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A High Throughput Fabrication Method to Produce a Silver Nanoparticles-Doped-Nanoclay Polymer Composite with Novel Synergistic Antibacterial Effects at the Material Interface Shaobo Cai, Behnam Pourdeyhimi, and Elizabeth G. Loboa ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 25 May 2017 Downloaded from http://pubs.acs.org on May 31, 2017

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ACS Applied Materials & Interfaces

A High Throughput Fabrication Method to Produce a Silver Nanoparticles-Doped-Nanoclay Polymer Composite with Novel Synergistic Antibacterial Effects at the Material Interface

Shaobo Cai1, Behnam Pourdeyhimi2, Elizabeth G. Loboa3, *

1. Department of Materials Science and Engineering at North Carolina State University, 3002 EB 1, Raleigh, NC 27695, USA; 2. The Nonwovens Institute at North Carolina State University, 2401 Research Drive, Raleigh, NC 27695, USA; 3. College of Engineering at University of Missouri, W1051 Thomas & Nell Lafferre Hall, Columbia, MO 65211, USA

KEYWORDS: high-throughput, silver nanoparticles-doped-nanoclay, synergistic antibacterial effect, reduced bacterial adhesion, minimal silver content, in-situ thermal reduction.

ACS Paragon Plus Environment

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ABSTRACT

In this study, we report a high throughput fabrication method at industrial pilot scale to produce a silver nanoparticles-doped-nanoclay/polylactic acid composite with a novel synergistic antibacterial effect. The obtained nanocomposite has a significantly lower affinity for bacterial adhesion, allowing the loading amount of silver nanoparticles to be tremendously reduced while maintaining satisfactory antibacterial efficacy at the material interface. This is a great advantage for many antibacterial applications where cost is a consideration. Further, unlike previously reported methods that require additional chemical reduction processes to produce the silver nanoparticles-doped-nanoclay, an in-situ preparation method was developed, in which silver nanoparticles were created simultaneously during the composite fabrication process by thermal reduction. This is the first report to show that altered material surface sub-micron structures created with the loading of nanoclay enables creation of a nanocomposite with significantly lower affinity for bacterial adhesion. This study provides a promising scalable approach to produce antibacterial polymeric products with minimal changes to industry standard equipment, fabrication processes or raw material input cost.


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1. Introduction: There is an increasing industrial demand for high performance materials with multiple enhanced properties that can be created using minimal changes to industry standard equipment, processes and input cost.1 Due to the relatively low cost and readily commercial availability, nanoclay has been widely used to improve the modulus, tensile strength, barrier properties, thermal properties and electrostatic capacities of polymers.2-8 It has been shown that loading a small amount of nanoclay into polymer results in significantly enhanced material properties.9-11 For materials of potential use in medical or healthcare related industrial sectors, the antibacterial capability of the material is also of great interest. However, it has been shown that direct loading of nanoclay into the polymer neither results in bacterial toxicity nor inhibits the growth of bacteria.12-14 Recently, investigators have shown that creation of certain micro- or nano-structures on material surfaces significantly alters their bacterial adhesion behavior.15-22 For example, for materials with surface structures at the micron scale, it was found that smaller size topologies decreased bacterial attachment.15,

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At the nanoscale, bacterial biofilm formation has been

observed to be more pronounced on material surfaces with a root-mean-square (RMS) roughness value of 10 nm compared to that of either 5 nm or 15 nm.23 Despite increasing interest in developing surface structured anti-bacterial-adhesion materials, reported methods often involve delicate fabrication processes.24-27 Though excellent antibacterial-adhesion properties have been obtained, many reported methods cannot be easily applied to large scale, low cost industrial production without considerable changes in standard equipment or production processes.


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Silver nanoparticles (silver NP) have also been extensively studied for antibacterial applications due to their wide-spectrum antibacterial activity.28-31 However, direct loading of silver NP into polymers often requires a high silver content to achieve satisfactory antibacterial effect. For example, it was reported that to obtain a 1-order (90%) decrease in bacterial number, at least 1 wt.% silver NP is required.29-30 This is a considerable problem when cost is a concern. Therefore, industrial application of the direct loading approach is very limited. The goal of this study was to develop a high throughput fabrication method at industrial pilot scale to produce a silver nanoparticles-doped-nanoclay (silver NP-nanoclay) polymer composite with a synergistic antibacterial effect. The developed method requires little change to industry standard equipment, processes or input cost. We hypothesized that at certain optimized fabrication conditions, loading nanoclay into polymer would alter the sub-micron structures at the material surface, enabling the resultant nanocomposite to exhibit significantly lower affinity for bacterial adhesion. We further hypothesized that the reduced bacterial adhesion would allow for the silver NP loading content to be greatly reduced, while still maintaining satisfactory antibacterial efficacy at the material interface. Finally, unlike previously reported methods that require additional chemical reduction processing to produce the silver NP-doped-nanoclay 14, 32, in our method, the silver NP were created simultaneously during the composite fabrication process by thermal reduction.


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2. Materials and Experiments: 2.1 Materials Polylactic acid (PLA) grade 6202D (NatureWorks LLC, Minnetonka, MN), a biodegradable and biocompatible polymer, was used as the matrix material for the nanocomposite. Nanomer I.44P nanoclay, control silver nanoparticles, and aqueous silver nitrate solution (0.1 M, 99%) were obtained from Sigma–Aldrich, USA. 2.2 Silver NP-nanoclay production A two-step process was developed to fabricate the silver NP-doped-nanoclay (silver NPnanoclay): silver ion-exchange and in-situ thermal reduction. In the first step, 10 g nanoclay was added into 100 ml 0.1 M silver nitrate solution and stirred vigorously for 30 min. The solution was stirred overnight at 60 °C for silver ion-exchange. The resulting mixture was centrifuged at 5000 rpm then washed three times with distilled water. The fabricated silver ion-exchangednanoclay was used immediately after drying. The in-situ thermal reduction of silver NP-nanoclay occurs simultaneously during the composite fabrication process due to the relatively high processing temperature. 2.3 Composite fabrication To produce the silver NP-nanoclay PLA nanocomposite, an industrial pilot scale meltblown unit mounted with a co-concentric spinneret (Biax Fiber-Film, Greenville, WI) was used for composite fabrication (Nonwovens Institute industrial pilot facilities, Raleigh, NC). Masterbatches containing 90 wt.% PLA and 10 wt.% additives were prepared by melt compounding in a counter-rotating twin-screw extruder (L/D = 28: 1) at Techmer PM (Clinton, TN). The masterbatches were melt diluted with pure PLA in the meltblown unit inside-built screw extruder to obtain the final additive concentrations. During the fabrication process,


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polymer melts were blown by high-velocity hot air streams and attenuated by drag force to form fine fibrous self-bonding polymer composite meshes. Before fabrication, materials were dried overnight to remove absorbed water. Systematically optimized fabrication parameters utilized in this study are provided in Table 1. Table 1. Optimized key fabrication parameters. Optimized Fabrication Parameters Die Temp.:

273 °C

Melt Temp.:

282 °C

Air Temp.:

254 °C

Air flow:

12 Psi

Throughput:

0.2 ghm

2.4 Composite characterizations The crystallinity of the samples was determined using a Rigaku SmartLab X-ray diffractometer (XRD). Samples were scanned from 2θ range 5 - 30° at an increment of 0.1°. The distribution of additives in the samples was obtained by transmission electron microscopy (TEM; JEOL 2000FX). To obtain the sections for TEM, samples were embedded in epoxy blocks and prepared using an ultramicrotome (Leica UC7 Cryo). The chemical compositions of the samples were analyzed using a Hitachi S3200N variable pressure scanning electron microscope (VPSEM) equipped with a Robinson backscattered electron detector and an Oxford Pentafet energy dispersive x-ray spectroscopy (EDX). During this test, the samples were not coated with any metals. An atomic force microscope (AFM; Bruker Dimension 3000) was used to visualize and quantify each sample’s surface roughness (root-mean-square roughness). 2.5 Antibacterial tests


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Two antibacterial tests, bacterial adhesion test and material interface antibacterial test, were performed in this study against two bacterial strains: Gram-negative Escherichia coli and Grampositive Staphylococcus aureus, individually. For both tests, sterilized disc specimens with area of 0.95 cm2 were placed in 48-well plates and pre-soaked in phosphate-buffered saline (PBS) for 24 h. 1 ml bacterial suspension with an initial concentration of 106 CFU ml-1 was added to each well. The bacterial adhesion test was modified from several previous reports

15, 24-25

. Specifically,

after 1-hour incubation, samples were gently rinsed three times with sterile PBS to remove unbound or loosely attached bacteria. Firmly attached bacteria were stained by immersing samples in SYTO® 9 (Thermo Fisher Scientific, Waltham, MA) solution following the manufacturer’s protocol and observed using fluorescent microscopy (Leica DM5500B). For quantitative analysis of adherent bacteria, rinsed discs were placed in 1 ml PBS with 1% Trion X-100 added and vigorously vortexed for 5 min to remove attached bacteria.

15, 24-25

Subsequently, the 1 ml PBS samples were serially diluted and diluents of 100 ul were plated onto soy agar plates (BD, Sparks, MD) for overnight incubation. The numbers of colonies on plates were quantified. SYTO® 9 staining was also performed on vortexed samples to ensure the complete removal of all attached bacteria. The material interface antibacterial test is a combination of the described bacterial adhesion test with a modified AATCC 100 antibacterial test (Assessment of Antimicrobial Finishes on Textile Materials). In this test, after 1-hour initial incubation and PBS rinsing, samples were transferred to new 48-well plates with 1ml fresh broth and incubated at 37 °C for an additional 48 hours. Bacterial numbers on the samples were then quantified using the same method described above.


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The following equation was used to calculate the percent of bacterial number reduction, for both the bacterial adhesion test and material interface antibacterial test: % = 100 ( − )/ where A is the number of bacteria recovered from the experimental samples, and B is the number of bacteria recovered from the control, pure PLA samples. 0% reduction indicates there was no difference in bacterial number between experimental samples and control samples; 100% reduction indicates no bacteria recovered from experimental samples. 2.6 Human skin cell viability test Sample cytotoxicity was investigated using both Live & Dead Assay (Molecular Probes, Eugene, OR) and quantitative AlamarBlue Assay (AbDSerotec, Raleigh, NC) with human dermal fibroblasts. Sterilized disc samples were seeded with human dermal fibroblasts at an initial density of 4 ×104 cells per sample. After up to 2 weeks of culture, cell seeded samples were stained with the Live & Dead Kit following manufacturer’s protocol and visualized by fluorescent microscopy (Leica DM5500B). For quantitative cytoviablity test, AlamarBlue reagent was added to the cell culture medium (Lonza, USA), incubated for 6 hours, and the absorbency measured using a microplate reader (TecanGENios, Tecan, Switzerland). A greater AlamarBlue reduction % indicates higher cell viability and proliferation. 2.7 Data analyses For all quantitative experiments, such as antibacterial tests, cytotoxicity tests and AFM RMS roughness analyses, at least 3 independent samples (i.e., disc specimens) were tested for each experimental group. For each sample, at least 3 technical replicates (i.e., soy agar plates) were performed (n = 9 total). Statistical differences between experimental groups were analyzed using


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Student’s t test. Quantitative data are presented as means +/- SEM. A value of P < 0.05 was considered significantly different between experimental groups that are labeled with “*”.


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3. Results and Discussion: 3.1 Composite fabrication As shown in Table 2, four types of samples were fabricated in this study. Pure PLA samples containing no additives were used as the negative control. 0.5 wt.% silver NP directly loaded samples were used as the positive control. Samples loaded with 1 wt.% pure nanoclay were used as another negative control. All controls were compared to nanocomposite samples containing 1 wt.% silver NP-doped-nanoclay (silver NP-nanoclay). Table 2. Sample abbreviations and corresponding additive loading content. Sample

Additive

Additive

Abbreviations

Type

Amount (wt.%)

Pure PLA

N/A

0

Silver NP

Silver nanoparticles

0.5%

Nanoclay

Nanoclay

1%

Silver NPNanoclay

Silver nanoparticles -doped nanoclay

1% (Nanoclay)

To obtain the silver NP-nanoclay composite, a two-step process was developed (Fig. 1). In the first step, silver ion-exchanged-nanoclay was produced following reported methods

14, 32-34

.

While previous methods often require additional chemical reduction processes to produce the silver NP-nanoclay

14, 32

, an in-situ preparation method was developed in this study, in which

well-dispersed silver ions were thermally reduced to silver nanoparticles

35-40

. The silver NP-

nanoclay was created simultaneously during the composite fabrication process due to the relatively high processing temperature (Fig. 1).


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Figure 1. Fabrication process of the nanocomposite and the in-situ thermal reduction of silver nanoparticles on nanoclay. 3.2 Composite characterizations A scanning electron microscope (SEM) equipped with a back-scattered electron detector was used to investigate the dispersion of silver NP on the sample surfaces. Heavy metals on the material surface show as bright spots under the back-scattered electron detector. As shown in Fig. 2 (arrows), multiple bright spots were observed for both silver NP positive control samples (Fig. 2a) and the silver NP-nanoclay composite (Fig. 2b). No such spots were observed for pure PLA or nanoclay samples (results not shown).


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Figure 2. Scanning electron microscope (SEM) with back-scattered electron detector images of a) silver NP samples and b) silver NP-nanoclay composite. Heavy metals show as bright spots (labeled with white arrows) under the back-scattered electron detector. To confirm the detected heavy metal nanoparticles (bright spots) were silver NP, energy dispersive X-ray spectroscopy (EDX) was used to analyze the elemental constituents of the samples. In the pure PLA samples, only carbon and oxygen signals were detected (Fig. 3a). With the loading of nanoclay, additional silicon and aluminum signals were observed (Fig. 3b). For silver NP positive control samples (Fig. 3c), weak but distinguishable silver peaks were noted (Fig. 3c’). However, in silver NP-nanoclay composite (Fig. 3d), the relative intensity of silver signals is rather low, which cannot be easily differentiated from the background noise (Fig. 3d’). This result may be due to the silver content in the nanocomposite being below the lowest detection limit of EDX for an element (around 0.5% atom percentage). The final silver content in the composite depends on the silver ion-exchange capacity of nanoclay, which varies from 1 wt.% to 10 wt.%, based on the weight of nanoclay

14, 33-34, 41-42

. With this assumption, the

maximum silver content in the 1 wt.% silver NP-nanoclay loaded composite is around 0.1 wt.%, apparently below the lowest detection limit of EDX.


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Figure 3. Energy-dispersive x-ray spectroscopy (EDX) spectra of a) pure PLA, b) nanoclay, c) silver NP and d) silver NP-nanoclay doped sample. Spectra labeled in black boxes in c) and d) were enlarged as c’) and d’) to show the detail information, respectively. Representative results of localized EDX analysis (from areas labeled with the red boxes) of e) silver NP and f) silver NP-nanoclay loaded samples. To address this problem, localized EDX analysis that focused on a small area of the sample surface around the “brighter spots” was performed. Using this approach, distinct silver signals were detected for both silver NP (Fig. 3e) and silver NP-nanoclay loaded samples (Fig. 3f), which confirmed that the detected “brighter spots” in Fig. 2 were silver nanoparticles.


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It is important to note that besides carbon, oxygen, silicon, aluminum and silver signals, no other elements such as nitrogen, chlorine or bromine were detected for the silver NP-nanoclay composite (Fig. 3e, f). The existence of chlorine or bromine can react with the silver cation to form AgBr or AgCl nanoparticles. The lack of these signals further confirmed that silver nanoparticles were obtained by thermal reduction during the relatively high temperature composite fabrication process. The dispersion of nanoclay and silver NP inside the PLA matrix was investigated by TEM (Fig. 4). The dark spots (labeled with red arrows) indicate silver nanoparticles, the dark lines represent the intersection of silicate layers, while the white background corresponds to the PLA matrix. Per the TEM images, the average size of the silver nanoparticles was found to range from approximately 50–100 nm in both control silver NP samples and silver NP-nanoclay nanocomposites.

Figure 4. Transmission electron microscopy (TEM) micrograph of a) silver NP, b) nanoclay and c) silver NP-nanoclay loaded samples. The silver nanoparticles are labeled with red arrows, and the dark lines represent the intersection of silicate layers. The white background corresponds to the PLA matrix. 3.3 Bacterial-adhesion test A bacterial adhesion test modified from previous reports

15, 24-25, 43

was performed to evaluate

the affinity of the fabricated nanocomposite to adhesion by Staphylococcus aureus and E. coli. It


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was found that after 1 hour incubation, the number of adherent bacteria on both nanoclay and silver NP-nanoclay loaded samples were significantly decreased when compared to that on pure PLA samples (Table 3). There was no significant difference in the number of adherent bacteria between silver NP samples and pure PLA samples. These results suggest that the reduced adhesion of Staphylococcus aureus and E. coli. on the composite material is because of the loading of nanoclay and is not affected by the addition of silver NP. This may be due to the loaded silver nanoparticles in the polymer not having sufficient time to generate noticeable antibacterial effect within the short time period

44-45

. As a result, despite the existence of silver

nanoparticles, the numbers of attached bacteria on the nanoclay sample and silver NP-nanoclay composite were basically the same. Table 3. Bacterial adhesion tests. Bacterial Number Reduction (%) Samples

Staphylococcus E. coli. aureus

Pure PLA

0

0

Silver NP

2.1

-0.6

Nanoclay

67.3

61.0

Silver NP- Clay

65.8

62.9

To confirm the above findings, fluorescent microscopy was used to observe the attached bacteria on the sample surface after incubation for 1 hour (Fig. 5). A similar trend was observed in that the number of attached bacteria on pure PLA samples was obviously greater than that on


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nanoclay or silver NP-nanoclay loaded samples. There was no noticeable difference in the number of attached bacteria between silver NP samples and pure PLA samples.

Figure 5. Fluorescent microscopy of attached bacteria on a) pure PLA, b) silver NP, c) nanoclay, and d) silver NP-nanoclay loaded samples. The Staphylococcus aureus bacteria were stained with SYTO® 9 dye, which shows as bright green punctate dots under fluorescent microscopy. The faded green lines in the background are PLA fibers that exhibit some autofluorescence. 3.4 Mechanism explanations It has been previously reported that the adhesion of bacteria onto a material surface occurs in three stages: transport, initial adhesion and final attachment. 46 The second stage occurs within 1 to 3 hour after contact between the material and the bacterial suspension, which is mainly affected by the physical properties of the material, such as surface topologies, electrostatic attractions and hydrophobic interactions.

26

Previous studies have shown that direct loading of

nanoclays into polymer neither results in any bacterial toxicity nor inhibits the growth of bacteria 12-14

.


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Combining the findings of these previous studies with our results, we hypothesized that the altered material affinity to bacteria with the loading of nanoclay is not due to the changes in chemical composition or toxicity, and is more likely due to the physical effects. Recently, investigators have shown that creation of surface structures altered the adhesion of bacteria to a material. 15-18 It has been reported that there is a strong dependence of bacterial attachment on the surface structural dimensions of the substrates.

23-27

Thus, we further hypothesized that loading

nanoclay into PLA altered the surface structures of the material, contributing to the decreased bacterial adhesion.


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Figure 6. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) images of (a, b) pure PLA, (c, d) silver NP and (e, f) nanoclay loaded PLA samples. The magnification of the SEM images in a), c) and e) was 40,000x. AFM images of b), d) and f) are acquired at a 45-degree angle to better visualize the three-dimensional morphology of the material surface.


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AFM images shown in b’), d’) and f’) are from a top view, in which the height of the material surface is indicated by varying color and shading. Scale bars = 1 um. Note the difference in surface peak height, size and shape between nanoclay loaded samples and pure PLA samples. g) Root-mean-square (RMS) roughness of the samples. Results demonstrate significantly greater surface roughness at the sub-micron scale for nanoclay-loaded samples. ‘*’ indicates a significant difference between the two sample groups (p

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