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Incorporation of reactive silver-tricalcium phosphate nanoparticles into polyamide 6 allows preparation of self-disinfecting fibers.

INTRODUCTION

Synthetic fibers - industrial and commercial - are omnipresent. In 2002, the global production of manufactured fibers was 36 million metric tons: Fiberglass pads build modern cars, polyesters form textile goods, and polyamide fibers are used as bristles for brooms or toothbrushes.

Providing antibacterial fibers is desirable as microbial contamination may imply serious health risks. Disinfection commonly involves germicides that need to be applied regularly and compromise human and the environment.

There are well-known problems with disinfectants besides the acquired resistances by bacteria (1). Many disinfectants are corrosive, irritate skin or mucosa, and can be inflammable or even explosive. If not disposed properly, they finally end up in a sewage plant and disrupt the cleaning sludge's growth or further proliferate into the environment.

A particular disadvantage of externally applied disinfectants is not related to their chemical composition but lies in the type of application. A disinfectant must be applied regularly. Between two applications, the concentration must be either dangerously high, or the agent is intermittently absent and thus germs may grow between two applications. An approach to overcome this limitation is to engineer a reactive agent into in a fiber/material.

Recently, the excellent antibacterial effect of silver-tricalcium phosphate (Ag/TCP) on clinically important bacteria Candida albicans and Pseudomonas aeruginosa has been shown on flat polymer coatings [2]. There, active, bacteria-triggered release has enabled to increase silver activity by 3-6 orders of magnitude. The antibacterial and antifungal properties of silver and silver-based compounds against a wide range of microorganisms are well known (2-8). Silver is highly effective against bacteria and has a relatively low toxicity to human cells (9), (10). The Reference Dose for Chronic Oral Exposure (RfD) published by the United States Environment Protection Agency (US-EPA) is 0.005 mg/kg/day (11). Only long-term exposition to silver in diverse forms such as elemental silver, silver dust, silver compounds, or colloidal silver through (homeopathic/alternative) pharmaceuticals or dietary supplements may lead to the so-called Argyria (a very rare, irreversible discoloration of the skin resulting from subepithelial silver deposits) (12). Similar toxic effects of metal-ions on living cells (oligodynamic effect) such as algae, molds, spores, fungi, viruses, and other prokaryotic and eukaryotic microorganisms are also observed with mercury, copper, tin, iron, or lead.

The present contribution investigates the bacteria-triggered release of antibacterial silver in common polymer fibers, using reactive Ag/TCP nanoparticles implemented in polyamide 6 (PA6) (Scheme 1). Therefore, our study translates active, triggered silver release from previously investigated simple surfaces into reactive polymer fibers.

[ILLUSTRATION OMITTED]

EXPERIMENTAL

For this study, Ag/TCP with a silver content of 1.3 wt% was produced by flame spray synthesis (13). These nanoparticles were extruded with bulk PA6 at a filler content of 2 wt%. Subsequent melt-spinning of the extrudates resulted in fibers that were collected as bundles and subjected to antibacterial testing, against Escherichia coli as a representative model bacterium. A second clinically relevant strain, Streptococcus sanguinis, a primary colonizer of the oral cavity leading to caries (14), was tested for the later development of self-disinfecting oral care products.

Preparation of the Ag/TCP Nanoparticles

Silver-tricalcium phosphate nanoparticles were prepared by flame spray synthesis (13), (15), (16). Briefly, calcium hydroxide (puriss. Ph. Eur., Riedel de Haen) dissolved in 2-ethylhexanoic acid (puriss. [greater than or equal to] 99%, Fluka) and tributyl phosphate (97%, Aldrich) were used as calcium and phosphor precursor, respectively (17). Silver acetate (purum. [greater than or equal to] 99%, Fluka) dissolved in 2-ethylhexanoic acid (puriss. [greater than or equal to] 99%, Fluka) was used as silver precursor (18). The liquid mixture with a molar Ca/P ratio = 1.5 and silver content of 2 wt% was obtained by mixing the corresponding amounts (5). This mixture was fed through a capillary into a methane-oxygen supporting flame, which ignited the dispersed mixture. The particles built during the combustion were collected on a baghouse filter unit (19).

Characterization of the Ag/TCP Nanoparticles

The specific surface area (SSA) of Ag/TCP nanoparticles was measured by nitrogen adsorption at 77 K according to Brunauer-Emmett-Teller (BET) after outgas-sing for 1 h at 150[degrees]C. The silver content in the Ag/TCP particles was measured after dissolution in concentrated nitric acid and detection employing flame atomic absorption spectroscopy (AAS). The Varian SpectrAA 220FS device was equipped with a slit width of 0.5 nm, lamp current 4.0 mA, air (PanGas) flow 13.5 L/min, acetylene (PanGas) flow 2.1 L/min, measuring the absorption at 328.1 nm. The hydrodynamic particle size distribution was measured on an X-ray disc centrifuge (BI-XDC, Brookhaven Instruments) using a 1.5% (wt/vol) of Ag/ TCP in EtOH (p.a., MERCK) dispersed by ultrasoninca-tion (UP400S, 24 kHz, Hielscher GmbH) at 200 W for 2 min (20).

Scanning electron microscopy (SEM) images were recorded on a Zeiss LEO Gemini 1530; transmission electron microscopy (TEM) images on a CM30 ST (Philips, LaB6 cathode, operated at 300 kV, point resolution ~4 [for all]). Scanning transmission electron microscopy (STEM) images were obtained with a high angle annular darkfield (HAADF) detector (Z contrast).

Preparation of the Fibers

The Ag/TCP powder and polyamide 6 pellets (Grilon F 34, EMS-Grivory) were dried at 100[degrees]C for 12 h in vacuum before processing. The extrusion was performed on a twin-screw 20D recycling extruder (Haake Polylab OS Rheodrive 16, Germany) with a maximum temperature of 270[degrees]C and a dwell time of 7 min. The screw geometry consisted of an inlet, a feeding, and a mixing zone. Ag/ TCP was dosed gravimetrically. The resulting pellets of 2 mm diameter were processed in a pilot melt spinning plant (Fourne Polymertechnik GmbH, Alfter-Impekoven, Germany) to obtain fibers. The compounds were spun using single screw extrusion (13 X 25D) and fed at 0.3 g/ min to a monofilament spinneret by a spin pump heated to 240[degrees]C. The extrudate was cooled in a quenching chamber with a length of 2 m and an air flow of 520 [m.sup.3]/min. The filaments were drawn by three heated godets maintained at constant temperatures to obtain a draw ratio of three. No spin finish was used for the whole process. The filaments were finally wound for storage and further analysis on a winder.

Silver Release

Simulated Body Fluid (SBF) was prepared according to Kobubo et al. (21). A bundle of fibers (330 mg) was immersed for 120 days in 45 ml SBF at 37[degrees]C. Another sample (775 mg) was placed in 50 ml 0.1 wt% citric acid for 42 days at 37[degrees]C to chemically simulate carrier (calcium phosphate) consumption as proposed by Loher et al. [21. As references, the same experiments were conducted with pure PA6 fibers. The silver release in the solutions was measured with AAS.

Antibacterial Experiments

Escherichia coli. Escherichia coli (strain C43) was grown in Difco[TM] LB broth (Chemie Brunschwig) for 4 h to a concentration of about [10.sup.8] CFU/ml (colony forming units per milliliter). This suspension was diluted to the required concentration with physiological saline (0.9 wt% NaCl in water).

Two different experiments were conducted with E.coli. (1) The chronological evolution of the antibacterial effect was investigated within the first 24 h. (2) The fiber bundles were exposed to new bacteria every 24 h for five times to test the long-term performance and overall capacity of the fibers to sustain anti-bacterial properties.

In the first experimental setup, bunches of 100 fibers (3.2 cm length) fixed with a wire were sterilized by UV radiation (50 W/[m.sup.2]) for 1 h, placed in Eppendorf tubes, and contaminated with 80 [micro]l of E. coli (~[10.sup.7] CFU), After 1, 14, and 24 h incubation, the bunches were washed in 30 ml of saline, sonicated (UP400S, 24 kHz, Hielscher GmbH: 50% amplitude, 100% pulse) for 20 s (to detach the bacteria from the fibers), and plated to a dilution of up to [10.sup.-4] in duplicate on trypticase soy agar contact plates (VWR). Incubation for 12 h at 37[degrees]C revealed the reduction to the control. Three samples for each time point and fiber type were used.

In the second experimental setup, the same type of fiber bunches as above were sterilized 1 h with UV light, incubated with 80 [micro]l E. coli ([10.sup.[6.7-7]] CFU) for 22 h, afterwards rinsed into 30 ml saline, sonicated for 20 s, and plated to a dilution of up to [10.sup.-4] in triplicate on trypticase soy agar contact plates (VWR). The bunches were removed and dried in a vacuum oven at 50[degrees]C for 1 h. Afterwards these bundles were used for the next cycle (UV, incubation with 80 [micro]l E. coli, and so forth).

Streptococcus sanguinis. Streptococcus sanguinis (DSM No.: 20068) was grown twice in an overnight culture in Schadler broth. A sample (15 ml) of the bacterial suspension ([~10.sup.8] CFU/ml) was vortexed, sonicated for 30 s, and centrifuged 5 min at 8,000 rpm. The bacterial pellet was resuspended in 2 ml saline and centrifuged again. The pellet was then suspended in 10 ml buffered saliva (8.58 ml saliva with 0.71 ml 67 mM [Na.sub.2][HPO.sub.4] buffer and 0.71 ml 67 mM [KH.sub.2][PO.sub.4] buffer) to a resulting pH of 7.0. The saliva was collected from two healthy people, filtered through a 70 [micro]m filter, sonicated at 80% amplitude for 30 s, and centrifuged at 15,000 rpm for 40 min at 4[degrees]C. The supernatant was collected and filtered through 0.45 [micro]m and 0.22 [micro]m filters.

For every time point (0, 8, 24 h) five bundles (3.2 cm long; 116 fibers for pure PA6, 100 fibers for Ag/TCP (different number to ensure the same surface area)) were incubated with each 80 [micro]l bacteria-saliva suspension ([10.sup.7.6] CFU); as control sample, one bundle of each type only with buffered saliva.

After the corresponding time, each bundle was placed in 12 ml saline, vortexed for 1 min and sonicated for 30 s to detach the bacteria from the fibers. A dilution row ([10.sup.-1] [-10.sup.-6]; for time points 8 h and 24 h also 100 [micro]l of [10.sup.0]) was plated in duplicate on human blood agar plates. The plates were incubated at 37[degrees]C under anaerobic conditions for 24 h.

Preparation of the human blood agar plates: 500 ml dd[H.sub.2]O mixed with 21.25 g BD BBL[TM] Columbia Agar Base were autoclaved and then cooled down to 56[degrees]C. A premix of 25 ml human blood, 5 ml hemin, and 250 [micro]l menadion was then added and finally poured into petri dishes.

RESULTS AND DISCUSSION

Ag/TCP Nanoparticles

The freshly prepared Ag/TCP powder consisted of a fine, white, free-flowing powder with very low pouring density, similar to the presently used fumed silica fillers. In agreement with Loher et al. (2), morphological investigations using scanning (SEM), transmission (TEM), and scanning transmission electron microscopy (STEM) (see Fig. 1) confirmed the presence of spherical particles with a primary particle size diameter of 20-50 nm.

[FIGURE 1 OMITTED]

Nitrogen adsorption at - 196[degrees]C according to the BET method revealed a specific surface area (SSA) of 80.8 [+ or -]0.3 g/[m.sup.2] in accord with recent reports on similar nanoparticles (5). Assuming spherical particles [17], the corresponding primary particle diameter ([d.sub.BET]) can be calculated by [d.sub.BET] = 6/[rho]/SSA. Here, the measured value of [d.sub.BET] = 24 nm ([rho]TCP = 3.14 g/[cm.sup.3]) correlated well to the above optically determined size range.

To distinguish between single particles and hard agglomerates, an X-Ray disk centrifuge (XDC) was used as suggested by Limbach et al. (22) and revealed a unim-odal size distribution of the hydrodynamic diameter around 65 nm (Fig. 1d).

Overall, the determined particle size was close to the range reported by Morones et al. (23), which affirmed direct interaction of nanoparticles with bacteria preferentially for particles with a diameter of 1-10 nm. Figure 1c shows the silver particles (diameter of 1-2 nm) decorating the primary TCP particles.

The phase composition was investigated by X-ray diffraction (XRD; Fig. 2) (13). The as prepared particles were amorphous and thus showed a broad signal in the XRD pattern as earlier reported by Loher et al. (17) for pure TCP and Ag/TCP (2). After sintering at 900[degrees]C, the particles underwent a crystallization process and the XRD pattern showed a good match to the [beta]-TCP reference (24), which further confirmed the purity of the material as excess of calcium or phosphate would have given rise to other phases (17). Flame atomic absorption spectroscopy (AAS) of Ag/TCP particles dissolved in concentrated nitric acid showed a total silver content of 1.3 wt%.

[FIGURE 2 OMITTED]

Extrudates and Fibers

In contrast to the transparent, snow-white pure polymer extrudates made of PA6, the silver containing Ag/TCP-PA6 extrudates showed a golden-yellow color (see Fig. 3). At several points, brown spots of 0.1 mm diameter were observed and attributed to agglomerates of Ag/ TCP. This demonstrates the difficulty of homogeneously dispersing the filler during the extrusion process (25). The same color observations held true for fibers produced from the extrudates.

[FIGURE 3 OMITTED]

The pure PA6 fibers had a diameter of 112 /[micro]m; the Ag/TCP-PA6 fibers were slightly thicker with a diameter of 125 /[micro]m. The Ag/TCP-PA6 fibers presented a rougher surface than the pure PA6 fibers.

Silver Release

To choose a relevant test environment, the silver release from the here developed reactive filler was measured during exposure to simulated body fluid (SBF). This protocol has been adopted from clinical research on implants and surgical devices (26). During immersion in SBF, the fibers' color gradually faded. Exposure during 120 days resulted in faint yellow fibers; longer exposure will result in fibers of comparable color to pure PA6 fibers. This observation suggests that the yellow color of silver containing fibers is actually caused by the Ag/TCP and not by polymer degradation.

AAS measurement of the SBF with Ag/TCP fibers in for 120 days showed a silver content of 150 ppm. In the citric acid with Ag/TCP fibers in for 42 days, 116 ppm were detected. This shows that a considerable part (>50%) can be washed-out during this time.

Antibacterial Experiments

Escherichia coli. Figure 4 shows the progression of bacteria on pure PA6 fibers and on Ag/TCP-PA6 fibers after 1, 8, and 24 h. On pure PA6 bristles proliferation of bacteria was observed from initially ~[10.sup.7] CFU growing to [10.sup.7.9] CFU within 8 h and to [10.sup.8.3] CFU within 24 h. On the Ag/TCP fibers instead, a reduction to [10.sup.4.2] CFU respectively to [10.sup.3.0] CFU was observed during the same timescale. Percentage-wise this corresponds to a reduction of bacterial load on the Ag/TCP fibers by 99.98% after 8 h, and 99.9996% after 24 h compared with controls.

[FIGURE 4 OMITTED]

Proliferation on pure PA6 could occur as residues of LB broth from the bacteria suspension remained on the bundles. A complete elimination within 24 h was not observed, as working at the detection limit, and single bacteria may have survived, as they did not had any interaction with the silver.

Figure 5 shows a reduction of 4-8 [log.sub.10] scales in every 22 h experiment. The effect does not fade after four experiments. 4-8 [log.sub.10] scales correspond to a difference of at least 99.99% between the two fiber types.

[FIGURE 5 OMITTED]

Streptococcus sanguinis. S. sanguinis play an important role in caries formation as a primary colonizer of the oral cavity. Similar results to the experiments with E. coli were obtained with 5. sanguinis (see Fig. 4). On the pure PA6 fibers, the number of CFU was reduced from initially [10.sup.5.6] CFU to [10.sup.5.5] CFU after 8 h and to [10.sup.4.9] CFU after 24 h. On the Ag/TCP fibers, reductions to [10.sup.5.1] CFU respectively to [10.sup.2.5] CFU were observed for the same time points. This corresponds to a difference between the two fiber types of 99.6% (2.4 [log.sub.10] scales) after 24 h.

CONCLUSION AND OUTLOOK

Our work demonstrates the successful application of a reactive, antibacterial Ag/TCP nanoparticle filler in common polymer fibers. These nanoparticles consist of 20-50 nm sized carriers, based on calcium phosphate, a major nutrient of bacteria. This bait is spiked with 1-2 nm silver particles. In the presence of growing bacteria, the TCP carrier particles are biodegraded and trigger the release of the antibacterial silver (8). Applying silver in this complex form allows significant increases in antibacterial efficiency due to a larger contact area (27) and the triggered release of the active silver exclusively when TCP is consumed by bacteria (2). This reduces the required amount of silver if compared to non-reactive systems.

The results show that Ag/TCP nanoparticles can effectively kill E. coli and S. sanguinis. A reduction of up to 99.99% has been verified and 100% seems possible within 24 h contact time. Should the occasion arise one could easily increase the amount of silver, either in the TCP by feeding more silver precursor into the production flame, or by dosing more Ag/TCP during polymer extrusion. A similar effect on other bacterial strains is expected, as silver shows a strong effect on many taxa (2-8). For application of Ag/TCP in commercial applications, more investigations on the stability of Ag/TCP-containing polymer have to be performed. The mechanical properties must be adjusted to meet the demands of real life, and the antibacterial effect must last for the lifespan of a product.

Regarding toxicology: Even if very little silver is used and possibly taken up by human organisms or spread in an ecosystem, one needs to keep in mind that the harmlessness of nanoparticles in general has not be proven conclusively yet (28). It should be considered, however, that one could easily eat (and fully incorporate) three toothbrushes with the examined Ag/TCP-PA6 bristles (containing 260 ppm silver) every week before exceeding the US-EPA threshold. The calcium phosphate carrier itself has been tested even in vivo (rabbits) at a dose of about 5 mg/kg bodyweight and did not show any indication for toxicity (29).

If these challenges can be mastered, commercial application of nano-silver-tricalcium phosphate seems an appropriate way to improve polymer characteristics and reducing in this way infections in a broad range of applications.

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Correspondence to: Wendelin J. Stark; e-mail: wendelin.stark@chem.ethz.ch

Published online in Wiley Online Library (wileyonlinelibrary.com).

[C] 2010 Society of Plastics Engineers

Lukas C. Gerber, (1) Dirk Mohn, (1) Giuseppino Fortunato, (2) Monika Astasov-Frauenhoffer, (3) Thomas Imfeld, (4) Tuomas Waltimo, (3) Matthias Zehnder, (4) Wendelin J. Stark (1)

(1) Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering, ETH Zurich, 8093 Zurich, Switzerland

(2) EMPA, Swiss Federal Laboratories for Materials Testing and Research, Advanced Fibers, 9014 St. Gallen, Switzerland

(3) Institute of Oral Microbiology and Preventive Dentistry, University of Basel Center of Dental Medicine, 4056 Basel, Switzerland

(4) Department of Preventive Dentistry, Periodontology, and Cariology University of Zurich Center of Dental Medicine, 8032 Zurich, Switzerland

DOI 10.1002/pen.21779
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Author:Gerber, Lukas C.; Mohn, Dirk; Fortunato, Giuseppino; Astasov-Frauenhoffer, Monika; Imfeld, Thomas; W
Publication:Polymer Engineering and Science
Article Type:Report
Geographic Code:4EXSI
Date:Jan 1, 2011
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