J Mater Sci (2014) 49:3407–3413 DOI 10.1007/s10853-014-8050-5
Fabrication of silver-loaded hollow mesoporous aluminosilica nanoparticles and their antibacterial activity Weijun Fang • Ling Ma • Jun Zheng Cheng Chen
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Received: 1 November 2013 / Accepted: 15 January 2014 / Published online: 28 January 2014 Ó Springer Science+Business Media New York 2014
Abstract A facile and an effective route for the preparation of silver-loaded hollow mesoporous aluminosilica ([Ag]-HMAS) nanoparticles is reported. In our fabrication process, the solid silica nanoparticles are first converted into high-quality hollow mesoporous aluminosilica particles, and then silver ions are induced into the matrix through an ion-exchange method. By EDX and ICP analysis, it is found that the final [Ag]-HMAS particles have high silver loading (15.8 % by weight), and the release amount of silver ions can be effectively controlled by altering the concentrations of Na? in the solution. The antibacterial properties of the [Ag]-HMAS particles against Escherichia coli (Gram-negative bacteria) and Bacillus subtilis (Gram-positive bacteria) are also investigated, both in liquid systems and on solid agar plates. The results show that the [Ag]-HMAS particles are highly effective against both Gram-negative and Gram-positive bacteria.
W. Fang (&) College of Basic Medicine, Anhui Medical University, Hefei 230032, Anhui, China e-mail:
[email protected] L. Ma Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, Fujian, China e-mail:
[email protected] J. Zheng C. Chen Center of Modern Experimental Technology, Anhui University, Hefei 230039, Anhui, China e-mail:
[email protected] C. Chen e-mail:
[email protected]
Introduction Infection with the bacteria is a serious problem for public health. Many antibacterial agents such as quaternary ammonium salts, phenolic compounds, and polymers have been used to reduce the risk of people’s health [1–7]. However, some of those organic antibacterial agents cannot be applied to medical devices, foods, living supplies, and so on, due to their toxicity and poor efficiency. Therefore, finding out a powerful antibacterial agent with good biologic compatibility is vital. It is well known that silver ions have strong antibacterial effects and a broad spectrum of antimicrobial activity, even at low concentrations and relatively nontoxic to human cells. Therefore, silver has been widely used in research and industry [8–11]. To improve their stabilities and longtime antibacterial activities, the most effective way is to load silver ions on porous materials. At present, zeolite [12–15], calcium phosphate [16–18], clay [19, 20], and silica [21–25] have been reported as supports for fabricating silver-containing antibacterial agents. Especially, hollow porous silica is expected to be a good candidate for this application owing to its unique hollow porous structures and large surface area [26, 27]. However, there is lack of studies using hollow porous materials as the carriers for silver ions to improve their antibacterial efficiency. Herein, we have developed an effective route to prepare silver-loaded hollow mesoporous aluminosilica ([Ag]HMAS) nanoparticles. In this process, we first employ ‘‘cationic surfactant assisted selective etching’’ method to obtain HMAS nanoparticles ([Na]-HMAS), and subsequently sodium ions in the [Na]-HMAS are replaced by silver ions through an ion-exchange method. By EDX, XPS, and ICP analysis, the final [Ag]-HMAS particles show high loading amount of silver and could achieve
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controlled release of the loaded Ag?. Finally, their antibacterial activity is also investigated by Escherichia coli and Bacillus subtilis, respectively.
Experimental section Chemicals TEOS was purchased from Alfa Aesar. AgNO3, cetyltrimethylammonium bromide (CTAB), Na2CO3, ethanol, ammonium aqueous solution (25–28 %), and NaAlO2 were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All the reagents were used as received without further purification. Synthesis of sSiO2 nanoparticles Solid SiO2 (sSiO2) were prepared using a modified Sto¨ber method. Typically, 74 mL of ethanol, 3.15 mL of ammonium aqueous solution (*28 %), and 10 mL of ultrapure water were mixed and further stirred for 1 h. The mixture was then heated up to 50 °C, and 6 mL of TEOS was added. After the reaction with stirred for 6 h, sSiO2 were obtained by centrifugation and washed with ethanol. Synthesis of [Na]-HMAS/CTAB nanoparticles 50 mg of sSiO2 were redispersed in 9.0 mL of water solution, and then 1.0 mL of water solution containing 12.5 mg of CTAB was added. After 10 min of stirring, 35 mg of NaAlO2 and 40 mg of Na2CO3 were added to the above mixture. The mixture was heated at 95 °C with continuous stirring for 4 h, and the products [Na]-HMAS/ CTAB nanoparticles were collected by centrifugation. Removal of CTAB from [Na]-HMAS/CTAB nanoparticles After drying under vacuum, the [Na]-HMAS/CTAB nanoparticles were then heated at a rate of 1.0 °C/min and maintained at 550 °C for 6 h in air to remove the remaining surfactant (CTAB).
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Determination of Ag? released from [Ag]-HMAS nanoparticles The releasing of Ag? was by shaking 1.0 mg of [Ag]HMAS nanoparticles with 1.0 mL of 0–100 mM NaNO3 solution for 24 h in the dark. The mixed solution was centrifuged, and the concentrations of Ag? in the supernatants were determined by ICP. Characterization Transmission electron microscopy (TEM) studies were performed on a TECNAI F-30 high-resolution TEM operating at 300 kV. Scanning electron microscopy (SEM) images were obtained on a Hitachi S4800 scanning electron microscope with a field emission electron gun. The surface area and pore size distribution of the final products were determined by Surface Area and Porosity Analyzer (Micromeritics Instrument Corp. ASAP2020). The chemical state of silver in our samples was characterized by XPS (PHI Quantum 2000). Measurements of antibacterial properties of [Ag]HMAS nanoparticles in LB liquid medium The antibacterial activities of the as-prepared [Ag]HMAS nanoparticles were valuated against E. coli BL21 and B. subtilis. The inoculation of these kinds of bacteria were prepared by growing strains in LuriaBertani (LB) liquid medium at 37 °C until a level of approximately 109 CFU/mL of bacteria was reached. Then, 400 lL of 109 CFU/mL bacterial suspension was added to 40 mL LB liquid medium containing different concentrations of [Ag]-HMAS nanoparticles (0, 50, 100, 200, 250, and 300 lg/mL) and incubated at 37 °C with continuous agitation (180 rpm). After incubated for 12 h, the bacterial growth inhibition was determined by measuring OD at 600 nm. Control experiments were also performed in the presence of [Na]-HMAS nanoparticles. Measurements of antibacterial properties of [Ag]HMAS nanoparticles on agar plates
Preparation of [Ag]-HMAS nanoparticles AgNO3 solution (2.0 mL) at a concentration of 1.25 mg/mL was mixed to 10 mg of [Na]-HMAS nanoparticles, and then the mixture was stirred in the dark at room temperature for 24 h. The final products [Ag]-HMAS were obtained by centrifugation, washed with ultrapure water, and dried in an oven at room temperature.
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To evaluate the bacterial growth inhibition with LB agar plates, 100 lL solution containing different concentrations of [Ag]-HMAS nanoparticles (0, 12.5, 25, and 50 lg/mL) were first plated onto the LB agar plates. Then, 100 lL of LB liquid medium with approximately 103 CFU of E. coli was plated onto the solidified agar plates and incubated at 37 °C for 20 h.
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Scheme 1 Synthetic routes of silver-loaded hollow aluminosilica nanoparticles ([Ag]-HMAS)
Fig. 1 SEM images and TEM images a SEM images of sSiO2, b SEM images of [Na]-HMAS particles, c and d TEM images of [Na]-HMAS particles
Results and discussion The design process of [Ag]-HMAS is depicted in Scheme 1. Solid silica nanoparticles (sSiO2) were prepared by Sto¨ber method and acted as the next reactive materials, and were dispersed into an aqueous mixture of Na2CO3, NaAlO2, and CTAB. The solid Sto¨ber silica nanoparticles could convert into [Na]-HMAS with thermal treatment of the mixture. Then, the [Ag]-HMAS nanoparticles were obtained through an ion-exchange process by immersion the [Na]-HMAS particles in AgNO3 solution. The sSiO2 nanoparticles were first characterized by SEM. As shown in Fig. 1a, the obtained sSiO2 is spherical in shape with an average diameter of 180 nm. The sSiO2 particles were then translated into [Na]-HMAS using the modified method reported by Zheng and coworkers [28]. The hollow structure of [Na]-HMAS can be demonstrated
Fig. 2 N2 adsorption/desorption isotherm and the pore size distribution (inset) of [Na]-HMAS particles
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Fig. 5 Desorption of the silver ions from [Ag]-HMAS particles in NaNO3 solutions
Fig. 3 Energy-dispersive X-ray spectroscopy (EDX) spectra of [Na]HMAS particles (a) and [Ag]-HMAS particles (b)
Fig. 4 High-resolution spectra of Ag 3d region for [Ag]-HMAS particles (top) and [Na]-HMAS particles (bottom)
by the contrast differences between the core and shell both in the SEM and in the TEM images (Fig. 1b, c). As revealed in the high-magnification TEM image (Fig. 1d), the shell of the [Na]-HMAS with a thickness of *33 nm exhibits a uniform and orderly mesoporous structure. The porosity of [Na]-HMAS was also investigated by N2
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Fig. 6 a Antimicrobial efficacy of [Ag]-HMAS particles against the Gram-negative bacteria E. coli in LB liquid medium. b Antimicrobial efficacy of [Ag]-HMAS particles against the Gram-positive bacteria B. subtilis in LB liquid medium
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Fig. 7 E. coli and B. subtilis grown on agar plates at different concentrations of [Ag]-HMAS particles. E. coli (top) a 0 lg/ml, b 12.5 lg/mL, c 25 lg/mL, and d 50 lg/mL; B. subtilis (bottom) e 0 lg/ml, f 12.5 lg/mL, g 25 lg/mL, and h 50 lg/mL
adsorption/desorption measurements. [Na]-HMAS shows a typical type IV isotherm feature (Fig. 2). The Brunauer– Emmett–Teller surface area, pore volume, and pore diameter of the [Na]-HMAS are 589.5 m2/g, 0.61 cm3/g, and 2.2 nm, respectively. In the hollow mesoporous aluminosilicate nanoparticles, the silicon atoms are partially substituted by tetrahedrally coordinated aluminum, producing a negative charge on the matrix. The alkaline metal cations, which are absorbed into the pores and balance the negative charge of the matrix, can be exchanged by transition metal ions, such as Ag?, Cu2?, and Zn2? [29–31]. Therefore, the content of the exchanged cations in the hollow mesoporous aluminosilicate nanoparticles is partially dependent on the aluminum percentage in the matrix. In order to obtain high-aluminum-containing nanocarriers, we modified the method reported by Zheng et al. for preparation of the [Na]-HMAS (See the experimental section for details). As revealed in Fig. 3a, the main composition elements in the [Na]-HMAS particles are Si, O, Al, and Na. The Al/Si molar ratio of 0.45 could be reached by EDX analysis, which is slightly higher than their reported results. Compared to other metal ions carriers, the [Na]-HMAS particles’ advantage lies in their hollow core, ordered mesoporous aluminosilicate shell, and a cation-exchange capability. Based on these unique characteristics, the [Na]HMAS particles are expected to be ideal metal ions carriers. It well known that silver ions have powerful antibacterial capability and a wide antibacterial spectrum.
Therefore, Ag? was selected as an exchange ion to fabricate the silver-loaded nano-antimicrobial agent. After Ag? was exchanged in the [Na]-HMAS particles framework, the amount of sodium in the matrix reduced to 3.6 % from 9.8 % (in weight), and the silver content could reach up to 18.3 % by EDX analysis (Fig. 3). However, not all the sodium ions in the matrix are replaced by silver ions. This may be because a part of Na? exists in the internal matrix framework during the synthesis process. The silver content of [Ag]-HMAS particles evaluated from inductively coupled plasma mass spectrometry (ICP) is 15.8 %, which is slightly lower than the value of EDX analysis. It probably resulted from the difference of the two methods. Silver ion was introduced through immersion of the [Na]-HMAS in AgNO3 solution since Ag? is known to replace Na? in the matrix. Figure 4 shows the high-resolution XPS spectra for both [Na]-HMAS particles and [Ag]-HMAS particles in Ag 3d region. There are two main peaks centered at 368.1 eV for Ag 3d5/2 and 374.2 eV for Ag 3d3/2 for the [Ag]-HMAS particles, suggesting that the silver in the [Ag]-HMAS particles is present in the oxidized state [32]. To examine whether Ag? could be released from the matrix, the [Ag]-HMAS particles were dispersed in different concentrations of NaNO3 solution. After 24 h of stirring, the mixed solution was centrifuged, and the concentrations of Ag? in the supernatants were determined by ICP. As shown in Fig. 5, the release amount of Ag? increases with increasing the concentrations of
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Na?, and almost all the Ag? are released at an Na? concentration of 80 mM, suggesting that the adsorbed silver ions could be exchanged by sodium ions in the solution. Subsequently, E. coli and B. subtilis, Gram-negative and Gram-positive bacteria, respectively, were used to investigate the antibacterial activity of the [Ag]-HMAS particles. Assays were carried out in the [Ag]-HMAS particles concentrations ranging from 50 to 300 lg/mL. The antimicrobial efficacies of the [Ag]-HMAS particles were also calculated for both the tested bacteria. As shown in Fig. 6, the [Na]-HMAS particles exhibit no bacterial inhibitory effects, whereas the [Ag]-HMAS particles show antibacterial properties at all the tested concentrations, suggesting that this antibacterial property is due to the presence of Ag? in the particles. The results also clearly indicate that at a given number of bacterial cells, the antibacterial efficacy for both the bacteria depends on the particles’ concentrations. At the concentration of 250 lg/mL, 100 % inhibitions of growth of the both bacteria are observed within 12 h. In addition, the antibacterial activity of the [Ag]HMAS particles was further studied on LB agar plates. Figure 7 shows the number of bacterial colonies grown in the presence of different amounts of [Ag]-HMAS particles when 100 lL of LB liquid media containing 103 bacterial cells was applied to each plate. It can be seen that the number of bacterial colonies decreases with increasing the amount of the [Ag]-HMAS particles used in the tests, and no bacterial colonies are observed in the presence of the samples at the concentration of 50 lg/mL for both E. coli and B. subtilis. Therefore, all the results suggest that the [Ag]-HMAS particles have excellent antibacterial activity against Gram-negative and Grampositive bacteria.
Conclusions [Na]-HMAS have been successfully adopted as novel vehicles for immobilization of silver ions. Silver ions could be easily adsorbed onto the hollow silica matrix by ionexchange method, and the silver loading amount in our vehicles is as high as 15.8 % in weight. The final [Ag]HMAS particles possess excellent antibacterial activities against both E. coli and B. subtilis. It is reasonable except that the [Ag]-HMAS particles have great potential use as an antibacterial agent. Acknowledgments We thank the Grants for Scientific Research of BSKY (0115027101) from Anhui Medical University, the Program for the Outstanding Young and Middle-aged Talents (0115027102) of Anhui Medical University for the financial support.
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