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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 Downloaded from http://pubs.acs.org on January 16, 2018

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TEMPO-oxidized bacterial cellulose pellicle with silver nanoparticles for wound dressing

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Chun-Nan Wu1#, Shih-Chang Fuh2#, Shin-Ping Lin3, Yen-Yi Lin3, Hung-Yueh Chen1,

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Jui-Ming Liu2, and Kuan-Chen Cheng1,3,4*

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Graduate Institute of Food Science and Technology, National Taiwan University, 1, Sec 4,

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Roosevelt Rd., Taipei 10617, Taiwan. 2

Division of Urology, Department of Surgery, Taoyuan General Hospital, Ministry of Health

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and Welfare, 1492, Chung-Shan Road, Taoyuan District, Taoyuan 330 ,Taiwan. 3

Institute of Biotechnology, National Taiwan University, 1, Sec 4, Roosevelt Rd., Taipei

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10617, Taiwan. 4

Department of Medical Research, China Medical University Hospital, China Medical

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University, 91, Hsueh-Shih Rd., Taichung 40402, Taiwan.

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# Chun-Nan Wu and Shih-Chang Fuh contributed equally to this work.

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ACS Paragon Plus Environment

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KEYWORDS: antibacterial, wound dressing, silver nanoparticle, TEMPO, bacterial

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cellulose

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ABSTRACT: Biocompatible bacterial cellulose pellicle (BCP) is a candidate for biomedical

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material such as wound dressing. However, due to lack of antibacterial activity, to grant BCP

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with the property is crucial for its biomedical application. In the present study, BCP was

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modified by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation using

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TEMPO/NaClO/NaBr system at pH 10 to form TEMPO-oxidized BCP (TOBCP) with

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anionic C6 carboxylate groups. The TOBCP was subsequently ion-exchanged in AgNO3

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solution and silver nanoparticles (AgNP) with diameter of ~16.5 nm were in situ synthesized

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on TOBC nanofiber surfaces by thermal reduction without using a reducing agent. FE-SEM,

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XRD, XPS, FTIR, and TGA were carried out to confirm morphology and structure of the

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pellicles with AgNP. The AgNP continuously released Ag+ with a rate of 12.2%/day at 37°C

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in 3 days. The TOBCP/AgNP exhibited high biocompatibility according to the result of in

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vitro cytotoxicity test (cell viability > 95% after 48 h of incubation) and showed significant

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antibacterial activities of 100% and 99.2% against E. coli and S. aureus, respectively. Hence,

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the highly biocompatible and highly antibacterial TOBCP/AgNP prepared in the present

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study is a promising candidate for wound dressing.

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INTRODUCTION

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Wound dressing is an important biomedical material used to protect wound and promote

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its healing. The wound dressing market is growing rapidly in the present healthcare system

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worldwide due to increasing chronic diseases.1,2 In general, an ideal wound dressing shows

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some biomedical properties such as biocompatibility, providing a moist environment,

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absorbing exudates, and high mechanical strengths. Bacterial cellulose pellicle (BCP) is a

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good candidate as functional wound dressing due to satisfaction with these unique

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properties.3,4

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Bacterial cellulose (BC) is the representative cellulose synthesized by microbes. It is

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mainly produced by Gluconacetobacter and has an appearance of white pellicle with an

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extremely high moisture content of ~99%.5 A pellicle is a wet biofilm that assembles

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surrounding cells at the air-liquid interface in a standing liquid culture.6,7 BCP has a unique

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microstructure of 3D network consisting of cellulose nanofibers cross-linked by numerous

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hydrogen bonds, resulting in high water retention capacity and high mechanical strengths.5,8

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When BCP is used as a wound dressing with some functional components, it could be a

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barrier to wound infection and advantageous for delivering drugs to an injured site.3 BCP has

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been commercially available as chronic wound dressing and temporary artificial skin.3,9,10

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Although BCP shows many superior properties described above, it lacks of antibacterial

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activity and thus could not decrease the possibility of bacterial infection disease such as

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bacterial sepsis for burns patients.11

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Silver ion (Ag+) was reported as an important component used for various antibacterial

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products due to its antibacterial effect and non-toxicity to human cells.12−14 It was, however,

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reported that high concentration of Ag+ may cause DNA damage response to mammalian 3



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cells.15 Silver nanoparticle (AgNP) has been demonstrated to gradually release Ag+ to inhibit

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bacterial growth in a moist environment at human body temperature.4,12−14,16,17 Therefore,

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when designing BCP containing AgNP as a biocompatible and antibacterial wound dressing,

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it is considered that surface-modified BCP nanofibers could absorb AgNP stronger, resulting

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in a controllable and smooth Ag+ release rate18.

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TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation is well

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known as a regioselective surface modification method which only oxidizes primary OH

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groups and is widely applied to the field of cellulose-based nanomaterials.19−25 By

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TEMPO-mediated oxidation, anionic carboxylate groups (–COO–Na+) are introduced to C6

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of cellulose nanofiber surfaces and thus adsorption of functional cationic ions (such as Ag+

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for antibacterial application) on the surfaces through ion-exchange can be achieved. Ifuku et

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al. used TEMPO/NaClO/NaBr system to prepare TEMPO-oxidized BCP (TOBCP) as a

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reaction template for AgNP synthesis26. The study focused on obtainment of AgNP and the

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TOBCP was only used as a template for synthesizing AgNP without describing their further

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application.

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In the present study, we prepared a novel wound dressing of TOBCP/AgNP, which

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possessed both high biocompatibility and high antibacterial activity. Characterization of the

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pellicles obtained after each preparation step was described as much as possible, including

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appearance, water retention value, FE-SEM images, XRD patterns, XPS spectra, FTIR

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spectra, TGA curves, Ag+ release behavior, in vitro cytotoxicity, and antibacterial activity

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tests. In particular, to our knowledge, cytotoxicities of TOBCP and TOBCP/AgNP have not

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been clearly reported to date although BCP is well-known biocompatible. These important

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results are helpful to development for not only antibacterial wound dressing but also other

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new antibacterial products for biomedical and hygiene applications in the future.

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EXPERIMENTAL SECTION

Materials.

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The Gluconacetobacter xylinus ATCC700178, Escherichia coli, and Staphylococcus

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aureus strains, and NIH3T3 cells were obtained from the Bioresource Collection and

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Research Center (Hsinchu, Taiwan). TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)

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was purchased from Sigma-Aldrich (St Louis, MO, USA). NaBr, 2 M NaClO solution, and

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AgNO3 were purchased from Wako Pure Chemicals (Osaka, Japan). All the chemicals were

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of laboratory grade and used as received. All the water used in the present study was double

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distilled.

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Production of BCP.

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Bacterial cellulose pellicles (BCP) were used as the starting materials for wound

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dressings and prepared according to the previously reported method.27 In brief, a cell

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suspension of G. xylinum (0.12 mL) was added to a CSL-Fru (corn steep liquor with fructose)

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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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9 cm formed at the interface between the medium and air was taken off from the dish. For

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purification, the obtained BCP was immersed in a 1.0 M NaOH solution at 80°C several

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times to remove the bacteria and impurity until the BCP became visibly white. The white and

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transparent BCP was further washed by tap water for 3 days to fully remove the residual

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impurity and followed by storage in water at 4°C.

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TEMPO-mediated oxidation of BCP.

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In the present study, the oxidation strategy was designed to combine high carboxylate

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content and to maintain the superior structure of the original BCP for application of wound

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dressing. A BCP was freeze-dried for 2 days and then cut into pieces 15 mm × 15 mm in size.

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The freeze-dried BCP pieces (~0.2 g cellulose) and 300 mL water were added to a 500 mL

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beaker. TEMPO (0.018 g) and NaBr (0.1 g) were added to the beaker and the solution was

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gently agitated for 1 h. The TEMPO-mediated oxidation was triggered by adding NaClO

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solution (10 mmol/g cellulose) to the solution. The pH value of the oxidation system was

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maintained at 10 by adding a 0.5 M NaOH solution using an autotitrator (836 Titrando;

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Metrohm, Herisau, Switzerland). The oxidation was for 1, 2, 4, or 8 h to determine an

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appropriate oxidation time for further preparation of wound dressing. For each oxidation time,

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the reaction was quenched by adding ethanol until the pH value of the system was not

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changed. The TEMPO-oxidized BCP (TOBCP, Figure 1a) were washed with water 10 times.

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The carboxylate group contents of the TOBCP treated for 1, 2, 4, and 8 h were determined to

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be 1.1, 1.4, 1.6, and 1.8 mmol/g cellulose, respectively, by electric conductivity titration with

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a 0.05 M NaOH solution. TOBCP treated for 1 h was selected as the starting material for

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preparing wound dressing due to its well-maintained structure as the original BCP (Figure 1b,

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cut into 15 mm × 15 mm in size). It is noticed that the C6 carboxylate groups of the TOBCP

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were –COO–Na+ and these pellicles were not specially coded as TOBCP/Na+ in the present

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study.

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In situ synthesis of silver nanoparticles on the TOBC nanofibers.

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To exchange sodium ion at the C6 carboxylate groups with silver ion (Ag+), the TOBCP

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were immersed in AgNO3 (1.5 mmol Ag+/g cellulose) solution in the dark for 24 h. The

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obtained products were washed with water at least 10 times to remove residue ions and coded

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as TOBCP/Ag+ (Figure 1b), which expressed the C6 silver carboxylate groups (–COO–Ag+)

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on the TOBC nanofiber surfaces. Thermal reduction of the TOBCP/Ag+ was carried out by

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placing the pellicles in a water bath at 100°C for 2 h. The color of the TOBCP/Ag+ gradually

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became deep yellow with increasing reduction time, indicating formation of silver

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nanoparticles (AgNP) in the pellicles. These TOBCP containing AgNP formed on the TOBC

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nanofiber surfaces were coded as TOBCP/AgNP (Figure 1b).

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Characterization of the pellicles.

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Water retention. Water retention values (WRV) of the pellicles were calculated using the

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following equation:

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WRV (%) = (Ww – Wd) / Wd

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where Ww and Wd are weights of the wet and freeze-dried (for 2 days) pellicles, respectively.

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Microstructural observation. Field emission scanning electron microscope (FE-SEM)

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images of the inner surfaces of the pellicles and AgNP were observed using a JSM-7800F

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Prime instrument (JEOL, Tokyo, Japan). The pellicle samples were coated with platinum

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using an auto fine coater (JEC-3000FC, JEOL, Tokyo, Japan) at 10 mA for 2 min. Diameter

× 100

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distribution of the AgNP was estimated by measuring diameters of 200 AgNP with the

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FE-SEM images at 200k magnification.

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Structural analyses. X-ray diffraction (XRD) patterns of the pellicles and the pellicle

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powders obtained by ball milling the pellicles were recorded using an X-ray diffractometer

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(Ultima IV, Rigaku, Tokyo, Japan) in reflection mode with monochromator-filtered Cu Kα

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radiation (λ 0.15418 nm) at 40 kV and 40 mA. X-ray photoelectron spectra (XPS) were

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obtained using an XPS spectrometer (Theta Probe, Thermo Scientific, UK) with

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monochromated Al Kα radiation at 20.0 eV pass energy. Fourier transform infrared (FTIR)

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spectra of the pellicle powders obtained by ball milling the pellicles were recorded using a

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FTIR spectrometer (Spectrum 100, Perkin Elmer, Shelton, USA) in transmission mode from

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400 to 4,000 cm-1 in wavenumber with a resolution of 4 cm-1. Thermogravimetric analysis

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(TGA) of the pellicles was performed using a thermogravimetric analyzer (TGA Q50, TA

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Instruments, New Castle, DE, USA) at 10°C min-1 from room temperature to 800°C in a

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nitrogen atmosphere.

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Ag+ release behavior. To investigating Ag+ release behavior, three TOBCP/AgNP were

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added to a 50 mL centrifuge tube containing 50 mL water and soaked at 37°C. The pellicles

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were taken from the tube and soaked in another tube containing 50 mL fresh water at 37°C

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every 24 h. The treatment was repeated for 16 days and the soaking solution obtained

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everyday was stored at 4°C. The Ag+ concentrations of the solutions were analyzed using an

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inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 8000, Perkin

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Elmer, Shelton, USA) and calculated to be cumulative release rates.

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In vitro cytotoxicity test.

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The NIH3T3 cells were incubated in Dulbecco’s modified Eagle's medium (DMEM)

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containing 10% fetal bovine serum (FBS) at 37°C in 5% CO2/95% air. The cells were

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subcultured every 3 days to refresh the medium and adjust the cell density to ~1.2 × 105

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cell/mL by trypsinization with phosphate-buffered saline (PBS) and 0.25% trypsin/DMEM.

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To evaluate the cytotoxicity of the pellicles, the NIH3T3 cells (200 µL) were seeded in a

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96-well tissue culture plate with an initial cell density of ~1.2 × 105 cell/mL per well. The

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sterilized pellicles were extracted by immersion of the pellicles (thickness ~1 mm) in DMEM

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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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where NIH3T3 cells incubated in the 10% FBS/DMEM all the time was used as the negative

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control. After 24 h, the medium in the 96-well plate was replaced by fresh medium

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containing the pellicle extracts followed by incubation for 24 h. After 48 h, the same

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procedure was repeated. The cytotoxicity of the pellicles on the NIH3T3 cells was evaluated

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via cell viability obtained by water-soluble tetrazolium salts (WST-1) assay. For the NIH3T3

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cells incubated in the medium containing pellicle extracts for 24 or 48 h, the medium was

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replaced by 5% WST-1/DMEM and the 96-well plate was placed in the dark for 30 min. The

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optical density (OD) values at 450 nm of the reacted liquid were measured using a Multiskan

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GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, USA). For each

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treatment condition, the test was repeated 5 times.

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Antibacterial activity test.

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Antibacterial activities of the pellicles prepared in the present study were carried out by

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disc diffusion and colony forming count method.4 Both the methods were investigated against

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E. coli and S. aureus , which were used as Gram-negative and Gram-positive bacteria models,

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respectively. (1) Disc diffusion method. The experiment was performed using a Luria– 9



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Bertani (LB) medium solid agar Petri dish with a width of 90 mm. The pellicles were cut into

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9 mm in diameter and placed on E. coli-cultured and S. aureus -cultured agar plates at 37°C

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for 24 h. Volume and concentration of the bacterial suspension for each dish were 0.1 mL and

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approximately 1.5 × 107 CFU mL-1, respectively. After bacterial incubation, the formed

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growth inhibition zones were observed. (2) Colony forming count method. Three square

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pellicles 15 mm in width and 1 mL of bacterial suspension (~1 × 107 CFU mL-1) were added

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to a 50 mL centrifuge tube. The mixture was incubated at 100 rpm and 37°C for 24 h. The

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tube was then filled with sterilized saline and vortexed. The 50 µL of diluted bacterial

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suspension was drawn from the tube and spread on a LB medium solid agar Petri dish

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described as above for 24 h. After bacterial incubation, the colony count was observed and

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antibacterial activity of the pellicle was simply evaluated by percentage reduction of the

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colony count (%), which was calculated using the following equation:

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[(ColonyC − ColonyT) / ColonyC] × 100%

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where ColonyC and ColonyT are colony count of the control and treated group, respectively.

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RESULTS AND DISCUSSION

Appearance of the pellicles.

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Figure 1a shows the effect of oxidation time on the structure of the TOBCP by

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TEMPO/NaClO/NaBr system at pH 10 and 10 mmol NaClO/g cellulose. Oxidation times and

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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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1.8 mmol/g cellulose, respectively. The TOBCP oxidized for 2 h was slightly more

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transparent than that oxidized for 1 h although the densities of the both pellicles were not

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significantly different. The result indicates the higher carboxylate group content resulted in 10


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higher repulsive forces between the TOBC nanofibers and higher swelling capacity in water.

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When the oxidation time was increased to 4 h, the densities of the more swollen TOBCP

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were much decreased, indicating its more unstable structure. Finally, the structure of the

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TOBCP oxidized for 8 h was significantly destroyed and only the sedimented fragments

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could be seen. Therefore, in the present study, the TOBCP oxidized for 1 h was used to

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prepare antibacterial wound dressings due to that the strong pellicle structure was completely

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maintained, showing sufficient mechanical resistance when used in a wet environment.

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Figure 1b shows the scheme of each treatment in the present study and their corresponding

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photos of the pellicles. The TOBCP was more transparent than the BCP because its structure

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was much swollen in water due to the carboxylate groups introduced on the BC nanofiber

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surfaces by TEMPO-mediated oxidation. The carboxylate groups were anionic and more

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hydrophilic than hydroxyl groups, resulting in repulsive forces between the BC nanofiber

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surfaces. Tollens’ reagent was used as a surface modifier to prepare a BCP wound dressing

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containing AgNP.11 By Ag(NH3)2+ ions in the Tollens’ reagent, the OH groups on the BC

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nanofiber surfaces were oxidized to aldehyde groups and further to carboxylate groups which

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are capable of stably absorbing the AgNP. Nevertheless, the OH groups on C2, C3, and C6 of

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the anhydroglucose units on the BC nanofiber surfaces were simultaneously oxidized. Due to

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C2-C3 cleavage occurring during oxidation, the pyranose rings were broken and thus the

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crystallinity and mechanical strengths of the BC nanofibers were decreased.18 Therefore, for

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development of antibacterial wound dressing with BCP and AgNP, a surface modification

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method combining oxidation of OH groups and maintenance of linear structure of cellulose

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chains is necessary. The appearance of the TOBCP/Ag+ obtained by ion-exchange of the

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TOBCP was almost the same with that of the TOBCP and thus mixing these two pellicles had

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to be avoided. The TOBCP/AgNP obtained by thermal reduction of the TOBCP/Ag+ for 2 h 11



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was deep yellow, indicating the formation of AgNP on the TOBC nanofiber surfaces.4,26 The

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color significantly became deeper with increasing reduction time due to the increased AgNP

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amount.

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Figure 1. Photos of the pellicles prepared in the present study. (a) The TOBCP with different

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oxidation times in water. The carboxylate group contents of the TOBCP oxidized for 1, 2, 4,

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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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preparation process and the samples obtained after each step.

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Water retention value.

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High water retention value (WRV) is the unique and superior property of BCP as a

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wound dressing and thus the WRV of the pellicles obtained by each treatment (shown in

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Figure 2 and Table 1) had to be maintained. The BCP used as the starting material showed a

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high WRV of 169% due to its 3D network and large surfaces of the BC nanofibers. The

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WRV of the TOBCP (194%) was higher than that of the BCP because the carboxylate groups

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were more hydrophilic than hydroxyl groups. Interestingly, the TOBCP/Ag+ showed a WRV

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of 173% which was lower than that of the TOBCP perhaps due to that the ion diameter of

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Ag+ (126 pm, 1 pm (picometer) = 1 × 10-12 m)28 was larger than that of Na+ (102 pm)29,

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retarding attraction of water by carboxylate groups. The TOBCP/AgNP showed a WRV

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(169%) slightly lower than the TOBCP/Ag+ and the same as the BCP. The carboxylate

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groups of the TOBCP/AgNP was mainly –COO–Na+ form attracting AgNP by electrostatic

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forces (see Figure 6) because Ag+ were reduced to AgNP and the TOBCP/AgNP was

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immersed in a nutral solution. Compared to the TOBCP/Ag+, the –COO–Na+ form was more

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hydrophilic than the –COO–Ag+ form due to ionic radius effect described as above. However,

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the presence of AgNP retarded water molecules to be attracted by the carboxylate groups

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more than Ag+. It is considered that the effect of AgNP was higher than that of ionic radius

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and thus the TOBCP/AgNP showed the WRV slightly lower than the TOBCP/Ag+. The result

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indicates that not only the TOBCP/AgNP maintained the high WRV of the BCP but the

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TOBCP and TOBCP/Ag+ showed higher WRV than the BCP. Furthermore, all of these

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pellicles also had very high water contents >99%, which was higher than those of the

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commercial wound dressings. It suggests that these pellicles might show high water vapor

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transmission rates (WVTR) to meet requirement of wound dressing application.30

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Water retention value (%)

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Figure 2. Water retention values of the pellicles.

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Microstructure observation.

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Figure 3 shows the FE-SEM images of the BCP (Figure 3a, 3e, and 3i) , TOBCP (Figure

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3b, 3f, and 3j), TOBCP/Ag+ (Figure 3c, 3g, and 3k), and TOBCP/AgNP (Figure 3d, 3h, and

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3l) with different magnifications. Thicknesses and densities of the pellicles were

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approximately 0.5 mm and 0.02 g cm-3, respectively. For all the treated pellicles, the unique

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3D network consisting of cellulose nanofibers was well maintained as that of the BCP. It

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indicates that all the treatments including TEMPO-mediated oxidation, iox-exchange, and

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thermal reduction were appropriate for preparation of wound dressings with superior

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microstructures. For the TOBCP/AgNP, the in situ synthesized AgNP on the TOBC

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nanofibers were clearly observed (Figure 3l). The AgNP existed singly or formed a 15



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aggregation where the each AgNP could be distinguished. The particle sizes and distribution

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of the AgNP (shown in Figure 4d) were determined by measuring diameters of 200 AgNP

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with the FE-SEM images. As a result, the FE-SEM images proved that for the TOBCP/AgNP,

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the 3D network of BC nanofibers was maintained and the AgNP were successfully

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synthesized on the TOBC nanofiber surfaces.

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Figure 3. FE-SEM images of the pellicles including BCP (a, e, j), TOBCP (b, f, j),

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TOBCP/Ag+ (c, g, k), and TOBCP/AgNP (d, h, l). The arrows in Figure 3l indicated the in

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situ synthesized AgNP.

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Structural analyses.

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Figure 4 shows the XRD patterns of the pellicles (Figure 4a), the pellicle powders

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(Figure 4b), and the TOBCP/AgNP powder from 35° to 80° (Figure 4c) in 2θ. In addition, for

315

comparison, AgNP size distribution by measuring 200 AgNP from the FE-SEM images was

316

shown in Figure 4d. For Figure 4a, all the pellicles showed characteristic peaks of native 17



317

cellulose Iα crystal structure and no significant difference was observed between the pellicles.

318

The peaks at 14.6°, 16.6° and 22.6° corresponded to the (1–10), (110), and (200)

319

crystallographic planes, respectively.31 Figure 4b shows a trend similar to Figure 4a besides

320

the small peaks at 34.5° for all the pellicles and a clear sharp peak at 38.1° only for the

321

TOBCP/AgNP. These two peaks corresponded the reflections including the (004)

322

crystallographic plane of cellulose nanofibers31 and the (111) reflections of the face centered

323

cubic structure of AgNP,32 respectively. The peak of AgNP only could be seen for

324

TOBCP/AgNP powder sample clearly indicates abundant AgNP were synthesized in the

325

TOBCP/AgNP. Figure 4c shows the XRD patterns of the TOBCP/AgNP powder (shown in

326

Figure 4b) from 35° to 80°. Besides the most significant peak at 38.1° explained as above, the

327

peaks at 44.2°, 64.5°, and 77.3° corresponded to (200), (220) and (311) reflections of the face

328

centered cubic structure of AgNP, respectively.32 Hence, except for the FE-SEM images as

329

morphological evidence, the XRD patterns also provided crystallographic evidence for the

330

maintained 3D network and the successfully in situ synthesized AgNP. Furthermore, as

331

shown in Figure 4d, the average diameter of the AgNP synthesized on the TOBC nanofibers

332

was 16.5 ± 4.0 nm by measuring 200 AgNP from the FE-SEM images, indicating a

333

homogeneous size distribution.

334

18


Page 18 of 44

Page 19 of 44

a

b

Intensity

Intensity

BCP TOBCP

BCP TOBCP

TOBCP/Ag+

TOBCP/Ag+ TOBCP/AgNP

TOBCP/AgNP 10

15

20

25

30

35

40

10

15

20

25

30

35

40

Diffraction angle 2θ (°)

c

d

(111)

40

average diameter = 16.5±4.0 nm (n = 200)

35

Fraction (%)

30

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 57 58 59 60

Biomacromolecules

(200)

25 20 15 10

(220)

(311) 5 0

35

40

45

50

55

60

65

70

75

80

0

5

Diffraction angle 2θ (°)

10

15

20

25

30

35

AgNP diameter (nm)

335

336

Figure 4. XRD patterns of the pellicles (a), powders obtained from the pellicles (b), and

337

TOBCP/AgNP powder from 35° to 80° (c). (d) AgNP size distribution from measurement of

338

200 AgNP by FE-SEM images.

339 19


40


340

To further confirm the synthesized AgNP, element analysis of the pellicles were carried

341

out by XPS (Figure 5a). The Na 1s peaks were observed for the TOBCP, TOBCP/Ag+, and

342

TOBCP/AgNP due to the –COO–Na+ groups introduced by TEMPO-mediated oxidation. The

343

Ag 3d peaks only appeared for the TOBCP/Ag+ and TOBCP/AgNP due to the introduced

344

silver element. The silver amounts of the TOBCP/Ag+ and TOBCP/AgNP were

345

approximately 0.9% and 0.7%, respectively. It is probably because detecting depth of XPS is

346

less than 10 nm and thus a part of the AgNP (particle size ~16.5 nm) interiors could not be

347

detected, resulting in a lower silver amount.33 For the TOBCP/Ag+, ~57% of the Na+ amount

348

was ion-exchanged by Ag+ and thus both the Na 1s and Ag 3d peaks could be simultaneously

349

observed. Distribution of the Ag element of pellicle surface (Figure 5b) and pellicle interior

350

(Figure 5c) were also investigated by analyzing Ag 3d5/2 peaks. For the TOBCP/Ag+ (solid

351

lines), the Ag+ (~367 eV) amount ratio of surface to interior was approximately 2:1,

352

indicating that ~33% Ag+ entered the TOBCP interior during ion-exchange. In addition, ~9%

353

and ~7% Ag+ amount of the surface and interior were reduced to AgNP (~368.5 eV),

354

respectively, due to that the surface was more easily heated than the interior. Therefore, for

355

the TOBCP/AgNP, AgNP amount ratio of surface to interior was approximately 18:7,

356

indicating not only the surface but also the interior containing a large amount of AgNP.

357

20


Page 20 of 44

Page 21 of 44

358 a

b

O 1s


Intensity (a.u.)

C 1s Ag 3d

Intensity (a.u.)

360

Na 1s

c


359 Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 57 58 59 60

Biomacromolecules

361 1200

362

1000

800

600

400

200

370

369

368

367

366

365

Binding energy (eV)

Binding energy (eV)

370

369

368

367

366

365

Binding energy (eV)

363

Figure 5. Typical XPS spectra of the pellicle surfaces (a), and surfaces (b) and interiors (c) of

364

the TOBCP/Ag+ and TOBCP/AgNP.

365

366

Figure 6 shows FTIR spectra of the pellicle powders from 1500 to 1800 cm-1 in

367

wavenumber. For the BCP, the peak at ~1650 cm-1 was corresponding to O–H bending of the

368

residual water.34 For the TOBCP, the peaks at ~1615 cm-1 and ~1735 cm-1 were

369

corresponding to C=O stretching of –COO–Na+ and –COOH groups, respectively.34 When

370

TEMPO-mediated oxidation used in the present study (TEMPO/NaClO/NaBr system at pH

371

10) is performed, the –CH2OH groups at C6 were oxidized to –COOH groups and then

372

neutralized by adding 0.5 M NaOH solution, resulting in –COO–Na+ groups. Thus, these –

373

COOH groups were considered not neutralized during oxidation. For the TOBCP/Ag+, the

374

peak at ~1615 cm-1 shifted to a lower wavenumber of ~1600 cm-1, indicating –COO–Ag+

375

groups were formed.26 The peak at ~1735 cm-1 showed a higher intensity than that of the

376

TOBCP due to the acidity of the AgNO3 solution used for ion-exchange, resulting in –COOH

377

groups from protonation of the –COO–Na+ groups. For the TOBCP/AgNP, the peak at ~1600

378

cm-1 shifted to a higher wavenumber of ~1610 cm-1, indicating that a combination of –COO– 21


Biomacromolecules

379

Na+ groups and AgNP was formed. The evidence for existence of the AgNP was well shown

380

by the results of FE-SEM images, XRD patterns, and XPS spectra as described above.

381

Theoretically, the peak at ~1610 cm-1 also included a part of –COO–Ag+ groups according to

382

the result of XPS. The peak at ~1735 cm-1 almost disappeared perhaps due to that pKa of the

383

–COOH groups decreases with increasing temperature during thermal reduction,35 resulting

384

in –COO–Na+ groups from deprotonation of the –COOH groups.

385

386 COO-Na+ + AgNP

387

388

COO-Ag +

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 57 58 59 60

Page 22 of 44

TOBCP/AgNP

COO-Na +

TOBCP/Ag+

389 TOBCP BCP

390 1800

391

392

1750

1700

1650

1600

1550

1500

-1

Wavenumber (cm )

Figure 6. FTIR spectra of the pellicle powders.

393

Figure 7 shows the results of thermal analysis of the pellicles from room temperature to

394

800°C, including thermogravimetric analysis (TGA) curves (Figure 7a) and derivative

395

thermogravimetric analysis (DTG) curves (Figure 7b). TGA result shows that all the pellicles

396

exhibited similar behavior of thermal degradation. Onset degradation temperature was coded

397

as Tonset and defined as the temperature corresponding to the intersection point of the two

398

tangent lines for the TGA curve in the range of 200–350°C. As the general native cellulose, 22


Page 23 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 57 58 59 60

Biomacromolecules

399

the Tonset of the BCP was ~325°C. For the TOBCP, the Tonset decreased to ~235°C due to the

400

formed –COO–Na+ at C6 of TOBC nanofiber suraces from TEMPO-mediated oxidation,

401

resulting in an early degradation.36 Interestingly, the TGA curves of the TOBCP/Ag+ and

402

TOBCP/AgNP simultaneously showed similar thermal behavior and the same residual weight

403

ratio at 800°C. In addition, both the Tonset of the two pellicles increased to ~295°C by

404

introducing silver element. It is a reasonable phenomenon due to thermal reduction of Ag+ of

405

the TOBCP/Ag+ occurring at ~100°C, resulting in complete formation of TOBCP/AgNP

406

from the TOBCP/Ag+ at >100°C. DTG result shows that the maximums of thermal

407

degradation rates of the BCP, TOBCP, TOBCP/Ag+, and TOBCP/AgNP occurred at ~357°C,

408

~297°C, ~330°C, and ~330°C, respectively, corresponding to the TGA result. To sum up, the

409

Tonset of the BCP (~325°C) decreased to ~235°C after TEMPO-mediated oxidation and then

410

increased to ~330°C after introduction of silver element which contributed considerable

411

thermal stability.

412

23


Biomacromolecules

413

a 100 90

414

BCP TOBCP TOBCP/Ag+ TOBCP/AgNP

80

416

70

Weight (%)

415

60 50 40 30

417

20 10

418

0 100 200 300 400 500 600 700 800

419

420

421

422

423

424

Temperature (°C)

b Derivative weight loss (%/°C)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 57 58 59 60

Page 24 of 44

2.5 BCP TOBCP TOBCP/Ag+ TOBCP/AgNP

2.0

1.5

1.0

0.5

0.0 100 200 300 400 500 600 700 800

Temperature (°C)

425

426

Figure 7. Thermogravimetric analysis (TGA) (a) and derivative thermogravimetric analysis

427

(DTG) (b) curves of the pellicles.

428

24


Page 25 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 57 58 59 60

429

Biomacromolecules

Ag+ release behavior.

430

Figure 8 shows the Ag+ release behavior including total release of Ag+ (Figure 8a) and

431

Ag+ release rate (Figure 8b). For comparison with the references (NaBH4-reduced AgNP in

432

BCP and NaBH4-reduced AgNP in TOBCP from oxidation at pH 6.8),4,17 the TOBCP/AgNP

433

were immersed in water at 37°C for 16 days to investigate short- and long-term Ag+ release

434

behavior. Total Ag+ content of the TOBCP/AgNP was determined to be approximately 0.068

435

g/g pellicle by XPS. As a result, the TOBCP/AgNP released Ag+ quite stably and rapidly.

436

The cumulative released Ag+ achieved to 97.0% at day 16 and thus all the Ag+ were almost

437

completely released. With the fourth day as the boundary between the two almost linear

438

curves, the average release rates of day 0–3 and day 5–16 were approximately 12.2%/day and

439

4.2%/day, respectively. It is perhaps due to that at day 0–3 most of the small AgNP and a part

440

of large AgNP surfaces released Ag+ simultaneously, while at day 5–16 mainly the residual

441

large AgNP released Ag+ stably. The initial Ag+ release rate in the present study (12.2%/day)

442

was slightly higher than that reported by Maneerung et al. (~10.7%/day of NaBH4-reduced

443

AgNP in BCP) and much higher than that reported by Feng et al. (~0.8%/day of

444

NaBH4-reduced AgNP in TOBCP from oxidation at pH 6.8).4,17 It is probably due to that the

445

BCP was much swollen at pH 10 than at pH 6.8 during oxidation, and distances between the

446

TOBC nanofibers were expanded by hot water during thermal reduction, increasing

447

possibility of releasing Ag+ to the outside of the TOBCP/AgNP. Although such high Ag+

448

release rate should show high antibacterial activity theoretically, its effect on human cell was

449

unknown. Therefore, biocompatibilities of the TOBCP/AgNP and other prepared pellicles

450

(BCP and TOBCP) were investigated.

451 25


Biomacromolecules

452

453

a 100 90

+

Total release of Ag (%)

454

455

456

457

80 70 60 50 40 30 20 10 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

458

Period (day)

b 459 Ag release rate (%/day)

460

461

+

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 57 58 59 60

462

463

16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

0 1 2 3 4 5 6 7 8 9 10111213141516 Period (day)

464

465

Figure 8. Total release of Ag+ (a) and Ag+ release rate (b) from the TOBCP/AgNP in water

466

at 37°C for 16 days.

467

26


Page 26 of 44

Page 27 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 57 58 59 60

468

Biomacromolecules

Cell viability.

469

Ifuku et al. used TEMPO/NaClO/NaBr system to prepare TEMPO-oxidized BCP

470

(TOBCP) as a reaction template for AgNP synthesis. AgNP were in situ synthesized on the

471

TOBC nanofiber surfaces by thermal reduction of Ag+, resulting in AgNP with uniform size

472

of ~13 nm.26 Nevertheless, the study focused on obtainment of AgNP and the TOBCP was

473

only used as a template for synthesizing AgNP without describing their further application.

474

TOBCP containing AgNP was prepared by TEMPO/NaClO/NaClO2 system at pH 6.8 and

475

reduction of Ag+ using reducing agents including NaBH4 and citric acid.17 However, although

476

the pellicles showed antibacterial activities, their carboxylate group contents from

477

TEMPO-mediated oxidation and cytotoxicity were not reported; Carboxylate group content is

478

a very important parameter to evaluate effect of TEMPO-mediated oxidation when compared

479

with other related reports, and to accurately calculate the AgNO3 amount for ion-exchange;

480

Cytotoxicity is used to calculate cell viability and further to reflect biocompatibility which is

481

the most basic property for a biomedical material. Furthermore, the reported Ag+ release rate

482

was quite low (only 0.8%/day), implying a low use efficiency of expensive silver resulting in

483

an undesired increase of preparation cost. It is considered perhaps due to lack of appropriate

484

control or clear amount of carboxylate group content from the oxidation, decreasing the use

485

efficiency. Therefore, it seems that biocompatibility and antibacterial activity of a

486

TOBCP/AgNP composite combining definite carboxylate group content and high Ag+ release

487

rate has not been reported.

488

In vitro cytotoxicity test could be used to determine cell viability which evaluates

489

biocompatibility of a material. In general, a material resulting in higher cell viability

490

possesses better biocompatibility.37 Biocompatibility, which could be briefly defined as the 27



491

property of a biomedical material to perform its desired function to therapy without

492

undesirable side effect occurring, is well known as the most important property for a

493

biomedical material.37,38 To our best knowledge, however, it seems that a study clearly

494

demonstrating biocompatibility of TOBCP or TOBCP/AgNP based on experimental evidence

495

has not been reported to date. Therefore, in vitro cytotoxicity was performed to demonstrate

496

the important issue in the present study although it is well known that BCP is a

497

highly-biocompatible biomedical material. Figure 9 shows cell viabilities of NIH3T3 cells

498

incubated in the different pellicle extracts at 37°C for 24 h (Figure 9a and Table 1) and 48 h

499

(Figure 9b and Table 1). The TOBCP/Ag+ was not subjected to the test due to the complete

500

sterilization using an autoclave at 121°C resulting in thermal reduction of Ag+ to partially

501

form TOBCP/AgNP. As a result, the BCP, TOBCP, and TOBCP/AgNP showed good

502

biocompatibilities after incubation for 48 h and no significant statistical difference between

503

the pellicles was found. It indicates that the TOBCP/AgNP showing high and stable Ag+

504

release rate is a promising biomedical material for wound dressing application. Furthermore,

505

biocompatibility of the TOBCP was also demonstrated and thus its potential for biomedical

506

applications is also expected.

507

508

509

28


Page 28 of 44

Page 29 of 44

510

511

a 120 110

513

514

515

Cell viability (% of control)

512

100 90 80 70 60 50 40 30 20 10

516

0

517

b 120 110

518

519

520

521

Cell viability (% of control)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 57 58 59 60

Biomacromolecules

100 90 80 70 60 50 40 30 20 10 0

522

523

Figure 9. Cell viabilities of NIH3T3 cells incubated in the BCP, TOBCP, and TOBCP/AgNP

524

extracts at 37°C for 24 h (a) and 48 h (b).

525

526 29



527

Antibacterial activity.

528

Figure 10 shows growth inhibition zones (GIZ) of the pellicles and filter papers

529

containing antibiotic (gentamicin sulfate) against E. coli and S. aureus by disc diffusion

530

method at 37°C for 24 h. The GIZ widths were determined by the largest distance differences,

531

which were between the radius of the pellicles and the distances of the colonies to the pellicle

532

centers. The GIZ widths of the antibiotic against to E. coli and S. aureus were approximately

533

4.2 and 2.9 mm, respectively, indicating that S. aureus was harder to inhibit than E. coli. In

534

addition, the same pellicle showed similar effects against E. coli and S. aureus, which were

535

lower than the antibiotic. For the BCP and TOBCP, no GIZ width was observed, indicating

536

almost no antibacterial activity against the two bacteria. The TOBCP/Ag+ was not subjected

537

to the test due to the complete sterilization using an autoclave at 121°C resulting in thermal

538

reduction of Ag+ to partially form TOBCP/AgNP. For the TOBCP/AgNP, the GIZ widths

539

against E. coli and S. aureus were both approximately 1.5 mm although some parts of the

540

later seemed slightly smaller. It indicates that the TOBCP/AgNP showed significant bacterial

541

activity against the two bacteria. Interestingly, silver mirror-like surface with fissures forming

542

on the TOBCP/AgNP could be clearly observed for the GIZ against E. coli probably due to

543

silver mirror reaction of Ag+ by E. coli. First, E. coli reduced the carboxylate groups to

544

aldehyde groups;39 Then, except for attacking E. coli, Ag+ (not reduced during thermal

545

reduction or released from AgNP) also formed silver ammonia complex ion ([Ag(NH3)2]+)

546

with nitrogen in the medium for incubation of E. coli;40 Finally, the [Ag(NH3)2]+ complex ion

547

oxidized the aldehyde groups to carboxyl groups and formed the silver mirror-like surface

548

with lower specific surface area than AgNP.40 It was considered that antibacterial activity of

549

the TOBCP/AgNP was lowered to some extent because the originally released Ag+ which

30


Page 30 of 44

Page 31 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 57 58 59 60

Biomacromolecules

550

attack E. coli were reduced to form the silver mirror-like surface, decreasing antibacterial

551

efficiency of Ag+.

552

553

554

555

556

557

558

559

560

561

562

563

Figure 10. Growth inhibition zones of the BCP, TOBCP, TOBCP/AgNP, and filter papers

564

containing antibiotic against E. coli (a) and S. aureus (b) at 37°C for 24 h.

565

31



566

Figure 11 (also Table 1 for the pellicle extracts) shows viabilities of E. coli and S.

567

aureus incubated in the different pellicle (or filter papers containing gentamicin sulfate)

568

extracts by colony forming count method at 37°C for 24 h. Lower viability of bacterium

569

represents higher antibacterial activity of the pellicle. The colonies incubated without

570

pellicles were used as the control groups. For E. coli (Figure 11a), the viabilities of the BCP

571

and TOBCP groups were slightly higher than that of the control group, indicating that they

572

showed no antibacterial activity against E. coli. Surprisingly, a viability of 0% was observed

573

for the TOBCP/AgNP group, which was the same as the result of the antibiotic group,

574

revealing that the TOBCP/AgNP could completely inhibit the growth of E. coli. For S. aureus

575

(Figure 11b), it clearly showed higher growth rates and higher resistances against

576

antibacterial factor than E. coli. The antibiotic could not completely inhibit the growth of S.

577

aureus and a viability of 0.05% was observed. For the BCP and TOBCP groups, the pellicles

578

not only were unable to inhibit the growth of S. aureus but also significantly increased its

579

growth rate, which were approximately 2.5 times as high as that of the control group. It

580

strongly indicates that antibacterial factor is considerably important for a wound dressing to

581

inhibit the growth of conventional bacteria. The S. aureus viability of the TOBCP/AgNP

582

group was very low to be only 0.79%, showing high antibacterial activity of the

583

TOBCP/AgNP against S. aureus .

584

In summary, the biocompatible TOBCP/AgNP possessed superior antibacterial activity

585

against E. coli and S. aureus, showing its potential for application of antibacterial wound

586

dressing. The antibacterial activity of the TOBCP/AgNP against S. aureus was lower than

587

that against E. coli, corresponding to the result reported by Feng et al.17 It is probably due to

588

that cellular wall of Gram-positive bacterium (S. aureus) has a thicker peptidoglycan layer

32


Page 32 of 44

Page 33 of 44

589

than Gram-negative bacterium (E. coli), forming a rigid resistance to decrease Ag+ amount

590

entering the cell interior.14

591

a

120 110 100 90 70 60

106.18 ±4.98

0 0.79 ±0.10

596

248.89 ±13.88

10

595

106.46 ±12.62

20

0

30

0.05 ±0.01

40

261.67 ±50.59

50

100 ±14.51

594

80

100 ±18.05

593

Viability (%)

592

0

b 350 300

597

250

598

599

Viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 57 58 59 60

Biomacromolecules

200 150 100

600

50 0

601

602

Figure 11. Viabilities of E. coli (a) and S. aureus (b) incubated in the different pellicle

603

extracts at 37°C for 24 h. Note the different scales of Y-axis in the figures. The value of the

604

viability was written on each group.

605

33



606

CONCLUSIONS

607

Highly biocompatible and highly antibacterial wound dressing of TOBCP/AgNP can be

608

prepared with BCP by TEMPO-mediated oxidation using TEMPO/NaClO/NaBr system at

609

pH 10, ion-exchange by Ag+, and thermal reduction of Ag+, resulting in synthesized AgNP on

610

the TOBC nanofiber surfaces. The appropriate oxidation time is determined to be 1 h

611

according to the well maintained structure and the carboxylate group content of the TOBCP

612

is approximately 1.1 mmol/g cellulose. The AgNO3 amount for ion-exchange can be

613

accurately calculated due to the definite carboxylate group content, avoiding waste of AgNO3.

614

Reduction of Ag+ by hot water bath instead of using a reducing agent results in AgNP with

615

uniform size of ~16.5 nm distributed in both surface and interior of the TOBCP/AgNP,

616

showing good efficiency of Ag+ reduction. Although the size of AgNP from thermal

617

reduction is larger than those from NaBH4 reduction,4,17,26 evaluating the quality of AgNP

618

should be carried out by Ag+ release behavior, biocompatibility, and antibacterial activity.

619

Ag+ release rate of the TOBCP/AgNP is 12.2%/day in 3 days, showing smooth and stable

620

Ag+ release behavior to simultaneously achieve high biocompatibility and high antibacterial

621

activity. High biocompatibility of the TOBCP/AgNP is clearly demonstrated according to the

622

NIH3T3 cell viability of 95.2±3.0% after 48 h of incubation and thus the TOBCP/AgNP can

623

be used for biomedical application. High antibacterial activity of the TOBCP/AgNP against E.

624

coli and S. aureus are 100% and 99.21%, respectively, similar to those of the reported

625

results.4,17 In addition, the TOBCP/AgNP shows a high WRV of 169% the same as that of the

626

BCP, revealing that the moist environment for promoting wound healing is well maintained

627

after the treatments during preparation. The future works are: (1) to investigate mechanical

628

strength of the TOBCP/AgNP to determine its application such as a coating, an adsorbing

34


Page 34 of 44

Page 35 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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 57 58 59 60

Biomacromolecules

629

membrane on a substrate, or as is; (2) to introduce curative drugs to the TOBCP/AgNP for

630

skin therapy.

631

632

Table 1. Water retention values, NIH3T3 cell viabilities, and antibacterial activities of the

633

pellicles.

Property (%)

BCP

TOBCP

TOBCP/Ag+

TOBCP/AgNP

Water retention value (WRV)

168.8±12.2

194.2±10.7

173±12.9

169.3±12.4

NIH3T3 cell viability after 24 h

95.0±3.4

99.9±6.6

－

100.7±5.4

NIH3T3 cell viability after 48 h

97.2±6.0

95.1±5.9

－

95.2±3.0

E. coli viability after 24 h (antibacterial activity)

106.5±12.6 (-6.5)

106.2±5.0 (-6.2)

－

0 (100)

S. aureus viability after 24 h (antibacterial activity)

261.7±50.6 (-161.7)

248.9±13.9 (-148.9)

－

0.8±0.1 (99.2)

634

635

35



636

AUTHOR INFORMATION

637

Corresponding Author

638

Kuan-Chen Cheng. Phone: +886 2 3366 1502, E-mail: [email protected]

639

ACKNOWLEDGMENT

640

This work was partially sponsored by “Aim for the Top University Plan” of National Taiwan

641

University and the Taoyuan General Hospital, Ministry of Health and Welfare, Taiwan, under

642

contract no. . The authors appreciate the researchers of National Taiwan

643

University (Taiwan) as follows: Prof. Hsi-Mei Lai (Department of Agricultural Chemistry)

644

for the autotitrator and Assoc. Prof. Kae-Kang Hwu (Department of Agronomy) for the ball

645

mill.

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Insert Table of Contents Graphic and Synopsis Here

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Bacterial cellulose pellicle (BCP)

CH2OH

Page 44 of 44

TEMPO-oxidized BCP with silver nanoparticles

COO-Na+ + AgNP

surface of BC nanofiber

100 nm

surface of BC nanofiber

100 nm

Antibacterial activity 99%

Highly biocompatible, Water retention value: 169%

Highly biocompatible, Water retention value: 169%


TEMPO-Oxidized Bacterial Cellulose Pellicle with ... - ACS Publications

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