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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Chitosan/phosvitin antibacterial films fabricated via layer-by-layer deposition
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Bin Zhou a,b , Ying Hu a,b , Jing Li a,b , Bin Li a,b,∗ a b
College of Food Science and technology, Huazhong Agriculture University, Wuhan 430070, China Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, Wuhan, China
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Article history: Received 24 October 2013 Received in revised form 1 December 2013 Accepted 9 December 2013 Available online xxx
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Keywords: Phosvitin Chitosan Layer-by-layer Electrospinning Bacterial inhibition activity
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1. Introduction
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Negatively charged phosvitin (PV) and positively charged chitosan (CS) were alternately deposited on negatively charged cellulose mats via layer-by-layer (LBL) self-assembly technique. The deposition of PV and CS was confirmed by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectra (FT-IR), and wide-angle X-ray diffraction (XRD). Morphologies of the LBL films coating mats were observed by scanning electron microscope (SEM). Thermal degradation properties were investigated by differential scanning calorimetry (DSC) and thermo-gravimetric analysis (TGA). Additionally, the result of microbial inhibition assay indicated that the composite nanofibrous mats had excellent antibacterial activity against Escherichia coli and Staphylococcus aureus, which could be used for antimicrobial packing, tissue engineering, wound dressing, etc. © 2013 Published by Elsevier B.V.
Electrostatic layer-by-layer (LBL) self-assembly technique, introduced by Decher and Hong, has been one of the most frequently utilized process for preparation of functional multilayer films [1–3]. The process is commenced by adsorbing a polyelectrolyte onto an oppositely charged surface, thereby reversing the surface charge. Further layers are added by repeating the process until the desired film thickness is reached [3,4]. In general, multilayer films with oppositely charged polyelectrolytes have enabled the engineering of functional coatings for a wide range of applications, from sensors and solar cells to biomaterial and tissue engineering [5]. Recently, nanofibrous mats obtained via electrospinning were used as templates for LBL deposition because of their unique characteristics such as ultra-thin fiber diameter, small pore size, high specific surface, three-dimensional structure, etc [6,7]. Up to now, various deposition materials have been utilized to fabricate the functional LBL structured composite films including proteins, polysaccharides, metal irons, particles, etc [8,9]. Composite materials could be more effective and attractive than single material. Nowadays, polyanion/polycation composites, include protein–polysaccharide, polysaccharide–polysaccharide,
∗ Corresponding author at: College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China. Q2 Tel.: +86 27 6373 0040/+86 27 8728 6847; fax: +86 27 8728 8636. E-mail address: [email protected] (B. Li).
protein–other polymers, and others, attracted intensive attentions and they could be good candidates as deposition materials to form LBL structured films to improve the properties of the templates. In the past decades, lots of antibacterial agents are selected to add into the nanofibrous mats. As antibacterial agents, the safety should be considered firstly, which would limit their application area greatly [10]. Chitosan is an attractive natural material owing to its good properties and abundance in nature, it is useful in many different applications. One of the most valuable properties of chitosan is its inherent antimicrobial activity which is mainly due to its polycationic nature. Its cationic amino group associates with anions on the bacterial cell wall, suppressing biosynthesis; moreover, CS disrupts the mass transport across the cell wall accelerating the death of bacteria [11]. In addition, phosvitin (PV) is a polyanionic phosphoglycoprotein present in egg yolk and represents about 7% of yolk proteins, of the known proteins, it is the most phosphorylated one [12,13]. This unique primary structure makes this protein one of the strongest metal (iron, calcium, etc.) chelating agents. And the iron binding capabilities contribute to the antimicrobial properties of phosvitin [14]. It can damage the outer membrane and kills E. coli, a model strain of Gram-negative bacteria, under thermal stress [15,16]. In this work, novel LBL films on nanofibrous polysaccharide template, specifically cellulose nanofibers hydrolyzed from electrospun cellulose acetate nanofibers. Chitosan and phosvitin were selected as deposition materials to be coated on cellulose nanofibers via electrostatic LBL self-assembly technique by alternate adsorption of positively charged chitosan and negatively charged phosvitin
0141-8130/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijbiomac.2013.12.016
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in aqueous media. The effect of the outermost layer variation, the number of deposition bilayers, and the composition of the multilayer on the formation of the LBL structured nanofibrous mats was investigated. Meanwhile, the bacterial inhibition experiments were performed under different temperature to determine the antimicrobial property of the resultant samples.
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2. Materials and methods
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2.1. Materials
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Cellulose acetate (CA, Mn = 3 × 104 ) was purchased from Sigma–Aldrich Co., USA. Chitosan (CS, Mw = 2.0 × 105 kDa) from shrimp shell with 92% deacetylation was provided by Zhejiang Yuhuan Ocean Biochemical Co., China. The other reagents were analytical grade purchased from China National Pharmaceutical Group Industry Corporation Ltd. All aqueous solutions were prepared using purified water with a resistance of 18.2 M cm. E. coli and S. aureus were obtained from China Center for Type Culture 140 Collection, Wuhan University (Wuhan, China).
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2.2. Preparation of PV from hen eggs
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Hen egg yolk phosvitin was isolated by modifying the Losso and Nakai method [17]. Egg yolk was lightly washed with distilled water and rolled on filter paper to remove adhering albumen. The yolk membrane was punctured with a needle and the contents were collected. Then, 100 g of yolk were diluted with 0.5 L of cold water at pH 5.0 and stirred at 4 ◦ C for 1 h. The precipitate was collected by centrifugation at 10,000g for 20 min at 4 ◦ C. The pellet was dissolved in 200 mL distilled water, stirred for 1 h and centrifuged at 10,000g for 20 min at 4 ◦ C. The pellet was extracted with 400 mL of hexane:ethanol (3:1, v:v) at 4 ◦ C for 3 h and centrifuged. The resulting cake was dried and extracted with 200 mL of 1.74 M NaCl overnight at 4 ◦ C. Then the suspension was centrifuged at 10,000g for 20 min at 4 ◦ C and the supernatant was dialyzed against distilled water for 24 h at 4 ◦ C and freeze dried.
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2.3. SDS-PAGE
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PV was dissolved in double distilled water, loaded and electrophoresed on a 12.5% SDS-PAGE gel to check its purity. SDS-PAGE was performed in 1.0 M Tris–HCl buffer (pH 6.8) for stacking gel (5% acrylamide) and 1.5 M Tris–HCl buffer (pH 8.8) for separating gel (12.5% acryamide). Migration was performed at 80 and 120 V in the stacking gel and separating gel, respectively. After electrophoresis, the gel was stained using the modified staining solution containing 0.05% Coomassie brilliant blue R-250, 0.1 mol/L aluminum nitrate, 25% isopropanal,10% acetic acid and 1.0% Triton X-100 [18,19].
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2.4. Electrospinning of CA nanofibrous mats and their hydrolysis
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Nanofibrous CA mats were fabricated by Deng’s method [20]. Briefly, 17 wt% CA solution prepared in a 2/1 (w/w) acetone/DMAc mixture. The CA solution was fed into a plastic syringe, which was driven by a syringe pump (LSP02-1B, Baoding Longer Precision Pump Co., Ltd., China). The positive electrode of a high voltage power supply (DW-P303-1ACD8, Tianjin Dongwen Co., China) was clamped to the metal needle tip of the syringe. A grounded cylindrical layer was used as a collector which rotated with a linear velocity of 100 m/min. The applied voltage was 16 kV and the tipto-collector distance was 20 cm. The ambient temperature and relative humidity were maintained at 25 ◦ C and 45%, respectively. The prepared fibrous mats were dried at 80 ◦ C in vacuum for 24 h to remove the trace solvent. Hydrolysis of the CA mats was performed in a 0.05 M NaOH aqueous solution at ambient temperature for 7
Scheme 1. (A) Schematic diagram illustrating the hydrolysis of CA nanofibrous mats and (B) schematic diagram illustrating the fabrication process of the LBL film-coating cellulose mats.
days following the previous report. And the schematic diagram of hydrolysis of CA was shown in Scheme 1A.
2.5. Formation of LBL structured multilayer on nanofibrous mats The concept for fabrication of LBL structured fibrous mats was shown in Scheme 1B. The concentration of the positively charged CS solutions was fixed as 1 mg/mL by dissolving them in a 0.5% (V/V) aqueous acetic acid solution and the pH of solutions was controlled at 5.0. The negatively charged PV solution was 1 mg/mL in pure water and the pH was adjusted to 6. The ionic strength of all dipping solutions was regulated by the addition of NaCl at a concentration of 0.1 mg/mL. First, nanofibrous cellulose mats were immersed into CS for 20 min followed by 2 min of rinsing in three pure water baths [21]. The mats were then immersed into the PV solution for 20 min, followed by the identical rinsing steps. The adsorption and rinsing steps were repeated until the desired number of deposition bilayers was obtained. Here, (CS/PV)n was used as a formula to label the LBL structured films, where n was the number of the CS/PV bilayers. The outermost layer was CS composite when n equaled to 5.5 and 10.5. The LBL films coated fibrous mats were dried at 80 ◦ C for 24 h under vacuum prior to further characterizations.
2.6. Characterization The morphology and composition of fibrous mats were examined by scanning electron microscopy (SEM) (S-4800, Hitachi Ltd., Japan). The diameters of the fibers were measured using an image analyzer (Adobe Photoshop CS5.0). Fourier transform infrared (FT-IR) spectra were recorded using a Nicolet170-SX instrument (Thermo Nicolet Ltd., USA) in the wavenumber range of 4000–400 cm−1 . The surface elemental composition of the samples was identified by X-ray photoelectron spectroscopy (XPS) using an axis ultra DLD apparatus (Kratos, U.K.). X-ray diffraction (XRD) was carried out using a diffract meter type D/max-rA (Rigaku Co., Japan) with Cu target and Ka radiation ( = 0.154 nm). -potential analysis was preformed using a Nano-25 zetasizer (Malven, England).
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Fig. 1. SEM images and diameter distribution histograms of nanofibrous mats ((a)–(f)): cellulose acetate, cellulose, (CS/PV)5 , (CS/PV)5.5 , (CS/PV)10 , and (CS/PV)10.5 .
2.7. Inhibition of bacterial activity
3.2. Fabrication of CS/PV nanofibrous mats
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The inhibition zone test was used to study the bacterial inhibition activity of nanofibrous mats. Gram-negative E. coli and Gram-positive S. aureus were selected as representative microorganism and cultivated in culture medium in an incubator. Unmodified cellulose mats were used as negative 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 micro-liters of 5.0–10.0 × 105 cfu/mL E. coli 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 or 50 ◦ C in an air-bathing thermostat shaker with a rotating speed of 120 r/min for 24 h, the bacteria inhibition zones were measured by a micrometer with a tolerance of 1 mm. All of the experiments were conducted in triplicate with data reported as error bar.
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3. Results and discussion
As previous works described, the primary driven force for LBL modification was the electrostatic deposition and hydrogen bonding between the template and the assembly layer [22]. Zeta potential of the raw materials was measured in order to determine the driven force of LBL coating (Table S1).The LBL films were fabricated from CS and PV on cellulose template. It can be seen that the cellulose nanofibrous template was negatively charged (∼−28.6 mV). CS was positively charged and it had -potential value of 25.8 mV, that was attributed to the protonation of amino group. Oppositely, PV is an acidic protein rich in phosphate groups bound mainly by serine [16]. The isoelectric point of PV is 4.0, and the -potential of PV was −22.4 mV under specific pH value. Furthermore, the charge of raw materials could be controlled via adjusting the pH value, to avoid assembling on the fibers too much to destroy the structure of the fibers. Based on this fact, the positively charged CS and negatively charged PV could be alternately deposited on the surface of negatively charged cellulose nanofibers through electrostatic interaction [23]. Scheme 1B presents the deposition of CS and PV.
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3.1. Preparation and SDS-PAGE of PV
3.3. Surface morphology analysis of CS/PV nanofibrous mats
The results of the SDS-PAGE patterns of standard PV and extracted PV are shown in Fig. S1. SDS-PAGE revealed that PV extracted from yielded a major band of about 35 kDa and a minor band of about 33 kDa, corresponding to -PV and ␣-PV, respectively. No other contaminated bands were found on the gel, indicating that PV was pure.
To investigate the influence of film-coating on the nanofibrous mats, LBL coated mats with different bilayer number were observed by SEM (Fig. 1). And the diameter distribution of nanofibers was shown in Fig. 1. The typical electrospun nanofibrous mats had a 3D structure with pores in micro and sub-micro size [24]. The CA mats were composed of loosely packed cylindrical fibers. Some
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Table 1 Element composition and content on the surface of cellulose mats and (CS/PV)10 , and (CS/PV)10.5 . Atomic fraction of element composition (%)
Nanofibrous mats
Cellulose (CS/PV)10 (CS/PV)10.5
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junctions among the CA fibers were caused by the trace remained solvent after electrospinning (Fig. 1a). After hydrolysis, the surface of the fibers become rough and porous (Fig. 1b) [25]. To study the impact of the number of coating bilayers on the formation of LBL films coated nanofibers, the cellulose fibers were coated with various bilayers of CS and PV. After LBL coating process, the space between the fibers remarkably reduced. Compared with the morphology of CA or cellulose fibrous mats, it could be observed that LBL films coated nanofibers had a few conglutinations, which revealed that the CS and PV solutions deposited on the surface of cellulose nanofibers successfully, and formed a thin layer of films but split into webs during the drying process, hence the surface of the composite mats was much rougher than that of cellulose nanofibrous mats.
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3.4. Surface composition measurement of CS/PV nanofibrous mats
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The chemical states of the elements are examined by the XPS measurement. The quantification function of XPS could determine the elemental compositions at a depth of up to 10 nm from the surface. To verify the deposition of CS and PV on the LBL films, XPS scans were carried out to further verify the surface composition of the resultant samples. The XPS spectra for cellulose fibrous mats, (CS/PV)10 and (CS/PV)10.5 LBL coated films were shown in Fig. 2A. As we all know, cellulose was consisted of carbon, oxygen, and hydrogen and both PV and CS contained nitrogen, however, CS did not contain phosphorus element, which was the characteristic element of PV. So, with regard to cellulose mats, the strong carbon and oxygen peaks were observed on the XPS scan. After CS and PV deposition, the presence of nitrogen, carbon, oxygen, and phosphorus was observed on the surface of composite mats. PV is a polyanionic phosphoglycoprotein present in egg yolk, of the known protein, it is the most phosphorylated one. So the characteristic elements of phosphorus verified that PV was deposited on the surface of the fibers successfully. As both PV and CS contained nitrogen, the presence of nitrogen peak cannot certify that CS was deposited on the surface of the fibers successfully. In order to further confirm LBL coating process in every layer, the ratio of N/P was measured (Table 1). From the obtained atomic concentration of the high resolution scans (N 1s and P 2p), the N/P ratio of (CS/PV)10 and (CS/PV)10.5 were obtained to be 4.41 and 5.18, respectively. The N/P ratio distinctly increased when the outmost layer was CS. These result proved that both CS and PV could successfully coat on the surface of the mats in every layer.
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3.5. FT-IR spectra of CS/PV nanofibrous mats
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FTIR reveals information about the molecular structure of chemical compounds and is useful for the characterization of biopolymers. Here, FTIR was used to verify the presence of CS and PV on cellulose nanofibers. The FTIR spectra for cellulose nanofibrous mats and LBL coated mats were depicted in Fig. 3. For the cellulose mats, assignment of the main absorption peaks are as follows: the characteristic band at 3422 cm−1 was attributed to the stretching of hydroxyl groups involved in intramolecular hydrogen bonding of the cellulose II crystal [26]; the band at 1066 cm−1 was assigned to the stretching of C–O [27]; asymmetric stretching of C–O–C bond of the glycosidic linkage and pyranose ring of cellulose were observed at around 1238, 1164 and 1052 cm−1 , respectively. And the 897 cm−1 was attributed to the C1 –H deformation vibrations of cellulose [28]. The two common bands of amino group observed at 1648 and 1535 cm−1 belongs to the amide I and amide II peaks, which indicates that PV and
Fig. 2. (A) XPS spectra of (CS/PV)10.5 (a), (CS/PV)10 (b), LBL coated films and cellulose fibrous mats (c), and (B) core-level spectra of N 1s narrow scans of (CS/PV)10.5 , (CS/PV)10 LBL coated films and cellulose fibrous mats ((a), (c), (e)), core-level spectra of P 2p narrow scans of (CS/PV)10.5 , (CS/PV)10 LBL coated films and cellulose fibrous mats ((b), (d), (f)).
CS were deposited on the surface of cellulose mats successfully [29]. 3.6. Crystalline property of CS/PV nanofibrous mats The crystalline phases of CS/PV nanofibrous mats with different layers are characterized by XRD measurements. For comparison, pure CS, CA and cellulose nanofibrous mats are also tested. As shown in Fig. 4, CA mats exhibited an obvious crystalline peak at 2 = 9.1◦ [30]. In addition, both cellulose nanofibrous mat and CS/PV nanofibrous mats with different layers exhibit the uniform cellulose structure with characteristic 2 values at (12.1◦ , 20.3◦ , and 21.8◦ ), respectively [31]. The diffraction peaks originated from CS and PV are hardly detected in the CS/PV samples. This may be due to the facts of: (i) the amorphous phase PV has no peak in the XRD pattern; (ii) the peaks of CS is difficult to be detected because of low CS loading content; or (iii)
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Fig. 3. FT-IR spectra of ((a)–(e)): (CS/PV)5 , (CS/PV)5.5 , (CS/PV)10 , (CS/PV)10.5 , and cellulose nanofibrous mats.
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the most intense of CS peaks is overlapped by those of cellulose.
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3.7. Thermal properties of CS/PV nanofibrous mats
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The thermal stability of cellulose mats and LBL films coating mats was evaluated by TGA and DSC analysis (Fig. 5). TGA provided the onset decomposition temperature, percentage of residue, highest weight loss temperature, and highest weight loss rates as the samples were heated [32]. The TG curves could be divided into three zones: the first weight loss is observed up to 100 ◦ C (∼10% weight loss), attributed to removal of adsorbed or bond water (I), the second weight loss step was corresponded to the decomposition of composite materials (II). For cellulose nanofibrous mats, it was observed at ca. 200 ◦ C, which was associated with the decomposition of cellulose (Fig. 5A). Neglecting the first weight loss stage, the onset decomposition temperature was recorded to investigate the thermal stability of nanofibrous mats. It can be seen that all LBL coated mats have the similar decomposition temperatures at
Fig. 5. (A) TGA thermograms of cellulose nanofibrous mats, LBL structure nanofibrous mats coated with: (CS/PV)10.5 , (CS/PV)10 , (CS/PV)5.5 , (CS/PV)5 , and cellulose fibrous mats; (B) Differential thermal gravimetry (DTG) of cellulose nanofibrous mats, LBL structure nanofibrous mats coated with: (CS/PV)10.5 , (CS/PV)10 , (CS/PV)5.5 , (CS/PV)5 , and cellulose fibrous mats; (C) DSC thermograms of LBL structure nanofibrous mats coated with: (CS/PV)10.5 , (CS/PV)10 , (CS/PV)5.5 , (CS/PV)5 ((a)–(d)), and cellulose fibrous mats (e).
Fig. 4. XRD patterns of CS (a), LBL structured mats coated with (CS/PV)10.5 , (CS/PV)10 , (CS/PV)5.5 , and (CS/PV)5 ((b)–(e)), cellulose fibrous mats (f), CA fibrous mats (g).
about 250 ◦ C as shown in Figs. 5A and B. This result indicated that (CS/PV) coated mats exhibited better thermal stability, no matter what component the outmost layer was. The last one was the carbonization of the degraded productions to ash(III) [33]. In this stage, with the increase of temperature the weight loss reached a maximum. Some significant properties shown in TGA curve could be also elucidated by analyzing of the DSC thermograms as reflected in Fig. 5C. The cellulose mats showed a broad exothermic event at ca. 200 ◦ C was associated with the degraded of cellulose mats. For LBL coated mats, the appearance of an obvious endothermic event at about 275 ◦ C may be attributed to the melting of CS. In addition, with the increase of the layer number, the thermal stability
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technique. Both the electrostatic attractions between cellulose fibers, CS and PV were applied in the LBL self-assembly processing. The deposition of CS and PV on the surface of cellulose mats was characterized by XPS, XRD, and FTIR. And the morphologies of composite nanofibrous mats became increasingly rough with increasing number of deposition layer. Moreover, the microbial inhibition assay demonstrated that the CS/PV composite mats had good antibacterial effects, which have potential application in the areas of antibacterial coating, tissue engineering, wound dressing, etc. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Grant no. 31371841). The authors greatly thank colleagues of Key Laboratory of Environment Correlative Dietology of Huazhong Agricultural University for offering many conveniences. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac. 2013.12.016. References
Fig. 6. Antimicrobial activities against E. coli and S. aureus of fibrous cellulose mats (control) and multilayer nanofibrous mats at 37 ◦ C (A) and 50 ◦ C (B).
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decreased slightly. All the results of DSC were consistent with the results of TGA.
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3.8. Antibacterial property of CS/PV nanofibrous mats
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Among the natural polymers CS and its derivatives are regarded as one of the most attractive for use in nanofibrous materials. One of the most valuable properties of CS is its inherent antimicrobial activity which is mainly due to its polycationic nature. CS inhibits the growth of a variety of pathogenic microorganisms—Gram-positive and Gram-negative bacteria, yeasts, and fungi [34–36]. In addition, PV exhibited good antimicrobial properties, due to chelating and surface activity (affinity for outer membrane) [14]. The antibacterial activity of LBL coated mats was determined by inhibition zone method against E. coli and S. aureus under 37 and 50 ◦ C (Fig. 6). Both under 37 and 50 ◦ C, cellulose mats hardly showed inhibitory effect, whereas composite mats revealed significant antibacterial activity and the antibacterial activity enhanced with increasing the numbers of bilayers, because the quantity of antibacterial agent was enhanced with increasing the numbers of bilayers. The LBL coated mats have a relatively weak antimicrobial activity for Gram-negative bacillus under 37 ◦ C, however, the antimicrobial activity for E. coli significantly enhanced due to the bacteriostatic effect of PV under thermal stress at 50 ◦ C. And these activities in conjunction with thermal stress seem to contribute to the loss of viability of E. coli [15]. 4. Conclusions In conclusion, we have fabricated CS and PV multilayer nanofibrous mats using electrospinning and electrostatic LBL deposition
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Chitosan/phosvitin antibacterial films fabricated via layer-by-layer deposition
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Bin Zhou a,b , Ying Hu a,b , Jing Li a,b , Bin Li a,b,∗ a b
College of Food Science and technology, Huazhong Agriculture University, Wuhan 430070, China Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, Wuhan, China
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Article history: Received 24 October 2013 Received in revised form 1 December 2013 Accepted 9 December 2013 Available online xxx
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Keywords: Phosvitin Chitosan Layer-by-layer Electrospinning Bacterial inhibition activity
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1. Introduction
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Negatively charged phosvitin (PV) and positively charged chitosan (CS) were alternately deposited on negatively charged cellulose mats via layer-by-layer (LBL) self-assembly technique. The deposition of PV and CS was confirmed by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectra (FT-IR), and wide-angle X-ray diffraction (XRD). Morphologies of the LBL films coating mats were observed by scanning electron microscope (SEM). Thermal degradation properties were investigated by differential scanning calorimetry (DSC) and thermo-gravimetric analysis (TGA). Additionally, the result of microbial inhibition assay indicated that the composite nanofibrous mats had excellent antibacterial activity against Escherichia coli and Staphylococcus aureus, which could be used for antimicrobial packing, tissue engineering, wound dressing, etc. © 2013 Published by Elsevier B.V.
Electrostatic layer-by-layer (LBL) self-assembly technique, introduced by Decher and Hong, has been one of the most frequently utilized process for preparation of functional multilayer films [1–3]. The process is commenced by adsorbing a polyelectrolyte onto an oppositely charged surface, thereby reversing the surface charge. Further layers are added by repeating the process until the desired film thickness is reached [3,4]. In general, multilayer films with oppositely charged polyelectrolytes have enabled the engineering of functional coatings for a wide range of applications, from sensors and solar cells to biomaterial and tissue engineering [5]. Recently, nanofibrous mats obtained via electrospinning were used as templates for LBL deposition because of their unique characteristics such as ultra-thin fiber diameter, small pore size, high specific surface, three-dimensional structure, etc [6,7]. Up to now, various deposition materials have been utilized to fabricate the functional LBL structured composite films including proteins, polysaccharides, metal irons, particles, etc [8,9]. Composite materials could be more effective and attractive than single material. Nowadays, polyanion/polycation composites, include protein–polysaccharide, polysaccharide–polysaccharide,
∗ Corresponding author at: College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China. Q2 Tel.: +86 27 6373 0040/+86 27 8728 6847; fax: +86 27 8728 8636. E-mail address: [email protected] (B. Li).
protein–other polymers, and others, attracted intensive attentions and they could be good candidates as deposition materials to form LBL structured films to improve the properties of the templates. In the past decades, lots of antibacterial agents are selected to add into the nanofibrous mats. As antibacterial agents, the safety should be considered firstly, which would limit their application area greatly [10]. Chitosan is an attractive natural material owing to its good properties and abundance in nature, it is useful in many different applications. One of the most valuable properties of chitosan is its inherent antimicrobial activity which is mainly due to its polycationic nature. Its cationic amino group associates with anions on the bacterial cell wall, suppressing biosynthesis; moreover, CS disrupts the mass transport across the cell wall accelerating the death of bacteria [11]. In addition, phosvitin (PV) is a polyanionic phosphoglycoprotein present in egg yolk and represents about 7% of yolk proteins, of the known proteins, it is the most phosphorylated one [12,13]. This unique primary structure makes this protein one of the strongest metal (iron, calcium, etc.) chelating agents. And the iron binding capabilities contribute to the antimicrobial properties of phosvitin [14]. It can damage the outer membrane and kills E. coli, a model strain of Gram-negative bacteria, under thermal stress [15,16]. In this work, novel LBL films on nanofibrous polysaccharide template, specifically cellulose nanofibers hydrolyzed from electrospun cellulose acetate nanofibers. Chitosan and phosvitin were selected as deposition materials to be coated on cellulose nanofibers via electrostatic LBL self-assembly technique by alternate adsorption of positively charged chitosan and negatively charged phosvitin
0141-8130/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijbiomac.2013.12.016
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in aqueous media. The effect of the outermost layer variation, the number of deposition bilayers, and the composition of the multilayer on the formation of the LBL structured nanofibrous mats was investigated. Meanwhile, the bacterial inhibition experiments were performed under different temperature to determine the antimicrobial property of the resultant samples.
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2. Materials and methods
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2.1. Materials
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Cellulose acetate (CA, Mn = 3 × 104 ) was purchased from Sigma–Aldrich Co., USA. Chitosan (CS, Mw = 2.0 × 105 kDa) from shrimp shell with 92% deacetylation was provided by Zhejiang Yuhuan Ocean Biochemical Co., China. The other reagents were analytical grade purchased from China National Pharmaceutical Group Industry Corporation Ltd. All aqueous solutions were prepared using purified water with a resistance of 18.2 M cm. E. coli and S. aureus were obtained from China Center for Type Culture 140 Collection, Wuhan University (Wuhan, China).
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2.2. Preparation of PV from hen eggs
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Hen egg yolk phosvitin was isolated by modifying the Losso and Nakai method [17]. Egg yolk was lightly washed with distilled water and rolled on filter paper to remove adhering albumen. The yolk membrane was punctured with a needle and the contents were collected. Then, 100 g of yolk were diluted with 0.5 L of cold water at pH 5.0 and stirred at 4 ◦ C for 1 h. The precipitate was collected by centrifugation at 10,000g for 20 min at 4 ◦ C. The pellet was dissolved in 200 mL distilled water, stirred for 1 h and centrifuged at 10,000g for 20 min at 4 ◦ C. The pellet was extracted with 400 mL of hexane:ethanol (3:1, v:v) at 4 ◦ C for 3 h and centrifuged. The resulting cake was dried and extracted with 200 mL of 1.74 M NaCl overnight at 4 ◦ C. Then the suspension was centrifuged at 10,000g for 20 min at 4 ◦ C and the supernatant was dialyzed against distilled water for 24 h at 4 ◦ C and freeze dried.
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2.3. SDS-PAGE
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PV was dissolved in double distilled water, loaded and electrophoresed on a 12.5% SDS-PAGE gel to check its purity. SDS-PAGE was performed in 1.0 M Tris–HCl buffer (pH 6.8) for stacking gel (5% acrylamide) and 1.5 M Tris–HCl buffer (pH 8.8) for separating gel (12.5% acryamide). Migration was performed at 80 and 120 V in the stacking gel and separating gel, respectively. After electrophoresis, the gel was stained using the modified staining solution containing 0.05% Coomassie brilliant blue R-250, 0.1 mol/L aluminum nitrate, 25% isopropanal,10% acetic acid and 1.0% Triton X-100 [18,19].
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2.4. Electrospinning of CA nanofibrous mats and their hydrolysis
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Nanofibrous CA mats were fabricated by Deng’s method [20]. Briefly, 17 wt% CA solution prepared in a 2/1 (w/w) acetone/DMAc mixture. The CA solution was fed into a plastic syringe, which was driven by a syringe pump (LSP02-1B, Baoding Longer Precision Pump Co., Ltd., China). The positive electrode of a high voltage power supply (DW-P303-1ACD8, Tianjin Dongwen Co., China) was clamped to the metal needle tip of the syringe. A grounded cylindrical layer was used as a collector which rotated with a linear velocity of 100 m/min. The applied voltage was 16 kV and the tipto-collector distance was 20 cm. The ambient temperature and relative humidity were maintained at 25 ◦ C and 45%, respectively. The prepared fibrous mats were dried at 80 ◦ C in vacuum for 24 h to remove the trace solvent. Hydrolysis of the CA mats was performed in a 0.05 M NaOH aqueous solution at ambient temperature for 7
Scheme 1. (A) Schematic diagram illustrating the hydrolysis of CA nanofibrous mats and (B) schematic diagram illustrating the fabrication process of the LBL film-coating cellulose mats.
days following the previous report. And the schematic diagram of hydrolysis of CA was shown in Scheme 1A.
2.5. Formation of LBL structured multilayer on nanofibrous mats The concept for fabrication of LBL structured fibrous mats was shown in Scheme 1B. The concentration of the positively charged CS solutions was fixed as 1 mg/mL by dissolving them in a 0.5% (V/V) aqueous acetic acid solution and the pH of solutions was controlled at 5.0. The negatively charged PV solution was 1 mg/mL in pure water and the pH was adjusted to 6. The ionic strength of all dipping solutions was regulated by the addition of NaCl at a concentration of 0.1 mg/mL. First, nanofibrous cellulose mats were immersed into CS for 20 min followed by 2 min of rinsing in three pure water baths [21]. The mats were then immersed into the PV solution for 20 min, followed by the identical rinsing steps. The adsorption and rinsing steps were repeated until the desired number of deposition bilayers was obtained. Here, (CS/PV)n was used as a formula to label the LBL structured films, where n was the number of the CS/PV bilayers. The outermost layer was CS composite when n equaled to 5.5 and 10.5. The LBL films coated fibrous mats were dried at 80 ◦ C for 24 h under vacuum prior to further characterizations.
2.6. Characterization The morphology and composition of fibrous mats were examined by scanning electron microscopy (SEM) (S-4800, Hitachi Ltd., Japan). The diameters of the fibers were measured using an image analyzer (Adobe Photoshop CS5.0). Fourier transform infrared (FT-IR) spectra were recorded using a Nicolet170-SX instrument (Thermo Nicolet Ltd., USA) in the wavenumber range of 4000–400 cm−1 . The surface elemental composition of the samples was identified by X-ray photoelectron spectroscopy (XPS) using an axis ultra DLD apparatus (Kratos, U.K.). X-ray diffraction (XRD) was carried out using a diffract meter type D/max-rA (Rigaku Co., Japan) with Cu target and Ka radiation ( = 0.154 nm). -potential analysis was preformed using a Nano-25 zetasizer (Malven, England).
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Fig. 1. SEM images and diameter distribution histograms of nanofibrous mats ((a)–(f)): cellulose acetate, cellulose, (CS/PV)5 , (CS/PV)5.5 , (CS/PV)10 , and (CS/PV)10.5 .
2.7. Inhibition of bacterial activity
3.2. Fabrication of CS/PV nanofibrous mats
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The inhibition zone test was used to study the bacterial inhibition activity of nanofibrous mats. Gram-negative E. coli and Gram-positive S. aureus were selected as representative microorganism and cultivated in culture medium in an incubator. Unmodified cellulose mats were used as negative 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 micro-liters of 5.0–10.0 × 105 cfu/mL E. coli 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 or 50 ◦ C in an air-bathing thermostat shaker with a rotating speed of 120 r/min for 24 h, the bacteria inhibition zones were measured by a micrometer with a tolerance of 1 mm. All of the experiments were conducted in triplicate with data reported as error bar.
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3. Results and discussion
As previous works described, the primary driven force for LBL modification was the electrostatic deposition and hydrogen bonding between the template and the assembly layer [22]. Zeta potential of the raw materials was measured in order to determine the driven force of LBL coating (Table S1).The LBL films were fabricated from CS and PV on cellulose template. It can be seen that the cellulose nanofibrous template was negatively charged (∼−28.6 mV). CS was positively charged and it had -potential value of 25.8 mV, that was attributed to the protonation of amino group. Oppositely, PV is an acidic protein rich in phosphate groups bound mainly by serine [16]. The isoelectric point of PV is 4.0, and the -potential of PV was −22.4 mV under specific pH value. Furthermore, the charge of raw materials could be controlled via adjusting the pH value, to avoid assembling on the fibers too much to destroy the structure of the fibers. Based on this fact, the positively charged CS and negatively charged PV could be alternately deposited on the surface of negatively charged cellulose nanofibers through electrostatic interaction [23]. Scheme 1B presents the deposition of CS and PV.
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3.1. Preparation and SDS-PAGE of PV
3.3. Surface morphology analysis of CS/PV nanofibrous mats
The results of the SDS-PAGE patterns of standard PV and extracted PV are shown in Fig. S1. SDS-PAGE revealed that PV extracted from yielded a major band of about 35 kDa and a minor band of about 33 kDa, corresponding to -PV and ␣-PV, respectively. No other contaminated bands were found on the gel, indicating that PV was pure.
To investigate the influence of film-coating on the nanofibrous mats, LBL coated mats with different bilayer number were observed by SEM (Fig. 1). And the diameter distribution of nanofibers was shown in Fig. 1. The typical electrospun nanofibrous mats had a 3D structure with pores in micro and sub-micro size [24]. The CA mats were composed of loosely packed cylindrical fibers. Some
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Table 1 Element composition and content on the surface of cellulose mats and (CS/PV)10 , and (CS/PV)10.5 . Atomic fraction of element composition (%)
Nanofibrous mats
Cellulose (CS/PV)10 (CS/PV)10.5
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O
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P
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54.01 29.44 29.52
– 10.02 9.28
– 2.27 1.79
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junctions among the CA fibers were caused by the trace remained solvent after electrospinning (Fig. 1a). After hydrolysis, the surface of the fibers become rough and porous (Fig. 1b) [25]. To study the impact of the number of coating bilayers on the formation of LBL films coated nanofibers, the cellulose fibers were coated with various bilayers of CS and PV. After LBL coating process, the space between the fibers remarkably reduced. Compared with the morphology of CA or cellulose fibrous mats, it could be observed that LBL films coated nanofibers had a few conglutinations, which revealed that the CS and PV solutions deposited on the surface of cellulose nanofibers successfully, and formed a thin layer of films but split into webs during the drying process, hence the surface of the composite mats was much rougher than that of cellulose nanofibrous mats.
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3.4. Surface composition measurement of CS/PV nanofibrous mats
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The chemical states of the elements are examined by the XPS measurement. The quantification function of XPS could determine the elemental compositions at a depth of up to 10 nm from the surface. To verify the deposition of CS and PV on the LBL films, XPS scans were carried out to further verify the surface composition of the resultant samples. The XPS spectra for cellulose fibrous mats, (CS/PV)10 and (CS/PV)10.5 LBL coated films were shown in Fig. 2A. As we all know, cellulose was consisted of carbon, oxygen, and hydrogen and both PV and CS contained nitrogen, however, CS did not contain phosphorus element, which was the characteristic element of PV. So, with regard to cellulose mats, the strong carbon and oxygen peaks were observed on the XPS scan. After CS and PV deposition, the presence of nitrogen, carbon, oxygen, and phosphorus was observed on the surface of composite mats. PV is a polyanionic phosphoglycoprotein present in egg yolk, of the known protein, it is the most phosphorylated one. So the characteristic elements of phosphorus verified that PV was deposited on the surface of the fibers successfully. As both PV and CS contained nitrogen, the presence of nitrogen peak cannot certify that CS was deposited on the surface of the fibers successfully. In order to further confirm LBL coating process in every layer, the ratio of N/P was measured (Table 1). From the obtained atomic concentration of the high resolution scans (N 1s and P 2p), the N/P ratio of (CS/PV)10 and (CS/PV)10.5 were obtained to be 4.41 and 5.18, respectively. The N/P ratio distinctly increased when the outmost layer was CS. These result proved that both CS and PV could successfully coat on the surface of the mats in every layer.
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3.5. FT-IR spectra of CS/PV nanofibrous mats
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FTIR reveals information about the molecular structure of chemical compounds and is useful for the characterization of biopolymers. Here, FTIR was used to verify the presence of CS and PV on cellulose nanofibers. The FTIR spectra for cellulose nanofibrous mats and LBL coated mats were depicted in Fig. 3. For the cellulose mats, assignment of the main absorption peaks are as follows: the characteristic band at 3422 cm−1 was attributed to the stretching of hydroxyl groups involved in intramolecular hydrogen bonding of the cellulose II crystal [26]; the band at 1066 cm−1 was assigned to the stretching of C–O [27]; asymmetric stretching of C–O–C bond of the glycosidic linkage and pyranose ring of cellulose were observed at around 1238, 1164 and 1052 cm−1 , respectively. And the 897 cm−1 was attributed to the C1 –H deformation vibrations of cellulose [28]. The two common bands of amino group observed at 1648 and 1535 cm−1 belongs to the amide I and amide II peaks, which indicates that PV and
Fig. 2. (A) XPS spectra of (CS/PV)10.5 (a), (CS/PV)10 (b), LBL coated films and cellulose fibrous mats (c), and (B) core-level spectra of N 1s narrow scans of (CS/PV)10.5 , (CS/PV)10 LBL coated films and cellulose fibrous mats ((a), (c), (e)), core-level spectra of P 2p narrow scans of (CS/PV)10.5 , (CS/PV)10 LBL coated films and cellulose fibrous mats ((b), (d), (f)).
CS were deposited on the surface of cellulose mats successfully [29]. 3.6. Crystalline property of CS/PV nanofibrous mats The crystalline phases of CS/PV nanofibrous mats with different layers are characterized by XRD measurements. For comparison, pure CS, CA and cellulose nanofibrous mats are also tested. As shown in Fig. 4, CA mats exhibited an obvious crystalline peak at 2 = 9.1◦ [30]. In addition, both cellulose nanofibrous mat and CS/PV nanofibrous mats with different layers exhibit the uniform cellulose structure with characteristic 2 values at (12.1◦ , 20.3◦ , and 21.8◦ ), respectively [31]. The diffraction peaks originated from CS and PV are hardly detected in the CS/PV samples. This may be due to the facts of: (i) the amorphous phase PV has no peak in the XRD pattern; (ii) the peaks of CS is difficult to be detected because of low CS loading content; or (iii)
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Fig. 3. FT-IR spectra of ((a)–(e)): (CS/PV)5 , (CS/PV)5.5 , (CS/PV)10 , (CS/PV)10.5 , and cellulose nanofibrous mats.
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the most intense of CS peaks is overlapped by those of cellulose.
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The thermal stability of cellulose mats and LBL films coating mats was evaluated by TGA and DSC analysis (Fig. 5). TGA provided the onset decomposition temperature, percentage of residue, highest weight loss temperature, and highest weight loss rates as the samples were heated [32]. The TG curves could be divided into three zones: the first weight loss is observed up to 100 ◦ C (∼10% weight loss), attributed to removal of adsorbed or bond water (I), the second weight loss step was corresponded to the decomposition of composite materials (II). For cellulose nanofibrous mats, it was observed at ca. 200 ◦ C, which was associated with the decomposition of cellulose (Fig. 5A). Neglecting the first weight loss stage, the onset decomposition temperature was recorded to investigate the thermal stability of nanofibrous mats. It can be seen that all LBL coated mats have the similar decomposition temperatures at
Fig. 5. (A) TGA thermograms of cellulose nanofibrous mats, LBL structure nanofibrous mats coated with: (CS/PV)10.5 , (CS/PV)10 , (CS/PV)5.5 , (CS/PV)5 , and cellulose fibrous mats; (B) Differential thermal gravimetry (DTG) of cellulose nanofibrous mats, LBL structure nanofibrous mats coated with: (CS/PV)10.5 , (CS/PV)10 , (CS/PV)5.5 , (CS/PV)5 , and cellulose fibrous mats; (C) DSC thermograms of LBL structure nanofibrous mats coated with: (CS/PV)10.5 , (CS/PV)10 , (CS/PV)5.5 , (CS/PV)5 ((a)–(d)), and cellulose fibrous mats (e).
Fig. 4. XRD patterns of CS (a), LBL structured mats coated with (CS/PV)10.5 , (CS/PV)10 , (CS/PV)5.5 , and (CS/PV)5 ((b)–(e)), cellulose fibrous mats (f), CA fibrous mats (g).
about 250 ◦ C as shown in Figs. 5A and B. This result indicated that (CS/PV) coated mats exhibited better thermal stability, no matter what component the outmost layer was. The last one was the carbonization of the degraded productions to ash(III) [33]. In this stage, with the increase of temperature the weight loss reached a maximum. Some significant properties shown in TGA curve could be also elucidated by analyzing of the DSC thermograms as reflected in Fig. 5C. The cellulose mats showed a broad exothermic event at ca. 200 ◦ C was associated with the degraded of cellulose mats. For LBL coated mats, the appearance of an obvious endothermic event at about 275 ◦ C may be attributed to the melting of CS. In addition, with the increase of the layer number, the thermal stability
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technique. Both the electrostatic attractions between cellulose fibers, CS and PV were applied in the LBL self-assembly processing. The deposition of CS and PV on the surface of cellulose mats was characterized by XPS, XRD, and FTIR. And the morphologies of composite nanofibrous mats became increasingly rough with increasing number of deposition layer. Moreover, the microbial inhibition assay demonstrated that the CS/PV composite mats had good antibacterial effects, which have potential application in the areas of antibacterial coating, tissue engineering, wound dressing, etc. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Grant no. 31371841). The authors greatly thank colleagues of Key Laboratory of Environment Correlative Dietology of Huazhong Agricultural University for offering many conveniences. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac. 2013.12.016. References
Fig. 6. Antimicrobial activities against E. coli and S. aureus of fibrous cellulose mats (control) and multilayer nanofibrous mats at 37 ◦ C (A) and 50 ◦ C (B).
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decreased slightly. All the results of DSC were consistent with the results of TGA.
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Among the natural polymers CS and its derivatives are regarded as one of the most attractive for use in nanofibrous materials. One of the most valuable properties of CS is its inherent antimicrobial activity which is mainly due to its polycationic nature. CS inhibits the growth of a variety of pathogenic microorganisms—Gram-positive and Gram-negative bacteria, yeasts, and fungi [34–36]. In addition, PV exhibited good antimicrobial properties, due to chelating and surface activity (affinity for outer membrane) [14]. The antibacterial activity of LBL coated mats was determined by inhibition zone method against E. coli and S. aureus under 37 and 50 ◦ C (Fig. 6). Both under 37 and 50 ◦ C, cellulose mats hardly showed inhibitory effect, whereas composite mats revealed significant antibacterial activity and the antibacterial activity enhanced with increasing the numbers of bilayers, because the quantity of antibacterial agent was enhanced with increasing the numbers of bilayers. The LBL coated mats have a relatively weak antimicrobial activity for Gram-negative bacillus under 37 ◦ C, however, the antimicrobial activity for E. coli significantly enhanced due to the bacteriostatic effect of PV under thermal stress at 50 ◦ C. And these activities in conjunction with thermal stress seem to contribute to the loss of viability of E. coli [15]. 4. Conclusions In conclusion, we have fabricated CS and PV multilayer nanofibrous mats using electrospinning and electrostatic LBL deposition
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