pubs.acs.org/Langmuir © 2009 American Chemical Society
Polyelectrolyte Multilayers with Intrinsic Antimicrobial Functionality: The Importance of Mobile Polycations Jenny A. Lichter and Michael F. Rubner* Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139 Received January 27, 2009. Revised Manuscript Received February 22, 2009 Cationic contact-killing is an important strategy for creating antimicrobial surfaces that prevent viable bacteria attachment. Recent studies have shown that highly swollen, compliant surfaces resist bacterial attachment and a sufficient density of mobile cationic charge can effectively disrupt bacterial cell membranes. Polyelectrolyte multilayers (PEMs), a popular coating system for surface modification, have been used to kill bacteria through the incorporation of contact-killing or leaching biocides. In this work, we show that manipulation of multilayer assembly and postassembly conditions (e.g., pH) to expose mobile cationic charge can create antimicrobial PEMs without the addition of specific biocidal species. As a model system, we explored PEMs comprising poly(allylamine hydrochloride) (PAH) and poly (sodium 4-styrene sulfonate) (SPS) assembled at high pH and subsequently immersed in low pH solutions. This system undergoes a reversible pH-dependent swelling transition, and we demonstrate that antimicrobial functionality at physiological pH conditions can be turned on and off with suitable pH treatment. In both airborne and waterborne bacteria assays, the viability of two strains of Gram positive Staphylococcus epidermidis (S. epidermidis), one biofilm forming and one nonbiofilm forming, and two strains of Gram negative Escherichia coli (E. coli) was effectively reduced on SPS/PAH multilayers displaying accessible cationic charge. To generalize our results, the pH assembly conditions of PEMs comprising poly(acrylic acid) (PAA) and PAH were also modified to introduce antibacterial capabilities.
Introduction There is an emerging understanding of the material properties, both physical and chemical, that can be manipulated to enhance the antimicrobial functionality of surfaces and thin film coatings. Prevention of bacterial attachment and subsequent bacterial infections has been approached in two general ways. In one approach, surfaces have been designed that, although not bactericidal, resist the attachment of bacteria. For example, researchers have found that, under certain conditions, control over hydrophobicity1 and surface roughness2 can modulate bacterial attachment. In addition, some argue that DVLO (Derjaguin, Landau, Verwey, and Overbeek) theory, evaluating a combination of Lifshitz-van der Waals and electrostatic interactions, can be utilized to optimize surfaces to minimize attachment.3 More recently, it has been demonstrated that control over the compliance of a surface can be used to dramatically decrease the amount of bacterial attachment, with highly hydrated, low modulus surfaces providing the most effective resistance.4-7 In the second approach, surfaces have been designed to kill bacteria before colonization by leaching antibacterial species such *Corresponding author. Mailing address: 77 Massachusetts Avenue, Room 13-5106, Cambridge, MA 02139. E-mail:
[email protected]. Telephone: (617) 253-6701. (1) Ciston, S.; Lueptow, R. M.; Gray, K. A. J. Membr. Sci. 2008, 320, 101–107. (2) Whitehead, K. A.; Verran, J. Food Bioprod. Process. 2006, 84, 253–259. (3) Hermansson, M. Colloids Surf., B 1999, 14, 105–119. (4) Lichter, J. A.; Thompson, M. T.; Delgadillo, M.; Nishikawa, T.; Rubner, M. F.; Van Vliet, K. J. Biomacromolecules 2008, 9, 1571–1578. (5) Richert, L.; Lavalle, P.; Payan, E.; Shu, X. Z.; Prestwich, G. D.; Stoltz, J. F.; Schaaf, P.; Voegel, J. C.; Picart, C. Langmuir 2004, 20, 448–458. (6) Bratskaya, S.; Marinin, D.; Simon, F.; Synytska, A.; Zschoche, S.; Busscher, H. J.; Jager, D.; van der Mei, H. C. Biomacromolecules 2007, 8, 2960–2968. (7) Boulmedais, F.; Frisch, B.; Etienne, O.; Lavalle, P.; Picart, C.; Ogier, J.; Voegel, J. C.; Schaaf, P.; Egles, C. Biomaterials 2004, 25, 2003–2011. (8) Jeon, H. J.; Yi, S. C.; Oh, S. G. Biomaterials 2003, 24, 4921–4928. (9) Lee, D.; Cohen, R. E.; Rubner, M. F. Langmuir 2005, 21, 9651–9659.
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as silver ions8-20 or antibiotics21-23 or through the creation of contact-killing surfaces, usually containing covalently attached cationic antimicrobials such as quaternary ammonium compounds,24-26 alkyl pyridiniums,27-29 or quaternary phosphonium.30 In the latter case, multiple mechanisms of action have (10) Dai, J. H.; Bruening, M. L. Nano Lett. 2002, 2, 497–501. (11) Yliniemi, K.; Vahvaselka, M.; Van Ingelgem, Y.; Baert, K.; Wilson, B. P.; Terryn, H.; Kontturi, K. J. Mater. Chem. 2008, 18, 199–206. (12) Yu, D. G.; Lin, W. C.; Yang, M. C. Bioconjugate Chem. 2007, 18, 1521–1529. (13) Fu, J. H.; Ji, J.; Fan, D. Z.; Shen, J. C. J. Biomed. Mater. Res., Part A 2006, 79A, 665–674. (14) Cui, X. Q.; Li, C. M.; Bao, H. F.; Zheng, X. T.; Lu, Z. S. J. Colloid Interface Sci. 2008, 327, 459–465. (15) Malcher, M.; Volodkin, D.; Heurtault, B.; Andre, P.; Schaaf, P.; Mohwald, H.; Voegel, J. C.; Sokolowski, A.; Ball, V.; Boulmedais, F.; Frisch, B. Langmuir 2008, 24, 10209–10215. (16) 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. (17) Choi, W. S.; Koo, H. Y.; Park, J. H.; Kim, D. Y. J. Am. Chem. Soc. 2005, 127, 16136–16142. (18) Lee, H.; Lee, Y.; Statz, A. R.; Rho, J.; Park, T. G.; Messersmith, P. B. Adv. Mater. 2008, 20, 1619-+. (19) Grunlan, J. C.; Choi, J. K.; Lin, A. Biomacromolecules 2005, 6, 1149–1153. (20) Yuan, W. Y.; Ji, J.; Fu, J. H.; Shen, J. C. J. Biomed. Mater. Res., Part B 2008, 85B, 556–563. (21) Kohnen, W.; Kolbenschlag, C.; Teske-Keiser, S.; Jansen, B. Biomaterials 2003, 24, 4865–4869. (22) Mao, Z. W.; Ma, L.; Gao, C. Y.; Shen, J. C. J. Controlled Release 2005, 104, 193–202. (23) Bhadra, D.; Gupta, G.; Bhadra, S.; Umamaheshwari, R. B.; Jain, N. K. J. Pharm. Pharm. Sci. 2004, 7, 241–251. (24) Li, Z.; Lee, D.; Sheng, X. X.; Cohen, R. E.; Rubner, M. F. Langmuir 2006, 22, 9820–9823. (25) Lee, S. B.; Koepsel, R. R.; Morley, S. W.; Matyjaszewski, K.; Sun, Y. J.; Russell, A. J. Biomacromolecules 2004, 5, 877–882. (26) Kurt, P.; Wood, L.; Ohman, D. E.; Wynne, K. J. Langmuir 2007, 23, 4719–4723. (27) Kugler, R.; Bouloussa, O.; Rondelez, F. Microbiology-Sgm 2005, 151, 1341–1348. (28) Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M. Biotechnol. Bioeng. 2002, 79, 465–471. (29) Cen, L.; Neoh, K. G.; Kang, E. T. Langmuir 2003, 19, 10295–10303. (30) Popa, A.; Davidescu, C. M.; Trif, R.; Ilia, G.; Iliescu, S.; Dehelean, G. React. Funct. Polym. 2003, 55, 151–158.
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been proposed for how these cationic species are able to disrupt the bacterial cell membrane,27,31 with some requiring hydrophobic chains of certain lengths to penetrate and burst the bacterial membrane. Nevertheless, it has been shown that high levels of positive charge are capable of conferring antimicrobial properties irrespective of hydrophobic chain length, perhaps by an ion exchange mechanism between the bacterial membrane and the charged surface.27,32 Understanding and engineering the optimal presentation of cationic charges is clearly important in the design of contactkilling antimicrobial surfaces. In the case of antibacterial cationic liposomes, a minimum zeta potential of +40 mV and a melted crystalline state (mobile state) as opposed to a rigid phase were necessary for antibacterial functionality.33 In studies of polycationic cytotoxicity with mammalian cells, the union of high linear charge density and chain mobility has led to higher levels of cell death. Multiple cationic attachment sites are necessary for membrane permeabilization, and as a result increasing the spacing between positively charged groups and/or the rigidity of a polymer chain should decrease the polycationic bioactivity.34 Specifically, testing the effects of decreasing linear charge density of polycations has confirmed these conclusions; as linear charge density decreased, cytotoxicity decreased.35,36 Likewise, the arrangement and flexibility of charges has proven to be important. Highly branched chains or flexible backbones led to higher cytoxic effects than globular or rigid chains.36 From these studies, it is clear that the antibacterial effect of polycations is dependent on the ability of multiple charges to attach to and interact with the cell membrane. These findings suggest the possibility of engineering a variety of polymer based positively charged surfaces to create a wide range of contact-killing materials. Polyelectrolyte multilayers (PEMs) have been investigated extensively as biomaterials and biomaterial interfaces for such diverse applications as drug release, biosensing, and cell and protein attachment control on implantable medical devices.37-40 Many researchers have also designed PEMs for bacterial contamination prevention. For example, antiadhesive PEMs incorporating poly(ethylene glycol)7 and other highly swellable, low modulus materials have been shown to greatly reduce the attachment of bacteria.4-6 Poly(L-lactic acid), a biocompatible biodegradeable synthetic polymer, has been coated with silver-loaded PEMs that improved surface hydrophilicity, hemocompatibility, cytocompatibilty, and antibacterial activity.12 Many other PEMs have been developed with leachable biocides such as silver,9-11,13-20,24,41 antibiotics,22,23,42 antimicrobial peptides,43,44 (31) Gilbert, P.; Moore, L. E. J. Appl. Microbiol. 2005, 99, 703–715. (32) Murata, H.; Koepsel, R. R.; Matyjaszewski, K.; Russell, A. J. Biomaterials 2007, 28, 4870–4879. (33) Bombelli, C.; Bordi, F.; Ferro, S.; Giansanti, L.; Jori, G.; Mancini, G.; Mazzuca, C.; Monti, D.; Ricchelli, F.; Sennato, S.; Venanzi, M. Mol. Pharmaceutics 2008, 5, 672–679. (34) Ryser, H. J. Nature (London) 1967, 215, 934–6. (35) Wilson, J. T.; Cui, W. X.; Chaikof, E. L. Nano Lett. 2008, 8, 1940–1948. (36) Fischer, D.; Li, Y. X.; Ahlemeyer, B.; Krieglstein, J.; Kissel, T. Biomaterials 2003, 24, 1121–1131. (37) Tang, Z. Y.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Adv. Mater. 2006, 18, 3203–3224. (38) Kim, B. S.; Choi, J. W. Biotechnol. Bioprocess Eng. 2007, 12, 323–332. (39) Picart, C. Curr. Med. Chem. 2008, 15, 685–697. (40) Campas, M.; O’Sullivan, C. Anal. Lett. 2003, 36, 2551–2569. (41) 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. (42) Chuang, H. F.; Smith, R. C.; Hammond, P. T. Biomacromolecules 2008, 9, 1660–1668. (43) 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. (44) Guyomard, A.; De, E.; Jouenne, T.; Malandain, J. J.; Muller, G.; Glindel, K. Adv. Funct. Mater. 2008, 18, 758–765.
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and other species.45-47 Stainless steel, an important orthopedic implant material, has been coated with PEMs containing quarternized polyethylamine-silver complex to create a cationic contactkilling surface with additional silver-leaching capabilities.41 Titanium, another prevalent orthopedic implant material, has been functionalized with chitosan-containing PEMs that confer cationic contact-killing properties.48 Other contact-killing PEMs have included guanidines,49 QACs,24 titania,20 and lysozymes.45,50 Chitosan, a natural biocompatible cationic polysaccharide, has been incorporated into many PEMs for antimicrobial functionality.6,20,48,51,52 Although the innate cationic charge of chitosan and its derivatives is known to be biocidal, it has been observed that differences in PEM assembly conditions can influence the film’s ultimate antimicrobial capabilities.5,53 This has been attributed to the amount of chitosan at the film surface, differences in film thickness, and variations in film rigidity, parameters all controlled by assembly conditions such as solution pH and/or ionic strength. To our knowledge, however, no one has investigated the effect of PEM assembly conditions on the actual mechanism of polycationic antibacterial action. The PEM assembly process distinguishes itself as a safe, waterbased, and versatile technique for creating coatings. Glass,54 plastics,12 metals,41 colloids,9 and even cells55,56 have been coated with PEMs for a huge variety of applications. One of the most useful aspects of PEMs is their sensitivity to assembly and postassembly conditions. For example, addition of salt to a polymer assembly solution can change both the chain conformation and the layer thickness.57 Chemical, thermal, or UV crosslinking can significantly alter the stability of a given PEM,58,59 and exposure to high ionic strength can controllably remove material and decrease thickness.60 Manipulating pH, both during and after multilayer assembly, is perhaps the richest method of PEM architecture control. We and others have shown that, with a given pair of polymers, manipulation of pH conditions during and/or after assembly can significantly alter layer thickness,61,62 swellability,63 elastic modulus,64 (45) Rudra, J. S.; Dave, K.; Haynie, D. T. J. Biomater. Sci., Polym. Ed. 2006, 17, 1301–1315. (46) Kim, B. S.; Park, S. W.; Hammond, P. T. ACS Nano 2008, 2, 386–392. (47) Nguyen, P. M.; Zacharia, N. S.; Verploegen, E.; Hammond, P. T. Chem. Mater. 2007, 19, 5524–5530. (48) Chua, P. H.; Neoh, K. G.; Kang, E. T.; Wang, W. Biomaterials 2008, 29, 1412–1421. (49) Pan, Y. F.; Xiao, H. N.; Zhao, G. L.; He, B. H. Polym. Bull. 2008, 61, 541–551. (50) Nepal, D.; Balasubramanian, S.; Simonian, A. L.; Davis, V. A. Nano Lett. 2008, 8, 1896–1901. (51) Feng, Y. H.; Han, Z. G.; Peng, J.; Lu, J.; Xue, B.; Li, L.; Ma, H. Y.; Wang, E. B. Mater. Lett. 2006, 60, 1588–1593. (52) Elsabee, M. Z.; Abdou, E. S.; Nagy, K. S. A.; Eweis, M. Carbohydr. Polym. 2008, 71, 187–195. (53) Fu, J. H.; Ji, J.; Yuan, W. Y.; Shen, J. C. Biomaterials 2005, 26, 6684–6692. (54) Hiller, J.; Mendelsohn, J. D.; Rubner, M. F. Nat. Mater. 2002, 1, 59–63. (55) Neu, B.; Voigt, A.; Mitlohner, R.; Leporatti, S.; Gao, C. Y.; Donath, E.; Kiesewetter, H.; Mohwald, H.; Meiselman, H. J.; Baumler, H. J. Microencapsulation 2001, 18, 385–395. (56) Donath, E.; Moya, S.; Neu, B.; Sukhorukov, G. B.; Georgieva, R.; Voigt, A.; Baumler, H.; Kiesewetter, H.; Mohwald, H. Chem.;Eur. J. 2002, 8, 5481–5485. (57) Decher, G.; Schlenoff, J. B. Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials; Wiley-VCH: Weinheim, 2003. (58) Yang, S. Y.; Rubner, M. F. J. Am. Chem. Soc. 2002, 124, 2100–2101. (59) Yang, S. Y.; Lee, D.; Cohen, R. E.; Rubner, M. F. Langmuir 2004, 20, 5978–5981. (60) Nolte, A. J.; Takane, N.; Hindman, E.; Gaynor, W.; Rubner, M. F.; Cohen, R. E. Macromolecules 2007, 40, 5479–5486. (61) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213–4219. (62) Yoo, D.; Shiratori, S. S.; Rubner, M. F. Macromolecules 1998, 31, 4309–4318. (63) Mendelsohn, J. D.; Yang, S. Y.; Hiller, J.; Hochbaum, A. I.; Rubner, M. F. Biomacromolecules 2003, 4, 96–106. (64) Thompson, M. T.; Berg, M. C.; Tobias, I. S.; Rubner, M. F.; Van Vliet, K. J. Biomaterials 2005, 26, 6836–6845.
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charge density,62 surface roughness, porosity,65,66 surface wettability,62 index of refraction,54 layer interpenetration,62 and bulk and surface composition.61,67 Adjustment of these material parameters can control, for example, cytophilicity63,64 and antireflection properties.54 Certain PEMs are pH responsive and therefore can be used for drug and dye uptake and release68,69 and nanochannel gating.70 Previously, we demonstrated that bacterial attachment could be reduced significantly, but not eliminated, by tuning assembly pH conditions to create a highly water swollen, low modulus surface.4 These PEMs acted by inhibiting attachment without killing bacteria. The addition of a bacteria killing capability would therefore further prevent surface contamination. By their nature, electrostatically associated PEMs contain significant cationic charge but are generally not considered to be antimicrobial without the addition of specific biocidal species (e.g., silver, chitosan, QACs, etc). In this work, we show how manipulation of assembly pH and postassembly conditions of typical synthetic polymers can be used to create surfaces with the necessary cationic charge density and chain mobility to confer contact-killing properties. Using a model PEM system comprising cationic poly(allylamine hydrochloride) (PAH) and anionic poly (sodium 4-styrene sulfonate) (SPS), we show that it is possible to turn an adherent, nontoxic coating into a highly bacteria-resistant and antibacterial PEM. This is achieved using specific pH conditions previously studied in our group: SPS/PAH multilayers assembled at high pH followed by a subsequent low pH treatment.71,72 This antibacterial and antiadherent functionality can be again switched off with subsequent pH treatment. Unlike most systems (such as grafted polymer surfaces) that require working at different pH conditions to adjust parameters such as the degree of ionization of functional groups, this system displays a “molecular memory effect,” allowing the creation of surfaces of identical chemical composition but with very different properties at neutral pH depending on the previous pH treatments.72 The polyelectrolyte multilayers discussed here are therefore an ideal platform for evaluating the effects of cationic surface charge and polymer chain and charge presentation on antibacterial properties. Two strains of Staphylococcus epidermidis (S. epidermidis), one biofilm forming and one nonbiofilm forming, and two strains of Esherichia coli (E. coli) were chosen as model Gram positive and Gram negative bacteria challenges, respectively. Both airborne and waterborne antibacterial capabilities were studied. Although this study focuses on the SPS/PAH PEM system, the general concept, adjusting assembly and/or postassembly conditions to create highly swellable systems with high cationic charge density and mobility, can be applied to other multilayers to produce antibacterial capabilities.
Materials and Methods PEM Assembly. Polyelectrolyte multilayers were assembled in the manner previously described.61,73 All polymer solutions (65) Mendelsohn, J. D.; Barrett, C. J.; Chan, V. V.; Pal, A. J.; Mayes, A. M.; Rubner, M. F. Langmuir 2000, 16, 5017–5023. (66) Tjipto, E.; Quinn, J. F.; Caruso, F. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 4341–4351. (67) Yuan, W. Y.; Dong, H.; Li, C. M.; Cui, X. Q.; Yu, L.; Lu, Z. S.; Zhou, Q. Langmuir 2007, 23, 13046–13052. (68) Chung, A. J.; Rubner, M. F. Langmuir 2002, 18, 1176–1183. (69) Zhu, Y. F.; Shi, J. L. Microporous Mesoporous Mater. 2007, 103, 243–249. (70) Lee, D.; Nolte, A. J.; Kunz, A. L.; Rubner, M. F.; Cohen, R. E. J. Am. Chem. Soc. 2006, 128, 8521–8529. (71) Itano, K.; Choi, J. Y.; Rubner, M. F. Macromolecules 2005, 38, 3450–3460. (72) Hiller, J.; Rubner, M. F. Macromolecules 2003, 36, 4078–4083. (73) Yang, S. Y.; Mendelsohn, J. D.; Rubner, M. F. Biomacromolecules 2003, 4, 987–994.
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were prepared with deionized water (0.01 M, based on the repeating unit molecular weight). SPS/PAH PEMs were assembled on plain glass slides (VWR) with SPS (poly(sodium 4-styrene sulfonate); Mw = 70 000 g/mol; Aldrich) and PAH (poly(allylamine hydrochloride); Mw = 70 000 g/mol; Polysciences) solutions that were pH adjusted using 1 M HCl and NaOH. The multilayers, assembled with the polymers adjusted to pH 4.0 or pH 9.3 (for pH 9.3 assembly, rinse baths were also adjusted to pH 9.3), contained 15.5 bilayers (with PAH on top). Note that 0.1 M NaCl was added to the pH 4.0 polymer solutions to ensure good film growth. Acid-treated SPS/PAH films were immersed in water at pH 2.5 for 15 min, rinsed thoroughly in deionized water, and dried with compressed air. An additional multilayer system included PAH assembled with the weak anionic polyelectrolyte PAA (poly(acrylic acid); Mw = >200 000 g/mol; 25% aqueous solution; Polysciences (previously labeled as Mw = 90 000 g/mol)). These other multilayers were assembled polyanion first on aminoalkylsilane coated glass (Sigma-Aldrich). PAA/PAH samples were made with both polymers adjusted to pH = 2.0 (9.5 or 10 bilayers), 4.0 (7.5 or 8 bilayers), and 6.5 (49.5 or 50 bilayers), with both PAA and PAH on top. Samples with PAA at pH 3.5 and PAH at pH 7.5 and 8.6 were also assembled with both PAA (5.5 bilayers) and PAH (6 bilayers) as the final layer. All PAA/PAH samples were assembled to have a final dry thickness of ∼50 nm. For Figure 1, a hydrogen-bonded system of PAA and poly(acrylamide) (PAAm) (Mw = 600 000-1 000 000 g/mol; Polysciences) was assembled on aminoalkylsilane-modified slides with 10.5 bilayers (PAA-topped). All polymer baths and rinse baths were adjusted to pH 3.0 during hydrogen-bonded PEM assembly. PAA/PAAm samples were lightly cross-linked at 100 °C for 24 h so they would remain intact in physiological conditions. For all ellispometry measurements, single-crystal polished silicon wafers (p-type, 1-50 MΩ cm, [100] orientation, WaferNet) were used as substrates. Note that PAA/PAH films built on silicon contained an additional 0.5 bilayer due to the negative charge of the silicon versus the positive charge of the aminoalkylsilane coated glass slides. For streaming zeta potential measurements, the PEMs were assembled on 0.02 in. thick polycarbonate (Grainger). The notation used to refer to PEMs throughout this paper is: P1 =P2
pH1 =pH2
where P1 is the polyanion, P2 is the polycation, pH1 is the pH of deposition of the polyanion, and pH2 is the pH of deposition of the polycation. Thickness and Swelling Measurements. Spectroscopic ellipsometry (M-2000D, J. A. Woollam Co., Inc.) was used to determine the thickness of PEMs. Swelling experiments, performed using in situ ellipsometry in the manner described previously,71 were conducted after equilibrating the samples for 10 min in deionized water. Rose Bengal Dye Uptake. Samples (coated with PEM on both sides) were immersed for 15 min in a 10-3 M solution of Rose Bengal adjusted to pH 5.5. The samples were rinsed twice with deionized water and dried with compressed air. Absorbance was measured with a Varian Cary 5E spectrophotometer. Streaming Zeta Potential Measurements. PEMs were assembled on polycarbonate slides cut from large sheets and placed (two at a time) in a 500 μm sample holder. Streaming zeta potential was measured at pH 6.2 with 1 mM KCl electrolyte using a ZetaCAD (Laval Laboratory, Inc.) streaming potential analyzer. Micron Particle Attachment. Carboxylate-modified fluorescent latex beads (1.0 μm sized; 2.5% aqueous suspension, Sigma) were diluted 1:1000 in deionized water and adjusted to pH 6.5. Samples were immersed in the suspension for 1 h, rinsed twice with deionized water, and dried with compressed air. Samples were imaged in fluorescent mode at 50 magnification with a Zeiss Axioplan 2 UV/visible microscope. At least five Langmuir 2009, 25(13), 7686–7694
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Article was used. Bacterial suspensions of 109 cells/mL were prepared as described for the waterborne attachment assays (centrifuged and measured for an optical density of 1 in PBS) and diluted to 2 106 cells/mL in sterile deionized water. Fine mists (d ∼ 5100 μm) were sprayed at 1 μL/cm2 3 spray for a total of 6 103 bacterial cells/cm2. SPS/PAH samples showed identical trends for both spray methods. Log kills were reported using the following convention: log kill = log(number of colonies on glass control) - log(number of colonies on sample). All experiments were performed in triplicate.
Results
Figure 1. Airborne S. epidermidis (14990) challenges showing no antibacterial activity on (a) PAA/PAH films (labeled with the pH of deposition and the identity of the top layer), (b) SPS/PAH films assembled at pH 4.0 with 0.1 M NaCl (PAH as top layer), and (c) PAA/PAAm films assembled at pH 3.0 and thermally cross-linked (PAA as top layer). All samples statistically identical to glass controls (Student’s t test, p < 0.05). pictures of each sample were collected. Results are presented as average number of particles per unit area. Zeta potential measurements on the latex particles were acquired with a Brookhaven Instruments Zeta-PALS analyzer. Aqueous Bacterial Attachment Assays (or Waterborne Bacterial Assays). Bacterial attachment was assayed as described previously.4 Briefly, monoclonal Staphylococcus epidermidis (S. epidermidis, ATCC # 14990 (nonbiofilm forming) or ATCC # 35984 (biofilm forming)) or Escherichia coli (E. coli, ATCC # 700728 or ATCC # 14948) was grown overnight in LBMiller broth (VWR). The incubation was centrifuged at 2700 rpm, decanted, and resuspended in 150 mM NaCl PBS (VWR) three times (10 min, 5 min, 5 min). The final resuspension was diluted to a density of 109 cells/mL measured via optical density. This resuspension was then diluted in sterile deionized water to 107 cells/mL. The PEMs were placed in the diluted bacterial suspension for 2 h at room temperature and rinsed thoroughly in three water baths. Samples were incubated under 1% LB agar (VWR) gel overnight, and colonies were counted to determine the ability of viable bacteria to attach to each sample. Samples with few colonies were hand counted. For densely populated slides, at least 10 digital images per sample were acquired with a 4 objective using an inverted optical microscope (Leica) and the total number of colonies per slide was extrapolated. Results were presented either as total number of colony density per area or as a normalized colony density calculated as follows: normalized colony density = average colony density for sample/average colony density for glass control. By definition, the normalized colony density of glass was equal to one. All experiments were performed in triplicate, except for one experiment with biofilm-forming S. epidermidis in which four samples were tested. Airborne Antibacterial Assays. Airborne assays were based on the protocol discussed by Klibanov et. al.74 Overnight bacterial cultures (∼109 cells/mL) were diluted 1:100 in sterile deionized water unless otherwise noted. The dilution was sprayed onto the sample surface using a gas chromatography sprayer (VWR International, cat. no. 21428-350). Samples were immediately placed in 100 mm Petri dishes (VWR) and covered with 1% LB agar gel overnight at 37 °C. Resulting colonies were counted as described above. For airborne studies on PAA/ PAAm and PAA/PAH samples, a slightly different protocol (74) Haldar, J.; Weight, A. K.; Klibanov, A. M. Nat. Protoc. 2007, 2, 2412–2417.
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Airborne Antimicrobial Assays. The antibacterial properties of cationic species have been extensively documented.24,27,28,32,75 Cationic polymers, both with and without hydrophobic segments, have been used to effectively kill a broad spectrum of bacteria. The typical cationic polymers present in PEMs have high linear charge density, but their usual form in a multilayer, strongly ionically coupled with a polyanionic species, generally does not produce any significant antibacterial activity even when the polycationic species is the top layer. Since the cationic functional groups are not “free” and mobile, they are unable to induce bacterial death. As shown in Figure 1, commonly used polymer combinations (SPS/PAH and PAA/PAH) assembled under typical conditions (with both polymers at the same pH) do not possess any airborne antibacterial properties against a model nonbiofilm forming S. epidermidis strain. The lightly cross-linked hydrogen bonded system PAA/PAAm, which likewise lacks free polycationic charge, also does not exhibit any biocidal activity (Figure 1c). To overcome the challenge of creating antimicrobial PEM surfaces, many researchers have incorporated antibacterial species, including silver ions,9-20,24,41 quaternary ammonium salts,24 chitosan,6,48,52,53 antibiotics,22,23,42 enzymes,45,50 and so on. However, as a surface modification process, PEM assembly offers a unique advantage. The tunability of the PEM assembly process can be exploited and surface parameters can be adjusted to create desired functionalities without the need of additional biocidal species. For example, adjusting assembly and postassembly pH can create multilayers with drastically different chain mobility restrictions (as indicated by hydrated swelling levels) and free cationic charge (charges not coupled with an anionic polymer), two factors that greatly influence microbiocidal abilities. One way to tune surface properties results from the pH-dependent degree of ionization of weak polyelectrolytes (e. g., poly(acrylic acid) (PAA) and PAH). A particularly interesting multilayer in this regard is the SPS/PAH multilayer asembled at high pH. This multilayer system has been shown to exhibit large, reversible swelling transitions with postassembly pH treatments.72 A schematic of the SPS/PAH 9.3/9.3 transition in low pH solutions is illustrated in Figure 2. This reversible change in swelling behavior is associated with the acid-activated opening of hydrophobic PAH clusters containing free amine groups and the reforming of those clusters at high pH (>pH 10).71 PAH, with a solution pKa ∼ 8.8,76 contains hydrophobic clusters composed of uncharged amines along the backbone in solution at pH 9.3. Hydrophobic clusters of PAH assembled into a film at high pH are opened at low pH ( 8.5) and subsequently treated at lower pH (
