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Biocomposites of nanofibrillated cellulose, polypyrrole and silver nanoparticles with electroconductive and antimicrobial properties Patrycja Bober, Jun Liu, Kirsi Susanna Mikkonen, Petri Ihalainen, Markus Pesonen, Carme Plumed-Ferrer, Atte von Wright, Tom Lindfors, Chunlin Xu, and Rose-Marie Latonen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm500939x • Publication Date (Web): 27 Aug 2014 Downloaded from http://pubs.acs.org on September 6, 2014
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Biocomposites of nanofibrillated cellulose, polypyrrole and silver nanoparticles with electroconductive and antimicrobial properties Patrycja Bober±,†,║, Jun Liu±,‡, Kirsi S. Mikkonen§, Petri Ihalainen¥, Markus Pesonen#, Carme Plumed-Ferrer¤, Atte von Wright¤, Tom Lindfors†,x, Chunlin Xu*,‡, Rose-Marie Latonen*,†
†
Process Chemistry Centre, Laboratory of Analytical Chemistry, Åbo Akademi University,
FI-20500 Turku, Finland ║
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, 162 06
Prague 6, Czech Republic ‡
Process Chemistry Centre, Laboratory of Wood and Paper Chemistry, Åbo Akademi University,
FI-20500 Turku, Finland §
Department of Food and Environmental Sciences, University of Helsinki, FI-00014, Helsinki,
Finland ¥
Center for Functional Materials, Laboratory of Physical Chemistry, Åbo Akademi University,
FI-20500 Turku, Finland 1 ACS Paragon Plus Environment
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#
Center for Functional Materials, Physics, Åbo Akademi University, FI-20500 Turku, Finland
¤
Institute of Public Health and Clinical Nutrition, University of Eastern Finland, FI-70211,
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Kuopio, Finland x
Academy of Finland, Hakaniemenranta 6, POB 131, FI-00531 Helsinki, Finland
±
These authors contributed equally to this work.
ABSTRACT In this work, flexible and free-standing composite films of nanofibrillated cellulose/polypyrrole (NFC/PPy) and NFC/PPy-silver nanoparticles (NFC/PPy-Ag) have been synthesized for the first time via in situ one-step chemical polymerization and applied in potential biomedical applications. Incorporation of NFC into PPy significantly improved its film formation ability resulting in composite materials with good mechanical and electrical properties. It is shown that the NFC/PPy-Ag composite films have strong inhibition effect against the growth of the Grampositive bacteria, e.g. Staphylococcus aureus. The electrical conductivity and strong antimicrobial activity makes it possible to use the silver composites in various applications aimed for biomedical treatments and diagnostics. Additionally, we report here the structural and morphological characterization of the composite materials with Fourier-transform infrared spectroscopy, atomic force microscopy, scanning and transmission electron microscopy techniques.
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KEYWORDS: polypyrrole, nanofibrillated cellulose, silver nanoparticles, composite, freestanding films, antimicrobial property INTRODUCTION Electrically conducting polymers have attracted a lot of attention due to the broad range of potential application areas especially in electrochemical biosensing and as biomedical materials for health care diagnostics.1 Polypyrrole (PPy) is one of the most studied conducting polymer possessing interesting properties, such as biocompatibility2 and tunable electrical conductivity which can be controlled by reversible doping. It can be synthesized from environmentally friendly aqueous solutions at neutral pH by both chemical and electrochemical polymerization, and it has been reported that cotton fibers coated with PPy have antibacterial properties against Escherichia coli (Gram-negative).3-5 Also other coatings based on PPy have shown inhibition towards bacteria adhesion.6,7 For example, the titanium alloy (Ti6Al7Nb) coated with PPy have antibacterial effects of as high as three times stronger than the titanium alloy without the PPy coating.7 Optimized conditions for oxidation of pyrrole with iron(III) chloride (FeCl3)8-11, ammonium peroxydisulphate ((NH4)2S2O8) 11-13, or cerium(IV) sulphate (Ce(SO4)2)14 have been extensively reported in the literature, in contrast to the conducting polymer-Ag composites, which have gained increasing attention only during the last few years.15-22 Silver exhibits the highest electrical conductivity among all the metals, σ = 6.3×105 S cm–1 (20°C),23 and is relatively cheap compared to other noble metals. The electrical conductivity of chemically synthesized PPy is typically 10−2 - 1 S cm−1,12,17,22 while the incorporation of large amounts of silver (70-80 wt%) in
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the PPy matrix may improve the conductivity of the PPy-Ag composites up to ca. 3-5 orders of magnitude (1000 S cm−1).21,24 In the composite material, the silver can be incorporated either as microspheres, nanocubes, nanofibers, nanorods, nanowires or microparticles.16,25,26 The direct chemical polymerization of pyrrole using silver nitrate (AgNO3) as oxidant is usually the most efficient way to prepare conducting PPy-Ag composites (Scheme 1).21 During the polymerization, the redox reaction between the positively charged and growing polymer chains results in the reduction of Ag+ to metallic silver on the PPy surface. Due to its antimicrobial properties, the incorporation of silver further improves the antimicrobial effect of PPy.4,27
Scheme 1. The oxidation of pyrrole with AgNO3 to electrically conductingPPy-nitrate. Metallic silver is simultaneously deposited on the PPy surface. Composite materials of conducting polymers and noble metals have beneficial properties, such as ionic and electrical conductivity, tunable electrical and optical activity, flexibility and capacitive properties which make them useful in many technological applications. However, in most cases the poor processability owing to the insolubility and infusibility of the conducting polymers limits their use in practical applications.28 To overcome this problem, the conducting polymers can be incorporated by chemical polymerization in electrically non-conducting and solution processable host materials resulting in the formation of electroconductive composites.29,30 For example, the poor mechanical properties of the conducting polymers can be
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considerably reinforced by nanofibrillated cellulose (NFC) possessing high mechanical strength. Previous studies have shown that it is possible to prepare composites containing conducting polymers and different types of fibers, such as wood cellulose fibers,31,32 bacterial cellulose33-35 or NFC.36-39 For instance, NFC prepared from kraft pulp by the 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) mediated oxidation method generally has dimensions of 3–10 nm in width and 100 nm–10 µm in length.40 The selective oxidation of the primary hydroxyl groups of cellulose into carboxylate groups by the TEMPO process results in electrostatic repulsion of the fibrils allowing the formation of stable dispersions of nano-sized cellulose fibers.40 The nanopaper prepared from NFC shows excellent mechanical properties, low coefficient of thermal expansion and high transmittance.42-44 Hence, the TEMPO oxidized NFC has been explored in nanocomposite films, e.g. of a commercial aqueous acrylic latex dispersion as a matrix due to its reinforcing property.45 Recently, composites of conducting polymers and cellulose have received increasing attention. It has been shown that NFC can be used as a template for the synthesis of electrically conducting polyaniline (PANI) to produce NFC-PANI hydrogels.46 This facile processing route is applicable also for a broad range of other applications. Moreover, individual NFC fibers were coated with PPy by in situ chemical polymerization to prepare electrically conducting composites for ionexchange and energy storage applications.47 NFC-PPy composites with tunable structural and electrochemical properties have also shown promising results in biomedical applications and as electrodes in structural batteries.38,48 We report here the synthesis of novel free-standing multifunctional biocomposites for biomedical applications consisting of nanofibrillated cellulose coated with PPy and silver 5 ACS Paragon Plus Environment
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nanoparticles (NFC/PPy-Ag). To the best of our knowledge, this has not been reported previously. It is shown that these composites are electroconductive and have antimicrobial properties. In this paper, we have studied the influence of the silver nanoparticle concentration on the electrical conductivity, chemical structure, morphology and antimicrobial properties of the NFC/PPy-Ag composites. Due to possible wound healing applications, the mechanical strength and oxygen permeability of the composite films have given special attention. MATERIALS AND METHODS Preparation of nanofibrillated cellulose. NFC was prepared from bleached birch Kraft pulp according to a previously published method.49 Briefly, cellulose fibers (1 g) were fully dispersed in 50 mL deionized water. A solution containing 16 mg TEMPO (0.1 mmol g-1 fibers) and 100 mg sodium bromide (1.0 mmol g-1 fibers) was prepared and mixed with the dispersed fiber suspension, and the pH of the slurry was adjusted to 10.0 by the addition of 0.5 M NaOH. The 10% sodium hypochlorite (NaClO) (10 mmol
-1
g fibers) solution was added dropwise to the
slurry within 3 h and the reaction was let to continue for 9 h while the pH of the reaction was kept at 10.5. The TEMPO-oxidized cellulose was precipitated in ethanol and thoroughly washed with deionized water by centrifugation. The oxidized cellulose (1.0 wt%) was fibrillated by a domestic blender (OBH Nordica 6658, Denmark) for 5 min (300 W) to yield the resulting NFC with a carboxylate content of 1.55 ± 0.8 mmol g-1 determined by conductometric titration. Preparation of composite films. The NFC/PPy-Ag and NFC/PPy composites were prepared by oxidation of pyrrole (10 mM, ≥98%, Sigma Aldrich) with AgNO3 (Sigma Aldrich) or iron(III) nitrate (Fe(NO3)3) (Sigma Aldrich), or their mixtures of various composition in 0.5 wt% aqueous
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NFC dispersions. The oxidant-to-pyrrole moleratio was 2.3. The concentrations of AgNO3 and Fe(NO3)3 and the percentage of them with respect to each other (p) used during polymerization are given in Table 1. Both the monomer and the oxidant/s were separately dissolved in the NFC suspension. After mixing the suspensions, the mixtures were left unstirred at room temperature for 24 h or for one week when the oxidation was done with AgNO3. The total volume of the suspensions was 20 ml. The formed suspensions were filtered by vacuum filtration (Durapore PVDF, hydrophilic, 0.22 µm pores, ∅=47 mm), rinsed with water and dried on the filter paper, first in air, and then over silica gel. The filter paper was easily removed from the films when they were dry. For measuring the tensile properties and oxygen permeability, free standing films of NFC and its composites (NFC/PPy-Ag, NFC/PPy) were prepared via filtration of the NFC dispersion (0.5 wt%) on a Glass Microanalysis Filter Holder (Sterlitech, USA) with nylon membrane filter (Sterlitech, USA, 0.1 µm pores, ∅=90 mm) and then dried in a vacuum desiccator at 40°C and at a pressure of 88 mbar for 4 h. Thermogravimetric analysis (TGA). The silver content in the composite materials was determined from powdered films as a residue with the air flow rate of 50 mL min–1 and the heating rate of 10°C min-1 up to 800°C with a PerkinElmer TGA 7 thermogravimetric analyzer. Electrical conductivity measurements. The conductivity of the free-standing films was measured with 4-point probe measurements, in a linear configuration, having a tip spacing of 1.82 mm. A suitable bias current of 10–10 to 10–3 A was applied over the films and the corresponding voltage was measured until a repeatable value was obtained. The conductivity of
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the samples was then calculated using finite-size corrections.50 All measurements were carried out in ambient conditions. Oxygen permeability (OP). The oxygen transmission rate (OTR) through NFC and the composite films was measured using the M8001 OTR Analyzer (Systech IIIinois, Oxfordshire, UK) with the detection limit of 0.008 cm3 (m-2·d-1). The OP values were obtained by multiplying the OTR with the thickness of the film (35-60 µm, the thinnest one being the NFC/PPy-Ag(100) film) and dividing it with the difference in oxygen gas partial pressure between the two sides of the film. All films were conditioned in a climate room at 23°C and 50% relative humidity (RH) and the measurements were performed at the same conditions. Microscopy. The surface morphologies of NFC/PPy and NFC/PPy-Ag free- standing films were characterized with the scanning electron microscopy (SEM) using the JEOL 6400 microscope. The bulk morphology of both the dried suspensions and of the free-standing films was also studied by transmission electron microscopy (TEM). To analyze the liquid samples, the sample suspensions were dried either on thin carbon films or holey carbon reinforced Formvar films. The fibers on the thin carbon film were observed by negative staining with 1% uranyl acetate. Cross-sectional images of the free-standing films were recorded after embedding them in Epon (Epoxy Embedding Medium kit, Sigma) and then cutting the films using ultramicrotome and diamond knifes to slices with 70 nm thickness. A JEOL JEM-1400 Plus TEM instrument operated at 80 kV was used for imaging. Topographical imaging and mapping of the Derjaguin–Muller–Toporov (DMT) modulus of the free-standing films was done using PEAKFORCE Quantitative Nanomechanical Mapping
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(Peakforce QNM).50 Peakforce QNM measurements were carried out at ambient conditions (RH = 15±2%, T = 26±1°C) with a Nanoscope V (MultimodeTM series, Bruker) atomic force microscope (AFM). The AFM microscope was placed on an active vibrationally damped table (MOD-1M, JRS Scientific Instruments, Switzerland) standing on a stone table to eliminate external vibrational noise. All the images (1024×1024 pixels) were obtained using TAP525A cantilevers (Bruker). Each cantilever used in the analysis was calibrated (spring constant and radius curvature of the tip) following the calibration procedures given in the Peakforce QNM user guide.51 The suggested modulus range (given by the manufacturer) for reliable measurements of the DMT modulus for the TAP525A cantilevers is between 1 GPa to 20 GPa.51 The Scanning Probe Image Processor (SPIP, Image Metrology, Denmark) software was used for analysis of the images. The root mean square (RMS) roughness values were calculated using 5 µm × 5 µm topographical images. The DMT modulus values are the peak values obtained from the histograms of the DMT modulus maps.52 FTIR spectroscopy. The FTIR-ATR spectra were recorded using a Harrick’s VideoMVPTM single reflection diamond ATR accessory (incidence angle: 45°) having a horizontal sampling area (diameter: 500 µm) and a built-in pressure applicator. The pure free-standing NFC film, the PPy powder and all the studied free-standing composite films were tightly pressed against the diamond crystal during the measurement. The VideoMVPTM ATR accessory was attached to the Bruker IFS 66S spectrometer equipped with a DTGS detector. Totally 32 interferograms were coadded for each spectrum (resolution: 4 cm-1). Mechanical properties. The tensile strength, elongation at break and Young’s modulus of the NFC and composite film specimens with ca. 5 mm width were determined at a strain rate of 5 9 ACS Paragon Plus Environment
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mm/min using the Instron universal testing machine (Instron-33R4465, Instron Corp., High Wycombe, England) equipped with a static load cell of 100 N by applying an initial grip distance of 20 mm. All the films were conditioned in a climate room (23°C and 50% RH) for three days before the analysis. The thickness of the specimens was measured as the average of three points using a micrometer (Lorentzen & Wettre, Kista, Sweden, precision 1 µm) and the width was measured with a digital caliper (Mahr GmbH 16ER, Germany, precision 10 µm). Ten specimens from three replicate films of each sample were tested. Antimicrobial properties. The antimicrobial activity of the nanomaterials was tested using agar plates and by checking the growth inhibition zones around the free-standing films. The antimicrobial test was done against both gram-positive (Listeria monocytogenes ATCC 7644, Staphylococcus aureus 298) and gram-negative bacteria (Salmonella infantis EELA 72), and also against yeast (Candida albicans EELA 188). All bacteria were grown in tryptone soy agar or broth at 37°C except for Candida that was grown in sabouraud dextrose agar or broth at 30°C. Pieces of the composite materials (appr. 1 cm2) were placed on top of each agar plate. Overnight cultures of each bacterium were mixed with 0.4 % bacteriological agar and poured on top of the agar plates as an overlay layer, covering also the nanomaterials. The plates were subsequently incubated for 24 h at their respective temperatures. All materials were obtained from Lab M (Lab M Limited, Bury, UK). RESULTS AND DISCUSSION Oxidation of pyrrole with AgNO3 has been reported as the most direct way to form electrically conducting PPy-Ag composites.21,24 The use of two oxidants,18 AgNO3 and Fe(NO3)3 mixed in
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the mole ratios of 0.25-4.0, were used in this work to control the amount of silver incorporated in the NFC/PPy-Ag composites. By increasing the concentration of the silver containing oxidant (AgNO3) it was possible to vary the silver content in the composite material from 0 to 33 wt% when Fe(NO3)3 or AgNO3 was used as the only oxidant, respectively (Table 1). It shows that the silver loading of the NFC/PPy films can easily be controlled with the simple approach with two oxidants resulting in the formation of flexible and free-standing films of NFC/PPy-Ag. Moreover, only one type of charge compensating counter ion (i.e. NO3-) is incorporated in the composite matrix during the polymerization simplifying the redox chemistry of the NFC/PPy-Ag composites. The preparation procedure of the NFC/PPy-Ag films is schematically shown in Figure 1.
Figure 1. The schematic illustration of the preparation of the free-standing flexible NFC/PPy-Ag composite film.
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Table 1. The concentration of Fe(NO3)3 and AgNO3 used for the oxidation of 0.01 M pyrrole in 0.5 wt% NFC suspension, the percentage of AgNO3 with respect to Fe(NO3)3 (p), weight percentage silver in the NFC/PPy-Ag composites (wAg) and their electrical conductivities (σ), oxygen gas transmission rate (OTR), oxygen permeability (OP) and RMS roughness. Sample
p (%)
[AgNO3] –1
(mmol L )
[Fe(NO3)3] –1
(mmol L )
wAg (wt%)
OTR
σ −1
(S cm )
3
-2
OP -1
(cm m ·d )
RMS
3
Roughness
(cm ·µm -2
-1
-1
m d ·kPa )
(nm)
NFC
-
-
-
0
