Antibacterial Properties of Silicone Membranes After a Simple Two-Step Immersion Process in Iodine and Silver Nitrate Solutions
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Biocontrol Science, 2018, Vol. 23, No. 3, 97－105 Original Antibacterial Properties of Silicone Membranes after a Simple Two-Step Immersion Process in Iodine and Silver Nitrate Solutions SHIHO AOKI, KOHEI YAMAKAWA, KENJI KUBO, JUNPEI TAKESHITA, MIKI TAKEUCHI, ＊ YUKA NOBUOKA, RISEI WADA, MIKIO KIKUCHI, AND JUN SAWAI Faculty of Applied Bioscience, Kanagawa Institute of Technology, 1030 Shimo-Ogino, Atsugi, Kanagawa 243-0292, Japan Received 6 July, 2017/Accepted 27 January, 2018 Silicone is widely used in packing materials, medical equipment, and separation membranes. Since microbial cells easily adhere to the surface of silicone materials and form biofilms, techniques for incorporating antimicrobial activity into silicone materials are in high demand. This study describes the preparation of silver（ Ag）/silicone composite membranes through a simple two-step immersion process, utilizing an iodine solution followed by a silver nitrate solution at room temperature. Scanning electron microscopy（ SEM） observations revealed that particles with sizes of several nanometers to several tens of nanometers were present on the silicone membrane surface; these particles were identified as silver iodide using energy-dispersive X-ray spectroscopy（ EDS）. The Ag/silicone membrane possessed excellent antibacterial efficacy against Escherichia coli and Staphylococcus aureus, and the antibacterial efficacy（ R） against both types of bacteria was R > 4, even after stomacher treatment or acidic treatment of pH 2-6 for 24 h. The mechanical strength of the silicone membrane was also maintained after antibacterial treatment, with Young’s modulus values of 7.9±1.2 MPa and 8.3±1.5 MPa for the untreated membrane and Ag/silicone membrane, respectively（ p > 0.05）. In addition, the reduction in permeation performance of the Ag/silicone membrane was only 20%, despite the antibacterial treatment on the membrane surface. This antibacterial treatment method of silicone membranes can be conducted at room temperature（ 25℃） without special equipment, and may be applied to other types of silicone materials. Key words ： Polydimethylsiloxane / Antibacterial activity / Biofilm / Silver / Iodine. INTRODUCTION microorganisms and biofilm formation, increasing the possibility of food poisoning and infections, degrading Polydimethylsiloxane（ PDMS）-based silicone materials appearance, and increasing the likelihood of manufac- are used widely in packing materials, cooking utensils, turing defects. Therefore, prevention of microbial adhe- and medical equipment（ Bae et al., 2015; Goveas et sion and biofilm formation on silicone materials is al., 2012; Hirahara et al., 2014; Shit and Shah, 2013）. important. In addition, silicone membranes have industrial applica- Recent studies have reported methods for incorpo- tions, such as in separation membranes for pervapora- rating antimicrobial activity into silicone materials. For tion（ Han et al., 2001; Zhao et al., 2013）. Silicone mate- example, Schierholz et al.（ 1994） incorporated the rials in food processing and medical institutions（ e.g., antibiotic rifampicin into silicone materials to prevent tubes, packing materials, and membranes） are often in microbial colonization. In their study, only high rifampicin contact with water, which promotes the adhesion of doses were administered over several weeks to prevent bacterial colonization. McBride et al.（ 2009） incorpo- ＊Corresponding author. Tel & Fax: +81-46-291-3193, E-mail : rated triclosan and prepared cast silicone membranes. sawa（i a）bio.kanagawa-it.ac.jp They reported that a prolonged antibacterial effect was 98 S. AOKI ET AL. obtained by grafting polyethylene glycol（ PEG） chains silicone used to construct the membrane. Fujimori et al. into the cast silicone membrane containing triclosan. In （2008） also doped AgI into nylon 6 by immersion in an addition, many researchers have reported the produc- iodide solution, followed by silver nitrate（ AgNO3） treat- tion of antimicrobial-coated silicone materials by surface ment to improve the fiber conductivity. In addition, the modification of silicone rubber, using methods such as composite fiber exhibited antibacterial activity. Effective immobilization of hydrogen peroxide-producing enzymes antimicrobial coating was thought to be possible by （e.g., cellobiose dehydrogenase） via ultrasound-assisted reacting Ag ion with the surface of the silicone membrane coating（ Lipovskt et al., 2015）; immobilization of short impregnated in advance with iodine. The present report peptides（ tryptophan-rich, arginine-rich, and lysine-rich） describes the preparation of Ag/silicone composite through surface grafting polymerization induced by membrane materials with high durability using a simple, plasma ultraviolet exposure（ Li et al., 2014）; electrostatic two-step immersion process̶first in an iodine solution layer-by-layer assembly of antifouling copolymers, and then in a AgNO3 solution̶at room temperature. In bactericidal derivatives, and cellobiose dehydrogenase addition, the antibacterial and mechanical properties （Vaterrodt et al., 2016）; immobilization of polysulfobe- were investigated as well the durability of the prepared taine polymers on crosslinked silicone layers through membranes. ozone treatment; covalent grafting of ultraviolet（ UV） -induced polymerization and thiol-ene click reactions（ Li MATERIALS AND METHODS et al., 2012）; and direct grafting of pol（y N-vinylimidazole） on silicone by gamma-ray irradiation（ Meléndez-Ortiz et Preparation of Ag/silicone composite membranes al., 2015）. Although several methods for incorporation The Ag/silicone composite membranes were prepared of organic agents have been reported, they require using the following method, which is a modification of the complicated steps, special equipment, or high energy procedure described by Fujimori et al.（ 2008）. A 0.3-mm levels. thick silicone membrane（ ASONE, Osaka, Japan） was Silver is a particularly suitable antibacterial agent cut into 50 mm×50 mm pieces. An iodine-potassium because of its oligodynamic effect at relatively low iodide solution was prepared by dissolving 0.03-0.15 M concentrations（ Clement and Jarrett, 1994）. Silver can I2 in 3.3 M KI solution. The silicone membrane was bind to microbial DNA, generate reactive oxygen species, immersed in the I2-KI for 1-24 h. The membrane was inhibit bacterial replication, and bind to the thiol groups removed, rinsed twice with sterile pure water, and then of metabolic enzymes in the bacterial electron-transport immersed in 0.25-0.75 M AgNO3 solution for 1-24 h. The chain, causing enzyme modification and inactivation membrane was removed, rinsed twice with sterile pure （Russell and Hugo, 1994）. Ciobanu et al.,（ 2015） and water, and disinfected with ethanol on a clean laboratory Popa et al.,（ 2015） used a thermal evaporation tech- bench. All reagents were purchased from Wako Pure nique（ 0.01 Pa and >1100℃） to coat Ag-doped Chemicals（ Osaka, Japan）. hydroxyapatite onto PDMS. Such an antibacterial composite was active against Candida albicans biofilms. Antibacterial efficacy test In addition, composite samples containing Ag could be Escherichia coli NBRC 3301 and Staphylococcus prepared using silver benzoate with PDMS（ Bovero et aureus subsp. aureus NBRC13276 were obtained from al., 2013） or by mixing Ag-loaded zeolite with PDMS the National Institute of Technology Evaluation Biological （Kaali et al., 2010）. Oktay and Kayaman-Apohan Resource Center（ NBRC, Kazusa, Japan）. They were （2013） prepared PDMS containing Ag by UV curing incubated in nutrient broth（ NB） with shaking（ 110 and sol-gel processes. Although these methods could strokes/min） for 20 h at 37℃. Cultures were washed produce effective antimicrobial-finished silicone mate- and resuspended in 1/500 NB at approximately 106 CFU/ rials, they required high energy, long reaction times, or ml. The bacterial suspension was stored in an ice water complicated preparation steps. bath before use. Antibacterial efficacy（ R） was evalu- Iodine molecules can accumulate at high concentra- ated according to JIS Z 2801（ 2001）. All growth media tions in silicone membranes according to studies on the were purchased from Eiken Chemicals（ Tokyo, Japan）. separation of iodine using these membranes（ Sawai et A value of R > 2 was considered to indicate antibacte- al., 2012a）. Nakamura et al.（ 2011） prepared iodine- rial efficacy. releasing silicone membranes using an adsorption process, which produced membranes with high anti- Scanning electron microscopy（ SEM） and elemen- bacterial activity. However, these membranes were not tary analysis durable because the iodine was easily released from the The surface of the Ag/silicone membrane was charac- membrane. Therefore, an Ag compound, such as silver terized using SEM and energy-dispersive X-ray spectros- iodine（ AgI）, was formulated with low solubility in the copy（ EDS; model SU9000, Hitachi High-Technologies ANTIBACTERIAL Ag/SILICONE MEMBRANE 99 Co., Tokyo, Japan）. The SEM was conducted at an properties（ a feed cell solution and a recovery cell solu- acceleration voltage of 0.5-30 kV and 1.7 mm working tion） were separated by a silicone membrane fixed with distance. The untreated silicone membrane was also a flange between two glass cells. A solution of penta- characterized by SEM（ JSF-7001F, Jeol Ltd., Tokyo, chlorophenol（ PCP: Wako Pure Chemicals） dissolved Japan）. in an acidic solvent was placed in the feed cell, and the recovery cell was filled with an alkaline solution. The PCP Tensile strength molecules dissolved in the feed solution, being proton-