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Langmuir 2006, 22, 9820-9823
Two-Level Antibacterial Coating with Both Release-Killing and Contact-Killing Capabilities Zhi Li,† Daeyeon Lee,‡ Xiaoxia Sheng,† Robert E. Cohen,*,‡ and Michael F. Rubner*,† Departments of Materials Science and Engineering and Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 ReceiVed July 27, 2006. In Final Form: September 29, 2006 Using a combination of an aqueous layer-by-layer deposition technique, nanoparticle surface modification chemistry, and nanoreactor chemistry, we constructed thin film coatings with two distinct layered functional regions: a reservoir for the loading and release of bactericidal chemicals and a nanoparticle surface cap with immobilized bactericides. This results in dual-functional bactericidal coatings bearing both chemical-releasing bacteria-killing capacity and contact bacteria-killing capacity. These dual-functional coatings showed very high initial bacteria-killing efficiency due to the release of Ag ions and retained significant antibacterial activity after the depletion of embedded Ag because of the immobilized quaternary ammonium salts.
Over the past few decades, considerable research effort has focused on creating antibacterial coatings on the surfaces of various objects such as garments and medical devices.1,2 With very recent developments in nanotechnology, elaborate and multifunctional surface coatings with precise architectural and chemical control on the nanoscale are becoming easily accessible. This offers a great opportunity to readdress the persistent challenge of obtaining effective and long-lasting antibacterial coatings. In the present work, through the use of a layer-by-layer (LbL) nanofabrication technique, nanoparticle surface modification chemistry, and nanoreactor chemistry, we constructed thin film coatings with two distinct layered regions: a reservoir for the loading and releasing of bactericidal chemicals and a nanoparticle surface cap with immobilized bactericides. This results in dualfunctional bactericidal surfaces bearing both chemical-releasing bacteria-killing capacity and contact bacteria-killing capacity. Typically, release-killing capacity is introduced to a surface by incorporating bacteria-killing chemicals such as antibiotics,3 phenols,4 and heavy metals5,6 using various methods such as spray or dip coating7 and hydrogel trapping.8 Alternatively, a surface can obtain contact bacteria-killing capacity through chemical modification with tethered bactericidal functionalities such as quaternary amine compounds,9-16 phosphonium salts,17,18 * To whom correspondence may be addressed. E-mail: recohen@ mit.edu;
[email protected]. † Department of Materials Science and Engineering. ‡ Department of Chemical Engineering. (1) Danese, P. N. Chem. Biol. 2002, 9, 873-880. (2) Lewis, K.; Klibanov, A. M. Trends Biotechnol. 2005, 23, 343-348. (3) Darouiche, R. O.; Mansouri, M. D.; Raad, I. I. Urology 2002, 59, 303-307. (4) Chung, D. W.; Papadakis, S. E.; Yam, K. L. Int. J. Food Sci. Technol. 2003, 38, 165-169. (5) Klueh, U.; Wagner, V.; Kelly, S.; Johnson, A.; Bryers, J. D. J. Biomed. Mater. Res., Part B 2000, 53, 621-631. (6) Johnson, J. R.; Roberts, P. L.; Olsen, R. J.; Moyer, K. A.; Stamm, W. E. J. Infect. Dis. 1990, 162, 1145-1150. (7) van de Belt, H.; Neut, D.; Schenk, W.; van Horn, J. R.; van der Mei, H. C.; Busscher, H. J. Acta Orthop. 2001, 72, 557-571. (8) Pugach, J. L.; DiTizio, V.; Mittelman, M. W.; Bruce, A. W.; DiCosmo F.; Khoury, A. E. J. Urol. 1999, 162, 883-887. (9) Isquith, A. J.; Abbott, E. A.; Walters, P. A. Appl. Microbiol. 1972, 24, 859-863. (10) Nurdin, N.; Helary, G.; Sauvet, G. J. Appl. Polym. Sci. 1993, 50, 663670. (11) Grapski, J. A.; Cooper, S. L. Biomaterials 2001, 22, 2239-2246. (12) Tiller, J. C.; Liao, C. J.; Lewis, K.; Klibanov, A. M. P. Natl. Acad. Sci. U.S.A. 2001, 98, 5981-5985. (13) Gottenbos, B.; van der Mei, H. C.; Klatter, F.; Nieuwenhuis, P.; Busscher, H. J. Biomaterials 2002, 23, 1417-1423.
and titanium oxide particles,19 which are able to kill bacteria upon contact. Each of these bacteria-killing mechanisms has its own advantages in certain circumstances but disadvantages in other settings. For instance, when the released antibacterial agent is depleted below the minimum inhibitory concentration (MIC), a chemical-releasing coating will lose its antibacterial efficiency dramatically.1 Immobilized antibacterial agents exhibit longterm durability and reduced development of drug-resistant mutation, and they are regarded as being environmentally friendly.2 These advantages contrast with the need for multistep surface modifications,15,16 direct bacteria contact, and a defectfree coating. Efforts toward new bacteria-resistant coating strategies that utilize the advantages of existing approaches while minimizing the disadvantages are of great importance. As an example, Tiller and co-workers presented surface coatings that can repel and kill bacteria at the same time,20 owing to the presence of both bacteria-repelling poly(ethylene glycol) as a top coating and embedded releasable silver nanoparticles (Ag NPs). Very recently, Sen and co-workers created dual-action antimicrobial coatings through spin coating on surfaces two-component composites consisting of a poly(4-vinyl-N-hexylpyidinium bromide) matrix and embedded silver bromide nanoparticles.21 Shown in Figure 1 is our scheme for creating a two-level dual-functional antibacterial coating with immobilized quaternary ammonium salts and releasable silver ions on a polystyrene surface. A reservoir region made of 20 bilayers of poly(allylamine hydrochloride) (PAH) (assembly pH 8.5) and poly(acrylic acid) (PAA) (assembly pH 3.5) and a cap region made of 10 bilayers of PAH (assembly pH 7.5) and silica nanoparticles (SiO2 NPs) (∼ 20 nm in diameter, assembly pH ∼9.5) can be constructed successfully through polyelectrolyte LbL deposition. As a facile, (14) Lee, S. B.; Koepsel, R. R.; Morley, S. W.; Matyjaszewski, K.; Sun, Y. J.; Russell, A. J. Biomacromolecules 2004, 5, 877-882. (15) Cen, L.; Neoh, K. G.; Kang, E. T. Langmuir 2003, 19, 10295-10303. (16) Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M. Biotechnol. Bioeng. 2002, 79, 465-471. (17) Kanazawa, A.; Ikeda, T.; Endo, T. J. Polym. Sci., Part A: Polym. Chem. 1993, 31, 1467-1472. (18) Popa, A.; Davidescu, C. M.; Trif, R.; Ilia, G.; Iliescu, S.; Dehelean, G. React. Funct. Polym. 2003, 55, 151-158. (19) Sunada, K.; Watanabe, T.; Hashimoto, K. J. Photochem. Photobiol., A 2003, 156, 227-233. (20) Ho, C. H.; Tobis, J.; Sprich, C.; Thomann, R.; Tiller, J. C. AdV. Mater. 2004, 16, 957-961. (21) Sambhy, V.; MacBride, M. M.; Peterson, B. R.; Sen, A. J. Am. Chem. Soc. 2006, 128, 9712-9718.
10.1021/la0622166 CCC: $33.50 © 2006 American Chemical Society Published on Web 10/31/2006
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Langmuir, Vol. 22, No. 24, 2006 9821
Figure 1. Scheme showing the design of a two-level dual-functional antibacterial coating with both quaternary ammonium salts and silver. The coating process begins with LbL deposition of a reservoir made of bilayers of PAH and PAA. (A) A cap region made of bilayers of PAH and SiO2 NPs is added to the top. (B) The SiO2 NP cap is modified with a quaternary ammonium silane, OQAS. (C) Ag+ can be loaded inside the coating using the available unreacted carboxylic acid groups in the LbL multilayers. Ag NPs are created in situ using the nanoreactor chemistry described previously.37,38
Figure 2. Cross-sectional TEM images showing the two-level antibacterial coatings with OQAS and silver. Ag NPs resulting from two Ag loading and reduction cycles were imbedded inside coatings by using a wet-phase reduction method using a dimethylamine borane (DMAB) complex solution.
aqueous-based nanofabrication technique based on the alternating adsorption of oppositely charged polyelectrolytes or nanoparticles,22,23 the LbL deposition technique has been used to create antimicrobial polyelectrolyte multilayers (PEM) that can release antimicrobial agents such as silver24-29 and cetrimide.26 Other PEMs have been constructed with bacteria-repelling or bacteriakilling properties using polymers such as PEGylated polypeptide and chitosan.30-32 As revealed in the cross-sectional TEM images (Figure 2), the packing of the SiO2 NPs in the cap region produces an (22) Decher, G. Science 1997, 277, 1232-1237. (23) Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials; Decher, G., Schlenoff, J. B., Eds,; Wiley-VCH: Weinheim, Germany, 2003. (24) Dai, J. H.; Bruening, M. L. Nano Lett. 2002, 2, 497-501. (25) Berg, M. C. Ph.D. Thesis, Massachusetts Institute of Technology, Cambridge, MA, 2005. (26) Grunlan, J. C.; Choi, J. K.; Lin, A. Biomacromolecules 2005, 6, 11491153. (27) Lee, D.; Cohen, R. E.; Rubner, M. F. Langmuir 2005, 21, 9651-9659. (28) Podsiadlo, P.; Paternel, S.; Rouillard, J. M.; Zhang, Z. F.; Lee, J.; Lee, J. W.; Gulari, L.; Kotov, N. A. Langmuir 2005, 21, 11915-11921. (29) Shi, Z.; Neoh, K. G.; Zhong, S. P.; Yung, L. Y. L.; Kang, E. T.; Wang, W. J. Biomed. Mater. Res., Part A 2006, 76A, 826-834. (30) Etienne, O.; Picart, C.; Taddei, C.; Haikel, Y.; Dimarcq, J. L.; Schaaf, P.; Voegel, J. C.; Ogier, J. A.; Egles, C. Antimicrob. Agents Chemother. 2004, 48, 3662-3669. (31) Boulmedais, F.; Frisch, B.; Etienne, O.; Lavalle, P.; Picart, C.; Ogier, J.; Voegel, J. C.; Schaaf, P.; Egles, C. Biomaterials 2004, 25, 2003-2011. (32) Fu, J. H.; Ji, J.; Yuan, W. Y.; Shen, J. C. Biomaterials 2005, 26, 66846692.
interconnected 3D nanoporosity that enables the transport of antibacterial agents between the reservoir region and the surroundings.33 In addition, as revealed by atomic force microscopy (AFM) (Supporting Information, Figure S1), the cap surface has 3D nanoscale roughness that arises from the random packing of SiO2 NPs as reported previously.33 This results in a significant increase of the exposed surface area compared to that in the original flat substrate. For example, the rms roughness value increased from ∼0.6 nm for the original polystyrene surface to ∼21.6 nm after a two-level coating with 10 layers of SiO2 NPs in the cap region. In addition, the SiO2 NPs in the high-surface-area cap can be chemically modified using well-established and versatile silica modification chemistry. In the case discussed below, the cap region reacts with [3-(trimethoxysilyl)propyl]octadecyl-dimethylammonium chloride (OQAS), which bears a quaternary ammonium salt structural unit coupled with a long hydrophobic alkyl chain (C18). This OQAS treatment transforms the rough surface from very hydrophilic (∼7° water advancing contact angle) to very hydrophobic (∼130° water advancing contact angle). In comparison, OQAS-modified flat glass has an advancing water contact angle of ∼105°, revealing the importance of surface roughness in enhancing hydrophobicity as described previously by Cassie, Wenzel, and others.34,35 OQAS and a series of other surface-immobilized hydrophobic cationic compounds are widely believed to kill bacteria by contact as a result of both the penetration and interruption of the bacterial membrane by their fatty alkyl chains and the increased osmotic pressure between the bacterial cytoplasm and the high-ionicstrength surroundings.12-16,36 Coated polystyrene substrates with OQAS-modified caps exhibited high antibacterial activity toward both waterborne and airborne bacteria (gram-negative Escherichia coli (E. coli) or gram-positive Staphylococcus epidermidis (S. epidermidis) as targets). In all cases, the surfaces were challenged with bacteria via previously reported protocols.16 Bacteriacontaminated surfaces were then covered with nutrition agars, followed by overnight culture. Bacteria colony-forming units (33) Cebeci, F. C.; Wu, Z. Z.; Zhai, L.; Cohen, R. E.; Rubner, M. F. Langmuir 2006, 22, 2856-2862. (34) Wenzel, R. N. Ind. Eng. Chem. 1936, 28, 988. (35) Cassie, A. B. D.; Baxter, S. Trans. Faraday Soc. 1944, 40, 546. (36) Kugler, R.; Bouloussa, O.; Rondelez, F. Microbiology (Reading, U.K.) 2005, 151, 1341-1348.
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Table 1. Percentage of Bacteria Killing Efficiency for Three Types of OQAS-Treated Surfaces, Tested with a Waterborne Test (wbt) or an Airborne Test (abt) killing efficiency test method
A (%)
Bb (%)
Cc (%)
E. coli (2 × cell/cm , wbt) E. coli (1 × 107 cell/cm3, abt) S. epidermidis (1 × 107 cell/cm3, abt)
92.3 89.0 83.0
99.2 99.2 99.1
>99.9 >99.9 >99.9
106
3
a
a Type A: OQAS-modified flat glass. b Type B: OQAS-modified polystyrene surface with a 10-layer SiO2 NP cap and an underlying reservoir made of 20 bilayers of PAH (pH 8.5)/PAA (pH 3.5). c Type C: Type B surface with two Ag loading and DMAB reduction cycles. Values in units of cell/cm3 represent different concentrations of bacterial solutions used to challenge surfaces. The percentage killing efficiency was calculated by comparing the colony-forming units (CFU) on a total area of 35 cm2 of a control surface and each type of coated surface. The control surfaces used are cell-culture-type polystyrene microscope slides.
(CFUs) were counted for both OQAS-modified surfaces and control surfaces to determine the bacteria killing efficiency. In the test conditions studied, killing efficiencies were greater than 99.0% (Table 1). OQAS-modified caps exhibited higher antibacterial activity than OQAS-modified flat glass surfaces, which showed around 90% killing efficiency under similar experimental conditions. This is presumably due to the increased bactericidal surface area that results from the 3D nanoscale roughness. Further investigation to understand the relationship between surface roughness and antibacterial activity is underway. Antibacterial reagents can be readily loaded directly through the OQAS-modified cap into the underlying PEM layers that contain free carboxylic acid binding groups.27 Silver (Ag), a well-known antiseptic agent, was loaded into the reservoir by simply dipping the coated substrate into a 10 mM silver acetate solution. Silver cations (Ag+) diffused into the reservoir and formed electrostatic pairs with carboxylate groups.37,38 The carboxylate-bound Ag+ ions could be reduced to zero-valent Ag NPs through our previously developed nanoreactor chemistry using a dimethylamine borane complex (DMAB) aqueous solution as the reductant.27,37,38 The reduction reaction also regenerated the carboxylic acid sites, rendering the reservoir capable of being “reloaded” through repeating Ag+/Ag NP loading and reduction cycles several times to obtain a desirable amount of Ag inside the coating. The whole process could be monitored through UVvis spectroscopy (Supporting Information, Figure S2). As revealed in the TEM cross-sectional images shown in Figure 2, the Ag NPs that formed through DMAB reduction were located primarily around interfaces, mostly in the cap region close to the reservoir/ cap interface and some near the reservoir/substrate interface. This tendency to form nanoparticles at interfaces when using nanoreactor chemistry has been observed previously.37 We note that reduction chemistry can also be carried out in the dry phase by using hydrogen gas as the reductant (Supporting Information, Figure S3).38 In this case, the nanoparticles exhibit less tendency to accumulate at interfaces. In the case of the wet-phase reduction, the multilayers are swollen with water, presumably providing more mobility to the nanoparticles or silver atoms that ultimately form the nanoparticles. We have not carried out experiments to find out why the nanoparticles under these conditions prefer to accumulate at the interfaces. We have also observed nanoparticles accumulating at interfaces on samples that have been reduced in the dry state.37 (37) Joly, S.; Kane, R.; Radzilowski, T.; Wang, T.; Wu, A.; Cohen, R. E.; Thomas, E. L.; Rubner, M. F. Langmuir 2000 16, 1354-1359. (38) Wang, T. C.; Rubner, M. F.; Cohen, R. E. Langmuir 2002, 18, 33703375.
Figure 3. Antibacterial assessment of four different coatings toward S. epidermidis as a function of incubation time in pH 7.4 PBS buffer at 37 °C. (A) Zone of inhibition (ZoI) for the four different coatings as a function of immersion time in PBS. (B) Killing efficiency in airborne tests for type II and type III coatings with different immersion times in PBS. See the text for an explanation of each type of coating.
The imbedded Ag (in the form of Ag+ or Ag NPs) was releasable through the OQAS-modified cap to the surrounding medium. This imparts to the coating a chemical-releasing-based antibacterial capability that can be clearly demonstrated via the popular Kirby-Bauer test.27 In that test, when a surface loaded with Ag+ or Ag NPs was placed on an agar plate spread with either E. coli or S. epidermidis bacteria, a zone of inhibition (ZoI) develops where no bacteria are capable of surviving. In contrast, no ZoI was observed for the coatings with only an OQAS treatment and no silver loading, confirming the absence of leaching of OQAS from the coatings. In addition, we also investigated the antibacterial efficiency toward waterborne or airborne bacteria (E. coli and S. epidermidis). For all studies, no bacteria CFUs were found for Ag-loaded coatings, whereas the CFU counts for the control polystyrene substrates of the same area varied between 480 and 8000 for the different bacterial tests carried out (Table 1). These results confirmed the releasing of Ag+ into the surroundings, consistent with the previously reported >99.99% bacteria-killing efficiency (E. coli or S. epidermidis) for silver-loaded PEM coatings.25,27 Having a coating with two distinct bactericidal mechanisms offers the opportunity to overcome certain disadvantages associated with each individual mechanism. For example, the continuous leaching of silver from the coating will eventually lead to depletion. To understand the effect of silver depletion further, four different coating types were investigated: (a) the type I coating contains an OQAS-modified SiO2 NP cap and a PEM reservoir with no Ag loading; (b) type II contains an OQASmodified cap and a reservoir with one loading of Ag+; and (c) type III contains a cap without OQAS modification and a reservoir with one loading of Ag+, (d) type IV contains an OQAS-modified cap and a reservoir with Ag NPs (two Ag loading and DMAB reduction cycles). Glass slides coated with these different types of coatings were immersed in phosphate buffer saline solutions (PBS, pH 7.4) and incubated at 37 °C with shaking. After every 24 h incubation period, samples were taken from the buffer solutions, and their antibacterial activity was measured against S. epidermidis using the airborne test and Kirby-Bauer
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methodology. As shown in Figure 3A, type I coatings showed zero ZoI values. For coatings with Ag (type II, III, and IV), ZoI values decreased with increasing immersion time in PBS buffer, indicating the loss of imbedded Ag to the buffer solutions.27 Consistent with our previous work on Ag-loaded PEMs,25,27 multilayer films with Ag NPs (type IV coating) showed long sustainability (more than 5 days), whereas PEMs with only Ag+ (type II and type III) lost their antibacterial effectiveness quickly. In fact, after 2 days of immersion, type II and type III exhibited zero ZoI values, suggesting a total loss of their ability to kill bacteria not directly in contact with the surface. However, because of immobilized OQAS, type II coatings were still able to kill more than 99% of airborne S. epidermidis after 3 days of immersion in PBS, whereas the type III coating lost its antibacterial ability as a result of silver depletion and the absence of any OQAS backup mechanism (Figure 3B). This proof-ofconcept experiment clearly demonstrates the advantages of having both the release-killing mechanism and the contact-killing mechanism. Compared with coatings (type I) solely based on contact killing, the dual-functional coatings (type II and type IV) showed a very high initial killing efficiency (>99.9%) due to Ag+ release, and they were able to kill bacteria with no direct surface contact. Compared with coatings (type III) solely based on release killing, significant antibacterial activity was retained (type II) as a result of the immobilized OQAS even after the depletion of the embedded silver. In conclusion, by using a two-level coating constructed through polyelectrolyte and nanoparticle multilayer deposition, one can
Langmuir, Vol. 22, No. 24, 2006 9823
separately control surface chemistry and chemical impregnation. Here we have created surface coatings that can kill bacteria not only through the release of imbedded silver ions but also via immobilized quaternary ammonium salts. This coating scheme is not limited only to silver and quaternary ammonium salts but should allow the incorporation of a variety of other antibacterial agents such as antibiotics and titanium oxide. Finally, we believe that this general coating strategy is applicable to other biological applications, where stimuli from both surface interactions (e.g. integrin ligands) and chemical uptake (e.g., chemokines) are critical to the fate of a biological entity approaching the surface, such as in cell adhesion or stem cell differentiation studies. Acknowledgment. This work was supported in part by the DARPA SDS Program and the MRSEC Program of the National Science Foundation under award number DMR 02-13282. This work also made use of the Shared CMSE Experimental Facilities supported in part by the MRSEC Program of the National Science Foundation under award number DMR 02-13282. We also acknowledge A. Nolte and Dr. Z. Wu for helpful discussions. Supporting Information Available: Experimental details, crosssectional TEM images showing the two-level antibacterial coatings with OQAS and silver NPs prepared through a dry-phase reduction method using hydrogen gas, and an AFM image revealing the high-surface-area SiO2 NP cap. This material is available free of charge via the Internet at http://pubs.acs.org. LA0622166
