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Bacterial adhesion inhibition of the quaternary ammonium functionalized silica nanoparticles
Colloids and Surfaces B: Biointerfaces 82 (2011) 651–656
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short communication
Bacterial adhesion inhibition of the quaternary ammonium functionalized silica nanoparticles Jooyoung Song, Hyeyoung Kong, Jyongsik Jang ∗ WCU Program of Chemical Convergence for Energy and Environment (C2E2), School of Chemical and Biological Engineering, College of Engineering, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
a r t i c l e
i n f o
Article history: Received 12 April 2010 Received in revised form 1 October 2010 Accepted 11 October 2010 Available online 20 October 2010 Keywords: Quaternary ammonium silane Nanoparticles Bacterial growth Antibacterial
a b s t r a c t Quaternary ammonium compounds have been considered as excellent antibacterial agents due to their effective biocidal activity, long term durability and environmentally friendly performance. In this work, 3-(trimethoxysilyl)-propyldimethyloctadecylammonium chloride as a quaternary ammonium silane was applied for the surface modification of silica nanoparticles. The quaternary ammonium silane provided silica surface with hydrophobicity and antibacterial properties. In addition, the glass surface which was coated with the surface modified silica nanoparticles presented bacterial growth inhibition activity. For comparison of bacterial growth resistance, hydrophobic silane (alkyl functionalized silane) modified silica nanoparticles and pristine silica nanoparticles were prepared. As a result of bacterial adhesion test, the quaternary ammonium functionalized silica nanoparticles exhibited the enhanced inhibition performance against growth of Gram-negative Escherichia coli (96.6%), Gram-positive Staphylococcus aureus (98.5%) and Deinococcus geothermalis (99.6%) compared to pristine silica nanoparticles. These bacteria resistances also were stronger than that of hydrophobically modified silica nanoparticles. It could be explained that the improved bacteria inhibition performance originated from the synergistic effect of hydrophobicity and antibacterial property of quaternary ammonium silane. Additionally, the antimicrobial efficacy of the fabricated nanoparticles increased with decreasing size of the nanoparticles. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Recently, adhesion and subsequent growth of microorganisms is a main concern of the biomedical device failure. A large number of infections are attributed to viable bacteria that adhered to medical devices and implants [1–4]. In contrast to plankton (free-floating bacteria), when the bacteria adhere to the surface, grow, and form biofilm, they are up to 1000 times more tolerant to disinfection treatment [4]. Therefore, it can be clearly anticipated that the bacterial growth is efficiently inhibited in the early step of bacterial adhesion. Several researchers reported that the bacterial adhesion was reduced on the hydrophobic surfaces because of weak binding energy at the interface between the bacterium and the hydrophobic surface [5–8]. However, hydrophobic treatments can neither perfectly protect the surface from bacterial adhesion nor eradicate the contacted bacteria; the sporadically adhered bacteria will grow and form biofilm eventually. The quaternary ammonium functionalized materials, as antibacterial agents, have received much attention because they provide effective protection against bacterial colonization
∗ Corresponding author. Tel.: +82 2 880 7069; fax: +82 2 888 1604. E-mail address: [email protected] (J. Jang). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.10.027
with long term durability and environmentally friendly performance [9–19]. Among the quaternary ammonium compounds, 3-(trimethoxysilyl)-propyldimethyloctadecylammonium chloride (quaternary ammonium silane (QAS)) excellently inhibits the adhesion and growth of bacteria due to its hydrophobicity and antibacterial property [16–19] The QAS-modified substrate surface can resist the bacterial adhesion with water-repelling hydrophobicity and eradicate the contacted bacteria with biocidal capability. Busscher et al. and Neoh groups reported that the QAS-treated substrates had antibacterial activities on the biofilm test [17–19]. However, relatively little attention has been paid to the study of the size control of QAS-modified materials in the viewpoint of nano scale. In the research field of nano-materials fabrication, the core–shell nanostructures have attracted a great deal of interest owing to their facile synthesis process and various potential applications [20–27]. Especially, as a core-part, silica nanoparticle has several advantages such as tunable surface functionality, low cytotoxicity, and easily controllable size [23–27]. In addition, the organic materials coated silica nanoparticles combine the functionality of organic shell with high colloidal stability of the silica core, which can expand their applications in the fields of magnetics [23,24], optics [25], and antimicrobials [26,27]. Herein, we report the fabrication of size controllable silicaQAS core–shell nanoparticles (NPs) and their bacterial growth
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resistance. Octadecyltrimethoxy silane (OdS) (alkyl functionalized silane) modified silica NPs were prepared in order to investigate the relationship between hydrophobicity and bacterial adhesion. For antimicrobial test, Gram-negative Escherichia coli (E. coli), Gram-positive Staphylococcus aureus (S. aureus), and Deinococcus geothermalis (D. geothermalis) were selected as detrimental bacteria. Based on the antimicrobial properties of the QAS compound, it can be expected that the fabricated silica-QAS core–shell NPs present excellent inhibition activity to the bacterial growth compared with pristine silica NPs and silica-OdS core–shell NPs. Furthermore, in order to evaluate the size-dependency of antibacterial efficacy of silica-QAS core–shell NPs, various sized (16, 28, and 54 nm) silica-QAS core–shell NPs were fabricated using different sized silica colloids as the core materials. 2. Experimental 2.1. Materials The 11 nm, 22 nm, and 44 nm sized silica nanoparticles were purchased from Aldrich (Milwaukee, WI). Glutaraldehyde, osmium tetroxide, octadecyltrimethoxy silane (OdS), and 3-(trimethoxysilyl)-propyldimethyloctadecylammonium chloride (QAS) were also purchased from Aldrich (Milwaukee, WI). For the bacterial growth test, E. coil (ATCC 11775), S. aureus (ATCC 12600), and D. geothermalis (DSM 11300) were purchased from Fisher Company. 2.2. Fabrication of the silane-modified silica nanoparticles First, 1 g of the silica nanopowder was dispersed in 100 ml of water–ethanol mixture solution (1:1, v/v) and added 0.5 ml of silane coupling agents (QAS or OdS) and the mixture was stirred at 25 ◦ C, 24 h. The silane-modified silica NPs were fabricated via covalent linkage between –Si(OCH)3 groups of silane coupling agent (QAS and OdS) and –OH groups of silica NPs under mild condition [28,29]. After silane treatment, the synthesized silane-modified silica nanoparticle was obtained by centrifugal precipitation and washed three times with distilled water to remove the residual reagents. 2.3. Characterization Photographs of transmission electron microscopy (TEM) were obtained with a JEOL JEM-200CX. Acceleration voltage for TEM was 200 kV. Field-emission scanning electron microscopy (FE-SEM) images were obtained using a JEOL 6700 at an acceleration voltage of 10 kV. In the sample preparation of TEM and FE-SEM characterizations, each silane-modified silica nanoparticles were dispersed in absolute ethanol and cast onto copper grid. The Z-potential of the surface-treated silica nanoparticles were measured with an ELS8000 at pH 7 and elemental analysis value was obtained using CHNS-932 (LECO Corp, US). For measurement of the water contact angles, each sample was pressed by hydraulic press to obtain disc shape and the diameter of disc was 1.3 mm. The water-contact angles of the pressed samples were measured with a Krüss DSA10 contact angle analyzer interfaced to drop shape analysis software.
(0.5 wt%) with sonication for 30 min. Then, the 2 mL of each suspension was drop casted onto the surface of glass slides and these slides were dried in 60 ◦ C oven. The pristine silica nanoparticles coated glass slide was prepared as a control. The coating thickness of the nanoparticles on the glass surface was ca. 15 m. Each coated glass slide was dipped into the aqueous suspension of each bacterium (E. coli and S. aureus) and incubated in shaker at 37 ◦ C. After 12 h, each glass slide was mildly washed with sterilized water and the slides were then incubated overnight at 37 ◦ C under growth agar. The test with D. geothermalis was proceeded similarly at 50 ◦ C. For accuracy of the growth resistance data, the entire test was repeated three times and the results were averaged. Although the surface of the glass slides was coated with the fabricated nanoparticles without crosslinking interaction, the coated nanoparticles were nearly not washed out from the surface of the glass slides in our experimental condition (Supporting information). 2.5. Antimicrobial tests of different sized silica-QAS core–shell nanoparticles The 0.5 wt% concentration solution of each 16, 28, 54 nm sized silica-QAS core–shell nanoparticles were prepared using methanol as solvent. Then, 1 mL of the prepared solution was coated on glass slides via drop casting method. The untreated glass slide was prepared as control. Aqueous suspension of S. aureus (106 –107 CFU/mL) was cast onto the prepared glass by aerosol spraying. After air-drying for 5 min, the glass slides were cultivated overnight at 37 ◦ C under autoclaved growth agar. Then, the grown bacterial colonies were inspected and counted to evaluate the antibacterial performances. 2.6. Field-emission scanning electron microscopy investigation The QAS-modified silica NPs, OdS-modified silica NPs, and pristine silica NPs were pressed by hydraulic press to obtain disc shape. Then, the bacterial suspension was drop casted to surface of disc shaped samples and cultivated overnight at appropriate temperature. Then, the bacteria were fixed in 2.5% glutaraldehyde for 2 h and rinsed with the distilled water several times. The bacteria were post-fixed for an additional 1 h with 1% osmium tetroxide in distilled water. After fixation, the samples were dehydrated with an ethanol series (20–100%), air-dried, and coated with platinum using sputter coater for FE-SEM observation. 3. Results and discussion 3.1. Fabrication of silane modified silica NPs The synthetic procedure of silane-modified silica NPs and the chemical structures of silane were illustrated in Scheme 1. The silica NPs (diameter: 44 nm) were treated with QAS or OdS in water–ethanol mixture solution (1:1, v/v) at room temperature for 24 h. After silane treatment, the samples were obtained by centrifugal precipitation and washed with distilled water several times to remove the residual reagents. The silane-modified silica NPs were fabricated via covalent linkage between –Si(OCH)3 groups of silane coupling agent (QAS and OdS) and –OH groups of silica NPs under mild condition [28,29].
2.4. Bacterial growth inhibitory on the glass surface 3.2. Characterization The E. coli and S. aureus were cultivated in sterilized LB broth and then incubated overnight at 37 ◦ C with a shaking incubator. The microorganism suspensions employed for the tests contained from 105 to 106 colony forming units (CFU). For preparation of the nanoparticles coated glasses, at first, the silane-modified silica nanoparticles (QAS and OdS) were dispersed in methanol solution
Fig. 1 represents FE-SEM and TEM images of the pristine silica and the silane-modified silica core–shell NPs. As shown in the FESEM images, the average diameter of both QAS and OdS treated NPs was increased to ca. 54 nm compared with the pristine silica cores (diameter: 44 nm). The TEM images clearly displayed that the silica
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Scheme 1. Schematic illustrations of the fabrication of silane-modified silica NPs and chemical structures of silane agents.
Table 1 Physical properties of pristine silica NPs, silica-OdS core–shell NPs, and silica-QAS core–shell NPs. Sample
Elemental analysis (wt%)
Pristine silica NPs Silica-OdS core–shell NPs Silica-QAS core–shell NPs
C
H
N
0.41 18.05 21.42
0.82 3.76 4.01
0.54 0.36 2.12
Z-potential (mV)
−34.72 −15.54 28.29
core was covered with a QAS and OdS shell and the shell thickness was ca. 5 nm (inset in Fig. 1) These results mean that both silane agents (QAS and OdS) are successfully anchored to the surface of silica NPs. The silane-modified silica NPs were further analyzed to obtain detailed information of their surface properties (Table 1). In the elemental analysis, the OdS-treated silica NPs showed the increment of % C and % H values compared to the pristine silica due to the alkyl-functional groups of OdS. The presence of QAS also can be verified from the increment in the % C, % H and % N values relative to the pristine silica NPs. It is noteworthy that the QASmodified silica has distinct increment of % N value than pristine silica owing to the nitrogen atoms of QAS. The Z-potential characterization was performed at pH 7. After surface modifications, the Z-potential value of silica-OdS core–shell NPs was shifted to −15.54 mV, whereas that of pristine silica was −34.72 mV. This shift
indicated that the negatively charged hydroxyl groups of silica surface were covered with thin OdS shell which had electrically neutral alkyl groups. On the other hand, the Z-potential value of silica-QAS core–shell NPs drastically changed to positive value (28.29 mV) due to the positively charged quaternary ammonium groups in the QAS compound. After then, in order to investigate the hydrophobicity of the NPs, the water-contact angles of each prepared sample were observed. For observation of the water-contact angles, the fabricated samples were pressed by hydraulic press to obtain disc shape and the pristine silica NPs were also identically prepared as a control material. As shown in Fig. 2, the water-contact angles of both QAS and OdS-modified silica NPs definitely increased compared to that of pristine silica NPs. It is well-known that the charged surface naturally shows hydrophilic property. However, the QAS and OdS modified silica NPs present hydrophobic property though they both have charged surface. It can be suggested that the hydrophobicity of the silane-modified silica nanoparticles (QAS and OdS) mainly originates from long alkyl groups (tail) of these silane compound which provides water-repelling property to the surface of silica NPs. Judging from these data, it was concluded that the surface modification of silica NPs with QAS and OdS was successfully proceeded. 3.3. Bacterial growth inhibition The bacterial growth inhibitory performances of silica-QAS and silica-OdS core–shell NPs were examined. The pristine silica
Fig. 1. FE-SEM and (inset) TEM images of the (a) pristine silica NPs, (b) silica-OdS core–shell NPs, and (c) silica-QAS core–shell NPs.
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Fig. 2. Water-contact images and numerical contact angle values of (a) pristine silica NPs, (b) OdS-modified silica NPs, and (c) QAS-modified silica NPs.
NPs were prepared as a hydrophilic control material. To evaluate the bacterial growth resistance of the prepared NPs coated surface, Gram-negative E. coli, Gram-positive S. aureus, and D. geothermalis were selected as the adhesive bacteria. It has been reported that D. geothermalis preferably adhere to hydrophilic surface than hydrophobic surface [30]. Therefore, it is expected that the fabricated hydrophobic core–shell NPs can repel this bacteria more effectively than other bacteria. For the bacterial growth test, the glass slides were coated with the silica-silane core–shell NPs via drop casting method with 2 mL of these NPs dispersed methanol solution (0.5 wt%). The water-contact angle values of the prepared nanoparticles coated glass surface were similar to that of the surface of the pressed nanoparticles. Then, the coated slides were dipped into the aqueous suspension of each bacterium (105 –106 CFU/mL) and incubated for 12 h with gently shaking. After washing with sterilized water, the slides were incubated overnight under growth agar at appropriate temperature. Fig. 3 presents the results of bacterial growth test. As can be seen in the graph, the average number of adhered bacteria onto the silica/OdS NPscoated glass surface reduced to ca. 29.4%, 23.7%, and 16.3% against Gram-negative E. coli, Gram-positive S. aureus, and D. geothermalis, respectively compared to that of pristine silica NPs (based on the CFU numbers of surface). Because binding energy at the interface between the bacterium and the hydrophobic surface was weaker [5–8], the numbers of adhered bacteria reduced on the coated surface with OdS-modified silica NPs than pristine silica NPs. In particular, the growth of D. geothermalis on the surface was most inhibited under this experimental condition among the tested bacteria.
On the other hand, the silica-QAS core–shell NPs strongly protected the surface from adhesive bacteria. The QAS-modified silica NPs had the reduced number of attached bacteria with an average number of ca. 3.4%, 1.5%, and 0.4% against E. coli, S. aureus, and D. geothermalis, respectively than that of pristine silica NPs. Although the QAS and OdS functionalized silica NPs had similar hydrophobicity (based on the values of water-contact angles), the silica-QAS NPs coated surface exhibited distinctly enhanced resistance to the bacterial growth than the OdS-modified silica NPs. The result can be explained that the improved bacterial growth inhibition of QASmodified silica NPs originates from the antimicrobial property of quaternary ammonium compounds. The quaternary ammonium groups of QAS provide bacteria-killing capability to the coated surface in addition to the bacteria-repelling hydrophobicity. As a result, the hydrophobic and biocidal QAS-modified silica NPs have enhanced bacteria resistant performances than the hydrophobic OdS-silica NPs. 3.4. FE-SEM investigation For the detailed investigation of the antibacterial performance of silica-silane core–shell NPs, the morphological changes of bacteria were observed after contact with the core–shell NPs. As shown in FE-SEM images (Fig. 4), the bacteria (E. coli, S. aureus, and D. geothermalis) incubated with the silica-OdS core–shell NPs were intact similar to the healthy bacteria on the untreated surface. However, the bacteria contacted with the silica-QAS core–shell NPs were seriously damaged in their outer membrane and lost their cellular integrity irrespective of bacterial species. Considering these results, it can be concluded that the OdS-modified silica NPs does not have any biocidal capability, whereas the QAS-modified silica has highly contact-active antibacterial performance. The positively charged quaternary ammonium groups of QAS interact with the lipid bilayer structures of bacterial membranes, leading to the destruction of bacteria [13]. Therefore, the silica-QAS core–shell NPs maintained quite clean state because of the synergistic effect of water-repelling hydrophobicity and antimicrobial properties. The bacterial adhesion is reduced and sparsely adhered bacteria are directly eradicated due to the water-repelling properties and the positive charge of the QAS-modified silica NPs. 3.5. Size dependence of antibacterial performance
Fig. 3. Relative number of viable bacteria on glass slides coated with pristine, OdSmodified, and QAS-modified silica NPs. The relative number of each species of bacteria adhered on the pristine silica NPs coated glass surface was defined as 100%.
In addition, the relationship between the size of the QASfunctionalized silica NPs and the antimicrobial efficacy was investigated. To determine the size dependency of antibacterial efficacy, the 16 and 28 nm-diameter silica-QAS core–shell NPs were fabricated using the 11 and 22 nm-diameter silica NPs as a core part (the detailed data were shown in Supporting information). Each
J. Song et al. / Colloids and Surfaces B: Biointerfaces 82 (2011) 651–656
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Fig. 4. FE-SEM images of untreated, incubated with silica-OdS core–shell NPs, and incubated with silica-QAS core–shell NPs of (top) E. coli, (middle) S. aureus, and (bottom) D. geothermalis (each scale bar represents 1 m).
glass slide was coated with 1 mL of the prepared 16, 28, and 54 nm sample (0.5 wt% in methanol solution) and uncoated glass slide was prepared as a control. Then, aqueous suspension of S. aureus (104 –105 CFU/mL) was casted onto the prepared slides by aerosol
spraying. The slides were then incubated overnight at 37 ◦ C under growth agar. Fig. 5 displays the antimicrobial results of silica-QAS core–shell NPs as a function of diameters against Gram-positive bacterium S. aureus. As shown in photographs, the silica-QAS coated
Fig. 5. Photographs of S. aureus colonies grown on glass slides (a) uncoated, (b) coated with 54 nm silica-QAS core–shell NPs, (c) 28 nm silica-QAS core–shell NPs, and (d) 16 nm silica-QAS core–shell NPs (each scale bar indicates 1 mm).
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J. Song et al. / Colloids and Surfaces B: Biointerfaces 82 (2011) 651–656
glass slides (Fig. 5b–d) effectively killed the sprayed bacteria compared to uncoated glass slide (Fig. 5a) because the QAS-modified silica NPs afforded the antimicrobial effect to the surface of glass slides and eradicated the contacted bacteria. Furthermore, as the size of the silica-QAS core–shell NPs decreased, the number of bacterial colonies on the glass slides reduced. It is well-known that the small antimicrobial agents have the enlarged surface area, leading to enhanced antibacterial performance based on same amount [26,27,31]. Similarly, the smaller silica-QAS core–shell NPs coated glass surface exhibits more effective antibacterial activity than the glass surface that coated with the bigger counterpart because the smaller particles covered larger area more compactly at equal quantity based on their enlarged surface area. These experimental results displayed that the diameter of the core–shell NPs could be controlled by changing the silica core size and the antimicrobial efficacy was definitely related to the surface area of the silica-QAS core–shell NPs. 4. Conclusion In conclusion, the QAS-modified silica NPs were successfully fabricated with strong covalent bonding between the silane agents and the surface of silica NPs. The fabricated silica-QAS core–shell NPs showed enhanced inhibition activity against growth of E. coli, S. aureus, and D. geothermalis compared to pristine and OdSmodified silica NPs. The silica-QAS core–shell NPs exhibited the excellent bacterial growth resistance based on the synergistic effect of hydrophobicity (water-repelling) and antibacterial property. Moreover, the antimicrobial efficacy of silica-QAS core–shell NPs increased with decreasing the size of the NPs. Based on these results, it is anticipated that the size controllable and bacterial growth resistant QAS-modified silica NPs can be applied in various application fields such as antiadhesion, bioadhesive, and sterilization area. Acknowledgements This research was supported by WCU (World Class University) program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R3110013).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfb.2010.10.027. References [1] J.A. Lichter, K.J. Van Vliet, M.F. Rubner, Macromolecules 42 (2009) 8573. [2] R.W. Huigens III, S.A. Rogers, A.T. Steinhauer, C. Melander, Org. Biomol. Chem. 7 (2009) 794. [3] S. Hou, E.A. Burton, K.A. Simon, D. Blodgett, Y.-Y. Luk, D. Ren, Appl. Environ. Microbiol. 73 (2007) 4300. [4] P. Tenke, C.R. Riedl, G.L. Jones, G.J. Williams, D. Stickler, E. Nagy, Int. J. Antimicrob. Agents (2004) S67. [5] S.C. Dexter, J. Colloid Interface Sci. 70 (1979) 346. [6] P.J. Eginton, H. Gibson, J. Holah, P.S. Handley, P. Gilbert, Colloids Surf. B: Biointerfaces 5 (1995) 153. [7] A. Hamza, V.A. Pham, T. Matsuura, J.P. Santerre, J. Membr. Sci. 131 (1997) 217. [8] C.I. Pereni, Q. Zhao, Y. Liu, E. Abel, Colloids Surf. B: Biointerfaces 48 (2006) 143. [9] J.A. Hornemann, A.A. Lysova, S.L. Codd, J.D. Seymour, S.C. Busse, P.S. Stewart, J.R. Brown, Biomacromolecules 9 (2008) 2322. [10] I. Yudovin-Farber, N. Beyth, A. Nyska, E.I. Weiss, J. Golenser, A.J. Domb, Biomacromolecuels 9 (2008) 3044. [11] M.J. Saif, J. Anwar, M.A. Munawar, Langmuir 25 (2009) 377. [12] C.Z. Chen, N.C. Beck-Tan, P. Dhurjati, T.K. Van Dyk, R.A. LaRossa, S.L. Cooper, Biomacromolecules 1 (2000) 473. [13] N. Nurdin, G. Helary, G. Sauvet, J. Appl. Polym. Sci. 50 (1993) 671. [14] C.H. Kim, K.S. Choi, J. Ind. Eng. Chem. 8 (2002) 71. [15] E.-R. Kenawy, F.I. Abdel-Hay, A.E.-R.R. El-Shanshoury, M.H. El-Newehy, J. Control. Release 50 (1998) 145. [16] Z. Li, D. Lee, X. Sheng, R.E. Cohen, M.F. Rubner, Langmuir 22 (2006) 9820. [17] J.J.H. Oosterhof, K.J.D.A. Buijssen, H.J. Busscher, B.F.A.M. Van der Lann, H.C. Van der Mei, Appl. Environ. Microbiol. 72 (2006) 3673. [18] B. Gotternbos, H.C. Van der Mei, F. Klatter, P. Nieuwenhuis, H.J. Busscher, Biomaterials 23 (2002) 1417. [19] Z. Shi, K.G. Neoh, E.T. Kang, Ind. Eng. Chem. Res. 46 (2007) 439. [20] F. Hu, K.G. Neoh, L. Cen, E.-T. Kang, Biomacromolecules 7 (2006) 809. [21] J. Jang, S. Kim, K.J. Lee, Chem. Commun. 26 (2007) 2689. [22] S. Shin, J. Jang, Chem. Commun. 41 (2007) 4230. [23] J.-Y. Hong, E. Kwon, J. Jang, Soft Matter 5 (2009) 951. ˜ [24] V. Salgueirino-Maceira, M.A. Correa-Duarte, Adv. Mater. 19 (2007) 4131. [25] U.A. Kober, M.R. Gallas, L.F. Campo, F.S. Rodembusch, V. Stefani, J. Sol–Gel Sci. Technol. 52 (2009) 305. [26] J. Song, H. Kong, J. Jang, Chem. Commun. 36 (2009) 5418. [27] J. Jang, Y. Kim, Chem. Commun. 34 (2008) 4016. [28] G. Schottner, Chem. Mater. 13 (2001) 3422. [29] J.A. Howarter, J.P. Youngblood, Langmuir 22 (2006) 11142. [30] M. Raulio, M. Järn, J. Ahola, J. Peltonen, J.B. Rosenholm, S. Tervakangas, J. Kolehmainen, T. Ruokolainen, P. Narko, M. Salkinoja-Salonen, J. Ind. Microbiol. Biotechnol. 35 (2008) 751. [31] H. Kong, J. Jang, Langmuir 24 (2008) 2051.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short communication
Bacterial adhesion inhibition of the quaternary ammonium functionalized silica nanoparticles Jooyoung Song, Hyeyoung Kong, Jyongsik Jang ∗ WCU Program of Chemical Convergence for Energy and Environment (C2E2), School of Chemical and Biological Engineering, College of Engineering, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
a r t i c l e
i n f o
Article history: Received 12 April 2010 Received in revised form 1 October 2010 Accepted 11 October 2010 Available online 20 October 2010 Keywords: Quaternary ammonium silane Nanoparticles Bacterial growth Antibacterial
a b s t r a c t Quaternary ammonium compounds have been considered as excellent antibacterial agents due to their effective biocidal activity, long term durability and environmentally friendly performance. In this work, 3-(trimethoxysilyl)-propyldimethyloctadecylammonium chloride as a quaternary ammonium silane was applied for the surface modification of silica nanoparticles. The quaternary ammonium silane provided silica surface with hydrophobicity and antibacterial properties. In addition, the glass surface which was coated with the surface modified silica nanoparticles presented bacterial growth inhibition activity. For comparison of bacterial growth resistance, hydrophobic silane (alkyl functionalized silane) modified silica nanoparticles and pristine silica nanoparticles were prepared. As a result of bacterial adhesion test, the quaternary ammonium functionalized silica nanoparticles exhibited the enhanced inhibition performance against growth of Gram-negative Escherichia coli (96.6%), Gram-positive Staphylococcus aureus (98.5%) and Deinococcus geothermalis (99.6%) compared to pristine silica nanoparticles. These bacteria resistances also were stronger than that of hydrophobically modified silica nanoparticles. It could be explained that the improved bacteria inhibition performance originated from the synergistic effect of hydrophobicity and antibacterial property of quaternary ammonium silane. Additionally, the antimicrobial efficacy of the fabricated nanoparticles increased with decreasing size of the nanoparticles. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Recently, adhesion and subsequent growth of microorganisms is a main concern of the biomedical device failure. A large number of infections are attributed to viable bacteria that adhered to medical devices and implants [1–4]. In contrast to plankton (free-floating bacteria), when the bacteria adhere to the surface, grow, and form biofilm, they are up to 1000 times more tolerant to disinfection treatment [4]. Therefore, it can be clearly anticipated that the bacterial growth is efficiently inhibited in the early step of bacterial adhesion. Several researchers reported that the bacterial adhesion was reduced on the hydrophobic surfaces because of weak binding energy at the interface between the bacterium and the hydrophobic surface [5–8]. However, hydrophobic treatments can neither perfectly protect the surface from bacterial adhesion nor eradicate the contacted bacteria; the sporadically adhered bacteria will grow and form biofilm eventually. The quaternary ammonium functionalized materials, as antibacterial agents, have received much attention because they provide effective protection against bacterial colonization
∗ Corresponding author. Tel.: +82 2 880 7069; fax: +82 2 888 1604. E-mail address: [email protected] (J. Jang). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.10.027
with long term durability and environmentally friendly performance [9–19]. Among the quaternary ammonium compounds, 3-(trimethoxysilyl)-propyldimethyloctadecylammonium chloride (quaternary ammonium silane (QAS)) excellently inhibits the adhesion and growth of bacteria due to its hydrophobicity and antibacterial property [16–19] The QAS-modified substrate surface can resist the bacterial adhesion with water-repelling hydrophobicity and eradicate the contacted bacteria with biocidal capability. Busscher et al. and Neoh groups reported that the QAS-treated substrates had antibacterial activities on the biofilm test [17–19]. However, relatively little attention has been paid to the study of the size control of QAS-modified materials in the viewpoint of nano scale. In the research field of nano-materials fabrication, the core–shell nanostructures have attracted a great deal of interest owing to their facile synthesis process and various potential applications [20–27]. Especially, as a core-part, silica nanoparticle has several advantages such as tunable surface functionality, low cytotoxicity, and easily controllable size [23–27]. In addition, the organic materials coated silica nanoparticles combine the functionality of organic shell with high colloidal stability of the silica core, which can expand their applications in the fields of magnetics [23,24], optics [25], and antimicrobials [26,27]. Herein, we report the fabrication of size controllable silicaQAS core–shell nanoparticles (NPs) and their bacterial growth
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resistance. Octadecyltrimethoxy silane (OdS) (alkyl functionalized silane) modified silica NPs were prepared in order to investigate the relationship between hydrophobicity and bacterial adhesion. For antimicrobial test, Gram-negative Escherichia coli (E. coli), Gram-positive Staphylococcus aureus (S. aureus), and Deinococcus geothermalis (D. geothermalis) were selected as detrimental bacteria. Based on the antimicrobial properties of the QAS compound, it can be expected that the fabricated silica-QAS core–shell NPs present excellent inhibition activity to the bacterial growth compared with pristine silica NPs and silica-OdS core–shell NPs. Furthermore, in order to evaluate the size-dependency of antibacterial efficacy of silica-QAS core–shell NPs, various sized (16, 28, and 54 nm) silica-QAS core–shell NPs were fabricated using different sized silica colloids as the core materials. 2. Experimental 2.1. Materials The 11 nm, 22 nm, and 44 nm sized silica nanoparticles were purchased from Aldrich (Milwaukee, WI). Glutaraldehyde, osmium tetroxide, octadecyltrimethoxy silane (OdS), and 3-(trimethoxysilyl)-propyldimethyloctadecylammonium chloride (QAS) were also purchased from Aldrich (Milwaukee, WI). For the bacterial growth test, E. coil (ATCC 11775), S. aureus (ATCC 12600), and D. geothermalis (DSM 11300) were purchased from Fisher Company. 2.2. Fabrication of the silane-modified silica nanoparticles First, 1 g of the silica nanopowder was dispersed in 100 ml of water–ethanol mixture solution (1:1, v/v) and added 0.5 ml of silane coupling agents (QAS or OdS) and the mixture was stirred at 25 ◦ C, 24 h. The silane-modified silica NPs were fabricated via covalent linkage between –Si(OCH)3 groups of silane coupling agent (QAS and OdS) and –OH groups of silica NPs under mild condition [28,29]. After silane treatment, the synthesized silane-modified silica nanoparticle was obtained by centrifugal precipitation and washed three times with distilled water to remove the residual reagents. 2.3. Characterization Photographs of transmission electron microscopy (TEM) were obtained with a JEOL JEM-200CX. Acceleration voltage for TEM was 200 kV. Field-emission scanning electron microscopy (FE-SEM) images were obtained using a JEOL 6700 at an acceleration voltage of 10 kV. In the sample preparation of TEM and FE-SEM characterizations, each silane-modified silica nanoparticles were dispersed in absolute ethanol and cast onto copper grid. The Z-potential of the surface-treated silica nanoparticles were measured with an ELS8000 at pH 7 and elemental analysis value was obtained using CHNS-932 (LECO Corp, US). For measurement of the water contact angles, each sample was pressed by hydraulic press to obtain disc shape and the diameter of disc was 1.3 mm. The water-contact angles of the pressed samples were measured with a Krüss DSA10 contact angle analyzer interfaced to drop shape analysis software.
(0.5 wt%) with sonication for 30 min. Then, the 2 mL of each suspension was drop casted onto the surface of glass slides and these slides were dried in 60 ◦ C oven. The pristine silica nanoparticles coated glass slide was prepared as a control. The coating thickness of the nanoparticles on the glass surface was ca. 15 m. Each coated glass slide was dipped into the aqueous suspension of each bacterium (E. coli and S. aureus) and incubated in shaker at 37 ◦ C. After 12 h, each glass slide was mildly washed with sterilized water and the slides were then incubated overnight at 37 ◦ C under growth agar. The test with D. geothermalis was proceeded similarly at 50 ◦ C. For accuracy of the growth resistance data, the entire test was repeated three times and the results were averaged. Although the surface of the glass slides was coated with the fabricated nanoparticles without crosslinking interaction, the coated nanoparticles were nearly not washed out from the surface of the glass slides in our experimental condition (Supporting information). 2.5. Antimicrobial tests of different sized silica-QAS core–shell nanoparticles The 0.5 wt% concentration solution of each 16, 28, 54 nm sized silica-QAS core–shell nanoparticles were prepared using methanol as solvent. Then, 1 mL of the prepared solution was coated on glass slides via drop casting method. The untreated glass slide was prepared as control. Aqueous suspension of S. aureus (106 –107 CFU/mL) was cast onto the prepared glass by aerosol spraying. After air-drying for 5 min, the glass slides were cultivated overnight at 37 ◦ C under autoclaved growth agar. Then, the grown bacterial colonies were inspected and counted to evaluate the antibacterial performances. 2.6. Field-emission scanning electron microscopy investigation The QAS-modified silica NPs, OdS-modified silica NPs, and pristine silica NPs were pressed by hydraulic press to obtain disc shape. Then, the bacterial suspension was drop casted to surface of disc shaped samples and cultivated overnight at appropriate temperature. Then, the bacteria were fixed in 2.5% glutaraldehyde for 2 h and rinsed with the distilled water several times. The bacteria were post-fixed for an additional 1 h with 1% osmium tetroxide in distilled water. After fixation, the samples were dehydrated with an ethanol series (20–100%), air-dried, and coated with platinum using sputter coater for FE-SEM observation. 3. Results and discussion 3.1. Fabrication of silane modified silica NPs The synthetic procedure of silane-modified silica NPs and the chemical structures of silane were illustrated in Scheme 1. The silica NPs (diameter: 44 nm) were treated with QAS or OdS in water–ethanol mixture solution (1:1, v/v) at room temperature for 24 h. After silane treatment, the samples were obtained by centrifugal precipitation and washed with distilled water several times to remove the residual reagents. The silane-modified silica NPs were fabricated via covalent linkage between –Si(OCH)3 groups of silane coupling agent (QAS and OdS) and –OH groups of silica NPs under mild condition [28,29].
2.4. Bacterial growth inhibitory on the glass surface 3.2. Characterization The E. coli and S. aureus were cultivated in sterilized LB broth and then incubated overnight at 37 ◦ C with a shaking incubator. The microorganism suspensions employed for the tests contained from 105 to 106 colony forming units (CFU). For preparation of the nanoparticles coated glasses, at first, the silane-modified silica nanoparticles (QAS and OdS) were dispersed in methanol solution
Fig. 1 represents FE-SEM and TEM images of the pristine silica and the silane-modified silica core–shell NPs. As shown in the FESEM images, the average diameter of both QAS and OdS treated NPs was increased to ca. 54 nm compared with the pristine silica cores (diameter: 44 nm). The TEM images clearly displayed that the silica
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Scheme 1. Schematic illustrations of the fabrication of silane-modified silica NPs and chemical structures of silane agents.
Table 1 Physical properties of pristine silica NPs, silica-OdS core–shell NPs, and silica-QAS core–shell NPs. Sample
Elemental analysis (wt%)
Pristine silica NPs Silica-OdS core–shell NPs Silica-QAS core–shell NPs
C
H
N
0.41 18.05 21.42
0.82 3.76 4.01
0.54 0.36 2.12
Z-potential (mV)
−34.72 −15.54 28.29
core was covered with a QAS and OdS shell and the shell thickness was ca. 5 nm (inset in Fig. 1) These results mean that both silane agents (QAS and OdS) are successfully anchored to the surface of silica NPs. The silane-modified silica NPs were further analyzed to obtain detailed information of their surface properties (Table 1). In the elemental analysis, the OdS-treated silica NPs showed the increment of % C and % H values compared to the pristine silica due to the alkyl-functional groups of OdS. The presence of QAS also can be verified from the increment in the % C, % H and % N values relative to the pristine silica NPs. It is noteworthy that the QASmodified silica has distinct increment of % N value than pristine silica owing to the nitrogen atoms of QAS. The Z-potential characterization was performed at pH 7. After surface modifications, the Z-potential value of silica-OdS core–shell NPs was shifted to −15.54 mV, whereas that of pristine silica was −34.72 mV. This shift
indicated that the negatively charged hydroxyl groups of silica surface were covered with thin OdS shell which had electrically neutral alkyl groups. On the other hand, the Z-potential value of silica-QAS core–shell NPs drastically changed to positive value (28.29 mV) due to the positively charged quaternary ammonium groups in the QAS compound. After then, in order to investigate the hydrophobicity of the NPs, the water-contact angles of each prepared sample were observed. For observation of the water-contact angles, the fabricated samples were pressed by hydraulic press to obtain disc shape and the pristine silica NPs were also identically prepared as a control material. As shown in Fig. 2, the water-contact angles of both QAS and OdS-modified silica NPs definitely increased compared to that of pristine silica NPs. It is well-known that the charged surface naturally shows hydrophilic property. However, the QAS and OdS modified silica NPs present hydrophobic property though they both have charged surface. It can be suggested that the hydrophobicity of the silane-modified silica nanoparticles (QAS and OdS) mainly originates from long alkyl groups (tail) of these silane compound which provides water-repelling property to the surface of silica NPs. Judging from these data, it was concluded that the surface modification of silica NPs with QAS and OdS was successfully proceeded. 3.3. Bacterial growth inhibition The bacterial growth inhibitory performances of silica-QAS and silica-OdS core–shell NPs were examined. The pristine silica
Fig. 1. FE-SEM and (inset) TEM images of the (a) pristine silica NPs, (b) silica-OdS core–shell NPs, and (c) silica-QAS core–shell NPs.
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Fig. 2. Water-contact images and numerical contact angle values of (a) pristine silica NPs, (b) OdS-modified silica NPs, and (c) QAS-modified silica NPs.
NPs were prepared as a hydrophilic control material. To evaluate the bacterial growth resistance of the prepared NPs coated surface, Gram-negative E. coli, Gram-positive S. aureus, and D. geothermalis were selected as the adhesive bacteria. It has been reported that D. geothermalis preferably adhere to hydrophilic surface than hydrophobic surface [30]. Therefore, it is expected that the fabricated hydrophobic core–shell NPs can repel this bacteria more effectively than other bacteria. For the bacterial growth test, the glass slides were coated with the silica-silane core–shell NPs via drop casting method with 2 mL of these NPs dispersed methanol solution (0.5 wt%). The water-contact angle values of the prepared nanoparticles coated glass surface were similar to that of the surface of the pressed nanoparticles. Then, the coated slides were dipped into the aqueous suspension of each bacterium (105 –106 CFU/mL) and incubated for 12 h with gently shaking. After washing with sterilized water, the slides were incubated overnight under growth agar at appropriate temperature. Fig. 3 presents the results of bacterial growth test. As can be seen in the graph, the average number of adhered bacteria onto the silica/OdS NPscoated glass surface reduced to ca. 29.4%, 23.7%, and 16.3% against Gram-negative E. coli, Gram-positive S. aureus, and D. geothermalis, respectively compared to that of pristine silica NPs (based on the CFU numbers of surface). Because binding energy at the interface between the bacterium and the hydrophobic surface was weaker [5–8], the numbers of adhered bacteria reduced on the coated surface with OdS-modified silica NPs than pristine silica NPs. In particular, the growth of D. geothermalis on the surface was most inhibited under this experimental condition among the tested bacteria.
On the other hand, the silica-QAS core–shell NPs strongly protected the surface from adhesive bacteria. The QAS-modified silica NPs had the reduced number of attached bacteria with an average number of ca. 3.4%, 1.5%, and 0.4% against E. coli, S. aureus, and D. geothermalis, respectively than that of pristine silica NPs. Although the QAS and OdS functionalized silica NPs had similar hydrophobicity (based on the values of water-contact angles), the silica-QAS NPs coated surface exhibited distinctly enhanced resistance to the bacterial growth than the OdS-modified silica NPs. The result can be explained that the improved bacterial growth inhibition of QASmodified silica NPs originates from the antimicrobial property of quaternary ammonium compounds. The quaternary ammonium groups of QAS provide bacteria-killing capability to the coated surface in addition to the bacteria-repelling hydrophobicity. As a result, the hydrophobic and biocidal QAS-modified silica NPs have enhanced bacteria resistant performances than the hydrophobic OdS-silica NPs. 3.4. FE-SEM investigation For the detailed investigation of the antibacterial performance of silica-silane core–shell NPs, the morphological changes of bacteria were observed after contact with the core–shell NPs. As shown in FE-SEM images (Fig. 4), the bacteria (E. coli, S. aureus, and D. geothermalis) incubated with the silica-OdS core–shell NPs were intact similar to the healthy bacteria on the untreated surface. However, the bacteria contacted with the silica-QAS core–shell NPs were seriously damaged in their outer membrane and lost their cellular integrity irrespective of bacterial species. Considering these results, it can be concluded that the OdS-modified silica NPs does not have any biocidal capability, whereas the QAS-modified silica has highly contact-active antibacterial performance. The positively charged quaternary ammonium groups of QAS interact with the lipid bilayer structures of bacterial membranes, leading to the destruction of bacteria [13]. Therefore, the silica-QAS core–shell NPs maintained quite clean state because of the synergistic effect of water-repelling hydrophobicity and antimicrobial properties. The bacterial adhesion is reduced and sparsely adhered bacteria are directly eradicated due to the water-repelling properties and the positive charge of the QAS-modified silica NPs. 3.5. Size dependence of antibacterial performance
Fig. 3. Relative number of viable bacteria on glass slides coated with pristine, OdSmodified, and QAS-modified silica NPs. The relative number of each species of bacteria adhered on the pristine silica NPs coated glass surface was defined as 100%.
In addition, the relationship between the size of the QASfunctionalized silica NPs and the antimicrobial efficacy was investigated. To determine the size dependency of antibacterial efficacy, the 16 and 28 nm-diameter silica-QAS core–shell NPs were fabricated using the 11 and 22 nm-diameter silica NPs as a core part (the detailed data were shown in Supporting information). Each
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Fig. 4. FE-SEM images of untreated, incubated with silica-OdS core–shell NPs, and incubated with silica-QAS core–shell NPs of (top) E. coli, (middle) S. aureus, and (bottom) D. geothermalis (each scale bar represents 1 m).
glass slide was coated with 1 mL of the prepared 16, 28, and 54 nm sample (0.5 wt% in methanol solution) and uncoated glass slide was prepared as a control. Then, aqueous suspension of S. aureus (104 –105 CFU/mL) was casted onto the prepared slides by aerosol
spraying. The slides were then incubated overnight at 37 ◦ C under growth agar. Fig. 5 displays the antimicrobial results of silica-QAS core–shell NPs as a function of diameters against Gram-positive bacterium S. aureus. As shown in photographs, the silica-QAS coated
Fig. 5. Photographs of S. aureus colonies grown on glass slides (a) uncoated, (b) coated with 54 nm silica-QAS core–shell NPs, (c) 28 nm silica-QAS core–shell NPs, and (d) 16 nm silica-QAS core–shell NPs (each scale bar indicates 1 mm).
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glass slides (Fig. 5b–d) effectively killed the sprayed bacteria compared to uncoated glass slide (Fig. 5a) because the QAS-modified silica NPs afforded the antimicrobial effect to the surface of glass slides and eradicated the contacted bacteria. Furthermore, as the size of the silica-QAS core–shell NPs decreased, the number of bacterial colonies on the glass slides reduced. It is well-known that the small antimicrobial agents have the enlarged surface area, leading to enhanced antibacterial performance based on same amount [26,27,31]. Similarly, the smaller silica-QAS core–shell NPs coated glass surface exhibits more effective antibacterial activity than the glass surface that coated with the bigger counterpart because the smaller particles covered larger area more compactly at equal quantity based on their enlarged surface area. These experimental results displayed that the diameter of the core–shell NPs could be controlled by changing the silica core size and the antimicrobial efficacy was definitely related to the surface area of the silica-QAS core–shell NPs. 4. Conclusion In conclusion, the QAS-modified silica NPs were successfully fabricated with strong covalent bonding between the silane agents and the surface of silica NPs. The fabricated silica-QAS core–shell NPs showed enhanced inhibition activity against growth of E. coli, S. aureus, and D. geothermalis compared to pristine and OdSmodified silica NPs. The silica-QAS core–shell NPs exhibited the excellent bacterial growth resistance based on the synergistic effect of hydrophobicity (water-repelling) and antibacterial property. Moreover, the antimicrobial efficacy of silica-QAS core–shell NPs increased with decreasing the size of the NPs. Based on these results, it is anticipated that the size controllable and bacterial growth resistant QAS-modified silica NPs can be applied in various application fields such as antiadhesion, bioadhesive, and sterilization area. Acknowledgements This research was supported by WCU (World Class University) program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R3110013).
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