pubs.acs.org/Langmuir © 2010 American Chemical Society
Photoactivated Antimicrobial Activity of Carbon Nanotube-Porphyrin Conjugates Indrani Banerjee, Dhananjoy Mondal, Jacob Martin, and Ravi S. Kane* Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, United States Received August 18, 2010. Revised Manuscript Received September 19, 2010 We report the design of antimicrobial nanocomposite films based on conjugates of multiwalled carbon nanotubes (MWNT) and protoporphyrin IX (PPIX) that are highly effective against Staphylococcus aureus (S. aureus) upon irradiation with visible light. S. aureus infections can lead to life-threatening situations, especially when caused by antibiotic-resistant strains. While the light-activated antimicrobial activity of porphyrins against such pathogens is wellknown, a facile way to incorporate porphyrins into coatings may lead to their more effective use. To that end, we decided to synthesize and characterize MWNT-PPIX conjugates which combine the biocidal capacity of porphyrins with the mechanical strength of MWNTs. The conjugates could effectively deactivate S. aureus cells in solution upon irradiation with visible light. We also designed large area nanocomposite films comprised of the MWNT-PPIX conjugates that showed potent antimicrobial activity. These MWNT-PPIX conjugates represent a facile strategy for the design of antimicrobial and antifouling coatings.
Introduction The contamination of surfaces by disease-causing pathogens is a serious concern in hospitals,1-3 food packaging and storage,4-7 water purification systems,8 marine and industrial equipment,8-12 and textiles,13-16 among others. The emergence of antibioticresistant bacteria increases the challenge of designing effective strategies for the prevention and treatment of infectious diseases. Photoactive agents represent a promising alternative to counteract infectious diseases caused by bacteria. These light-activated antimicrobial agents generally function by generating lethal reactive oxygen species (ROS) upon exposure to light. ROS can be generated in the form of superoxide anions or hydroxyl radicals (type I) or singlet oxygen (type II).17 Unlike antibiotics, which usually have a single-target mechanism leading to biocidal action, ROS acts using a multitargeted mechanism, thereby
reducing the probability of emergence of resistance against ROS.18 Numerous light-activated antimicrobial agents have been reported, including titania and its alloys,13,19-22 methylene blue,23 toluidine blue,18,24 and porphyrins.25-27 Porphyrins have been extensively studied over the past decade for their potent biocidal capacity.25-28 Usually, porphyrins are more toxic toward Gram-positive bacteria, such as S. aureus, which have weaker cell walls compared to Gram-negative bacteria such as E. coli.15,26-29 Previous papers report that porphyrins function by producing singlet oxygen via the type II pathway.28,30 Recently, Bozja et al.15 fabricated photoactive nylon fabrics with a covalently attached porphyrin compound (protoporphyrin IX (PPIX)) that were found to be effective against S. aureus. These materials may be useful for manufacturing antimicrobial clothing, such as laboratory coats. Effective methods for incorporating porphyrins into thin coatings that are compatible with a larger variety of surfaces may lead to a more effective and widespread
*Corresponding author. E-mail:
[email protected].
(1) Donlan, R. M. Clin. Infect. Dis. 2001, 33, 1387–1392. (2) Pavithra, D.; Doble, M. Biomed. Mater. (Bristol, U. K.) 2008, 3. (3) Cole, N.; Hume, E. B. H.; Vijay, A. K.; Sankaridurg, P.; Kumar, N.; Willcox, M. D. P. Invest. Ophthalmol. Visual Sci. 2010, 51, 390–395. (4) Meyer, B. Int. Biodeterior. Biodegrad. 2003, 51, 249-253. (5) Li, X. H.; Xing, Y.; Jiang, Y. H.; Ding, Y. L.; Li, W. L. Int. J. Food Sci. Technol. 2009, 44, 2161–2168. (6) Conte, A.; Buonocore, G. G.; Bevilacqua, A.; Sinigaglia, M.; Del Nobile, M. A. J. Food Prot. 2006, 69, 866–870. (7) Kenawy, E. R.; Worley, S. D.; Broughton, R. Biomacromolecules 2007, 8, 1359–1384. (8) Asuri, P.; Karajanagi, S. S.; Kane, R. S.; Dordick, J. S. Small 2007, 3, 50–53. (9) Yebra, D. M.; Kiil, S.; Dam-Johansen, K. Prog. Org. Coat. 2004, 50, 75–104. (10) Chambers, L. D.; Stokes, K. R.; Walsh, F. C.; Wood, R. J. K. Surf. Coat. Technol. 2006, 201, 3642–3652. (11) Flemming, H. C. Appl. Microbiol. Biotechnol. 2002, 59, 629–640. (12) Almeida, E.; Diamantino, T. C.; de Sousa, O. Prog. Org. Coat. 2007, 59, 2–20. (13) Yang, C.; Liang, G. L.; Xu, K. M.; Gao, P.; Xu, B. J. Mater. Sci. 2009, 44, 1894–1901. (14) Meilert, K. T.; Laub, D.; Kiwi, J. J. Mol. Catal. A: Chem. 2005, 237, 101– 108. (15) Bozja, J.; Sherrill, J.; Michielsen, S.; Stojiljkovic, I. J. Polym. Sci., Part A: Polym. Chem. 2003, 41, 2297–2303. (16) Qi, K. H.; Daoud, W. A.; Xin, J. H.; Mak, C. L.; Tang, W. Z.; Cheung, W. P. J. Mater. Chem. 2006, 16, 4567–4574. (17) Page, K.; Wilson, M.; Parkin, I. P. J. Mater. Chem. 2009, 19, 3819–3831.
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(18) Gil-Tomas, J.; Tubby, S.; Parkin, I. P.; Narband, N.; Dekker, L.; Nair, S. P.; Wilson, M.; Street, C. J. Mater. Chem. 2007, 17, 3739–3746. (19) Matsunaga, T.; Tomoda, R.; Nakajima, T.; Wake, H. FEMS Microbiol. Lett. 1985, 29, 211–214. (20) Fujishima, A.; Zhang, X. T.; Tryk, D. A. Surf. Sci. Rep. 2008, 63, 515– 582. (21) Lin, H.; Xu, Z.; Wang, X.; Long, J.; Su, W.; Fu, X.; Lin, Q. J. Biomed. Mater. Res., Part B 2008, 87, 425–31. (22) Krishna, V.; Pumprueg, S.; Lee, S. H.; Zhao, J.; Sigmund, W.; Koopman, B.; Moudgil, B. M. Process Saf. Environ. Prot. 2005, 83, 393–397. (23) Perni, S.; Piccirillo, C.; Pratten, J.; Prokopovich, P.; Chrzanowski, W.; Parkin, I. P.; Wilson, M. Biomaterials 2009, 30, 89–93. (24) Decraene, V.; Pratten, J.; Wilson, M. Appl. Environ. Microbiol. 2006, 72, 4436–4439. (25) Stojiljkovic, I.; Evavold, B. D.; Kumar, V. Expert Opin. Invest. Drugs 2001, 10, 309–320. (26) Yu, K. G.; Li, D. H.; Zhou, C. H.; Diao, J. L. Chin. Chem. Lett. 2009, 20, 411–414. (27) Banfi, S.; Caruso, E.; Buccafurni, L.; Battini, V.; Zazzaron, S.; Barbieri, P.; Orlandi, V. J. Photochem. Photobiol., B 2006, 85, 28–38. (28) Malik, Z.; Hanania, J.; Nitzan, Y. J. Photochem. Photobiol., B 1990, 5, 281– 293. (29) Parsons, C.; McCoy, C. P.; Gorman, S. P.; Jones, D. S.; Bell, S. E. J.; Brady, C.; McGlinchey, S. M. Biomaterials 2009, 30, 597–602. (30) Cannistraro, S.; Vandevorst, A.; Jori, G. Photochem. Photobiol. 1978, 28, 257–259.
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Figure 1. Schematic representation of the synthesis of MWNT-PPIX from PPIX and acid-functionalized MWNTs.
use of porphyrins in hospitals, the food industry, and community settings. Carbon nanotubes are emerging as attractive scaffolds for the immobilization of antimicrobial agents.31,32 Nanotubes can be used to form ultrathin, flexible, and transparent films.33 Moreover, the high aspect ratio of carbon nanotubes leads to their efficient entanglement and retention within polymeric coatings.8 While photoinduced ROS generation has been reported for carbonaceous nanomaterials including carbon nanotubes,34-37 we reasoned that the efficiency could be significantly enhanced by functionalization of the nanotubes with porphyrins, molecules that are highly efficient in generating ROS. Researchers have reported covalent functionalization of single-walled carbon nanotubes (SWNTs) as well as MWNTs with porphyrins.38-41 These prior studies, however, have focused on electron/energy transfer between the carbon nanotubes and porphyrins and their applications as light harvesting materials for photonic devices and solar energy utilization.39-41 To our knowledge, the potential of such conjugates to form antimicrobial films/coatings has not been explored. In this work, we have synthesized MWNT-PPIX conjugates (Figure 1) and investigated their antimicrobial activity. The conjugates showed potent bactericidal activity against S. aureus cells in solution upon irradiation with visible light. Large area nanocomposite films composed of MWNT-PPIX conjugates were fabricated and also showed effective bactericidal activity. (31) Dinu, C. Z.; Zhu, G.; Bale, S. S.; Anand, G.; Reeder, P. J.; Sanford, K.; Whited, G.; Kane, R. S.; Dordick, J. S. Adv. Funct. Mater. 2010, 20, 392–398. (32) Nepal, D.; Balasubramanian, S.; Simonian, A. L.; Davis, V. A. Nano Lett. 2008, 8, 1896–1901. (33) Wu, Z.; Chen, Z.; Du, X.; Logan, J. M.; Sippel, J.; Nikolou, M.; Kamaras, K.; Reynolds, J. R.; Tanner, D. B.; Hebard, A. F.; Rinzler, A. G. Science 2004, 305, 1273–6. (34) Zheng, M.; Rostovtsev, V. V. J. Am. Chem. Soc. 2006, 128, 7702–7703. (35) Joshi, A.; Punyani, S.; Bale, S. S.; Yang, H. C.; Borca-Tasciuc, T.; Kane, R. S. Nature Nanotechnol. 2008, 3, 41–45. (36) Bosi, S.; Da Ros, T.; Spalluto, G.; Prato, M. Eur. J. Med. Chem. 2003, 38, 913–23. (37) Brunet, L.; Lyon, D. Y.; Hotze, E. M.; Alvarez, P. J.; Wiesner, M. R. Environ. Sci. Technol. 2009, 43, 4355–60. (38) Gudipati, C. S.; Greenlief, C. M.; Johnson, J. A.; Prayongpan, P.; Wooley, K. L. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 6193–6208. (39) Yu, J. X.; Mathew, S.; Flavel, B. S.; Johnston, M. R.; Shapter, J. G. J. Am. Chem. Soc. 2008, 130, 8788–8796. (40) Guo, Z.; Du, F.; Ren, D. M.; Chen, Y. S.; Zheng, J. Y.; Liu, Z. B.; Tian, J. G. J. Mater. Chem. 2006, 16, 3021–3030. (41) Schumacher, J. F.; Carman, M. L.; Estes, T. G.; Feinberg, A. W.; Wilson, L. H.; Callow, M. E.; Callow, J. A.; Finlay, J. A.; Brennan, A. B. Biofouling 2007, 23, 55–62.
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These MWNT-PPIX conjugates may be used to design antimicrobial and antifouling coatings in a simple and efficient way.
Experimental Methods Materials. PPIX and 1-hydroxybenzotriazole (HOBt) were purchased from Sigma Chemical Co. (St. Louis, MO). 1-[3(Dimethylamino)propyl]-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), anhydrous N,N-dimethylformamide (DMF), and dichloromethane (DCM) were purchased from Acros Chemicals (Somerville, NJ). MWNTs were purchased from Cheap Tubes, Inc. All other chemicals were obtained from commercial sources and used without further purification. 1H NMR spectra was recorded on a Varian 500 MHz spectrometer. Chemical shifts are reported in parts per million (ppm) relative to trimethylsilane (TMS) for the 1H NMR spectra. Synthesis of the tert-Butyloxy Carbamate (Boc)-Protected Ethylenediamine Derivative of PPIX (PPIX-EDBoc) (2).
PPIX (500 mg, 0.89 mmol) was dissolved in ∼50 mL of dry DMF at ambient temperature. To this solution, 300 mg of N-Bocethylenediamine (1.88 mmol) was added followed by 100 mg of NHS (0.89 mmol) and 475 mg of EDC (2.7 mmol). The solution immediately turned dark reddish-brown, and a precipitate began to form after 1 h. The mixture was gently stirred for 24 h, after which the precipitate was filtered and washed with ether and dried under vacuum for the removal of residual solvent. The small, crystalline flakes were left on the filter paper, covered with aluminum foil, and used for the next reaction without any further purification. The yield was 647 mg (86%, 0.76 mmol). Mass spectra (ESI) m/z 847.52 (M þ H)þ; 869.50 (M þ Na)þ. 1H NMR (CDCl3, 500 MHz): δ 10.25 (1 H, s), 10.19 (2 H, s), 10.11 (1 H, s), 8.30 (2 H, dd, J = 13, J = 15 Hz), 6.41 (2 H, dd, J = 18, J = 2 Hz), 6.17 (2 H, dd, J = 11, J = 2), 4.40 (6 H, m), 3.74 (3 H, s), 3.72 (3 H, s), 3.65 (3 H, s), 3.64 (3 H, s), 3.02-3.21 (8 H, m), 2.75-2.86 (8 H, m), 1.56 (18 H, s).
Synthesis of the Ethylenediamine Derivative of PPIX (PPIX-ED) (3). The tert-butyloxy carbamate groups in PPIXEDBoc derivative (2, 235 mg, 0.28 mmol) were deprotected via stirring in a mixture of trifluoroacetic acid (TFA, 4 mL) and DCM (20 mL) for 3 h at room temperature. TFA was removed under reduced pressure, followed by coevaporation with toluene three times. The paste obtained was precipitated with anhydrous diethyl ether overnight at low temperature. The precipitate was filtered out and dried under vacuum to give 3 (180 mg, 0.28 mmol). Mass spectra (ESI) m/z 647.40 (M þ H)þ; 669.37 (M þ Na)þ. 1H NMR (MeOH-d4, 500 MHz): δ 10.05-9.85 (4 H, m), 8.35-8.20 (2 H, m), 6.38 (2 H, d, J = 17 Hz), 6.27 (2 H, d, J = 10 Hz), Langmuir 2010, 26(22), 17369–17374
Banerjee et al. 4.41 (6 H, br s), 3.70-3.25 (12 H, m), 3.21-3.14 (6 H, m), 2.53 (4 H, br s). Preparation of Acid-Functionalized MWNTs. 50 mg of asreceived MWNTs was added to a mixture of 150 mL of sulfuric acid and 50 mL of nitric acid and ultrasonicated for ca. 4 h. The nanotube suspension in acid was first diluted in Milli-Q water and then filtered through a 0.8 μm polycarbonate membrane (Millipore), followed by several washes with Milli-Q water. Residual solvent was removed by lyophilization to obtain a dry powder that was used for further reactions. Preparation of MWNT-PPIX (4). A suspension of 50.0 mg of acid-functionalized MWNTs in 50 mL of thionyl chloride (SOCl2) was refluxed for 24 h under a nitrogen atmosphere using a procedure similar to that used by Li et al.42 SOCl2 was removed under vacuum, and then a solution containing 500 mg of PPIXED (0.77 mmol) in ∼80 mL of anhydrous DMF was added. After stirring at 130 °C for 3 days, the reaction mixture was diluted with ethanol and then filtered through a 0.22 μm pore-sized polytetrafluoroethylene membrane filter (Millipore). After washing with tetrahydrofuran (THF) and DCM to remove the excess PPIXED, the black solid was dried under vacuum to give MWNTPPIX (4). Characterization of the nitrogen content of the sample by elemental analysis (carried out at Atlantic Microlab Inc.) was used to confirm the attachment of PPIX to the MWNTs. UV-vis and Fluorescence Spectroscopy. The optical properties were characterized using UV-vis and fluorescence spectroscopy. The MWNT-PPIX, PPIX-ED, and acid-functionalized MWNTs were dissolved in DMF by sonication for ca. 2 min to a known concentration. The UV-vis absorption spectra were recorded using a Hitachi U2910 spectrophotometer. Fluorescence spectra were measured using a Spex Fluorolog Tau 3 spectrofluorimeter, with an excitation wavelength of 404 nm. Bacterial Culture. S. aureus (ATCC 33807) cells were grown in nutrient broth (NB, Difco) overnight at 37 °C. 200 μL of this preinoculum culture was added to 10 mL of autoclaved NB media and grown at 37 °C for 4-5 h to harvest the cells at a density of ca. 109 cells/mL. The cells were washed and resuspended in saline. Fluorescence-Based Live/Dead Assay. For the fluorescence-based assay we used a live/dead assay kit from Invitrogen which contains propidium iodide (PI) and SYTO 9. While SYTO 9 can enter through cell membranes and stain both live and dead cells, PI can only enter through damaged cell membranes. MWNTPPIX was dispersed in saline by sonication for ca. 5 min. 100 μL of the S. aureus suspension containing 109 cells/mL was incubated with and without 0.1 mg/mL MWNT-PPIX in 96-well plates with or without exposure to light for 15 min. A compact fluorescence lamp (Sunlite Co.) with an output spectrum that mimics daylight was used for light irradiation. The lamp was placed at a distance of ca. 10 cm from the 96-well plates. We also used a short pass 850 nm filter (Andover Corp.) to make sure that the cells were not exposed to infrared radiation. In addition, the 96-well plates were placed on ice baths to prevent heating of the cell solution during the irradiation. Additional control experiments were carried out by treating the cells with acid-functionalized MWNTs (without PPIX). After the experiments, the cells were stained using the protocol mentioned in the kit and imaged using a fluorescence microscope (Olympus). Both fluorescent and bright field images were obtained at three random locations and analyzed further.
Antimicrobial Tests in Solution Using a Plating Technique. The experimental protocol for light irradiation and the controls (in dark) was similar to that for the fluoresecence-based live/dead assay. Various concentrations of MWNT-PPIX and varying periods of light irradiation were used. After the experiment, the cells were diluted ca. 104-105 times in saline and spread on NB agar plates (in duplicates). The plates were incubated (42) Li, H. P.; Martin, R. B.; Harruff, B. A.; Carino, R. A.; Allard, L. F.; Sun, Y. P. Adv. Mater. 2004, 16, 896–900.
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Figure 2. UV-vis (a) and fluorescence spectra (b) of MWNTPPIX (black), PPIX-ED (dark gray), and acid-functionalized MWNT (light gray) in DMF at room temperature. overnight in the dark at 37 °C. The number of colonies grown on the plates was counted.
Preparation and Antimicrobial Tests of the Nanocomposite Films. MWNT-PPIX conjugates and acid-functionalized
MWNTs were dispersed in Milli-Q water by sonication. 400 μL of the solutions was mixed with 10-15 mL of water and vacuumfiltered through 47 mm diameter nitrocellulose membranes (0.45 μm pore size, Millipore) and dried under vacuum for ca. 2 h to remove moisture. The stock solution of S. aureus (109 cells/mL) in saline was diluted by a factor of about 5 104. 50 μL of the diluted bacterial suspension was spread on agar plates, and the films were placed on top of the NB agar such that the surface containing the conjugates came into contact with the agar. Since the agar is hydrated, the films readily come into conformal contact with the surface. The agar plate was irradiated under light in an inverted position so that the film surface containing the conjugates or acidfunctionalized MWNTs was exposed to light. After irradiation with visible light, the plates were incubated overnight at 37 °C, and the number of colonies formed in the region below the films was counted. The same experiments were performed with plates stored in the dark.
Results and Discussion Synthesis and Characterization of MWNT-PPIX Conjugates. Figure 1 summarizes the procedure used to synthesize MWNT-PPIX conjugates. In brief, PPIX was first allowed to react with mono Boc-protected ethylenediamine (ED) in the presence of EDC and NHS. The Boc protecting group was removed using TFA to give ethylenediamine-functionalized PPIX. MWNTs were acid-functionalized by treating them with a mixture of concentrated sulfuric acid and concentrated nitric acid (see Experimental Methods section for details). The acid-functionalized MWNTs were refluxed in thionyl chloride and then allowed to react with ED-functionalized PPIX to yield MWNT-PPIX conjugates (Figure 1). The MWNT-PPIX conjugates were dispersible in several solvents including water, DMF, and THF, following slight sonication for 2-5 min. Characterization by UV-vis spectroscopy (Figure 2a) further confirmed the attachment of PPIX to the MWNTs. While acid-functionalized MWNTs had an essentially featureless absorption throughout the visible range, the DOI: 10.1021/la103298e
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Figure 3. Characterization of photoinduced toxicity to S. aureus using fluorescence microscopy. (a-e) Merged fluorescence micrographs showing S. aureus cells stained with PI (red) and SYTO 9 (white). Images (a) and (b) show cells incubated with 0.1 mg/mL MWNT-PPIX for 15 min under light and in the dark, respectively. Images (c) and (d) show cells incubated with 0.04 mg/mL MWNT under light and in the dark, respectively, and image (e) shows cells irradiated with light for 15 min without any treatment with MWNT-PPIX or MWNTs. (f) Survival percentage of S. aureus from the fluorescence images when treated with MWNT-PPIX, MWNT, or left untreated in the dark (black bars) or under light for 15 min (white bars).
MWNT-PPIX conjugates showed an intense peak at ca. 409 nm and less intense peaks at higher wavelengths that are characteristic of PPIX. The slight red shift (404 to 409 nm) of the peaks seen in the conjugates as compared to PPIX may be due to the interaction of PPIX with the MWNTs,40 consistent with the formation of the conjugates. The MWNT-PPIX conjugates were further characterized by fluorescence spectroscopy (Figure 2b). As seen in Figure 2b, while the fluorescence spectrum for the MWNTPPIX conjugates was similar to that for PPIX-ED, no significant fluorescence was observed for the acid-functionalized MWNTs. These results further confirm the attachment of PPIX to the MWNTs. Fluorescence Assays Confirming Photoinduced Bacterial Cell Membrane Damage. We tested the photoinduced toxicity of MWNT-PPIX conjugates to S. aureus cells. We chose S. aureus for these experiments as a model Gram-positive bacterium because of the threat posed by the emergence of antibioticresistant strains such as methicillin-resistant S. aureus (MRSA). The bacterial cells were incubated with MWNT-PPIX conjugates and then exposed to light from a compact fluorescence lamp with an output spectrum that mimics daylight (see Experimental Methods section). A short-pass (850 nm) filter was used to prevent exposure of the samples to light at higher wavelengths. The viability of the cells was then monitored using a live/dead 17372 DOI: 10.1021/la103298e
assay kit (see Experimental Methods section) which monitors membrane damage, since ROS are known to cause damage to the cell membrane. The assay uses SYTO 9 and PI. While SYTO 9 is membrane-permeable and stains both live and dead cells, PI can only enter cells that have damaged membranes. As seen in Figure 3a, almost all of the cells treated with MWNT-PPIX conjugates and exposed to visible light for 15 min were stained red with PI, indicating damaged cell membranes. In contrast, most of the cells that were exposed to the conjugates in the dark (Figure 3b) or to acid-functionalized MWNTs in light or dark (Figure 3, c and d, respectively) or to light in the absence of conjugates (Figure 3e) were stained only with SYTO 9 (shown as white in Figure 3), indicating that there was no significant damage to their cell membranes. The survival percentage;the percentage of cells stained with only SYTO 9;for the different treatment conditions was calculated from fluorescence images and is plotted in Figure 3f. As seen in the figure, the survival percentage was only about 7% when the cells were treated with MWNT-PPIX under light. Antimicrobial Activity of MWNT-PPIX in Solution. Though cell wall damage indicates possible cell death, in some cases when bacteria are exposed to favorable growth conditions, they can recover and start multiplying.43 Hence, to test whether the conjugates are capable of causing permanent damage to the Langmuir 2010, 26(22), 17369–17374
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Figure 5. Plot showing the survival percentage of S. aureus treated with films containing acid-functionalized MWNT or MWNTPPIX in the dark (black bars) and on irradiation with light for 1 h (white bars).
S. aureus cells, we used standard plating techniques to determine the number of colony forming units (CFUs) for cells treated with MWNT-PPIX conjugates or acid-functionalized MWNTs (see Experimental Methods section for details). Figure 4a plots the survival percentage, where the CFUs after treatment are normalized relative to those for control cells stored in the dark without any exposure to MWNT-PPIX conjugates or acid-functionalized MWNTs. As seen in Figure 4a, only a few viable S. aureus cells remain following incubation with MWNTPPIX conjugates (0.1 mg/mL) and exposure to visible light for 15 min. In contrast, there was no significant decrease in the number of CFUs for cells exposed to MWNT-PPIX conjugates in the dark and for cells exposed to an equivalent amount of acidfunctionalized MWNTs or to saline solution (with and without irradiation; Figure 4a). Collectively, these results indicate the efficient photoinduced porphyrin-mediated bactericidal effect for the MWNT-PPIX conjugates on S. aureus cells. Next, we tested the effect of the concentration of the conjugates on their bactericidal efficiency following irradiation with visible light for 15 min (Figure 4b). The experiments were performed for concentrations of the conjugates ranging from 0.01 to 0.1 mg/mL. It was observed that the survival percentage was ca. 50% for concentrations of MWNT-PPIX conjugates as low as 0.04 mg/mL
and was less than 5% when the concentration was increased to 0.1 mg/mL. The effect of the time of light exposure on the survival percentage was also studied. The bacteria were treated with a 0.1 mg/mL concentration of the MWNT-PPIX conjugates and exposed to light for 5, 10, and 15 min (see Figure 4c). The survival percentage was ca. 50% after only 5 min exposure to visible light and was less than 10% after 10 min of irradiation. These results indicate that the conjugates are highly effective in deactivating S. aureus, even at low concentrations of MWNT-PPIX and short irradiation times. In contrast to the strong photoinduced bactericidal effects of MWNT-PPIX on S. aureus cells, no significant bactericidal effect was seen on E. coli cells, even after using high concentrations of the conjugates (as high as 4 mg/mL) and irradiation times as long as 2 h. In an earlier report, Bozja et al.15 reported that E. coli cells are resistant to photoinduced inactivation by PPIX. This resistance was attributed to a more resistant cell wall structure with an additional outer membrane and wider periplasmic space, present in E. coli and other Gram-negative bacteria, which acts as an extra shield protecting the bacteria against the ROS generated by porphyrins.15 Antimicrobial Activity of MWNT-PPIX Films. A major advantage of carbon nanotubes is their ability to form flexible macroscopic films using different techniques such as filtration44 or layer-by-layer assembly.32 We therefore reasoned that MWNTPPIX conjugates could be used to form highly active thin antimicrobial nanocomposite films. To that end, we used vacuum filtration to form thin MWNT-PPIX coatings on nitrocellulose filter membranes. The conjugates were first sonicated in deionized water to make a uniform suspension and then filtered through nitrocellulose membranes to yield uniform films. The bactericidal activity of the resultant films was measured by placing them in intimate contact with agar plates previously inoculated with S. aureus. On light exposure for 1 h on the plates followed by overnight incubation at 37 °C, it was observed that the number of bacterial colonies that appeared under the area covered by the films were ca. 15-20% of the number of colonies that appeared on an equivalent area which was not covered by the films (control). In contrast, no reduction in the number of colonies was seen when plates with the films were stored in the dark. Moreover, no decrease in the number of colonies was seen using coatings formed with acid-functionalized MWNTs alone (Figure 5). These results again indicate that the observed bactericidal effect is photoinduced and porphyrin-mediated. Moreover, these bactericidal effects were observed in the presence of growth media
(43) Milovic, N. M.; Wang, J.; Lewis, K.; Klibanov, A. M. Biotechnol. Bioeng. 2005, 90, 715–722.
(44) Kang, S.; Pinault, M.; Pfefferle, L. D.; Elimelech, M. Langmuir 2007, 23, 8670–8673.
Figure 4. Plots showing the survival of S. aureus cells exposed to MWNT-PPIX and acid-functionalized MWNTs in saline buffer: (a) Survival percentage of S. aureus cells when left untreated or when treated with either acid-functionalized MWNTs or MWNT-PPIX in dark (indicated by black bars) or in the presence of light (indicated by white bars) for 15 min. (b) Survival percentage of S. aureus when treated with increasing concentrations of MWNT-PPIX in the presence of light for 15 min. (c) Survival percentage of S. aureus when treated with 0.1 mg/mL MWNT-PPIX and exposed to light for 0, 1, 5, 10, and 15 min.
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which has a more complex composition than saline, suggesting that the nanocomposite films based on MWNT-PPIX conjugates may be suitable candidates for use in a practical setting.
Conclusions In summary, we have demonstrated the potent photoinduced antimicrobial effect of porphyrin-conjugated MWNTs. The conjugates are highly effective in small amounts and on short time scales. While polymers constitute an alternative scaffold for antimicrobial agents, they are susceptible to degradation by ROS.17 The MWNT-PPIX conjugates combine the mechanical strength of carbon nanotubes with the antimicrobial capacity of the porphyrins, thus providing a means to fabricate antimicrobial surfaces and coatings which can be used extensively for various
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purposes. Moreover, because of the multitarget biocidal effects of ROS, pathogens are not as likely to develop resistance against these conjugates as they are against antibiotics. The reported work should therefore contribute to efforts to produce more effective antimicrobial materials. One potential limitation of the films is that Gram-negative bacteria may have an inherent resistance to ROS damage. In future work, we will explore the ability to engineer MWNT-PPIX conjugates to overcome this potential pitfall. Acknowledgment. We acknowledge support from a NYSTAR faculty development award. Any opinions, findings, conclusions, or recommendations expressed are those of the authors and do not necessarily reflect the views of NYSTAR.
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