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Short Communication

Quaternary ammonium polyethylenimine nanoparticles for treating bacterial contaminated water Shady Farah a,1 , Oren Aviv a,b,1 , 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 and 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 13 November 2014 Received in revised form 29 January 2015 Accepted 2 March 2015 Available online xxx Keywords: Antimicrobial nanoparticles Polyethylenimine Antimicrobial surface Water disinfection

a b s t r a c t This study highlights the potential application of antimicrobial quaternary ammonium nanomaterials for water disinfection. Quaternary ammonium polyethylenimine (QA-PEI) nanoparticles (NPs) were synthesized by polyethylenimine crosslinking and alkylation with octyl iodide followed by methyl iodide quaternization. Particles modified with octyldodecyl alkyl chains were also prepared and evaluated. The antimicrobial activity of QA-PEI NPs was studied after anchoring in non-leaching polymeric coatings and also in aqueous suspension. Particles at different loadings (w/w) were embedded in polyethylene vinyl acetate and polyethylene methacrylic acid coatings and tested for antimicrobial activity against four representative strains of bacteria in static and dynamic modes. Coatings embedded with fluorescent labelled particles tracked by Axioscope fluorescence microscope during the antimicrobial test indicates no particles leaching out. Coatings loaded with 5% w/w QA-PEI exhibited strong antibacterial activity. Aqueous suspension was tested and found effective for bacterial decontamination at 0.1 ppm and maintains its activity for several weeks. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Waterborne diseases remain a major cause of death in many countries. Over one billion people lack access to safe water [1,2]. Chemical disinfectants commonly used by the water industry such as free chlorine, chloramines and ozone can react with various constituents in natural water to form disinfection byproducts (DBPs), many of which are carcinogens [3]. The rapid growth in nanotechnology has generated interest in new materials and composites for environmental applications [4]. Recently, several nanomaterials have been reported to possess antimicrobial activity, including chitosan, silver nanoparticles (nAg), photocatalytic TiO2 , fullerol, aqueous fullerene nanoparticles (nC60) and carbon nanotubes (CNT) [5–10]. Unlike conventional chemical disinfectants, these antimicrobial nanomaterials are not oxidants or consumable, and not expected to produce harmful side effects. If properly incorporated into treatment processes, they have the potential to replace or enhance conventional

∗ Corresponding author. Tel.: +972 26757573; fax: +972 26757076. E-mail address: [email protected] (A.J. Domb). 1 Equal contribution.

disinfection methods [11,12]. However, several challenges exist for efficient application of antimicrobial nanomaterials in drinking water treatment, primarily concerning dispersion and retention of nanomaterials and the sustainability of antimicrobial activity [4]. Quaternary ammonium (QA) functionalized materials, as antibacterial agents, have received much attention as they provide effective protection against bacterial colonization with long term durability and environmentally friendly performance. They demonstrated an ability to kill a wide spectrum of bacteria by contact [13,14]. In the present work, we report preparation of active antimicrobial surfaces using different methods, based on our previously described QA polyethylenimine nanoparticles (QA-PEI NPs) C8 alkylated [15,16], and C18 modified, where these NPs embedded in polyethylene vinyl acetate (PEVA) and polyethylene methacrylic acid (PEMA) coatings and these coatings were tested for their antibacterial activity against representative bacteria, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Heterotrophic plate count, in static and dynamic modes, for potential application in self-sterilization coatings and large water containers. In this study, QA-PEI NPs were also analyzed for their activity in aqueous suspension in comparison to aging period for waste water reuse as in reuse systems.

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amine groups were prepared and tested (Scheme 1) (Scheme 1S, SI). 2.2.2. QA-PEI NPs analysis IR spectra were recorded on Perkin-Elmer System 2000 FTIR. Particles size and Zeta-potential were 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. Thermal analysis was determined on a Metler TA 4000-DSC differential scanning calorimeter, calibrated with zink and indium standards, at a heating rate of 10 ◦ C/min (typical sample weight was 10 mg).

Scheme 1. Chemical structure of C8 /C18 alkylated quaternary ammonium polyethylenimine (QA-PEI) crosslinked nanoparticles (Scheme 1S Supporting Information, SI).

2. Materials and methods 2.1. Materials Polyethylenimine (PEI), molecular weight of 600 K–1 MDa was purchased from Fluka (Rehovot, Israel). Iodooctane, diiodopentane, iodomethane, sodium bicarbonate, anhydrous dichloromethane and dansyl chloride were all purchased from Sigma–Aldrich (Rehovot, Israel). Ethanol, ethyl acetate and chloroform (HPLC grade) were purchased from J.T. Baker, Holland. All solvents and reagents were of analytical grade. Tween 20 and span 60 purchased from Dexcel Pharma (Jerusalem, Israel). PEVA 28 (28% VA) and 46 (46% VA), PEMA were purchased from DuPontTM Elvax®, Switzerland. Bacterial strains: clinically isolated Escherichia coli and Pseudomonas aeruginosa (Maurice and Gabriela Goldschleger School of Dental Medicine at Tel-Aviv University, Israel). Staphylococcus aureus ATCC 8639 were used in this study. Heterotrophic plate count bacteria, HPC (mixed bacteria from tap water).

2.2. Methods 2.2.1. Preparation of QA-PEI NPs The synthesis of QA-PEI nanoparticle was previously described [15–17]. In brief, 10 g of PEI dissolved in 100 ml ethanol was crosslinked with diiodopentane (4% per primary amine in PEI) under reflux for 24 h. N-alkylation with iodooctane was conducted at amine:iodooctane 1:0.25 molar ratio under reflux for 24 h. After 24 h, excess sodium bicarbonate (1.25:1 mole ratio, NaHCO3 /iodooctane) was added to neutralize released HI; neutralization reaction was continued for 3 h under the same conditions. Thereafter, methylation of these particles was carried out by reacting with methyl iodide at 1:3 mole ratios (PEI/methyl iodide) at 42 ◦ C for 48 h. HI released was neutralized by adding molar equivalent of sodium bicarbonate per PEI, and the reaction was continued under the same conditions for additional 24 h. Formed NaI salt, excess of unreacted NaHCO3 , and traces of the unreacted iodooctane and methyl iodide were removed by washing with hexane and DDW. The average yield was 80% (mol/mol) (Scheme 1) (Scheme 1S, Supporting Information, SI). QA-PEI particles with more hydrophobic nature with C18 alkylated, were also prepared by the same procedure except an extra alkylation step for 24 h were performed before C8 alkylation step. Particles with ratio between alkylation agents C18 :C8 1:9, 2:8 and 3:7 respectively, of total 25% alkylated

2.2.3. Degree of alkylation and quaternization Degree of alkylation of PEI particles is the percent of substituted ethyleneimine units with an appropriate alkyl halide. The degree of alkylation of QA-PEI NPs was determined as follows: Degree of Alkylation% = † The

(Carbon/Nitrogen)Found †

(Carbon/Nitrogen)Calculated

× 100

(1)

values were calculated by using ChemDraw Ultra 7.0

Degree of quaternization is the percent of quaternerized polyethylenimine based particle. The degree of quaternization with an appropriate alkyl halide and with methyl iodide of QA-PEI NPs was determined as follows: Degree of Quaternization% = † The

Halide contentFound †

Halide contentCalculated

× 100

(2)

values were calculated by using ChemDraw Ultra 7.0

2.2.4. Fluorescence labeling of QA-PEI Previously freeze-dried PEI (2 g, 12.2 mmol of ␧-NH2 ) was dissolved in 10 ml of anhydrous dichloromethane. To this solution 1 ml (0.12 mmol equivalent to 1% mol/mol to ␧-NH2 ) of dansyl chloride in anhydrous dichloromethane was added. The mixture was stirred at room temperature for 3 h. To this solution 265 ␮l of dibromopentane at 1:0.04 mole ratio (monomer units of PEI/dibromopentane) were added. Crosslinking was continued at reflux conditions for 24 h. During crosslinking step, labeled PEI was precipitated, filtered and washed with several amounts of dichoromethane. The crude was dried overnight on NaOH pellets. Further steps such as alkylation and methylation were repeated as described in the typical procedure (see Section 2.2.1). These labeled particles were used for tracking the particles onto the surface of the polymeric coating using Axioscope fluorescence microscope. Labeling of the quaternary ammonium polyethylenimine with the above dye did not affect the biological activity of the polymer. 2.2.5. Quaternary ammonium (QA) coatings preparation 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®), QA-PEI particles were suspended in polymers solutions at 1% w/v in chloroform or ethyl acetate. Stable homogenous suspension with minimal particle aggregates was obtained by mixing the QA-PEI 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. Coatings with 5, 15 and 30% w/w particles were prepared. These coatings were made by two methods: cast or spray coating. For cast coating, 24 wells plates were used, each well was filled with 200 ␮l of the QA-PEI suspension followed by fast evaporation at 50 ◦ C to prevent particle precipitation or aggregation (Fig. 1S, SI).

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Spray coating method, 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 (Fig. 1S, SI). Particles suspension was mixed during spraying process to avoid undesired particles participation. This method was also applied for large volume container coatings. 2.2.6. SEM evaluation of PEI particles and surface coatings For QA-particles visualization, a suspension of 0.01% w/v of QAPEI particles in DDW was prepared using probe sonicator for 1 min (Vibra cell, at amplitude 60 Hz), then suspension droplet was spread over a silica plate using capillary followed by fast water evaporation, resulted with individual particles separated. For polymeric coatings embedded QA-PEI NPs covering plastic surfaces visualization, coated surfaces were cut into 1 cm × 1 cm pieces. Both samples were then Au/Pd coated to thickness of about 10 nm using a sputtering deposition machine (Polarone E5100) and were imaged using scanning electron microscopy (SEM), FEI E-SEM Quanta 2000 at constant acceleration voltage of 5 kV. 2.2.7. Antimicrobial activity analysis For 24 wells plate, 1 ml of bacterial suspension inlet fresh prepared (Appendix I 2.2.7, SI) was transferred into each polymeric coated well (duplicate for each type of coating) and into control’s wells, free QA NPs polymer coatings and uncoated wells (Blank). 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 2.2.7, SI). 3. Results and discussion 3.1. NPs synthesis and characterization QA-PEI NPs were prepared by PEI crosslinking with diiodopentane followed by 25% alkylation of the reactive sites, with octyl iodide, and methyl iodide for nitrogen methylation as given in Section 2.2.1. FTIR spectra of the particles indicated peaks at 3400 cm−1 (N–H), 2950 and 2850 cm−1 (C–H), 1617 cm−1 (N–H2 ), 1460 cm−1 (C–H), and 967 cm−1 for quaternary amine. The QA-PEI particles were analyzed for their size by Zetasizer and SEM visualized. Particles were found spherically shaped with nanometric size: 160–190 nm. Elemental analysis confirmed the expected structure of the product (found: %C 32.93, %H 6.62, %N 6.38 and %I 45.43, calculated: %C 32.55%, H 6.34%, N 6.07% and I 55.03,) and indicated that these particles were ∼83% of nitrogens quaterarnized (see Eq. (2)). These NPs possess positive charge of 65.0 ± 2.3 mV, as determined

3

by zeta potential analysis. Extending the quaternarization process from 48 to 72 h with excess of methyl iodide resulted with slight increase quaternarization% up to 90% as determined by elemental analysis (found: %C 32.68, %H 6.80, %N 6.41 and %I 49.38) and were positively charged particles with 70.5 ± 3.5 mV. Thermal analysis by DSC showed that these crosslinked particles have Tg peak at 145 ◦ C while melt at 250 ◦ C, and decompose at 325 ◦ C. To assess the potential use of the above described antimicrobial nanomaterials for water disinfection field, it was decided to examine their antimicrobial activity embedded within non-leaching polymeric coatings for self-sterilization coatings as for water’s storage containers as also to study their activity for waste water reuse as in reuse systems. Accordingly, particles were studied for their activity in aqueous suspension with microbial inlet. Suspension activity was also studied in comparison to aging period. 3.2. Polymer coatings embedded with QA-PEI NPs preparation Coatings embedded QA-particles was prepared as detailed in Section 2.2.5, were made by two methods: cast or spray coating. Coatings with different particle loading % (w/w) were prepared and microscopically analyzed, both methods showed homogeneous coatings (Fig. 1A and B). SEM evaluation of the polymeric coating showed good distribution of the QA-PEI NPs while minimal particles aggregation was found, while the majority of the particles found on the top of the polymeric coating, a small fraction was found partially or completely buried inside the coating (Fig. 1C and D). Spray method was selected for further studies due to ease to control and durability of coating, this method was also applied for inner coating of bottles of 0.25–1 L for large volume containers water treatment test. Fluorescent QA-PEI particles were prepared by labeling with dansyl chloride at 1% mol/mol to ␧-NH2 and tracked in the polymeric coating and in incubation medium using Axioscope fluorescence microscope (Fig. 2S, SI). No particles leaching from the polymeric coating was noticed during the antimicrobial test, as no change in the particles distribution with absent of fluorescent signal in the replaced medium attributed to the antimicrobial activity to surface contact between the bacteria and fixed QA-PEI NPs. 3.3. Antimicrobial activity in polymeric coatings Polymer coatings embedded with QA-PEI NPs at different concentration (w/w), 15% and 30% were prepared and tested against Staphylococcus aureus, Pseudomonas aerogenous, Escherichia coli and Heterotrophic plate count bacteria in 24 wells plate under static and dynamic conditions. The results showed that coatings incorporate

Fig. 1. Photo of polymeric coatings embedded with 5% w/w quaternary ammonium polyethylene imine (QA-PEI) nanoparticles prepared either by: (A) solvent cast or (B) spray coating of particles suspension in 1% w/v solutions of polyethylene vinyl acetate (PEVA) 28%. (C) SEM figure of the purple square in photo B, QA-PEI particles imbedded in PEVA, ×10,000 magnification, 5 ␮m scale bar. Minimal particles aggregation is obvious. (D) SEM figure, zoom in, in the purple square in photo C, QA-PEI particles imbedded in PEVA, ×150,000 magnification, 300 nm scale bar. QA-PEI particles are fixed on and in the polymeric coating. (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.)

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Fig. 2. Antimicrobial activity of polyethylene vinyl acetate (PEVA) coatings embedded with quaternary ammonium polyethylenimine (QA-PEI) nanoparticles analyzed at: (A) dynamic conditions and (B) static condition against Heterotrophic plate count (HPC), Staphylococcus aureus (S. Aureus), Escherichia coli (E. Coli) and Pseudomonas aerogenous (P. Aeruginosa) bacteria. Analysis was done in duplicate in two 24 wells plates.

of 15% QA-PEI NPs possess bactericide activity against Staphylococcus aureus, Escherichia coli and Heterotrophic plate count, while only coatings embedded with 30% w/w particle showed bactericide activity against Pseudomonas aerogenous in dynamic condition (Fig. 2A). In static condition, the 30% w/w QA-particle embedded coatings showed bacterostatic behavior against Pseudomonas aerogenous, while blank wells or free QA-particles coatings showed increased bacteria proliferation, ∼1010 CFU/ml. Similar activity was found, as in dynamic mode, against the other tested bacteria at 15% w/w (Fig. 2B). The difference in the activity against Pseudomonas aerogenous can be explained by the analytical conditions where in static mode there is no water stirring which induces adsorption of bacterial extracellular debris onto the antimicrobial surface, leading to less contact between QA NPs surface and the surface of the bacteria, which is critical as reported in the estimated mechanism of action resulting in biofilm formation [18]. Moreover, Pseudomonas aerogenous is known for its intrinsic resistance to many front-line antibiotics, mainly due to its low outer membrane permeability and active efflux of antibiotics [19]. The proposed mechanism of QA compounds is penetration of the alkyl groups of the QA moieties to the cell wall and disruption of the bacterial cell wall resulting in lysis [20]. Here, we assume that NPs modified with longer alkyl chain, could show better results against resistance bacteria such

as Pseudomonas aerogenous, where elongation of the alkyl chains may improve the interaction with bacterial cell wall resulting in enhanced antimicrobial performance. Accordingly, modified NPs, C18 alkylated QA-PEI NPs were synthesized and tested for their antimicrobial activity in polymeric coating. 3.4. C18 alkyl chain modified QA-PEI NPs QA-PEI NPs modified with C18 alkyl chains were synthesized with ratios C18 :C8 1:9, 2:8 and 3:7 respectively of total 25% amine groups alkylated (C8 + C18 ). The chemical structure of C18 alkylated particles was confirmed by FTIR and it was found very similar to the above described C8 alkylated particles. C18 Alkylated particles were designed to be more hydrophobic as shown by the increase of C/N elemental ratio, while C18 :C8 3:7 particles possessed the highest C/N value and accordingly the lowest surface charge (Table 1). Polymeric coatings embedded with the C18 :C8 3:7 alkylated QA-particles were prepared by spray coating and evaluated for their antimicrobial activity after 72 h of stagnation against Pseudomonas aerogenous, Staphylococcus aureus and Heterotrophic plate count bacteria with 1 layer coating incorporate 5% or 15% w/w particles, in comparison to QA-PEI particles C8 alkylated. It was found that polymeric coatings embedded with 5% w/w C18 :C8 3:7 QA-PEI

1.00E+09

HPC

1.00E+08

S.Aureus

P.Aeruginosa

1.00E+07 1.00E+06 1.00E+05 CFU/ml

1.00E+04 1.00E+03 1.00E+02 1.00E+01 P.Aeruginosa S.Aureus

1.00E+00 Inlet

Control 1Blank

Control 2 Coatings Free QA

HPC 5% w/w QA-PEI (C8)

5% w/w QA-PEI (3:7, C18:C8)

15% w/w QA-PEI (C8)

15% w/w QA-PEI (3:7, C18:C8)

Fig. 3. Antimicrobial activity of quaternary ammonium polyethyleneimine particles (QA-PEI), 25% alkylated with 3:7 C18 :C8 compared to QA-PEI with 25% C8 alkylated particles, embedded in polyethylene vinyl acetate (PEVA) coatings against Heterotrophic plate count (HPC), Staphylococcus aureus (S. Aureus) and Pseudomonas aerogenous (P. Aeruginosa) bacteria. Analysis was done in duplicate in two 24 wells plates.

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Table 1 Characterization of modified QA-PEI particles. Batch name

ShF-2-150 ShF-2-150A ShF-2-150B ShF-2-150C

PEI:CX I (mol/mol)

*

1:0.25 1:0.25** 1:0.25** 1:0.25**

PEI:CH3 I (mol/mol), Step time

1:3, 48 h 1:3, 48 h 1:3, 48 h 1:3, 48 h

Elemental analysisa

%C

%H

%N

%I

32.93 37.49 31.06 31.55

6.62 7.31 6.16 6.11

6.38 7.66 6.39 5.59

45.43 40.28 41.55 39.14

C/Nb

Alkylation%␣

Quaternaization%␤

Zeta Potential (mV)c

5.16 4.89 4.86 5.64

25 21.9 21.0 23.5

82.5 74.2 77.6 74.1

65.0 ± 2.3 64.8 ± 1.1 68.9 ± 1.3 48.4 ± 3.8

C, H, N and I content of QA-PEI particles (%C, %H, %N± 0.3%, %I ± 0.5%). C/N was determined by elemental analysis of the final particles after alkylation with octyl iodide or/and octyldodecyl, and subsequent methylation with methyl iodide. c Zeta potential of QA-PEI particles. 1% w/v suspension in water, samples were analyzed in triplicate. * Crosslinked PEI particles were reacted with 25% mol/mol alkyl iodide (CX I): PEI particles were reacted with iodooctane (C8 I) for 24 h. ** Crosslinked PEI particles were reacted with 25% mol/mol alkyl iodide (CX I): PEI particles were reacted first with iodooctadecane (C18 I) for 24 h followed by reacting with iodooctane (C8 I) for another 24 h at the following ratio: ShF-2-150A (1:9, C18 :C8 ), ShF-2-150B (2:8, C18 :C8 ) and ShF-2-150 C (3:7, C18 :C8 ). ␣ Alkylation % calculated according to Eq. (1). ␤ Quaternization % calculated according to Eq. (2). a

b

Inlet

1.00E+05

Outlet After 0 hr Outlet After 24 hr QA-PEI Activity After 0 hr QA-PEI Activity After 24 hr

1.00E+04

1.00E+03 CFU/ml 1.00E+02

1.00E+01

1.00E+00

Inlet / Outlet

0.01

0.1

1

Particles Concentration (ppm) Fig. 4. Activity of quaternary ammonium polyethyleneimine (QA-PEI) nanoparticles in aqueous suspension against Heterotrophic plate count bacteria at different ppm concentrations. Particles show activity above 0.1 ppm.

NPs were highly effective against the three tested bacteria, while coatings embedded with C8 alkylated QA-PEI particles were inactive against Pseudomonas aerogenous and showed moderate activity against Staphylococcus aureus at the same NPs loading% (Fig. 3). This activity improvement could be attributed to the increase in: alkyl chains length and also in hydrophobic nature, were according to the suggested mechanism, both required for efficient contact between QA NPs surface and bacteria cell wall [20]. The effectiveness of QA-PEI embedded coatings was evaluated also in bottles of 0.25–1 L, representing large volume containers loaded with contaminated water with Heterotrophic plate count bacteria with an inlet of 104 CFU/ml. Coatings of 5% w/w QA-PEI in polyethylene vinyl acetate were prepared by spray coating and tested against Heterotrophic plate count bacteria. These coatings evaluated after 24 and 72 h of stagnation. It was found that developed coatings were highly active bactericide surfaces, as identified with complete bacterial inhibition also in large container such as 1 L. 3.5. Activity of QA-PEI NPs in aqueous suspension A 5.0 × 103 CFU/ml of Heterotrophic plate count bacterial inlet was added into water suspension containing QA-PEI NPs at

concentrations of 0.01, 0.1, 1 and 10 ppm. QA-PEI concentration above 0.1 ppm was found active in correspondence to the concentration (Fig. 4). It was also found that these particles lose their antibacterial activity with time when left suspended in water, 1 ppm after 21 days and 10 ppm after 46 days.

4. Conclusions Strong antimicrobial activity was observed for QA-PEI NPs embedded in polymeric coatings or dispersed in water at concentrations as low as 0.1 ppm. QA-PEI derivatives with a mixture of octyl and octadecanyl at a 7:3 ratio possessed the highest antibacterial activity against the resistant bacteria Pseudomonas aerogenous due to alkyl chains permeability and hydrophobicity improvements. PEVA coatings containing 5% w/w QA-PEI NPs showed high antimicrobial activity against representative strains of Gram positive and Gram negative bacteria. Effectiveness of theses coatings in water containers was demonstrated. It is anticipated that these QA-PEI NPs will find application in water purification systems as disinfectants and for preventing accumulation of micro-organisms onto device surfaces as well as in bioadhesives and self-sterilizing surfaces.

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