materials Article
In Situ Synthesis of Silver Nanoparticles on the Polyelectrolyte-Coated Sericin/PVA Film for Enhanced Antibacterial Application Rui Cai 1 , Gang Tao 2 , Huawei He 2,3, * ID , Pengchao Guo 2 , Meirong Yang 2 , Chaoxiang Ding 2 , Hua Zuo 4 , Lingyan Wang 1 , Ping Zhao 2 and Yejing Wang 1, * 1 2
3 4
*
College of Biotechnology, Southwest University, Beibei, Chongqing 400715, China; [email protected] (R.C.); [email protected] (L.W.) State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400715, China; [email protected] (G.T.); [email protected] (P.G.); [email protected] (M.Y.); [email protected] (C.D.); [email protected] (P.Z.) Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Beibei, Chongqing 400715, China College of Pharmaceutical Sciences, Southwest University, Beibei, Chongqing 400715, China; [email protected] Correspondence: [email protected] (H.H.); [email protected] (Y.W.); Tel.: +86-23-6825-1575 (Y.W.)
Received: 3 July 2017; Accepted: 17 August 2017; Published: 18 August 2017
Abstract: To develop silk sericin (SS) as a potential antibacterial biomaterial, a novel composite of polyelectrolyte multilayers (PEMs) coated sericin/poly(vinyl alcohol) (SS/PVA) film modified with silver nanoparticles (AgNPs) has been developed using a layer-by-layer assembly technique and ultraviolet-assisted AgNPs synthesis method. Ag ions were enriched by PEMs via the electrostatic attraction between Ag ions and PEMs, and then reduced to AgNPs in situ with the assistance of ultraviolet irradiation. PEMs facilitated the high-density growth of AgNPs and protected the synthesized AgNPs due to the formation of a 3D matrix, and thus endowed SS/PVA film with highly effective and durable antibacterial activity. Scanning electron microscopy, energy dispersive spectroscopy, X-ray diffractometry, Fourier transfer infrared spectroscopy, water contact angle, mechanical property and thermogravimetric analysis were applied to characterize SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films, respectively. AgNPs-PEMs-SS/PVA film has exhibited good mechanical performance, hydrophilicity, water absorption capability as well as excellent and durable antibacterial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa and good stability and degradability. This study has developed a simple method to design and prepare AgNPs-PEMs-SS/PVA film for potential antibacterial application. Keywords: sericin; poly(vinyl alcohol); polyelectrolyte multilayers; AgNPs; antibacterial activity
1. Introduction Silk sericin (SS) is a globular protein produced by Bombyx mori (silkworm) [1], which contains 18 amino acids including the essential amino acids required by the human body [2]. It is about 20–30% of the total weight of silk cocoon. The main role of sericin is to envelop the fibroin [3]. As a natural protein, silk sericin is biocompatible and biodegradable. Since sericin has a variety of additional properties like gelling ability, water-holding capacity and skin adhesion [4], it has been widely applied in the biomedical field. Moreover, silk sericin could promote the adhesion and proliferation of human skin and accelerate burn or scald wound healing [5–7]. Therefore, it is regarded as a good candidate for wound dressing material. However, sericin itself is frangible and has a poor mechanical performance,
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which is not favorable for its application in wound healing. In our previous study, we have prepared sericin/poly(vinyl alcohol) (SS/PVA) blend film to improve the mechanical property of sericin [8]. Bacterial infection is a huge challenge for wound healing. To resolve this problem, it is necessary to functionalize the SS/PVA film with good antibacterial activity. Antimicrobial surface coating or modification has attracted more and more attention in biomedical and industrial fields [9–12]. With the rapid development of nanotechnology, a lot of novel nanomaterials have been created with exciting chemical and physical properties [13,14]. As an essential metal nanometer material, silver nanoparticles (AgNPs) have a broad range of applications, from electronics [15] and catalysis [16] to infection prevention [17] and medical diagnosis [18]. It is an effective broad-spectrum antimicrobial agent [19–21] and rarely leads to the development of resistant microbes [22]. Compared with other antibacterial agents, AgNP has low cytotoxicity to human cells [23,24]. In addition, AgNP is an effective anti-inflammatory agent and is used to promote wound healing [25]. To date, a number of chemical and physical methods have been developed to synthesize AgNPs [26–29]. In the traditional methods, toxic chemical reagents and expensive instruments such as ultrasound microwave [30] and γ-radiation [31] are required for AgNPs synthesis. Hence, it is necessary to develop a green and economical method to prepare AgNPs. Ultraviolet (UV)-assisted AgNPs synthesis is one of the most simple and environmentally friendly methods [32]. Studies have shown that AgNPs are commonly immobilized on the material’s surface [33,34]. In this case, Ag+ binding sites are limited on the surface of material, so only limited AgNPs could be synthesized. In addition, the synthesized AgNPs are unstable due to the weak van der Waals interactions, which may reduce its bactericidal efficiency. Polymers such as poly(vinyl pyrrolidone), poly(acrylic acid) and polyamide could provide more binding sites for Ag+ [35,36]. Layer-by-layer self-assembly is a simple technique to construct polyelectrolyte multilayers (PEMs) [37,38]. It is based on the sequential adsorption of oppositely charged components, in which two oppositely charged polymers are deposited on the surface of material by means of electrostatic attraction [39–42]. PEMs could provide more Ag+ binding sites and create a three-dimensional (3D) space for the growth of AgNPs, thus improving the loading of AgNPs and protecting the entrapped AgNPs. In this study, we have established a poly(acrylic acid) (PAA)/poly(dimethyl diallyl ammonium chloride) (PDDA)/PAA multilayered structure on the surface of SS/PVA film, which acted as a three-dimensional matrix to allow the high density synthesis of AgNPs in situ via the UV-assisted AgNPs synthesis and protect the synthesized AgNPs from oxidation and falling off. Scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffractometry (XRD), Fourier transfer infrared spectroscopy (FT-IR), water contact angle, mechanical property and thermogravimetric analysis (TGA) were performed to characterize the prepared films. Antimicrobial assays were conducted to investigate the antimicrobial performance of AgNPs-PEMs-SS/PVA film against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). This novel composite film has exhibited great potential in biomedical applications for its excellent performance and durable antimicrobial activity. 2. Results and Discussion 2.1. Preparation of PEMs-SS/PVA Film Ito et al. have prepared a highly flexible, strong adhesion, and transferrable AgNPs-loaded polymer nanosheet using the layer-by-layer assembly technique [43]. Here, poly (ethyleneimine) (PEI) is a cationic polymer and could adhere on SS/PVA film surface via the electric interaction between PEI and the negatively charged groups exposed on the surface of SS/PVA film. PAA and PDDA are two weak polyelectrolytes with negative and positive charges, respectively, which are used to construct a stable PEM on the surface of SS/PVA film for the adsorption of Ag+ . The alternative treatment with PAA and PDDA resulted in the formation of a three-dimensional matrix on the surface of SS/PVA blend film. AgNPs were synthesized in situ on the film surface by UV irradiation. PEMs provided a
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three-dimensional space for the high-density growth of AgNPs and protected the synthesized AgNPs, antibacterial activity. The preparation antibacterial characterization AgNPs-PEMs-SS/PVA thus endowing SS/PVA film with highlyand effective and durable antibacterialof activity. The preparation antibacterial activity. The preparation and antibacterial characterization of AgNPs-PEMs-SS/PVA film were described in Figure 1. and characterization film antibacterial were described in Figure 1. of AgNPs-PEMs-SS/PVA film were described in Figure 1.
Figure 1.1.Schematic diagram of the preparation and antibacterial of AgNPs-PEMs-SS/ Schematic diagram of the preparation and characterization antibacterial characterization of Figure 1. Schematic diagram of the preparation and antibacterial characterization of AgNPs-PEMs-SS/ PVA film. AgNPs-PEMs-SS/PVA film. PVA film.
2.2. SEM, 2.2. SEM, EDS EDS and and XRD XRD Analysis Analysis 2.2. SEM, EDS and XRD Analysis SEM images images were shown shown in Figure Figure 2. The The surface morphology morphology of SS/PVA SS/PVA film SEM film was was uniform, uniform, SEM images were were shown in in Figure 2. 2. The surface surface morphology of of SS/PVA film was uniform, which showed good structural integrity without any cracks and interface layers (Figure 2a), which showed good structural integrity without any cracks and interface layers (Figure 2a), indicating which showed good structural integrity without any cracks and interface layers (Figure 2a), indicating and PVA has good compatibility. Figurethe 2b showed the seemed film surface sericin andsericin PVA has good compatibility. Figure 2b showed surface to beseemed coveredto bybe a indicating sericin and PVA has good compatibility. Figure 2b film showed the film surface seemed to be covered by a layer of membranous-like substance after PEM coating on the surface of SS/PVA film. layer of membranous-like substance after substance PEM coating on PEM the surface of SS/PVA film. Figure 2c showed covered by a layer of membranous-like after coating on the surface of SS/PVA film. Figure 2c showed the cross-sectional structurefilm, of PEMs-SS/PVA film, formed suggesting PEMs formed a the cross-sectional structure of PEMs-SS/PVA suggesting PEMs a three-dimensional Figure 2c showed the cross-sectional structure of PEMs-SS/PVA film, suggesting PEMs formed a three-dimensional matrix. Figure 2d–f showed the morphologies of films AgNPs-SS/PVA films matrix. Figure 2d–f showed the morphologies of AgNPs-SS/PVA without and withwithout PEMs, three-dimensional matrix. Figure 2d–f showed the morphologies of AgNPs-SS/PVA films without and with PEMs, respectively. High-density AgNPs were observed on the surface of PEMs-SS/PVA respectively. High-density AgNPs were observed theobserved surface of film, whereas and with PEMs, respectively. High-density AgNPs on were onPEMs-SS/PVA the surface of PEMs-SS/PVA film, awhereas only a of small amount of AgNPs wasofonSS/PVA the surface of SS/PVA filmunder without only small amount AgNPs was on the surface film without PEMs the PEMs same film, whereas only a small amount of AgNPs was on the surface of SS/PVA film without PEMs under the same reaction condition. The result indicated that PEM coating promoted the synthesis of reaction The result indicated that PEM coating the promoted synthesis of under thecondition. same reaction condition. The result indicated thatpromoted PEM coating thehigh-density synthesis of + + high-density AgNPs. Previous studies indicate that the carboxyl groups of PAA greatly increase Ag AgNPs. Previous studies indicate that indicate the carboxyl groups of PAA greatly increase Ag increase adsorption high-density AgNPs. Previous studies that the carboxyl groups of PAA greatly Ag+ + 0 + 0 adsorption and thus the transformation of Ag to Ag0irradiation under UV irradiation [44]. With UV and thus promote thepromote transformation of Ag to Ag under With UV irradiation + to UV adsorption and thus promote the transformation of Ag Ag under UV [44]. irradiation [44]. With UV irradiation time increased to 30 min, AgNPs density on the surface of PEMs-SS/PVA film time increased to 30 min, AgNPs density the surface of PEMs-SS/PVA filmofsignificantly increased irradiation time increased to 30 min, on AgNPs density on the surface PEMs-SS/PVA film significantly increased (Figure 2g,h). The long-lasting antibacterial activity of AgNPs material is (Figure 2g,h). The long-lasting antibacterial activity of AgNPs material is mainly dependent on the significantly increased (Figure 2g,h). The long-lasting antibacterial activity of AgNPs materiallife is mainly dependent on the life of AgNPs. film Hence, filmantibacterial was expected to have of AgNPs. Hence, AgNPs-PEMs-SS/PVA wasAgNPs-PEMs-SS/PVA expected to have durable activity for mainly dependent on the life of AgNPs. Hence, AgNPs-PEMs-SS/PVA film was expected to have durable antibacterial activity for its high density AgNPs. its high density AgNPs. durable antibacterial activity for its high density AgNPs.
Figure 2. Cont.
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Figure 2.2. Surface morphologies of of SS/PVA SS/PVA (a); (a); PEMs-SS/PVA PEMs-SS/PVA (b); structure of of Figure Surface morphologies (b); cross-sectional cross-sectional structure PEMs-SS/PVA films (c); SS/PVA film in silver nitrate solution irradiated by UV light for 10 min (d); PEMs-SS/PVA films (c); SS/PVA film in silver nitrate solution irradiated by UV light for 10 min PEMs-SS/PVA film film in silver nitrate solution irradiated by UV for 10for min min (d); PEMs-SS/PVA in silver nitrate solution irradiated bylight UV light 10(e,f) minand (e,f)30 and 30(g,h). min (f,h) are magnification of (e,g), respectively; EDS and (g,h). (f,h)the are high the high magnification of (e,g), respectively; EDSspectrum spectrumof of PEMs-SS/PVA PEMs-SS/PVA film film and AgNPs-PEMs-SS/PVA film (i); XRD XRD patterns patterns of of sericin sericin (j1), (j1), SS/PVA SS/PVA film film (j2), (j2), PEMs-SS/PVA PEMs-SS/PVA film (j3) AgNPs-PEMs-SS/PVA film (i); film (j3) and AgNPs-PEMs-SS/PVA film (j4) (j). and AgNPs-PEMs-SS/PVA film (j4) (j).
The EDS spectrum showed the peaks of carbon, nitrogen, chlorine and oxygen element on The EDS spectrum showed the peaks of carbon, nitrogen, chlorine and oxygen element on PEMs-SS/PVA film (Figure 2i). The peaks of carbon, nitrogen, chlorine and oxygen were attributed PEMs-SS/PVA film (Figure 2i). The peaks of carbon, nitrogen, chlorine and oxygen were attributed to PAA, PDDA, PVA and sericin, respectively. The chlorine peak was mainly from PDDA. The EDS to PAA, PDDA, PVA and sericin, respectively. The chlorine peak was mainly from PDDA. The EDS spectrum of AgNPs-PEMs-SS/PVA film showed a distinct peak of the silver element except carbon, spectrum of AgNPs-PEMs-SS/PVA film showed a distinct peak of the silver element except carbon, nitrogen, chlorine, oxygen and chlorine peak. nitrogen, chlorine, oxygen and chlorine peak. XRD patterns were shown in Figure 2j. A broad peak located at 2θ = 19.2° was discovered in XRD patterns were shown in Figure 2j. A broad peak located at 2θ = 19.2◦ was discovered in sericin sericin pattern, which was consistent with the previous report [45]. The other three patterns had a pattern, which was consistent with the previous report [45]. The other three patterns had a characteristic characteristic broad peak located at 2θ = 19.8°, which could be assigned to the crystal structure of broad peak located at 2θ = 19.8◦ , which could be assigned to the crystal structure of PVA. No significant PVA. No significant change occurred on the pattern of SS/PVA film after PEM coating, UV change occurred on the pattern of SS/PVA film after PEM coating, UV irradiation and AgNPs irradiation and AgNPs modification, suggesting these treatments did not affect the crystal structure modification, suggesting these treatments did not affect the crystal structure of SS/PVA film. After of SS/PVA film. After AgNPs modification, one peak appeared at 2θ = 38.2°, which could be ascribed AgNPs modification, one peak appeared at 2θ = 38.2◦ , which could be ascribed to the crystal planes to the crystal planes (111) of the face-centered cubic structure of AgNPs. Another peak appeared at (111) of the face-centered cubic structure of AgNPs. Another peak appeared at 2θ = 32.2◦ , which could 2θ = 32.2°, which could be attributed to the crystal planes (111) of Ag2O. UV irradiation facilitates the+ be attributed to the crystal planes (111) of Ag2 O. UV irradiation facilitates the transformation of Ag transformation of Ag+ to Ag0. At the same time, it will produce a small part of ozone. Ag2O may be to Ag0 . At the same time, it will produce a small part of ozone. Ag2 O may be derived from the derived from the oxidation of AgNPs on the film surface exposed to the UV-generated ozone [46,47] oxidation of AgNPs on the film surface exposed to the UV-generated ozone [46,47] or air in aqueous or air in aqueous solutions [48]. EDS and XRD results suggested that AgNPs were successfully solutions [48]. EDS and XRD results suggested that AgNPs were successfully synthesized and evenly synthesized and evenly distributed on the surface of PEMs-SS/PVA film. The high density and good distributed on the surface of PEMs-SS/PVA film. The high density and good crystallinity of AgNPs crystallinity of AgNPs may greatly increase the antibacterial activity of PEMs-SS/PVA film. may greatly increase the antibacterial activity of PEMs-SS/PVA film. 2.3. FT-IR FT-IR Analysis Analysis 2.3. FT-IR spectrum spectrum is is aa classical classical method method to to reveal reveal the the structure structure of of aa substance substance [49]. [49]. Figure Figure 33 showed showed FT-IR the FT-IR spectra of sericin (a), SS/PVA (b), PEMs-SS/PVA (c) and AgNPs-PEMs-SS/PVA films the FT-IR spectra of sericin (a), SS/PVA (b), PEMs-SS/PVA (c) and AgNPs-PEMs-SS/PVA films (d). (d). −1 were discovered in all samples, which may be Three characteristic peaks at 1640, 1521 and 1244 cm − 1 Three characteristic peaks at 1640, 1521 and 1244 cm were discovered in all samples, which may assigned to to C=O stretching vibration C-N stretching stretching and and N–H N-H be assigned C=O stretching vibrationofofamide amideI Ipeak, peak,aa combination combination of of C–N bendingvibration vibrationof ofamide amideIIIIpeak peakand anda acombination combination N-H bending and C-N stretching vibration bending ofof N–H bending and C–N stretching vibration of −1 and 3340 cm−1, which of amide III, respectively. Pure sericin and PVA have broad bands at 3274 cm − 1 − 1 amide III, respectively. Pure sericin and PVA have broad bands at 3274 cm and 3340 cm , which are are associated amine N-H stretching vibration sericinand andthe theO–H O-Hstretching stretchingvibration vibration of of associated withwith the the amine N–H stretching vibration ofof sericin PVA, respectively. respectively. After After blending blending with with PVA, PVA, the the N–H N-H stretching stretching vibration vibration band band of of sericin sericin slightly slightly PVA, −1, indicating that sericin and PVA had good compatibility. After PEM shifted from 3274 to 3320 cm − 1 shifted from 3274 to 3320 cm , indicating that sericin and PVA had good compatibility. After PEM −1 modification, aa weak could be ascribed to the vibration band 1 , which modification, weakpeak peakoccurred occurredatat1476 1476cm cm,−which could be ascribed tocycle the cycle vibration of PDDA [50]. The confirmed the presence of PEMs on the of SS/PVA film.film. No band of PDDA [50]. results The results confirmed the presence of PEMs on surface the surface of SS/PVA significant change was observed on the C-O vibration band of AgNPs-PEMs-SS/PVA film. This may No significant change was observed on the C–O vibration band of AgNPs-PEMs-SS/PVA film. This be because the carboxyl groups were involved in the adsorption of Ag+, but did not participate in the formation of AgNPs. The -CH2 characteristic peak of PVA at 2940 cm−1 may mask the -CH3 and -CH2 vibration band of PDDA at 2870 cm−1 and 2945 cm−1, respectively.
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may be because the carboxyl groups were involved in the adsorption of Ag+ , but did not participate in the formation of AgNPs. The –CH2 characteristic peak of PVA at 2940 cm−1 may mask the –CH3 and –CH band of PDDA at 2870 cm−1 and 2945 cm−1 , respectively. Materials 2017, 10, 967 5 of 15 2 vibration
Figure 3. 3. FT-IR spectra of sericin (a); SS/PVA (b); PEMs-SS/PVA (c) and(c) AgNPs-PEMs-SS/PVA films (d). Figure FT-IR spectra of sericin (a); SS/PVA (b); PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films (d).
2.4. Wettability Measurement and Water Uptake Ability
2.4. Wettability Measurement Waterbehavior Uptake Ability Figure 4a–c showed theand wetting of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA filmsFigure assessed byshowed water contact anglebehavior measurement, respectively. The water angle of SS/PVA 4a–c the wetting of SS/PVA, PEMs-SS/PVA andcontact AgNPs-PEMs-SS/PVA film was 21.3°, indicating that SS/PVA film had excellent hydrophilicity. After PEM the films assessed by water contact angle measurement, respectively. The water contact anglecoating, of SS/PVA water contact angle of SS/PVA film increased to 58°, indicating its good hydrophilicity. PEM coating film was 21.3◦ , indicating that SS/PVA film had excellent hydrophilicity. After PEM coating, the water on SS/PVA film reduce porositytoof58SS/PVA film, thus resulting in the increase water ◦ , indicating contact angle of may SS/PVA filmthe increased its good hydrophilicity. PEMofcoating contact angle of SS/PVA film. In addition, time delay from making the film to water contact angle on SS/PVA film may reduce the porosity of SS/PVA film, thus resulting in the increase of water measurement could result in surface saturation with hydrocarbons and thus cause the increase of contact angle of SS/PVA film. In addition, time delay from making the film to water contact angle water contact angle. film had water contact angle of 62°. Thethe water contact measurement could AgNPs-PEMs-SS/PVA result in surface saturation witha hydrocarbons and thus cause increase of angle was hardly changed before and after AgNPs modification, indicating that AgNPs modification ◦ water contact angle. AgNPs-PEMs-SS/PVA film had a water contact angle of 62 . The water contact did affect thechanged hydrophilicity of PEMs-SS/PVA film. Karumuri et al. have reported that water anglenot was hardly before and after AgNPs modification, indicating that AgNPs modification contact angle has slightly gone down with AgNPs deposition, but the small difference does not did not affect the hydrophilicity of PEMs-SS/PVA film. Karumuri et al. have reported that water affect the hydrophilicity [51]. contact angle has slightly gone down with AgNPs deposition, but the small difference does not affect The swelling [51]. properties of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films were the hydrophilicity 2) were immersed in PBS buffer (pH 7.4) for 12 h, 24 h examined (Figure 4d). All samples (1 × 1 cm The swelling properties of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films were and 48 h, respectively. The swelling ratios of these films about 1.8(pH without 2 examined (Figure 4d). All samples (1 × 1 cm ) were immersedwere in PBS buffer 7.4) forsignificant 12 h, 24 h differences, indicatingThe these filmsratios had of good hygroscopicity. The propertiesdifferences, were not and 48 h, respectively. swelling these films were about 1.8swelling without significant significantly different after 12 h, 24 h and 48 h because the total accessible surface of these films had indicating these films had good hygroscopicity. The swelling properties were not significantly different been completely saturated by water molecules quickly. These results indicated that SS/PVA, after 12 h, 24 h and 48 h because the total accessible surface of these films had been completely PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films had good hydrophilicity and water absorption saturated by water molecules quickly. These results indicated that SS/PVA, PEMs-SS/PVA and capability. AgNPs-PEMs-SS/PVA films had good hydrophilicity and water absorption capability.
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Figure 4. Water contact angles of SS/PVA (a); PEMs-SS/PVA (b); and AgNPs-PEMs-SS/PVA films (c);
Figure 4. Water contact angles of SS/PVA (a); PEMs-SS/PVA (b); and AgNPs-PEMs-SS/PVA films (c); swelling ratios of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films (d). swelling of SS/PVA, PEMs-SS/PVA andPEMs-SS/PVA AgNPs-PEMs-SS/PVA films (d). Figure ratios 4. Water contact angles of SS/PVA (a); (b); and AgNPs-PEMs-SS/PVA films (c); swelling ratios of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films (d). 2.5. Mechanical Property
2.5. Mechanical Property The tensile strength and elongation at break of these films were measured, as shown in Figure 5. 2.5. Mechanical Property SS/PVA film had a tensile strength of 2.45 at MPa, which much lower thanmeasured, that of the modified The tensile strength and elongation break of was these films were as shown in The PEM tensile strength and elongation at break of these films were measured, as shown in Figure 5. films. coating and AgNPs modification resulted in the increase of the tensile strength about Figure 5. SS/PVA film had a tensile strength of 2.45 MPa, which was much lower than that of SS/PVA a tensilewith strength MPa, film, whichsuggesting was muchthat lower than that ofand the AgNPs modified three film timeshad compared that of of 2.45 SS/PVA PEM coating the modified films. PEM coating and AgNPs modification resulted in the increase of the tensile modification increased the stiffness of SS/PVA film. Itinmay attributed to tensile the increase of film films. PEM coating and AgNPs modification resulted the be increase of the strength about strength about three times compared with that of SS/PVA film, suggesting that PEM coating and thickness. Elongation at break represents the flexibility of material [52]. Although the elongation at three times compared with that of SS/PVA film, suggesting that PEM coating and AgNPs AgNPs modification increased the stiffness ofhad SS/PVA film. It may gone be attributed towith the that increase of break (strain) of AgNPs-PEMs-SS/PVA film gone down a little compared of modification increased the stiffness of SS/PVA film. It may be attributed to the increase of film film thickness. Elongation atwas break represents the flexibility of material [52]. Although the elongation SS/PVA Elongation film, the change not statistically strain of AgNPs-PEMs-SS/PVA film was at thickness. at break represents the different. flexibilityThe of material [52]. Although the elongation at break break (strain) of AgNPs-PEMs-SS/PVA film had gone down a little gone compared with that 43.80(strain) ± 3.2%,of which still could support its application as an antibacterial biomaterial. PEM coating AgNPs-PEMs-SS/PVA film had gone down a little gone compared with that of of SS/PVA film, the change was not statistically different. The strain of AgNPs-PEMs-SS/PVA film was and AgNPs modification increased the film thickness, resulting in a slight decrease of the film SS/PVA film, the change was not statistically different. thus The strain of AgNPs-PEMs-SS/PVA film was flexibility. Inwhich general, mechanical performance of AgNPs-PEMs-SS/PVA film was ablePEM toPEM support 43.80 ± ±3.2%, still could support its as an an antibacterial antibacterialbiomaterial. biomaterial. coating 43.80 3.2%,which stillthe could support its application application as coating its application as an antibacterial biomaterial. and AgNPs modification increased the film thickness, thus resulting in a slight decrease of the film and AgNPs modification increased the film thickness, thus resulting in a slight decrease of the film flexibility. InIn general, filmwas was able support flexibility. general,the themechanical mechanicalperformance performance of AgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA film able to to support its its application asasananantibacterial application antibacterialbiomaterial. biomaterial.
Figure 5. Mechanical properties of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films: (a) tensile strength and (b) elongation at break. Data are the average value plus standard deviation (SD) from three independent experiments. Statistical significance is assessed using an unpaired t-test. * indicates p < 0.05. Figure 5. Mechanical properties of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films:
Figure 5. Mechanical properties of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films: (a) tensile (a) tensile and (b) at elongation at break. Data are thevalue average plus standard strength andstrength (b) elongation break. Data are the average plusvalue standard deviationdeviation (SD) from (SD) from three independent experiments. Statistical significance is assessed using an unpaired t-test. three independent experiments. Statistical significance is assessed using an unpaired t-test. * indicates * indicates p < 0.05. p < 0.05.
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2.6. TGA Analysis 2.6. TGA Analysis TGA reflects that the mechanism led to a unique thermal behavior. The result showed SS/PVA TGA reflects that the mechanism led to a unique thermal behavior. The result showed SS/PVA film underwent a multi-steps degradation process (Figure 6). In 0–100 °C, the initial weight loss was film underwent a multi-steps degradation process (Figure 6). In 0–100 ◦ C, the initial weight loss was due to the presence of moisture. While increasing temperature to 200 °C, the observed weight loss due to the presence of moisture. While increasing temperature to 200 ◦ C, the observed weight loss may be attributed to the degradation of the exposed side chains and the breakdown of main chain may be attributed to the degradation of the exposed side chains and the breakdown of main chain groups of sericin. After blending with PVA, water evaporation caused the reduction of initial weight groups of sericin. After blending with PVA, water evaporation caused the reduction of initial weight from room temperature to 180 °C. At this stage, the weight loss of SS/PVA film was slower than that from room temperature to 180 ◦ C. At this stage, the weight loss of SS/PVA film was slower than that of sericin, which may be due to the fact that SS/PVA film contained more bound water than sericin of sericin, which may be due to the fact that SS/PVA film contained more bound water than sericin itself did. The second stage of weight loss occurred from 200 to 600 °C. This may be ascribed to the itself did. The second stage of weight loss occurred from 200 to 600 ◦ C. This may be ascribed to the degradation of the exposed side chains and the breakdown of main chain groups of sericin and PVA. degradation of the exposed side chains and the breakdown of main chain groups of sericin and PVA. The weight loss of SS/PVA film reached an equilibrium at around 620 °C, which was higher than that The weight loss of SS/PVA film reached an equilibrium at around 620 ◦ C, which was higher than that of sericin. SS/PVA film had about 3% residual weight, suggesting that the hydrogen-bond of sericin. SS/PVA film had about 3% residual weight, suggesting that the hydrogen-bond interaction interaction between sericin and PVA may be favorable to the thermal stability of SS/PVA blend film. between sericin and PVA may be favorable to the thermal stability of SS/PVA blend film. After PEM After PEM coating, the equilibrium temperature increased to around 730 °C. PEMs-SS/PVA film had coating, the equilibrium temperature increased to around 730 ◦ C. PEMs-SS/PVA film had about 4% about 4% residual weight, indicating that PEM coating could enhance the thermostability of SS/PVA residual weight, indicating that PEM coating could enhance the thermostability of SS/PVA film and film and delay its thermal degradation. After AgNPs modification, the residual weight increased to delay its thermal degradation. After AgNPs modification, the residual weight increased to about 7%, about 7%, and the equilibrium temperature increased to around 650 °C, implying that AgNPs may and the equilibrium temperature increased to around 650 ◦ C, implying that AgNPs may improve the improve the thermostability of PEMs-SS/PVA film and reduce the weight loss. The result also thermostability of PEMs-SS/PVA film and reduce the weight loss. The result also indicated that PEMs indicated that PEMs and AgNPs were successfully modified on the surface of SS/PVA film. and AgNPs were successfully modified on the surface of SS/PVA film.
Figure AgNPs-PEMs-SS/PVAfilms. films. Figure6. 6. TGA TGA curves curves of of sericin, sericin, SS/PVA, SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA
2.7. Inhibition Zone Assay of AgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA films were assessed against Gram-negative Gram-negative The antibacterial effects of bacteria (E. coli and P. aeruginosa) and Gram-positive Gram-positive bacteria bacteria (S. (S. aureus), aureus), respectively respectively (Figure (Figure 7). 7). SS/PVA and SS/PVA and PEMs-SS/PVA PEMs-SS/PVAfilms filmsdid didnot notexhibit exhibit significant significant bactericidal bactericidal effects effects against against either either E. coli, coli, P. aeruginosa aeruginosa or or S. S. aureus. aureus. However, However, after after AgNPs AgNPs modification, modification, the the film film could could effectively effectively kill bacteria P. form evident evident inhibition inhibitionzones zoneswith withan anapproximate approximateaverage averagediameter diameter cm. The diameters and form ofof 2.52.5 cm. The diameters of of the inhibition zones were summarizedininTable Table1.1.The Theresult resultsuggested suggestedthat thatAgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA the inhibition zones were summarized film obviously inhibited the growth growth of of E. E. coli coli (a), (a), S. S. aureus aureus (b) (b) and and P. P.aeruginosa aeruginosa(c). (c).
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Figure 7. AgNPs-PEMs-SS/PVA films against E. coli (a); Figure 7. Antibacterial Antibacterialeffects effectsof ofSS/PVA, SS/PVA,PEMs-SS/PVA, PEMs-SS/PVA, AgNPs-PEMs-SS/PVA films against E. coli S. aureus (b) and P. aeruginosa (c); Growth curves assays of E. coli (d); S. aureus (e) and P. aeruginosa (a); S. aureus (b) and P. aeruginosa (c); Growth curves assays of E. coli (d); S. aureus (e) and P. aeruginosa (f). (f). f1); Bacteria Bacteria in in the the presence presence of ofSS/PVA SS/PVA (d2, (d2, e2, e2, f2), f2), PEMs-SS/PVA PEMs-SS/PVA Bacteria without without treatment treatment (d1, (d1, e1, e1, f1); Bacteria (d3, e3, e3, f3) f3) and and AgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA films films (d4, (d4, e4, e4, f4). f4). (d3, Table 1.1. Diameters Diameters of ofinhibition inhibitionzones zonesofofSS/PVA, SS/PVA,PEMs-SS/PVA PEMs-SS/PVA and and AgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA films Table P.aeruginosa. aeruginosa. against E. coli, S. aureus and P.
Bacteria Bacteria E. coil E. coil P. aeruginosa P. aeruginosa S.S.aureus aureus
Control (cm) PEMs-SS/PVA (cm) AgNPs-PEMs-SS/PVA (cm) Control (cm) PEMs-SS/PVA (cm) AgNPs-PEMs-SS/PVA (cm) 1.55 ± 0.02 1.55 ± 0.03 2.17 ± 0.42 1.55 ± 0.02 1.55 ± 0.03 2.17 ± 0.42 1.55 ± 0.01 1.55 ± 0.02 2.49 ± 0.41 1.55 ± 0.01 1.55 ± 0.02 2.49 ± 0.41 1.55 ± 0.03 1.551.55 ± 0.04 2.82 1.55 ± 0.03 ± 0.04 2.82±±0.26 0.26
2.8. Bacterial Curve Assay Assay 2.8. Bacterial Growth Growth Curve Bacterial growth growth curve curve experiments experiments were were carried carried out out to to further further evaluate evaluate the the antibacterial antibacterial effects effects Bacterial of AgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVAfilms. films.The The optical density of bacteria nm )(OD 600) was used to of optical density of bacteria at 600at nm600 (OD 600 was used to evaluate evaluate bacterial growth in the presence of the films. As shown in Figure 7d–f, lag phase of bacterial growth in the presence of the films. As shown in Figure 7d–f, the lag phase ofthe E. coli, S. aureus E. coli, S. aureus and P. aeruginosa was less than 1 h in the presence of SS/PVA and PEMs-SS/PVA and P. aeruginosa was less than 1 h in the presence of SS/PVA and PEMs-SS/PVA films. However, in the films. However, in the presence offilms, AgNPs-PEMs-SS/PVA lag phase coli, P. aeruginosa presence of AgNPs-PEMs-SS/PVA the lag phase of E. films, coli, P. the aeruginosa andofS.E. aureus significantly and S. aureus significantly prolonged to 26 h, 22 h and 30 h, respectively. For AgNPs film prolonged to 26 h, 22 h and 30 h, respectively. For AgNPs film without PEMs, the lag phase of without bacteria PEMs, the lag phase of bacteria generally prolongs to 12 h [8]. Compared to AgNPs film without generally prolongs to 12 h [8]. Compared to AgNPs film without PEMs, AgNPs-PEMs-SS/PVA PEMs, AgNPs-PEMs-SS/PVA film hadeffect a more inhibitory on thedemonstrated bacterial growth. film had a more significant inhibitory on significant the bacterial growth. effect The results that The results demonstrated that AgNPs-PEMs-SS/PVA film had excellent antimicrobial activity. AgNPs-PEMs-SS/PVA film had excellent antimicrobial activity. 2.9. Antimicrobial Stability Analysis Analysis 2.9. Antimicrobial Stability Figure 8a,b 8a,b showed showed the the antimicrobial antimicrobial stability stability analysis analysis of of AgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA films films against against Figure E. coli coli and and S. S. aureus, aureus, after after the the films films were were treated treated in in different differentpHs pHs(5, (5,7.4, 7.4,9) 9)for for24 24h. h.OD OD600 600 was to E. was used used to evaluate bacterial growth in the presence of the treated AgNPs-PEMs-SS/PVA films. After 18 h, evaluate bacterial growth in the presence of the treated AgNPs-PEMs-SS/PVA films. After 18 h, the film the film stillconsiderable showed considerable antimicrobial activity with compared that ofThe theantimicrobial control. The still showed antimicrobial activity compared that of with the control. antimicrobial activity of the film underwas pHhigher 5 condition was thatunder of the activity of the film treated under pHtreated 5 condition than that of higher the filmthan treated pHfilm 7.4 treated under pH 7.4 or pH 9 conditions. This may be due to the fact that AgNPs-PEMs-SS/PVA film or pH 9 conditions. This may be due to the fact that AgNPs-PEMs-SS/PVA film was more stable and was more stable and AgNPs loss was less under pH 5 condition than that under other conditions. The result indicated that AgNPs-PEMs-SS/PVA film had durable and steady antibacterial activity.
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AgNPs loss was less under pH 5 condition than that under other conditions. The result indicated that Materials 2017, 10, 967 9 of 15 AgNPs-PEMs-SS/PVA film had durable and steady antibacterial activity.
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Figure 8. films against E. coli (a) and S. aureus (b). Figure 8. Durable Durablebactericidal bactericidalactivity activityofofAgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA films against E. coli (a) and S. aureus DataData are are the the average value plus standard deviation (b). average value plus standard deviation(SD) (SD)from fromthree three independent independent experiments. experiments. Figure 8. Durable bactericidal activity of AgNPs-PEMs-SS/PVA films against E. coli (a) and S. aureus (b). Statistical significance is assessed using an unpaired unpaired t-test. t-test. ** indicates pp
In Situ Synthesis of Silver Nanoparticles on the Polyelectrolyte-Coated Sericin/PVA Film for Enhanced Antibacterial Application Rui Cai 1 , Gang Tao 2 , Huawei He 2,3, * ID , Pengchao Guo 2 , Meirong Yang 2 , Chaoxiang Ding 2 , Hua Zuo 4 , Lingyan Wang 1 , Ping Zhao 2 and Yejing Wang 1, * 1 2
3 4
*
College of Biotechnology, Southwest University, Beibei, Chongqing 400715, China; [email protected] (R.C.); [email protected] (L.W.) State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400715, China; [email protected] (G.T.); [email protected] (P.G.); [email protected] (M.Y.); [email protected] (C.D.); [email protected] (P.Z.) Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Beibei, Chongqing 400715, China College of Pharmaceutical Sciences, Southwest University, Beibei, Chongqing 400715, China; [email protected] Correspondence: [email protected] (H.H.); [email protected] (Y.W.); Tel.: +86-23-6825-1575 (Y.W.)
Received: 3 July 2017; Accepted: 17 August 2017; Published: 18 August 2017
Abstract: To develop silk sericin (SS) as a potential antibacterial biomaterial, a novel composite of polyelectrolyte multilayers (PEMs) coated sericin/poly(vinyl alcohol) (SS/PVA) film modified with silver nanoparticles (AgNPs) has been developed using a layer-by-layer assembly technique and ultraviolet-assisted AgNPs synthesis method. Ag ions were enriched by PEMs via the electrostatic attraction between Ag ions and PEMs, and then reduced to AgNPs in situ with the assistance of ultraviolet irradiation. PEMs facilitated the high-density growth of AgNPs and protected the synthesized AgNPs due to the formation of a 3D matrix, and thus endowed SS/PVA film with highly effective and durable antibacterial activity. Scanning electron microscopy, energy dispersive spectroscopy, X-ray diffractometry, Fourier transfer infrared spectroscopy, water contact angle, mechanical property and thermogravimetric analysis were applied to characterize SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films, respectively. AgNPs-PEMs-SS/PVA film has exhibited good mechanical performance, hydrophilicity, water absorption capability as well as excellent and durable antibacterial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa and good stability and degradability. This study has developed a simple method to design and prepare AgNPs-PEMs-SS/PVA film for potential antibacterial application. Keywords: sericin; poly(vinyl alcohol); polyelectrolyte multilayers; AgNPs; antibacterial activity
1. Introduction Silk sericin (SS) is a globular protein produced by Bombyx mori (silkworm) [1], which contains 18 amino acids including the essential amino acids required by the human body [2]. It is about 20–30% of the total weight of silk cocoon. The main role of sericin is to envelop the fibroin [3]. As a natural protein, silk sericin is biocompatible and biodegradable. Since sericin has a variety of additional properties like gelling ability, water-holding capacity and skin adhesion [4], it has been widely applied in the biomedical field. Moreover, silk sericin could promote the adhesion and proliferation of human skin and accelerate burn or scald wound healing [5–7]. Therefore, it is regarded as a good candidate for wound dressing material. However, sericin itself is frangible and has a poor mechanical performance,
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which is not favorable for its application in wound healing. In our previous study, we have prepared sericin/poly(vinyl alcohol) (SS/PVA) blend film to improve the mechanical property of sericin [8]. Bacterial infection is a huge challenge for wound healing. To resolve this problem, it is necessary to functionalize the SS/PVA film with good antibacterial activity. Antimicrobial surface coating or modification has attracted more and more attention in biomedical and industrial fields [9–12]. With the rapid development of nanotechnology, a lot of novel nanomaterials have been created with exciting chemical and physical properties [13,14]. As an essential metal nanometer material, silver nanoparticles (AgNPs) have a broad range of applications, from electronics [15] and catalysis [16] to infection prevention [17] and medical diagnosis [18]. It is an effective broad-spectrum antimicrobial agent [19–21] and rarely leads to the development of resistant microbes [22]. Compared with other antibacterial agents, AgNP has low cytotoxicity to human cells [23,24]. In addition, AgNP is an effective anti-inflammatory agent and is used to promote wound healing [25]. To date, a number of chemical and physical methods have been developed to synthesize AgNPs [26–29]. In the traditional methods, toxic chemical reagents and expensive instruments such as ultrasound microwave [30] and γ-radiation [31] are required for AgNPs synthesis. Hence, it is necessary to develop a green and economical method to prepare AgNPs. Ultraviolet (UV)-assisted AgNPs synthesis is one of the most simple and environmentally friendly methods [32]. Studies have shown that AgNPs are commonly immobilized on the material’s surface [33,34]. In this case, Ag+ binding sites are limited on the surface of material, so only limited AgNPs could be synthesized. In addition, the synthesized AgNPs are unstable due to the weak van der Waals interactions, which may reduce its bactericidal efficiency. Polymers such as poly(vinyl pyrrolidone), poly(acrylic acid) and polyamide could provide more binding sites for Ag+ [35,36]. Layer-by-layer self-assembly is a simple technique to construct polyelectrolyte multilayers (PEMs) [37,38]. It is based on the sequential adsorption of oppositely charged components, in which two oppositely charged polymers are deposited on the surface of material by means of electrostatic attraction [39–42]. PEMs could provide more Ag+ binding sites and create a three-dimensional (3D) space for the growth of AgNPs, thus improving the loading of AgNPs and protecting the entrapped AgNPs. In this study, we have established a poly(acrylic acid) (PAA)/poly(dimethyl diallyl ammonium chloride) (PDDA)/PAA multilayered structure on the surface of SS/PVA film, which acted as a three-dimensional matrix to allow the high density synthesis of AgNPs in situ via the UV-assisted AgNPs synthesis and protect the synthesized AgNPs from oxidation and falling off. Scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffractometry (XRD), Fourier transfer infrared spectroscopy (FT-IR), water contact angle, mechanical property and thermogravimetric analysis (TGA) were performed to characterize the prepared films. Antimicrobial assays were conducted to investigate the antimicrobial performance of AgNPs-PEMs-SS/PVA film against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). This novel composite film has exhibited great potential in biomedical applications for its excellent performance and durable antimicrobial activity. 2. Results and Discussion 2.1. Preparation of PEMs-SS/PVA Film Ito et al. have prepared a highly flexible, strong adhesion, and transferrable AgNPs-loaded polymer nanosheet using the layer-by-layer assembly technique [43]. Here, poly (ethyleneimine) (PEI) is a cationic polymer and could adhere on SS/PVA film surface via the electric interaction between PEI and the negatively charged groups exposed on the surface of SS/PVA film. PAA and PDDA are two weak polyelectrolytes with negative and positive charges, respectively, which are used to construct a stable PEM on the surface of SS/PVA film for the adsorption of Ag+ . The alternative treatment with PAA and PDDA resulted in the formation of a three-dimensional matrix on the surface of SS/PVA blend film. AgNPs were synthesized in situ on the film surface by UV irradiation. PEMs provided a
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three-dimensional space for the high-density growth of AgNPs and protected the synthesized AgNPs, antibacterial activity. The preparation antibacterial characterization AgNPs-PEMs-SS/PVA thus endowing SS/PVA film with highlyand effective and durable antibacterialof activity. The preparation antibacterial activity. The preparation and antibacterial characterization of AgNPs-PEMs-SS/PVA film were described in Figure 1. and characterization film antibacterial were described in Figure 1. of AgNPs-PEMs-SS/PVA film were described in Figure 1.
Figure 1.1.Schematic diagram of the preparation and antibacterial of AgNPs-PEMs-SS/ Schematic diagram of the preparation and characterization antibacterial characterization of Figure 1. Schematic diagram of the preparation and antibacterial characterization of AgNPs-PEMs-SS/ PVA film. AgNPs-PEMs-SS/PVA film. PVA film.
2.2. SEM, 2.2. SEM, EDS EDS and and XRD XRD Analysis Analysis 2.2. SEM, EDS and XRD Analysis SEM images images were shown shown in Figure Figure 2. The The surface morphology morphology of SS/PVA SS/PVA film SEM film was was uniform, uniform, SEM images were were shown in in Figure 2. 2. The surface surface morphology of of SS/PVA film was uniform, which showed good structural integrity without any cracks and interface layers (Figure 2a), which showed good structural integrity without any cracks and interface layers (Figure 2a), indicating which showed good structural integrity without any cracks and interface layers (Figure 2a), indicating and PVA has good compatibility. Figurethe 2b showed the seemed film surface sericin andsericin PVA has good compatibility. Figure 2b showed surface to beseemed coveredto bybe a indicating sericin and PVA has good compatibility. Figure 2b film showed the film surface seemed to be covered by a layer of membranous-like substance after PEM coating on the surface of SS/PVA film. layer of membranous-like substance after substance PEM coating on PEM the surface of SS/PVA film. Figure 2c showed covered by a layer of membranous-like after coating on the surface of SS/PVA film. Figure 2c showed the cross-sectional structurefilm, of PEMs-SS/PVA film, formed suggesting PEMs formed a the cross-sectional structure of PEMs-SS/PVA suggesting PEMs a three-dimensional Figure 2c showed the cross-sectional structure of PEMs-SS/PVA film, suggesting PEMs formed a three-dimensional matrix. Figure 2d–f showed the morphologies of films AgNPs-SS/PVA films matrix. Figure 2d–f showed the morphologies of AgNPs-SS/PVA without and withwithout PEMs, three-dimensional matrix. Figure 2d–f showed the morphologies of AgNPs-SS/PVA films without and with PEMs, respectively. High-density AgNPs were observed on the surface of PEMs-SS/PVA respectively. High-density AgNPs were observed theobserved surface of film, whereas and with PEMs, respectively. High-density AgNPs on were onPEMs-SS/PVA the surface of PEMs-SS/PVA film, awhereas only a of small amount of AgNPs wasofonSS/PVA the surface of SS/PVA filmunder without only small amount AgNPs was on the surface film without PEMs the PEMs same film, whereas only a small amount of AgNPs was on the surface of SS/PVA film without PEMs under the same reaction condition. The result indicated that PEM coating promoted the synthesis of reaction The result indicated that PEM coating the promoted synthesis of under thecondition. same reaction condition. The result indicated thatpromoted PEM coating thehigh-density synthesis of + + high-density AgNPs. Previous studies indicate that the carboxyl groups of PAA greatly increase Ag AgNPs. Previous studies indicate that indicate the carboxyl groups of PAA greatly increase Ag increase adsorption high-density AgNPs. Previous studies that the carboxyl groups of PAA greatly Ag+ + 0 + 0 adsorption and thus the transformation of Ag to Ag0irradiation under UV irradiation [44]. With UV and thus promote thepromote transformation of Ag to Ag under With UV irradiation + to UV adsorption and thus promote the transformation of Ag Ag under UV [44]. irradiation [44]. With UV irradiation time increased to 30 min, AgNPs density on the surface of PEMs-SS/PVA film time increased to 30 min, AgNPs density the surface of PEMs-SS/PVA filmofsignificantly increased irradiation time increased to 30 min, on AgNPs density on the surface PEMs-SS/PVA film significantly increased (Figure 2g,h). The long-lasting antibacterial activity of AgNPs material is (Figure 2g,h). The long-lasting antibacterial activity of AgNPs material is mainly dependent on the significantly increased (Figure 2g,h). The long-lasting antibacterial activity of AgNPs materiallife is mainly dependent on the life of AgNPs. film Hence, filmantibacterial was expected to have of AgNPs. Hence, AgNPs-PEMs-SS/PVA wasAgNPs-PEMs-SS/PVA expected to have durable activity for mainly dependent on the life of AgNPs. Hence, AgNPs-PEMs-SS/PVA film was expected to have durable antibacterial activity for its high density AgNPs. its high density AgNPs. durable antibacterial activity for its high density AgNPs.
Figure 2. Cont.
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Figure 2.2. Surface morphologies of of SS/PVA SS/PVA (a); (a); PEMs-SS/PVA PEMs-SS/PVA (b); structure of of Figure Surface morphologies (b); cross-sectional cross-sectional structure PEMs-SS/PVA films (c); SS/PVA film in silver nitrate solution irradiated by UV light for 10 min (d); PEMs-SS/PVA films (c); SS/PVA film in silver nitrate solution irradiated by UV light for 10 min PEMs-SS/PVA film film in silver nitrate solution irradiated by UV for 10for min min (d); PEMs-SS/PVA in silver nitrate solution irradiated bylight UV light 10(e,f) minand (e,f)30 and 30(g,h). min (f,h) are magnification of (e,g), respectively; EDS and (g,h). (f,h)the are high the high magnification of (e,g), respectively; EDSspectrum spectrumof of PEMs-SS/PVA PEMs-SS/PVA film film and AgNPs-PEMs-SS/PVA film (i); XRD XRD patterns patterns of of sericin sericin (j1), (j1), SS/PVA SS/PVA film film (j2), (j2), PEMs-SS/PVA PEMs-SS/PVA film (j3) AgNPs-PEMs-SS/PVA film (i); film (j3) and AgNPs-PEMs-SS/PVA film (j4) (j). and AgNPs-PEMs-SS/PVA film (j4) (j).
The EDS spectrum showed the peaks of carbon, nitrogen, chlorine and oxygen element on The EDS spectrum showed the peaks of carbon, nitrogen, chlorine and oxygen element on PEMs-SS/PVA film (Figure 2i). The peaks of carbon, nitrogen, chlorine and oxygen were attributed PEMs-SS/PVA film (Figure 2i). The peaks of carbon, nitrogen, chlorine and oxygen were attributed to PAA, PDDA, PVA and sericin, respectively. The chlorine peak was mainly from PDDA. The EDS to PAA, PDDA, PVA and sericin, respectively. The chlorine peak was mainly from PDDA. The EDS spectrum of AgNPs-PEMs-SS/PVA film showed a distinct peak of the silver element except carbon, spectrum of AgNPs-PEMs-SS/PVA film showed a distinct peak of the silver element except carbon, nitrogen, chlorine, oxygen and chlorine peak. nitrogen, chlorine, oxygen and chlorine peak. XRD patterns were shown in Figure 2j. A broad peak located at 2θ = 19.2° was discovered in XRD patterns were shown in Figure 2j. A broad peak located at 2θ = 19.2◦ was discovered in sericin sericin pattern, which was consistent with the previous report [45]. The other three patterns had a pattern, which was consistent with the previous report [45]. The other three patterns had a characteristic characteristic broad peak located at 2θ = 19.8°, which could be assigned to the crystal structure of broad peak located at 2θ = 19.8◦ , which could be assigned to the crystal structure of PVA. No significant PVA. No significant change occurred on the pattern of SS/PVA film after PEM coating, UV change occurred on the pattern of SS/PVA film after PEM coating, UV irradiation and AgNPs irradiation and AgNPs modification, suggesting these treatments did not affect the crystal structure modification, suggesting these treatments did not affect the crystal structure of SS/PVA film. After of SS/PVA film. After AgNPs modification, one peak appeared at 2θ = 38.2°, which could be ascribed AgNPs modification, one peak appeared at 2θ = 38.2◦ , which could be ascribed to the crystal planes to the crystal planes (111) of the face-centered cubic structure of AgNPs. Another peak appeared at (111) of the face-centered cubic structure of AgNPs. Another peak appeared at 2θ = 32.2◦ , which could 2θ = 32.2°, which could be attributed to the crystal planes (111) of Ag2O. UV irradiation facilitates the+ be attributed to the crystal planes (111) of Ag2 O. UV irradiation facilitates the transformation of Ag transformation of Ag+ to Ag0. At the same time, it will produce a small part of ozone. Ag2O may be to Ag0 . At the same time, it will produce a small part of ozone. Ag2 O may be derived from the derived from the oxidation of AgNPs on the film surface exposed to the UV-generated ozone [46,47] oxidation of AgNPs on the film surface exposed to the UV-generated ozone [46,47] or air in aqueous or air in aqueous solutions [48]. EDS and XRD results suggested that AgNPs were successfully solutions [48]. EDS and XRD results suggested that AgNPs were successfully synthesized and evenly synthesized and evenly distributed on the surface of PEMs-SS/PVA film. The high density and good distributed on the surface of PEMs-SS/PVA film. The high density and good crystallinity of AgNPs crystallinity of AgNPs may greatly increase the antibacterial activity of PEMs-SS/PVA film. may greatly increase the antibacterial activity of PEMs-SS/PVA film. 2.3. FT-IR FT-IR Analysis Analysis 2.3. FT-IR spectrum spectrum is is aa classical classical method method to to reveal reveal the the structure structure of of aa substance substance [49]. [49]. Figure Figure 33 showed showed FT-IR the FT-IR spectra of sericin (a), SS/PVA (b), PEMs-SS/PVA (c) and AgNPs-PEMs-SS/PVA films the FT-IR spectra of sericin (a), SS/PVA (b), PEMs-SS/PVA (c) and AgNPs-PEMs-SS/PVA films (d). (d). −1 were discovered in all samples, which may be Three characteristic peaks at 1640, 1521 and 1244 cm − 1 Three characteristic peaks at 1640, 1521 and 1244 cm were discovered in all samples, which may assigned to to C=O stretching vibration C-N stretching stretching and and N–H N-H be assigned C=O stretching vibrationofofamide amideI Ipeak, peak,aa combination combination of of C–N bendingvibration vibrationof ofamide amideIIIIpeak peakand anda acombination combination N-H bending and C-N stretching vibration bending ofof N–H bending and C–N stretching vibration of −1 and 3340 cm−1, which of amide III, respectively. Pure sericin and PVA have broad bands at 3274 cm − 1 − 1 amide III, respectively. Pure sericin and PVA have broad bands at 3274 cm and 3340 cm , which are are associated amine N-H stretching vibration sericinand andthe theO–H O-Hstretching stretchingvibration vibration of of associated withwith the the amine N–H stretching vibration ofof sericin PVA, respectively. respectively. After After blending blending with with PVA, PVA, the the N–H N-H stretching stretching vibration vibration band band of of sericin sericin slightly slightly PVA, −1, indicating that sericin and PVA had good compatibility. After PEM shifted from 3274 to 3320 cm − 1 shifted from 3274 to 3320 cm , indicating that sericin and PVA had good compatibility. After PEM −1 modification, aa weak could be ascribed to the vibration band 1 , which modification, weakpeak peakoccurred occurredatat1476 1476cm cm,−which could be ascribed tocycle the cycle vibration of PDDA [50]. The confirmed the presence of PEMs on the of SS/PVA film.film. No band of PDDA [50]. results The results confirmed the presence of PEMs on surface the surface of SS/PVA significant change was observed on the C-O vibration band of AgNPs-PEMs-SS/PVA film. This may No significant change was observed on the C–O vibration band of AgNPs-PEMs-SS/PVA film. This be because the carboxyl groups were involved in the adsorption of Ag+, but did not participate in the formation of AgNPs. The -CH2 characteristic peak of PVA at 2940 cm−1 may mask the -CH3 and -CH2 vibration band of PDDA at 2870 cm−1 and 2945 cm−1, respectively.
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may be because the carboxyl groups were involved in the adsorption of Ag+ , but did not participate in the formation of AgNPs. The –CH2 characteristic peak of PVA at 2940 cm−1 may mask the –CH3 and –CH band of PDDA at 2870 cm−1 and 2945 cm−1 , respectively. Materials 2017, 10, 967 5 of 15 2 vibration
Figure 3. 3. FT-IR spectra of sericin (a); SS/PVA (b); PEMs-SS/PVA (c) and(c) AgNPs-PEMs-SS/PVA films (d). Figure FT-IR spectra of sericin (a); SS/PVA (b); PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films (d).
2.4. Wettability Measurement and Water Uptake Ability
2.4. Wettability Measurement Waterbehavior Uptake Ability Figure 4a–c showed theand wetting of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA filmsFigure assessed byshowed water contact anglebehavior measurement, respectively. The water angle of SS/PVA 4a–c the wetting of SS/PVA, PEMs-SS/PVA andcontact AgNPs-PEMs-SS/PVA film was 21.3°, indicating that SS/PVA film had excellent hydrophilicity. After PEM the films assessed by water contact angle measurement, respectively. The water contact anglecoating, of SS/PVA water contact angle of SS/PVA film increased to 58°, indicating its good hydrophilicity. PEM coating film was 21.3◦ , indicating that SS/PVA film had excellent hydrophilicity. After PEM coating, the water on SS/PVA film reduce porositytoof58SS/PVA film, thus resulting in the increase water ◦ , indicating contact angle of may SS/PVA filmthe increased its good hydrophilicity. PEMofcoating contact angle of SS/PVA film. In addition, time delay from making the film to water contact angle on SS/PVA film may reduce the porosity of SS/PVA film, thus resulting in the increase of water measurement could result in surface saturation with hydrocarbons and thus cause the increase of contact angle of SS/PVA film. In addition, time delay from making the film to water contact angle water contact angle. film had water contact angle of 62°. Thethe water contact measurement could AgNPs-PEMs-SS/PVA result in surface saturation witha hydrocarbons and thus cause increase of angle was hardly changed before and after AgNPs modification, indicating that AgNPs modification ◦ water contact angle. AgNPs-PEMs-SS/PVA film had a water contact angle of 62 . The water contact did affect thechanged hydrophilicity of PEMs-SS/PVA film. Karumuri et al. have reported that water anglenot was hardly before and after AgNPs modification, indicating that AgNPs modification contact angle has slightly gone down with AgNPs deposition, but the small difference does not did not affect the hydrophilicity of PEMs-SS/PVA film. Karumuri et al. have reported that water affect the hydrophilicity [51]. contact angle has slightly gone down with AgNPs deposition, but the small difference does not affect The swelling [51]. properties of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films were the hydrophilicity 2) were immersed in PBS buffer (pH 7.4) for 12 h, 24 h examined (Figure 4d). All samples (1 × 1 cm The swelling properties of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films were and 48 h, respectively. The swelling ratios of these films about 1.8(pH without 2 examined (Figure 4d). All samples (1 × 1 cm ) were immersedwere in PBS buffer 7.4) forsignificant 12 h, 24 h differences, indicatingThe these filmsratios had of good hygroscopicity. The propertiesdifferences, were not and 48 h, respectively. swelling these films were about 1.8swelling without significant significantly different after 12 h, 24 h and 48 h because the total accessible surface of these films had indicating these films had good hygroscopicity. The swelling properties were not significantly different been completely saturated by water molecules quickly. These results indicated that SS/PVA, after 12 h, 24 h and 48 h because the total accessible surface of these films had been completely PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films had good hydrophilicity and water absorption saturated by water molecules quickly. These results indicated that SS/PVA, PEMs-SS/PVA and capability. AgNPs-PEMs-SS/PVA films had good hydrophilicity and water absorption capability.
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Figure 4. Water contact angles of SS/PVA (a); PEMs-SS/PVA (b); and AgNPs-PEMs-SS/PVA films (c);
Figure 4. Water contact angles of SS/PVA (a); PEMs-SS/PVA (b); and AgNPs-PEMs-SS/PVA films (c); swelling ratios of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films (d). swelling of SS/PVA, PEMs-SS/PVA andPEMs-SS/PVA AgNPs-PEMs-SS/PVA films (d). Figure ratios 4. Water contact angles of SS/PVA (a); (b); and AgNPs-PEMs-SS/PVA films (c); swelling ratios of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films (d). 2.5. Mechanical Property
2.5. Mechanical Property The tensile strength and elongation at break of these films were measured, as shown in Figure 5. 2.5. Mechanical Property SS/PVA film had a tensile strength of 2.45 at MPa, which much lower thanmeasured, that of the modified The tensile strength and elongation break of was these films were as shown in The PEM tensile strength and elongation at break of these films were measured, as shown in Figure 5. films. coating and AgNPs modification resulted in the increase of the tensile strength about Figure 5. SS/PVA film had a tensile strength of 2.45 MPa, which was much lower than that of SS/PVA a tensilewith strength MPa, film, whichsuggesting was muchthat lower than that ofand the AgNPs modified three film timeshad compared that of of 2.45 SS/PVA PEM coating the modified films. PEM coating and AgNPs modification resulted in the increase of the tensile modification increased the stiffness of SS/PVA film. Itinmay attributed to tensile the increase of film films. PEM coating and AgNPs modification resulted the be increase of the strength about strength about three times compared with that of SS/PVA film, suggesting that PEM coating and thickness. Elongation at break represents the flexibility of material [52]. Although the elongation at three times compared with that of SS/PVA film, suggesting that PEM coating and AgNPs AgNPs modification increased the stiffness ofhad SS/PVA film. It may gone be attributed towith the that increase of break (strain) of AgNPs-PEMs-SS/PVA film gone down a little compared of modification increased the stiffness of SS/PVA film. It may be attributed to the increase of film film thickness. Elongation atwas break represents the flexibility of material [52]. Although the elongation SS/PVA Elongation film, the change not statistically strain of AgNPs-PEMs-SS/PVA film was at thickness. at break represents the different. flexibilityThe of material [52]. Although the elongation at break break (strain) of AgNPs-PEMs-SS/PVA film had gone down a little gone compared with that 43.80(strain) ± 3.2%,of which still could support its application as an antibacterial biomaterial. PEM coating AgNPs-PEMs-SS/PVA film had gone down a little gone compared with that of of SS/PVA film, the change was not statistically different. The strain of AgNPs-PEMs-SS/PVA film was and AgNPs modification increased the film thickness, resulting in a slight decrease of the film SS/PVA film, the change was not statistically different. thus The strain of AgNPs-PEMs-SS/PVA film was flexibility. Inwhich general, mechanical performance of AgNPs-PEMs-SS/PVA film was ablePEM toPEM support 43.80 ± ±3.2%, still could support its as an an antibacterial antibacterialbiomaterial. biomaterial. coating 43.80 3.2%,which stillthe could support its application application as coating its application as an antibacterial biomaterial. and AgNPs modification increased the film thickness, thus resulting in a slight decrease of the film and AgNPs modification increased the film thickness, thus resulting in a slight decrease of the film flexibility. InIn general, filmwas was able support flexibility. general,the themechanical mechanicalperformance performance of AgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA film able to to support its its application asasananantibacterial application antibacterialbiomaterial. biomaterial.
Figure 5. Mechanical properties of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films: (a) tensile strength and (b) elongation at break. Data are the average value plus standard deviation (SD) from three independent experiments. Statistical significance is assessed using an unpaired t-test. * indicates p < 0.05. Figure 5. Mechanical properties of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films:
Figure 5. Mechanical properties of SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films: (a) tensile (a) tensile and (b) at elongation at break. Data are thevalue average plus standard strength andstrength (b) elongation break. Data are the average plusvalue standard deviationdeviation (SD) from (SD) from three independent experiments. Statistical significance is assessed using an unpaired t-test. three independent experiments. Statistical significance is assessed using an unpaired t-test. * indicates * indicates p < 0.05. p < 0.05.
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2.6. TGA Analysis 2.6. TGA Analysis TGA reflects that the mechanism led to a unique thermal behavior. The result showed SS/PVA TGA reflects that the mechanism led to a unique thermal behavior. The result showed SS/PVA film underwent a multi-steps degradation process (Figure 6). In 0–100 °C, the initial weight loss was film underwent a multi-steps degradation process (Figure 6). In 0–100 ◦ C, the initial weight loss was due to the presence of moisture. While increasing temperature to 200 °C, the observed weight loss due to the presence of moisture. While increasing temperature to 200 ◦ C, the observed weight loss may be attributed to the degradation of the exposed side chains and the breakdown of main chain may be attributed to the degradation of the exposed side chains and the breakdown of main chain groups of sericin. After blending with PVA, water evaporation caused the reduction of initial weight groups of sericin. After blending with PVA, water evaporation caused the reduction of initial weight from room temperature to 180 °C. At this stage, the weight loss of SS/PVA film was slower than that from room temperature to 180 ◦ C. At this stage, the weight loss of SS/PVA film was slower than that of sericin, which may be due to the fact that SS/PVA film contained more bound water than sericin of sericin, which may be due to the fact that SS/PVA film contained more bound water than sericin itself did. The second stage of weight loss occurred from 200 to 600 °C. This may be ascribed to the itself did. The second stage of weight loss occurred from 200 to 600 ◦ C. This may be ascribed to the degradation of the exposed side chains and the breakdown of main chain groups of sericin and PVA. degradation of the exposed side chains and the breakdown of main chain groups of sericin and PVA. The weight loss of SS/PVA film reached an equilibrium at around 620 °C, which was higher than that The weight loss of SS/PVA film reached an equilibrium at around 620 ◦ C, which was higher than that of sericin. SS/PVA film had about 3% residual weight, suggesting that the hydrogen-bond of sericin. SS/PVA film had about 3% residual weight, suggesting that the hydrogen-bond interaction interaction between sericin and PVA may be favorable to the thermal stability of SS/PVA blend film. between sericin and PVA may be favorable to the thermal stability of SS/PVA blend film. After PEM After PEM coating, the equilibrium temperature increased to around 730 °C. PEMs-SS/PVA film had coating, the equilibrium temperature increased to around 730 ◦ C. PEMs-SS/PVA film had about 4% about 4% residual weight, indicating that PEM coating could enhance the thermostability of SS/PVA residual weight, indicating that PEM coating could enhance the thermostability of SS/PVA film and film and delay its thermal degradation. After AgNPs modification, the residual weight increased to delay its thermal degradation. After AgNPs modification, the residual weight increased to about 7%, about 7%, and the equilibrium temperature increased to around 650 °C, implying that AgNPs may and the equilibrium temperature increased to around 650 ◦ C, implying that AgNPs may improve the improve the thermostability of PEMs-SS/PVA film and reduce the weight loss. The result also thermostability of PEMs-SS/PVA film and reduce the weight loss. The result also indicated that PEMs indicated that PEMs and AgNPs were successfully modified on the surface of SS/PVA film. and AgNPs were successfully modified on the surface of SS/PVA film.
Figure AgNPs-PEMs-SS/PVAfilms. films. Figure6. 6. TGA TGA curves curves of of sericin, sericin, SS/PVA, SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA
2.7. Inhibition Zone Assay of AgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA films were assessed against Gram-negative Gram-negative The antibacterial effects of bacteria (E. coli and P. aeruginosa) and Gram-positive Gram-positive bacteria bacteria (S. (S. aureus), aureus), respectively respectively (Figure (Figure 7). 7). SS/PVA and SS/PVA and PEMs-SS/PVA PEMs-SS/PVAfilms filmsdid didnot notexhibit exhibit significant significant bactericidal bactericidal effects effects against against either either E. coli, coli, P. aeruginosa aeruginosa or or S. S. aureus. aureus. However, However, after after AgNPs AgNPs modification, modification, the the film film could could effectively effectively kill bacteria P. form evident evident inhibition inhibitionzones zoneswith withan anapproximate approximateaverage averagediameter diameter cm. The diameters and form ofof 2.52.5 cm. The diameters of of the inhibition zones were summarizedininTable Table1.1.The Theresult resultsuggested suggestedthat thatAgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA the inhibition zones were summarized film obviously inhibited the growth growth of of E. E. coli coli (a), (a), S. S. aureus aureus (b) (b) and and P. P.aeruginosa aeruginosa(c). (c).
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Figure 7. AgNPs-PEMs-SS/PVA films against E. coli (a); Figure 7. Antibacterial Antibacterialeffects effectsof ofSS/PVA, SS/PVA,PEMs-SS/PVA, PEMs-SS/PVA, AgNPs-PEMs-SS/PVA films against E. coli S. aureus (b) and P. aeruginosa (c); Growth curves assays of E. coli (d); S. aureus (e) and P. aeruginosa (a); S. aureus (b) and P. aeruginosa (c); Growth curves assays of E. coli (d); S. aureus (e) and P. aeruginosa (f). (f). f1); Bacteria Bacteria in in the the presence presence of ofSS/PVA SS/PVA (d2, (d2, e2, e2, f2), f2), PEMs-SS/PVA PEMs-SS/PVA Bacteria without without treatment treatment (d1, (d1, e1, e1, f1); Bacteria (d3, e3, e3, f3) f3) and and AgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA films films (d4, (d4, e4, e4, f4). f4). (d3, Table 1.1. Diameters Diameters of ofinhibition inhibitionzones zonesofofSS/PVA, SS/PVA,PEMs-SS/PVA PEMs-SS/PVA and and AgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA films Table P.aeruginosa. aeruginosa. against E. coli, S. aureus and P.
Bacteria Bacteria E. coil E. coil P. aeruginosa P. aeruginosa S.S.aureus aureus
Control (cm) PEMs-SS/PVA (cm) AgNPs-PEMs-SS/PVA (cm) Control (cm) PEMs-SS/PVA (cm) AgNPs-PEMs-SS/PVA (cm) 1.55 ± 0.02 1.55 ± 0.03 2.17 ± 0.42 1.55 ± 0.02 1.55 ± 0.03 2.17 ± 0.42 1.55 ± 0.01 1.55 ± 0.02 2.49 ± 0.41 1.55 ± 0.01 1.55 ± 0.02 2.49 ± 0.41 1.55 ± 0.03 1.551.55 ± 0.04 2.82 1.55 ± 0.03 ± 0.04 2.82±±0.26 0.26
2.8. Bacterial Curve Assay Assay 2.8. Bacterial Growth Growth Curve Bacterial growth growth curve curve experiments experiments were were carried carried out out to to further further evaluate evaluate the the antibacterial antibacterial effects effects Bacterial of AgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVAfilms. films.The The optical density of bacteria nm )(OD 600) was used to of optical density of bacteria at 600at nm600 (OD 600 was used to evaluate evaluate bacterial growth in the presence of the films. As shown in Figure 7d–f, lag phase of bacterial growth in the presence of the films. As shown in Figure 7d–f, the lag phase ofthe E. coli, S. aureus E. coli, S. aureus and P. aeruginosa was less than 1 h in the presence of SS/PVA and PEMs-SS/PVA and P. aeruginosa was less than 1 h in the presence of SS/PVA and PEMs-SS/PVA films. However, in the films. However, in the presence offilms, AgNPs-PEMs-SS/PVA lag phase coli, P. aeruginosa presence of AgNPs-PEMs-SS/PVA the lag phase of E. films, coli, P. the aeruginosa andofS.E. aureus significantly and S. aureus significantly prolonged to 26 h, 22 h and 30 h, respectively. For AgNPs film prolonged to 26 h, 22 h and 30 h, respectively. For AgNPs film without PEMs, the lag phase of without bacteria PEMs, the lag phase of bacteria generally prolongs to 12 h [8]. Compared to AgNPs film without generally prolongs to 12 h [8]. Compared to AgNPs film without PEMs, AgNPs-PEMs-SS/PVA PEMs, AgNPs-PEMs-SS/PVA film hadeffect a more inhibitory on thedemonstrated bacterial growth. film had a more significant inhibitory on significant the bacterial growth. effect The results that The results demonstrated that AgNPs-PEMs-SS/PVA film had excellent antimicrobial activity. AgNPs-PEMs-SS/PVA film had excellent antimicrobial activity. 2.9. Antimicrobial Stability Analysis Analysis 2.9. Antimicrobial Stability Figure 8a,b 8a,b showed showed the the antimicrobial antimicrobial stability stability analysis analysis of of AgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA films films against against Figure E. coli coli and and S. S. aureus, aureus, after after the the films films were were treated treated in in different differentpHs pHs(5, (5,7.4, 7.4,9) 9)for for24 24h. h.OD OD600 600 was to E. was used used to evaluate bacterial growth in the presence of the treated AgNPs-PEMs-SS/PVA films. After 18 h, evaluate bacterial growth in the presence of the treated AgNPs-PEMs-SS/PVA films. After 18 h, the film the film stillconsiderable showed considerable antimicrobial activity with compared that ofThe theantimicrobial control. The still showed antimicrobial activity compared that of with the control. antimicrobial activity of the film underwas pHhigher 5 condition was thatunder of the activity of the film treated under pHtreated 5 condition than that of higher the filmthan treated pHfilm 7.4 treated under pH 7.4 or pH 9 conditions. This may be due to the fact that AgNPs-PEMs-SS/PVA film or pH 9 conditions. This may be due to the fact that AgNPs-PEMs-SS/PVA film was more stable and was more stable and AgNPs loss was less under pH 5 condition than that under other conditions. The result indicated that AgNPs-PEMs-SS/PVA film had durable and steady antibacterial activity.
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AgNPs loss was less under pH 5 condition than that under other conditions. The result indicated that Materials 2017, 10, 967 9 of 15 AgNPs-PEMs-SS/PVA film had durable and steady antibacterial activity.
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Figure 8. films against E. coli (a) and S. aureus (b). Figure 8. Durable Durablebactericidal bactericidalactivity activityofofAgNPs-PEMs-SS/PVA AgNPs-PEMs-SS/PVA films against E. coli (a) and S. aureus DataData are are the the average value plus standard deviation (b). average value plus standard deviation(SD) (SD)from fromthree three independent independent experiments. experiments. Figure 8. Durable bactericidal activity of AgNPs-PEMs-SS/PVA films against E. coli (a) and S. aureus (b). Statistical significance is assessed using an unpaired unpaired t-test. t-test. ** indicates pp
