Antimicrobial durability of air filters coated with airborne Sophora flavescens nanoparticles

Science of the Total Environment 444 (2013) 110–114

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Antimicrobial durability of air filters coated with airborne Sophora flavescens nanoparticles Eui-seok Chong a, Gi Byoung Hwang a, Chu Won Nho b, Bo Mi Kwon a, Jung Eun Lee c, SungChul Seo d, Gwi-Nam Bae a,⁎, Jae Hee Jung a,⁎⁎ a

Center for Environment, Health, and Welfare Research, Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Republic of Korea Functional Food Center, Korea Institute of Science and Technology (KIST Gangneung Institute), Gangneung, Gangwon-do 210-340, Republic of Korea Biosafety Research Team, National Institute of Environmental Research, Kyungseo-Dong, Seo-Gu, Incheon 404-170, Republic of Korea d Department of Environmental Health, College of Medicine, Korea University, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-701, Republic of Korea b c

G R A P H I C A L

A B S T R A C T

Variations in (a) the concentrations of major antimicrobial chemical compounds on S. flavescens nanoparticle-coated filters: kurarinone, kuraridin, and sophoraflavanone-G and (b) the inactivation rate of antimicrobial filters as a function of time.

a r t i c l e

i n f o

Article history: Received 22 June 2012 Received in revised form 24 October 2012 Accepted 21 November 2012 Available online 21 December 2012 Keywords: Natural product Nanoparticles Antimicrobial filter Durability Sophora flavescens

a b s t r a c t Airborne biological particles containing viruses, bacteria, and/or fungi can be toxic and cause infections and allergy symptoms. Recently, natural materials such as tea tree oil and Sophora flavescens have shown promising antimicrobial activity when applied as air filter media. Although many of these studies demonstrated excellent antimicrobial efficacy, only a few of them considered external environmental effects such as the surrounding humidity, temperature, and natural degradation of chemicals, all of which can affect the antimicrobial performance of these natural materials. In this study, we investigated the antimicrobial durability of air filters containing airborne nanoparticles from S. flavescens for 5 months. Antimicrobial tests and quantitative chemical analyses were performed every 30 days. Morphological changes in the nanoparticles were also evaluated by scanning electron microscopy. The major antimicrobial compounds remained stable and active for ~ 90 days at room temperature. After about 90 days, the quantities of major antimicrobial compounds decreased noticeably with a consequent decrease in

⁎ Corresponding author. Tel.: +82 2 958 5676; fax: +82 2 958 5805. ⁎⁎ Corresponding author. Tel.: +82 2 958 5718; fax: +82 2 958 5805. E-mail addresses: [email protected] (G.-N. Bae), [email protected] (J.H. Jung). 0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2012.11.075

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antimicrobial activity. These results are promising for the implementation of new technologies using natural antimicrobial products and provide useful information regarding the average life expectancy of antimicrobial filters using nanoparticles of S. flavescens. © 2012 Elsevier B.V. All rights reserved.

1. Introduction With the increase in indoor human activities resulting from modern social life, many researchers have begun to consider the importance of indoor air quality (IAQ) (Daisey et al., 2003; Jones, 1999; Spengler and Sexton, 1983). In particular, the biological terrorism attack with anthrax in the US in 2001 and the pandemic outbreak of the influenza A (H1N1) virus in 2009 have emphasized the importance of controlling airborne biological particles. Bioaerosols can cause various diseases and infections, allergies, and toxic reactions (Burge, 1990; Clark et al., 1983; Dales et al., 2000) and can spread easily with air currents. Therefore, the implementation of bioaerosol controls has become an important issue for safeguarding human health against hazardous bioaerosols. Techniques utilizing ultraviolet (UV) irradiance, electric ionization, and thermal treatment have been suggested as means of controlling the concentration and viability of bioaerosols (Hwang et al., 2010; Jung et al., 2009; Lee et al., 2008; Lin and Li, 2002; Peccia and Hernandez, 2004). In particular, filtration technology is one of the most widely used methods of controlling aerosols and hydrosols. However, microorganisms collected on a filter surface can stay alive and grow if provided with nutrients and a suitable environment (McFeters and Stuart, 1972), resulting in possible contamination of air conditioning, ventilation, and/or water supply systems. Such situations have led to outbreaks of Legionnaires' disease and Pontiac fever (Brown et al., 1999; Castellani Pastoris et al., 1997; Kool et al., 1999). Therefore, various materials such as silver and copper nanoparticles, carbon nanotubes (CNTs), and biocidal chemicals have been used to bestow antimicrobial activities to the filter medium. Previous studies have shown that silver nanoparticles can alter the structure of bacterial membranes, resulting in death of the bacteria (Sondi and Salopek-Sondi, 2004). Copper nanoparticles also work as an antimicrobial material and have shown a higher degree of bacterial inactivation than silver nanoparticles (Yoon et al., 2007; Yun et al., 2009). However, many warnings have been raised regarding the risk of exposure to nanoparticles. People exposed to nanomaterials have exhibited various symptoms including peribronchial inflammation, mucosal inflammation and necrosis and skin irritation (Bermudez et al., 2004; Hoet et al., 2004; Lam et al., 2004; Warheit et al., 2007). Arora et al. (2008) reported that human skin carcinoma (A431) and human fibrosarcoma (HT-1080) cells exposed to 7–20 nm silver particles for 24 h showed reduced cell viability and suffered oxidative stress. This study also demonstrated that some silver nanoparticles could enter cells and cause DNA fragmentation and higher caspase-3 activity. Under the same conditions, with mouse fibroblasts and liver cells, silver nanoparticles caused cell death, oxidative stress, and apoptosis (Arora et al., 2009). Human abdominal full-thickness skin exposed to polyvinyl pyrrolidone-coated 25-nm silver particles for 24 h was permeated and the skin damaged in an in vitro diffusion cell system (Larese et al., 2009). Male C57BL/6N mice injected with 29-nm silver particles showed altered gene expression associated with oxidative stress in the caudate, frontal cortex and hippocampus regions of the brain (Rahman et al., 2009). Rats exposed to 1 or 5 mg/kg of single-walled carbon nanotubes (SWCNTs) via intratracheal instillation developed multifocal macrophage-containing granulomas in the lung (Warheit et al., 2004). Additionally, the presence of new drug-resistant bacterial strains highlights the urgent need to develop novel antimicrobial agents (Naimi et al., 2003). Recently, several natural products applicable to filter media have shown promising antimicrobial properties with relatively less human toxicity (Carson et al., 2006; Pibiri et al., 2006). The antimicrobial

properties of various natural products such as tea tree oil and Sophora flavescens have been evaluated with regard to filtration and bioaerosol control and have been shown to efficiently inactivate microorganisms (Jung et al., 2011a; Pibiri et al., 2006; Pyankov et al., 2008). However, most of these previous studies did not account for environmental effects. Most natural products are organic compounds consisting of various volatile chemicals. Therefore, external environmental factors such as humidity, temperature, and exposure time can affect the antimicrobial characteristics of a natural product. Recently, Hwang et al. (2012) studied the short-term effects of humid airflow on antimicrobial filters using S. flavescens nanoparticles. Increasing relative humidity (RH) induced coalescence and morphological changes in S. flavescens nanoparticles with a consequent reduction in the rate of bacterial inactivation. We evaluated the antimicrobial durability of S. flavescens nanoparticles deposited onto a filter material for 5 months and determined the mechanism of the antimicrobial durability by analyzing the chemical composition and morphology of the particles as a function of time. 2. Materials and methods 2.1. Preparation of ethanolic plant extract for nebulization A traditional herbal medicine extracted from the perennial herb S. flavescens Ait., which is widely distributed in northeast Asia (De Naeyer et al., 2004), was used. Whole S. flavescens plants were purchased in June 2010 from Kyungdong Market in Seoul, Korea. A reference specimen was stored at the Functional Food Center at the Korea Institute of Science and Technology's Gangneung Institute at Gangneung, Korea. A freeze-dried powder of S. flavescens ethanolic extract was suspended in pure ethanol (111727; Merck, Darmstadt, Germany). Before nebulization/aerosolization, the suspension was sonicated for 10 min to ensure uniformity and filtered through a cellulose acetate membrane filter with 0.25-μm pores (National Scientific, Rockwood, TN, USA) to remove insoluble residue. 2.2. Preparation of antimicrobial filters Fig. 1 shows a schematic of the nebulization and thermal drying process used for generating natural-product nanoparticles. Twenty milliliters (0.625% w/v) of ethanolic S. flavescens extract was loaded into a single-jet Collison nebulizer (BGI, Waltham, MA, USA) to generate airborne natural-product particles. The nebulizer was supplied with an airflow of 1 L/min that had been filtered through a diffusion dryer and high-efficiency particulate air (HEPA) filter. The airflow was controlled by mass flow controllers (MFC; 1179A Mass-Flo; MKS Instruments, Andover, MA). The aerosolized droplets from the outlet of the nebulizer were passed through an active carbon adsorbent to remove ethanol vapor and diluted with 9 L/min of air. The whole mixed airflow was passed through a thermal quartz tube (inner diameter, 29 mm; length, 700 mm; thickness, 1 mm) as described previously (Jung et al., 2011a) to eliminate liquid ethanol. The inside temperature of the quartz tube was 200 °C and the residence time of the particles was 1.4 s. The size and number of nanoparticles ultimately generated were measured using a scanning mobility particle sizer (SMPS) system, consisting of a differential mobility analyzer (DMA 3081; TSI Inc., Shoreview, MN) and a condensation particle counter (CPC 3010; TSI Inc.). The measured particle size distribution was log-normal with a geometric mean diameter (GMD) of 110.7 nm, a peak diameter of 109.4 nm and a geometric

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Fig. 1. Experimental set-up used for the nebulization and thermal drying process used to generate natural-product nanoparticles.

standard deviation (GSD) of ~1.64. The nanoparticles were deposited continuously for 3 min (~50 μg nanoparticles/cm2 of cross-sectional filter area) onto a polyurethane resin fiber filter with a fiber diameter of 10–20 μm, thickness of 0.3 mm, and packing density of about 33%. The quantity of the natural product deposited on the filters was determined by weighing the filters before and after the particle deposition process with a microbalance (Mettler MT5; Mettler-Toledo International Inc., Seoul, Republic of Korea) with an accuracy of 1 μg. 2.3. Filter storage conditions The produced antimicrobial filters were stored in a clean diffusion dryer box packed with silica beads. The temperature of the storage box was kept at ~25 °C at 20±2.5% RH. The filters were stored for 30, 60, 90, 120, and 150 days. 2.4. Preparation of test bacterial suspensions Two bacteria (Gram-positive Staphylococcus epidermidis (KCTC 1917) and Gram-negative Escherichia coli (ATCC 8739)) were used in this study. Cultures of S. epidermidis were grown in nutrient broth (NB; Becton Dickinson, Franklin Lakes, NJ) at 37 °C for 24 h. E. coli cultures were grown in tryptic soy broth (TSB; Becton Dickinson) at 37 °C for 18 h. Stationary-phase organisms were harvested by centrifugation (5000 ×g, 10 min). The bacterial pellets were washed carefully three times with sterilized deionized water (SDW) using a centrifuge (5000 ×g, 10 min) to remove residual particles. A 30-mL aliquot with a bacterial titer of approximately 5 × 107 colony forming units (CFU)/ mL was placed in a six-jet Collison nebulizer (BGI).

soy agar for E. coli), followed by incubation at 37 °C for 24 h (12 h for E. coli). Colonies that grew on the plate were then enumerated. The inactivation rate was calculated using the following equation: Inactivation rateð% Þ ¼

  CFUexperiment 1−  100; CFUcontrol

ð1Þ

where CFUexperiment and CFUcontrol are the concentrations of bacterial colonies from the antimicrobial and pristine (control) filters, respectively. 2.6. Chemical components and morphological analyses The nanoparticles of S. flavescens deposited on the filters were extracted three times with pure ethanol. The major antimicrobial components of the S. flavescens extract were kurarinone, sophoraflavanone G, and kuraridin, as described previously (Jung et al., 2011b; Kang et al., 2000; Kuroyanagi et al., 1999). The extract was separated by high-performance liquid chromatography (HPLC) using a 1200 Series HPLC instrument (Agilent Technologies, Santa Clara, CA, USA) in order to determine the amount of each compound in the extract. The separation was performed with a C18-type MGII 5-μm (Shiseido, Tokyo, Japan), 4.6× 250-mm column (Fine Chemicals, Markham, ON, Canada) eluted with a MeCNH2O gradient of 20:80 to 100:0 in 45 min at a

2.5. Bacterial inactivation tests Fig. 2 shows a schematic detailing the bioaerosol inactivation tests, including bioaerosol generation (Jung et al., 2011c). Using the nebulizer, test bacteria were aerosolized at 5 L/min in the dry and filtered airstream. The total concentration of bacterial bioaerosols deposited on the antimicrobial and control filters was approximately 6 × 10 5 particles/cm 2filter. There was a 10-minute exposure before removing the filter from the holder for microbial analysis. Each filter was placed into 5 mL of phosphate-buffered saline (PBS; pH 7.4) with 0.01% Tween 80 and sonicated for 10 min to ensure that the bacteria were transmitted from the filter to the PBS buffer. The bacterial suspension was serially diluted and plated onto nutrient agar (tryptic

Fig. 2. Schematic showing the bioaerosol inactivation tests and bioaerosol generation.

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Fig. 3. Variation in the inactivation rate of antimicrobial filters as a function of time.

flow rate of 1 mL/min. Morphological variation of nanoparticles deposited on the filters was observed using a NOVA scanning electron microscope (SEM; 200 NANO SEM; FEI, Hillsboro, OR, USA). 3. Results Fig. 3 shows the inactivation rate changes for S. epidermidis and E. coli on S. flavescens nanoparticle-deposited filters as a function of time. The initial inactivation rates were 98.0 ± 5.0% for S. epidermidis and 89.1 ± 6.0% for E. coli. This initial inactivation rate remained stable (filters exhibited a low standard deviation) for approximately 60 days for both S. epidermidis and E. coli. Although inactivation rates were similar after 90 days, standard deviations increased significantly (85.7 ± 9.0% for S. epidermidis, 86.7 ± 18.1% for E. coli). After 150 days, inactivation rates dropped to 23.1 ± 6.6% for S. epidermidis and 14.0 ± 4.5% for E. coli. Fig. 4 shows the variation in the quantities of the major antimicrobial chemicals (kurarinone, kuraridin, and sophoraflavanone G) in the nanoparticles on the antimicrobial filters. According to Jung et al. (2011a), chemical composition remains unchanged when the extract of S. flavescens is converted to nanoparticles by nebulization and thermal drying. The content of major antimicrobial chemical compounds began to decrease after 90 days (28 wt.% kurarinone, 8 wt.% kuraridine, and 5 wt.% sophoraflavanone G). The SEM micrographs in Fig. 5 show the morphology of naturalproduct nanoparticles deposited on the surface of antimicrobial filters

Fig. 5. SEM micrographs show the filter surface: (a) initially and (b) after 120 days.

in the (a) initial stage and (b) after 120 days. According to Hwang et al. (2012), morphological changes in S. flavescens nanoparticles on the filter surface can affect the antimicrobial activity of the filter. However, in this case, only slight morphological changes were observed, as shown in Fig. 5. 4. Discussion

Fig. 4. Variation in the concentrations of major antimicrobial chemical compounds on S. flavescens nanoparticle-coated filters: kurarinone, kuraridin, and sophoraflavanone G.

The similar decreasing trends shown in Figs. 3 and 4 suggest that the degradation of the major antimicrobial compounds might be the cause of the observed decrease in the bacterial inactivation rate as a function of time. The observed decrease in the concentration of antimicrobial compounds might be attributed to oxidation. Liu et al. (2009) demonstrated that thermal energy can degrade kurarinone. According to their study, 25.54% and 16.0% of the total kurarinone were degraded after 2 h at 80 °C and at 60 °C, respectively, while only slight changes were observed after 48 h at 25 °C. Therefore, these chemical reactions progress slowly under standard conditions, i.e., room temperature, 20% RH, and atmospheric pressure. However, the nanostructure of the natural-product particles can also give rise to characteristic behaviors that are different than those observed in bulk solid or liquid phases. Differences between nanoscale and bulk materials can affect not only mechanical but also thermodynamic, chemical, and optical properties (Nishide et al., 2000; Subramania et al., 1999; Wang et al., 1999). Since nanoparticles have a much greater specific surface

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area, chemical reactions at the particle surface can progress more quickly than analogous reactions in bulk states. However, experimental evidence is needed to define the exact mechanisms or reasons for the rapid decrease in the levels of antimicrobial compounds after 90 days. The major antimicrobial compounds remained stable and active for 90 days. After this time, however, rapid degradation of the major antimicrobial compounds was observed at ambient conditions with an associated drop in antimicrobial activity of the filter. These results could provide useful data on the average life of antimicrobial filters that employ natural-product nanoparticles for the control of indoor environments and contribute to research and development of functional materials using natural products. To obtain a more detailed account of the antimicrobial durability of filters containing natural-product nanoparticles, similar evaluations should be performed under other environmental conditions. In addition, further studies are necessary to establish standard methods for testing antimicrobial durability of filters and filter materials. Acknowledgments This research was supported by the Converging Research Center funded by the Ministry of Education, Science and Technology (2012K001370). References Arora S, Jain J, Rajwade JM, Paknikar KM. Cellular responses induced by silver nanoparticles: in vitro studies. Toxicol Lett 2008;179:93-100. Arora S, Jain J, Rajwade JM, Paknikar KM. Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells. Toxicol Appl Pharmacol 2009;236:310–8. Bermudez E, Mangum JB, Wong BA, Asgharian B, Hext PM, Warheit DB, et al. Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles. Toxicol Sci 2004;77:347–57. Brown CM, Nuorti PJ, Breiman RF, Hathcock AL, Fields BS, Lipman HB, et al. A community outbreak of Legionnaires' disease linked to hospital cooling towers: an epidemiological method to calculate dose of exposure. Int J Epidemiol 1999;28:353–9. Burge H. Bioaerosols: prevalence and health effects in the indoor environment. J Allergy Clin Immunol 1990;86(5):687–701. Carson CF, Hammer KA, Riley TV. Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. Clin Microbiol Rev 2006;19:50–62. Clark S, Rylander R, Larsson L. Airborne bacteria, endotoxin and fungi in dust in poultry and swine confinement buildings. Am Ind Hyg Assoc J 1983;44(7):537–41. Daisey JM, Angell WJ. Apte MG. Indoor air quality, ventilation and health symptoms in schools: an analysis of existing information. Indoor Air 2003;13:53–64. Dales RE, Cakmak S, Burnett RT, Judek S, Coates F, Brook RJ. Influence of ambient fungal spores on emergency visits for asthma to a regional children's hospital. Am J Respir Crit Care Med 2000;162:2087–90. De Naeyer A, Berghe WV, Pocock V, Milligan S, Haegeman G, De Keukeleire D. Estrogenic and anticarcinogenic properties of kurarinone, a lavandulyl flavanone from the roots of Sophora flavescens. J Nat Prod 2004;67(11):1829–32. Hoet PH, Brüske-Hohlfeld I, Salata OV. Review: nanoparticles—known and unknown health risks. J Nanobiotechnol 2004;2:12–26. Hwang GB, Jung JH, Jeong TG, Lee BU. Effect of hybrid UV-thermal energy stimuli on inactivation of S. epidermidis and B. subtilis bacterial bioaerosols. Sci Total Environ 2010;408(23):5903–9. Hwang GB, Jung JH, Nho CW, Lee BU, Lee SJ, Bae GN. Short-term effect of humid airflow on antimicrobial air filters using Sophora flavescens nanoparticles. Sci Total Environ 2012;421–422:273–9. Jones AP. Indoor air quality and health. Atmos Environ 1999;33:4535–64. Jung JH, Lee JE, Kim SS. Thermal effects on bacterial bioaerosols in continuous air flow. Sci Total Environ 2009;407:301–17.

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