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Biomacromolecules 2011, 12, 6–9
Light-Activated Covalent Coating of Cotton with Bactericidal Hydrophobic Polycations Bryan B. Hsu† and Alexander M. Klibanov*,†,‡ Departments of Chemistry and Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States Received August 10, 2010; Revised Manuscript Received November 18, 2010
A methodology is developed and validated whereby a cotton fabric is impregnated with a photosensitive hydrophobic N-alkyl-polyethylenimine, followed by its covalent immobilization triggered by ultraviolet light. The resultant fabric efficiently kills on contact waterborne pathogenic bacteria E. coli and S. aureus.
Introduction Communicable diseases are a major public health threat and the leading cause of death in children worldwide.1 They contribute significantly to nosocomial infections in hospitals through contaminated staff, surfaces, and medical devices.2,3 Populations of developing nations are particularly vulnerable to pathogenic microbes because of inferior municipal infrastructures and standards of living;4 their implementation of existing technologies to combat transmission of these diseases often fails because of limited resources.5 Biocidal materials can be created by impregnating, adsorbing, or covalently attaching microbicidal agents to their surfaces.6,7 We have employed the last approach by covalently immobilizing N-alkylated poly(vinyl pyridines) and polyethylenimines (PEIs), thus making them permanently microbicidal and nonleaching;8-10 however, this procedure requires a multistep and surface-specific immobilization and may be problematic for large-scale applications. To address this issue, we also have developed an alternative procedure, whereby surfaces are noncovalently coated by hydrophobic polycations, for example, N,N-dodecyl,methylPEI.11 This “painting”, while simple, is potentially liable to environmental deterioration.12 Herein we describe the next generation microbicidal coating that combines the permanence and resilience of covalent attachment with the ease of painting. The new photosensitive hydrophobic polycationic salt can be synthesized elsewhere and then shipped to a field location for a simple coating procedure requiring no skilled personnel. Ultraviolet (UV) light was selected as the immobilization trigger because of its simplicity and the abundance of sunlight in most developing countries. As described below, a cotton fabric is simply soaked in a solution of a designed N-alkyl-PEI, followed by a UV irradiation, to result in a permanently bactericidal textile.
Materials and Methods Synthesis. Unless otherwise noted, all chemicals were from SigmaAldrich Chemical and used without further purification. All 1H NMR spectra were obtained with a Varian Inova-500 spectrometer. As shown in Scheme 1A, following drying under vacuum for 3 days, 1.6 g of branched PEI (1) (Mw ) 750 kDa) was dissolved in 7.7 mL of anhydrous N,N-dimethylformamide with ultrasonication. After stirring * Corresponding author. E-mail:
[email protected]. † Department of Chemistry. ‡ Department of Biological Engineering.
with an additional 1.8 mL of anhydrous CH2Cl2 and 120 µL of N,Ndiisopropylethylamine for 30 min at room temperature, 1.8 mL of 0.5 M trityl (Tr) chloride in anhydrous CH2Cl2 was added dropwise at -10 °C in a NaCl/ice bath under argon.13,14 This mixture was stirred and returned to room temperature over 20 h to yield a white precipitate. The volatiles were removed under vacuum, and the mixture was solubilized in CH3OH and dialyzed twice from a regenerated cellulose membrane bag (Spectrum Laboratories, 3.5 kDa MWCO) against 1 L of CH3OH at room temperature for 18 h. After solvent removal under vacuum, the yield of N-Tr-PEI (2) was 1.2 g, with 2.2% of the amino groups tritylated. 1H NMR (500 MHz, CD3OD, δ): 2.25-3.00 (br, 4H, -NCH2CH2-), 7.09 (s, 2H, ortho-Ar), 7.18 (s, 1H, para-Ar), 7.35 (s, 2H, meta-Ar). As shown in Scheme 1B, 2 was dissolved in 15 mL of tert-amyl alcohol and stirred with 4.6 g of K2CO3 and 20 mL of 1-bromododecane at 95 °C for 96 h.11,15 After cooling to room temperature and centrifugation at 6000 rpm for 30 min, the supernatant was dialyzed twice against 2 L of hexane for 18 h. The solvent was removed under vacuum, and the yield of N,N-dodecyl,Tr-PEI (3) was 6.0 g. 1H NMR (500 MHz, CD2Cl2, δ): 0.88 (t, 3H, N-CH2-(CH2)10-CH3), 1.2-1.9 (br, 20H, N-CH2-(CH2)10-CH3), 2.4-4.2 (br, 6H, N-CH2-(CH2)10-CH3 and -NCH2CH2-), 7.20 (s, 2H, ortho-Ar), 7.27 (s, 1H, para-Ar), 7.48 (s, 2H, meta-Ar). As shown in Scheme 1C, 2.0 g of 3 was dissolved in 40 mL of CH2Cl2 and stirred with 40 mL of CF3COOH for 1 h at room temperature, followed by the addition of 40 mL of CH3OH and stirring for an additional 30 min.14,16,17 The volatiles were removed under vacuum, and 100 mL of a neutralizing solution (0.5% (w/v) tetraethylammonium bromide and 5% (v/v) pyridine in CHCl3) was added and stirred for 15 min. This solution was dialyzed twice against 2 L of the neutralizing solution for 12 h and then thrice against 2 L of CHCl3 for 12 h. The yield of deprotected N-dodecylPEI (4) was 1.6 g. 1H NMR (500 MHz, CD2Cl2, δ): 0.88 (t, 3H, N-CH2-(CH2)10-CH3), 1.2-1.9 (br, 20H, N-CH2-(CH2)10-CH3), 2.4-4.2 (br, 6H, N-CH2-(CH2)10-CH3 and -NCH2CH2-). As shown in Scheme 1D, 0.75 g of 4 in 5 mL of anhydrous CH2Cl2 was stirred with 8.8 µL of N,N-diisopropylethylamine for 5 min and then combined with 5 mL of 1% (w/v) N-succinimidyl-6(4′-azido-2′-nitrophenylamino)hexanoate (SANPAH) (Thermo Scientific) in anhydrous CH2Cl2. This mixture was stirred at room temperature in the dark for 1 h before dialysis against 1 L of CH2Cl2 for 14 h, after which time the solvent was removed under vacuum. The yield of N,N-ANPAH,dodecyl-PEI (5), where ANPAH is 6-(4′azido-2′-nitrophenylamino)hexanoyl), was 0.73 g. 1H NMR (500 MHz, CD2Cl2, δ): 0.88 (t, 3H, N-CH2-(CH2)10-CH3), 1.2-1.9 (br, 20H, N-CH2-(CH2)10-CH3), 2.4-4.2 (br, 6H, N-CH2-(CH2)10-CH3 and -NCH2CH2-), 6.8-7.9 (3H, ArN3).
10.1021/bm100934c 2011 American Chemical Society Published on Web 12/03/2010
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Scheme 1. Synthetic Scheme for Making of N,N,N-ANPAH,dodecyl,methyl-PEI (6)a
a Abbreviated chemical groups are trityl (triphenylmethyl, Tr) and 6-(4′-azido-2′-nitrophenylamino)hexanoyl (ANPAH). See Materials and Methods for details.
As shown in Scheme 1E, 0.73 g of 5 was dissolved in 15 mL of anhydrous CH2Cl2 and stirred with 2 mL of CH3I at 37 °C in the dark for 24 h.11,15 The volatiles were removed under vacuum. The yield of N,N,N-ANPAH,dodecyl,methyl-PEI (6) was 0.83 g. (The physical appearance of the polymer is shown in Figure 1A.) 1H NMR (500 MHz, CD2Cl2, δ): 0.88 (t, 3H, N-CH2-(CH2)10-CH3), 1.2-1.9 (br, 20H, N-CH2-(CH2)10-CH3), 2.4-4.2 (br, 9H, N-CH2-(CH2)10-CH3, N-CH3 and -NCH2CH2-), 6.8-7.9 (3H, ArN3). Covalent Modification of Cotton. Woven undyed cotton sheets (ITW TexWipe) were cut into 2.5 × 2.5 cm pieces, ultrasonicated in isopropyl alcohol for 5 min, dried at 80 °C, and autoclaved (Figure 1C). For photocoating, the cotton pieces were dipped in a 50 mg/mL solution of 6 in CH2Cl2 (Figure 1B) for 1 min, allowed to dry in darkness, and irradiated on both sides at 365 nm with an 8 W handheld UV lamp (UVP EL series) at a 1 cm distance for 30 min. The resultant pieces of cotton were washed in CH2Cl2 and allowed to dry; then, the same procedure was repeated two more times. To maximize the removal of the unbound polymer, the 6-photocoated cotton was washed further. The resultant cotton sample gained a yellowish tint (Figure 1D). Covalent immobilization of N,N-hexyl,methyl-PEI to cotton was performed as previously described.8 Surface Characterization. The surface elemental analysis of plain cotton and 6-photocoated cotton by XPS was performed on a Kratos HS-AXIS spectrometer with an Al anode by Arkema. The quaternary ammonium groups were quantified as previously described.8 In brief, 1 cm2 samples of plain and 6-photocoated cotton were shaken in 1 mL of 1% aqueous fluorescein solution for 5 min before shaking twice with 50 mL of water for 10 min at 250 rpm. The surface-bound fluorescein was displaced with 0.9 mL of 0.25% cetyltrimethylammonium chloride at 250 rpm for 10 min. After the addition of 0.1 mL of 0.1 M phosphate buffer (pH 8.0) to the detergent solution, the absorbance was measured at 501 nm; the previously determined10 extinction coefficient of 77 000 M-1 cm-1 was used for quantification. Waterborne Bactericidal Assay. E. coli (the Coli Genetic Stock Center, CGSC4401) and S. aureus (ATCC 33807) were cultured and prepared as previously described.15 Bactericidal efficiency was determined using 2.5 × 2.5 cm pieces of cotton immersed in, and shaken with, 10 mL of a 4 × 104 cells/mL bacterial suspension in phosphate-buffered saline (PBS) at 250 rpm. Incubations with E. coli and S. aureus were performed at 37 °C for 2 h and at room temperature for 4 h, respectively. Assays with plain and 6-photocoated cotton were used to determine their respective effects on bacterial viability, whereas otherwise identical conditions without
cotton determined the initial bacterial viability. Of these, five-fold serial dilutions were plated onto yeast-dextrose broth (YDB) agar plates and grown overnight at 37 °C. The viability of bacteria adhered to plain cotton, to N,N-hexyl,methylPEI derivatized cotton (included herein as a positive control), and to 6-photocoated cotton was determined immediately postassay. Cotton samples tested against S. aureus were briefly dipped thrice in 40 mL of PBS to displace any nonadhered cells9 and incubated with 20 mL of YDB at 250 rpm and 37 °C. At 0.5 h intervals, these suspensions were briefly vortexed, and 100 µL aliquots were plated onto YDBagar plates and grown overnight at 37 °C.
Results and Discussion We selected branched PEI (1) as the starting point of our polycationic coating because of its high amine density and a 1:2:1 ratio of primary, secondary, and tertiary amino groups.18 Our previous studies6 have revealed that high microbicidal activity requires not only high positive charge but also substantial hydrophobicity (i.e., alkyl chain length).11,19 For photo cross-linking, we chose an aryl azide group with a ∼1.8 nm spacer arm because of its extension beyond proximal alkyl groups and preferential reactivity toward nucleophiles,20 such as cellulosic -OH groups. To this end, we derivatized the primary amino groups13 of 1 with a stoichiometrically small fraction of trityl chloride (Scheme 1), which yielded 2 with some 2% of the total amines protected. Subsequent alkylation of the free amino groups of 2 with 1-bromododecane yielded 3. In preparation for the photo cross-linker 6-(4′-azido-2′-nitrophenylamino)hexanoyl (ANPAH), 3 was deprotected under acidic conditions to give 4, whose primary amines were reacted with the NHS-ester precursor of ANPAH to produce 5. Positive charges were maximized by methylating the nucleophilic amino groups into quaternary ammonium groups, yielding the final photosensitive polymer 6 (Figure 1A). Plain cotton fabric was then dipped into a 50 mg/ mL solution of 6 in CH2Cl2 (Figure 1B), dried in the dark, and derivatized by exposure to UV light, followed by thorough washing with fresh CH2Cl2. Comparison of plain cotton (Figure 1C) with that 6-photocoated thrice (Figure 1D) shows the latter’s yellow color and thus qualitatively confirms the coating. For characterization of the coated surface, we used X-ray photoelectron spectroscopy (XPS). Elemental analysis of plain cotton (first row of Table 1) shows significant quantities of
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Figure 2. Waterborne bactericidal efficiencies of plain cotton (the left pair of bars) and of that photocoated with 6 (the right pair of bars) against E. coli (white bars) and S. aureus (gray bars).
Figure 1. N,N,N-ANPAH,dodecyl,methyl-PEI (6) prepared by us herein is shown as a solid (A) and as a 50 mg/mL solution in CH2Cl2 (B). Plain and 6-photocoated cotton samples are depicted in (C) and (D), respectively. See Materials and Methods for details. Table 1. Elemental Surface Compositions of Plain Cotton and of That 6-Photocoated Thrice, As Determined by Means of X-ray Photoelectron Spectroscopy (XPS) elemental composition (atomic %) sample
C
O
N
Ca
I
plain cotton 6-photocoated cotton
54.4 71.0
44.5 26.1
N.D.a22 1.8
1.1 N.D.
N.D. 1.1
a
N.D.: not detectable (i.e., < 0.5 atomic %).
Table 2. Quaternary Ammonium Group Densities of Branched N-alkyl-PEIs Covalently Immobilized onto Glass Slides and Cotton Fabric quaternary ammonium group density (nmol/cm2) alkyl group
cotton
glass
hexyl (C6) dodecyl (C12)
17.3 ( 0.4 11.0 ( 0.9c
a
6.8 ( 1.9b 4.8 ( 1.5b
a Covalently derivatized with N,N-hexyl,methyl-PEI; see ref 8. b Covalently derivatized with N,N-alkyl,methyl-PEI; see ref 19. c Covalently photocoated with 6.
carbon, oxygen, and calcium. (The Ca is likely a residual contaminant from the manufacturer’s washing procedure.21) The measured C/O ratio of 1.22 agrees well with the theoretical value of 1.20 for cellulose (C6H10O5)n. In comparison, the elemental composition of the 6-photocoated cotton (second row of Table 1) reveals substantial quantities of nitrogen and iodine as well as an over a two-fold jump in the C/O ratio to 2.72, confirming the presence of covalently immobilized 6. To quantify the polymer on the surface, we titrated the quaternary ammonium groups with fluorescein.8 Whereas the plain cotton showed only a low, nonspecific fluorescein absorption of 0.14 ( 0.02 nmol/cm2, the 6-photocoated cotton exhibits nearly a 100-fold greater value (first column, second row of Table 2). To put this value in perspective, we compared and found it to be similar to that for covalently immobilized N,Nhexyl,methyl-PEI (first row of column 1 in Table 2). Separately, previous studies19 with branched N,N-alkyl,methyl-PEI (with
alkyl being hexyl or dodecyl) covalently immobilized on glass found somewhat lower levels of N-quaternization (column 2 of Table 2). The overall lesser density of the latter on glass is likely due to cotton’s greater surface roughness. The ultimate merit of the photocoating procedure developed herein rests on how bactericidal the treated cotton fabric becomes. We addressed that issue with representative waterborne human pathogenic bacteria, E. coli and S. aureus (Gramnegative and Gram-positive, respectively) using two different tests. Under our assay conditions, 195 ( 22 and 92 ( 13 colonyforming units (CFU) were observed in plated five-fold serial dilutions of E. coli and S. aureus suspensions, respectively. Upon incubation of the bacteria with plain cotton, their titers were only slightly reduced (Figure 2), presumably due to entrapment of some bacteria within the cotton fibers. However, following the same incubation with 6-photocoated cotton, essentially no viable bacteria were detected (Figure 2). We elaborated on this finding further in another test by assessing the viability of bacteria adhered to the cotton samples. Immediately after the waterborne bactericidal assay with S. aureus, we transferred the tested cotton samples into fresh media and monitored the bacterial concentration under growth conditions. As a positive control, we included cotton derivatized with covalently immobilized N,N-hexyl,methyl-PEI, which had undergone identical bacterial treatment and showed 100% bactericidal activity against waterborne S. aureus. As seen in Figure 3, there was vigorous bacterial growth on plain cotton, indicating that the bacteria adhered to, or entrapped within, the cotton fibers remain viable. In contrast, both the N,N-hexyl,methyl-PEIderivatized and 6-photocoated cotton samples exhibited no significant concentrations of bacteria over the same 2 h period, indicating that their surfaces inactivated the cells adhered to them. These observations are consistent with our previous studies, showing that hydrophobic polycations covalently immobilized23 or “painted”11 onto solid surfaces kill bacteria on contact.
Conclusions Herein we have designed and synthesized a photosensitive N-alkyl-PEI that shares the permanence of covalently attached hydrophobic polycations but is applied as easily as painting. The imbedded polymer can be cross-linked to a cotton fabric in a simple procedure triggered by UV light with the resultant quaternization densities and, importantly, bactericidal efficien-
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Figure 3. Replicative ability of S. aureus adhered to plain cotton (white bars), N,N-hexyl,methyl-PEI-derivatized cotton (gray bars), and 6-photocoated cotton (black bars) after a waterborne bactericidal assay. See Materials and Methods for details.
cies similar to those in the previously described labor-intensive and multistep covalent attachment procedure. Acknowledgment. This work was financially supported by Vestergaard-Frandsen SA (Switzerland) and by the U.S. Army through the Institute of Soldier Nanotechnologies at the Massachusetts Institute of Technology under contract DAAD-1902-D0002 with the Army Research Office. We thank Dr. Sebastien Gouin for insightful discussions.
References and Notes (1) Bryce, J.; Boschi-Pinto, C.; Shibuya, K.; Black, R. E. WHO estimates of the causes of death in children. Lancet 2005, 365, 1147–1152. (2) Rosenthal, V. D.; Maki, D. G.; Salomao, R.; Alvarez-Moreno, C.; Mehta, Y.; Higuera, F.; Cuellar, L. E.; Arikan, O. A.; Abouqal, R.; Leblebicioglu, H. Device-associated nosocomial infections in 55 intensive care units of 8 developing countries. Ann. Intern. Med. 2006, 145, 582–591. (3) Boyce, J. M.; Potter-Bynoe, G.; Chenevert, C.; King, T. Environmental contamination due to methicillin-resistant Staphylococcus aureus: possible infection control implications. Infect. Control Hosp. Epidemiol. 1997, 18, 622–627. (4) Harris, E. Building scientific capacity in developing countries. EMBO Rep. 2004, 5, 7–11. (5) Madu, C. N. Transferring technology to developing countries - critical factors for success. Long Range Plann. 1989, 22, 115–124.
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(6) Klibanov, A. M. Permanently microbicidal materials coatings. J. Mater. Chem. 2007, 17, 2479–2482. (7) Goddard, J. M.; Hotchkiss, J. H. Polymer surface modification for the attachment of bioactive compounds. Prog. Polym. Sci. 2007, 32, 698–725. (8) Lin, J.; Qiu, S. Y.; Lewis, K.; Klibanov, A. M. Mechanism of bactericidal and fungicidal activities of textiles covalently modified with alkylated polyethylenimine. Biotechnol. Bioeng. 2003, 83, 168– 172. (9) Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M. Polymer surfaces derivatized with poly(vinyl-N-hexylpyridinium) kill airborne and waterborne bacteria. Biotechnol. Bioeng. 2002, 79, 465–471. (10) Tiller, J. C.; Liao, C. J.; Lewis, K.; Klibanov, A. M. Designing surfaces that kill bacteria on contact. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5981–5985. (11) Park, D.; Wang, J.; Klibanov, A. M. One-step, painting-like coating procedures to make surfaces highly and permanently bactericidal. Biotechnol. Prog. 2006, 22, 584–589. (12) Mukherjee, K.; Rivera, J. J.; Klibanov, A. M. Practical aspects of hydrophobic polycationic bactericidal “paints”. Appl. Biochem. Biotechnol. 2008, 151, 61–70. (13) Krakowiak, K. E.; Bradshaw, J. S. Selective protection of the primary amine functions of linear tetraamines using the trityl group. Synth. Commun. 1998, 28, 3451–3459. (14) Wuts, P. G. M.; Greene, T. W. Greene’s ProtectiVe Groups in Organic Synthesis, 4th ed.; Wiley: Hoboken, NJ, 2007. (15) Haldar, J.; Weight, A. K.; Klibanov, A. M. Preparation, application and testing of permanent antibacterial and antiviral coatings. Nature Protoc. 2007, 2, 2412–2417. (16) Canle, M.; Demirtas, I.; Maskill, H. Substituent effects upon rates of deamination and base strengths of substituted N-tritylamines. J. Chem. Soc., Perkin Trans. 2 2001, 1748–1752. (17) Theodorou, V.; Ragoussis, V.; Strongilos, A.; Zelepos, E.; Eleftheriou, A.; Dimitriou, M. A convenient method for the preparation of primary amines using tritylamine. Tetrahedron Lett. 2005, 46, 1357–1360. (18) Dick, C. R.; Ham, G. E. Characterization of polyethylenimine. J. Macromol. Sci., Chem. 1970, A-4, 1301–1314. (19) Lin, J.; Qiu, S. Y.; Lewis, K.; Klibanov, A. M. Bactericidal properties of flat surfaces and nanoparticles derivatized with alkylated polyethylenimines. Biotechnol. Prog. 2002, 18, 1082–1086. (20) Scriven, E. F. V. Azides and Nitrenes: ReactiVity and Utility; Academic Press: Orlando, FL, 1984. (21) Lyon, B. ITW Texwipe, productions manager. Personal communication, 2010. (22) Despotopoulou, M. Arekema, Inc., materials analysis manager. Personal communication, 2010. (23) Milovic, N. M.; Wang, J.; Lewis, K.; Klibanov, A. M. Immobilized N-alkylated polyethylenimine avidly kills bacteria by rupturing cell membranes with no resistance developed. Biotechnol. Bioeng. 2005, 90, 715–722.
BM100934C
