Acs Biomac 7b01660

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Article
TEMPO-oxidized bacterial cellulose pellicle
with silver nanoparticles for wound dressing
Chun-Nan Wu, Shih-Chang Fuh, Shin-Ping Lin, Yen-Yi Lin,
Hung-Yueh Chen, Jui-Ming Liu, and Kuan-Chen Cheng
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01660 • Publication Date (Web): 15 Jan 2018
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Page 1 of 44 Biomacromolecules
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4 1 TEMPO-oxidized bacterial cellulose pellicle with
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6 2 silver nanoparticles for wound dressing
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8 3 Chun-Nan Wu1#, Shih-Chang Fuh2#, Shin-Ping Lin3, Yen-Yi Lin3, Hung-Yueh Chen1,
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10 4 Jui-Ming Liu2, and Kuan-Chen Cheng1,3,4*
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13 5 1
Graduate Institute of Food Science and Technology, National Taiwan University, 1, Sec 4,
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6 Roosevelt Rd., Taipei 10617, Taiwan.
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19 7 Division of Urology, Department of Surgery, Taoyuan General Hospital, Ministry of Health
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21 8 and Welfare, 1492, Chung-Shan Road, Taoyuan District, Taoyuan 330 ,Taiwan.
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Institute of Biotechnology, National Taiwan University, 1, Sec 4, Roosevelt Rd., Taipei
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26 10 10617, Taiwan.
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30 11 Department of Medical Research, China Medical University Hospital, China Medical
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32 12 University, 91, Hsueh-Shih Rd., Taichung 40402, Taiwan.
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35 13 # Chun-Nan Wu and Shih-Chang Fuh contributed equally to this work.
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Biomacromolecules Page 2 of 44
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3 15 KEYWORDS: antibacterial, wound dressing, silver nanoparticle, TEMPO, bacterial
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5 16 cellulose
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8 17 ABSTRACT: Biocompatible bacterial cellulose pellicle (BCP) is a candidate for biomedical
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18 material such as wound dressing. However, due to lack of antibacterial activity, to grant BCP
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13 19 with the property is crucial for its biomedical application. In the present study, BCP was
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15 20 modified by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation using
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17 21 TEMPO/NaClO/NaBr system at pH 10 to form TEMPO-oxidized BCP (TOBCP) with
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19 22 anionic C6 carboxylate groups. The TOBCP was subsequently ion-exchanged in AgNO3
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21 23 solution and silver nanoparticles (AgNP) with diameter of ~16.5 nm were in situ synthesized
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24 on TOBC nanofiber surfaces by thermal reduction without using a reducing agent. FE-SEM,
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26 25 XRD, XPS, FTIR, and TGA were carried out to confirm morphology and structure of the
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28 26 pellicles with AgNP. The AgNP continuously released Ag+ with a rate of 12.2%/day at 37°C
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30 27 in 3 days. The TOBCP/AgNP exhibited high biocompatibility according to the result of in
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32 28 vitro cytotoxicity test (cell viability > 95% after 48 h of incubation) and showed significant
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34 29 antibacterial activities of 100% and 99.2% against E. coli and S. aureus, respectively. Hence,
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30 the highly biocompatible and highly antibacterial TOBCP/AgNP prepared in the present
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39 31 study is a promising candidate for wound dressing.
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1
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3 33 INTRODUCTION
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6 34 Wound dressing is an important biomedical material used to protect wound and promote
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8 35 its healing. The wound dressing market is growing rapidly in the present healthcare system
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36 worldwide due to increasing chronic diseases.1,2 In general, an ideal wound dressing shows
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13 37 some biomedical properties such as biocompatibility, providing a moist environment,
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15 38 absorbing exudates, and high mechanical strengths. Bacterial cellulose pellicle (BCP) is a
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17 39 good candidate as functional wound dressing due to satisfaction with these unique
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19 40 properties.3,4
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22 41 Bacterial cellulose (BC) is the representative cellulose synthesized by microbes. It is
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42 mainly produced by Gluconacetobacter and has an appearance of white pellicle with an
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27 43 extremely high moisture content of ~99%.5 A pellicle is a wet biofilm that assembles
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29 44 surrounding cells at the air-liquid interface in a standing liquid culture.6,7 BCP has a unique
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31 45 microstructure of 3D network consisting of cellulose nanofibers cross-linked by numerous
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33 46 hydrogen bonds, resulting in high water retention capacity and high mechanical strengths.5,8
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35 47 When BCP is used as a wound dressing with some functional components, it could be a
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48 barrier to wound infection and advantageous for delivering drugs to an injured site.3 BCP has
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40 49 been commercially available as chronic wound dressing and temporary artificial skin.3,9,10
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42 50 Although BCP shows many superior properties described above, it lacks of antibacterial
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44 51 activity and thus could not decrease the possibility of bacterial infection disease such as
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46 52 bacterial sepsis for burns patients.11
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53 Silver ion (Ag+) was reported as an important component used for various antibacterial
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52 54 products due to its antibacterial effect and non-toxicity to human cells.12−14 It was, however,
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54 55 reported that high concentration of Ag+ may cause DNA damage response to mammalian
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3 56 cells.15 Silver nanoparticle (AgNP) has been demonstrated to gradually release Ag+ to inhibit
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5 57 bacterial growth in a moist environment at human body temperature.4,12−14,16,17 Therefore,
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7 58 when designing BCP containing AgNP as a biocompatible and antibacterial wound dressing,
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9 59 it is considered that surface-modified BCP nanofibers could absorb AgNP stronger, resulting
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60 in a controllable and smooth Ag+ release rate18.
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15 61 TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation is well
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17 62 known as a regioselective surface modification method which only oxidizes primary OH
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19 63 groups and is widely applied to the field of cellulose-based nanomaterials.19−25 By
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21 64 TEMPO-mediated oxidation, anionic carboxylate groups (–COO–Na+) are introduced to C6
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65 of cellulose nanofiber surfaces and thus adsorption of functional cationic ions (such as Ag+
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26 66 for antibacterial application) on the surfaces through ion-exchange can be achieved. Ifuku et
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28 67 al. used TEMPO/NaClO/NaBr system to prepare TEMPO-oxidized BCP (TOBCP) as a
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30 68 reaction template for AgNP synthesis26. The study focused on obtainment of AgNP and the
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32 69 TOBCP was only used as a template for synthesizing AgNP without describing their further
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34 70 application.
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71 In the present study, we prepared a novel wound dressing of TOBCP/AgNP, which
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40 72 possessed both high biocompatibility and high antibacterial activity. Characterization of the
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42 73 pellicles obtained after each preparation step was described as much as possible, including
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44 74 appearance, water retention value, FE-SEM images, XRD patterns, XPS spectra, FTIR
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46 75 spectra, TGA curves, Ag+ release behavior, in vitro cytotoxicity, and antibacterial activity
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48 76 tests. In particular, to our knowledge, cytotoxicities of TOBCP and TOBCP/AgNP have not
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77 been clearly reported to date although BCP is well-known biocompatible. These important
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3 78 results are helpful to development for not only antibacterial wound dressing but also other
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5 79 new antibacterial products for biomedical and hygiene applications in the future.
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8 80 EXPERIMENTAL SECTION
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12 81 Materials.
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15 82 The Gluconacetobacter xylinus ATCC700178, Escherichia coli, and Staphylococcus
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17 83 aureus strains, and NIH3T3 cells were obtained from the Bioresource Collection and
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19 84 Research Center (Hsinchu, Taiwan). TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)
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21 85 was purchased from Sigma-Aldrich (St Louis, MO, USA). NaBr, 2 M NaClO solution, and
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86 AgNO3 were purchased from Wako Pure Chemicals (Osaka, Japan). All the chemicals were
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26 87 of laboratory grade and used as received. All the water used in the present study was double
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28 88 distilled.
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31 89 Production of BCP.
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34 90 Bacterial cellulose pellicles (BCP) were used as the starting materials for wound
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91 dressings and prepared according to the previously reported method.27 In brief, a cell
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39 92 suspension of G. xylinum (0.12 mL) was added to a CSL-Fru (corn steep liquor with fructose)
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41 93 medium (20 mL) in a Petri dish and incubated at 28°C for 10 days. A BCP with a diameter of
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43 94 9 cm formed at the interface between the medium and air was taken off from the dish. For
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45 95 purification, the obtained BCP was immersed in a 1.0 M NaOH solution at 80°C several
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47 96 times to remove the bacteria and impurity until the BCP became visibly white. The white and
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97 transparent BCP was further washed by tap water for 3 days to fully remove the residual
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52 98 impurity and followed by storage in water at 4°C.
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13 102 TEMPO-mediated oxidation of BCP.
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16 103 In the present study, the oxidation strategy was designed to combine high carboxylate
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18 104 content and to maintain the superior structure of the original BCP for application of wound
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20 105 dressing. A BCP was freeze-dried for 2 days and then cut into pieces 15 mm × 15 mm in size.
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106 The freeze-dried BCP pieces (~0.2 g cellulose) and 300 mL water were added to a 500 mL
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25 107 beaker. TEMPO (0.018 g) and NaBr (0.1 g) were added to the beaker and the solution was
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27 108 gently agitated for 1 h. The TEMPO-mediated oxidation was triggered by adding NaClO
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29 109 solution (10 mmol/g cellulose) to the solution. The pH value of the oxidation system was
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31 110 maintained at 10 by adding a 0.5 M NaOH solution using an autotitrator (836 Titrando;
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33 111 Metrohm, Herisau, Switzerland). The oxidation was for 1, 2, 4, or 8 h to determine an
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35 112 appropriate oxidation time for further preparation of wound dressing. For each oxidation time,
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113 the reaction was quenched by adding ethanol until the pH value of the system was not
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40 114 changed. The TEMPO-oxidized BCP (TOBCP, Figure 1a) were washed with water 10 times.
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42 115 The carboxylate group contents of the TOBCP treated for 1, 2, 4, and 8 h were determined to
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44 116 be 1.1, 1.4, 1.6, and 1.8 mmol/g cellulose, respectively, by electric conductivity titration with
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46 117 a 0.05 M NaOH solution. TOBCP treated for 1 h was selected as the starting material for
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48 118 preparing wound dressing due to its well-maintained structure as the original BCP (Figure 1b,
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119 cut into 15 mm × 15 mm in size). It is noticed that the C6 carboxylate groups of the TOBCP
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3 120 were –COO–Na+ and these pellicles were not specially coded as TOBCP/Na+ in the present
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5 121 study.
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8 122 In situ synthesis of silver nanoparticles on the TOBC nanofibers.
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12 123 To exchange sodium ion at the C6 carboxylate groups with silver ion (Ag+), the TOBCP
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14 124 were immersed in AgNO3 (1.5 mmol Ag+/g cellulose) solution in the dark for 24 h. The
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16 125 obtained products were washed with water at least 10 times to remove residue ions and coded
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18 126 as TOBCP/Ag+ (Figure 1b), which expressed the C6 silver carboxylate groups (–COO–Ag+)
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20 127 on the TOBC nanofiber surfaces. Thermal reduction of the TOBCP/Ag+ was carried out by
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22 128 placing the pellicles in a water bath at 100°C for 2 h. The color of the TOBCP/Ag+ gradually
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129 became deep yellow with increasing reduction time, indicating formation of silver
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27 130 nanoparticles (AgNP) in the pellicles. These TOBCP containing AgNP formed on the TOBC
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29 131 nanofiber surfaces were coded as TOBCP/AgNP (Figure 1b).
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32 132 Characterization of the pellicles.
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35 133 Water retention. Water retention values (WRV) of the pellicles were calculated using the
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134 following equation:
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41 135 WRV (%) = (Ww – Wd) / Wd × 100
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44 136 where Ww and Wd are weights of the wet and freeze-dried (for 2 days) pellicles, respectively.
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47 137 Microstructural observation. Field emission scanning electron microscope (FE-SEM)
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138 images of the inner surfaces of the pellicles and AgNP were observed using a JSM-7800F
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52 139 Prime instrument (JEOL, Tokyo, Japan). The pellicle samples were coated with platinum
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54 140 using an auto fine coater (JEC-3000FC, JEOL, Tokyo, Japan) at 10 mA for 2 min. Diameter
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3 141 distribution of the AgNP was estimated by measuring diameters of 200 AgNP with the
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5 142 FE-SEM images at 200k magnification.
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8 143 Structural analyses. X-ray diffraction (XRD) patterns of the pellicles and the pellicle
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144 powders obtained by ball milling the pellicles were recorded using an X-ray diffractometer
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13 145 (Ultima IV, Rigaku, Tokyo, Japan) in reflection mode with monochromator-filtered Cu Kα
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15 146 radiation (λ 0.15418 nm) at 40 kV and 40 mA. X-ray photoelectron spectra (XPS) were
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17 147 obtained using an XPS spectrometer (Theta Probe, Thermo Scientific, UK) with
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19 148 monochromated Al Kα radiation at 20.0 eV pass energy. Fourier transform infrared (FTIR)
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21 149 spectra of the pellicle powders obtained by ball milling the pellicles were recorded using a
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150 FTIR spectrometer (Spectrum 100, Perkin Elmer, Shelton, USA) in transmission mode from
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26 151 400 to 4,000 cm-1 in wavenumber with a resolution of 4 cm-1. Thermogravimetric analysis
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28 152 (TGA) of the pellicles was performed using a thermogravimetric analyzer (TGA Q50, TA
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30 153 Instruments, New Castle, DE, USA) at 10°C min-1 from room temperature to 800°C in a
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32 154 nitrogen atmosphere.
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35 155 Ag+ release behavior. To investigating Ag+ release behavior, three TOBCP/AgNP were
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156 added to a 50 mL centrifuge tube containing 50 mL water and soaked at 37°C. The pellicles
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40 157 were taken from the tube and soaked in another tube containing 50 mL fresh water at 37°C
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42 158 every 24 h. The treatment was repeated for 16 days and the soaking solution obtained
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44 159 everyday was stored at 4°C. The Ag+ concentrations of the solutions were analyzed using an
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46 160 inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 8000, Perkin
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48 161 Elmer, Shelton, USA) and calculated to be cumulative release rates.
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52 162 In vitro cytotoxicity test.
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3 163 The NIH3T3 cells were incubated in Dulbecco’s modified Eagle's medium (DMEM)
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5 164 containing 10% fetal bovine serum (FBS) at 37°C in 5% CO2/95% air. The cells were
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7 165 subcultured every 3 days to refresh the medium and adjust the cell density to ~1.2 × 105
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9 166 cell/mL by trypsinization with phosphate-buffered saline (PBS) and 0.25% trypsin/DMEM.
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167 To evaluate the cytotoxicity of the pellicles, the NIH3T3 cells (200 µL) were seeded in a
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14 168 96-well tissue culture plate with an initial cell density of ~1.2 × 105 cell/mL per well. The
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16 169 sterilized pellicles were extracted by immersion of the pellicles (thickness ~1 mm) in DMEM
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18 170 at a 1.25 cm2/mL extraction ratio at 37°C for 24 h according to ISO 10993-12. The well
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20 171 where NIH3T3 cells incubated in the 10% FBS/DMEM all the time was used as the negative
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22 172 control. After 24 h, the medium in the 96-well plate was replaced by fresh medium
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173 containing the pellicle extracts followed by incubation for 24 h. After 48 h, the same
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27 174 procedure was repeated. The cytotoxicity of the pellicles on the NIH3T3 cells was evaluated
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29 175 via cell viability obtained by water-soluble tetrazolium salts (WST-1) assay. For the NIH3T3
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31 176 cells incubated in the medium containing pellicle extracts for 24 or 48 h, the medium was
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33 177 replaced by 5% WST-1/DMEM and the 96-well plate was placed in the dark for 30 min. The
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35 178 optical density (OD) values at 450 nm of the reacted liquid were measured using a Multiskan
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179 GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, USA). For each
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40 180 treatment condition, the test was repeated 5 times.
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43 181 Antibacterial activity test.
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46 182 Antibacterial activities of the pellicles prepared in the present study were carried out by
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48 183 disc diffusion and colony forming count method.4 Both the methods were investigated against
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184 E. coli and S. aureus , which were used as Gram-negative and Gram-positive bacteria models,
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53 185 respectively. (1) Disc diffusion method. The experiment was performed using a Luria–
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3 186 Bertani (LB) medium solid agar Petri dish with a width of 90 mm. The pellicles were cut into
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5 187 9 mm in diameter and placed on E. coli-cultured and S. aureus -cultured agar plates at 37°C
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7 188 for 24 h. Volume and concentration of the bacterial suspension for each dish were 0.1 mL and
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9 189 approximately 1.5 × 107 CFU mL-1, respectively. After bacterial incubation, the formed
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190 growth inhibition zones were observed. (2) Colony forming count method. Three square
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14 191 pellicles 15 mm in width and 1 mL of bacterial suspension (~1 × 107 CFU mL-1) were added
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16 192 to a 50 mL centrifuge tube. The mixture was incubated at 100 rpm and 37°C for 24 h. The
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18 193 tube was then filled with sterilized saline and vortexed. The 50 µL of diluted bacterial
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20 194 suspension was drawn from the tube and spread on a LB medium solid agar Petri dish
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22 195 described as above for 24 h. After bacterial incubation, the colony count was observed and
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196 antibacterial activity of the pellicle was simply evaluated by percentage reduction of the
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27 197 colony count (%), which was calculated using the following equation:
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30 198 [(ColonyC − ColonyT) / ColonyC] × 100%
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33 199 where ColonyC and ColonyT are colony count of the control and treated group, respectively.
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200 RESULTS AND DISCUSSION
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40 201 Appearance of the pellicles.
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43 202 Figure 1a shows the effect of oxidation time on the structure of the TOBCP by
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45 203 TEMPO/NaClO/NaBr system at pH 10 and 10 mmol NaClO/g cellulose. Oxidation times and
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47 204 carboxylate group contents of the TOBCP from left to right were 1, 2, 4, 8 h, and 1.1, 1.4, 1.6,
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205 1.8 mmol/g cellulose, respectively. The TOBCP oxidized for 2 h was slightly more
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52 206 transparent than that oxidized for 1 h although the densities of the both pellicles were not
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54 207 significantly different. The result indicates the higher carboxylate group content resulted in
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3 208 higher repulsive forces between the TOBC nanofibers and higher swelling capacity in water.
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5 209 When the oxidation time was increased to 4 h, the densities of the more swollen TOBCP
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7 210 were much decreased, indicating its more unstable structure. Finally, the structure of the
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9 211 TOBCP oxidized for 8 h was significantly destroyed and only the sedimented fragments
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212 could be seen. Therefore, in the present study, the TOBCP oxidized for 1 h was used to
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14 213 prepare antibacterial wound dressings due to that the strong pellicle structure was completely
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16 214 maintained, showing sufficient mechanical resistance when used in a wet environment.
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18 215 Figure 1b shows the scheme of each treatment in the present study and their corresponding
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20 216 photos of the pellicles. The TOBCP was more transparent than the BCP because its structure
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22 217 was much swollen in water due to the carboxylate groups introduced on the BC nanofiber
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218 surfaces by TEMPO-mediated oxidation. The carboxylate groups were anionic and more
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27 219 hydrophilic than hydroxyl groups, resulting in repulsive forces between the BC nanofiber
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29 220 surfaces. Tollens’ reagent was used as a surface modifier to prepare a BCP wound dressing
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31 221 containing AgNP.11 By Ag(NH3)2+ ions in the Tollens’ reagent, the OH groups on the BC
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33 222 nanofiber surfaces were oxidized to aldehyde groups and further to carboxylate groups which
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35 223 are capable of stably absorbing the AgNP. Nevertheless, the OH groups on C2, C3, and C6 of
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224 the anhydroglucose units on the BC nanofiber surfaces were simultaneously oxidized. Due to
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40 225 C2-C3 cleavage occurring during oxidation, the pyranose rings were broken and thus the
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42 226 crystallinity and mechanical strengths of the BC nanofibers were decreased.18 Therefore, for
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44 227 development of antibacterial wound dressing with BCP and AgNP, a surface modification
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46 228 method combining oxidation of OH groups and maintenance of linear structure of cellulose
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48 229 chains is necessary. The appearance of the TOBCP/Ag+ obtained by ion-exchange of the
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230 TOBCP was almost the same with that of the TOBCP and thus mixing these two pellicles had
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53 231 to be avoided. The TOBCP/AgNP obtained by thermal reduction of the TOBCP/Ag+ for 2 h
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3 232 was deep yellow, indicating the formation of AgNP on the TOBC nanofiber surfaces.4,26 The
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5 233 color significantly became deeper with increasing reduction time due to the increased AgNP
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246 Figure 1. Photos of the pellicles prepared in the present study. (a) The TOBCP with different
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38 247 oxidation times in water. The carboxylate group contents of the TOBCP oxidized for 1, 2, 4,
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40 248 and 8 h were 1.1, 1.4, 1.6, and 1.8 mmol/g cellulose, respectively. (b) The scheme of the
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42 249 preparation process and the samples obtained after each step.
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3 251 Water retention value.
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6 252 High water retention value (WRV) is the unique and superior property of BCP as a
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8 253 wound dressing and thus the WRV of the pellicles obtained by each treatment (shown in
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254 Figure 2 and Table 1) had to be maintained. The BCP used as the starting material showed a
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13 255 high WRV of 169% due to its 3D network and large surfaces of the BC nanofibers. The
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15 256 WRV of the TOBCP (194%) was higher than that of the BCP because the carboxylate groups
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17 257 were more hydrophilic than hydroxyl groups. Interestingly, the TOBCP/Ag+ showed a WRV
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19 258 of 173% which was lower than that of the TOBCP perhaps due to that the ion diameter of
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21 259 Ag+ (126 pm, 1 pm (picometer) = 1 × 10-12 m)28 was larger than that of Na+ (102 pm)29,
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260 retarding attraction of water by carboxylate groups. The TOBCP/AgNP showed a WRV
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26 261 (169%) slightly lower than the TOBCP/Ag+ and the same as the BCP. The carboxylate
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28 262 groups of the TOBCP/AgNP was mainly –COO–Na+ form attracting AgNP by electrostatic
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30 263 forces (see Figure 6) because Ag+ were reduced to AgNP and the TOBCP/AgNP was
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32 264 immersed in a nutral solution. Compared to the TOBCP/Ag+, the –COO–Na+ form was more
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34 265 hydrophilic than the –COO–Ag+ form due to ionic radius effect described as above. However,
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266 the presence of AgNP retarded water molecules to be attracted by the carboxylate groups
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39 267 more than Ag+. It is considered that the effect of AgNP was higher than that of ionic radius
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41 268 and thus the TOBCP/AgNP showed the WRV slightly lower than the TOBCP/Ag+. The result
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43 269 indicates that not only the TOBCP/AgNP maintained the high WRV of the BCP but the
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45 270 TOBCP and TOBCP/Ag+ showed higher WRV than the BCP. Furthermore, all of these
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47 271 pellicles also had very high water contents >99%, which was higher than those of the
48
49 272 commercial wound dressings. It suggests that these pellicles might show high water vapor
50
51
52
273 transmission rates (WVTR) to meet requirement of wound dressing application.30
53
54
55
56 14
57
58
59
1
2
3 274
4 250
5
6 275
7
Water retention value (%)

200
8
9
276
10
150
11
12
13 277
100
14
15
16 278
17 50
18
19 279
20 0
21
22
280
23
24
25
26 281 Figure 2. Water retention values of the pellicles.
27
28
29 282
30
31
32 283 Microstructure observation.
33
34
35
284 Figure 3 shows the FE-SEM images of the BCP (Figure 3a, 3e, and 3i) , TOBCP (Figure
36
37
38 285 3b, 3f, and 3j), TOBCP/Ag+ (Figure 3c, 3g, and 3k), and TOBCP/AgNP (Figure 3d, 3h, and
39
40 286 3l) with different magnifications. Thicknesses and densities of the pellicles were
41
42 287 approximately 0.5 mm and 0.02 g cm-3, respectively. For all the treated pellicles, the unique
43
44 288 3D network consisting of cellulose nanofibers was well maintained as that of the BCP. It
45
46 289 indicates that all the treatments including TEMPO-mediated oxidation, iox-exchange, and
47
48
290 thermal reduction were appropriate for preparation of wound dressings with superior
49
50
51 291 microstructures. For the TOBCP/AgNP, the in situ synthesized AgNP on the TOBC
52
53 292 nanofibers were clearly observed (Figure 3l). The AgNP existed singly or formed a
54
55
56 15
57
58
59
1
2
3 293 aggregation where the each AgNP could be distinguished. The particle sizes and distribution
4
5 294 of the AgNP (shown in Figure 4d) were determined by measuring diameters of 200 AgNP
6
7 295 with the FE-SEM images. As a result, the FE-SEM images proved that for the TOBCP/AgNP,
8
9 296 the 3D network of BC nanofibers was maintained and the AgNP were successfully
10
11
12
297 synthesized on the TOBC nanofiber surfaces.
13
14
15 298
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56 16
57
58
59
1
2
3 299
4
5
6 300
7
8
9
301
10
11
12
13 302
14
15
16 303
17
18
19 304
20
21
22
305
23
24
25
26 306
27
28
29 307
30
31
32 308
33
34
35
309 Figure 3. FE-SEM images of the pellicles including BCP (a, e, j), TOBCP (b, f, j),
36
37
38 310 TOBCP/Ag+ (c, g, k), and TOBCP/AgNP (d, h, l). The arrows in Figure 3l indicated the in
39
40 311 situ synthesized AgNP.
41
42
43 312 Structural analyses.
44
45
46 313 Figure 4 shows the XRD patterns of the pellicles (Figure 4a), the pellicle powders
47
48
314 (Figure 4b), and the TOBCP/AgNP powder from 35° to 80° (Figure 4c) in 2θ. In addition, for
49
50
51 315 comparison, AgNP size distribution by measuring 200 AgNP from the FE-SEM images was
52
53 316 shown in Figure 4d. For Figure 4a, all the pellicles showed characteristic peaks of native
54
55
56 17
57
58
59
1
2
3 317 cellulose Iα crystal structure and no significant difference was observed between the pellicles.
4
5 318 The peaks at 14.6°, 16.6° and 22.6° corresponded to the (1–10), (110), and (200)
6
7 319 crystallographic planes, respectively.31 Figure 4b shows a trend similar to Figure 4a besides
8
9 320 the small peaks at 34.5° for all the pellicles and a clear sharp peak at 38.1° only for the
10
11
12
321 TOBCP/AgNP. These two peaks corresponded the reflections including the (004)
13
14 322 crystallographic plane of cellulose nanofibers31 and the (111) reflections of the face centered
15
16 323 cubic structure of AgNP,32 respectively. The peak of AgNP only could be seen for
17
18 324 TOBCP/AgNP powder sample clearly indicates abundant AgNP were synthesized in the
19
20 325 TOBCP/AgNP. Figure 4c shows the XRD patterns of the TOBCP/AgNP powder (shown in
21
22 326 Figure 4b) from 35° to 80°. Besides the most significant peak at 38.1° explained as above, the
23
24
25
327 peaks at 44.2°, 64.5°, and 77.3° corresponded to (200), (220) and (311) reflections of the face
26
27 328 centered cubic structure of AgNP, respectively.32 Hence, except for the FE-SEM images as
28
29 329 morphological evidence, the XRD patterns also provided crystallographic evidence for the
30
31 330 maintained 3D network and the successfully in situ synthesized AgNP. Furthermore, as
32
33 331 shown in Figure 4d, the average diameter of the AgNP synthesized on the TOBC nanofibers
34
35 332 was 16.5 ± 4.0 nm by measuring 200 AgNP from the FE-SEM images, indicating a
36
37
38 333 homogeneous size distribution.
39
40
41 334
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56 18
57
58
59
1
2
3
4
5 a b
6
7
8
9
10 BCP
Intensity
Intensity
11 BCP
12
13 TOBCP
TOBCP
14
15 TOBCP/Ag+ TOBCP/Ag+
16
17 TOBCP/AgNP
18 TOBCP/AgNP
19
20 10 15 20 25 30 35 40 10 15 20 25 30 35 40
21
22 Diffraction angle 2θ (°)
23 40
24
25
c (111)
d average diameter = 16.5±4.0 nm
35
26 (n = 200)
27 30
28
29
Fraction (%)
25
30
Intensity
31 20
32
33 15
34 (200)
35 (220) 10
36 (311)
37 5
38
39 0
35 40 45 50 55 60 65 70 75 80 0 5 10 15 20 25 30 35 40
40
41 Diffraction angle 2θ (°) AgNP diameter (nm)
42 335
43
44
45
46
336 Figure 4. XRD patterns of the pellicles (a), powders obtained from the pellicles (b), and
47
48 337 TOBCP/AgNP powder from 35° to 80° (c). (d) AgNP size distribution from measurement of
49
50 338 200 AgNP by FE-SEM images.
51
52
53 339
54
55
56 19
57
58
59
1
2
3 340 To further confirm the synthesized AgNP, element analysis of the pellicles were carried
4
5 341 out by XPS (Figure 5a). The Na 1s peaks were observed for the TOBCP, TOBCP/Ag+, and
6
7 342 TOBCP/AgNP due to the –COO–Na+ groups introduced by TEMPO-mediated oxidation. The
8
9 343 Ag 3d peaks only appeared for the TOBCP/Ag+ and TOBCP/AgNP due to the introduced
10
11
12
344 silver element. The silver amounts of the TOBCP/Ag+ and TOBCP/AgNP were
13
14 345 approximately 0.9% and 0.7%, respectively. It is probably because detecting depth of XPS is
15
16 346 less than 10 nm and thus a part of the AgNP (particle size ~16.5 nm) interiors could not be
17
18 347 detected, resulting in a lower silver amount.33 For the TOBCP/Ag+, ~57% of the Na+ amount
19
20 348 was ion-exchanged by Ag+ and thus both the Na 1s and Ag 3d peaks could be simultaneously
21
22 349 observed. Distribution of the Ag element of pellicle surface (Figure 5b) and pellicle interior
23
24
25
350 (Figure 5c) were also investigated by analyzing Ag 3d5/2 peaks. For the TOBCP/Ag+ (solid
26
27 351 lines), the Ag+ (~367 eV) amount ratio of surface to interior was approximately 2:1,
28
29 352 indicating that ~33% Ag+ entered the TOBCP interior during ion-exchange. In addition, ~9%
30
31 353 and ~7% Ag+ amount of the surface and interior were reduced to AgNP (~368.5 eV),
32
33 354 respectively, due to that the surface was more easily heated than the interior. Therefore, for
34
35 355 the TOBCP/AgNP, AgNP amount ratio of surface to interior was approximately 18:7,
36
37
356 indicating not only the surface but also the interior containing a large amount of AgNP.
38
39
40
41 357
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56 20
57
58
59
1
2
3 358
4
5 a b c
O 1s
TOBCP/Ag+ TOBCP/Ag+
6 359 TOBCP/AgNP TOBCP/AgNP
7
Na 1s
8 C 1s
Intensity (a.u.)
Intensity (a.u.)
Intensity (a.u.)
9 Ag 3d
360
10
11
12
13 361
14
15 1200 1000 800 600 400 200 370 369 368 367 366 365 370 369 368 367 366 365
16 362 Binding energy (eV) Binding energy (eV) Binding energy (eV)
17
18
19 363 Figure 5. Typical XPS spectra of the pellicle surfaces (a), and surfaces (b) and interiors (c) of
20
21 364 the TOBCP/Ag+ and TOBCP/AgNP.
22
23
24
25 365
26
27
28 366 Figure 6 shows FTIR spectra of the pellicle powders from 1500 to 1800 cm-1 in
29
30 367 wavenumber. For the BCP, the peak at ~1650 cm-1 was corresponding to O–H bending of the
31
32 368 residual water.34 For the TOBCP, the peaks at ~1615 cm-1 and ~1735 cm-1 were
33
34 369 corresponding to C=O stretching of –COO–Na+ and –COOH groups, respectively.34 When
35
36
370 TEMPO-mediated oxidation used in the present study (TEMPO/NaClO/NaBr system at pH
37
38
39 371 10) is performed, the –CH2OH groups at C6 were oxidized to –COOH groups and then
40
41 372 neutralized by adding 0.5 M NaOH solution, resulting in –COO–Na+ groups. Thus, these –
42
43 373 COOH groups were considered not neutralized during oxidation. For the TOBCP/Ag+, the
44
45 374 peak at ~1615 cm-1 shifted to a lower wavenumber of ~1600 cm-1, indicating –COO–Ag+
46
47 375 groups were formed.26 The peak at ~1735 cm-1 showed a higher intensity than that of the
48
49
376 TOBCP due to the acidity of the AgNO3 solution used for ion-exchange, resulting in –COOH
50
51
52 377 groups from protonation of the –COO–Na+ groups. For the TOBCP/AgNP, the peak at ~1600
53
54 378 cm-1 shifted to a higher wavenumber of ~1610 cm-1, indicating that a combination of –COO–
55
56 21
57
58
59
1
2
3 379 Na+ groups and AgNP was formed. The evidence for existence of the AgNP was well shown
4
5 380 by the results of FE-SEM images, XRD patterns, and XPS spectra as described above.
6
7 381 Theoretically, the peak at ~1610 cm-1 also included a part of –COO–Ag+ groups according to
8
9 382 the result of XPS. The peak at ~1735 cm-1 almost disappeared perhaps due to that pKa of the
10
11
12
383 –COOH groups decreases with increasing temperature during thermal reduction,35 resulting
13
14 384 in –COO–Na+ groups from deprotonation of the –COOH groups.
15
16
17 385
18
19
20 386
21 COO-Na+ + AgNP
22
23
387
24
25 COO-Ag + TOBCP/AgNP
Absorbance
26
27 388
COO-Na + TOBCP/Ag+
28
29
30 389
31 TOBCP
32
33 390
BCP
34
35
1800 1750 1700 1650 1600 1550 1500
36
37
391 -1
Wavenumber (cm )
38
39
40 392 Figure 6. FTIR spectra of the pellicle powders.
41
42
43 393 Figure 7 shows the results of thermal analysis of the pellicles from room temperature to
44
45 394 800°C, including thermogravimetric analysis (TGA) curves (Figure 7a) and derivative
46
47 395 thermogravimetric analysis (DTG) curves (Figure 7b). TGA result shows that all the pellicles
48
49
50
396 exhibited similar behavior of thermal degradation. Onset degradation temperature was coded
51
52 397 as Tonset and defined as the temperature corresponding to the intersection point of the two
53
54 398 tangent lines for the TGA curve in the range of 200–350°C. As the general native cellulose,
55
56 22
57
58
59
1
2
3 399 the Tonset of the BCP was ~325°C. For the TOBCP, the Tonset decreased to ~235°C due to the
4
5 400 formed –COO–Na+ at C6 of TOBC nanofiber suraces from TEMPO-mediated oxidation,
6
7 401 resulting in an early degradation.36 Interestingly, the TGA curves of the TOBCP/Ag+ and
8
9 402 TOBCP/AgNP simultaneously showed similar thermal behavior and the same residual weight
10
11
12
403 ratio at 800°C. In addition, both the Tonset of the two pellicles increased to ~295°C by
13
14 404 introducing silver element. It is a reasonable phenomenon due to thermal reduction of Ag+ of
15
16 405 the TOBCP/Ag+ occurring at ~100°C, resulting in complete formation of TOBCP/AgNP
17
18 406 from the TOBCP/Ag+ at >100°C. DTG result shows that the maximums of thermal
19
20 407 degradation rates of the BCP, TOBCP, TOBCP/Ag+, and TOBCP/AgNP occurred at ~357°C,
21
22 408 ~297°C, ~330°C, and ~330°C, respectively, corresponding to the TGA result. To sum up, the
23
24
25
409 Tonset of the BCP (~325°C) decreased to ~235°C after TEMPO-mediated oxidation and then
26
27 410 increased to ~330°C after introduction of silver element which contributed considerable
28
29 411 thermal stability.
30
31
32 412
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56 23
57
58
59
1
2
3 413
4
5 a 100
6 414 90 BCP
7 80
TOBCP
8 TOBCP/Ag+
TOBCP/AgNP
9 70
415
10
Weight (%)
60
11
12 50
13 416
40
14
15 30
16 417
20
17
18 10
19 418 0
20 100 200 300 400 500 600 700 800
21
22 Temperature (°C)
419
23
24 b 2.5
25 BCP
Derivative weight loss (%/°C)
26 420 TOBCP
2.0
27 TOBCP/Ag+
28 TOBCP/AgNP
29 421 1.5
30
31
32 422 1.0
33
34
35 0.5
423
36
37
38 0.0
39 424
100 200 300 400 500 600 700 800
40
41 Temperature (°C)
42 425
43
44
45 426 Figure 7. Thermogravimetric analysis (TGA) (a) and derivative thermogravimetric analysis
46
47
427 (DTG) (b) curves of the pellicles.
48
49
50
51 428
52
53
54
55
56 24
57
58
59
1
2
3 429 Ag+ release behavior.
4
5
6 430 Figure 8 shows the Ag+ release behavior including total release of Ag+ (Figure 8a) and
7
8 431 Ag+ release rate (Figure 8b). For comparison with the references (NaBH4-reduced AgNP in
9
10
432 BCP and NaBH4-reduced AgNP in TOBCP from oxidation at pH 6.8),4,17 the TOBCP/AgNP
11
12
13 433 were immersed in water at 37°C for 16 days to investigate short- and long-term Ag+ release
14
15 434 behavior. Total Ag+ content of the TOBCP/AgNP was determined to be approximately 0.068
16
17 435 g/g pellicle by XPS. As a result, the TOBCP/AgNP released Ag+ quite stably and rapidly.
18
19 436 The cumulative released Ag+ achieved to 97.0% at day 16 and thus all the Ag+ were almost
20
21 437 completely released. With the fourth day as the boundary between the two almost linear
22
23
438 curves, the average release rates of day 0–3 and day 5–16 were approximately 12.2%/day and
24
25
26 439 4.2%/day, respectively. It is perhaps due to that at day 0–3 most of the small AgNP and a part
27
28 440 of large AgNP surfaces released Ag+ simultaneously, while at day 5–16 mainly the residual
29
30 441 large AgNP released Ag+ stably. The initial Ag+ release rate in the present study (12.2%/day)
31
32 442 was slightly higher than that reported by Maneerung et al. (~10.7%/day of NaBH4-reduced
33
34 443 AgNP in BCP) and much higher than that reported by Feng et al. (~0.8%/day of
35
36
444 NaBH4-reduced AgNP in TOBCP from oxidation at pH 6.8).4,17 It is probably due to that the
37
38
39 445 BCP was much swollen at pH 10 than at pH 6.8 during oxidation, and distances between the
40
41 446 TOBC nanofibers were expanded by hot water during thermal reduction, increasing
42
43 447 possibility of releasing Ag+ to the outside of the TOBCP/AgNP. Although such high Ag+
44
45 448 release rate should show high antibacterial activity theoretically, its effect on human cell was
46
47 449 unknown. Therefore, biocompatibilities of the TOBCP/AgNP and other prepared pellicles
48
49 450 (BCP and TOBCP) were investigated.
50
51
52
53 451
54
55
56 25
57
58
59
1
2
3 452
4
5
6 453
7
8
a 100
9 90
454
10 80
Total release of Ag (%)

11
70
12
+
13 455 60
14 50
15
40
16 456
17 30
18 20
19 457
20 10
21 0
22 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
458
23 Period (day)
24
25
b 16
15
26 459 14
27 13
Ag release rate (%/day)
28 12
29 460 11
10
30
9
31 8
32 461 7
33 6
34 5
+
35 4
462 3
36
2
37 1
38 0
39 463 0 1 2 3 4 5 6 7 8 9 10111213141516
40 Period (day)
41
42 464
43
44
45 465 Figure 8. Total release of Ag+ (a) and Ag+ release rate (b) from the TOBCP/AgNP in water
46
47
466 at 37°C for 16 days.
48
49
50
51 467
52
53
54
55
56 26
57
58
59
1
2
3 468 Cell viability.
4
5
6 469 Ifuku et al. used TEMPO/NaClO/NaBr system to prepare TEMPO-oxidized BCP
7
8 470 (TOBCP) as a reaction template for AgNP synthesis. AgNP were in situ synthesized on the
9
10
471 TOBC nanofiber surfaces by thermal reduction of Ag+, resulting in AgNP with uniform size
11
12
13 472 of ~13 nm.26 Nevertheless, the study focused on obtainment of AgNP and the TOBCP was
14
15 473 only used as a template for synthesizing AgNP without describing their further application.
16
17 474 TOBCP containing AgNP was prepared by TEMPO/NaClO/NaClO2 system at pH 6.8 and
18
19 475 reduction of Ag+ using reducing agents including NaBH4 and citric acid.17 However, although
20
21 476 the pellicles showed antibacterial activities, their carboxylate group contents from
22
23
477 TEMPO-mediated oxidation and cytotoxicity were not reported; Carboxylate group content is
24
25
26 478 a very important parameter to evaluate effect of TEMPO-mediated oxidation when compared
27
28 479 with other related reports, and to accurately calculate the AgNO3 amount for ion-exchange;
29
30 480 Cytotoxicity is used to calculate cell viability and further to reflect biocompatibility which is
31
32 481 the most basic property for a biomedical material. Furthermore, the reported Ag+ release rate
33
34 482 was quite low (only 0.8%/day), implying a low use efficiency of expensive silver resulting in
35
36
483 an undesired increase of preparation cost. It is considered perhaps due to lack of appropriate
37
38
39 484 control or clear amount of carboxylate group content from the oxidation, decreasing the use
40
41 485 efficiency. Therefore, it seems that biocompatibility and antibacterial activity of a
42
43 486 TOBCP/AgNP composite combining definite carboxylate group content and high Ag+ release
44
45 487 rate has not been reported.
46
47
48 488 In vitro cytotoxicity test could be used to determine cell viability which evaluates
49
50
489 biocompatibility of a material. In general, a material resulting in higher cell viability
51
52
53 490 possesses better biocompatibility.37 Biocompatibility, which could be briefly defined as the
54
55
56 27
57
58
59
1
2
3 491 property of a biomedical material to perform its desired function to therapy without
4
5 492 undesirable side effect occurring, is well known as the most important property for a
6
7 493 biomedical material.37,38 To our best knowledge, however, it seems that a study clearly
8
9 494 demonstrating biocompatibility of TOBCP or TOBCP/AgNP based on experimental evidence
10
11
12
495 has not been reported to date. Therefore, in vitro cytotoxicity was performed to demonstrate
13
14 496 the important issue in the present study although it is well known that BCP is a
15
16 497 highly-biocompatible biomedical material. Figure 9 shows cell viabilities of NIH3T3 cells
17
18 498 incubated in the different pellicle extracts at 37°C for 24 h (Figure 9a and Table 1) and 48 h
19
20 499 (Figure 9b and Table 1). The TOBCP/Ag+ was not subjected to the test due to the complete
21
22 500 sterilization using an autoclave at 121°C resulting in thermal reduction of Ag+ to partially
23
24
25
501 form TOBCP/AgNP. As a result, the BCP, TOBCP, and TOBCP/AgNP showed good
26
27 502 biocompatibilities after incubation for 48 h and no significant statistical difference between
28
29 503 the pellicles was found. It indicates that the TOBCP/AgNP showing high and stable Ag+
30
31 504 release rate is a promising biomedical material for wound dressing application. Furthermore,
32
33 505 biocompatibility of the TOBCP was also demonstrated and thus its potential for biomedical
34
35 506 applications is also expected.
36
37
38
39 507
40
41
42 508
43
44
45 509
46
47
48
49
50
51
52
53
54
55
56 28
57
58
59
1
2
3 510
4
5
6 511
7
8 a 120
9 110
512
10 100
Cell viability (% of control)

11
90
12
13 513 80
14 70
15 60
16 514
50
17
18 40
19 515 30
20 20
21
10
22
516 0
23
24
25
26 517 b 120
27 110
28
100
29 518
Cell viability (% of control)
30 90
31 80
32 519 70
33
60
34
35 50
520
36 40
37 30
38
521 20
39
40 10
41 0
42 522
43
44
45 523 Figure 9. Cell viabilities of NIH3T3 cells incubated in the BCP, TOBCP, and TOBCP/AgNP
46
47
524 extracts at 37°C for 24 h (a) and 48 h (b).
48
49
50
51 525
52
53
54 526
55
56 29
57
58
59
1
2
3 527 Antibacterial activity.
4
5
6 528 Figure 10 shows growth inhibition zones (GIZ) of the pellicles and filter papers
7
8 529 containing antibiotic (gentamicin sulfate) against E. coli and S. aureus by disc diffusion
9
10
530 method at 37°C for 24 h. The GIZ widths were determined by the largest distance differences,
11
12
13 531 which were between the radius of the pellicles and the distances of the colonies to the pellicle
14
15 532 centers. The GIZ widths of the antibiotic against to E. coli and S. aureus were approximately
16
17 533 4.2 and 2.9 mm, respectively, indicating that S. aureus was harder to inhibit than E. coli. In
18
19 534 addition, the same pellicle showed similar effects against E. coli and S. aureus, which were
20
21 535 lower than the antibiotic. For the BCP and TOBCP, no GIZ width was observed, indicating
22
23
536 almost no antibacterial activity against the two bacteria. The TOBCP/Ag+ was not subjected
24
25
26 537 to the test due to the complete sterilization using an autoclave at 121°C resulting in thermal
27
28 538 reduction of Ag+ to partially form TOBCP/AgNP. For the TOBCP/AgNP, the GIZ widths
29
30 539 against E. coli and S. aureus were both approximately 1.5 mm although some parts of the
31
32 540 later seemed slightly smaller. It indicates that the TOBCP/AgNP showed significant bacterial
33
34 541 activity against the two bacteria. Interestingly, silver mirror-like surface with fissures forming
35
36
542 on the TOBCP/AgNP could be clearly observed for the GIZ against E. coli probably due to
37
38
39 543 silver mirror reaction of Ag+ by E. coli. First, E. coli reduced the carboxylate groups to
40
41 544 aldehyde groups;39 Then, except for attacking E. coli, Ag+ (not reduced during thermal
42
43 545 reduction or released from AgNP) also formed silver ammonia complex ion ([Ag(NH3)2]+)
44
45 546 with nitrogen in the medium for incubation of E. coli;40 Finally, the [Ag(NH3)2]+ complex ion
46
47 547 oxidized the aldehyde groups to carboxyl groups and formed the silver mirror-like surface
48
49 548 with lower specific surface area than AgNP.40 It was considered that antibacterial activity of
50
51
52
549 the TOBCP/AgNP was lowered to some extent because the originally released Ag+ which
53
54
55
56 30
57
58
59
1
2
3 550 attack E. coli were reduced to form the silver mirror-like surface, decreasing antibacterial
4
5 551 efficiency of Ag+.
6
7
8 552
9
10
11
12 553
13
14
15 554
16
17
18 555
19
20
21 556
22
23
24
25 557
26
27
28 558
29
30
31 559
32
33
34 560
35
36
37
38 561
39
40
41 562
42
43
44 563 Figure 10. Growth inhibition zones of the BCP, TOBCP, TOBCP/AgNP, and filter papers
45
46 564 containing antibiotic against E. coli (a) and S. aureus (b) at 37°C for 24 h.
47
48
49
50 565
51
52
53
54
55
56 31
57
58
59
1
2
3 566 Figure 11 (also Table 1 for the pellicle extracts) shows viabilities of E. coli and S.
4
5 567 aureus incubated in the different pellicle (or filter papers containing gentamicin sulfate)
6
7 568 extracts by colony forming count method at 37°C for 24 h. Lower viability of bacterium
8
9 569 represents higher antibacterial activity of the pellicle. The colonies incubated without
10
11
12
570 pellicles were used as the control groups. For E. coli (Figure 11a), the viabilities of the BCP
13
14 571 and TOBCP groups were slightly higher than that of the control group, indicating that they
15
16 572 showed no antibacterial activity against E. coli. Surprisingly, a viability of 0% was observed
17
18 573 for the TOBCP/AgNP group, which was the same as the result of the antibiotic group,
19
20 574 revealing that the TOBCP/AgNP could completely inhibit the growth of E. coli. For S. aureus
21
22 575 (Figure 11b), it clearly showed higher growth rates and higher resistances against
23
24
25
576 antibacterial factor than E. coli. The antibiotic could not completely inhibit the growth of S.
26
27 577 aureus and a viability of 0.05% was observed. For the BCP and TOBCP groups, the pellicles
28
29 578 not only were unable to inhibit the growth of S. aureus but also significantly increased its
30
31 579 growth rate, which were approximately 2.5 times as high as that of the control group. It
32
33 580 strongly indicates that antibacterial factor is considerably important for a wound dressing to
34
35 581 inhibit the growth of conventional bacteria. The S. aureus viability of the TOBCP/AgNP
36
37
582 group was very low to be only 0.79%, showing high antibacterial activity of the
38
39
40 583 TOBCP/AgNP against S. aureus .
41
42
43 584 In summary, the biocompatible TOBCP/AgNP possessed superior antibacterial activity
44
45 585 against E. coli and S. aureus, showing its potential for application of antibacterial wound
46
47 586 dressing. The antibacterial activity of the TOBCP/AgNP against S. aureus was lower than
48
49 587 that against E. coli, corresponding to the result reported by Feng et al.17 It is probably due to
50
51
52
588 that cellular wall of Gram-positive bacterium (S. aureus) has a thicker peptidoglycan layer
53
54
55
56 32
57
58
59
1
2
3 589 than Gram-negative bacterium (E. coli), forming a rigid resistance to decrease Ag+ amount
4
5 590 entering the cell interior.14
6
7
8 591
a 120
9 110
10 100
11 90
12 592
80
13
Viability (%) 70
14
15 593 60
16 50
17 40
106.46 ±12.62
18
106.18 ±4.98
594 30 100 ±14.51
19
20
20
21 595
10 0
0
22 0
23
24
25 596
26
b 350
27 300
28 597
29 250
30
Viability (%)
31 598 200
32
33
150
34 599
35
261.67 ±50.59
248.89 ±13.88
100
36
100 ±18.05
0.05 ±0.01
0.79 ±0.10
37
38 600 50
39
40 0
41 601
42
43
44 602 Figure 11. Viabilities of E. coli (a) and S. aureus (b) incubated in the different pellicle
45
46 603 extracts at 37°C for 24 h. Note the different scales of Y-axis in the figures. The value of the
47
48
604 viability was written on each group.
49
50
51
52 605
53
54
55
56 33
57
58
59
1
2
3 606 CONCLUSIONS
4
5
6 607 Highly biocompatible and highly antibacterial wound dressing of TOBCP/AgNP can be
7
8 608 prepared with BCP by TEMPO-mediated oxidation using TEMPO/NaClO/NaBr system at
9
10
609 pH 10, ion-exchange by Ag+, and thermal reduction of Ag+, resulting in synthesized AgNP on
11
12
13 610 the TOBC nanofiber surfaces. The appropriate oxidation time is determined to be 1 h
14
15 611 according to the well maintained structure and the carboxylate group content of the TOBCP
16
17 612 is approximately 1.1 mmol/g cellulose. The AgNO3 amount for ion-exchange can be
18
19 613 accurately calculated due to the definite carboxylate group content, avoiding waste of AgNO3.
20
21 614 Reduction of Ag+ by hot water bath instead of using a reducing agent results in AgNP with
22
23
615 uniform size of ~16.5 nm distributed in both surface and interior of the TOBCP/AgNP,
24
25
26 616 showing good efficiency of Ag+ reduction. Although the size of AgNP from thermal
27
28 617 reduction is larger than those from NaBH4 reduction,4,17,26 evaluating the quality of AgNP
29
30 618 should be carried out by Ag+ release behavior, biocompatibility, and antibacterial activity.
31
32 619 Ag+ release rate of the TOBCP/AgNP is 12.2%/day in 3 days, showing smooth and stable
33
34 620 Ag+ release behavior to simultaneously achieve high biocompatibility and high antibacterial
35
36
621 activity. High biocompatibility of the TOBCP/AgNP is clearly demonstrated according to the
37
38
39 622 NIH3T3 cell viability of 95.2±3.0% after 48 h of incubation and thus the TOBCP/AgNP can
40
41 623 be used for biomedical application. High antibacterial activity of the TOBCP/AgNP against E.
42
43 624 coli and S. aureus are 100% and 99.21%, respectively, similar to those of the reported
44
45 625 results.4,17 In addition, the TOBCP/AgNP shows a high WRV of 169% the same as that of the
46
47 626 BCP, revealing that the moist environment for promoting wound healing is well maintained
48
49 627 after the treatments during preparation. The future works are: (1) to investigate mechanical
50
51
52
628 strength of the TOBCP/AgNP to determine its application such as a coating, an adsorbing
53
54
55
56 34
57
58
59
1
2
3 629 membrane on a substrate, or as is; (2) to introduce curative drugs to the TOBCP/AgNP for
4
5 630 skin therapy.
6
7
8 631
9
10
11
12 632 Table 1. Water retention values, NIH3T3 cell viabilities, and antibacterial activities of the
13
14 633 pellicles.
15
16
17 Property (%) BCP TOBCP TOBCP/Ag+ TOBCP/AgNP
18
19 Water retention value (WRV) 168.8±12.2 194.2±10.7 173±12.9 169.3±12.4
20
21 NIH3T3 cell viability after 24 h 95.0±3.4 99.9±6.6 － 100.7±5.4
22
NIH3T3 cell viability after 48 h 97.2±6.0 95.1±5.9 － 95.2±3.0
23
24
E. coli viability after 24 h
25 (antibacterial activity)
106.5±12.6 (-6.5) 106.2±5.0 (-6.2) － 0 (100)
26
27 S. aureus viability after 24 h
261.7±50.6 (-161.7) 248.9±13.9 (-148.9) － 0.8±0.1 (99.2)
28 (antibacterial activity)
29
30
31 634
32
33
34
35 635
36
37
38
39
40
41
42
43
44
45
46
47
48
49
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51
52
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55
56 35
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1
2
3 636 AUTHOR INFORMATION
4
5
6 637 Corresponding Author
7
8
9
638 Kuan-Chen Cheng. Phone: +886 2 3366 1502, E-mail: kccheng@ntu.edu.tw
10
11
12
13 639 ACKNOWLEDGMENT
14
15
16 640 This work was partially sponsored by “Aim for the Top University Plan” of National Taiwan
17
18 641 University and the Taoyuan General Hospital, Ministry of Health and Welfare, Taiwan, under
19
20 642 contract no. <PTH10530>. The authors appreciate the researchers of National Taiwan
21
22
643 University (Taiwan) as follows: Prof. Hsi-Mei Lai (Department of Agricultural Chemistry)
23
24
25 644 for the autotitrator and Assoc. Prof. Kae-Kang Hwu (Department of Agronomy) for the ball
26
27 645 mill.
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29
30 646
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3 Bacterial cellulose pellicle (BCP) TEMPO-oxidized BCP with silver nanoparticles
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9 CH2OH COO-Na+ + AgNP
10
11
100 nm 100 nm
12 surface of BC nanofiber surface of BC nanofiber
13 Antibacterial activity <0% Antibacterial activity >99%

14 Highly biocompatible, Water retention value: 169% Highly biocompatible, Water retention value: 169%
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Water retention value (%)

Total release of Ag (%)

Cell viability (% of control)

13 Antibacterial activity <0% Antibacterial activity >99%

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