Antimicrobial activity of silver nanoparticles in situ growth on TEMPO

Antimicrobial activity of silver nanoparticles in situ growth on TEMPOmediated oxidized bacterial cellulose Jin Feng, Qingshan Shi, Wenru Li, Xiulin Shu, Aimei Chen, Xiaobao Xie & Xiaomo Huang Cellulose ISSN 0969-0239 Volume 21 Number 6 Cellulose (2014) 21:4557-4567 DOI 10.1007/s10570-014-0449-2

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Author's personal copy Cellulose (2014) 21:4557–4567 DOI 10.1007/s10570-014-0449-2

ORIGINAL PAPER

Antimicrobial activity of silver nanoparticles in situ growth on TEMPO-mediated oxidized bacterial cellulose Jin Feng • Qingshan Shi • Wenru Li • Xiulin Shu • Aimei Chen • Xiaobao Xie Xiaomo Huang

•

Received: 4 March 2014 / Accepted: 16 September 2014 / Published online: 27 September 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract In order to improve the antimicrobial activity of bacterial cellulose (BC), the silver nanoparticles (Ag NPs) were in situ fabricated on the BC membranes, affording BC and Ag hybrid antimicrobial materials, BC ? Ag, which possesses excellent antimicrobial performance. Typically, carboxyl groups were firstly introduced into BC by TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation. Then, the carboxyl-functionalized BC was performed with ion-exchange reaction to change the sodium ions into Ag? by immersing in AgNO3 aqueous solution, generating Ag? anchored BC. Finally, two types of distinct reductive reagents including NaBH4 and sodium citrate were employed to transform Ag? into Ag NPs to fabricate BC ? Ag. The diameters of Ag NPs were determined to be 3.8 nm for NaBH4-reduced BC ? Ag, and 22.0 nm for sodium citrate-reduced one, respectively. The silver content of BC ? Ag were determined to be 1.944 and 2.895 wt% for NaBH4-reduced sample and sodium citrate-reduced one, respectively. Two types

J. Feng Q. Shi (&) W. Li X. Shu A. Chen X. Xie X. Huang State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangdong Institute of Microbiology, Guangzhou 510070, Guangdong, People’s Republic of China e-mail: [email protected]

of BC ? Ag both showed a slow and persistent Ag? release profile, but the NaBH4-reduced one released much more Ag? than that of sodium citrate under the same measurement condition. In-depth antibacterial analysis via the disc diffusion and colony forming count method disclosed that BC ? Ag exhibited strong bactericidal effects against both Escherichia coli and Staphylococcus aureus. And the antibacterial activity of NaBH4-reduced BC ? Ag was higher than the sodium citrate-reduced one. Overall, this study would further improve the antibacterial efficiency of BC ? Ag. Keywords Bacterial cellulose Silver nanoparticles TEMPO Selective oxidation Antimicrobial activity Abbreviations BC Bacterial cellulose TEMPO 2,2,6,6- tetramethylpiperidine-1oxyl radical BC ? Ag Silver nanoparticles on carboxylfunctionalized BC composites ROS Reactive oxygen species DP Degree of polymerization E. coli Escherichia coli S. aureus Staphylococcus aureus SH Schenk and Hildebrandt medium SEM Scanning electron microscopy XRD X-ray diffraction

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ICP-MS FTIR TEM LB SPR

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Inductively coupled plasma mass spectrometry Fourier transform infrared Transmission electron microscopy Luria–Bertani medium Surface plasmon resonance

Introduction Silver and its complex materials were well-accepted to present strong inhibitory and bactericidal effects as well as a broad spectrum of antimicrobial activities (Carr et al. 1973; Pana´cˇek et al. 2006; Holladay et al. 2006; Marambio-Jones and Hoek 2010). Some research demonstrated that elemental silver and silver compounds in aqueous solution could release silver ions, which resulted in their specific bactericidal functions (Sanpui et al. 2008). Some other work was endeavored to elucidate the interactions of silver ions with three main components of the bacterial cell including the peptidoglycan cell wall and the plasma membrane (Li et al. 2010); bacterial DNA (Pal et al. 2007); and bacterial proteins especially enzymes involved in vital cellular processes such as the electron transport chain (Pal et al. 2007; Li et al. 2011), which probably led to the bactericidal effect of silver. In addition, silver nanoparticles (Ag NPs) were found to present stronger antibacterial effects than free silver ions (Choi et al. 2008). Further exploration revealed that the intrinsic antibacterial effects of Ag NPs were not only derived from the elution and diffusion of Ag? (Chaloupka et al. 2010), the physical interactions between Ag NPs and bacteria cell walls were proposed to cause the cellular lysis of bacteria (Morones et al. 2005). The substantial evidence showed that Ag NPs could mediate the generation of reactive oxygen species (ROS), which might explain the bactericidal behavior of Ag NPs (Su et al. 2009). Recently, hybrid polymeric materials fabricated from Ag NPs and polymers have attracted much attention, due to their potential applications in electronic, catalytic, antimicrobial agents (Kong and Jang 2006; He et al. 2009). In the past few years, natural polymers have intrigued extensive research interest for their potential applications in biomedical materials and devices due to their native advantages over their synthetic polymer

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counterparts. Among these, bacterial cellulose (BC), one of the most promising biopolymers, was synthesized by genera Gluconacetobacter (Petersen and Gatenholm 2011; Czaja et al. 2006). BC is significantly different from plant cellulose. Firstly, BC is entirely free of lignin and hemicelluloses. Secondly, BC possesses natural refined three dimensional nanofibrils network (Czaja et al. 2007). Therefore, it displays several unique properties, including high porosity, water-holding capability, water absorption, mechanical properties, and biocompatibility which makes BC suitable for modern wound dressing based (Czaja et al. 2006). Recently, many efforts have been devoted to the fabrication of Ag NPs on BC membranes. In a typical process, Ag NPs were synthesized by the in situ formation of Ag? on BC membrane by immersing it into AgNO3 solution, followed by and then in situ reduction with reducing agents (Maneerung et al. 2008; Yang et al. 2012; Barud et al. 2011). However, the binding interactions between Ag? and BC fibers were observed to be too weak, resulting in low yield and sparse distribution of Ag NPs on BC fibers. Therefore, a stronger interaction between Ag? and cellulose fibers was the pressing need to prepare densely immobilized silver nanoparticles with higher yield (Ifuku et al. 2009; Cao et al. 2013). Typically, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) has been extensively utilized to introduce aldehyde and carboxyl functional groups into polysaccharides (Saito et al. 2006). The C6 primary hydroxyl groups of cellulose are oxidized to carboxyl functionalities by the TEMPO-mediated oxidation (Isogai et al. 2011b). There are two types of TEMPO-mediated oxidation systems: one as TEMPO/NaBr/NaClO system under weakly alkaline condition, and the other as TEMPO/NaClO/NaClO2 system under weakly acidic condition (Saito et al. 2005, 2010). TEMPO-oxidized wood celluloses by TEMPO/NaClO/NaClO2 in water at pH 6.8 has higher tensile strength than those prepared from TEMPO/NaBr/NaClO oxidation in water at pH 10, which results from the higher DP (degree of polymerization) of the former one (Saito et al. 2009). Additionally, the crystallinity indices and the crystal sizes of cellulose kept constant upon the TEMPOmediated oxidation (Isogai et al. 2011a). Up to now, some strategies were explored concerning the fabrication of Ag NPs on TEMPO-mediated oxidized bacterial cellulose (Ifuku et al. 2009) and jute

Author's personal copy Cellulose (2014) 21:4557–4567

fibers (Cao et al. 2013). They employed TEMPO/ NaBr/NaClO system to oxidize the cellulose, but the antimicrobial exploration for the complex materials of Ag NPs on oxidized bacterial cellulose was still blank. In the current work, TEMPO/NaClO/NaClO2 system was selected to oxidize the BC to prepare carboxylfunctionalized BC. Then, Ag NPs were in situ reduced from the anchored Ag? within the matrix of carboxylfunctionalized BC by reducing agents, affording hybrid polymeric BC ? Ag. Finally, the structural characterization and antimicrobial activities of the resulting BC ? Ag were further investigated. We anticipate that this work would probably expand the research horizons of functionalized bacterial cellulose and further improve the antibacterial efficiency.

Experimental Materials Gluconacetobacter intermedius BC-41 was stocked in our laboratory. Escherichia coli (E. coli) ATCC 8739 and Staphylococcus aureus (S. aureus) ATCC 6538 were supplied by Guangdong Institute of Microbiology (Guangzhou, China). All chemical reagents were purchased from Shanghai Aladdin Chemical Regent Inc (China) and used as received. Production of BC G.intermedius BC-41 was routinely grown in Schenk and Hildebrandt (SH) medium, contains glucose 20 g/ L, peptone 5 g/L, yeast 5 g/L, Na2HPO412H2O 2.7 g/ L, citric acid 1.5 g/L, with pH 5.0. G.intermedius BC41 was maintained on SH agar slant for 3 days at 28 °C to get its activation and then inoculated into the 50 mL SH culture medium. And the inoculum was cultured for 24 h at 28 °C with rotation speed of 120 r/ min. After that, 10 mL of cell suspension was introduced into a 500-mL Erlenmeyer flask containing 100 mL of SH culture medium, and then incubated statically at 28 °C for 10 days. Purification of BC After incubation, the BC membrane had been produced on the surface of the liquid culture broth. The BC membrane was collected and flushed for twice

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with distilled water to remove the residual medium. And then the BC membrane was dipped into 0.1 M NaOH aqueous solution and heated at 100 °C water bath until the membrane became milky translucent. Subsequently, the BC membrane was washed by distilled water repeatedly until the pH value of the washing liquid became neutral. TEMPO-mediated oxidation of BC The BC membrane (wet weight 11 g, U = 9 cm) was suspended in 0.1 M sodium phosphate buffer (90 mL, pH 6.8) dissolving TEMPO (0.016 g) and sodium chlorite (80 %, 1.13 g) in an airtight flask. And the flask was shaken at 40 °C for 30 min. Then, 1 mL sodium hypochlorite solution (ClO-: 7.5 %) was added at one step to the flask. The flask was immediately stoppered, and then shaken at 40 °C for 5 h. After the oxidation, the reaction was quenched by the addition of 5 mL of ethanol, followed by shaking for another 30 min. The oxidized BC was washed thoroughly with distilled water. Subsequently, the carboxyl-functionalized BC was dipped into 0.05 M NaOH aqueous solution and shaken for 1 h. After that, the oxidized BC was rinsed completely with distilled water until the pH of the washing liquid was neutral and finally immersed in the distilled water prior to use. (Saito et al. 2010). Preparation of BC ? Ag The fabrication of BC ? Ag was achieved by immersing carboxyl-functionalized BC into 50 mL of 0.001 M AgNO3 solution at 40 °C and kept in dark overnight, followed by a thorough rinsing with distilled water. Finally, according to the following procedures, two types of reducing agents were employed to fabricate the resultant Ag NPs anchored BC, BC ? Ag: (a)

The Ag? modified-BC was reduced in an ice water bath of 0.1 M NaBH4 for 1 h. After that, the membrane was rinsed with a large amount of distilled water to remove the excess chemical residues and then freeze-dried. Proposed mechanism of the reaction process could be expressed as follows (Creighton et al. 1979): 2Agþ + 2NaBH4 + 6H2 O ! 2Ag0 + 2H3 BO3 + 2Naþ + 7H2 "

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(b)

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The Ag? tethered modified-BC was reduced in 100 mL distilled water and heated to boiling, then 1 mL sodium citrate (0.1 M) was added. The solution was kept on boiling for 1 h and then cooled to the room temperature. Subsequently, the membrane was washed with distilled water to remove the excess chemical agents and freeze-dried. Proposed mechanism of reaction process could be expressed as follows (Lee and Meisel 1982):

Bruker FTIR Equinox 55 spectrometer with an ATR attachment in the range of 400–4,000 cm-1 with a resolution of 4 cm-1.

4Agþ þ C6 H5 O7 Na3 þ 2H2 O ! 4Ag0

Transmission electron microscopy (TEM)

þC6 H5 O7 H3 þ 3Naþ þ Hþ þ O2 " :

Characterization Scanning electron microscopy (SEM) SEM analysis was performed using a Hitachi H-3000 N scanning electron microscope. The samples cut from the freeze-dried BC and carboxyl-functionalized BC were coated with gold film before analysis. X-ray diffraction (XRD) The membranes were cut into a square sample with a size of 2 cm 9 2 cm, and placed on a glass slide. XRD patterns were taken by a Rigaku D/max 2500v/ pc X-ray diffractometer using Cu Ka radiation (Ka = 0.15405 nm) at a scan rate of 0.5°/min, using a voltage of 36 kV and a current of 20 mA. Ag? exchange treatment with carboxyl-functionalized BC

UV–Vis UV–Vis spectra were recorded by a UV-5200 PC spectrophotometer (Shanghai Metash Instrument co Ltd., China).

Small pieces of undried BC ? Ag were milled into fine thin slices in liquid nitrogen. The samples were prepared by dropping 10 lL of fine thin slices BC ? Ag dispersions on a copper grid and dried at room temperature after removing excess solution using filter paper. TEM images were recorded by using a Hitachi H-7650 transmission electron microscope. The particle size of Ag NPs was measured by the image analysis of TEM micrograph using Image J software. The average size of Ag Nps was determined from the measurement of more than 150 particles. And the histogram of size distribution was established by Origin 7.5 software. (Yang et al. 2012; Nam and Condon 2014). The silver contents The silver content of BC ? Ag was quantified by ICPMS (Agilent 7700, Japan) with 0.1 g of sample dissolved in 10 M nitric acid. The release of silver ions

The carboxyl-functionalized BC was immersed in 50 mL AgNO3 solution (0.001 M) at 40 °C and kept in dark overnight, followed by a thorough rinsing with distilled water. The formed Ag? anchored BC was finally obtained by freeze-dried. Finally, 0.05 g Ag? anchored BC was added nitric acid. The content of Ag? was measured by an Agilent 7700 inductively coupled plasma mass spectrometry (ICP-MS).

The silver release profile from BC ? Ag was tested by dialysis experiments. The dialysis tubes (Spectra/Por Biotech; cellulose ester; MWCO 3500) filled with 0.05 g sample within 10 mL distilled water. The dialysis tube was then immersed in 100 mL distilled, and carried out under slow stirring with a magnetic stirrer at room temperature (Kittler et al. 2010). The concentration of Ag? was determined by ICP-MS (Agilent 7700, Japan).

Fourier transform infrared (FTIR) spectroscopy

Antimicrobial activity studies

The series of BC derivatives were freeze dried. And the IR spectra of all samples were obtained using a

Antimicrobial activities of BC ? Ag were investigated against E. coli ATCC 8739 as a model for Gram-

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negative bacteria and S. aureus ATCC 6538 as a model for Gram-positive bacteria. Two methods as followed were employed to evaluate the antimicrobial activities. The disc diffusion method It was performed in Luria-Bertani (LB) medium solid agar Petri dish. The sample membranes were cut into disk-liake pieces with a diameter of 1.0 cm and sterilized upon UV treatment for 1 h. After sterilization, the discs were placed on E. coli-cultured agar plate and S. aureus-cultured agar plate, which were incubated for 24 h at 37 °C. Clear zones of inhibition formed around the discs were measured. The colony forming count method The samples were dispensed into 5 mL steriled saline water (0.8 wt%) saline water containing about 105– 106 cfu/mL of E. coli or S. aureus (logarithmic phase cells grown), and then shaken at 37 °C. 1.0 mL of the suspension was taken out from the test tube after incubation for 24 h, and diluted 10-fold (to ensure the bacterial colonies grown could be counted easily and correctly). The diluted dispersion was plated on LB agar plates in triplicate and incubated at 37 °C for 24 h. The number of bacterial colonies on each plate was counted. The killing rate (g) is relative to the viable bacteria counts as follows: g ¼ YX Y 100%, where Y is the number of microorganism colonies on the control tube (a sterile 0.8 wt% saline water without sample) and X is the number of microorganism colonies on the samples (Ouyang et al. 2009).

Results and discussion

Fig. 1 SEM images of BC (a) and carboxyl-functionalized BC (b)

2014; Saito et al. 2009; Saito and Isogai 2004). The crystal sizes were nearly unchanged before and after the oxidation treatment. After the soaking treatment in AgNO3 solution, the silver content of Ag? modified carboxyl-functionalized BC was determined to be 3.6 wt%, whereas, it was just 1.26 wt% for the primitive BC due to the lack of anchor groups. It meant that the carboxyl-functionalized BC possessed higher affinity with Ag? than that of BC.

Characterization of carboxyl-functionalized BC Characterization of BC ? Ag SEM analyses were performed to investigate the morphology of the original BC and the carboxylfunctionalized BC. There is no significant difference between them (Fig. 1a, b). They both displayed a similar three-dimensional fiber network nanostructure with the width of fiber determined to be 40–80 nm. Figure 2 showed X-ray diffraction patterns of the BC before and after the TEMPO treatment. The results were similar and typical for the cellulose I. (French

FTIR spectra for a series of BC derivatives FTIR-ATR spectra of a series of BC derivatives were shown in Fig. 3. The BC had no obvious absorption band from 1,500 to 2,000 cm-1(Fig. 3a). As shown in Fig. 3b, the signal at 1,596 cm-1 attributing to the carbonyl groups occurred in consistent with the carboxyl-functionalized BC, which indicated that the

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Fig. 2 XRD patterns of BC (a) and carboxyl-functionalized BC (b)

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Fig. 4 UV–visible spectra recorded for hybrid BC ? Ag membranes prepared from NaBH4 (a) and sodium citrate (b)

reduced one, the signal slightly shifted from 1,596 cm-1 to 1,593 cm-1 (Fig. 3e). Thus, these results indicated some difference between Ag NPs reduced by NaBH4 and sodium citrate. UV–Vis

Fig. 3 FTIR spectra of BC (a), carboxyl-functionalized BC (b), carboxyl-functionalized BC upon incubation with AgNO3 (c), NaBH4-reduced BC ? Ag (d) and sodium citrate-reduced BC ? Ag (e)

hydroxyl groups at the C6 position of BC were successfully converted to carboxyl groups (Ifuku et al. 2009). Upon treatment with AgNO3, the sodium salt was changed to silver slat. The absorption band of carboxyl-functionalized BC treated with AgNO3 was still around 1,596 cm-1 (Fig. 3c). When Ag? was reduced into Ag NPs by NaBH4 or sodium citrate, the stretching vibration band for carbonyl groups was changed. For the NaBH4 reduced one, the absorption peak of carbonyl groups was slightly shifted from 1,596 to 1,601 cm-1 (Fig. 3d). For the sodium citrate

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The carboxyl-functionalized BC also has a distinctive 3D nanostructure with large pores. The majority of Ag? was readily penetrated into the oxidized BC through their pores. The absorbed Ag? were bound to oxidized BC via electrostatic interactions, the electron-rich oxygen atoms of polar carboxyl groups carboxyl-functionalized BC were expected to interact with electropositive transition metal cations. In this work, there were two different methods to reduce the silver ions into Ag NPs by sodium citrate or NaBH4. For the NaBH4 pathway, the original colorless membrane turned brown upon reduction treatment in aqueous solution of NaBH4. For the sodium citrate way, the color of BC ? Ag changed drastically to yellow after sodium citrate treatment. As shown in Fig. 4, typical absorption peaks for Ag NPs were observed for the resultant BC ? Ag, which was related to the surface plasmon resonance (SPR) of conducting electron (or free electron) on the surface of Ag NPs (Lu et al. 2009). The maxima were observed at 392 nm for the NaBH4-reduced sample and at 416 nm for the sodium citrate-reduced sample. The red-shift and broadening of absorption probably resulted from the increase of particle size and distribution for Ag NPs (Lu et al. 2009).


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Fig. 5 TEM images and particle size distribution histograms for NaBH4-reduced BC ? Ag (a, c) and sodium citrate-reduced BC ? Ag (b, d)

Electron microscopic analysis The size and size distribution of Ag NPs formed on the carboxyl-functionalized BC were analyzed by transmission electron microscopy (TEM) (Figs. 5a, b), and the histogram based on the TEM images further illustrated their average size and size distribution (Figs. 5c, d). Well dispersed and regular spherical Ag NPs were obtained via the NaBH4-reduced pathway. The diameter (d) and standard deviation (r) were estimated to be 3.8 nm and 2.7 nm, respectively. (Figures 5a, c) As shown in Figs. 5b, d, spherical Ag

NPs with much larger size (d = 22.0 nm) and wider distribution (r = 14.0 nm) were obtained via the sodium citrate-reduced profile. These differences concerning the formed Ag NPs agreed with the results of UV–Vis absorption spectra. Thus, we can envisage that the reduction ability of reducing agents significantly influenced the nucleation rate of Ag NPs within functionalized BC membrane. The size of Ag NPs obtained by the NaBH4-reduced pathway was much smaller than that obtained by many conventional methods (Maneerung et al. 2008; Yang et al. 2012; Ifuku et al. 2009).

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Fig. 6 Cumulative Ag? release profile for BC ? Ag prepared from NaBH4 (a) and sodium citrate (b)

Determination of silver contents The silver contents of BC ? Ag were determined by elemental analysis using ICP-MS. The silver content of NaBH4-reduced sample is 1.944 wt%, and the data for sodium citrate-reduced sample is 2.895 wt%. Combined with the analysis of particle size and the silver loading content, we found that the Ag NPs within BC ? Ag prepared from the NaBH4-reduced sample exhibited smaller size and relatively lower silver loading content. The relatively lower content of NaBH4-reduced Ag NPs may possible result from their smaller size. The smaller size of Ag PNs was easier washed away, when the distilled water removed the excess chemicals in BC ? Ag preparation process. In this study, the silver loading content of BC ? Ag was higher than no modified BC (Yang et al. 2012). The release of silver ions To interrogate the silver release profile from hybrid BC ? Ag, the dialysis experiments were conducted. Previous research found that the silver release was just in the form of Ag? but not Ag NPs (Kittler et al. 2010). As displayed in Fig. 6, the release rate of NaBH4reduced sample was much faster than that of sodium citrate-reduced one. In the initial 3 days, Ag? diffused out of the dialysis tube quickly. Up to 16 days, the cumulative Ag? release for the NaBH4-reduced sample was 3.3 %, which was much larger than 0.75 % for the sodium citrate-reduced sample due to larger

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Fig. 7 Photographs of the inhibition zone of the samples against E. coli and S. aureus (a: BC, b: carboxyl-functionalized BC, c: NaBH4-reduced BC ? Ag, d: sodium citrate-reduced BC ? Ag)

specific surface area for Ag NPs with smaller size (Martinez-Castanon et al. 2008). The majority of Ag? came from the oxidation of the zero-valent Ag NPs, by the typical reaction with dissolved O2, mediated by protons and other components in the surrounding fluid phase. [Eqs. (1), (2)] (Liu and Hurt 2010):

Author's personal copy Cellulose (2014) 21:4557–4567 Table 1 The antibacterial activity of the samples

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Strains

Samples

Sample concentrations (mg/mL)

The viable colonies Killing of bacteria after 24 h ratio (%) contact (cfu/mL)

E. coli

Blank

0

1.41 9 106

–

BC

2,000

1.47 9 107

–

Carboxyl-functionalized BC

2,000

8.70 9 106

–

NaBH4-reduced BC ? Ag

250

1

[99.99

Sodium citrate-reduced BC ? Ag

250

4

S. aureus

[99.99 4

Blank

0

2.1 9 10

–

BC

2,000

1.29 9 106

–

Carboxyl-functionalized BC

2,000

3.13 9 105

–

NaBH4-reduced BC ? Ag

1,000

4.07 9 102

98.06

Sodium citrate-reduced BC ? Ag

1,000

5.33 9 103

74.62

Hþ

Ag0 þ O2 ! Ag O2 ! Agþ þ reactive oxygen intermediates

ð1Þ

Hþ

Reactive oxygen intermediates þ Ag0 ! Agþ þ H2 O ð2Þ ?

Unfortunately, the total release content of Ag was not high due to the following two proposed processes: the cellulose fibers with nanostructural networks acted as physical barriers to impede the Ag? diffusion out of the membrane; meanwhile, the carboxyl groups within carboxyl-functionalized BC could probably capture free Ag? again to reduce the release content. Antimicrobial activity studies The antibacterial activities of BC ? Ag against E. coli and S. aureus were evaluated by the disc diffusion method and the colony forming count method. As demonstrated in Fig. 7, the photographs for the inhibition zone of BC, carboxyl-functionalized BC, NaBH4reduced BC ? Ag and sodium citrate-reduced sample against E. coli and S. aureus were presented respectively. No inhibition zone for two types of bacterial strains was observed for BC and carboxyl-functionalized BC, which demonstrated their good biocompatibility. Whereas for BC ? Ag, they showed obvious antibacterial effects. The width of the inhibition zone for the NaBH4-reduced sample was about 6.1 mm for E. coli, and 7.7 mm for S. aureus, however, for the sodium citrate-reduced sample, it was 3.7 and 3.2 mm respectively. Further quantitation analysis was performed for the samples upon 24 h incubation with bacteria as shown

in Table 1. There was no obvious bactericidal activity for BC and carboxyl-functionalized BC according to the viable counts. It should be noted that the killing rate of NaBH4-reduced BC ? Ag was slightly higher than that of sodium citrate-reduced sample. The NaBH4-reduced sample released much more Ag? than that of sodium citrate-reduced sample, meanwhile, the smaller size Ag NPs with larger surface area would be favorable for the interaction between Ag NPs and bacteria, affording higher bactericidal effect than the larger size Ag NPs (Martinez-Castanon et al. 2008). Based on above two evaluation methods, BC ? Ag exhibited good antimicrobial activity for both E. coli (Gram-negative) and S. aureus (Grampositive). The antimicrobial activity against S. aureus was lower than that against E.coli, which was probably due to the lower interaction efficacy of Ag NPs against S. aureus for the difference of membrane structure.

Conclusions In summary, the fabrication of Ag NPs on carboxylfunctionalized BC was achieved, affording hybrid BC ? Ag membranes. Firstly, BC was treated with TEMPO/NaClO2/NaClO to oxidize the C6 hydroxyl groups of BC into carboxyl groups. Followed by the ion exchange reaction, the carboxyl-functionalized BC membranes were tightly modified with Ag?. Finally, Ag NPs were in situ reduced within the carboxyl-functionalized BC membranes via two types of distinct reductive reagents, NaBH4 or sodium citrate, forming hybrid BC ? Ag membranes. The

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average diameter of Ag NPs for the NaBH4-treated sample was determined to be 3.8 nm, and the silver loading content was 1.944 wt%; for the sodium citrate-reduced sample, it was 22.0 nm and 2.895 wt%, respectively. Two kinds of hybrid BC ? Ag membranes both displayed a slow and persistent Ag? release profile. But, they still exhibited significantly efficient antimicrobial activities against two kinds of bacterial strains, E. coli and S. aureus. The hybrid BC ? Ag membranes described in this work would be promising in the application of antimicrobial dressing and coating. Acknowledgments This work was funded by the Cooperation Projects of Foshan City and Chinese Academy (No. 2012HY100115), Strategic Cooperation Projects Guangdong Province and Chinese Academy (No. 2011B090300018), the Scientific and Technological Project of Guangdong Province (No. 2011B010400039, 2011B070500020) and the Scientific and Technological Project of Guangzhou City (No. 11A24060559).

References Barud HS, Regiani T, Marques RF, Lustri WR, Messaddeq Y, Ribeiro SJ (2011) Antimicrobial bacterial cellulose-silver nanoparticles composite membranes. J Nanomater 2011:10 Cao X, Ding B, Yu J, Al-Deyab SS (2013) In situ growth of silver nanoparticles on TEMPO-oxidized jute fibers by microwave heating. Carbohydr Polym 92:571–576 Carr HS, Wlodkowski TJ, Rosenkranz HS (1973) Silver sulfadiazine: in vitro antibacterial activity. Antimicrob Agents Chemother 4:585–587 Chaloupka K, Malam Y, Seifalian AM (2010) Nanosilver as a new generation of nanoproduct in biomedical applications. Trends Biotechnol 28:580–588 Choi O, Deng KK, Kim N-J, Ross L Jr, Surampalli RY, Hu Z (2008) The inhibitory effects of silver nanoparticles, silver ions, and silver chloride colloids on microbial growth. Water Res 42:3066–3074 Creighton JA, Blatchford CG, Albrecht MG (1979) Plasma resonance enhancement of Raman scattering by pyridine adsorbed on silver or gold sol particles of size comparable to the excitation wavelength. J Chem Soc Faraday Trans 2 Mol Chem Phys 75:790–798 Czaja W, Krystynowicz A, Bielecki S, Brown RM Jr (2006) Microbial cellulose—the natural power to heal wounds. Biomaterials 27:145–151 Czaja WK, Young DJ, Kawecki M, Brown RM (2007) The future prospects of microbial cellulose in biomedical applications. Biomacromolecules 8:1–12 French AD (2014) Idealized powder diffraction patterns for cellulose polymorphs. Cellulose 21:885–896 He D, Hu B, Yao Q-F, Wang K, Yu S-H (2009) Large-scale synthesis of flexible free-standing SERS substrates with high sensitivity: electrospun PVA nanofibers embedded

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Cellulose (2014) 21:4557–4567 with controlled alignment of silver nanoparticles. ACS Nano 3:3993–4002 Holladay RJ, Christensen H, Moeller WD (2006) Treatment of humans with colloidal silver composition. US Patents Ifuku S, Tsuji M, Morimoto M, Saimoto H, Yano H (2009) Synthesis of silver nanoparticles templated by TEMPOmediated oxidized bacterial cellulose nanofibers. Biomacromolecules 10:2714–2717 Isogai A, Saito T, Fukuzumi H (2011a) TEMPO-oxidized cellulose nanofibers. Nanoscale 3:71–85 Isogai T, Saito T, Isogai A (2011b) Wood cellulose nanofibrils prepared by TEMPO electro-mediated oxidation. Cellulose 18:421–431 Kittler S, Greulich C, Diendorf J, Koller M, Epple M (2010) Toxicity of silver nanoparticles increases during storage because of slow dissolution under release of silver ions. Chem Mater 22:4548–4554 Kong H, Jang J (2006) One-step fabrication of silver nanoparticle embedded polymer nanofibers by radical-mediated dispersion polymerization. Chem Commun 28:3010–3012 Lee P, Meisel D (1982) Adsorption and surface-enhanced Raman of dyes on silver and gold sols. J Phys Chem 86:3391–3395 Li W-R, Xie X-B, Shi Q-S, Zeng H-Y, Ouyang Y-S, Chen Y-B (2010) Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli. Appl Microbiol Biotechnol 85:1115–1122 Li W-R, Xie X-B, Shi Q-S, Duan S-S, Ouyang Y-S, Chen Y-B (2011) Antibacterial effect of silver nanoparticles on Staphylococcus aureus. Biometals 24:135–141 Liu J, Hurt RH (2010) Ion release kinetics and particle persistence in aqueous nano-silver colloids. Environ Sci Technol 44:2169–2175 Lu X, Rycenga M, Skrabalak SE, Wiley B, Xia Y (2009) Chemical synthesis of novel plasmonic nanoparticles. Annu Rev Phys Chem 60:167–192 Maneerung T, Tokura S, Rujiravanit R (2008) Impregnation of silver nanoparticles into bacterial cellulose for antimicrobial wound dressing. Carbohydr Polym 72:43–51 Marambio-Jones C, Hoek EM (2010) A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J Nanopart Res 12:1531–1551 Martinez-Castanon G, Nino-Martinez N, Martinez-Gutierrez F, Martinez-Mendoza J, Ruiz F (2008) Synthesis and antibacterial activity of silver nanoparticles with different sizes. J Nanopart Res 10:1343–1348 Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Ramı´rez JT, Yacaman MJ (2005) The bactericidal effect of silver nanoparticles. Nanotechnology 16:2346 Nam S, Condon BD (2014) Internally dispersed synthesis of uniform silver nanoparticles via in situ reduction of [Ag (NH3) 2]? along natural microfibrillar substructures of cotton fiber. Cellulose. doi:10.1007/s10570-014-0270-y Ouyang Y, Xie Y, Tan S, Shi Q, Chen Y (2009) Structure and antibacterial activity of Ce3? exchanged montmorillonites. J Rare Earths 27:858–863 Pal S, Tak YK, Song JM (2007) Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli. Appl Environ Microbiol 73:1712–1720

Author's personal copy Cellulose (2014) 21:4557–4567 Pana´cˇek A, Kvitek L, Prucek R, Kolar M, Vecerova R, Pizurova N, Sharma VK, Nevecˇna´ TJ, Zboril R (2006) Silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity. J Phys Chem B 110:16248–16253 Petersen N, Gatenholm P (2011) Bacterial cellulose-based materials and medical devices: current state and perspectives. Appl Microbiol Biotechnol 91:1277–1286 Saito T, Isogai A (2004) TEMPO-Mediated oxidation of native cellulose. The effect of oxidation conditions on chemical and crystal structures of the water-insoluble fractions. Biomacromolecules 5:1983–1989 Saito T, Shibata I, Isogai A, Suguri N, Sumikawa N (2005) Distribution of carboxylate groups introduced into cotton linters by the TEMPO-mediated oxidation. Carbohydr Polym 61:414–419 Saito T, Okita Y, Nge T, Sugiyama J, Isogai A (2006) TEMPOmediated oxidation of native cellulose: microscopic analysis of fibrous fractions in the oxidized products. Carbohydr Polym 65:435–440

4567 Saito T, Hirota M, Tamura N, Kimura S, Fukuzumi H, Heux L, Isogai A (2009) Individualization of nano-sized plant cellulose fibrils by direct surface carboxylation using TEMPO catalyst under neutral conditions. Biomacromolecules 10:1992–1996 Saito T, Hirota M, Tamura N, Isogai A (2010) Oxidation of bleached wood pulp by TEMPO/NaClO/NaClO2 system: effect of the oxidation conditions on carboxylate content and degree of polymerization. J Wood Sci 56:227–232 Sanpui P, Murugadoss A, Prasad P, Ghosh SS, Chattopadhyay A (2008) The antibacterial properties of a novel chitosan–Agnanoparticle composite. Int J Food Microbiol 124:142–146 Su H-L, Chou C-C, Hung D-J, Lin S-H, Pao I, Lin J-H, Huang F-L, Dong R-X, Lin J-J (2009) The disruption of bacterial membrane integrity through ROS generation induced by nanohybrids of silver and clay. Biomaterials 30:5979–5987 Yang G, Xie J, Hong F, Cao Z, Yang X (2012) Antimicrobial activity of silver nanoparticle impregnated bacterial cellulose membrane: effect of fermentation carbon sources of bacterial cellulose. Carbohydr Polym 87:839–845

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