Antimicrobial Behavior of Polyelectrolyte−Surfactant Thin Film

pubs.acs.org/Langmuir © 2009 American Chemical Society

Antimicrobial Behavior of Polyelectrolyte-Surfactant Thin Film Assemblies Charlene M. Dvoracek,† Galina Sukhonosova,† Michael J. Benedik,‡ and Jaime C. Grunlan*,† †

Department of Mechanical Engineering, Texas A&M University, College Station, Texas 77843, and ‡ Department of Biology, Texas A&M University, College Station, Texas 77843 Received April 2, 2009. Revised Manuscript Received May 18, 2009

Layer-by-layer (LbL) assembly, a technique that alternately deposites cationic and anionic materials, has proven to be a powerful technique for assembling thin films with a variety of properties and applications. The present work incorporates the antimicrobial agent cetyltrimethylammonium bromide (CTAB) in the cationic layer and uses poly (acrylic acid) (PAA) as the anionic layer. When the films are exposed to a humid environment, these agents diffuse out of the film, inhibiting bacterial growth in neighboring regions. Film growth, microstructure, and antimicrobial efficacy are studied here, with 10-bilayer films yielding thicknesses on the order of 2 μm. Various factors are shown to influence the antimicrobial efficacy including time, temperature, secondary ingredients, and number of bilayers. As more layers are deposited, antimicrobial efficacy is increased because more CTAB is able to diffuse throughout the film, and higher amounts of antimicrobials are released. Additionally, inclusion of the cationic poly(diallyldimethylammonium chloride) (PDDA) in the cationic layer in conjunction with CTAB increases film uniformity, and as a result, antimicrobial effectiveness is enhanced. These thin films provide the ability to render a surface antimicrobial and may be useful for bandages or sterilization of disposable objects (e.g., surgical marker).

Introduction Langmuir’s discovery that surfaces will adsorb only a single layer of ions initiated the field of thin films with thicknesses in the nanometer to micrometer range.1 Iler built upon this concept by creating multilayer films with positively and negatively charged particles.2 In the early nineties, Decher further developed this idea by creating the formal layer-by-layer (LbL) assembly process.3-5 In this process, a substrate is alternately dipped into aqueous solutions containing charged ingredients, as shown in Figure 1, building a film through electrostatic attractions. Hydrogen bonding and other types of van der Waals attractions can also be used to build LbL assemblies,6-8 but electrostatic-based deposition remains the predominant form.2,9-12 Each positive and negative pair deposited is known as a bilayer (BL), with each bilayer typically 1-100 nm thick.9,13 These films are highly tailorable by altering pH,11,14 ionic strength,10,14 chemistry,15 and molecular weight.12,16 Additionally, film properties can be tailored by *To whom correspondence should be addressed. Tel: +1 979 845 3027. Fax: +1 979 862 3989. E-mail: [email protected]. (1) Langmuir, I. Method of substance detection. General Electric Co.; U.S. Patent 2232539, Feb 1941. (2) Iler, R. K. J. Colloid Interface Sci. 1966, 21, 569–594. (3) Decher, G.; Hong, J. D. Int. J. Phys. Chem. 1991, 95, 1430–1434. (4) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210, 831–835. (5) Hammond, P. T. Adv. Mater. 2004, 16, 1271–1293. (6) DeLongchamp, D. M.; Hammond, P. T. Langmuir 2004, 20, 5403–5411. (7) Fu, Y.; Bai, S. L.; Cui, S. X.; Qiu, D. L.; Wang, Z. Q.; Zhang, X. Macromolecules 2002, 35, 9451–9458. (8) Kotov, N. A. Nanostruct. Mater. 1999, 12, 789–796. (9) Decher, G.; Schlenoff, J. B. Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials; Wiley-VCH: Weinheim, Germany, 2003. (10) McAloney, R. A.; Sinyor, M.; Dudnik, V.; Goh, M. C. Langmuir 2001, 17, 6655–6663. (11) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213–4219. (12) Sui, Z. J.; Salloum, D.; Schlenoff, J. B. Langmuir 2003, 19, 2491–2495. (13) Jan, C. J.; Walton, M. D.; McConnell, E. P.; Jang, W. S.; Kim, Y. S.; Grunlan, J. C. Carbon 2006, 44, 1974–1981. (14) Schoning, M. J.; H. A., M.; Poghossian, A. J. Solid State Electrochem. 2009, 13, 115–122. (15) Mermut, O.; Barrett, C. J. J. Phys. Chem. B 2003, 107, 2525–2530. (16) Zhang, H. Y.; Wang, D.; Wang, Z. Q.; Zhang, X. Eur. Polym. J. 2007, 43, 2784–2791.

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adding small concentrations of additives to the deposition solution. These additives include clay,17-19 viruses,20 colloidal particles,21,22 or antimicrobial agents.23,24 The layer-by-layer technique has been used to make thin films for antireflection,25 gas barrier,18 battery electrolytes,26 and microcapsule drug delivery.27,28 The incorporation of small molecules or nanoparticles, added to either the cationic or anionic mixtures, can impart different characteristics or properties to the film23 or to the surroundings, including diffusion of different types of biomolecules from the films.29,30 For example, the addition of antiseptic agents could be useful in applications such as food packaging31-34 and wound dressing.35 Silver particles are known to kill a broad array of infectious bacteria, making them (17) Eckle, M.; Decher, G. Nano Lett. 2001, 1, 45–49. (18) Jang, W. S.; Rawson, I.; Grunlan, J. C. Thin Solid Films 2008, 516, 4819– 4825. (19) Kotov, N. Mater. Perform. 2008, 47, 20–21. (20) Yoo, P. J.; Nam, K. T.; Qi, J. F.; Lee, S. K.; Park, J.; Belcher, A. M.; Hammond, P. T. Nat. Mater. 2006, 5, 234–240. (21) Dawidczyk, T. J.; Walton, M. D.; Jang, W.-S.; Grunlan, J. C. Langmuir 2008, 24, 8314–8318. (22) Sukhorukov, G. B.; Donath, E.; Lichtenfeld, H.; Knippel, E.; Knippel, M.; Budde, A.; Mohwald, H. Colloids Surf., A 1998, 137, 253–266. (23) Grunlan, J. C.; Choi, J. K.; Lin, A. Biomacromolecules 2005, 6, 1149– 1153. (24) 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. (25) Fujita, S.; Shiratori, S. Jpn. J. Appl. Phys., Part 1 2004, 43, 2346–2351. (26) Lowman, G. M.; Tokuhisa, H.; Lutkenhaus, J. L.; Hammond, P. T. Langmuir 2004, 20, 9791–9795. (27) Antipov, A. A.; Sukhorukov, G. B.; Donath, E.; Mohwald, H. J. Phys. Chem. B 2001, 105, 2281–2284. (28) Wood, K. C.; Little, S. R.; Langer, R.; Hammond, P. T. Angew. Chem., Int. Ed. 2005, 44, 6704–6708. (29) Pilbat, A. M.; Szegletes, Z.; Kota, Z.; Ball, V.; Schaaf, P.; Voegel, J. C.; Szalontai, B. Langmuir 2007, 23, 8236–8242. (30) 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. (31) Becerril, R.; Gomez-Lus, R.; Goni, P.; Lopez, P.; Nerin, C. Anal. Bioanal. Chem. 2007, 388, 1003–1011. (32) Han, J. H. Food Technol. 2000, 54, 56–65. (33) Quintavalla, S.; Vicini, L. Meat Sci. 2002, 62, 373–380. (34) Tripathi, S.; Mehrotra, G. K.; Dutta, P. K. E-Polymers; No. 93; 2008. (35) Sant, S. B.; Gill, K. S.; Burrell, R. E. Philod. Mag. Lett. 2000, 80, 249.

Published on Web 06/18/2009

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Figure 1. Schematic of the LbL process involving alternate dipping in cationic and anionic solutions, with rinsing and drying between each deposition. A schematic of the resulting thin film made from CTAB molecules and poly(acrylic acid), as the positively and negatively charged molecules, is also shown.

the most widely used antimicrobial agent.36-40 Other widely used antiseptics include iodine,41 quaternary ammonium compounds,42,43 essential oils,44-47 antibiotics,48,49 and cetyltrimethylammonium bromide (CTAB),23,50,51 which is the focus of this work. The antimicrobial agents can be incorporated in their ionic form in layer-by-layer assemblies because their counterions (Br- in the case of CTAB) are displaced during deposition. Evidence suggests that antimicrobial action occurs primarily in this charged state,52,53 eliminating the need for an activation step. This could provide greater effectiveness at lower concentration than in other systems where antiseptics are incorporated as uncharged solids or salts.23

In the present study, CTAB is incorporated into the cationic layers of a film, using LbL assembly. CTAB has been shown previously to demonstrate greater antimicrobial efficacy in LbL films than silver;23 this partnered with its potential for growth made it suitable for further investigation. Growth trends of the various recipes for films were studied, and the final film crosssections and surfaces were examined. Next, the effects of the number of bilayers, antimicrobial concentration, incubation temperature, chemistry, and time delay after deposition were explored with respect to antimicrobial effectiveness. Efficacy of these films, measured with a Kirby-Bauer-like test, is strongest with more bilayers, lower testing temperatures, and when using PDDA in combination with CTAB in the cationic layers.

(36) Chambers, H. F. In Goodman and Gilman’s The Pharmacological Basis of Theraputics, 10th ed; Hardman, J. G., Gilman, A. G., Ed.; McGraw-Hill: New York, 2001. (37) Balogh, L.; Swanson, D. R.; Tomalia, D. A.; Hagnauer, G. L.; McManus, A. T. Nano Lett. 2001, 1, 18–21. (38) Kumar, R.; Munstedt, H. Biomaterials 2005, 26, 2081–2088. (39) Redmond, S. M.; Rand, S. C.; Tang, H. X.; Martin, D. C.; Balogh, P.; Balogh, L. Abstracts of Papers of the American Chemical Society; American Chemical Society: Washington, DC, 2000; Vol. 220, pp U285-U286. (40) Sondi, I.; Salopek-Sondi, B. J. Colloid Interface Sci. 2004, 275, 177–182. (41) Touitou, E.; Deutsch, J.; Matar, S. Int. J. Pharm. 1994, 103, 199–202. (42) Gottenbos, B.; van der Mei, H. C.; Klatter, F.; Nieuwenhuis, P.; Busscher, H. J. Biomaterials 2002, 23, 1417–1423. (43) Tiller, J. C.; Liao, C. J.; Lewis, K.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5981–5985. (44) Lopez, P.; Sanchez, C.; Batlle, R.; Nerin, C. J. Agric. Food Chem. 2005, 53, 6939–6946. (45) Lopez, P.; Sanchez, C.; Batlle, R.; Nerin, C. J. Agric. Food Chem. 2007, 55, 8814–8824. (46) Rodriguez, A.; Batlle, R.; Nerin, C. Prog. Org. Coat. 2007, 60, 33–38. (47) Rodriguez, A.; Nerin, C.; Batlle, R. J. Agric. Food Chem. 2008, 56, 6364– 6369. (48) Donelli, G.; Francolini, I.; Piozzi, A.; Di Rosa, R.; Marconi, W. J. Chemother. 2002, 14, 501–507. (49) Gu, H. W.; Ho, P. L.; Tong, E.; Wang, L.; Xu, B. Nano Lett. 2003, 3, 1261– 1263. (50) Evans, D. J.; Allison, D. G.; Brown, M. R. W.; Gilbert, P. J. Antimicrob. Chemother. 1990, 26, 473–478. (51) Brown, M. R. W.; Collier, P. J.; Gilbert, P. Antimicrob. Agents Chemother. 1990, 34, 1623–1628. (52) Dibrov, P.; Dzioba, J.; Gosink, K. K.; Hase, C. C. Antimicrob. Agents Chemother. 2002, 46, 2668–2670. (53) Simonetti, N.; Simonetti, G.; Bougnol, F.; Scalzo, M. Appl. Environ. Microbiol. 1992, 58, 3834–3836.

Materials and Methods

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The anionic deposition solution consisted of 0.2 wt % poly (acrylic acid) (PAA) (Aldrich, St. Louis, MO) with a molecular weight (Mw) of 100,000-200,000 g/mol in deionized water (18.2 MΩ). Cationic solutions contained 0.2 wt % poly(diallyldimethylammonium chloride) (PDDA) (Aldrich, St. Louis, MO) unless otherwise noted. The antimicrobial agent, cetyltrimethylammonium bromide (Aldrich, St. Louis, MO), was added to the cationic solution at various molarities to determine its maximum effectiveness. All solutions were used at their natural pH; typical values are 2.7 for 0.2 wt % PAA, 4.8 for 0.2 wt % PDDA, and 5.1 for 0.2 wt % PDDA with 5 mM CTAB. Substrates used in different applications were 175 μm poly(ethylene terephthalate) (PET) (trade name ST505 by DuPont Teijin, Tekra Corp., New Berlin, WI), polystyrene (PS) (Goodfellow, Oakdale, PA), and silicon wafers polished on one side (University Wafer, South Boston, MA). Bacterial growth media were from Difco LB Broth solidified with 1.5% bacteriological agar (United States Biologicals, Swampscott, MA). Escherichia coli (E. coli) K-12, lab strain MB 458, (Fh galK16 galE15 relA1 rpsL150 spoT1 mcrB1) and Staphylococcus aureus (S. aureus) wild type strain, lab strain MB 1594, were the bacteria used in testing. Film Deposition. In all cases, the substrate was negatively charged, either by using a substrate with an inherent negative charge or corona treatment (BD-20C Corona Treater, ElectroTechnic Products Inc., Chicago, IL) of an uncharged polymer substrate, such as PET. Corona treatment oxidizes the polymeric DOI: 10.1021/la901161z

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Article surface, providing better cationic adhesion and higher repeatability.54,55 Prior to corona treatment, the PET and PS films were rinsed with methanol and deionized water, and then dried with filtered air, while glass and silicon substrates were rinsed with acetone instead of methanol. The cleaned substrates were then dipped alternately in positive and negative solutions to build the film. The initial dip in each solution was five minutes, with subsequent dips of one minute each. Between each layer, the films were rinsed with deionized water and blown dry with air. Specimens were stored in a desiccator prior to testing. Film Growth. A Maxtek Research Quartz Crystal Microbalance (RQCM) from Infinicon (East Syracuse, NY) with a frequency range of 3.8-6 MHz was used in conjunction with 5 MHz quartz crystals. The crystal, in its holder, was dipped alternately in the positive (PDDA + CTAB or CTAB only) and negative (0.2 wt % PAA) solutions, with frequency (which is later correlated to mass) measured at every layer. A Dektak 3 Stylus Profilometer (Neutronix-Quintel, Morgan Hill, CA) was also used to more directly measure thickness. Films evaluated using profilometry were deposited onto glass slides. This method gives an absolute measurement of thickness, but it is not very accurate for film thicknesses below 1 μm. The profilometry readings were taken upon the completion of each film, while the QCM measurements were used to monitor the film’s growth. Film Characterization. Film surfaces were imaged with a Nanosurf EasyScan 2 atomic force microscope (AFM) (Nanoscience Instruments, Inc., Phoenix, AZ) in dynamic mode with an ACL-A cantilever tip. Sample preparation for the AFM involved the deposition of our thin films onto silicon wafers. The AFM was used to characterize film roughness and uniformity. Cross-sections of the assemblies were imaged with a JEOL 1200 EX TEM (JEOL USA Inc., Peabody, MA) at an accelerating voltage of 100 kV. PS substrates were used instead of PET to facilitate sectioning. After deposition, the film and substrate were embedded in epoxy resin with a 1:1 anhydride/epoxide (A/E) ratio. This epoxy comprised Areldite 502 and Quetal 651 as the epoxy resin, along with a dodecenylsuccinic anhydride (DDSA) hardener and a benzyldimethylamine (BDMA) accelerator. Using ultramicrotomy, specimens were sectioned down to 70-110 nm thicknesses. These sections were vapor stained on nickel grids using a RuO4 staining solution prepared by adding 1 mL of 10 w/v % sodium hypochlorite solution to 0.02 g of RuCl3.56 Antimicrobial Effectiveness. The effectiveness of the antimicrobial films were tested using the Kirby-Bauer test, where the LbL-coated (or bare PET) disks are used in place of antibiotic filter disks.57 The double membrane encapsulated gram negative bacterium E. coli and the single membrane gram positive bacterium S. aureus were chosen as representatives of the major pathogenic groups for these tests. Gram positive bacteria are generally more susceptible to membrane perturbing agents such as CTAB than are gram negative bacteria. Luria broth and Luria broth plates were used throughout for microbial growth. Films were deposited on PET substrates, with bare PET samples evaluated as controls. The zone of inhibition (ZOI) of PET alone was zero, indicating that it is not inhibitory. Antimicrobial properties of various films were tested under a variety of conditions that include film composition, number of bilayers, testing temperature, and age of a given film. ZOI was recorded for each condition as the average of 3 radial measurements, from the rim of the disk to the beginning of bacterial growth, with 2 disks per condition, as shown in Figure 2.

(54) Owens, D. K. J. Appl. Polym. Sci. 1975, 19, 265–271. (55) Zhang, D.; Sun, Q.; Wadsworth, L. C. Polym. Eng. Sci. 1998, 38, 965–970. (56) Brown, G. M.; Butler, J. H. Polymer 1997, 38, 3937–3945. (57) Benson, H. J. Microbiological Applications: A Laboratory Manual in General Microbiology, 3rd ed.; Wm. C. Brown Company Publishers: Duboque, IA, 1980.

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Figure 2. Result of a Kirby-Bauer test, in which PET disks covered with a given assembly are placed on a bacteria-swabbed agar plate and incubated. The resulting ring of no bacterial growth is the zone of inhibition, which is the measure of antimicrobial efficacy.

Results and Discussion Film Deposition and Microstructure. Figure 3a shows film deposition monitored using QCM. A control system (without the antimicrobial agent) is included to show the influence of CTAB on growth. With the addition of CTAB, growth proceeds at a much higher rate. Additionally, weight variation between PDDA +CTAB/PAA and the CTAB/PAA systems is minimal, suggesting that CTAB deposits to a much greater extent than PDDA. This is not surprising considering that CTAB is a much smaller molecule and likely has greater mobility in solution. Film thickness was measured using profilometry at 7, 10, 15, and 20 bilayers, as shown in Figure 3b. Additionally, ellipsometry was performed to confirm thicknesses up to 5 bilayers and is included in Supporting Information. The growth trend here confirms the trend obtained using QCM. Since the PDDA/PAA films exhibit much slower (thinner) growth, they were too thin for measurement using profilometry at less than 20 BL and were not analyzed. Again, film growth in the CTAB/PAA system was greater than PDDA+CTAB/PAA. It has been shown that the addition of salts to LbL solutions yields much thicker films.58 In this system, CTAB is a salt, increasing ionic strength and screening charges on the polymers. Similarly, when PDDA is removed from the cationic solution, the charge density decreases, and the resulting film is slightly thicker. In the absence of PDDA, rougher films with larger domain structure are generated. It is also possible that CTAB molecules, consisting of a 16-carbon tail and cationic ammonium headgroup, deposit as something resembling a lipid bilayer found in cell walls (shown schematically in Figure 1).59 This would account for the ability of singly charged CTAB to generate the charge inversion necessary to grow in the absence of a highly charged cation such as PDDA. The change in growth slope around the third bilayer is indicative of two growth modes: initially, islandic expansion followed by the typical vertical layer adsorption.60 In films with only single ingredients in each layer, film composition in weight or mole percent can be determined from QCM data since this method obtains the weight of each layer deposited. For this analysis, CTAB molecular weight was calculated without bromide because this is removed in aqueus solution. In the case of PAA, repeat unit molecular weight was used. Calculations revealed that CTAB/PAA films are 20.1 mol % ( 0.86 mol % CTAB and 79.9 mol % ( 0.86 mol % PAA. The surfaces of the films were analyzed using atomic force microscopy, as shown in Figure 4. Comparison of CTAB+ (58) Schlenoff, J. B.; Ly, H.; Li, M. J. Am. Chem. Soc. 1998, 120, 7626–7634. (59) Becker, W. M.; Kleinsmith, L. J.; Hardin, J. The World of the cell, 6th ed.; Pearson Education, Inc., publishing as Benjamin Cummings: San Fransisco, CA, 2006. (60) Ostrander, J. W.; Mamedov, A. A.; Kotov, N. A. J. Am. Chem. Soc. 2001, 123, 1101–1110.

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Figure 3. Film mass as a function of the number of layers deposited, as measured with QCM (a). Profilometry measurements are also shown to confirm the linear growth trend suggested by QCM (b).

Figure 4. AFM height images of CTAB/PAA (a) and PDDA+CTAB/PAA (b) 10BL films deposited on silicon wafers. The corresponding phase images (c,d) for each of these systems are directly beneath the height images.

PDDA/PAA and CTAB/PAA films reveals a definite structural difference. The system without the polymer in the cationic layer has a much rougher surface. The range of surface height is halved with the inclusion of PDDA. Also worth noting is that this variation in surface height seen in the CTAB/PAA system is on the order of the overall surface height seen previously using profilometry. This data suggests a higher amount of CTAB aggregation without PDDA, revealing better dispersion with the addition of the polymer to the cationic layer. Attempts to use infrared microscopy were inconclusive, but this was expected because the IR spot size of 10-15 μm does not provide high enough resolution to distinguish the regions in these films. The corresponding phase images, which are more akin to frictional contrast, do an even better job of highlighting the order induced by the presence of PDDA in the assembly (Figure 4d). Phase Langmuir 2009, 25(17), 10322–10328

contrast is much less pronounced in the surface image of the assembly without PDDA (Figure 4c), which suggests there is less physical/chemical distinction between the bumps and the regions between them. Figure 5 shows TEM cross-sections of 10 BL films deposited on polystyrene, with and without PDDA in the cationic layer. It should be noted that these images do not represent the full film thickness because of low film strength that results in fracture during sectioning. The films in these micrographs have a mottled appearance, indicating that the layers of the film intertwine and diffuse among each other rather than laying down discretely. These high levels of diffusion during film deposition suggest that CTAB will easily diffuse through the film during use, increasing antimicrobial efficacy. Additionally, the film created with PDDA in the cationic layer (Figure 5b) shows better uniformity because DOI: 10.1021/la901161z

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Figure 7. Zone of inhibition as a function of the number of bilayers of PDDA+CTAB/PAA. Error bars reflect maximium and minimum zones of inhibition. Figure 5. TEM cross-sections of CTAB/PAA (a) and PDDA +CTAB/PAA (b) 10 BL films on polystyrene.

Figure 6. Zone of inhibition for 10- and 20-bilayer films with or without PDDA in the cationic layers. Additional bilayers do not enhance PDDA+CTAB/PAA efficacy, but they do increase the efficacy of CTAB/PAA films.

the PDDA spatially separates CTAB during deposition. While this does not seem to affect initial antimicrobial activity, this may increase film longevity because diffusion out of the film involves the breakup of smaller aggregates. This affects film longevity, consequently affecting reliability, because diffusion out of the film is less sporadic. It is these nano/microstructural characteristics that influence the antimicrobial behavior of these films, as described in the next section. Antimicrobial Efficacy. All antimicrobial testing was done using the Kirby-Bauer protocol.57 Films were deposited on PET substrates, with bare PET samples evaluated as controls. In both the E. coli and S. aureus tests, the ZOI of the PET alone was zero, indicating that it is not inhibitory. Antimicrobial properties of various films were tested under a variety of conditions that include film composition, number of bilayers, testing temperature, and age of a given film. The antimicrobial effectiveness of both PDDA+CTAB/PAA and CTAB/PAA were evaluated with both 10 and 20 bilayers of deposition. At 10 BL, films with PDDA exhibited a greater ZOI (i.e., greater antimicrobial efficacy) than without. The results were similar at 20 BL, as shown in Figure 6. As was discussed in the preceding section, PDDA in the cationic layer creates improved 10326 DOI: 10.1021/la901161z

dispersion of CTAB in solution. With improved dispersion of CTAB molecules, the antimicrobial range is also increased. It is likely that the presence of PDDA allows more CTAB to diffuse out of a given assembly, effectively making the film behave as though it has more biocidal agent. This is why films show increasing efficacy beyond 10 BL of deposition (i.e., more bilayers mean more antimicrobials capable of diffusing out of the film). At this testing temperature (37 °C), the maximum ZOI observed is approximately 2.3 mm in the case of S. aureus. Increases in ancimicrobial efficacy above this point are not observed because of insufficient time for antimicrobial diffusion prior to microbial growth. It is important to see whether or not the antimicrobial agent diffuses out of merely the top bilayer or if CTAB in the lower layers of the film diffuse out as well. By testing various films with different numbers of bilayers, the ability of the bottom layers to contribute to the overall antimicrobial action was tested, as shown in Figure 7. Equal amounts of CTAB were deposited in each of the 9.5 (9 bilayers plus one extra cationic antimicrobial layer) and 10 bilayer films, and the antimicrobial effects are similar. Additionally, S. aureus ZOI levels off very quickly with number of bilayers, whereas E. coli seems to show increasing ZOI. This may be due to the fact that S. aureus is more sensitive to CTAB and therefore more quickly becomes governed by diffusion rather than release concentration. To further investigate the abilities of CTAB to diffuse through the film, a 10 BL film was constructed with no antimicrobial in the top five bilayers (5BL CTAB +PDDA/PAA followed by 5BL PDDA/PAA). These results, shown in Figure 8, demonstrate that CTAB diffuses through multiple layers. In fact, a comparison of the 5 BL film and the 10 BL film with CTAB only in the lower five bilayers shows equivalent results within error. These results show that the PAA layers do not hinder CTAB diffusion. PAA simply acts to provide charge inversion for electrostatic deposition (Figure 3). At lower temperatures, bacteria grows more slowly, allowing CTAB more time to diffuse out into the test plates during the period of bacteria proliferation. In addition to body temperature (37 °C), antimicrobial testing was performed at temperatures of 23 and 18.2 °C. At decreasing temperatures, bacterial growth rates are reduced, resulting in the larger ZOI observed with both E. coli and S. aureus, as shown in Figure 9, due to the longer time allowed for CTAB to diffuse. These data suggest that the reason these films do not experience ZOIs greater than 2.5 mm at body temperature is because the antimicrobial cannot travel further than this distance during the bacterial growth period. Once Langmuir 2009, 25(17), 10322–10328

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Figure 8. Zone of inhibition to E. coli and S. aureus for assemblies of varying composition. The ability of CTAB to diffuse through the system was evaluated by building a film with CTAB only in the lower 5 BL using a PDDA+CTAB/PAA film. Error bars reflect maximium and minimum zones of inhibition.

Figure 9. Zone of inhibition as a function of temperature for 10 BL PDDA+CTAB/PAA films. The larger ZOI at lower temperature is attributed to slower bacterial growth and longer time for CTAB diffusion. Error bars reflect maximum and minimum ZOI.

bacteria have established themselves, there will be no subsequent loss of density. Time Delay. The duration of the film efficacy was examined by storing the films in a desiccator for varying lengths of time before testing, as shown in Figure 10. These stored films showed decreased effectiveness initially, but the films maintained significant antimicrobial efficacy over the course of four weeks. It is possible that these films rearrange to some equilibrium state after deposition. While antimicrobial activity may not be lost, some CTAB molecules may complex, decreasing their ability to diffuse out. This would explain the initial decrease and eventual leveling of efficacy. It seems that these films can be stored in dry environments for long periods of time without significant loss of antimicrobial efficacy. For antimicrobial films, it is important to know how long films will remain active once in use. In this case, duration of efficacy was tested by performing these Kirby-Bauer tests over multiple days, where disks were exposed to the moist environment of an agar plate in the incubater for a varying number of days prior to exposure to bacteria. Each day, the antimicrobial disks were removed and placed onto newly swabbed plates. New, clean disks Langmuir 2009, 25(17), 10322–10328

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Figure 11. Zone of inhibition as a function of days of exposure to the Luria Broth plates (used for Kirby-Bauer testing) for 10 BL films. Both PDDA+CTAB/PAA and CTAB/PAA films were evaluated to determine how long antimicrobial release will be sustained when in use. Error bars reflect maximum and minimum ZOI.

Figure 10. Zone of inhibition as a function of storage time for 10 BL PDDA+CTAB/PAA films. Prior to testing, films were stored in a dry environment. Error bars reflect ZOI.

were exposed to bacterial growth each day, but all disks were cut from the same initial film and exposed to agar media without bacteria prior to testing. This use of clean disks for each day ensured that daily removal from the test plate would not influence the results. These 10 BL films of PDDA+CTAB/PAA released CTAB strongly over two days, as shown in Figure 11. Results from this test demonstrate a decreasing effectiveness, with loss of reliability after five days for samples with PDDA in the cationic layer and after four days for samples without. Samples with only CTAB in the cationic layer (i.e., no PDDA) show some antimicrobial action at longer times. Since PDDA acts as a dispersing agent, as seen in both TEM micrographs (Figure 5) and AFM images (Figure 4), discrepancies with films not containing PDDA may be due to a somewhat variable CTAB concentration from disk to disk (i.e., PDDA-containing films are more homogeneous and consistent).

Conclusions The introduction of antimicrobial agents into LbL films allows them to exhibit antimicrobial behavior. Additionally, this DOI: 10.1021/la901161z

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work shows that the polycation is not necessary to build the antimicrobial films, but it does improve film uniformity. The lower uniformity of films without PDDA results in decreased film homogeneity and antimicrobial efficacy. A key learning from this work was that the CTAB molecules easily diffuse through the entire LbL film thickness. Using PDDA-PAA as a base for CTAB incorporation produced the highest thickness growth rate, suggesting looser polymer packing that facilitates molecular movement through the system. Comparison of film activity at varying temperatures demonstrated higher bacterial killing abilities at lower temperatures where CTAB had longer time to diffuse out into the system before bacterial growth. These observations, in combination with longevity studies, show that the best films are effective over a 4-6 day period of activity upon continuous

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exposure to healthy bacteria. This study provides a method to improve antimicrobial efficacy and to use LbL assembly to produce films with other types of biological activity (e.g., drug delivery or enzyme stability). Acknowledgment. We acknowledge financial support for this work from the National Science Foundation Graduate Research Fellowship and Texas Engineering Experiment Station (TEES). Supporting Information Available: Film growth of three different systems of varying polyelectrolyte strength combinations. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2009, 25(17), 10322–10328