International Journal of Biological Macromolecules 62 (2013) 608–613
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Preparation of TEMPO-oxidized cellulose/amino acid/nanosilver biocomposite film and its antibacterial activity Mingming Huang a , Feiran Chen b , Zhenyou Jiang c , Yiqun Li b,∗ a b c
College of Life Science and Technology, Jinan University, Guangzhou 510632, PR China Department of Chemistry, Jinan University, Guangzhou 510632, PR China School of Medicine, Jinan University, Guangzhou 510632, PR China
a r t i c l e
i n f o
Article history: Received 28 July 2013 Received in revised form 8 October 2013 Accepted 12 October 2013 Available online 17 October 2013 Keywords: TEMPO oxidized cellulose Silver nanoparticles Antibacterial activity
a b s t r a c t Novel biocomposite films were prepared by employing amino acid phenylalanine (Phe) or tryptophan (Try) functionalized TEMPO-oxidized cellulose (TOC) with silver nanoparticles (AgNPs) produced in situ by reduction with silver nitrate and sodium borohydride via homogeneous and heterogeneous approaches. The as-prepared biocomposite films, i.e. TOC–Phe–AgNPs and TOC–Try–AgNPs, were characterized by digital photographs, FTIR, Elemental Analysis, SEM, and TEM and the results confirmed the formation of the desired films. The in vitro antibacterial activity of two resulting composite film of TOC–Phe–AgNPs and TOC–Try–AgNPs was evaluated against Staphylococcus aureus and Escherichia coli bacteria by agar diffusion test. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Cellulose, which is the most abundant renewable polymer resource available on earth, is considered as an almost inexhaustible raw material source for the increasing demand for environment friendly and biocompatible materials [1]. However, due to the extensive network of inter- and intra-molecular hydrogen bonding between its fibrils, cellulose is extremely difficult to dissolve in water and most common organic solvents, meanwhile limited its economically feasible and environment friendly chemical processes required applications [1,2]. Therefore, chemical modifications of cellulose were interesting routes for preparing cellulose derivatives with specific properties [3–5]. Numerous efforts are underway seeking new ways to expand the application range of cellulose while retaining its prominent properties. Among all of them, (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO)mediated oxidation [6–8] of native cellulose appears of particular interest because its mild operative conditions, simplicity, efficiency, and high regioselectivity: only the C6 primary hydroxyl groups presenting on the surfaces of cellulosic microfibrils is converted to C6 carboxylate groups. The existed carboxylic groups can serve as templates to bind various molecules of interest for wide specific potential applications, opening thus new horizons for various applications of oxidized cellulose. For example, importantly, installed carboxyl groups allow the use of conjugation techniques,
∗ Corresponding author. Tel.: +8615521332351. E-mail addresses: [email protected], [email protected] (Y. Li). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.018
such as 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide (EDAC/NHS) activation, for irreversible linking amino acid or proteins onto cellulose [9–12]. Many kinds of preparations of TEMPO-oxidized cellulose have been reported, however, there are very few trials on TEMPOoxidized cellulose containing amino acid molecules, which can server as N,O-bidentate ligand to form a complex with transition metal silver [13,14], etc. Silver ions and silver compounds have been known to have strong antimicrobial activity [15–17] against nearly 650 types of bacteria and have potential applications in various fields like antibacterial filters, wound dressing materials, etc. The silver nanoparticles (AgNPs) show efficient antimicrobial properties compared to other salts due to their extremely large surface area, which provides better contact with microorganisms. The AgNPs are considered to be a slow release source of silver ions, which react with the groups of proteins and interfere with DNA replication [18]. Up to present time, a large number of researches got reported on immobilizing silver nanoparticles on to cellulose and other artificial polymers with potency in drug delivery systems as well as antibacterial applications. Only reports with very scanty information are available on immobilization of silver nanoparticles onto TOC and its derivatives. In the past decade, ionic liquids (ILs) have received enormous interest as solvents for biopolymer dissolution and regeneration owing to their solvent properties and process benefits. For example, it has been reported that ionic liquids 1-n-butyl-3methylimidazolium chloride ([bmim]Cl) was found that it could act as the non-derivatizing solvent for cellulose [19]. Dissolution
M. Huang et al. / International Journal of Biological Macromolecules 62 (2013) 608–613
609
Scheme 1. Sequential scheme of the preparation of amino acid modified TEMPO-oxidized cellulose/silver nanoparticles composite film.
of cellulose and its derivatives in ionic liquids can largely overcome this limitation so that the preparation of cellulose composites and derivatives is becoming more and more convenient. Extensive research has been carried out and much progress achieved in this area for the production of some advanced materials [20]. On the basis of the above points, we have recently studied the preparation of all cellulose composite materials based on cellulose using functionalized ionic liquid 1-hydroxylethly-3-methyl methylimidazolium chloride ([HOC2 mim]Cl) as well as other ionic liquid as solvent [21]. The amino acid functionalized TEMPOoxidized cellulose (TOC–AA) derivatives that are soluble in ionic liquids, so it can be efficiently used for the preparation of high quality films to immobilize AgNPs to construct the antibacterial activity of TOC–AA–AgNPs biocomposite materials. In the present work, effort has been made to prepare new two biocomposite films of amino acid functionalized TOC–AgNPs (TOC–AA–AgNPs). The sequential protocols were based on the steps schematically shown in Scheme 1. Initially, oxidation of cellulose was performed by the TEMPO–NaBr–NaClO system. Then, l-phenylalanine (Phe) or l-tryptophan (Try) was introduced to the TOC matrix using EDAC/NHS as activator, respectively. After that, the amino acid modified TOC and AgNO3 was dissolved in ionic liquid [bmim]Cl to form homogenous mixture (homogeneous system), or suspension in AgNO3 aqueous solution (heterogeneous system), and AgNPs were formed in situ as using NaBH4 as a reducing reagent. Finally, the resulting amino acid functionalized TOC/AgNPs composite (TOC–AA–AgNPs) were prepared successfully. Moreover, the in vitro antibacterial activities were evaluated against bacteria including two Staphylococcus aureus and Escherichia coli microorganisms by agar diffusion test.
analyses were performed on a Perkin Elmer EA2400II elemental analyzer. The elemental silver content of the composite was determined by Perkin Elmer Optima 2000DV inductively coupled plasma (ICP) spectroscopy. Scanning electron microscopy (SEM) was performed with a Philips XL 30ESEM instrument. Transmission electron microscopy (TEM) was performed with a Philips Tecnai instrument operating at 40–100 kV. Microcrystalline cellulose (MCC), [bmim]Cl, NaBH4 , TEMPO, EDAC, AgNO3 and other chemicals were reagent grade and used as purchased. 2.2. Preparation of TEMPO-oxidized cellulose Oxidized cellulose was prepared similarly according to literature method [22,23]. A typical procedure was as follows: Microcrystalline cellulose (0.648 g, i.e. 4 mmol of anhydroglucose units) was dispersed in Na2 CO3 /NaHCO3 buffer solutions (80 ml, pH = 10) for 1 min under moderate stirring. TEMPO (10 mg, 0.065 mmol) and NaBr (0.20 g, 1.9 mmol) were added in the suspension and maintain at 0 ◦ C (use an ice bath.) The sodium hypochlorite solution which was stored at 0 ◦ C (21%, 4.10 ml, 13.20 mmol, pH = 10 adjusted by 0.5 M aqueous HCl) was added every 30 min (four times) and stirring vigorous. When the cellulose almost had disappeared, 10 ml of methanol was added to quench the reaction. The solution was acidified to pH = 3 by adding 0.5 M HCl. Then centrifuged and abandoned the insoluble cellulose. The oxidized cellulose was precipitated by adding five volumes ethanol and centrifuged. The precipitate was washed with ethanol/water (v/v = 9/1) and centrifuged several times, followed dialysed in distilled water for 4 h (water changed every hour), and finally, dried at 40 ◦ C in a vacuum oven.
2. Experimental 2.1. Apparatus and reagents
2.3. Preparation of amino acids functionalized TEMPO-oxidized cellulose
FTIR spectra were recorded on a Bruker Equinox-55 spectrometer as KBr pellets in the range 400–4000 cm−1 . Elemental
The coupling of oxidized cellulose with amino acids is a modified method according to the literature [24,25]. Initially, 10 mg
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Fig. 1. The digital pictures of resulting TOC–Phe–AgNPs biocomposite films (a), TOC–Try–AgNPs biocomposite films (b).
of dried oxidized cellulose was added to 50 ml of deionized water, 23 ml of N-hydroxy-succinimide (NHS) aqueous solution (50 mg ml−1 ) was added after cellulose homogeneously dispersed under moderate magnetic stirring. 12 ml fresh N-ethylN -(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) aqueous solution (10 mg ml−1 ) was added quickly and continually stirring vigorously at room temperature for 30 min, pH was maintained at 7.5–8 by adding 0.5 M NaOH and/or HCl. Then the suspension was dialyzed under distilled water for 8 h (water changed every hour) to remove excess EDAC, NHS and by-product urea. Secondly, 100 mg selected amino acid (l–Phenylanaline, Phe or l–Tryptophan, Try) was added to the 100 ml distilled water of oxidized cellulose under moderate magnetic stirring 24 h at room temperature. The suspension was then dialyzed thoroughly in distilled water for 72 h (water changed every 4–8 h) to remove unbound amino acid. Then the oxidized cellulose bound amino acid was successively obtained by freeze drying.
2.4. Preparation of amino acid modified TEMPO-oxidized cellulose–silver nanoparticles composite film Homogeneous approach: Amino acid modified oxidized cellulose (0.1 g) was dissolved completely in ionic liquid [bmim]Cl (5.0 g) at 80 ◦ C in the oil bath under vacuum for 30 min till the mixture turn to a clear amber solution. Then, AgNO3 (1 × 10−5 mol) was added to the ionic liquid solution by stirring for 10 min at 80 ◦ C. After that, NaBH4 (1 × 10−3 mol) was added slowly under stirring till the mixture turned to dark brown color and kept at this temperature for another 10 min to ensure to form Ag nanoparticles completely. After then, the mixture was poured to a glass plate and rinsed with a large amount of distilled water to remove the excess chemical to produce hydrogel. Finally, the resulting composite film was obtained by freeze drying. The dark brown color of the composite film indicated formation of silver nano-particles. The Ag content of TOC–Phe–AgNPs and TOC–Try–AgNPs was found to be 17.7 mg/g and 17.6 mg/g by ICP, respectively. Heterogeneous approach: TOC–Phe or TOC–Try composite film was immersed in 4 × 10−4 M AgNO3 aqueous solution (50 ml). Then 0.2 M NaBH4 (10 ml) was added dropwise slowly. Upon completion of the reaction, the mixture was maintained for another 1 h under room temperature. Finally, the resulting composite film was treated by the process as given above. The Ag content of TOC–Phe–AgNPs and TOC–Try–AgNPs was found to be 10.1 mg/g and 8.7 mg/g by ICP, respectively. The digital photographs of as-prepared TOC–Phe–AgNPs biocomposite films and TOC–Try–AgNPs biocomposite films are shown in Fig. 1.
2.5. Antibacterial assay of amino acid modified oxidized cellulose–silver nanoparticles composite film Two kinds of bacterial: E. coli and S. aureus were used here for the antibacterial activity tests. E. coli and S. aureus bacterial suspensions was spread onto LB agar plates with Phe and Trp modified cellulose–silver nanoparticles biocomposite film, composite film without nano-Ag were used as control. After incubating at 40 ◦ C for 24 h, the zone of inhibition was observed. 3. Results and discussion 3.1. Fourier transfer infrared spectra and elemental analysis Fig. 2 shows the FTIR spectra of TEMPO oxidized cellulose (TOC) before and after functionalization with amino acids. Curve (a) contained a peak at 1740 cm−1 for the C=O band of carboxylate group TOC. This band is decreased after bounding with amino acids Try or Phe (b, c) due to the formation of amide bond. Curve b and c (particularly c) contained a strong characteristic absorption at 1600–1660 cm−1 which was assigned to the C=O band of the amide, confirming the modification of TOC with amino acid. Curve d and e are FTIR spectra of free Thr and Phe used as reference. The N content was found up to be 0.3 mmol g−1 for TOC–The and 0.15 mmol g−1 for TOC–Try, respectively, by elemental
Fig. 2. FTIR comparative spectra of (a) TEMPO oxidized cellulose (TOC), (b) Try modified TOC (TOC–Try), (c) Phe modified TOC (TOC–Phe), (d) Free Phe, and (e) Free Try.
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Fig. 3. TEM of Phe modified TOC with AgNPs (TOC–Phe–AgNPs, (a)), Trp modified TOC with AgNPs (TOC–Try–AgNPs, (b)), heterogeneous system gets TOC–Phe–AgNPs (c) and TOC–Try–AgNPs (d).
microanalysis, which further indicated the amino acid was introduced to the TOC molecular skeleton. 3.2. Morphology study The morphology of TOC–Phe–AgNPs and TOC–Try–AgNPs was studied by TEM and SEM. TEM micrographs (Fig. 3a and b) reveal that the silver particles distributed on TOC–Phe and TOC–Try are quasi-spherical in shape and consistently distributed in the range of 35 nm. As it can be observed from TEM images of TOC–Phe–AgNPs (Fig. 3c) and TOC–Try–AgNPs (Fig. 3d) prepared from heterogeneous process, silver nanoparticles are not homogenously dispersed on composite film and it is found that nanoparticles have irregular shapes.
The introduction of ligands onto polymeric skeleton is of special interest, because it enables the creation of a specific environment to control the growth of metal nanoparticle seeds. When a linear polymer is used as a stabilizing agent, modification of the functional groups can offer a specific reactive field around the silver nanoparticles that allows the particle size and shape control in the preparation. As an example, PVP, in which existed the C–N and C=O functional moieties, has been successfully used to control the size and shape of silver nanoclusters via coordinative bonding between C–N and C=O/Ag ions. [26,27] In homogeneous system, quasi-sphere silver nanoparticles were fabricated using TOC–AA as a promising template in which its molecular backbone played coordinated and stabilizer role. TOC–AA also contained the C–N and C=O functional groups which also play the role of controlling the size
CO2H CO2H CO2H CO2 H
TMPO-Oxidation
EDAS/NHS R
cellulose molecular
CO2H NH2
R O
CO2H
R
NH
NH
O
R NaBH4
O
R
CO2H O
CO2H NH
R
Ag0 O
CO2H NH
O
R
CO2H NH Ag
R
Ag+ cordinated with terminal amino acid
0
O
CO2H
R
+ NH Ag
O
R
CO2H NH
Ag+ O
CO2H NH
Ag+
CO2H NH
Ag0
Growth of AgNPs
Scheme 2. Schematic illustration of the controlled generation of AgNPs on a TOC–AA molecular skeleton in homogeneous system.
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M. Huang et al. / International Journal of Biological Macromolecules 62 (2013) 608–613
Fig. 4. SEM of TOC (a), TOC–AgNPs (b), TOC–Phe–AgNPs (c) and TOC–Try–AgNPs (d).
and shape of AgNPs according the model suggested by Ma et al. [27]: First, the complex of TOC–AA/Ag+ ions via coordinative bonding (the ligand of C–N and C=O in TOC–AA contributes electronic density to the sp orbital of silver ions) was constructed. Second,
the complex promotes silver nucleation, which tends to produce small silver particles. Third, the steric effect of TOC–AA covering the silver surface via physical and chemical bonding inhibits particle–particle contact and thus the agglomeration of the powder.
Fig. 5. The digital photographs of antibacterial activities of TOC–Phe–AgNPs against S. aureus (a), TOC–Phe–AgNPs against E. coli (b), TOC–Try–AgNPs against S. aureus (c), TOC–Try–AgNPs against E. coli (d).
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But in the case of heterogeneous process, TOC–AA is insoluble and just immersed in AgNO3 aqueous solution. Thus, silver ions to be reduced almost completely deposited on the surface of the AA–TOC to form irregular shapes. The plausible process of controlled generation of quasi-sphere AgNPs is depicted in Scheme 2. Representative SEM images of TOC, TOC–AgNPs, TOC–Phe–AgNPs and TOC–Try–AgNPs, which prepared in homogeneous approach, was shown in Fig. 4. The AgNPs are distributed in the TOC, TOC–Phe and TOC–Try matrix, as is most readily visible in images b, c and d of Fig. 4. But no apparent change in the morphology was observed in images c and d. (Fig. 4c–d).
TOC–Phe–AgNPs and TOC–Try–AgNPs nanocomposites exhibited an excellent antibacterial activity against both S. aureus and E. coli bacteria.
3.3. Antibacterial activities
References
The in vitro antibacterial activities of TOC–Phe–AgNPs and TOC–Try–AgNPs nanocomposite films were performed against two bacteria: S. aureus and E. coli bacteria. Each experiment was repeated for at least three times and best results of the digital photographs of the antibacterial effects are shown in Fig. 5. Also, by comparison, TOC–Phe–AgNPs and TOC–Try–AgNPs nanocomposite films prepared by heterogeneous process showed less antibacterial activities than those of films prepared by homogeneous process. These results were relevant to the shapes of silver nanoparticles immobilized on composite film. The quasi-sphere AgNPs provide better contact with microorganisms due to high surface area and high fraction of surface atoms, leading to incorporating more AgNPs inside the bacteria and promoting its efficacy in a sustained manner. From the images, no inhibition zone was observed for the TOC–Phe and TOC–Try as control, implying that the TOC–Phe and TOC–Try composite films do not have any antibacterial properties. These results clearly indicate that the antibacterial activity is only due to the silver nanoparticles which were impregnated inside amino acid modified TEMPO-oxidized cellulose and not due to the biocomposite films itself.
[1] D. Klemm, B. Heublein, H.P. Fink, A. Bohn, Angewandte Chemie International Edition 44 (2005) 3358–3393. [2] V. Finkenstadt, R. Millane, Macromolecules 31 (1998) 7776–7783. [3] L. Feng, Z.-l. Chen, Journal of Molecular Liquids 142 (2008) 1–5. [4] A. Pinkert, K.N. Marsh, S. Pang, M.P. Staiger, Chemical Reviews 109 (2009) 6712–6728. [5] M. Gericke, J. Trygg, P. Fardim, Chemical Reviews (2013). [6] A. Isogai, T. Saito, H. Fukuzumi, Nanoscale 3 (2011) 71–85. [7] T. Saito, M. Hirota, N. Tamura, S. Kimura, H. Fukuzumi, L. Heux, A. Isogai, Biomacromolecules 10 (2009) 1992–1996. [8] Y. Okita, T. Saito, A. Isogai, Biomacromolecules 11 (2010) 1696–1700. [9] L. Ma, C. Gao, Z. Mao, J. Zhou, J. Shen, Biomaterials 25 (2004) 2997–3004. [10] E. Lasseuguette, Cellulose 15 (2008) 571–580. [11] H. Orelma, L.-s. Johansson, I. Filpponen, O.J. Rojas, J. Laine, Biomacromolecules 13 (2012) 2802–2810. [12] J.V. Staros, R.W. Wright, D.M. Swingle, Analytical Biochemistry 156 (1986) 220–222. [13] I.R. Nabiev, V.A. Savchenko, E.S. Efremov, Journal of Raman Spectroscopy 14 (1983) 375–379. [14] J. Suh, M. Moskovits, Journal of the American Chemical Society 108 (1986) 4711–4718. [15] V.K. Sharma, R.A. Yngard, Y. Lin, Advances in Colloid and Interface Science 145 (2009) 83–96. [16] S.-M. Li, N. Jia, M.-G. Ma, Z. Zhang, Q.-H. Liu, R.-C. Sun, Carbohydrate Polymers 86 (2011) 441–447. [17] R. Srivastava, D.K. Tiwari, P.K. Dutta, International Journal of Biological Macromolecules 49 (2011) 863–870. [18] T. Angelova, N. Rangelova, R. Yuryev, N. Georgieva, R. Müller, Materials Science and Engineering C 32 (2012) 1241–1246. [19] R.P. Swatloski, S.K. Spear, J.D. Holbrey, R.D. Rogers, Journal of the American Chemical Society 124 (2002) 4974–4975. [20] S. Zhu, Y. Wu, Q. Chen, Z. Yu, C. Wang, S. Jin, Y. Ding, G. Wu, Green Chemistry 8 (2006) 325–327. [21] H. Ma, B. Zhou, H.-S. Li, Y.-Q. Li, S.-Y. Ou, Carbohydrate Polymers 84 (2011) 383–389. [22] L. Li, S. Zhao, J. Zhang, Z.X. Zhang, H. Hu, Z. Xin, J.K. Kim, Fibers and Polymers 14 (2013) 352–357. [23] C. Tahiri, M.R. Vignon, Cellulose 7 (2000) 177–188. [24] C. AndreiaáCateto, RSC Advances 1 (2011) 1695–1697. [25] K. Jiang, L.S. Schadler, R.W. Siegel, X. Zhang, H. Zhang, M. Terrones, Journal of Materials Chemistry 14 (2004) 37–39. [26] Z. Zhang, B. Zhao, L. Hu, Journal of Solid State Chemistry 121 (1996) 105–110. [27] B. Yin, H. Ma, S. Wang, S. Chen, Journal of Physical Chemistry B 107 (2003) 8898–8904.
4. Conclusion Herein, novel two nanocomposite films of TOC–Phe–AgNPs and TOC–Try–AgNPs with excellent antimicrobial properties have been successfully synthesized in ionic liquid [bmim]Cl homogeneous system and aqueous heterogeneous system. FTIR, ICP and TEM results implied that the obtained samples were amino acid functionalized TEMPO oxidized cellulose–silver nanoparticles nanocomposites. SEM images showed that the silver nanoparticles were dispersed in the amino acid functionalized TEMPO oxidized cellulose (TOC–AA) matrix and the strong interaction was formed between the cellulose and silver particles. The as-prepared
Acknowledgment We are grateful to the National Natural Science Foundation of China (Nos. 21372099 and 21072077), the Guangdong Natural Science Foundation (Nos. 10151063201000051 and 8151063201000016), and the Guangzhou Science & Technology Project (2010Y1-C511) for financial support.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Preparation of TEMPO-oxidized cellulose/amino acid/nanosilver biocomposite film and its antibacterial activity Mingming Huang a , Feiran Chen b , Zhenyou Jiang c , Yiqun Li b,∗ a b c
College of Life Science and Technology, Jinan University, Guangzhou 510632, PR China Department of Chemistry, Jinan University, Guangzhou 510632, PR China School of Medicine, Jinan University, Guangzhou 510632, PR China
a r t i c l e
i n f o
Article history: Received 28 July 2013 Received in revised form 8 October 2013 Accepted 12 October 2013 Available online 17 October 2013 Keywords: TEMPO oxidized cellulose Silver nanoparticles Antibacterial activity
a b s t r a c t Novel biocomposite films were prepared by employing amino acid phenylalanine (Phe) or tryptophan (Try) functionalized TEMPO-oxidized cellulose (TOC) with silver nanoparticles (AgNPs) produced in situ by reduction with silver nitrate and sodium borohydride via homogeneous and heterogeneous approaches. The as-prepared biocomposite films, i.e. TOC–Phe–AgNPs and TOC–Try–AgNPs, were characterized by digital photographs, FTIR, Elemental Analysis, SEM, and TEM and the results confirmed the formation of the desired films. The in vitro antibacterial activity of two resulting composite film of TOC–Phe–AgNPs and TOC–Try–AgNPs was evaluated against Staphylococcus aureus and Escherichia coli bacteria by agar diffusion test. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Cellulose, which is the most abundant renewable polymer resource available on earth, is considered as an almost inexhaustible raw material source for the increasing demand for environment friendly and biocompatible materials [1]. However, due to the extensive network of inter- and intra-molecular hydrogen bonding between its fibrils, cellulose is extremely difficult to dissolve in water and most common organic solvents, meanwhile limited its economically feasible and environment friendly chemical processes required applications [1,2]. Therefore, chemical modifications of cellulose were interesting routes for preparing cellulose derivatives with specific properties [3–5]. Numerous efforts are underway seeking new ways to expand the application range of cellulose while retaining its prominent properties. Among all of them, (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO)mediated oxidation [6–8] of native cellulose appears of particular interest because its mild operative conditions, simplicity, efficiency, and high regioselectivity: only the C6 primary hydroxyl groups presenting on the surfaces of cellulosic microfibrils is converted to C6 carboxylate groups. The existed carboxylic groups can serve as templates to bind various molecules of interest for wide specific potential applications, opening thus new horizons for various applications of oxidized cellulose. For example, importantly, installed carboxyl groups allow the use of conjugation techniques,
∗ Corresponding author. Tel.: +8615521332351. E-mail addresses: [email protected], [email protected] (Y. Li). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.018
such as 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide (EDAC/NHS) activation, for irreversible linking amino acid or proteins onto cellulose [9–12]. Many kinds of preparations of TEMPO-oxidized cellulose have been reported, however, there are very few trials on TEMPOoxidized cellulose containing amino acid molecules, which can server as N,O-bidentate ligand to form a complex with transition metal silver [13,14], etc. Silver ions and silver compounds have been known to have strong antimicrobial activity [15–17] against nearly 650 types of bacteria and have potential applications in various fields like antibacterial filters, wound dressing materials, etc. The silver nanoparticles (AgNPs) show efficient antimicrobial properties compared to other salts due to their extremely large surface area, which provides better contact with microorganisms. The AgNPs are considered to be a slow release source of silver ions, which react with the groups of proteins and interfere with DNA replication [18]. Up to present time, a large number of researches got reported on immobilizing silver nanoparticles on to cellulose and other artificial polymers with potency in drug delivery systems as well as antibacterial applications. Only reports with very scanty information are available on immobilization of silver nanoparticles onto TOC and its derivatives. In the past decade, ionic liquids (ILs) have received enormous interest as solvents for biopolymer dissolution and regeneration owing to their solvent properties and process benefits. For example, it has been reported that ionic liquids 1-n-butyl-3methylimidazolium chloride ([bmim]Cl) was found that it could act as the non-derivatizing solvent for cellulose [19]. Dissolution
M. Huang et al. / International Journal of Biological Macromolecules 62 (2013) 608–613
609
Scheme 1. Sequential scheme of the preparation of amino acid modified TEMPO-oxidized cellulose/silver nanoparticles composite film.
of cellulose and its derivatives in ionic liquids can largely overcome this limitation so that the preparation of cellulose composites and derivatives is becoming more and more convenient. Extensive research has been carried out and much progress achieved in this area for the production of some advanced materials [20]. On the basis of the above points, we have recently studied the preparation of all cellulose composite materials based on cellulose using functionalized ionic liquid 1-hydroxylethly-3-methyl methylimidazolium chloride ([HOC2 mim]Cl) as well as other ionic liquid as solvent [21]. The amino acid functionalized TEMPOoxidized cellulose (TOC–AA) derivatives that are soluble in ionic liquids, so it can be efficiently used for the preparation of high quality films to immobilize AgNPs to construct the antibacterial activity of TOC–AA–AgNPs biocomposite materials. In the present work, effort has been made to prepare new two biocomposite films of amino acid functionalized TOC–AgNPs (TOC–AA–AgNPs). The sequential protocols were based on the steps schematically shown in Scheme 1. Initially, oxidation of cellulose was performed by the TEMPO–NaBr–NaClO system. Then, l-phenylalanine (Phe) or l-tryptophan (Try) was introduced to the TOC matrix using EDAC/NHS as activator, respectively. After that, the amino acid modified TOC and AgNO3 was dissolved in ionic liquid [bmim]Cl to form homogenous mixture (homogeneous system), or suspension in AgNO3 aqueous solution (heterogeneous system), and AgNPs were formed in situ as using NaBH4 as a reducing reagent. Finally, the resulting amino acid functionalized TOC/AgNPs composite (TOC–AA–AgNPs) were prepared successfully. Moreover, the in vitro antibacterial activities were evaluated against bacteria including two Staphylococcus aureus and Escherichia coli microorganisms by agar diffusion test.
analyses were performed on a Perkin Elmer EA2400II elemental analyzer. The elemental silver content of the composite was determined by Perkin Elmer Optima 2000DV inductively coupled plasma (ICP) spectroscopy. Scanning electron microscopy (SEM) was performed with a Philips XL 30ESEM instrument. Transmission electron microscopy (TEM) was performed with a Philips Tecnai instrument operating at 40–100 kV. Microcrystalline cellulose (MCC), [bmim]Cl, NaBH4 , TEMPO, EDAC, AgNO3 and other chemicals were reagent grade and used as purchased. 2.2. Preparation of TEMPO-oxidized cellulose Oxidized cellulose was prepared similarly according to literature method [22,23]. A typical procedure was as follows: Microcrystalline cellulose (0.648 g, i.e. 4 mmol of anhydroglucose units) was dispersed in Na2 CO3 /NaHCO3 buffer solutions (80 ml, pH = 10) for 1 min under moderate stirring. TEMPO (10 mg, 0.065 mmol) and NaBr (0.20 g, 1.9 mmol) were added in the suspension and maintain at 0 ◦ C (use an ice bath.) The sodium hypochlorite solution which was stored at 0 ◦ C (21%, 4.10 ml, 13.20 mmol, pH = 10 adjusted by 0.5 M aqueous HCl) was added every 30 min (four times) and stirring vigorous. When the cellulose almost had disappeared, 10 ml of methanol was added to quench the reaction. The solution was acidified to pH = 3 by adding 0.5 M HCl. Then centrifuged and abandoned the insoluble cellulose. The oxidized cellulose was precipitated by adding five volumes ethanol and centrifuged. The precipitate was washed with ethanol/water (v/v = 9/1) and centrifuged several times, followed dialysed in distilled water for 4 h (water changed every hour), and finally, dried at 40 ◦ C in a vacuum oven.
2. Experimental 2.1. Apparatus and reagents
2.3. Preparation of amino acids functionalized TEMPO-oxidized cellulose
FTIR spectra were recorded on a Bruker Equinox-55 spectrometer as KBr pellets in the range 400–4000 cm−1 . Elemental
The coupling of oxidized cellulose with amino acids is a modified method according to the literature [24,25]. Initially, 10 mg
610
M. Huang et al. / International Journal of Biological Macromolecules 62 (2013) 608–613
Fig. 1. The digital pictures of resulting TOC–Phe–AgNPs biocomposite films (a), TOC–Try–AgNPs biocomposite films (b).
of dried oxidized cellulose was added to 50 ml of deionized water, 23 ml of N-hydroxy-succinimide (NHS) aqueous solution (50 mg ml−1 ) was added after cellulose homogeneously dispersed under moderate magnetic stirring. 12 ml fresh N-ethylN -(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) aqueous solution (10 mg ml−1 ) was added quickly and continually stirring vigorously at room temperature for 30 min, pH was maintained at 7.5–8 by adding 0.5 M NaOH and/or HCl. Then the suspension was dialyzed under distilled water for 8 h (water changed every hour) to remove excess EDAC, NHS and by-product urea. Secondly, 100 mg selected amino acid (l–Phenylanaline, Phe or l–Tryptophan, Try) was added to the 100 ml distilled water of oxidized cellulose under moderate magnetic stirring 24 h at room temperature. The suspension was then dialyzed thoroughly in distilled water for 72 h (water changed every 4–8 h) to remove unbound amino acid. Then the oxidized cellulose bound amino acid was successively obtained by freeze drying.
2.4. Preparation of amino acid modified TEMPO-oxidized cellulose–silver nanoparticles composite film Homogeneous approach: Amino acid modified oxidized cellulose (0.1 g) was dissolved completely in ionic liquid [bmim]Cl (5.0 g) at 80 ◦ C in the oil bath under vacuum for 30 min till the mixture turn to a clear amber solution. Then, AgNO3 (1 × 10−5 mol) was added to the ionic liquid solution by stirring for 10 min at 80 ◦ C. After that, NaBH4 (1 × 10−3 mol) was added slowly under stirring till the mixture turned to dark brown color and kept at this temperature for another 10 min to ensure to form Ag nanoparticles completely. After then, the mixture was poured to a glass plate and rinsed with a large amount of distilled water to remove the excess chemical to produce hydrogel. Finally, the resulting composite film was obtained by freeze drying. The dark brown color of the composite film indicated formation of silver nano-particles. The Ag content of TOC–Phe–AgNPs and TOC–Try–AgNPs was found to be 17.7 mg/g and 17.6 mg/g by ICP, respectively. Heterogeneous approach: TOC–Phe or TOC–Try composite film was immersed in 4 × 10−4 M AgNO3 aqueous solution (50 ml). Then 0.2 M NaBH4 (10 ml) was added dropwise slowly. Upon completion of the reaction, the mixture was maintained for another 1 h under room temperature. Finally, the resulting composite film was treated by the process as given above. The Ag content of TOC–Phe–AgNPs and TOC–Try–AgNPs was found to be 10.1 mg/g and 8.7 mg/g by ICP, respectively. The digital photographs of as-prepared TOC–Phe–AgNPs biocomposite films and TOC–Try–AgNPs biocomposite films are shown in Fig. 1.
2.5. Antibacterial assay of amino acid modified oxidized cellulose–silver nanoparticles composite film Two kinds of bacterial: E. coli and S. aureus were used here for the antibacterial activity tests. E. coli and S. aureus bacterial suspensions was spread onto LB agar plates with Phe and Trp modified cellulose–silver nanoparticles biocomposite film, composite film without nano-Ag were used as control. After incubating at 40 ◦ C for 24 h, the zone of inhibition was observed. 3. Results and discussion 3.1. Fourier transfer infrared spectra and elemental analysis Fig. 2 shows the FTIR spectra of TEMPO oxidized cellulose (TOC) before and after functionalization with amino acids. Curve (a) contained a peak at 1740 cm−1 for the C=O band of carboxylate group TOC. This band is decreased after bounding with amino acids Try or Phe (b, c) due to the formation of amide bond. Curve b and c (particularly c) contained a strong characteristic absorption at 1600–1660 cm−1 which was assigned to the C=O band of the amide, confirming the modification of TOC with amino acid. Curve d and e are FTIR spectra of free Thr and Phe used as reference. The N content was found up to be 0.3 mmol g−1 for TOC–The and 0.15 mmol g−1 for TOC–Try, respectively, by elemental
Fig. 2. FTIR comparative spectra of (a) TEMPO oxidized cellulose (TOC), (b) Try modified TOC (TOC–Try), (c) Phe modified TOC (TOC–Phe), (d) Free Phe, and (e) Free Try.
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Fig. 3. TEM of Phe modified TOC with AgNPs (TOC–Phe–AgNPs, (a)), Trp modified TOC with AgNPs (TOC–Try–AgNPs, (b)), heterogeneous system gets TOC–Phe–AgNPs (c) and TOC–Try–AgNPs (d).
microanalysis, which further indicated the amino acid was introduced to the TOC molecular skeleton. 3.2. Morphology study The morphology of TOC–Phe–AgNPs and TOC–Try–AgNPs was studied by TEM and SEM. TEM micrographs (Fig. 3a and b) reveal that the silver particles distributed on TOC–Phe and TOC–Try are quasi-spherical in shape and consistently distributed in the range of 35 nm. As it can be observed from TEM images of TOC–Phe–AgNPs (Fig. 3c) and TOC–Try–AgNPs (Fig. 3d) prepared from heterogeneous process, silver nanoparticles are not homogenously dispersed on composite film and it is found that nanoparticles have irregular shapes.
The introduction of ligands onto polymeric skeleton is of special interest, because it enables the creation of a specific environment to control the growth of metal nanoparticle seeds. When a linear polymer is used as a stabilizing agent, modification of the functional groups can offer a specific reactive field around the silver nanoparticles that allows the particle size and shape control in the preparation. As an example, PVP, in which existed the C–N and C=O functional moieties, has been successfully used to control the size and shape of silver nanoclusters via coordinative bonding between C–N and C=O/Ag ions. [26,27] In homogeneous system, quasi-sphere silver nanoparticles were fabricated using TOC–AA as a promising template in which its molecular backbone played coordinated and stabilizer role. TOC–AA also contained the C–N and C=O functional groups which also play the role of controlling the size
CO2H CO2H CO2H CO2 H
TMPO-Oxidation
EDAS/NHS R
cellulose molecular
CO2H NH2
R O
CO2H
R
NH
NH
O
R NaBH4
O
R
CO2H O
CO2H NH
R
Ag0 O
CO2H NH
O
R
CO2H NH Ag
R
Ag+ cordinated with terminal amino acid
0
O
CO2H
R
+ NH Ag
O
R
CO2H NH
Ag+ O
CO2H NH
Ag+
CO2H NH
Ag0
Growth of AgNPs
Scheme 2. Schematic illustration of the controlled generation of AgNPs on a TOC–AA molecular skeleton in homogeneous system.
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Fig. 4. SEM of TOC (a), TOC–AgNPs (b), TOC–Phe–AgNPs (c) and TOC–Try–AgNPs (d).
and shape of AgNPs according the model suggested by Ma et al. [27]: First, the complex of TOC–AA/Ag+ ions via coordinative bonding (the ligand of C–N and C=O in TOC–AA contributes electronic density to the sp orbital of silver ions) was constructed. Second,
the complex promotes silver nucleation, which tends to produce small silver particles. Third, the steric effect of TOC–AA covering the silver surface via physical and chemical bonding inhibits particle–particle contact and thus the agglomeration of the powder.
Fig. 5. The digital photographs of antibacterial activities of TOC–Phe–AgNPs against S. aureus (a), TOC–Phe–AgNPs against E. coli (b), TOC–Try–AgNPs against S. aureus (c), TOC–Try–AgNPs against E. coli (d).
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But in the case of heterogeneous process, TOC–AA is insoluble and just immersed in AgNO3 aqueous solution. Thus, silver ions to be reduced almost completely deposited on the surface of the AA–TOC to form irregular shapes. The plausible process of controlled generation of quasi-sphere AgNPs is depicted in Scheme 2. Representative SEM images of TOC, TOC–AgNPs, TOC–Phe–AgNPs and TOC–Try–AgNPs, which prepared in homogeneous approach, was shown in Fig. 4. The AgNPs are distributed in the TOC, TOC–Phe and TOC–Try matrix, as is most readily visible in images b, c and d of Fig. 4. But no apparent change in the morphology was observed in images c and d. (Fig. 4c–d).
TOC–Phe–AgNPs and TOC–Try–AgNPs nanocomposites exhibited an excellent antibacterial activity against both S. aureus and E. coli bacteria.
3.3. Antibacterial activities
References
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4. Conclusion Herein, novel two nanocomposite films of TOC–Phe–AgNPs and TOC–Try–AgNPs with excellent antimicrobial properties have been successfully synthesized in ionic liquid [bmim]Cl homogeneous system and aqueous heterogeneous system. FTIR, ICP and TEM results implied that the obtained samples were amino acid functionalized TEMPO oxidized cellulose–silver nanoparticles nanocomposites. SEM images showed that the silver nanoparticles were dispersed in the amino acid functionalized TEMPO oxidized cellulose (TOC–AA) matrix and the strong interaction was formed between the cellulose and silver particles. The as-prepared
Acknowledgment We are grateful to the National Natural Science Foundation of China (Nos. 21372099 and 21072077), the Guangdong Natural Science Foundation (Nos. 10151063201000051 and 8151063201000016), and the Guangzhou Science & Technology Project (2010Y1-C511) for financial support.
