Macromolecular Rapid Communications
Communication
Novel Bioactive Amino-Functionalized Cellulose Nanofibers Katrin Roemhild, Cornelia Wiegand, Uta-Christina Hipler, Thomas Heinze*
Amino-cellulose-based nanofibers are prepared by electrospinning of blended solutions of 6-deoxy-6-trisaminoethyl-amino (TEAE) cellulose and polyvinyl alcohol (PVA). The TEAE cellulose with a degree of substitution of 0.67 is synthesized via a nucleophilic displacement reaction starting from cellulose-p-toluenesulfonic acid ester. Several solution characteristics such as polymer concentration, electrical conductivity, and surface tension as well as setup parameters are investigated to optimize the ability of nanofiber formation. These parameters are evaluated using the rheological studies of the solutions. The nanofibers obtained are characterized by scanning electron microscopy (SEM) and show a high antimicrobial activity against Staphylococcus aureus and Klebsiella pneumoniae.
1. Introduction Polymers bearing amino groups gain increasing interest due to their promising properties for biomedical applications.[1–4] The water-soluble synthetic polymers, polyethyleneimine and poly(amidoamine)s, are the materials of choice for polyplex formation in gene delivery although they possess a certain toxicity.[5] On the contrary, polysaccharides are nontoxic and can be easily modified by various chemical reactions.[6] An efficient path to obtain Dr. K. Roemhild Center of Excellence for Polysaccharide Research, Thuringian Institute of Textile and Plastics Research e.V., Breitscheidstraße 97, 07407 Rudolstadt, Germany Dr. C. Wiegand, Dr. U.-C. Hipler Department of Dermatology, University Medical Center Jena, Erfurter Str. 35, 07743 Jena, Germany Prof. T. Heinze Center of Excellence for Polysaccharide Research, Institute of Organic Chemistry and Macromolecular Chemistry, Friedrich Schiller University of Jena, Humboldtstraße 10, 07743 Jena, Germany Fax: +49 (0) 3641 9 48272 E-mail: Thomas.Heinze@uni-jena Macromol. Rapid Commun. 2013, 34, 1767−1771 © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
amino group-containing and water-soluble cellulose of varying structures is nucleophilic displacement reaction of p-toluenesulfonic acid ester of the biopolymer with diand oligoamines, leading to water-soluble 6-deoxy-6-(ωaminoalkyl)aminocellulose.[7,8] By using the hydrodynamic technique of analytical ultracentrifugation as a probe, it could be shown that the 6-deoxy-6-aminocelluloses form multiple oligomeric species. This protein-like behavior appears consistent with their important functional ability to oligomerize and further substantiates the high potential of carbohydrates in nanoscience.[9] Very recently, spherical nanoparticles with sizes from 80 to 200 nm were obtained by self-assembly of highly functionalized 6-deoxy-6-(ω-aminoalkyl)aminocellulosecarbamates. The particles are very stable, nontoxic, and possess primary amino groups that are accessible to further modifications in aqueous suspension. Incorporation of the nanoparticles in human foreskin fibroblasts hTERTBJ1 and breast carcinoma MCF-7 cells occurs without any transfection reagent.[10] The electrospinning technique is a widely used method for the production of nanofiber, which opens up a way to realize high effective surface areas.[11] Such fibers are made from different polymers and find interest in various
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application fields[11–14] namely in biomedical, composite, filter, catalytic, and textile applications. In general, the fiber-forming ability depends on both the specific solution or melt characteristics like rheologic parameters, surface tension, or electric conductivity and the spinning conditions like the applied voltage, the electrode distance, or the humidity of the surrounding area, for example.[15] Nevertheless, electrospinning of polyelectrolytes from aqueous solutions is not successful in the majority of cases. Especially, amino cellulose derivatives exhibit very special rheological and self-structuring properties[9] that may hinder the formation of fibers in the electrical field. Commonly applied procedures for such problematic polymers are, on the one hand, the addition of cosolvents or salts to reduce repulsive forces between ionic groups of the polymer chain.[16] On the other hand, water-soluble polymers showing a stable-fiber-forming behavior like polyvinyl alcohol (PVA) or polyethylene oxide are used to assist fiber formation.[17–19] Wound infection is a key factor in compromised wound healing as bacteria maintain an inflammatory environment that hinders re-epithelization.[20–25] Often chronic wounds are polymicrobial, and infections involve mixed populations of aerobic and anaerobic bacteria. Staphylococcus aureus is considered to be the most problematic germ in traumatic, surgical, and burn wound infections but other microorganisms such as Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae may also play a role in chronic wound infection.[26,27] Mostly antibiotics are used to reduce the bacterial burden and battle infection. However, as widespread application of systemic and topical antibiotics has been associated with the emergence of resistant bacterial strains such as methicillin resistant Staphylococcus aureus (MRSA), the need for alternative antimicrobials is evident. Antimicrobial polymers have raised increasing interest in the area of health care and hygienic applications as they may be incorporated into fibers, coated on glass surfaces, or applied onto plastics, reducing biofouling and adherence of pathogenic microorganisms.[28] The aim of our work was to study the ability to form nanofibers of amino cellulose by means of electrospinning and to evaluate the products regarding their antimicrobial activities.
2. Experimental Section 2.1. Materials Polyvinyl alcohol [PVA; 92% hydrolyzed, 4% in H2O (20 °C): 17.00 ± 2.50 cP] was obtained from Celanese. 6-Deoxy-6trisaminoethyl-amino (TEAE) cellulose was synthesized starting from microcrystalline cellulose (Avicel PH 101, Fluka) via cellulose-p-toluenesulfonic acid ester (tosylate) with tosyl chloride
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according to Rahn et al.[20] Briefly, the cellulose tosylate with a degree of substitution (DS) of tosyl moieties of 0.99 was allowed to react with 59.10 mL trisaminoethyl amine (25 mol mol−1 AGU), yielding TEAE cellulose with a DS of 0.67 determined from the elemental analysis (C: 48.53%; H: 8.05%; N: 13.10%; S: 2.06%). There is a residual DS of tosyl groups of 0.19. The chitosan used was purchased from BioLog company Biotechnology und Logistics GmbH, Germany, as Chitosan 85/120/ A1 and has a deacetylation degree of 85%, an Mw of 220 000 Da, and a polydispersity index (PDI) of 1.7 (SEC: multidetector calibration, dn/dc: 0.163).[21] S. aureus ATCC 6538 and K. pneumoniae ATCC 4352 were purchased from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany). For cultivation of bacteria, special peptone and “lab-lemco” powder for preparation of casobouillon were obtained from Oxoid (UK). Columbia agar plates with 5% sheep blood were purchased from Biomeriéux (France). NaCl solution was obtained from Fresenius Kabi Deutschland GmbH (Germany) and Tween 20 was purchased from Roth (Germany).
2.2. Analytical Methods The polymer content of the mixed polymer solutions in distilled water was adjusted to 7.9% and 9.5% and both contain ≈0.5% TEAE cellulose. The electrical conductivities of the solvents were measured with a conductivity meter (703, Knick at 23 °C) and the surface tension with a Gibertini digital surface tensiometer. The rheological characterization of the polymer solutions was carried out using a rheometer HAAKE Rheostress 100 equipped with a cylinder-measuring system and a thermostat at 25 °C. The measurement was performed in the rotation mode. Determination of the solid contents of the solutions was performed by means of weighing precipitated films, after exhaustive solvent extraction and drying. Morphology of the electrospun fibers (webs) was studied with a scanning electron microscope (Auriga Crossbeam, Carl Zeiss, Germany) at a voltage of 3 kV and a working distance of 4.7 mm after 5 min gold sputtering.
2.3. Antibacterial Activity Determination of antimicrobial activity was performed according to JIS L 1902:2002[29] as previously reported.[30,31] In brief, 20 mL caso-bouillon was inoculated either with S. aureus or K. pneumoniae and cultivated for 24 h at 37 °C. For experiments, 400 mg samples of the polypropylene (PP) web with the electrospun fibers were incubated with 200 μL of the bacterial suspension for 24 h at 37 °C. Polyester was used as growth control. For bacteria quantification, the incubated samples were extracted in 10 mL 0.9% NaCl solution with Tween 20. Serial dilutions were plated on Columbia agar plates, incubated for 24 h at 37 °C and the colonies counted afterward. Total microbial count of the samples in [cfu] and microbial growth in [%] were calculated. Growth reduction compared with the starting value was determined according to JIS L 1902:2002.[29]
Microbial growth [%] =
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Sample [cfu] × 100 Control [cfu]
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Table 1. Composition and properties of the polymer solutions of 6-deoxy-6-triaminotriethylamino (TEAE) cellulose and polyvinyl alcohol (PVA) used.
Sample
Polymer composition
Polymer content [%]
Conductivity [μs cm−1]
Surface tension [mN m−1]
E-mat 1
TEAE cellulose/PVA 1/15
7.9
865
49.5
E-mat 2
TEAE cellulose/PVA 1/18
9.5
943
49.5
E-mat 3a)
Chitosan/PVA 1/18
9.6
1366
n.d.
E-mat PVA 1
PVA
10.1
1072
n.d.
E-mat PVA 2
PVA
13.1
1213
58.4
a)In
acetic acid.
growth reduction [log cfu] = log control [cfu] − log sample [cfu] Rating : no antibacterial activity = growth reduction < 0.5 [log cfu] slight antibacterial activity = growth reduction > 0.5 [log cfu] and < 1.0 [log cfu] significant antibacterbial activity = growth reduction > 1.0 [log cfu] and = 3.0 [log cfu] strong antibacterial activity = growth reduction > 3.0 [log cfu]
2.4. Electrospinning Process For the electrospinning process, a Nanospider Lab 500 spinning instrument (Elmarco s.r.o, Czech Republic) with a pike spinning electrode was used. The fibers were continuously formed in the electrical field by drawing them from a film that the lower part of the rotating electrode steadily withdraws from the polymer solution placed in a bottom reservoir and collected on a PP-support web, fixed at a distance of 15 cm above this electrode. Spinning was performed at an ambient humidity of 30%–40% at 19 °C and an applied voltage of 70 kV from bottom to top electrode, where the whole setup was placed in an electrically insulated housing. The content of electrospun fibers on the PP web was determined by weighing before and after spinning.
sensitive to solution properties, especially viscosity. The solubility of TEAE is restricted to low concentrations and therefore, PVA was added as modifier. These blend solutions were prepared by mixing the 1% aqueous solution of TEAE cellulose and a 15% (E-mat 1) or 18% solution of PVA (E-mat 2) in water in the ratio 1:1. The high concentration of PVA has been deduced from previous studies, in which an aqueous PVA solution did not show sufficient fiber forming at concentrations below 10%. Beside rheological properties, especially conductivity and surface tension contribute significantly to the fiberforming ability.[32] As summarized in Table 1, the characteristics of both solutions of TEAE cellulose and PVA are at least similar to that of pure PVA solution in terms of their conductivity despite differences in composition. Differences are obvious due to the surface tension values of pure PVA solution and solutions, indicating of both polymers as a result of surface activity of the TEAE cellulose. The most important difference is the rheological behavior of the blend solutions. Whereas pure PVA solutions with a concentration of 10% or 13% polymer show Newton-like rheological behavior, the TEAE-cellulose-mixed blend solutions have a slight but significant shear-thinning effect. It is rather remarkable that despite the lower polymer content of 9.5% of the E-mat 2 blend solution, the viscosity level is slightly higher than that of the 10% PVA solution (Figure 1).
3. Results and Discussion Initial studies were done with pure 1% aqueous solution of TEAE cellulose under different conditions. However, droplets were formed rather than stable elongated species. Processing the low-viscous solution initially failed since the electrospinning process is known to be very
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Figure 1. Rheological behavior of 6-deoxy-trisaminoethylamino (TEAE)-cellulose-mixed blend solutions compared with the pure solution of polyvinyl alcohol (PVA) at 25 °C.
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Figure 2. SEM pictures of the different nanofiber webs: E-mat 1: 6-deoxy-6-trisaminoethylamino (TEAE) cellulose/polyvinyl alcohol (PVA) 1/15; E-mat 2: TEAE cellulose/PVA 1/18; E-mat PVA: PVA.
Applying a voltage of 70 kV and a plate distance of 15 cm, the blends of TEAE cellulose/PVA solutions could be electrospun to create a coating on a PP mesh constructed of defect-free nanofibers with diameters ranging from 50 to 160 nm. The nanofiber contents on the PP support were 19.7% (E-mat 1) and 13.4% (E-mat 2) after 20 min time of spinning. Compared with the pure PVA web, spun from a 13% solution (60–500 nm), the fibers are thinner as determined by the fiber diameter analysis. In both cases, the pure PVA E-mat and the TEAE-cellulose-containing mats, freshly spun fibers converge and flow together prior to consolidation resulting in thicker fibers. The scanning electron microscopy (SEM) images even sporadically show species formed by superposing two individual fibers at which the phase boundary is still visible as small groove mark. Irregular fiber morphologies are then expressed in form of oblique or random fiber length distributions that do not accurately reflect the initial fiber diameters (Figure 2). The electrospun fiber samples E-mat 1 and E-mat 2 achieved a strong reduction of S. aureus growth in the
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test. Moreover, these samples exhibited a significant antibacterial efficacy against the gram-negative K. pneumoniae. The differences in the bactericidal capacity against the bacteria species are most likely due to the differences in the cell wall properties of gram-positive and gramnegative bacteria.[33] The samples E-mat PVA and E-mat 3 only marginally affected bacterial growth of both, S. aureus and K. pneumonia (Figure 3).
4. Conclusion TEAE celluloses blended with PVA were successfully electrospun to nanofibrous webs with fiber diameters between 50 and 160 nm blending. The electrospinning conditions were optimized regarding the blend ratio, the polymer concentration, the voltage and the two working electrodes distance. Contrary to E-mat PVA and chitosan-containing E-mat 3, the TEAE-containing nanofiber nonwovens showed a strong reduction of S. aureus growth and a significant antibacterial efficacy against the gram-negative K. pneumoniae.
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[7] [8] [9]
[10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] Figure 3. Inhibition of the growth of a) Staphylococcus aureus and b) Klebsiella pneumoniae by different nanofiber webs.
[21]
[22] Received: August 2, 2013; Revised: September 26, 2013; Published online: October 22, 2013; DOI: 10.1002/marc.201300588
Keywords: amino cellulose; antimicrobial activity; electrospinning; nanofibers
[23] [24] [25] [26] [27]
[1] N. T. K. Thanh, L. A. W. Green, Nano Today 2010, 5, 213. [2] C. Lemarchand, R. Gref, P. Couvreur, Eur. J. Pharm. Biopharm. 2004, 58, 27. [3] S. Dong, M. Roman, J. Am. Chem. Soc. 2007, 129, 13810. [4] J. L. Cohen, S. Schubert, P. R. Wich, L. Cui, J. A. Cohen, J. L. Mynar, J. M. J. Frechet, Bioconjugate Chem. 2011, 22, 1056. [5] M. Liu, J. Chen, Y. P. Cheng, Y. N. Xue, R. X. Zhuo, S. W. Huang, Macromol. Biosci. 2010, 10, 384. [6] Th. Heinze, T. Liebert, Celluloses and Polyoses/Hemicelluloses, in Polymer Science: A Comprehensive Reference, Vol. 10
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[28] [29] [30] [31] [32] [33]
(Eds: K. Matyjaszewski, M. Möller), Elsevier, Amsterdam, NL 2012, pp. 83–152. J. Tiller, P. Berlin, D. Klemm, Biotechnol. Appl. Biochem. 1999, 30, 155. P. Berlin, D. Klemm, J. Tiller, R. Rieseler, Macromol. Chem. Phys. 2000, 201, 2070. Th. Heinze, M. Nikolajski, St. Daus, T. M. D. Besong, N. Michaelis, P. Berlin, G. A. Morris, A. J. Rowe, St. E. Harding, Angew. Chem Int. Ed. 2011, 50, 8602. M. Nikolajski, J. Wotschadlo, J. H. Clement, Th. Heinze, Macromol. Biosci. 2012, 12, 920. A. Greiner, J. H. Wendorff, Angew. Chem Int. Ed. 2007, 46, 5670. D. H. Reneker, I. Chun, Nanotechnology 1996, 7, 216. A. Frenot, I. S. Chronakis, Curr. Opin. Colloid Interface Sci. 2003, 8, 64. J. Xie, X. Li, Y. Xia, Macromol. Rapid Commun. 2008, 29, 1775. Z.-M. Huang, Y.-Z. Zhang, M. Kotaki, S. Ramakrishna, Compos. Sci. Technol. 2003, 63, 2223. M. G. McKee, M. T. Hunley, J. M. Layman, T. E. Long, Macromolecules 2006, 39, 575. K. Desai, K. Kit, J. Li, S. Zivanovic, Biomacromolecules, 2008, 9(3), 1000. J. H. Park, H. W. Lee, D. K. Chae, W. Oh, J. D. Yun, Y. Deng, J. H. Yeum, Colloid. Polym. Sci. 2009, 287, 943. S. Sundarrajan, S. Ramakrishna, Int. J. Green Nanotechnol. 2011, 3(3), 244. K. Rahn, M. Diamantoglou, H. Berghmans, D. Klemm, T. Heinze, Angew. Makromol. Chem. 1996, 238, 143. M. X. Weinhold, J. C. M. Sauvageau, N. Keddig, M. Matzke, B. Tartsch, I. Grunwald, C. Kuebel, B. Jastorff, J. Thoeming, Green Chem. 2009, 11, 498. R. G. Sibbald, H. Orsted, G. S. Schultz, P. Coutts, D. Keast, Ostomy Wound Manage. 2003, 49, 23. V. Falanga, Wound Rep. Reg. 2000, 8(5), 347. R. Warriner, R. Burrell, Adv. Skin Wound Care 2005, 18(8), 2. J. B. Wright, K. Lam, A. G. Buret, M. E. Olson, R. E. Burrell, Wound Rep. Reg. 2002, 10, 141. J. Dissemond, E. N. Schmid, S. Esser, M. Witthoff, M. Goos, Hautarzt 2004, 55, 280. P. G. Bowler, B. I. Duerden, D. G. Armstrong, Clin. Microbiol. Rev. 2001, 14(2), 244. E. R. Kenawy, S. D. Worley, R. Broughton, Biomacromolecules 2007, 8, 1359. Japanese Industrial Standard JIS L 1902:2002, Testing for antibacterial activity and efficacy on textile products. C. Wiegand, T. Heinze, U.-C. Hipler, Wound Rep. Reg. 2009, 17(4), 511. C. Wiegand, M. Abel, P. Ruth, U.-C. Hipler, Skin Pharmacol. Physiol. 2012, 25, 288. J. M. Deitzel, J. Kleinmeyer, D. Harris, N. C. Beck Tan, Polymer 2001, 42, 261. G. McDonnell, A. D. Russel, Clin. Microbiol. Rev. 1999, 12, 147.
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Communication
Novel Bioactive Amino-Functionalized Cellulose Nanofibers Katrin Roemhild, Cornelia Wiegand, Uta-Christina Hipler, Thomas Heinze*
Amino-cellulose-based nanofibers are prepared by electrospinning of blended solutions of 6-deoxy-6-trisaminoethyl-amino (TEAE) cellulose and polyvinyl alcohol (PVA). The TEAE cellulose with a degree of substitution of 0.67 is synthesized via a nucleophilic displacement reaction starting from cellulose-p-toluenesulfonic acid ester. Several solution characteristics such as polymer concentration, electrical conductivity, and surface tension as well as setup parameters are investigated to optimize the ability of nanofiber formation. These parameters are evaluated using the rheological studies of the solutions. The nanofibers obtained are characterized by scanning electron microscopy (SEM) and show a high antimicrobial activity against Staphylococcus aureus and Klebsiella pneumoniae.
1. Introduction Polymers bearing amino groups gain increasing interest due to their promising properties for biomedical applications.[1–4] The water-soluble synthetic polymers, polyethyleneimine and poly(amidoamine)s, are the materials of choice for polyplex formation in gene delivery although they possess a certain toxicity.[5] On the contrary, polysaccharides are nontoxic and can be easily modified by various chemical reactions.[6] An efficient path to obtain Dr. K. Roemhild Center of Excellence for Polysaccharide Research, Thuringian Institute of Textile and Plastics Research e.V., Breitscheidstraße 97, 07407 Rudolstadt, Germany Dr. C. Wiegand, Dr. U.-C. Hipler Department of Dermatology, University Medical Center Jena, Erfurter Str. 35, 07743 Jena, Germany Prof. T. Heinze Center of Excellence for Polysaccharide Research, Institute of Organic Chemistry and Macromolecular Chemistry, Friedrich Schiller University of Jena, Humboldtstraße 10, 07743 Jena, Germany Fax: +49 (0) 3641 9 48272 E-mail: Thomas.Heinze@uni-jena Macromol. Rapid Commun. 2013, 34, 1767−1771 © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
amino group-containing and water-soluble cellulose of varying structures is nucleophilic displacement reaction of p-toluenesulfonic acid ester of the biopolymer with diand oligoamines, leading to water-soluble 6-deoxy-6-(ωaminoalkyl)aminocellulose.[7,8] By using the hydrodynamic technique of analytical ultracentrifugation as a probe, it could be shown that the 6-deoxy-6-aminocelluloses form multiple oligomeric species. This protein-like behavior appears consistent with their important functional ability to oligomerize and further substantiates the high potential of carbohydrates in nanoscience.[9] Very recently, spherical nanoparticles with sizes from 80 to 200 nm were obtained by self-assembly of highly functionalized 6-deoxy-6-(ω-aminoalkyl)aminocellulosecarbamates. The particles are very stable, nontoxic, and possess primary amino groups that are accessible to further modifications in aqueous suspension. Incorporation of the nanoparticles in human foreskin fibroblasts hTERTBJ1 and breast carcinoma MCF-7 cells occurs without any transfection reagent.[10] The electrospinning technique is a widely used method for the production of nanofiber, which opens up a way to realize high effective surface areas.[11] Such fibers are made from different polymers and find interest in various
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DOI: 10.1002/marc.201300588
1767
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K. Roemhild et al.
www.mrc-journal.de
application fields[11–14] namely in biomedical, composite, filter, catalytic, and textile applications. In general, the fiber-forming ability depends on both the specific solution or melt characteristics like rheologic parameters, surface tension, or electric conductivity and the spinning conditions like the applied voltage, the electrode distance, or the humidity of the surrounding area, for example.[15] Nevertheless, electrospinning of polyelectrolytes from aqueous solutions is not successful in the majority of cases. Especially, amino cellulose derivatives exhibit very special rheological and self-structuring properties[9] that may hinder the formation of fibers in the electrical field. Commonly applied procedures for such problematic polymers are, on the one hand, the addition of cosolvents or salts to reduce repulsive forces between ionic groups of the polymer chain.[16] On the other hand, water-soluble polymers showing a stable-fiber-forming behavior like polyvinyl alcohol (PVA) or polyethylene oxide are used to assist fiber formation.[17–19] Wound infection is a key factor in compromised wound healing as bacteria maintain an inflammatory environment that hinders re-epithelization.[20–25] Often chronic wounds are polymicrobial, and infections involve mixed populations of aerobic and anaerobic bacteria. Staphylococcus aureus is considered to be the most problematic germ in traumatic, surgical, and burn wound infections but other microorganisms such as Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae may also play a role in chronic wound infection.[26,27] Mostly antibiotics are used to reduce the bacterial burden and battle infection. However, as widespread application of systemic and topical antibiotics has been associated with the emergence of resistant bacterial strains such as methicillin resistant Staphylococcus aureus (MRSA), the need for alternative antimicrobials is evident. Antimicrobial polymers have raised increasing interest in the area of health care and hygienic applications as they may be incorporated into fibers, coated on glass surfaces, or applied onto plastics, reducing biofouling and adherence of pathogenic microorganisms.[28] The aim of our work was to study the ability to form nanofibers of amino cellulose by means of electrospinning and to evaluate the products regarding their antimicrobial activities.
2. Experimental Section 2.1. Materials Polyvinyl alcohol [PVA; 92% hydrolyzed, 4% in H2O (20 °C): 17.00 ± 2.50 cP] was obtained from Celanese. 6-Deoxy-6trisaminoethyl-amino (TEAE) cellulose was synthesized starting from microcrystalline cellulose (Avicel PH 101, Fluka) via cellulose-p-toluenesulfonic acid ester (tosylate) with tosyl chloride
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according to Rahn et al.[20] Briefly, the cellulose tosylate with a degree of substitution (DS) of tosyl moieties of 0.99 was allowed to react with 59.10 mL trisaminoethyl amine (25 mol mol−1 AGU), yielding TEAE cellulose with a DS of 0.67 determined from the elemental analysis (C: 48.53%; H: 8.05%; N: 13.10%; S: 2.06%). There is a residual DS of tosyl groups of 0.19. The chitosan used was purchased from BioLog company Biotechnology und Logistics GmbH, Germany, as Chitosan 85/120/ A1 and has a deacetylation degree of 85%, an Mw of 220 000 Da, and a polydispersity index (PDI) of 1.7 (SEC: multidetector calibration, dn/dc: 0.163).[21] S. aureus ATCC 6538 and K. pneumoniae ATCC 4352 were purchased from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany). For cultivation of bacteria, special peptone and “lab-lemco” powder for preparation of casobouillon were obtained from Oxoid (UK). Columbia agar plates with 5% sheep blood were purchased from Biomeriéux (France). NaCl solution was obtained from Fresenius Kabi Deutschland GmbH (Germany) and Tween 20 was purchased from Roth (Germany).
2.2. Analytical Methods The polymer content of the mixed polymer solutions in distilled water was adjusted to 7.9% and 9.5% and both contain ≈0.5% TEAE cellulose. The electrical conductivities of the solvents were measured with a conductivity meter (703, Knick at 23 °C) and the surface tension with a Gibertini digital surface tensiometer. The rheological characterization of the polymer solutions was carried out using a rheometer HAAKE Rheostress 100 equipped with a cylinder-measuring system and a thermostat at 25 °C. The measurement was performed in the rotation mode. Determination of the solid contents of the solutions was performed by means of weighing precipitated films, after exhaustive solvent extraction and drying. Morphology of the electrospun fibers (webs) was studied with a scanning electron microscope (Auriga Crossbeam, Carl Zeiss, Germany) at a voltage of 3 kV and a working distance of 4.7 mm after 5 min gold sputtering.
2.3. Antibacterial Activity Determination of antimicrobial activity was performed according to JIS L 1902:2002[29] as previously reported.[30,31] In brief, 20 mL caso-bouillon was inoculated either with S. aureus or K. pneumoniae and cultivated for 24 h at 37 °C. For experiments, 400 mg samples of the polypropylene (PP) web with the electrospun fibers were incubated with 200 μL of the bacterial suspension for 24 h at 37 °C. Polyester was used as growth control. For bacteria quantification, the incubated samples were extracted in 10 mL 0.9% NaCl solution with Tween 20. Serial dilutions were plated on Columbia agar plates, incubated for 24 h at 37 °C and the colonies counted afterward. Total microbial count of the samples in [cfu] and microbial growth in [%] were calculated. Growth reduction compared with the starting value was determined according to JIS L 1902:2002.[29]
Microbial growth [%] =
Macromol. Rapid Commun. 2013, 34, 1767−1771 © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Sample [cfu] × 100 Control [cfu]
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Table 1. Composition and properties of the polymer solutions of 6-deoxy-6-triaminotriethylamino (TEAE) cellulose and polyvinyl alcohol (PVA) used.
Sample
Polymer composition
Polymer content [%]
Conductivity [μs cm−1]
Surface tension [mN m−1]
E-mat 1
TEAE cellulose/PVA 1/15
7.9
865
49.5
E-mat 2
TEAE cellulose/PVA 1/18
9.5
943
49.5
E-mat 3a)
Chitosan/PVA 1/18
9.6
1366
n.d.
E-mat PVA 1
PVA
10.1
1072
n.d.
E-mat PVA 2
PVA
13.1
1213
58.4
a)In
acetic acid.
growth reduction [log cfu] = log control [cfu] − log sample [cfu] Rating : no antibacterial activity = growth reduction < 0.5 [log cfu] slight antibacterial activity = growth reduction > 0.5 [log cfu] and < 1.0 [log cfu] significant antibacterbial activity = growth reduction > 1.0 [log cfu] and = 3.0 [log cfu] strong antibacterial activity = growth reduction > 3.0 [log cfu]
2.4. Electrospinning Process For the electrospinning process, a Nanospider Lab 500 spinning instrument (Elmarco s.r.o, Czech Republic) with a pike spinning electrode was used. The fibers were continuously formed in the electrical field by drawing them from a film that the lower part of the rotating electrode steadily withdraws from the polymer solution placed in a bottom reservoir and collected on a PP-support web, fixed at a distance of 15 cm above this electrode. Spinning was performed at an ambient humidity of 30%–40% at 19 °C and an applied voltage of 70 kV from bottom to top electrode, where the whole setup was placed in an electrically insulated housing. The content of electrospun fibers on the PP web was determined by weighing before and after spinning.
sensitive to solution properties, especially viscosity. The solubility of TEAE is restricted to low concentrations and therefore, PVA was added as modifier. These blend solutions were prepared by mixing the 1% aqueous solution of TEAE cellulose and a 15% (E-mat 1) or 18% solution of PVA (E-mat 2) in water in the ratio 1:1. The high concentration of PVA has been deduced from previous studies, in which an aqueous PVA solution did not show sufficient fiber forming at concentrations below 10%. Beside rheological properties, especially conductivity and surface tension contribute significantly to the fiberforming ability.[32] As summarized in Table 1, the characteristics of both solutions of TEAE cellulose and PVA are at least similar to that of pure PVA solution in terms of their conductivity despite differences in composition. Differences are obvious due to the surface tension values of pure PVA solution and solutions, indicating of both polymers as a result of surface activity of the TEAE cellulose. The most important difference is the rheological behavior of the blend solutions. Whereas pure PVA solutions with a concentration of 10% or 13% polymer show Newton-like rheological behavior, the TEAE-cellulose-mixed blend solutions have a slight but significant shear-thinning effect. It is rather remarkable that despite the lower polymer content of 9.5% of the E-mat 2 blend solution, the viscosity level is slightly higher than that of the 10% PVA solution (Figure 1).
3. Results and Discussion Initial studies were done with pure 1% aqueous solution of TEAE cellulose under different conditions. However, droplets were formed rather than stable elongated species. Processing the low-viscous solution initially failed since the electrospinning process is known to be very
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Figure 1. Rheological behavior of 6-deoxy-trisaminoethylamino (TEAE)-cellulose-mixed blend solutions compared with the pure solution of polyvinyl alcohol (PVA) at 25 °C.
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Figure 2. SEM pictures of the different nanofiber webs: E-mat 1: 6-deoxy-6-trisaminoethylamino (TEAE) cellulose/polyvinyl alcohol (PVA) 1/15; E-mat 2: TEAE cellulose/PVA 1/18; E-mat PVA: PVA.
Applying a voltage of 70 kV and a plate distance of 15 cm, the blends of TEAE cellulose/PVA solutions could be electrospun to create a coating on a PP mesh constructed of defect-free nanofibers with diameters ranging from 50 to 160 nm. The nanofiber contents on the PP support were 19.7% (E-mat 1) and 13.4% (E-mat 2) after 20 min time of spinning. Compared with the pure PVA web, spun from a 13% solution (60–500 nm), the fibers are thinner as determined by the fiber diameter analysis. In both cases, the pure PVA E-mat and the TEAE-cellulose-containing mats, freshly spun fibers converge and flow together prior to consolidation resulting in thicker fibers. The scanning electron microscopy (SEM) images even sporadically show species formed by superposing two individual fibers at which the phase boundary is still visible as small groove mark. Irregular fiber morphologies are then expressed in form of oblique or random fiber length distributions that do not accurately reflect the initial fiber diameters (Figure 2). The electrospun fiber samples E-mat 1 and E-mat 2 achieved a strong reduction of S. aureus growth in the
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test. Moreover, these samples exhibited a significant antibacterial efficacy against the gram-negative K. pneumoniae. The differences in the bactericidal capacity against the bacteria species are most likely due to the differences in the cell wall properties of gram-positive and gramnegative bacteria.[33] The samples E-mat PVA and E-mat 3 only marginally affected bacterial growth of both, S. aureus and K. pneumonia (Figure 3).
4. Conclusion TEAE celluloses blended with PVA were successfully electrospun to nanofibrous webs with fiber diameters between 50 and 160 nm blending. The electrospinning conditions were optimized regarding the blend ratio, the polymer concentration, the voltage and the two working electrodes distance. Contrary to E-mat PVA and chitosan-containing E-mat 3, the TEAE-containing nanofiber nonwovens showed a strong reduction of S. aureus growth and a significant antibacterial efficacy against the gram-negative K. pneumoniae.
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[7] [8] [9]
[10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] Figure 3. Inhibition of the growth of a) Staphylococcus aureus and b) Klebsiella pneumoniae by different nanofiber webs.
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[22] Received: August 2, 2013; Revised: September 26, 2013; Published online: October 22, 2013; DOI: 10.1002/marc.201300588
Keywords: amino cellulose; antimicrobial activity; electrospinning; nanofibers
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