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Quaternary ammonium poly(diethylaminoethyl methacrylate) possessing antimicrobial activity Shady Farah a , Oren Aviv a,b , Natalia Laout b , Stanislav Ratner b , Nurit Beyth c , Abraham J. Domb a,∗ a Institute of Drug Research, School of Pharmacy, Faculty of Medicine, Center for Nanoscience & Nanotechnology and The Alex Grass Center for Drug Design and Synthesis, The Hebrew University of Jerusalem, Jerusalem 91120, Israel b Strauss-Water Co, R&D Laboratories, Petach Tikva, Israel c Department of Prosthodontics, Faculty of Dentistry, The Hebrew University-Hadassah, Jerusalem 91120, Israel
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Article history: Received 21 November 2014 Received in revised form 25 January 2015 Accepted 28 January 2015 Available online xxx Keywords: Diethylaminoethyl methacrylate Quaternary ammonium (QA) polymers Antimicrobial nanoparticles Antimicrobial surfaces
a b s t r a c t Quaternary ammonium (QA) methacrylate monomers and polymers were synthesized from a Nalkylation of N,N-diethylaminoethyl methacrylate (DEAEM) monomer. Linear copolymers, and for the first time reported crosslinked nanoparticles (NPs), based QA-PDEAEM were prepared by radical polymerization of the quaternized QA-DEAEM monomers with either methyl methacrylate (MMA) or a divinyl monomer. QA-PDEAEM NPs of 50–70 nm were embedded in polyethylene vinyl acetate coating. QA-polymers with N-C8 and N-C18 alkyl chains and copolymers with methyl methacrylate were prepared at different molar ratios and examined for their antimicrobial effectiveness. These coatings exhibited strong antibacterial activity against four representative Gram-positive and Gram-negative bacteria. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The growing global concern about emerging infectious diseases has greatly stimulated research on polymeric biocides [1–3]. Chemical biocides play an important role in the preservation of products as diverse as cutting fluids, crop protection agents, foods and beverages, fabrics, cosmetics, and medicines [4–6]. N,N-diethylaminoethyl methacrylate (DEAEM) is a common vinyl monomer that can be copolymerized with acrylamide, acrylic acid, vinyl-2-pyrrolidone (NVP), etc., to prepare polymers with specific functions, such as flocculants and surfactants in waste water [7,8]. Furthermore, DEAEM is widely used to modify the properties of bulk plastic materials by grafting or copolymerization [7]. Polymers containing quaternary ammonium (QA) groups in a side chain or in a main chain have a broad spectrum of antimicrobial activity, and they are effective against both Gram-positive and Gram negative bacteria as well as against viruses, fungi, and algae [1,9–12]. There have been, however, very few reports on the antimicrobial polymeric ammonium salts prepared from quaternary DEAEM monomer [7,13,14].
Cationic polymers were obtained by the quaternization of the polymer, which limits the quaternization yield due to steric and electrostatic hindrance [15–17]. This polymeric group exhibits diversify of positive activity, while modifications and quaternarization degree improvement are highly encouraged for enhancing antimicrobial properties [18–20]. In this study, QA acrylate monomers were prepared from the alkylation of DEAEM, then they were polymerized. Coatings, and for the first time reported nanoparticles (NPs), based QA-PDEAEM were prepared by homo and copolymerizaion of these QA acrylate monomers, and their antibacterial activities were evaluated against four representative bacterial strains: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Heterotrophic plate count (HPC). The study outcome provide a wide range of manipulation for developing NPs and modified polymeric coatings based QA-PDEAEM for preventing accumulation of micro-organisms onto device surfaces as well as in bioadhesives and self sterilizing surfaces. 2. Materials and methods 2.1. Materials and microorganisms
∗ Corresponding author. Tel.: +972 26757573; fax: +972 26757076. E-mail address: [email protected] (A.J. Domb).
N,N-diethylaminoethyl methacrylate (DEAEM), 2,2 -azobis(2methylpropionitrile) (AIBN), divinylbenzene (DVB), ethylene glycol
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dimethacrylate (EDGMA), methyl methacrylate (MMA), iodooctane and octyldodecyl iodide were purchased from Sigma-Aldrich (Rehovot, Israel). Sodium bicarbonate, ethanol, chloroform, ethyl acetate and methanol (HPLC grade) were purchased from J.T. Baker, Holland. All solvents and reagents were of analytical grade. Polysorbate 20 (Tween 20) and sorbitan monooleate 60 (span 60) purchased from Dexcel Pharma (Jerusalem, Israel). Polyethylene vinyl acetate 28 (28% VA) and 46 (46% VA), polyethylene methacrylic acid were purchased from DuPontTM Elvax® , Switzerland. Bacterial strains and growth conditions: clinically isolated E. coli and P. aeruginosa (Maurice and Gabriela Goldschleger School of Dental Medicine at Tel-Aviv University, Israel). S. aureus ATCC 8639 were used in this study. Heterotrophic plate count bacteriaHPC (mixed bacteria from tap water). The bacteria were cultured aerobically overnight in 5 ml of brain–heart infusion (BHI) broth (Difco, Detroit, MI), at 37 ◦ C.
2.2. Methods 2.2.1. Quaternary ammonium—Polydiethylaminoethyl methacrylate synthesis (QA-PDEAEM) QA-PDEAEM polymer were prepared using two methods, differ mainly in their steps order; monomer polymerization to form PDEAEM followed by quaternary ammonium formation (QA-DEAEM) followed by vinyl radical polymerization to form QAPDEAEM, Scheme 1. In the first route, polymerization of DEAEM was carried out at reflux for 24 h in ethanol (40 g monomers per 100 ml ethanol) with 20 mg (0.05% w/w) of recrystallized AIBN with nitrogen continuously bubbled through the reaction medium. The reaction was quenched in cold water and the PDEAEM precipitate was isolated by decantation and dried by lyophilization. Then, 10 g of the dried polymer (PDEAEM) was dissolved in ethanol (10 ml) and N-alkylation was conducted using iodooctane at 1:1.5 mole ratio (monomer to alkylation agent, 14.62 ml). Alkylation step was carried out under reflux for 24 h. Excess of NaHCO3 (1.25 equimolar, 0.065 mol, 5.7 g) was added to neutralize HI released for 3 h under the same conditions. Formed NaI salt and excess of unreacted NaHCO3 were discarded by decantation, washed with hexane and double-deionized water (DDW), and lyophilized to form smooth powder, QA-PDEAEM. In the second route, 10 g of DEAEM monomers were first reacted with iodooctane at 1:1.5 ratio for 24 h in ethanol for monomer quarter ammonium formation, QADEAEM, then the resulted QA-monomer was decanted by hexane to discard unreacted iodooctane and dried completely at 50 ◦ C. QADEAEM monomer polymerization and purification was carried out similar to route I first step, QA-PDEAEM.
2.2.2. Quaternary ammonium—polydiethylaminoethyl methacrylate nanoparticles synthesis (QA-PDEAEM NPs) Ten grams of QA-DEAEM monomers (prepared in route II, step I) were dissolved in 100 ml ethanol and 1% w/w crosslinker DVB or EDGMA was added and the reaction mixture was stirred for 10 min followed by adding 10 mg (0.1% w/w) of recrystallized AIBN with nitrogen continuously bubbled through the reaction medium under reflux conditions for 24 h with stirring at 1000 rpm, Scheme 1. The reaction mixture was quenched in cold water, and the precipitate was isolated by decantation, lyophilized and well grinded using mortar and pestle followed by multiple washings with 100 ml methanol:DDW 20:80 mixture and DDW to remove unreacted AIBN and QA-DEAEM monomers, respectively, using center flask filtration followed second lyophilization for overnight. The final resultant bulky material was ground to obtain a fine powder, QAPDEAEM NPs.
2.2.3. QA-DEAEM monomers, QA-PDEAEM polymers and NPs analysis IR spectra were recorded on a Perkin Elmer System 2000 FTIR. Particles size measured using a Zetasizer 2000 (Malvern, UK), measurements were done in double-deionized water (DDW) in triplicate. Hydrophobicity (C/N) was estimated by elemental microanalysis of nitrogen (% N), carbon (% C) and iodine (% I) using a Perkin-Elmer 2400/II CHN analyzer. Zeta-potential of QA-PDEAEM polymers and particles were measured at 1% w/v in water (pH 5.5) in triplicate using Zetasizer 2000 (Malvern, UK). For QAPDEAEM NPs SEM visualization: particles were first Au/Pd coated to thickness of about 10 nm using a sputtering deposition machine (Polarone E5100) and were imaged using High resolution scanning electron microscope (HR SEM) Sirion (FEI company) at constant acceleration voltage of 2 kV. Linear polymers were characterized for their average molecular weight by GPC water-system. Monomers and polymers structure were confirmed 1 H NMR spectra (D2 O or CDCl3 ) were obtained on a Varian 300-MHz spectrometer in 5 mm o.d. tubes. D2 O/CDCl3 -containing tetramethylsilane served as solvent and shift reference. Degree of quaternary ammonium formation was determined by 1 H NMR and elemental analysis as follow: ((Halide contentFound /Halide contentCalculated ) × 100) [21]. 2.2.4. Preparation of quaternary ammonium (QA) coatings For crosslinked QA-PDEAEM NPs different polymer coatings were applied and evaluated using polyethylene vinyl acetate (PEVA, 28% and 46% VA, DuPontTM Elvax® ) and polyethylene methacrylic acid (PEMA, DuPontTM Elvax® ) in 24 wells plate, QA-PDEAEM NPs were suspended in polymer solutions at 1% w/v in chloroform or ethyl acetate. Stable homogenous suspension with minimal particle aggregates was obtained by mixing the NPs with Tween 20 and Span 60 at 1:1 and 1:0.3 w/w, respectively, followed by 10 min probe sonication with an amplitude of 60 Hz. QA-particles suspension were sprayed over, using spray painting machine with constant air pressure of 0.4 atm and 20 cm distance from the sprayed object. Particles suspension were mixed during spraying process to avoid precipitation. Coatings with 5, 15 and 30% w/w QA-particles were prepared, while the same method were repeated without QA-particles for control coatings. For non-crosslinked QA-PDEAEM, coatings were prepared in wells by fast solvent evaporation. QA-PDEAEM was dissolved in chloroform (10 mg/ml) and each well was filled with 0.2 ml of the polymer solution. The well plate was evaporated in the fume hood for 5 min to form a stable film of polymer. 2.2.5. Antimicrobial activity analysis The materials were tested in 24 wells plate, for each well 1 ml of bacterial suspension inlet fresh prepared (Appendix I 2.2.5) was transferred into each polymeric coated well (duplicate for each type of coating) and into control’s wells, free QA coatings. Plates were incubated at 35 ◦ C temperature for 6 days in different conditions: static and dynamic modes, without/or with stirring, respectively, and then results were collected using Count of Bacterial Concentration Method (Appendix I 2.2.5). 3. Results and discussion Quaternary ammonium poly(diethylaminoethyl methacrylate) (QA-PDEAEM) was prepared either by polymerization of DEAEM monomers to form PDEAEM followed by quaternaization (Scheme 1, Route I) or monomer quaternaization (QA-DEAEM), followed by vinyl radical polymerization to form QA-PDEAEM, (Scheme 1, Route II). With the first method, polymerization of DEAEM was done at reflux for 24 h in ethanol with 0.05% w/w AIBN as an initiator. The reaction mixture was quenched in cold water to precipitate the polymer having a molecular weight of 32,000 Da.
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Scheme 1. Preparation of quaternary ammonium PDEAEM polymers and nanoparticles.
The dried polymer was N-alkylated with excess iodooctane in ethanol at reflux for 24 h, followed by neutralization with NaHCO3 . The polymer was isolated by precipitation in water, washed with hexane and DDW, and lyophilized to form fine powder of poly(Noctyl-N,N-diethyl aminoethyl methacrylate). In the second method DEAEM monomers were first reacted with excess iodooctane in refluxing ethanol for monomer quaternarization, then the resultant QA-monomer was washed with hexane to discard unreacted iodooctane. The QA monomer structure was
confirmed by FT-IR (Fig. 1) and 1 H NMR (Fig. 2B). The zeta potential was positive at 53.0 ± 4.2 mV. 1 H NMR analysis showed two hydrogen double bond peaks at 5.79 and 6.16 ppm and the absence of starting monomer peaks between 2 and 3 ppm (Fig. 2A and B). QAPDEAEM prepared by the first method possessed a low percentage quaternization as shown by peak integrals of 1 H NMR spectrum, indicating ∼36% of the nitrogen groups were quaternarized (Fig. 2C and D). These results were also confirmed by zeta potential and elemental analysis (Table 1).
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Fig. 1. FTIR spectra of diethylaminoethyl methacrylate monomer (DEAEM) and its quaternarized derivative with C8 (QA-DEAEM). The change in the nitrogen bonds is marked with arrows at 3350 cm−1 and 967 cm−1 for quaternary nitrogen. Shifts in (C H) bonds peeks were noticed at 2966 cm−1 and 2860 cm−1 due to added octyl chains. Double bond identified at 1644 cm−1 (C C) with no change.
C18 alkyl chain modifications and crosslinked NPs preparation. QA-PDEAEM was modified by partial alkylation of PDEAEM for 24 h with octadecyliodide (C18 I, 10–30% mol/mol) to increase hydrophobic properties of the polymer, followed by another alkylation step with iodooctane (C8 I, 90–70% mol/mol, respectively),
to fit reactants molar ratio of 1:1 for alkylation reagents:PDEAEM tertiary amine. Poly(QA-DEAEM) NPs were prepared by copolymerization of a QA-DEAEM monomer with 1% w/w crosslinker 1,4-divinylbenzene (DVB) or ethylene glycol dimethacrylate (EDGMA) and 0.1% w/w
Fig. 2. 1 H NMR spectra of: (A), (D2 O, 300 MHz), diethylaminoethyl methacrylate monomer (starting material). (B), (D2 O, 300 MHz), synthesized C8 quaternarized monomer (QA-DEAEM), double bond peaks at 5.79 and 6.16 ppm. Absence of starting monomer peaks between 2 and 3 ppm. (C), (CDCl3 , 300 MHz), quaternary ammonium poly(diethylaminoethyl methacrylate) (QA-PDEAEM) prepared by Method I, Scheme 1. The peak integrals indicating ∼36% of nitrogen groups were quaternarized. (D), (CDCl3 , 300 MHz). Quaternary ammonium poly(diethylaminoethyl methacrylate) (QA-PDEAEM) prepared by Method II, Scheme 1.
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Table 1 Characterization of diethylaminoethyl methacrylate (DEAEM) materials. Material
Specification
Elemental analysisa %C
%H
%N
%I
DEAEM monomer* QA-DEAEM monomer** QA-PDEAEM*** QA-PDEAEM*** QA-PDEAEM particles crosslinked with 1% DVB**** QA-PDEAEM particles crosslinked with 1% EDGMA****
Starting material Route II, step 1 Route I Route II Route II Route II
63.64 46.98 37.17 50.80 53.05 52.17
9.87 8.74 5.64 8.62 9.32 9.09
7.45 3.68 2.40 3.29 3.75 3.72
0.46 32.70 10.88 29.93 19.23 24.84
* ** *** **** a b c
Zeta potential (mV)b
Particles size (nm)c
– 53.0 ± 18.4 ± 56.7 ± 29.3 ± 37.4 ±
– – – – 50.8 ± 8.6 70.4 ± 6.3
4.2 4.5 2.1 3.8 0.2
DEAEM—diethylaminoethyl methacrylate monomer. QA-DEAEM—octyl quaternarized derivative of diethylaminoethyl methacrylate monomer. QA-PDEAEM—uncrosslinked polymers. Crosslinked nanoparticles (NPs) were prepared by polymerization of QA-DEAEM monomer with DVB or EGDMA. The standard deviation of the results is in the range of ±0.3%. Zeta potential of DEAEM polymers and particles. Measured using 1% of particles suspended in water (pH 5.5) result are average of three measurements. Particles size (diameter) of QA-PDEAEM nanoparticles (NPs) as found by Zetasizer DLS, Malvern.
Fig. 3. Spherical crosslinked nanoparticles (NPs) of quaternary ammonium polydiethylaminoethyl methacrylate (QA-PDEAEM). (A) 1% w/w ethylene glycoldimethacrylate (EDGMA) crosslinked particles, magnification 200,000×, scale bar 500 nm. (B) 1% 1,4-divinylbenzene (DVB) crosslinked particles, magnification 240,000×, scale bar 500 nm. Particles were first gold coated using a sputtering deposition machine and then imaged by ultra high resolution SEM (Magellan 400 L) at constant acceleration voltage of 2 kV.
AIBN in refluxing for 24 h (Scheme 1). The reaction mixture was quenched in cold water, and the precipitate was isolated by decantation, lyophilized and well grinded followed by multiple washings to remove unreacted AIBN/QA-DEAEM monomers and followed second lyophilization for overnight. The final resultant bulky material was ground to obtain a fine powder of particle size 50.8 ± 8.6 nm for DVB crosslinking. Particles were prepared using
EDGMA as a crosslinker and were identified with a slightly larger particle size of 70.4 ± 6.3 nm (Fig. 3). Both particles were identified with a positive charge as found by zeta potential (Table 1). The antimicrobial activity of QA-PDEAEM polymers and QAPDEAEM NPs was performed against four representative strains for Gram-positive and Gram-negative bacteria in static and dynamic modes. Coated wells for antimicrobial tests were prepared by
HPC E.Coli P.Aeruginosa
1.00E+09
S.Aureus
1.00E+08 1.00E+07 CFU/ml
1.00E+06 1.00E+05 1.00E+04
S.Aureus
1.00E+03
P.Aeruginosa
1.00E+02
E.Coli
1.00E+01
HPC
1.00E+00 Inlet
Control
QA-PDEAEM QA-PDEAEM 100% C8 70%:30% C8:C18
QA-PDEAEM Coatings
Fig. 4. Antimicrobial activity of uncrosslinked QA-PDEAEM coatings of 1 layer with 100% C8 alkyl chains or modified 70:30 C8 :C18 prepared by Rout I, Scheme 1. Activity was determined at static conditions against Heterotrophic plate count (HPC), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) bacteria. Analysis was done in triplicate in two 24 wells plates. Wells without polymeric coating served as control.
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HPC E.Coli P.Aeruginosa 1.00E+06
S.Aureus
1.00E+05 CFU/ml 1.00E+04
1.00E+03 S.Aureus
1.00E+02
P.Aeruginosa E.Coli
1.00E+01
HPC
1.00E+00 Inlet
Control
5% QAPDEAEM
15% QAPDEAEM
30% QAPDEAEM
%Loading of QA-PDEAEM Nanoparticles
Fig. 5. Antimicrobial activity of polyethylene vinyl acetate coatings embedded with QA-PDEAEM nanoparticles (NPs) at different % loadings. NPs prepared by 1% crosslinker of EDGMA. Activity was analyzed at static conditions against Heterotrophic plate count (HPC), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) bacteria. Analysis was done in triplicate in two 24 wells plates. Wells with polyethylene vinyl acetate coatings free QA-PDEAEM NPs served as control.
adding particles suspended in polyethylene vinyl acetate at different particle % loading by mixing the QA-PDEAEM particles with polysorbate 20 (Tween 20) and sorbitan monooleate 60 (Span 60) at 1:1 and 1:0.3 w/w, respectively, followed by 10 min probe sonication with an amplitude of 60 Hz. Particles crosslinked with EDGMA were selected for antimicrobial testing due to their higher positive charge. For uncrosslinked QA-PDEAEM, coatings were prepared in wells by fast solvent evaporation followed by antimicrobial activity testing. For the static mode, the QA-PDEAEM coatings showed high antimicrobial activity against the four strains for both the C8 and the 30% C18 modified polymers (Fig. 4). Coatings containing 5% particles exhibited antimicrobial activity with activity increasing as the particle content increased to 15% w/v (Fig. 5). Antimicrobial analysis in dynamic mode resulted in no noticeable change in resultant activity (data not shown). Solubility and copolymerization. Insolubility of PDEAEM polymer coatings is essential for self-sterilization surface applications; otherwise, the coating is washed out by water. The solubility of QAPDEAEM polymers in water of increased pH (pH = 7) was 18 mg/ml at 100% C8 and 8 mg/ml for the 70:30 C8 :C18 . Copolymerization of QA-DEAEM with the hydrophobic monomer MMA at 10%-30% w/w content resulted in insoluble polymers, and no change in the resultant activity (data not shown). 4. Conclusions Soluble polymers and crosslinked NPs containing QA-DEAEM were prepared and tested for their antimicrobial activity. Coatings of copolymers containing QA-DEAEM units possessed high antimicrobial activity against four representative strains of bacteria. Coating of polyethylene vinyl acetate loaded with 5% or higher QA-PDEAEM crosslinked NPs displayed antimicrobial activity. Copolymerization with MMA or exchanging 30% of the C8 with C18 did not affect the antimicrobial activity, however this did decrease their water solubility (pH 7); insoluble polymers were found for MMA copolymerization. These coatings may have applications in
coating of water containers to prevent bacterial growth and as an additive for making self-sterilizing plastics and bioadhesives. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015. 01.051. References [1] S. Farah, W. Khan, I. Farber, D. Kesler-Shvero, N. Beyth, E. Weiss, A.J. Domb, Polym. Adv. Technol. 24 (2013) 446. [2] E.-R. Kenawy, F.I. Abdel-Hay, A. Abou EL-Magd, Y. Mahmoud, J. Bioact. Compat. Polym. 20 (2005) 95. [3] G.L. Woo, M.L. Yang, H.Q. Yin, F. Jaffer, M.W. Mitelman, J.P. Santerre, J. Biomed. Mater. Res. 59 (2002) 35. [4] P.L. Taylor, A.L. Ussher, R.E. Burrll, Biomaterials 26 (2005) 7221. [5] Y. Sun, T. Chen, S.D. Worley, G. Sun, J. Polym. Sci. A: Polym. Chem. 39 (2001) 3073. [6] L. Massi, F. Guittard, S. Geribaldi, R. Levy, Y. Duccini, Int. J. Antimicrob. Agents 21 (2003) 20. [7] L. Guiqian, W. Dingcai, F. Ruowen, React. Funct. Polym. 67 (2007) 355. [8] H. Parviainen, M. Hiltunen, S.L. Maunu, J. Appl. Polym. Sci. (2014), http://dx.doi.org/10.1002/APP.40448. [9] S. Borman, Sci. Technol. 79 (2001) 13. [10] S. Farah, O. Aviv, N. Laout, S. Ratner, N. Beyth, A.J. Domb, Polym. Adv. Technol. 25 (2014) 689. [11] B. Gao, X. Zhang, Y. Zhu, J. Biomater. Sci. Polym. Ed. 18 (2007) 531. [12] J. Lin, S. Qiu, K. Lewis, A.M. Klibanov, Biotechnol. Bioeng. 83 (2003) 168. [13] M. Makhloufia, T. Benaissa, A. Derdour, D.E. Henni, E-J. Chem. 7 (2010) S61. [14] Y.H. Xiao, J.H. Chen, M. Fang, X.D. Xing, H. Wang, Y.J. Wang, F. Li, J. Oral Sci. 3 (2008) 323. [15] G. Sauvet, S. Dupond, K. Kazmierski, J. Chojnowski, J. Appl. Polym. Sci. 75 (2000) 1005. [16] E.-R. Kenawy, F.I. Abdel-Hay, A.E.-R.R. El-Shanshoury, M.H. El-Newehy, J. Polym. Sci. A: Polym. Chem. 40 (2002) 2384. [17] E.-R. Kenawy, Y.A.-G. Mahmoud, Macromol. Biosci. 3 (2003) 107. [18] A. Munoz-Bonilla, M. Fernández-García, Prog. Polym. Sci. 37 (2012) 281. [19] J. Hasan, R. Crawford, E. Ivanova, Trends Biotechnol. 31 (2013) 295. [20] L. Timofeeva, N. Kleshcheva, Appl. Microbiol. Biotechnol. 89 (2011) 475. [21] I. Yudovin-Farber, N. Beyth, E.I. Weiss, A.J. Domb, J. Nanopart. Res. 12 (2010) 591.
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Short communication
Quaternary ammonium poly(diethylaminoethyl methacrylate) possessing antimicrobial activity Shady Farah a , Oren Aviv a,b , Natalia Laout b , Stanislav Ratner b , Nurit Beyth c , Abraham J. Domb a,∗ a Institute of Drug Research, School of Pharmacy, Faculty of Medicine, Center for Nanoscience & Nanotechnology and The Alex Grass Center for Drug Design and Synthesis, The Hebrew University of Jerusalem, Jerusalem 91120, Israel b Strauss-Water Co, R&D Laboratories, Petach Tikva, Israel c Department of Prosthodontics, Faculty of Dentistry, The Hebrew University-Hadassah, Jerusalem 91120, Israel
a r t i c l e
i n f o
Article history: Received 21 November 2014 Received in revised form 25 January 2015 Accepted 28 January 2015 Available online xxx Keywords: Diethylaminoethyl methacrylate Quaternary ammonium (QA) polymers Antimicrobial nanoparticles Antimicrobial surfaces
a b s t r a c t Quaternary ammonium (QA) methacrylate monomers and polymers were synthesized from a Nalkylation of N,N-diethylaminoethyl methacrylate (DEAEM) monomer. Linear copolymers, and for the first time reported crosslinked nanoparticles (NPs), based QA-PDEAEM were prepared by radical polymerization of the quaternized QA-DEAEM monomers with either methyl methacrylate (MMA) or a divinyl monomer. QA-PDEAEM NPs of 50–70 nm were embedded in polyethylene vinyl acetate coating. QA-polymers with N-C8 and N-C18 alkyl chains and copolymers with methyl methacrylate were prepared at different molar ratios and examined for their antimicrobial effectiveness. These coatings exhibited strong antibacterial activity against four representative Gram-positive and Gram-negative bacteria. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The growing global concern about emerging infectious diseases has greatly stimulated research on polymeric biocides [1–3]. Chemical biocides play an important role in the preservation of products as diverse as cutting fluids, crop protection agents, foods and beverages, fabrics, cosmetics, and medicines [4–6]. N,N-diethylaminoethyl methacrylate (DEAEM) is a common vinyl monomer that can be copolymerized with acrylamide, acrylic acid, vinyl-2-pyrrolidone (NVP), etc., to prepare polymers with specific functions, such as flocculants and surfactants in waste water [7,8]. Furthermore, DEAEM is widely used to modify the properties of bulk plastic materials by grafting or copolymerization [7]. Polymers containing quaternary ammonium (QA) groups in a side chain or in a main chain have a broad spectrum of antimicrobial activity, and they are effective against both Gram-positive and Gram negative bacteria as well as against viruses, fungi, and algae [1,9–12]. There have been, however, very few reports on the antimicrobial polymeric ammonium salts prepared from quaternary DEAEM monomer [7,13,14].
Cationic polymers were obtained by the quaternization of the polymer, which limits the quaternization yield due to steric and electrostatic hindrance [15–17]. This polymeric group exhibits diversify of positive activity, while modifications and quaternarization degree improvement are highly encouraged for enhancing antimicrobial properties [18–20]. In this study, QA acrylate monomers were prepared from the alkylation of DEAEM, then they were polymerized. Coatings, and for the first time reported nanoparticles (NPs), based QA-PDEAEM were prepared by homo and copolymerizaion of these QA acrylate monomers, and their antibacterial activities were evaluated against four representative bacterial strains: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Heterotrophic plate count (HPC). The study outcome provide a wide range of manipulation for developing NPs and modified polymeric coatings based QA-PDEAEM for preventing accumulation of micro-organisms onto device surfaces as well as in bioadhesives and self sterilizing surfaces. 2. Materials and methods 2.1. Materials and microorganisms
∗ Corresponding author. Tel.: +972 26757573; fax: +972 26757076. E-mail address: [email protected] (A.J. Domb).
N,N-diethylaminoethyl methacrylate (DEAEM), 2,2 -azobis(2methylpropionitrile) (AIBN), divinylbenzene (DVB), ethylene glycol
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dimethacrylate (EDGMA), methyl methacrylate (MMA), iodooctane and octyldodecyl iodide were purchased from Sigma-Aldrich (Rehovot, Israel). Sodium bicarbonate, ethanol, chloroform, ethyl acetate and methanol (HPLC grade) were purchased from J.T. Baker, Holland. All solvents and reagents were of analytical grade. Polysorbate 20 (Tween 20) and sorbitan monooleate 60 (span 60) purchased from Dexcel Pharma (Jerusalem, Israel). Polyethylene vinyl acetate 28 (28% VA) and 46 (46% VA), polyethylene methacrylic acid were purchased from DuPontTM Elvax® , Switzerland. Bacterial strains and growth conditions: clinically isolated E. coli and P. aeruginosa (Maurice and Gabriela Goldschleger School of Dental Medicine at Tel-Aviv University, Israel). S. aureus ATCC 8639 were used in this study. Heterotrophic plate count bacteriaHPC (mixed bacteria from tap water). The bacteria were cultured aerobically overnight in 5 ml of brain–heart infusion (BHI) broth (Difco, Detroit, MI), at 37 ◦ C.
2.2. Methods 2.2.1. Quaternary ammonium—Polydiethylaminoethyl methacrylate synthesis (QA-PDEAEM) QA-PDEAEM polymer were prepared using two methods, differ mainly in their steps order; monomer polymerization to form PDEAEM followed by quaternary ammonium formation (QA-DEAEM) followed by vinyl radical polymerization to form QAPDEAEM, Scheme 1. In the first route, polymerization of DEAEM was carried out at reflux for 24 h in ethanol (40 g monomers per 100 ml ethanol) with 20 mg (0.05% w/w) of recrystallized AIBN with nitrogen continuously bubbled through the reaction medium. The reaction was quenched in cold water and the PDEAEM precipitate was isolated by decantation and dried by lyophilization. Then, 10 g of the dried polymer (PDEAEM) was dissolved in ethanol (10 ml) and N-alkylation was conducted using iodooctane at 1:1.5 mole ratio (monomer to alkylation agent, 14.62 ml). Alkylation step was carried out under reflux for 24 h. Excess of NaHCO3 (1.25 equimolar, 0.065 mol, 5.7 g) was added to neutralize HI released for 3 h under the same conditions. Formed NaI salt and excess of unreacted NaHCO3 were discarded by decantation, washed with hexane and double-deionized water (DDW), and lyophilized to form smooth powder, QA-PDEAEM. In the second route, 10 g of DEAEM monomers were first reacted with iodooctane at 1:1.5 ratio for 24 h in ethanol for monomer quarter ammonium formation, QADEAEM, then the resulted QA-monomer was decanted by hexane to discard unreacted iodooctane and dried completely at 50 ◦ C. QADEAEM monomer polymerization and purification was carried out similar to route I first step, QA-PDEAEM.
2.2.2. Quaternary ammonium—polydiethylaminoethyl methacrylate nanoparticles synthesis (QA-PDEAEM NPs) Ten grams of QA-DEAEM monomers (prepared in route II, step I) were dissolved in 100 ml ethanol and 1% w/w crosslinker DVB or EDGMA was added and the reaction mixture was stirred for 10 min followed by adding 10 mg (0.1% w/w) of recrystallized AIBN with nitrogen continuously bubbled through the reaction medium under reflux conditions for 24 h with stirring at 1000 rpm, Scheme 1. The reaction mixture was quenched in cold water, and the precipitate was isolated by decantation, lyophilized and well grinded using mortar and pestle followed by multiple washings with 100 ml methanol:DDW 20:80 mixture and DDW to remove unreacted AIBN and QA-DEAEM monomers, respectively, using center flask filtration followed second lyophilization for overnight. The final resultant bulky material was ground to obtain a fine powder, QAPDEAEM NPs.
2.2.3. QA-DEAEM monomers, QA-PDEAEM polymers and NPs analysis IR spectra were recorded on a Perkin Elmer System 2000 FTIR. Particles size measured using a Zetasizer 2000 (Malvern, UK), measurements were done in double-deionized water (DDW) in triplicate. Hydrophobicity (C/N) was estimated by elemental microanalysis of nitrogen (% N), carbon (% C) and iodine (% I) using a Perkin-Elmer 2400/II CHN analyzer. Zeta-potential of QA-PDEAEM polymers and particles were measured at 1% w/v in water (pH 5.5) in triplicate using Zetasizer 2000 (Malvern, UK). For QAPDEAEM NPs SEM visualization: particles were first Au/Pd coated to thickness of about 10 nm using a sputtering deposition machine (Polarone E5100) and were imaged using High resolution scanning electron microscope (HR SEM) Sirion (FEI company) at constant acceleration voltage of 2 kV. Linear polymers were characterized for their average molecular weight by GPC water-system. Monomers and polymers structure were confirmed 1 H NMR spectra (D2 O or CDCl3 ) were obtained on a Varian 300-MHz spectrometer in 5 mm o.d. tubes. D2 O/CDCl3 -containing tetramethylsilane served as solvent and shift reference. Degree of quaternary ammonium formation was determined by 1 H NMR and elemental analysis as follow: ((Halide contentFound /Halide contentCalculated ) × 100) [21]. 2.2.4. Preparation of quaternary ammonium (QA) coatings For crosslinked QA-PDEAEM NPs different polymer coatings were applied and evaluated using polyethylene vinyl acetate (PEVA, 28% and 46% VA, DuPontTM Elvax® ) and polyethylene methacrylic acid (PEMA, DuPontTM Elvax® ) in 24 wells plate, QA-PDEAEM NPs were suspended in polymer solutions at 1% w/v in chloroform or ethyl acetate. Stable homogenous suspension with minimal particle aggregates was obtained by mixing the NPs with Tween 20 and Span 60 at 1:1 and 1:0.3 w/w, respectively, followed by 10 min probe sonication with an amplitude of 60 Hz. QA-particles suspension were sprayed over, using spray painting machine with constant air pressure of 0.4 atm and 20 cm distance from the sprayed object. Particles suspension were mixed during spraying process to avoid precipitation. Coatings with 5, 15 and 30% w/w QA-particles were prepared, while the same method were repeated without QA-particles for control coatings. For non-crosslinked QA-PDEAEM, coatings were prepared in wells by fast solvent evaporation. QA-PDEAEM was dissolved in chloroform (10 mg/ml) and each well was filled with 0.2 ml of the polymer solution. The well plate was evaporated in the fume hood for 5 min to form a stable film of polymer. 2.2.5. Antimicrobial activity analysis The materials were tested in 24 wells plate, for each well 1 ml of bacterial suspension inlet fresh prepared (Appendix I 2.2.5) was transferred into each polymeric coated well (duplicate for each type of coating) and into control’s wells, free QA coatings. Plates were incubated at 35 ◦ C temperature for 6 days in different conditions: static and dynamic modes, without/or with stirring, respectively, and then results were collected using Count of Bacterial Concentration Method (Appendix I 2.2.5). 3. Results and discussion Quaternary ammonium poly(diethylaminoethyl methacrylate) (QA-PDEAEM) was prepared either by polymerization of DEAEM monomers to form PDEAEM followed by quaternaization (Scheme 1, Route I) or monomer quaternaization (QA-DEAEM), followed by vinyl radical polymerization to form QA-PDEAEM, (Scheme 1, Route II). With the first method, polymerization of DEAEM was done at reflux for 24 h in ethanol with 0.05% w/w AIBN as an initiator. The reaction mixture was quenched in cold water to precipitate the polymer having a molecular weight of 32,000 Da.
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Scheme 1. Preparation of quaternary ammonium PDEAEM polymers and nanoparticles.
The dried polymer was N-alkylated with excess iodooctane in ethanol at reflux for 24 h, followed by neutralization with NaHCO3 . The polymer was isolated by precipitation in water, washed with hexane and DDW, and lyophilized to form fine powder of poly(Noctyl-N,N-diethyl aminoethyl methacrylate). In the second method DEAEM monomers were first reacted with excess iodooctane in refluxing ethanol for monomer quaternarization, then the resultant QA-monomer was washed with hexane to discard unreacted iodooctane. The QA monomer structure was
confirmed by FT-IR (Fig. 1) and 1 H NMR (Fig. 2B). The zeta potential was positive at 53.0 ± 4.2 mV. 1 H NMR analysis showed two hydrogen double bond peaks at 5.79 and 6.16 ppm and the absence of starting monomer peaks between 2 and 3 ppm (Fig. 2A and B). QAPDEAEM prepared by the first method possessed a low percentage quaternization as shown by peak integrals of 1 H NMR spectrum, indicating ∼36% of the nitrogen groups were quaternarized (Fig. 2C and D). These results were also confirmed by zeta potential and elemental analysis (Table 1).
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Fig. 1. FTIR spectra of diethylaminoethyl methacrylate monomer (DEAEM) and its quaternarized derivative with C8 (QA-DEAEM). The change in the nitrogen bonds is marked with arrows at 3350 cm−1 and 967 cm−1 for quaternary nitrogen. Shifts in (C H) bonds peeks were noticed at 2966 cm−1 and 2860 cm−1 due to added octyl chains. Double bond identified at 1644 cm−1 (C C) with no change.
C18 alkyl chain modifications and crosslinked NPs preparation. QA-PDEAEM was modified by partial alkylation of PDEAEM for 24 h with octadecyliodide (C18 I, 10–30% mol/mol) to increase hydrophobic properties of the polymer, followed by another alkylation step with iodooctane (C8 I, 90–70% mol/mol, respectively),
to fit reactants molar ratio of 1:1 for alkylation reagents:PDEAEM tertiary amine. Poly(QA-DEAEM) NPs were prepared by copolymerization of a QA-DEAEM monomer with 1% w/w crosslinker 1,4-divinylbenzene (DVB) or ethylene glycol dimethacrylate (EDGMA) and 0.1% w/w
Fig. 2. 1 H NMR spectra of: (A), (D2 O, 300 MHz), diethylaminoethyl methacrylate monomer (starting material). (B), (D2 O, 300 MHz), synthesized C8 quaternarized monomer (QA-DEAEM), double bond peaks at 5.79 and 6.16 ppm. Absence of starting monomer peaks between 2 and 3 ppm. (C), (CDCl3 , 300 MHz), quaternary ammonium poly(diethylaminoethyl methacrylate) (QA-PDEAEM) prepared by Method I, Scheme 1. The peak integrals indicating ∼36% of nitrogen groups were quaternarized. (D), (CDCl3 , 300 MHz). Quaternary ammonium poly(diethylaminoethyl methacrylate) (QA-PDEAEM) prepared by Method II, Scheme 1.
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Table 1 Characterization of diethylaminoethyl methacrylate (DEAEM) materials. Material
Specification
Elemental analysisa %C
%H
%N
%I
DEAEM monomer* QA-DEAEM monomer** QA-PDEAEM*** QA-PDEAEM*** QA-PDEAEM particles crosslinked with 1% DVB**** QA-PDEAEM particles crosslinked with 1% EDGMA****
Starting material Route II, step 1 Route I Route II Route II Route II
63.64 46.98 37.17 50.80 53.05 52.17
9.87 8.74 5.64 8.62 9.32 9.09
7.45 3.68 2.40 3.29 3.75 3.72
0.46 32.70 10.88 29.93 19.23 24.84
* ** *** **** a b c
Zeta potential (mV)b
Particles size (nm)c
– 53.0 ± 18.4 ± 56.7 ± 29.3 ± 37.4 ±
– – – – 50.8 ± 8.6 70.4 ± 6.3
4.2 4.5 2.1 3.8 0.2
DEAEM—diethylaminoethyl methacrylate monomer. QA-DEAEM—octyl quaternarized derivative of diethylaminoethyl methacrylate monomer. QA-PDEAEM—uncrosslinked polymers. Crosslinked nanoparticles (NPs) were prepared by polymerization of QA-DEAEM monomer with DVB or EGDMA. The standard deviation of the results is in the range of ±0.3%. Zeta potential of DEAEM polymers and particles. Measured using 1% of particles suspended in water (pH 5.5) result are average of three measurements. Particles size (diameter) of QA-PDEAEM nanoparticles (NPs) as found by Zetasizer DLS, Malvern.
Fig. 3. Spherical crosslinked nanoparticles (NPs) of quaternary ammonium polydiethylaminoethyl methacrylate (QA-PDEAEM). (A) 1% w/w ethylene glycoldimethacrylate (EDGMA) crosslinked particles, magnification 200,000×, scale bar 500 nm. (B) 1% 1,4-divinylbenzene (DVB) crosslinked particles, magnification 240,000×, scale bar 500 nm. Particles were first gold coated using a sputtering deposition machine and then imaged by ultra high resolution SEM (Magellan 400 L) at constant acceleration voltage of 2 kV.
AIBN in refluxing for 24 h (Scheme 1). The reaction mixture was quenched in cold water, and the precipitate was isolated by decantation, lyophilized and well grinded followed by multiple washings to remove unreacted AIBN/QA-DEAEM monomers and followed second lyophilization for overnight. The final resultant bulky material was ground to obtain a fine powder of particle size 50.8 ± 8.6 nm for DVB crosslinking. Particles were prepared using
EDGMA as a crosslinker and were identified with a slightly larger particle size of 70.4 ± 6.3 nm (Fig. 3). Both particles were identified with a positive charge as found by zeta potential (Table 1). The antimicrobial activity of QA-PDEAEM polymers and QAPDEAEM NPs was performed against four representative strains for Gram-positive and Gram-negative bacteria in static and dynamic modes. Coated wells for antimicrobial tests were prepared by
HPC E.Coli P.Aeruginosa
1.00E+09
S.Aureus
1.00E+08 1.00E+07 CFU/ml
1.00E+06 1.00E+05 1.00E+04
S.Aureus
1.00E+03
P.Aeruginosa
1.00E+02
E.Coli
1.00E+01
HPC
1.00E+00 Inlet
Control
QA-PDEAEM QA-PDEAEM 100% C8 70%:30% C8:C18
QA-PDEAEM Coatings
Fig. 4. Antimicrobial activity of uncrosslinked QA-PDEAEM coatings of 1 layer with 100% C8 alkyl chains or modified 70:30 C8 :C18 prepared by Rout I, Scheme 1. Activity was determined at static conditions against Heterotrophic plate count (HPC), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) bacteria. Analysis was done in triplicate in two 24 wells plates. Wells without polymeric coating served as control.
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HPC E.Coli P.Aeruginosa 1.00E+06
S.Aureus
1.00E+05 CFU/ml 1.00E+04
1.00E+03 S.Aureus
1.00E+02
P.Aeruginosa E.Coli
1.00E+01
HPC
1.00E+00 Inlet
Control
5% QAPDEAEM
15% QAPDEAEM
30% QAPDEAEM
%Loading of QA-PDEAEM Nanoparticles
Fig. 5. Antimicrobial activity of polyethylene vinyl acetate coatings embedded with QA-PDEAEM nanoparticles (NPs) at different % loadings. NPs prepared by 1% crosslinker of EDGMA. Activity was analyzed at static conditions against Heterotrophic plate count (HPC), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) bacteria. Analysis was done in triplicate in two 24 wells plates. Wells with polyethylene vinyl acetate coatings free QA-PDEAEM NPs served as control.
adding particles suspended in polyethylene vinyl acetate at different particle % loading by mixing the QA-PDEAEM particles with polysorbate 20 (Tween 20) and sorbitan monooleate 60 (Span 60) at 1:1 and 1:0.3 w/w, respectively, followed by 10 min probe sonication with an amplitude of 60 Hz. Particles crosslinked with EDGMA were selected for antimicrobial testing due to their higher positive charge. For uncrosslinked QA-PDEAEM, coatings were prepared in wells by fast solvent evaporation followed by antimicrobial activity testing. For the static mode, the QA-PDEAEM coatings showed high antimicrobial activity against the four strains for both the C8 and the 30% C18 modified polymers (Fig. 4). Coatings containing 5% particles exhibited antimicrobial activity with activity increasing as the particle content increased to 15% w/v (Fig. 5). Antimicrobial analysis in dynamic mode resulted in no noticeable change in resultant activity (data not shown). Solubility and copolymerization. Insolubility of PDEAEM polymer coatings is essential for self-sterilization surface applications; otherwise, the coating is washed out by water. The solubility of QAPDEAEM polymers in water of increased pH (pH = 7) was 18 mg/ml at 100% C8 and 8 mg/ml for the 70:30 C8 :C18 . Copolymerization of QA-DEAEM with the hydrophobic monomer MMA at 10%-30% w/w content resulted in insoluble polymers, and no change in the resultant activity (data not shown). 4. Conclusions Soluble polymers and crosslinked NPs containing QA-DEAEM were prepared and tested for their antimicrobial activity. Coatings of copolymers containing QA-DEAEM units possessed high antimicrobial activity against four representative strains of bacteria. Coating of polyethylene vinyl acetate loaded with 5% or higher QA-PDEAEM crosslinked NPs displayed antimicrobial activity. Copolymerization with MMA or exchanging 30% of the C8 with C18 did not affect the antimicrobial activity, however this did decrease their water solubility (pH 7); insoluble polymers were found for MMA copolymerization. These coatings may have applications in
coating of water containers to prevent bacterial growth and as an additive for making self-sterilizing plastics and bioadhesives. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015. 01.051. References [1] S. Farah, W. Khan, I. Farber, D. Kesler-Shvero, N. Beyth, E. Weiss, A.J. Domb, Polym. Adv. Technol. 24 (2013) 446. [2] E.-R. Kenawy, F.I. Abdel-Hay, A. Abou EL-Magd, Y. Mahmoud, J. Bioact. Compat. Polym. 20 (2005) 95. [3] G.L. Woo, M.L. Yang, H.Q. Yin, F. Jaffer, M.W. Mitelman, J.P. Santerre, J. Biomed. Mater. Res. 59 (2002) 35. [4] P.L. Taylor, A.L. Ussher, R.E. Burrll, Biomaterials 26 (2005) 7221. [5] Y. Sun, T. Chen, S.D. Worley, G. Sun, J. Polym. Sci. A: Polym. Chem. 39 (2001) 3073. [6] L. Massi, F. Guittard, S. Geribaldi, R. Levy, Y. Duccini, Int. J. Antimicrob. Agents 21 (2003) 20. [7] L. Guiqian, W. Dingcai, F. Ruowen, React. Funct. Polym. 67 (2007) 355. [8] H. Parviainen, M. Hiltunen, S.L. Maunu, J. Appl. Polym. Sci. (2014), http://dx.doi.org/10.1002/APP.40448. [9] S. Borman, Sci. Technol. 79 (2001) 13. [10] S. Farah, O. Aviv, N. Laout, S. Ratner, N. Beyth, A.J. Domb, Polym. Adv. Technol. 25 (2014) 689. [11] B. Gao, X. Zhang, Y. Zhu, J. Biomater. Sci. Polym. Ed. 18 (2007) 531. [12] J. Lin, S. Qiu, K. Lewis, A.M. Klibanov, Biotechnol. Bioeng. 83 (2003) 168. [13] M. Makhloufia, T. Benaissa, A. Derdour, D.E. Henni, E-J. Chem. 7 (2010) S61. [14] Y.H. Xiao, J.H. Chen, M. Fang, X.D. Xing, H. Wang, Y.J. Wang, F. Li, J. Oral Sci. 3 (2008) 323. [15] G. Sauvet, S. Dupond, K. Kazmierski, J. Chojnowski, J. Appl. Polym. Sci. 75 (2000) 1005. [16] E.-R. Kenawy, F.I. Abdel-Hay, A.E.-R.R. El-Shanshoury, M.H. El-Newehy, J. Polym. Sci. A: Polym. Chem. 40 (2002) 2384. [17] E.-R. Kenawy, Y.A.-G. Mahmoud, Macromol. Biosci. 3 (2003) 107. [18] A. Munoz-Bonilla, M. Fernández-García, Prog. Polym. Sci. 37 (2012) 281. [19] J. Hasan, R. Crawford, E. Ivanova, Trends Biotechnol. 31 (2013) 295. [20] L. Timofeeva, N. Kleshcheva, Appl. Microbiol. Biotechnol. 89 (2011) 475. [21] I. Yudovin-Farber, N. Beyth, E.I. Weiss, A.J. Domb, J. Nanopart. Res. 12 (2010) 591.
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