the non-viable, necrotic cells, positive for FITC-Annexin V binding and for PI uptake. The experiment was repeated three times. Group
% of total UR
PAN (Ag/OVA)5 (Ag/OVA)5.5 (Ag/OVA)10 (Ag/OVA)10.5
5.67 3.81 4.03 3.91 5.87
LR ± ± ± ± ±
1.00 1.45 1.98 2.00 2.01
18.16 7.48 5.77 13.77 17.71
UL ± ± ± ± ±
4.23 3.12 2.14 4.51 5.10
was observed between the component of the outmost layer and the change of cytotoxicity. It could be induced that a small amount of assembled Ag and OVA possessed favorable biocompatibility. At first, the cytotoxicity of the LBL film-coating mats reduced because of the adding of OVA, but then enhanced as the silver ions increased. The results of antimicrobial activities of the LBL film-coating PAN nanofibrous mats against E. coil and S. aureus were shown
Fig. 3. XRD patterns of (a) pure PAN powders, and LBL structured nanofibrous mats coated with: (b) (Ag/OVA)10 and (c) (Ag/OVA)10.5 .
11.45 10.59 10.64 11.03 12.55
LL ± ± ± ± ±
5.12 3.98 2.11 4.42 5.14
64.72 78.12 79.56 71.29 63.87
UR + LR ± ± ± ± ±
9.14 6.14 4.96 5.44 6.41
23.83 11.29 9.8 17.68 23.58
± ± ± ± ±
5.12 6.46 3.12 4.22 8.42
in Fig. 5. It could be observed that the pure PAN nanofibrous mats had no antibacterial effects, since the diameter of the inhibition zone was 6 mm. Nanofibrous mats coated with Ag and OVA exhibited obvious antibacterial activity against E. coil and S.
Fig. 4. TGA thermograms of (a) PAN nanofibrous mats and LBL structured nanofibrous mats coated with: (b) (Ag/OVA)5 , (c) (Ag/OVA)5.5 , (d) (Ag/OVA)10 , and (e) (Ag/OVA)10.5 .
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Fig. 5. Antimicrobial activities against E. coli and S. aureus of (a) fibrous PAN mats, (b) AgNO3 filter paper, and LBL structured mats coated with: (c) (Ag/OVA)5 , (d) (Ag/OVA)5.5 , (e) (Ag/OVA)10 , and (f) (Ag/OVA)10.5. The experiment was repeated three times.
aureus. The inhibition ability against S. aureus was slightly better compared with that against E. coil. This result was identical with previous reports [38]. Compared with the positive controls, the antibacterial effects of the Ag/OVA coated mats were even better. It could be attributed to the immobilization and protection effect of the nanofibrous mats to the LBL coated composition. It could be speculated that the LBL film-coating mats with Ag on the outmost layer {(Ag/OVA)5.5 and (Ag/OVA)10.5 } would exhibit better antibacterial effect than the ones whose outmost layer was OVA {(Ag/OVA)5 and (Ag/OVA)10 }. However, the antibacterial effect did not increase with the increase of Ag coated times. This pattern was similar to that of the cellular compatibility of the mats. 4. Conclusions Ag/OVA films coated PAN nanofibrous mats were successfully fabricated by electrospinning and LBL self-assembly technique. Both the strong attractive interactions between silver ions and the CN groups of PAN and the electrostatic attractions between Ag and OVA were applied in the LBL self-assembly processing. The XPS and XRD results evidenced that Ag and OVA were deposited onto the surface of PAN templates The addition of Ag/OVA resulted in larger fiber diameter and better thermal stability which could broaden the applications in food packaging, catalysis, sensors, textiles, tissue engineering and antimicrobial wound dressing. Additionally, flow cytometry assay suggested that the number of coating bilayers was a critical point affecting the cytotoxicity of the fibrous mats. The toxicity of the nanofibrous mats had been improved after LBL modification. The addition of silver ions onto the nanofibrous mats led to excellent antibacterial activity against E. coil and S. aureus. Acknowledgment Financial support from contract grant sponsors: The National Natural Science Foundation of China (Grant Nos. 31071607 and 31101365). References [1] B. Geller, Status and prospects for development of polyacrylonitrile fibre production. A review, Fibre Chem. 34 (2002) 151–161. [2] Z.M. Huang, Y.Z. Zhang, M. Kotaki, S. Ramakrishna, A review on polymer nanofibers by electrospinning and their applications in nanocomposites, Compos. Sci. Technol. 63 (2003) 2223–2253.
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