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montmorillonite nanocomposite films and antibacterial activity
Carbohydrate Polymers 171 (2017) 202–210
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Photochemical synthesis of silver nanoparticles on chitosans/montmorillonite nanocomposite films and antibacterial activity Juliana S. Gabriel, Virgínia A.M. Gonzaga, Alessandra L. Poli, Carla C. Schmitt ∗ Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos SP, Caixa Postal 780, 13560-970, Brazil
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Article history: Received 21 February 2017 Received in revised form 25 April 2017 Accepted 5 May 2017 Available online 5 May 2017 Keywords: Silver nanoparticles Chitosan derivatives Montmorillonite Nanocomposite films UV irradiation Antibacterial activity
a b s t r a c t Silver nanoparticles (AgNPs) were synthetized on chitosans/montmorillonite nanocomposite films by photochemical method. Nanocomposites were prepared using chitosans with different molar masses and deacetylation degrees, as well as modified with diethylaminoethyl (DEAE) and dodecyl groups. AgNPs formation on the films was followed by the appearance of the plasmon band around 440 nm as a function of irradiation time. TEM images revealed AgNPs with spherical morphology for all nanocomposites. For nanocomposites using modified chitosans, the AgNPs synthesis occurred quickly (1.5 h) while for the others films it was above 11 h. The film of modified chitosan with dodecyl and DEAE groups presented smaller and more uniform nanoparticles size along mixture of exfoliated and intercalated structures. This modified chitosan is an amphiphilic compound that can act controlling the size/shape of the AgNPs. The results of antibacterial activity suggested that all nanocomposite-AgNPs films inhibited the growth of Escherichia coli and Bacillus subtilis. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Over the past few years, silver nanoparticles (AgNPs) have become a target of great interest due to their remarkable electronic, optical, mechanical, magnetic, chemical and antimicrobial properties (Dick, McFarland, Haynes, & Van Duyne, 2002; Ghosh & Maiti, 1996; Kanmani & Rhim, 2014; Severin, Kirstein, Sokolov, & Rabe, 2009; Thuc et al., 2016). In regard with size and shape-based of AgNPs, several applications ranging from catalysts, biomedical field, disinfectant sprays, ink, food package and textile products (Chernousova & Epple, 2013; Emam & Ahamed, 2016; Thuc et al., 2016; Zoya, 2012). Hence, a myriad of methods for AgNPs synthesis have been reported, including biological, chemical, photochemical and electrochemical processes (Bhaduri et al., 2013; Lengke, Fleet, & Southam, 2007; Patra et al., 2014; Thuc et al., 2016). However, the chemical reduction using hazardous reducing agents, such as sodium borohydrate, formaldehyde and ammonia is typically employed for AgNPs preparation, limiting the use of AgNPs for biological or medical applications (Emam & Ahamed, 2016; El-
∗ Corresponding author. E-mail addresses: [email protected] (J.S. Gabriel), [email protected] (V.A.M. Gonzaga), [email protected] (A.L. Poli), [email protected] (C.C. Schmitt). http://dx.doi.org/10.1016/j.carbpol.2017.05.021 0144-8617/© 2017 Elsevier Ltd. All rights reserved.
Nour, Eftaiha, Al-Warthan, & Ammar, 2010; Lombardo, Poli, Castro, Perussi, & Schmitt, 2016; Wojtysiak & Kudelski, 2012). On the other hand, the number of publications about green synthesis of the AgNPs that use environmentally friendly compounds as reducing agents has risen. Among these processes, the green irradiation methods including laser, gamma, ultrasonic wave, ion and ultraviolet (UV) radiation of silver salts in aqueous solution have been used as alternative routes to broaden the range of applications (Shameli et al., 2010; Son, Youk, & Park, 2006; Zhou et al., 2012). In addition, the synthesis of AgNPs was also described using clay suspension, in which the clay lamellae behave as nanoreactors of Ag+ to Ag0 (Patakfalvi & Dékány, 2004). Furthermore, several polysaccharides, such as glucose, dextrose, starch and chitosan (Ji, Liu, Zhang, Xiong, & Sun, 2016; Oluwafemi et al., 2016; Thomas et al., 2009) have been investigated as reducers and/or stabilizing agents for AgNPs preparation owing to their ability for coordinating with metal ions (Emam & Ahamed, 2016). These polymer-Ag+ complexes can be reduced by different experimental conditions, to produce smalls AgNPs with narrow size distribution (Emam & Ahamed, 2016). Regarding natural polymers, chitosan, the main derivative of chitin, has been extensively studied as a cationic biopolymer of high bioactivity, biodegradability, biocompatibility and low toxicity, in addition to its antimicrobial activity (Araiza, Alcouffe, Rochas, Montembault, & David, 2010; Epure, Griffon, Pollet, & Avérous, 2011; Youssef, Yousef, El-Sayed, & Kamel, 2015). These properties
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Table 1 Inhibition zones for nanocomposite films and examples of absence (a) and presence (b) of these zones. These images were obtained to Ch83 and Ch83/10%SWy-2/AgNPs films, respectively, against B. subtilis. E. coli
B. subtilis
Sample
d (mm)
Sample
d (mm)
Ch83 Ch83/10%SWy-2 Ch30/10%SWy-2 Ch30d/10%SWy-2 Ch30-DEAE/10%SWy-2 Ch30-DEAE-Dod/10%SWy-2 Ch83/AgNPs Ch83/2.5%SWy-2/AgNPs Ch83/5%SWy-2/AgNPs Ch83/10%SWy-2/AgNPs Ch30/10%SWy-2/AgNPs Ch30d/10%SWy-2/AgNPs Ch30-DEAE/10%SWy-2/AgNPs Ch30-DEAE-Dod/10%SWy-2/AgNPs
N.I N.I N.I N.I N.I N.I 8.7 ± 0.6 7.6 ± 0.3 8.7 ± 0.6 8.3 ± 0.6 7.5 ± 0.5 7.8 ± 0.2 8.3 ± 0.3 8.0 ± 0.3
Ch83 Ch83/10%SWy-2 Ch30/10%SWy-2 Ch30d/10%SWy-2 Ch30-DEAE/10%SWy-2 Ch30-DEAE-Dod/10%SWy-2 Ch83/AgNPs Ch83/2.5%SWy-2/AgNPs Ch83/5%SWy-2/AgNPs Ch83/10%SWy-2/AgNPs Ch30/10%SWy-2/AgNPs Ch30d/10%SWy-2/AgNPs Ch30-DEAE/10%SWy-2/AgNPs Ch30-DEAE-Dod/10%SWy-2/AgNPs
N.I N.I N.I N.I N.I N.I 8.5 ± 0.7 8.3 ± 0.5 8.7 ± 0.6 7.3 ± 0.6 8.0 ± 0.3 8.3 ± 0.5 8.0 ± 0.2 9.0 ± 0.3
N.I.: No Inhibition. The error was obtained by three independent experiments. The results were reported as mean ± standard deviation.
combined with its ability to form films, allows the application of this polysaccharide in food packaging, bone substitutes and artificial skin (Shameli et al., 2010). In order to obtain superior stability of chitosan-based nanocomposites, the addition of a natural multilayer silicate known as montmorillonite clay significantly enhances the chemical and/or mechanical stability, in comparison with the polymer itself (Wang et al., 2005; Xie et al., 2013; Xu, Ren, & Hhanna, 2006). Montmorillonite clay is formed by a single octahedral sheet of magnesia or alumina located between two silica tetrahedral sheets (Xu et al., 2006). Moreover, the characteristics of chitosan/montmorillonite nanocomposites coupled with AgNPs properties can result in versatile materials for a wide range of applications. Several approaches concerning photochemical synthesis of AgNPs in solutions of polymers or nanocomposites have been extensively reported in the literature (Krishnan et al., 2015; Shameli et al., 2010; Thomas, Yallapu, Sreedhar, & Bajpai, 2009). However, the metal nanoparticles synthesis can also be performed in solid material directly on films using irradiation, as described by Sakamoto, Fujistuka, and Majima, 2009, which studied the fabrication of Au/Cu bimetallic nanoparticles in poly(vinyl alcohol) films by UV light irradiation. Herein we report AgNPs synthesis on chitosan/clay nanocomposite films by UV irradiation after the films preparation. The influence of clay concentration and modification of chitosan structure was also evaluated in the AgNPs formation. The characterization of samples was performed using X-ray diffraction analyses (XRD), UV–vis spectroscopy and transmission electron microscopy (TEM). Finally, the antibacterial activities of materials were investigated against Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis).
2. Materials and methods 2.1. Materials The chitosan (Ch83) used in this work was purchased from Aldrich Chemical Co with a deacetylation degree of 85% and a viscosity-average molar mass of 83,000 g mol−1 . Chitosan with
low molecular weight (Ch30) 30,000 g mol−1 (deacetylation degree of 85%) was obtained according to the method reported by Tommeraas, Varum, Christensen, and Smidsrod, (2001). It was also prepared chitosan with a deacetylation degree of 98% (Ch30d) and a viscosity-average molar mass of 30,000 g mol−1 (Tiera et al., 2006). Modified chitosans (Fig. 1) used in this work were prepared according to the method reported earlier (Gabriel, Tiera, & Tiera, 2015). Ch30-DEAE is a hydrophilic chitosan derivative with 40% of substitution degree by diethylaminoethyl groups (DEAE). Ch30DEAE-Dod is an amphiphilic chitosan derivative with 40 and 6% of substitution degree by DEAE and dodecyl groups (Dod), respectively. The substitution degrees were determined by 1 HNMR spectroscopy (data not shown). The SWy-2 montmorillonite clay was kindly supplied by Source Clays Repository of Clay Minerals Society, University of Missouri (Columbia, MO). The SWy-2 clay was purified according to the method described by Gessner, Schmitt, and Neumann, (1994). Glacial acetic acid (Synth, Brazil), silver nitrate (TEC-LAB, Brazil) and distilled water were also used in this work.
2.2. Nanocomposite films preparation SWy-2 dispersions with 2.5 wt%, 5 wt% and 10 wt% based on chitosan were prepared by dispersing appropriate amounts of SWy2 into 15 mL of 0.25 mol L−1 aqueous acetic acid solution under stirring for 24 h. Then, 0.2 g of Ch83 powder and 1.0 mL of silver nitrate solution (0.05 mol L−1 ) were added into the clay dispersions. The mixtures were stirred continuously for 24 h. Afterwards, these solutions were cast onto polystyrene Petri dishes with dimensions of 60 mm × 15 mm. The films were dried in an oven at 30 ◦ C, and peeled from the Petri dishes. Additionally, the nanocomposite films containing Ch30, Ch30d, Ch30-DEAE and Ch30-DEAE-Dod were prepared using only 10 wt% of SWy-2 based on polymer by the methodology described above. Accordingly, AgNPs were synthesized by photochemical method. The nanocomposite films were placed in a UV light irradiation chamber containing sixteen UV germicidal lamps at 25 ◦ C up to 24 h. At the same position, the emission of lamps was 254 nm
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Fig. 1. Molecular structures of modified chitosans (a) Ch30-DEAE and (b) Ch30-DEAE-Dod.
2.4. Antibacterial activity
Fig. 2. Emission spectrum of irradiating lamp and absorption spectra of SWy-2 and Ch83/10%SWy-2/AgNO3 , Ch30/10%SWy-2/AgNO3 , Ch30-DEAE-Dod/10%SWy2/AgNO3 films.
with power of 18603 mW m−2 , as measured by SPR-01 Spectroradiometer (Luzchem, Ottawa, Canada). The emission spectrum of the lamps and the absorption spectra of the SWy-2 and nanocomposite films are shown in Fig. 2.
2.3. Characterization The formation of AgNPs was monitored by transmittance of films using a Shimadzu UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan) with an integrating sphere attachment (ISR-240A), in the range of 200–700 nm. The X-ray diffraction analysis was performed on a Rigaku Rotaflex − RU 200 B diffractometer (Rigaku, Tokyo, Japan) with Cu radiation ( = 0,154 nm) at 50 kV, 100 mA. The basal spacing between clay layers was calculated using Bragg’s equation (Callister, 2006). The AgNPs morphology and size distribution as well as the dispersion level of SWy-2 in nanocomposite films were examined by Scanning Transmission Electron Microscopy (STEM) using FEITECNAI G2 −F20 microscope (FEI, USA) equipped with an energy dispersive spectrometer (TECHNA, Italy). The samples were previously cut by Reichert Ultracut S. (LEICA, Austria) and placed on carbon coated Cu minigrids (CF-200 Cu, Electron Microscopy Sciences, USA).
2.4.1. Disk diffusion method Escherichia coli and Bacillus subtilis The disk diffusion method was chosen to evaluate the antibacterial activity of films (Tan et al., 2016). For the antibacterial tests, the non pathogenic bacteria Escherichia coli (DSM 5451-0599001) and Bacillus subtilis (DMS 1970-0110-001) were selected as model of Gram-negative and Gram-positive bacteria, respectively. Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) were incubated in 15.0 mL of sterile growth medium overnight at 37 ◦ C with 200 rpm (preculture). Then, 100 L of preculture solution (107 cells mL−1 ) of E. coli and B. subtilis were inoculated and spread on different agar plates. Afterwards, the samples disks with 6.0 mm of diameter were placed onto the medium plates and they were incubated at 37 ◦ C for 24 h. The antibacterial activity was indicated for the presence of inhibition zones around the samples.
3. Results and discussion 3.1. UV–vis spectroscopy The formation of AgNPs in the films containing different clay concentrations was followed by UV–vis spectra as a function of irradiation time. Fig. 3a-d shows the UV-vis spectra of the Ch83/2.5%SWy-2/AgNPs, Ch83/5%SWy2/AgNPs, Ch83/10%SWy-2/AgNPs nanocomposites and the reference Ch83/AgNPs (no clay content), during the irradiation process. The UV–vis spectra of nanocomposite films based on Ch83 present the plasmon absorption band around 440 nm, indicating the formation of AgNPs. This band becomes narrower and more intense with irradiation time. Furthermore, the presence of another absorption band around 250–300 nm indicates the simultaneous degradation of Ch83, forming radicals (Andrady, Torikai, & Kobatake, 1996). According to Sionkowska, Planecka, Lewandowska, Kaczmarek, and Szarszewska, (2013), the appearance of the band in the 290 nm during the UV irradiation of chitosan films, indicates the formation of new chromophores. For the nanocomposite films with lower clay content (2.5 and 5 wt%), the AgNPs maximum concentration was obtained after 24 h of irradiation (Fig. 3a,b). On the other hand, for Ch83/10%SWy2/AgNPs nanocomposite film, which presents the largest clay content, it can be observed that the AgNPs maximum concentration occurred after 11 h of irradiation (Fig. 3c), indicating the fastest formation of AgNPs. Moreover, the plasmon band is more intense than the degradation band
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Fig. 3. UV–vis spectra of the nanocomposites (a) Ch83/2.5%SWy-2/AgNPs (b) Ch83/5%SWy-2/AgNPs (c) Ch83/10%SWy-2/AgNPs, (d) Ch83/AgNPs, (e) Ch30/10%SWy-2/AgNPs, Ch30d/10%SWy-2/AgNPs and Ch83/10%SWy-2/AgNPs (f) Ch30-DEAE/10%SWy-2/AgNPs and (g) Ch30-DEAE-Dod/10%SWy-2/AgNPs.
(300 nm) of Ch83, suggesting that the formation process of AgNPs was predominant. However, for Ch83/AgNPs film without clay, the band at 300 nm assigned to degradation photoproducts is superior in comparison to plasmon band (Fig. 3d). According to these results, SWy-2 can be considered as a stabilizer against UV degradation. This occurs due to its charge transfer band in the range of 241–243 nm (Karickhoff & Bailey, 1973) (Fig. 2). Lombardo, Poli, Neumann, Machado, and Schimitt, (2013) observed the stabilization by clay against photooxidative degradation of poly(ethyleneoxide)/montmorillonite. SWy-2 can scatter and absorb part of the light UV, decreasing the absorption by chitosan. The AgNPs synthesis was also evaluated on nanocomposite films prepared using chitosans with lower molar mass (Ch30), higher degree of deacetylation (Ch30d) and modified (Ch30DEAE/10%SWy-2/AgNPs and Ch30-DEAE-Dod/10%SWy-2/AgNPs). The UV–vis spectra of Ch30/10%SWy-2/AgNPs, Ch30d/10%SWy2/AgNPs and Ch83/10%SWy-2/AgNPs nanocomposites are shown in Fig. 3e. It is worth noting, that the increase of approximately 10%
of chitosan deacetylation degree did not influence the formation of AgNPs, since absorption spectra of Ch30/10%SWy-2/AgNPs and Ch30d/10%SWy-2/AgNPs films are similar. However, it is possible to note that the intensity of plasmon absorption band of Ch83/10%SWy-2/AgNPs nanocomposite is higher than the others. This difference may be related to molar masses of chitosans. Hence, the increase of polymer chain length is responsible for the high concentration of silver nanoparticles formed. This result corroborates with Luo, Zhang, Zeng, Zeng, and Wang, (2005) that reported a larger amount of silver nanoparticles formed with the increase of poly(ethylene glycol) molar mass. For nanocomposites using modified chitosans (Ch30DEAE/10%SWy-2/AgNPs and Ch30-DEAE-Dod/10%SWy-2/AgNPs), the synthesis of AgNPs occurred quickly, in approximately 1.5 h, while for all the others nanocomposite films it was over 11 h (Fig. 3f, g). According to some studies (Mucha & Pawlak, 2002; Tan et al., 2016), the mechanism of chitosan degradation under UV light occurs by two pathways (Fig. 4). This is in agreement with pre-
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Fig. 4. Chitosan photodegradation.
vious findings with regard to the influence of the viscosity-average molar mass of the polymer, its lower molar mass results in fewer amounts of glycosidic bonds in polymer chain and consequently, lower formation of radicals responsible for the Ag+ reduction. Thus, a possible mechanism of AgNPs formation involves the production of radicals and macroradicals from chitosan chains, followed by reduction of Ag+ to Ag0 . Then, Ag0 species interact with Ag+ ions forming clusters that absorb other Ag0 species for the AgNPs formation (Latif, Al-Rubeaan, & Saeb, 2014). In this system, the Ag+ ions interact with polysaccharide by chelation (Emam & Ahamed, 2016). According to Druet, Achari, and Isaad (2015), the chelation of metalic ions by chitosan occurs in free amine groups and in oxygen atoms, even in acidic aqueous solutions. Thereby, the increase of amine groups amount in chitosan, enhance its complexation ability. Therefore, the time difference in AgNPs synthesis (from above 11 h to 1.5 h) can be attributed to the expressive increase of amine groups on chitosan derivatives. The insertion of 40% of DEAE groups in polymer chains increase significantly their chelation sites, allowing the polymer chain to interact with a larger amount of Ag+ . Thus, this insertion facilitates the Ag+ reduction after UV irradiation process. 3.2. X-ray diffraction analyses XRD was used to check the cristallinity of chitosan and the interlamellar spacing of SWy-2 by analyzing the diffractogram on 2 from 3◦ to 50◦ . The XRD patterns for SWy-2, Ch83 film and nanocomposites are presented in Fig. 5. The diffraction pattern of SWy-2 (Fig. 5a) presents a reflection peak (001) at 2 = 7.0◦ , which corresponds to an interlamellar spacing of 12.6 Å. Ch83 shows char-
acteristic diffraction peaks on approximately 2 = 7◦ , 11◦ and 18◦ , which the first two peaks correspond to hydrated crystalline structures and the third peak to an amorphous structure of chitosan (Rhim, Hong, Park, & Perry, 2006). In the nanocomposite films (Fig. 5b,c), the 001 reflection peak shifted from 7.0◦ to 4.0–6.3◦ (interlamellar spacing of ˚ ˚ suggesting the formation of intercalated structures 22.1 A–14.0 A), for the majority of samples. For nanocomposite films of Ch83, which different clay concentrations are employed, the interlayer space increased for lower clay content (Fig. 5b). Considering the other films, the greatest interlamellar distances were observed for nanocomposites based on lower molar mass chitosan (Ch30/10%SWy-2/AgNPs) or modified chitosans (Ch30DEAE/10%SWy-2/AgNPs and Ch30-DEAE-Dod/10%SWy-2/AgNPs), indicating that the molar mass (Ch83 and Ch30) as well as the polymer chemical structure influenced in the level of clay intercalation (Fig. 5c). Moreover, the X-ray diffraction for nanocomposite films suggests that the ordered structure of the clay was disturbed, since 001 peaks intensity are lower and wider (Lombardo et al., 2016). Thus, both of these results revealed the occurrence of intercalated along with some exfoliated structures. On the other hand, the 001 reflection peak disappeared for Ch30d/10%SWy-2/AgNPs, indicating that exfoliated structure was formed with the full expansion of SWy-2 interlayer space. 3.3. Scanning transmission electron microscopy TEM analyses were used to determine the shape and the size distribution of the AgNPs, as well as the dispersion state of SWy-2 in the nanocomposites. As can be seen in Fig. 6, TEM images revealed
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Fig. 5. X-ray diffraction and the interlamellar distance d for (a) SWy-2 and Ch83 film, (b) nanocomposite films prepared with Ch83 and (c) nanocomposite films prepared with Ch30, Ch30d and modified chitosans.
for all samples, small black spots that are attributed to the AgNPs. Moreover, AgNPs presented spherical morphology independently of chitosan or clay amount used in the preparation of the films. According to Tunc¸ and Duman, (2010), clay mineral tactoids (darkest areas) in TEM images demonstrate intercalated structures of the clay in the nanocomposites, while for exfoliated structures the clay layers are completely and uniformed dispersed in polymeric matrix. The particle sizes obtained from TEM for all the nanocomposites were around 2.7–6.3 nm. It is possible to observe in Fig. 6a–i, the clay concentration influenced in the uniformity of AgNPs in the films based on Ch83. The Ch83/2.5%SWy-2/AgNPs (Fig. 6b), and Ch83/5%SWy-2/AgNPs (Fig. 6e) exhibited a larger formation and a more uniform size of AgNPs, while for Ch83/10%SWy-2/AgNPs, the increase of SWy-2 concentration (10 wt%), resulted in a nonuniform nanoparticles size with a broad size distribution (2–20 nm), and aggregation of some particles (Fig. 6h). In addition, concerning the SWy-2 dispersion, was noticed that in low clay concentrations (2.5 and 5 wt%), intercalated and a small amount of exfoliated structures were formed (Fig. 6a, d). For the Ch83/10%SWy-2/AgNPs (Fig. 6g), intercalated structure with aggregates formation were noted, indicating the lowest clay dispersion. These results are in agreement with XDR analyses.
On the other hand, Ch30/10%SWy-2/AgNPs and Ch30d/10%SWy-2/AgNPs films (Fig. 7a–f) presented a greater uniformity of nanoparticles size with a narrower size distribution (1.5–6.3 nm) in comparison to Ch83/10%SWy-2/AgNPs, this behavior is attributed to lower molar mass of chitosans and better exfoliation of clay. The increase of chitosan deacetylation degree promoted the formation of largest AgNPs (6.3 nm) with a broad size distribution (2–11 nm) and the presence of some aggregated nanoparticles (Figs. 6e, f). Furthermore, the absence of clay tactoids for Ch30/10%SWy-2/AgNPs and Ch30d/10%SWy-2/AgNPs (Fig. 7a, d) suggest that exfoliated structures were predominant. Among Ch30-DEAE/10%SWy-2/AgNPs (Fig. 7g–i) and Ch30DEAE-Dod/10%SWy-2/AgNPs (Fig. 7j-l) nanocomposites, TEM micrographs reveal that smaller and the most uniform nanoparticles size have been formed in the film of modified chitosan with dodecyl group (Ch30-DEAE-Dod/10%SWy-2/AgNPs). The modified chitosan Ch30-DEAE-Dod is an amphiphilic compound (containing hydrophilic and hydrophobic groups) that can act as a surfactant, controlling the size/shape of the AgNPs (Sakamoto, Fujistuka, & Majima, 2009). Moreover, SWy-2 tactoids were not observed, indicating a better dispersion of silicate lamellae in these nanocomposite films (Fig. 7g, j).
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Fig. 6. TEM images and particle size distribution of nanocomposites (a-c) Ch83/2.5%SWy-2/AgNPs, (d-f) Ch83/5%SWy-2/AgNPs and (g-i) Ch83/10%SWy-2/AgNPs.
3.4. Antibacterial activity 3.4.1. Disk diffusion method The zones of inhibition were obtained with diameters ranging from 6.0 to –9.0 mm against E. coli and B. subtilis. The tests were performed three times for each sample and the results are shown in Table 1. The Petri dishes with disks of nanocomposites without AgNPs (Ch83, Ch83/10%SWy-2, Ch30/10%SWy-2, Ch30d/10%SWy-2, Ch30-DEAE/10%SWy-2 and Ch30-DEAE-Dod/10%SWy-2) showed a dense population of bacterial colonies of E. coli and B. subtilis surrounded the film disks. However, these films inhibited the growth of both bacteria by direct contact, no bacterial development was observed under these disks incubated for 24 h. The presence of SWy-2 did not modify this activity. The absence of inhibition zone ˜ and de la was also reported by Leceta, Guerrero, Ibarburu, Duenas, Caba (2013). It can be explained by the limitation of the chitosan diffusion in agar plate. The Petri dishes with nanocomposites-AgNPs disks presented inhibition zones around of circular films. These results indicated that the films are equally effective to both E. coli and B. subtilis. The antibacterial activity of AgNPs occurs due to release of Ag+ from AgNPs surface by oxidative reaction. These Ag+ ions interact with functional groups of proteins and enzymes contributing to inactivation and interruption of cell process (Sohrabnezhad, Pourahmad, Mehdipour Moghaddam, & Sadeghi, 2015). According to Krishnan
et al. (2015), smaller nanoparticles are more effective against bacteria due to the higher ability of them in release of Ag+ ions. The synthesis of AgNPs occurred quickly and directly on films of modified chitosans/clay by UV irradiation, promoting the formation of smaller AgNPs and avoids the agglomerates formation. This methodology allows the homogeneous distribution of the AgNPs on the material due to the immobilization of silver ions (Ag+ ) on the polymer matrix before the photochemical reaction. In the literature was found a method, in which AgNPs are synthetized in solution for further preparation of films (Krishnan et al., 2015). This sort of method generates AgNP agglomerates due to the drying process. 4. Conclusions AgNPs were synthetized on films of chitosans/montmorillonite nanocomposites by photochemical method. The AgNPs formation was characterized by the appearance of the plasmon absorption band around 440 nm. TEM images revealed AgNPs with spherical morphology for all films. The increase of clay concentration resulted in faster synthesis of AgNPs and nonuniform nanoparticles size with aggregation of some particles. For 2.5 wt% and 5 wt% of clay, the AgNPs exhibited more uniform size and mixture of intercalated/exfoliated structures. Nanocomposites based on lower molar mass chitosan Ch30 had predominant exfoliated structure.
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Fig. 7. TEM images and particle size distribution of nanocomposites (a-c) Ch30/10%SWy-2/AgNPs, (d-f) Ch30d/10%SWy-2/AgNPs, (g-i) Ch30-DEAE/10%SWy-2/AgNPs and (j-l) Ch30-DEAE-Dod/10%SWy-2/AgNPs.
For nanocomposites prepared using modified chitosans, the synthesis of AgNPs occurred quickly, in approximately 1.5 h, while for all other nanocomposite films it was above 11 h. The film of modified chitosan with dodecyl groups presented smaller and more uniform nanoparticles size along mixture of exfoliated and intercalated structures. This modified chitosan is an amphiphilic compound (containing hydrophilic and hydrophobic groups) that can act as a surfactant, controlling the size/shape of the AgNPs. The antibacterial activity of nanocomposite films was determined against the growth of E. coli and B. subtilis. Films prepared in the absence of AgNPs showed no inhibition zones, indicating that
AgNPs are responsible for the antibacterial activity. The presence of clay did not influence the bactericidal activity. The formation of inhibition zones suggested that all the films containing AgNPs prevented the growth of both bacteria. Acknowledgements This work was supported by CNPq (308940/2013-0 and 401434/2014-1) and J.S.G would like to thank CAPES and CNPq (200672/2015-0) for a graduate fellowship. The authors are grateful to Prof. Luc Avérous for assistance with antibacterial tests and
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Photochemical synthesis of silver nanoparticles on chitosans/montmorillonite nanocomposite films and antibacterial activity Juliana S. Gabriel, Virgínia A.M. Gonzaga, Alessandra L. Poli, Carla C. Schmitt ∗ Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos SP, Caixa Postal 780, 13560-970, Brazil
a r t i c l e
i n f o
Article history: Received 21 February 2017 Received in revised form 25 April 2017 Accepted 5 May 2017 Available online 5 May 2017 Keywords: Silver nanoparticles Chitosan derivatives Montmorillonite Nanocomposite films UV irradiation Antibacterial activity
a b s t r a c t Silver nanoparticles (AgNPs) were synthetized on chitosans/montmorillonite nanocomposite films by photochemical method. Nanocomposites were prepared using chitosans with different molar masses and deacetylation degrees, as well as modified with diethylaminoethyl (DEAE) and dodecyl groups. AgNPs formation on the films was followed by the appearance of the plasmon band around 440 nm as a function of irradiation time. TEM images revealed AgNPs with spherical morphology for all nanocomposites. For nanocomposites using modified chitosans, the AgNPs synthesis occurred quickly (1.5 h) while for the others films it was above 11 h. The film of modified chitosan with dodecyl and DEAE groups presented smaller and more uniform nanoparticles size along mixture of exfoliated and intercalated structures. This modified chitosan is an amphiphilic compound that can act controlling the size/shape of the AgNPs. The results of antibacterial activity suggested that all nanocomposite-AgNPs films inhibited the growth of Escherichia coli and Bacillus subtilis. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Over the past few years, silver nanoparticles (AgNPs) have become a target of great interest due to their remarkable electronic, optical, mechanical, magnetic, chemical and antimicrobial properties (Dick, McFarland, Haynes, & Van Duyne, 2002; Ghosh & Maiti, 1996; Kanmani & Rhim, 2014; Severin, Kirstein, Sokolov, & Rabe, 2009; Thuc et al., 2016). In regard with size and shape-based of AgNPs, several applications ranging from catalysts, biomedical field, disinfectant sprays, ink, food package and textile products (Chernousova & Epple, 2013; Emam & Ahamed, 2016; Thuc et al., 2016; Zoya, 2012). Hence, a myriad of methods for AgNPs synthesis have been reported, including biological, chemical, photochemical and electrochemical processes (Bhaduri et al., 2013; Lengke, Fleet, & Southam, 2007; Patra et al., 2014; Thuc et al., 2016). However, the chemical reduction using hazardous reducing agents, such as sodium borohydrate, formaldehyde and ammonia is typically employed for AgNPs preparation, limiting the use of AgNPs for biological or medical applications (Emam & Ahamed, 2016; El-
∗ Corresponding author. E-mail addresses: [email protected] (J.S. Gabriel), [email protected] (V.A.M. Gonzaga), [email protected] (A.L. Poli), [email protected] (C.C. Schmitt). http://dx.doi.org/10.1016/j.carbpol.2017.05.021 0144-8617/© 2017 Elsevier Ltd. All rights reserved.
Nour, Eftaiha, Al-Warthan, & Ammar, 2010; Lombardo, Poli, Castro, Perussi, & Schmitt, 2016; Wojtysiak & Kudelski, 2012). On the other hand, the number of publications about green synthesis of the AgNPs that use environmentally friendly compounds as reducing agents has risen. Among these processes, the green irradiation methods including laser, gamma, ultrasonic wave, ion and ultraviolet (UV) radiation of silver salts in aqueous solution have been used as alternative routes to broaden the range of applications (Shameli et al., 2010; Son, Youk, & Park, 2006; Zhou et al., 2012). In addition, the synthesis of AgNPs was also described using clay suspension, in which the clay lamellae behave as nanoreactors of Ag+ to Ag0 (Patakfalvi & Dékány, 2004). Furthermore, several polysaccharides, such as glucose, dextrose, starch and chitosan (Ji, Liu, Zhang, Xiong, & Sun, 2016; Oluwafemi et al., 2016; Thomas et al., 2009) have been investigated as reducers and/or stabilizing agents for AgNPs preparation owing to their ability for coordinating with metal ions (Emam & Ahamed, 2016). These polymer-Ag+ complexes can be reduced by different experimental conditions, to produce smalls AgNPs with narrow size distribution (Emam & Ahamed, 2016). Regarding natural polymers, chitosan, the main derivative of chitin, has been extensively studied as a cationic biopolymer of high bioactivity, biodegradability, biocompatibility and low toxicity, in addition to its antimicrobial activity (Araiza, Alcouffe, Rochas, Montembault, & David, 2010; Epure, Griffon, Pollet, & Avérous, 2011; Youssef, Yousef, El-Sayed, & Kamel, 2015). These properties
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Table 1 Inhibition zones for nanocomposite films and examples of absence (a) and presence (b) of these zones. These images were obtained to Ch83 and Ch83/10%SWy-2/AgNPs films, respectively, against B. subtilis. E. coli
B. subtilis
Sample
d (mm)
Sample
d (mm)
Ch83 Ch83/10%SWy-2 Ch30/10%SWy-2 Ch30d/10%SWy-2 Ch30-DEAE/10%SWy-2 Ch30-DEAE-Dod/10%SWy-2 Ch83/AgNPs Ch83/2.5%SWy-2/AgNPs Ch83/5%SWy-2/AgNPs Ch83/10%SWy-2/AgNPs Ch30/10%SWy-2/AgNPs Ch30d/10%SWy-2/AgNPs Ch30-DEAE/10%SWy-2/AgNPs Ch30-DEAE-Dod/10%SWy-2/AgNPs
N.I N.I N.I N.I N.I N.I 8.7 ± 0.6 7.6 ± 0.3 8.7 ± 0.6 8.3 ± 0.6 7.5 ± 0.5 7.8 ± 0.2 8.3 ± 0.3 8.0 ± 0.3
Ch83 Ch83/10%SWy-2 Ch30/10%SWy-2 Ch30d/10%SWy-2 Ch30-DEAE/10%SWy-2 Ch30-DEAE-Dod/10%SWy-2 Ch83/AgNPs Ch83/2.5%SWy-2/AgNPs Ch83/5%SWy-2/AgNPs Ch83/10%SWy-2/AgNPs Ch30/10%SWy-2/AgNPs Ch30d/10%SWy-2/AgNPs Ch30-DEAE/10%SWy-2/AgNPs Ch30-DEAE-Dod/10%SWy-2/AgNPs
N.I N.I N.I N.I N.I N.I 8.5 ± 0.7 8.3 ± 0.5 8.7 ± 0.6 7.3 ± 0.6 8.0 ± 0.3 8.3 ± 0.5 8.0 ± 0.2 9.0 ± 0.3
N.I.: No Inhibition. The error was obtained by three independent experiments. The results were reported as mean ± standard deviation.
combined with its ability to form films, allows the application of this polysaccharide in food packaging, bone substitutes and artificial skin (Shameli et al., 2010). In order to obtain superior stability of chitosan-based nanocomposites, the addition of a natural multilayer silicate known as montmorillonite clay significantly enhances the chemical and/or mechanical stability, in comparison with the polymer itself (Wang et al., 2005; Xie et al., 2013; Xu, Ren, & Hhanna, 2006). Montmorillonite clay is formed by a single octahedral sheet of magnesia or alumina located between two silica tetrahedral sheets (Xu et al., 2006). Moreover, the characteristics of chitosan/montmorillonite nanocomposites coupled with AgNPs properties can result in versatile materials for a wide range of applications. Several approaches concerning photochemical synthesis of AgNPs in solutions of polymers or nanocomposites have been extensively reported in the literature (Krishnan et al., 2015; Shameli et al., 2010; Thomas, Yallapu, Sreedhar, & Bajpai, 2009). However, the metal nanoparticles synthesis can also be performed in solid material directly on films using irradiation, as described by Sakamoto, Fujistuka, and Majima, 2009, which studied the fabrication of Au/Cu bimetallic nanoparticles in poly(vinyl alcohol) films by UV light irradiation. Herein we report AgNPs synthesis on chitosan/clay nanocomposite films by UV irradiation after the films preparation. The influence of clay concentration and modification of chitosan structure was also evaluated in the AgNPs formation. The characterization of samples was performed using X-ray diffraction analyses (XRD), UV–vis spectroscopy and transmission electron microscopy (TEM). Finally, the antibacterial activities of materials were investigated against Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis).
2. Materials and methods 2.1. Materials The chitosan (Ch83) used in this work was purchased from Aldrich Chemical Co with a deacetylation degree of 85% and a viscosity-average molar mass of 83,000 g mol−1 . Chitosan with
low molecular weight (Ch30) 30,000 g mol−1 (deacetylation degree of 85%) was obtained according to the method reported by Tommeraas, Varum, Christensen, and Smidsrod, (2001). It was also prepared chitosan with a deacetylation degree of 98% (Ch30d) and a viscosity-average molar mass of 30,000 g mol−1 (Tiera et al., 2006). Modified chitosans (Fig. 1) used in this work were prepared according to the method reported earlier (Gabriel, Tiera, & Tiera, 2015). Ch30-DEAE is a hydrophilic chitosan derivative with 40% of substitution degree by diethylaminoethyl groups (DEAE). Ch30DEAE-Dod is an amphiphilic chitosan derivative with 40 and 6% of substitution degree by DEAE and dodecyl groups (Dod), respectively. The substitution degrees were determined by 1 HNMR spectroscopy (data not shown). The SWy-2 montmorillonite clay was kindly supplied by Source Clays Repository of Clay Minerals Society, University of Missouri (Columbia, MO). The SWy-2 clay was purified according to the method described by Gessner, Schmitt, and Neumann, (1994). Glacial acetic acid (Synth, Brazil), silver nitrate (TEC-LAB, Brazil) and distilled water were also used in this work.
2.2. Nanocomposite films preparation SWy-2 dispersions with 2.5 wt%, 5 wt% and 10 wt% based on chitosan were prepared by dispersing appropriate amounts of SWy2 into 15 mL of 0.25 mol L−1 aqueous acetic acid solution under stirring for 24 h. Then, 0.2 g of Ch83 powder and 1.0 mL of silver nitrate solution (0.05 mol L−1 ) were added into the clay dispersions. The mixtures were stirred continuously for 24 h. Afterwards, these solutions were cast onto polystyrene Petri dishes with dimensions of 60 mm × 15 mm. The films were dried in an oven at 30 ◦ C, and peeled from the Petri dishes. Additionally, the nanocomposite films containing Ch30, Ch30d, Ch30-DEAE and Ch30-DEAE-Dod were prepared using only 10 wt% of SWy-2 based on polymer by the methodology described above. Accordingly, AgNPs were synthesized by photochemical method. The nanocomposite films were placed in a UV light irradiation chamber containing sixteen UV germicidal lamps at 25 ◦ C up to 24 h. At the same position, the emission of lamps was 254 nm
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Fig. 1. Molecular structures of modified chitosans (a) Ch30-DEAE and (b) Ch30-DEAE-Dod.
2.4. Antibacterial activity
Fig. 2. Emission spectrum of irradiating lamp and absorption spectra of SWy-2 and Ch83/10%SWy-2/AgNO3 , Ch30/10%SWy-2/AgNO3 , Ch30-DEAE-Dod/10%SWy2/AgNO3 films.
with power of 18603 mW m−2 , as measured by SPR-01 Spectroradiometer (Luzchem, Ottawa, Canada). The emission spectrum of the lamps and the absorption spectra of the SWy-2 and nanocomposite films are shown in Fig. 2.
2.3. Characterization The formation of AgNPs was monitored by transmittance of films using a Shimadzu UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan) with an integrating sphere attachment (ISR-240A), in the range of 200–700 nm. The X-ray diffraction analysis was performed on a Rigaku Rotaflex − RU 200 B diffractometer (Rigaku, Tokyo, Japan) with Cu radiation ( = 0,154 nm) at 50 kV, 100 mA. The basal spacing between clay layers was calculated using Bragg’s equation (Callister, 2006). The AgNPs morphology and size distribution as well as the dispersion level of SWy-2 in nanocomposite films were examined by Scanning Transmission Electron Microscopy (STEM) using FEITECNAI G2 −F20 microscope (FEI, USA) equipped with an energy dispersive spectrometer (TECHNA, Italy). The samples were previously cut by Reichert Ultracut S. (LEICA, Austria) and placed on carbon coated Cu minigrids (CF-200 Cu, Electron Microscopy Sciences, USA).
2.4.1. Disk diffusion method Escherichia coli and Bacillus subtilis The disk diffusion method was chosen to evaluate the antibacterial activity of films (Tan et al., 2016). For the antibacterial tests, the non pathogenic bacteria Escherichia coli (DSM 5451-0599001) and Bacillus subtilis (DMS 1970-0110-001) were selected as model of Gram-negative and Gram-positive bacteria, respectively. Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) were incubated in 15.0 mL of sterile growth medium overnight at 37 ◦ C with 200 rpm (preculture). Then, 100 L of preculture solution (107 cells mL−1 ) of E. coli and B. subtilis were inoculated and spread on different agar plates. Afterwards, the samples disks with 6.0 mm of diameter were placed onto the medium plates and they were incubated at 37 ◦ C for 24 h. The antibacterial activity was indicated for the presence of inhibition zones around the samples.
3. Results and discussion 3.1. UV–vis spectroscopy The formation of AgNPs in the films containing different clay concentrations was followed by UV–vis spectra as a function of irradiation time. Fig. 3a-d shows the UV-vis spectra of the Ch83/2.5%SWy-2/AgNPs, Ch83/5%SWy2/AgNPs, Ch83/10%SWy-2/AgNPs nanocomposites and the reference Ch83/AgNPs (no clay content), during the irradiation process. The UV–vis spectra of nanocomposite films based on Ch83 present the plasmon absorption band around 440 nm, indicating the formation of AgNPs. This band becomes narrower and more intense with irradiation time. Furthermore, the presence of another absorption band around 250–300 nm indicates the simultaneous degradation of Ch83, forming radicals (Andrady, Torikai, & Kobatake, 1996). According to Sionkowska, Planecka, Lewandowska, Kaczmarek, and Szarszewska, (2013), the appearance of the band in the 290 nm during the UV irradiation of chitosan films, indicates the formation of new chromophores. For the nanocomposite films with lower clay content (2.5 and 5 wt%), the AgNPs maximum concentration was obtained after 24 h of irradiation (Fig. 3a,b). On the other hand, for Ch83/10%SWy2/AgNPs nanocomposite film, which presents the largest clay content, it can be observed that the AgNPs maximum concentration occurred after 11 h of irradiation (Fig. 3c), indicating the fastest formation of AgNPs. Moreover, the plasmon band is more intense than the degradation band
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Fig. 3. UV–vis spectra of the nanocomposites (a) Ch83/2.5%SWy-2/AgNPs (b) Ch83/5%SWy-2/AgNPs (c) Ch83/10%SWy-2/AgNPs, (d) Ch83/AgNPs, (e) Ch30/10%SWy-2/AgNPs, Ch30d/10%SWy-2/AgNPs and Ch83/10%SWy-2/AgNPs (f) Ch30-DEAE/10%SWy-2/AgNPs and (g) Ch30-DEAE-Dod/10%SWy-2/AgNPs.
(300 nm) of Ch83, suggesting that the formation process of AgNPs was predominant. However, for Ch83/AgNPs film without clay, the band at 300 nm assigned to degradation photoproducts is superior in comparison to plasmon band (Fig. 3d). According to these results, SWy-2 can be considered as a stabilizer against UV degradation. This occurs due to its charge transfer band in the range of 241–243 nm (Karickhoff & Bailey, 1973) (Fig. 2). Lombardo, Poli, Neumann, Machado, and Schimitt, (2013) observed the stabilization by clay against photooxidative degradation of poly(ethyleneoxide)/montmorillonite. SWy-2 can scatter and absorb part of the light UV, decreasing the absorption by chitosan. The AgNPs synthesis was also evaluated on nanocomposite films prepared using chitosans with lower molar mass (Ch30), higher degree of deacetylation (Ch30d) and modified (Ch30DEAE/10%SWy-2/AgNPs and Ch30-DEAE-Dod/10%SWy-2/AgNPs). The UV–vis spectra of Ch30/10%SWy-2/AgNPs, Ch30d/10%SWy2/AgNPs and Ch83/10%SWy-2/AgNPs nanocomposites are shown in Fig. 3e. It is worth noting, that the increase of approximately 10%
of chitosan deacetylation degree did not influence the formation of AgNPs, since absorption spectra of Ch30/10%SWy-2/AgNPs and Ch30d/10%SWy-2/AgNPs films are similar. However, it is possible to note that the intensity of plasmon absorption band of Ch83/10%SWy-2/AgNPs nanocomposite is higher than the others. This difference may be related to molar masses of chitosans. Hence, the increase of polymer chain length is responsible for the high concentration of silver nanoparticles formed. This result corroborates with Luo, Zhang, Zeng, Zeng, and Wang, (2005) that reported a larger amount of silver nanoparticles formed with the increase of poly(ethylene glycol) molar mass. For nanocomposites using modified chitosans (Ch30DEAE/10%SWy-2/AgNPs and Ch30-DEAE-Dod/10%SWy-2/AgNPs), the synthesis of AgNPs occurred quickly, in approximately 1.5 h, while for all the others nanocomposite films it was over 11 h (Fig. 3f, g). According to some studies (Mucha & Pawlak, 2002; Tan et al., 2016), the mechanism of chitosan degradation under UV light occurs by two pathways (Fig. 4). This is in agreement with pre-
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Fig. 4. Chitosan photodegradation.
vious findings with regard to the influence of the viscosity-average molar mass of the polymer, its lower molar mass results in fewer amounts of glycosidic bonds in polymer chain and consequently, lower formation of radicals responsible for the Ag+ reduction. Thus, a possible mechanism of AgNPs formation involves the production of radicals and macroradicals from chitosan chains, followed by reduction of Ag+ to Ag0 . Then, Ag0 species interact with Ag+ ions forming clusters that absorb other Ag0 species for the AgNPs formation (Latif, Al-Rubeaan, & Saeb, 2014). In this system, the Ag+ ions interact with polysaccharide by chelation (Emam & Ahamed, 2016). According to Druet, Achari, and Isaad (2015), the chelation of metalic ions by chitosan occurs in free amine groups and in oxygen atoms, even in acidic aqueous solutions. Thereby, the increase of amine groups amount in chitosan, enhance its complexation ability. Therefore, the time difference in AgNPs synthesis (from above 11 h to 1.5 h) can be attributed to the expressive increase of amine groups on chitosan derivatives. The insertion of 40% of DEAE groups in polymer chains increase significantly their chelation sites, allowing the polymer chain to interact with a larger amount of Ag+ . Thus, this insertion facilitates the Ag+ reduction after UV irradiation process. 3.2. X-ray diffraction analyses XRD was used to check the cristallinity of chitosan and the interlamellar spacing of SWy-2 by analyzing the diffractogram on 2 from 3◦ to 50◦ . The XRD patterns for SWy-2, Ch83 film and nanocomposites are presented in Fig. 5. The diffraction pattern of SWy-2 (Fig. 5a) presents a reflection peak (001) at 2 = 7.0◦ , which corresponds to an interlamellar spacing of 12.6 Å. Ch83 shows char-
acteristic diffraction peaks on approximately 2 = 7◦ , 11◦ and 18◦ , which the first two peaks correspond to hydrated crystalline structures and the third peak to an amorphous structure of chitosan (Rhim, Hong, Park, & Perry, 2006). In the nanocomposite films (Fig. 5b,c), the 001 reflection peak shifted from 7.0◦ to 4.0–6.3◦ (interlamellar spacing of ˚ ˚ suggesting the formation of intercalated structures 22.1 A–14.0 A), for the majority of samples. For nanocomposite films of Ch83, which different clay concentrations are employed, the interlayer space increased for lower clay content (Fig. 5b). Considering the other films, the greatest interlamellar distances were observed for nanocomposites based on lower molar mass chitosan (Ch30/10%SWy-2/AgNPs) or modified chitosans (Ch30DEAE/10%SWy-2/AgNPs and Ch30-DEAE-Dod/10%SWy-2/AgNPs), indicating that the molar mass (Ch83 and Ch30) as well as the polymer chemical structure influenced in the level of clay intercalation (Fig. 5c). Moreover, the X-ray diffraction for nanocomposite films suggests that the ordered structure of the clay was disturbed, since 001 peaks intensity are lower and wider (Lombardo et al., 2016). Thus, both of these results revealed the occurrence of intercalated along with some exfoliated structures. On the other hand, the 001 reflection peak disappeared for Ch30d/10%SWy-2/AgNPs, indicating that exfoliated structure was formed with the full expansion of SWy-2 interlayer space. 3.3. Scanning transmission electron microscopy TEM analyses were used to determine the shape and the size distribution of the AgNPs, as well as the dispersion state of SWy-2 in the nanocomposites. As can be seen in Fig. 6, TEM images revealed
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Fig. 5. X-ray diffraction and the interlamellar distance d for (a) SWy-2 and Ch83 film, (b) nanocomposite films prepared with Ch83 and (c) nanocomposite films prepared with Ch30, Ch30d and modified chitosans.
for all samples, small black spots that are attributed to the AgNPs. Moreover, AgNPs presented spherical morphology independently of chitosan or clay amount used in the preparation of the films. According to Tunc¸ and Duman, (2010), clay mineral tactoids (darkest areas) in TEM images demonstrate intercalated structures of the clay in the nanocomposites, while for exfoliated structures the clay layers are completely and uniformed dispersed in polymeric matrix. The particle sizes obtained from TEM for all the nanocomposites were around 2.7–6.3 nm. It is possible to observe in Fig. 6a–i, the clay concentration influenced in the uniformity of AgNPs in the films based on Ch83. The Ch83/2.5%SWy-2/AgNPs (Fig. 6b), and Ch83/5%SWy-2/AgNPs (Fig. 6e) exhibited a larger formation and a more uniform size of AgNPs, while for Ch83/10%SWy-2/AgNPs, the increase of SWy-2 concentration (10 wt%), resulted in a nonuniform nanoparticles size with a broad size distribution (2–20 nm), and aggregation of some particles (Fig. 6h). In addition, concerning the SWy-2 dispersion, was noticed that in low clay concentrations (2.5 and 5 wt%), intercalated and a small amount of exfoliated structures were formed (Fig. 6a, d). For the Ch83/10%SWy-2/AgNPs (Fig. 6g), intercalated structure with aggregates formation were noted, indicating the lowest clay dispersion. These results are in agreement with XDR analyses.
On the other hand, Ch30/10%SWy-2/AgNPs and Ch30d/10%SWy-2/AgNPs films (Fig. 7a–f) presented a greater uniformity of nanoparticles size with a narrower size distribution (1.5–6.3 nm) in comparison to Ch83/10%SWy-2/AgNPs, this behavior is attributed to lower molar mass of chitosans and better exfoliation of clay. The increase of chitosan deacetylation degree promoted the formation of largest AgNPs (6.3 nm) with a broad size distribution (2–11 nm) and the presence of some aggregated nanoparticles (Figs. 6e, f). Furthermore, the absence of clay tactoids for Ch30/10%SWy-2/AgNPs and Ch30d/10%SWy-2/AgNPs (Fig. 7a, d) suggest that exfoliated structures were predominant. Among Ch30-DEAE/10%SWy-2/AgNPs (Fig. 7g–i) and Ch30DEAE-Dod/10%SWy-2/AgNPs (Fig. 7j-l) nanocomposites, TEM micrographs reveal that smaller and the most uniform nanoparticles size have been formed in the film of modified chitosan with dodecyl group (Ch30-DEAE-Dod/10%SWy-2/AgNPs). The modified chitosan Ch30-DEAE-Dod is an amphiphilic compound (containing hydrophilic and hydrophobic groups) that can act as a surfactant, controlling the size/shape of the AgNPs (Sakamoto, Fujistuka, & Majima, 2009). Moreover, SWy-2 tactoids were not observed, indicating a better dispersion of silicate lamellae in these nanocomposite films (Fig. 7g, j).
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Fig. 6. TEM images and particle size distribution of nanocomposites (a-c) Ch83/2.5%SWy-2/AgNPs, (d-f) Ch83/5%SWy-2/AgNPs and (g-i) Ch83/10%SWy-2/AgNPs.
3.4. Antibacterial activity 3.4.1. Disk diffusion method The zones of inhibition were obtained with diameters ranging from 6.0 to –9.0 mm against E. coli and B. subtilis. The tests were performed three times for each sample and the results are shown in Table 1. The Petri dishes with disks of nanocomposites without AgNPs (Ch83, Ch83/10%SWy-2, Ch30/10%SWy-2, Ch30d/10%SWy-2, Ch30-DEAE/10%SWy-2 and Ch30-DEAE-Dod/10%SWy-2) showed a dense population of bacterial colonies of E. coli and B. subtilis surrounded the film disks. However, these films inhibited the growth of both bacteria by direct contact, no bacterial development was observed under these disks incubated for 24 h. The presence of SWy-2 did not modify this activity. The absence of inhibition zone ˜ and de la was also reported by Leceta, Guerrero, Ibarburu, Duenas, Caba (2013). It can be explained by the limitation of the chitosan diffusion in agar plate. The Petri dishes with nanocomposites-AgNPs disks presented inhibition zones around of circular films. These results indicated that the films are equally effective to both E. coli and B. subtilis. The antibacterial activity of AgNPs occurs due to release of Ag+ from AgNPs surface by oxidative reaction. These Ag+ ions interact with functional groups of proteins and enzymes contributing to inactivation and interruption of cell process (Sohrabnezhad, Pourahmad, Mehdipour Moghaddam, & Sadeghi, 2015). According to Krishnan
et al. (2015), smaller nanoparticles are more effective against bacteria due to the higher ability of them in release of Ag+ ions. The synthesis of AgNPs occurred quickly and directly on films of modified chitosans/clay by UV irradiation, promoting the formation of smaller AgNPs and avoids the agglomerates formation. This methodology allows the homogeneous distribution of the AgNPs on the material due to the immobilization of silver ions (Ag+ ) on the polymer matrix before the photochemical reaction. In the literature was found a method, in which AgNPs are synthetized in solution for further preparation of films (Krishnan et al., 2015). This sort of method generates AgNP agglomerates due to the drying process. 4. Conclusions AgNPs were synthetized on films of chitosans/montmorillonite nanocomposites by photochemical method. The AgNPs formation was characterized by the appearance of the plasmon absorption band around 440 nm. TEM images revealed AgNPs with spherical morphology for all films. The increase of clay concentration resulted in faster synthesis of AgNPs and nonuniform nanoparticles size with aggregation of some particles. For 2.5 wt% and 5 wt% of clay, the AgNPs exhibited more uniform size and mixture of intercalated/exfoliated structures. Nanocomposites based on lower molar mass chitosan Ch30 had predominant exfoliated structure.
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Fig. 7. TEM images and particle size distribution of nanocomposites (a-c) Ch30/10%SWy-2/AgNPs, (d-f) Ch30d/10%SWy-2/AgNPs, (g-i) Ch30-DEAE/10%SWy-2/AgNPs and (j-l) Ch30-DEAE-Dod/10%SWy-2/AgNPs.
For nanocomposites prepared using modified chitosans, the synthesis of AgNPs occurred quickly, in approximately 1.5 h, while for all other nanocomposite films it was above 11 h. The film of modified chitosan with dodecyl groups presented smaller and more uniform nanoparticles size along mixture of exfoliated and intercalated structures. This modified chitosan is an amphiphilic compound (containing hydrophilic and hydrophobic groups) that can act as a surfactant, controlling the size/shape of the AgNPs. The antibacterial activity of nanocomposite films was determined against the growth of E. coli and B. subtilis. Films prepared in the absence of AgNPs showed no inhibition zones, indicating that
AgNPs are responsible for the antibacterial activity. The presence of clay did not influence the bactericidal activity. The formation of inhibition zones suggested that all the films containing AgNPs prevented the growth of both bacteria. Acknowledgements This work was supported by CNPq (308940/2013-0 and 401434/2014-1) and J.S.G would like to thank CAPES and CNPq (200672/2015-0) for a graduate fellowship. The authors are grateful to Prof. Luc Avérous for assistance with antibacterial tests and
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