Hybrid calcium carbonate/polymer microparticles containing silver nanoparticles as antibacterial agents
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We report here on synthesis and characterization of novel hybrid material consisting of silver nanoparticles (nAgs) embedded in calcium carbonate microparticles (μ-CaCO3) serving as carriers for sustained release. nAgs are commonly used as antimicrobial agents in many commercial products (textiles, cosmetics, and drugs). Although they are considered to be safe, their interactions with human organisms are still not fully understood; therefore it is important to apply them with caution and limit their presence in the environment. The synthesis of the new material was based on the co-precipitation of CaCO3 and nAg in the presence of poly(sodium 4-styrenesulfonate). Such designed system enables sustained release of nAg to the environment. This hybrid colloidal material (nAg/μ-CaCO3) was characterized by microscopic and spectroscopic methods. The release of nAg from μ-CaCO3 microparticles was followed in water at various pH values. Microbiological tests confirmed the effectiveness of these microparticles as an antibacterial agent. Importantly, the material can be stored as a dry powder and subsequently re-suspended in water without the risk of losing its antimicrobial activity. nAg/μ-CaCO3 was applied here to insure bacteriostatic properties of down feathers that may significantly prolong their lifetime in typical applications. Such microparticles may be also used as, e.g., components of coatings and paints protecting various surfaces against microorganism colonization.
KeywordsSilver nanoparticles Antimicrobial agent Calcium carbonate microparticles Controlled release
Silver, in the form of nanoparticles (nAg) and ions, is a common antimicrobial agent. It interacts with cell components (e.g., DNA, RNA, and ribosomal units), deactivating them and effectively stopping microbial life processes. It has been observed that nAg can puncture cell membranes and penetrate deep into the cell interior. This, in combination with their slow dissolution and ability to release Ag+ ions, results in excellent antimicrobial activity even at nanomolar concentrations (Sondi and Salopek-Sondi 2004; Yamanaka et al. 2005; Feng et al. 2000; Lok et al. 2006). nAg are effective agents even against multidrug-resistant strains (Lara et al. 2009). These exceptional properties of nAg resulted in wide range of their practical applications. They have been applied as an antibiotic additive in the production of biomedical devices (Roe et al. 2008; Cao and Liu 2010), wound dressings (Nguyen et al. 2011; Tian et al. 2007), water (Lv et al. 2009; Jain and Pradeep 2005) and air (Yoon et al. 2008) filters, textiles (Tang et al. 2011; Lee et al. 2003), food packaging (Tankhiwale and Bajpai 2009), cosmetics (Kokura et al. 2010), and drugs (Shahverdi et al. 2007).
However, it has been shown that the growing number of application of nAg may lead to the build up of silver concentrations in the environment to such a level at which it can not only cause health problems in humans (Kim et al. 2011) but also may be harmful for various components of the environment (Lee et al. 2007) and may even affect the industrially relevant processes (Brar et al. 2010). Therefore, it is important to use nAg rationally and to control their release to the environment. The current paper presents our approach to this problem. We have proposed to embed nAg into the biologically inert calcium carbonate microparticle (μ-CaCO3) matrix (Lei et al. 2006). Several other attempts to immobilize nAg with polymers have been already proposed (Benetti et al. 2010). They include immobilization on glass and silica (Cai et al. 2001; Lv et al. 2008), on natural fibers (Tang et al. 2011; Lee et al. 2003), on hydroxyapatite (Nirmala et al. 2011), and even on the surface of steel (Chen et al. 2010). However, to the best of our knowledge, the colloidal system containing silver nanoparticles trapped inside calcium carbonate microparticles (nAg/μ-CaCO3) has not been fabricated so far. Such application of μ-CaCO3 microparticles is in line with their most common usage as sacrificial templates for fabrication of hollow polymeric microcapsules (Volodkin et al. 2004) carrying various organic molecules such as low molecular weight drugs (Tong et al. 2011) or proteins (Petrov et al. 2005). The approach proposed here should insure long-lasting antibacterial activity of the material due to the sustained release of silver on one hand and limit the contact of nAg with human body on the other. In addition, this material can be easily stored as a water dispersible powder and its surface charge can be readily modified for various applications. This new material may find applications as a component of coatings and paints protecting the surfaces against microorganism colonization.
Materials and methods
Poly(sodium 4-styrenesulfonate) (PSS, Aldrich, Mw ≈ 70,000 g/mol), poly(allylamine hydrochloride) (PAH, Aldrich, Mw ≈ 15,000 g/mol). Silver nitrate, trisodium citrate, calcium nitrate, sodium carbonate, acetone, sulfuric acid (98 %), nitric acid (65 %), hydrogen peroxide (30 %), ethylenediaminetetraacetic acid (EDTA), disodium phosphate, and citric acid were obtained from POCH Gliwice, Poland. Single-side polished silicon plates were obtained from the Institute of Electronic Materials Technology (Warsaw, Poland) and cleaned before the use in freshly prepared “piranha solution” (a mixture of 30 % solution of H2O2 and concentrated H2SO4 in 1:3 ratio) (Caution: “piranha solution” should be handled with extreme care!). Deionized water was used in all experiments. Buffers were prepared by mixing appropriate amounts of 0.1 M citric acid and 0.2 Na2HPO4.
Synthesis of nAg/μ-CaCO3 material and its surface modification
In the first step, nAg were obtained by modified Lee and Meisel method (1982). Shortly, 45 mg of silver nitrate was dissolved in 250 ml of water and then 5 ml of 1 % trisodium citrate was added. The reaction mixture was placed in an ultrasonic bath and heated to 75 °C for 60 min. In the second step, 20 ml of the obtained nAg colloid was simultaneously mixed with 50 ml of 0.03 M Ca(NO3) solution and 50 ml of 0.03 Na2CO3 with addition of PSS (4.8 g/l). The mixture was sonicated for 5 min at 25 °C. The obtained white colloid was washed with deionized water and centrifuged at 4,000 rpm for 5 min to remove excess of silver. The washing process was repeated three times and the obtained product was dried under vacuum.
For surface modification, nAg/μ-CaCO3 particles were dispersed in PAH solution (1 g/l in 0.1 M NaCl) and stirred for 15 min. They were subsequently centrifuged and washed with deionized water.
Characterization of nAg
UV–Vis spectrum of the obtained nAg suspension was acquired. The size of the nanoparticles was determined by atomic force microscopy (AFM, Picoforce, Bruker, USA) working in tapping mode. Standard silicon cantilevers (Bruker) with nominal spring constants equal to 40 N/m were used for the measurements. For that purpose, a silicon plate was first immersed in PAH solution (1 g/l in 0.1 M NaCl) and sonicated for 5 min. The plate was subsequently rinsed with deionized water and dried under stream of argon. A drop of the colloidal suspension of nAg was deposited on such prepared silicon support and left for drying before the imaging.
Characterization of nAg/μ-CaCO3 material
Scanning electron microscopy images were obtained for nAg/μ-CaCO3 using Hitachi S-4700 microscope with field emission. The samples were coated with sputtered gold layer before imaging.
To check if silver in the form of nanoparticles is embedded in μ-CaCO3, the matrix was dissolved in 0.2 M EDTA and the UV–Vis spectrum of the obtained solution was measured.
Total content of silver in nAg/μ-CaCO3 was measured by atomic absorption spectrometry (AAS) using PerkinElmer Aanalyst 300 instrument with flame atomizer. The samples were transferred into solution by dissolving in 0.12 M HNO3. The appropriate calibration procedure was performed using samples of AgNO3 in 0.12 M HNO3. The measurements were also performed for the starting nAg dispersion and supernatant collected after centrifugation of the reaction mixture.
Accelerated release of nAg from nAg/μ-CaCO3
The samples of nAg/μ-CaCO3 (440 mg) were dispersed in 8 ml of the appropriate buffer solutions with pH values equal to 5.4 and 6.8. They were vigorously stirred for a given period of time and then centrifuged. Supernatants were collected for AAS analyses and the next portions of fresh buffer solutions were added each time. The procedure was continued for 14 days.
Treatment of down feathers
The nAg/μ-CaCO3 were introduced to the feathers by their immersion in the 0.6 % dispersion for 30 min. Then the feathers were dried under a stream of argon. The samples were investigated by optical microscopy (Nikon Eclipse LV 100) and SEM (Hitachi S-4700 s microscope) to confirm the attachment of nAg/μ-CaCO3 to the feathers.
In vitro antimicrobial activity
To prove the antimicrobial activity of the obtained materials, the microbiological tests were carried out. Various bacterial species were placed in physiological salt media to which equal amounts of nAg/μ-CaCO3 dispersion were added and left for 24 h. The bacterial and fungal species were naturally occurring skin microbes and were acquired from several healthy people. The microbial concentration was expressed in McFarland scale (°McF) based on the nephelometric measurements. One McFarland scale is equivalent to turbidity of standard BaSO4 colloid in concentration of 4.80 × 10−5 M. nAg/μ-CaCO3 as well as down/nAg/μ-CaCO3 and sole nAg dispersion systems were tested. The initial turbidity was acquired before the addition of the antimicrobial materials or down feathers. The concentration of the antimicrobial hybrid material was kept constant in each test and it was allowed to sediment before the measurements of the microbial concentration. The feathers, after the incubation, were removed from the dispersion. In the case of sole nAg dispersion, the total turbidity (with contribution of nAg) was measured. The respective microbial concentrations were taken as mean values of a few measurements.
Results and discussion
Successful two-step synthesis of novel antimicrobial material consisting of nAg embedded in μ-CaCO3 microparticle matrix was achieved. The nAg were obtained by modified method developed earlier by Lee and Meisel. The method is based on the reduction of silver nitrate by sodium citrate in hot water (Lee and Meisel 1982). It is important to note that the process is carried out in aqueous medium and with the use of low toxicity reagents. Citrate ions play also a second function—they protect Ag nanoparticles form aggregation, thus keeping their size relatively small. This is very important considering the fact that the antimicrobial activity of nAg rises significantly with the reduction of their sizes (Panáček et al. 2006).
Novel hybrid antibacterial microparticles, nAg/μ-CaCO3, consisting of nAg incorporated into calcium carbonate/polymer microparticles (μ-CaCO3), were obtained in a simple synthetic procedure carried out in aqueous environment and involving non-toxic compounds. Originally prepared nAg with diameters in the range of 30–50 nm were used in co-precipitation process which results in the formation of nAg/μ-CaCO3 spherical microparticles. The obtained material enabled sustained release of nAg, thus limiting the possible environmental problems. Surface charge of the matrix can be readily modified by adsorption of polycations expanding potential applications of nAg/μ-CaCO3 to various surfaces. This novel material can be safely stored as a white powder without the risk of losing its antimicrobial activity, in contrast to the sole nAg which tend to aggregate when dried. nAg/μ-CaCO3 system was shown to be efficient in limiting the proliferation of microorganisms that makes it suitable for applications involving contact with human skin. As an example, the successful application of nAg/μ-CaCO3 for the protection of down feathers against microorganisms was presented.
The authors would like to acknowledge the financial support of EU Programme “Doctus” MPC.ZS.4110-63.4/2008. The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (Contract No. POIG.02.01.00-12-023/08). The P2M program from the ESF is also gratefully acknowledged.
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