Intrinsic Hydrophobic Antibacterial Thin Film from Renewable

Research Article pubs.acs.org/journal/ascecg

Intrinsic Hydrophobic Antibacterial Thin Film from Renewable Resources: Application in the Development of Anti-Biofilm Urinary Catheters Krishnamoorthy Lalitha,† Miryala Sandeep,‡ Yadavali Siva Prasad,† Vellaisamy Sridharan,† C. Uma Maheswari,† C. S. Srinandan,‡ and Subbiah Nagarajan*,†

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†

Organic Synthesis Group, Department of Chemistry and CeNTAB, School of Chemical and Biotechnology, SASTRA University, Thanjavur 613401, Tamil Nadu, India ‡ Biofilm Biology Lab, Centre for Research on Infectious Diseases, School of Chemical and Biotechnology, SASTRA University, Thanjavur 613401, Tamil Nadu, India S Supporting Information *

ABSTRACT: The use of renewable resources to develop functional materials is increasing in order to meet the sustainability challenges. In an era of inexorable evolution of antimicrobial resistance, there is a substantial increase in demand for the development of efficient antimicrobial thin film coating from renewable resources for public bacterial threats, food, biomedical, and industrial applications. In the present investigation, we have used cardanol, a phenolic compound having unsaturated hydrophobic tail isolated from cashew fruits, and linseed oil, a vegetable oil and an important biobased building block, which are cheap and easy to regenerate. This study reports the synthesis of cardanol based metal complexes having unsaturated hydrophobic unit and acrylated epoxidized linseed oil (AELO) prepared via epoxidation of double bonds followed by acrylation. The double bond present in the metal complexes and AELO is prone to form assembled thin film under atmospheric conditions, without the need of any initiators. Assembled thin film is one of the important aspects of nanotechnology holding a wide range of applications. 1H NMR and FT-IR analysis revealed the existence of a strong interaction between ligand and metal, which paves a way to develop a nonleachable metal based thin film coating. The leaching behavior of thin film coating was investigated under various aggressive conditions with the aid of UV−vis spectroscopy. The mechanical properties of assembled thin film coating material composed of cardanol-based metal complex and AELO are described using oscillatory rheology. Morphological and SAXD analysis clearly revealed the formation of the assembled structure in thin films. Thermal response of these materials has been investigated using TGA and DSC measurements. Intrinsic hydrophobic character was identified by contact angle measurement. Antimicrobial and biofilm inhibitory behavior of synthesized compounds and thin films were investigated against various human pathogenic bacterial strains. The assembled thin film coated catheter tube completely inhibits the biofilm formation of uropathogenic Escherichia coli (UPEC). Thus, the developed thin film coating material holds promise to be used as metal enabled, nonleachable coating materials for public bacterial threats, and food and biomedical applications. In particular, this material can be potentially used for developing urinary catheter tubes with antibacterial properties. KEYWORDS: Renewable resource, Assembled thin film, Cardanol, Antibacterial, Antibiofilm, Linseed oil

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INTRODUCTION Over the last few decades, there has been a growing interest in the use of natural raw material derived from renewable resources to produce valuable products, especially for the manufacturing of thin film coatings, surfactants, soaps, cosmetic products, lubricants, and paints.1 In particular, thin film coating materials derived from renewable natural resources have attained substantial interest among scientists due to the finite availability of petroleum-based resources and economic and environmental prospects.2 In recent years, an intensive investigation has been done to develop sustainable materials from vegetable oils,3,4 carbohydrates,5−7terpenes,8,9 rosins,10 phenols,11−13 and other renewable raw materials14−16 Generally, drying oil when exposed © 2016 American Chemical Society

to air forms an excellent scratch free coating by a simple air oxidation process via a well-known free radical mechanism.17 Although vegetable oils and phenolic lipids possess double bonds, which are actively used as reactive sites for coating, they can be further functionalized to derive promising nonleachable antibacterial thin film coating material for various applications discussed below. Bacterial infections are considered as a major health issue causing an increased number of health risks, especially in hospitals, public toilets, railway stations, schools, sanitary Received: July 30, 2016 Revised: October 16, 2016 Published: November 7, 2016 436

DOI: 10.1021/acssuschemeng.6b01806 ACS Sustainable Chem. Eng. 2017, 5, 436−449

Research Article

ACS Sustainable Chemistry & Engineering facilities, the biomedical field, etc.18−20 The number of fatalities has been rising every year as a result of bacterial infections, which has attained a considerable concern worldwide.21 In the last six decades, antibiotics has been a better choice for the treatment of bacterial infections;22 however, because of overuse, bacteria have evolved resistance thereby reducing the efficacy of antibiotics.23 Moreover, bacteria predominantly attach to biotic or abiotic surfaces and display tolerance toward adverse environmental conditions and antimicrobial reagents by forming a biofilm community. Biofilm formation is considered as one of the main strategies for long-term persistent bacterial survival in a variety of sites including hostile environments.24,25 Planktonic bacteria adhere to the surfaces and develop into three-dimensional biofilm structures; antimicrobial treatment of such an environment poses additional challenges.26 A number of strategies developed to tackle microbial infections have been discussed in the literature, which includes surface modified antimicrobial peptides, metal ion release systems, drug release systems, silver and gold based surface coating, polymer modified surfaces, etc.27−30 However, the reported strategies have concerns about stability, efficacy, safety, and economic viability. There is a need to develop economically viable, environmentally safer and nonleachable antimicrobial thin film coating material from readily available renewable raw material to overcome microbial threats in public, biomedical, and industrial environments. Nanobiomedical thin film techniques are more attractive to overcome the limitations of existing tools.31,32 Zinc and copper play vital roles in various biological processes, and researchers have exponentially explored the antimicrobial activity of copper and zinc complexes.33−35 At the commercial level, zinc and copper have been blended in consumer products and present as zinc pyrithione in shampoo,36 zinc lactate and zinc sulfate in toothpaste and mouthwash,37 and zinc acetate and zinc gluconate in mineral supplements.38 Moreover, EPA has approved a wide variety of copper based antimicrobial material with public health benefits such as antimicrobial copper-alloy touch surfaces, etc.39 Most of the previous work in this area is concerned more on metal nanoparticles and metal complexes of copper, silver, gold, and platinum.40−42 Metal incorporated in coating materials could possibly inhibit biofilm formation of bacteria and could be employed as antimicrobial and antifouling agents. There are several disadvantages on the commercial use of metal blended coatings such as leachability, stability of coated surface, and availability of metal in the environment.43−47 However, to overcome the existing limitations on the development of an antimicrobial thin film surface, we discuss the synthesis and assembly of zinc and copper based Schiff base complexes derived from a renewable resource, cardanol in acrylated epoxidized linseed oil (AELO), and its subsequent use in the development of antimicrobial thin film coating materials.

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shifts (δ) are reported in parts per million (ppm) with respect to internal standard TMS, and coupling constants (J) are given in Hz. Proton multiplicity is assigned using the following abbreviations: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m). Electrospray ionization mass spectra (ESI-MS) were carried out in positive mode with a Thermo Fisher LCQ Advantage Max. instrument by dissolving the solid sample in methanol. The infrared spectrum was obtained using FTIR Shimadzu 8000 Spectrometer as KBr pellets in the spectral range of 4000 to 400 cm−1. UV/vis spectra were recorded on Thermo Scientific Evolution 220 UV/visible spectrophotometer. The spectra were recorded in the continuous mode between 200 and 700 nm, with a wavelength increment of 1 nm and a bandwidth of 1 nm. Purification of Cardanol. The major constituent present in cashew nut shell liquid (CNSL) is cardanol 1a, a biobased non-isoprene lipid, comprising a phenolic lipid mixture: 5% of 3-n-pentadecylphenol (3-PDP), 50% of 3-(8Z-pentadecenyl) phenol, 16% of 3-(8Z, 11Zpentadecadienyl) phenol, and 29% of 3-(8Z,11Z,14-pentadecatrienyl)phenol. CNSL was distilled at a temperature between 210 and 280 °C, under a pressure from 2 to 8 mmHg to get cardanol.48−51 Cardanol was obtained as a pale yellow liquid, which darkens during storage. After a second distillation, a mixture of cardanol mono-, di-, and triene was obtained and used for the preparation of antimicrobial thin film coating material. Hydrogenated cardanol, 1b can be easily obtained as a pure compound by hydrogenation of the double bonds present in the side chain of distilled cardanol. Synthesis. General Procedure for the Synthesis of 2-Hydroxy-4alkylbenzaldehydes 2a and 2b. To a solution of 3-alkyl phenol (4 mmol) in acetonitrile were added anhydrous magnesium chloride (6 mmol), triethylamine (15 mmol) ), dry paraformaldehyde (35 mmol), and the mixture was heated under reflux for about 12−15 h. After the completion of the reaction, as monitored by TLC, the reaction mixture was cooled to room temperature and 5% aq. HCl was added. The crude product was extracted with ethyl acetate, dried over anhydrous Na2SO4 and pure product was isolated by column chromatography using 95:5 v/v hexane-ethyl acetate as eluent. Compound 2a. Isolated as a yellow liquid; yield = 88%. 1H NMR (300 MHz, CDCl3): δ 0.88 (t, J = 7.2 Hz, 3H), 1.25−1.30 (m, 16H), 1.59−1.64 (m, 4H), 1.95−2.05 (m, 2H), 2.61 (t, J = 7.5 Hz, 2H), 5.33− 5.39 (m, 2H), 6.80 (s, 1H), 6.83 (d, J = 7.8 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 9.83 (s, 1H), 11.05 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 195.8, 161.8, 153.8, 133.6, 130.0, 129.7, 120.5, 118.9, 117.1, 36.4, 32.6, 31.8, 30.7, 29.7, 29.7, 29.7, 29.6, 29.4, 29.3, 29.3, 29.2, 29.2, 29.0, 27.2, 27.2, 22. 7, 14.1. Compound 2b. Isolated as a white crystalline solid; yield = 92%. 1 H NMR (300 MHz, CDCl3): δ 0.88 (t, J = 6.9 Hz, 3H), 1.29−1.34 (m, 24H), 1.57−1.66 (m, 2H), 2.61 (t, J = 7.8 Hz, 2H), 6.80 (s, 1H), 6.83 (dd, J = 7.8, 1.5 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 9.83 (s, 1H), 11.05 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 195.8, 161.8, 153.8, 133.6, 120.5, 118.8, 117.1, 36.5, 31.9, 30.7, 29.7, 29.7, 29.5, 29.4, 29.4, 29.3, 22.7, 14.1. General Procedure for the Synthesis of Schiff Base Ligands 3a and 3b. To the solution of 2-hydroxy-4-alkylbenzaldehydes 2a or 2b (15.0 mmol) in methanol (10 mL) was added allylamine (15.0 mmol), and the reaction mixture was refluxed for 2 h. After completion of the reaction, as identified using TLC, the solvent was removed under vacuum and washed the entire contents using diethyl ether. The resulting yellow oil was dissolved in dichloromethane and dried over anhydrous Na2SO4. The solvent was removed under vacuum to yield the products. Compound 3a. Isolated as a viscous yellow liquid; yield = 96%. IR (KBr) cm−1: 3300−3500 (OH stretch), 2930, 2857 (CH stretch), 1626 (CN stretch), 1569 (CC stretch), 1453, 1398 (CH3, CH2 bend), 1263 (CN stretch). 1H NMR (CDCl3, 300 MHz): δ 13.29 (br s, 1H), 8.16 (s, 1H, −CHN), 7.01 (d, J = 7.8 Hz, 1H, ArH), 6.69 (s, 1H, Ar−H), 6.57 (d, J = 7.5 Hz, 1H, ArH), 5.95−5.80 (m, 1H, CHCH2), 5.30−5.25 (m, 2H, alk-H), 5.14−5.02 (m, 2H, CH CH2), 4.07 (d, J = 5.1 Hz, 2H, CH2CHCH2), 2.46 (t, J = 7.5 Hz, 2H, −CH2), 1.93−1.91 (m, 2H, −CH2), 1.55−1.43 (m, 4H, −CH2), 1.13−1.08 (m, 16H, −CH2), 0.78 (t, J = 6.9 Hz, 3H, −CH3). 13C NMR (CDCl3, 75 MHz): δ 165.4, 161.4, 148.0, 135.1, 131.2, 130.3, 129.8,

EXPERIMENTAL SECTION

Materials and General Methods. All reagents and solvents needed for the synthesis were purchased from Sigma-Aldrich, Merck, Alfa Aesar, and Avra chemicals and were used as such without further purification. LR grade solvents were used to purify the compounds and distilled solvents were used, when necessary. The reaction progress was monitored by thin-layer chromatography using precoated silica gel plates purchased from Merck and visualized by UV detection or molecular iodine. Column chromatography was performed on silica gel (100−200 mesh) purchased from Avra chemicals, India. Characterization Methods. 1H- and 13C NMR spectra were recorded on a Bruker Avance 300 MHz instrument in either CDCl3 or CDCl3 with a few drops of DMSO-d6 at room temperature. Chemical 437


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ACS Sustainable Chemistry & Engineering 119.0, 116.8, 116.7, 116.1, 61.3, 36.2, 32.7, 32.0, 31.9, 31.1, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.1, 29.0, 27.3, 27.3, 22.8, 14.2. Compound 3b. Obtained as a pale yellow solid; yield = 97%. IR (KBr) cm−1: 3300−3500 (OH stretch), 2920, 2848 (CH stretch), 1631 (CN stretch), 1511 (CC stretch), 1463, 1400 (CH3, CH2 bend), 1289 (CN stretch). 1H NMR (CDCl3, 300 MHz): δ 13.49 (br s, 1H, −OH), 8.25 (s, 1H, −CHN), 7.08 (d, J = 7.8 Hz, 1H, ArH), 6.72 (d, J = 0.9 Hz, 1H, ArH), 6.63 (dd, J = 7.8, 1.2 Hz, 1H, ArH), 6.01−5.88 (m, 1H, −CHCH2), 5.19−5.08 (m, 2H, −CHCH2), 4.16−4.14 (m, 2H, −CH2CHCH2), 2.50 (t, J = 7.4 Hz, 2H, −CH2), 1.18 (m, 26H, −CH2), 0.81 (t, J = 6.6 Hz, 3H, −CH3). 13 C NMR (CDCl3, 75 MHz): δ 165.3, 160.2, 148.2, 135.0, 131.1, 119.0, 116.7, 116.7, 116.2, 61.2, 36.1, 31.9, 31.0, 29.7, 29.6, 29.5, 29.4, 29.3, 22.7, 14.1. General Procedure for the Synthesis of Zinc Complexes 4a and 4b. To the solution of Schiff base ligands 3a or 3b (10.0 mmol) and triethylamine (10.0 mmol) in methanol, zinc acetate (5.0 mmol) was added slowly and the mixture was refluxed for 2 h. The complete consumption of Schiff base ligand was identified by TLC. After complete complexation, the solution was allowed to cool to room temperature and the solvent was removed under vacuum. The resulting viscous pale yellow oil was dissolved in dichloromethane and washed well with water. The organic layer was separated and dried under sodium sulfate. The solvent was removed under reduced pressure to yield the corresponding zinc complexes. Compound 4a. Yellow liquid. IR (KBr) cm−1: 2923, 2851 (CH stretch), 1614 (CN stretch), 1525 (CC stretch), 1436, 1396 (CH3, CH2 bend), 1258 (CN stretch). 1H NMR (CDCl3, 300 MHz): δ 8.08 (s, 1H), 6.99 (d, J = 7.8 Hz, 1H), 6.67 (s, 1H), 6.44 (d, J = 7.2 Hz, 1H), 6.06−5.85 (m, 1H), 5.38−5.02 (m, 4H), 4.13 (s, 2H), 2.59−2.49 (m, 2H), 2.01−1.98 (m, 2H), 1.71−1.58 (m, 4H), 1.46−1.25 (m, 16H), 0.93−0.86 (m, 3H). 13C NMR (CDCl3, 75 MHz) δ = 195.8, 170.7,165.3, 150.9, 135.5, 134.6, 133.6, 131.3, 130.3, 129.9, 129.8, 122.3, 119.1, 118.9, 117.1, 116.6, 116.4, 116.2, 115.7, 62.5, 60.5, 46.3, 36.1, 32.6, 31.8, 31.7, 30.9, 30.6, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 28.9, 28.8, 27.2, 22.6, 21.8, 14.1, 8.7. HRMS (ESI) m/z calculated for C50H76N2O2Zn [M + H]+: 801.5266. Found: 801.4960. Compound 4b. Obtained as white solid. IR (KBr) cm−1: 2918, 2850 (CH stretch), 1620 (CN stretch), 1524 (CC stretch), 1478, 1433, 1402 (CH3, CH2 bend), 1196 (CN stretch). 1H NMR (CDCl3, 300 MHz): δ 8.10 (s, 1H), 6.99 (d, J = 8.1 Hz, 1H), 6.67 (s, 1H), 6.43 (d, J = 7.8 Hz, 1H), 6.08−5.81 (m, 1H), 5.26−5.06 (m, 2H), 4.16 (dd, J = 30.0, 4.2 Hz, 2H), 2.59−2.51 (m, 2H), 1.45−1.20 (m, 26H), 0.9 (m, 3H). 13C NMR (CDCl3, 75 MHz): δ 170.9, 170.7, 151.0, 135.4, 133.5, 122.4, 119.1, 116.0, 115.5, 62.6, 36.1, 31.9, 30.5, 29.7, 29.6, 29.5, 29.3, 22.7, 14.1. HRMS (ESI) m/z calculated for C50H80N2O2Zn [M + H]+: 805.5579. Found: 805.0579. General Procedure for the Synthesis of Copper Complexes 4c and 4d. To the Schiff base ligand 3a or 3b (10.0 mmol) dissolved in methanol (4.0 mL) were added triethylamine (10.0 mmol) and copper acetate (5.0 mmol), and the mixture was refluxed for 2 h. After the progress of the reaction was monitored using TLC, the crude product was extracted using dichloromethane and dried using anhydrous Na2SO4. Compound 4c. Obtained as a green viscous liquid. HRMS (ESI) m/z calculated for C50H76N2O2Cu [M + MeOH]+: 831.5454. Found: 831.6335. IR (KBr) cm−1: 2925, 2853 (CH stretch), 1614 (CN stretch), 1524 (CC stretch), 1432, 1395 (CH3, CH2 bend), 1319 (CN stretch). Compound 4d. Obtained as a green solid; HRMS (ESI) m/z calculated for C50H80N2O2Cu [M + H]+: 804.5583. Found: 803.8623. IR (KBr) cm−1: 2918, 2850 (CH stretch), 1619 (CN stretch), 1528 (CC stretch), 1484, 1432, 1398 (CH3, CH2 bend), 1205, 1146 (CN stretch). General Procedure for the Synthesis of Epoxidised Linseed Oil (ELO). Epoxidation of linseed oil was carried out in a three necked round-bottom flask fitted with a magnetic stirrer and temperature sensor. To a mixture of linseed oil (7.95 g, 10 mmol) and formic acid (4.6 g, 100 mmol) was added slowly hydrogen peroxide (12.24 g, 108 mmol), and the mixture was refluxed at 60 °C for 4 h. After the

completion of the reaction, as identified by TLC, the reaction mixture was cooled to room temperature, filtered, and washed several times with distilled water until a pH of 7.0 was obtained. The viscous liquid thus obtained was dissolved in DCM, dried under Na2SO4, filtered, and the solvent was removed under reduced pressure to get the epoxidized linseed oil. 1H NMR spectra of ELO is given in the Supporting Information. Acrylation of Epoxidised Linseed Oil. Epoxidised linseed oil was acrylated via ring opening of the oxirane group using acrylic acid in the presence of triethyl amine as a catalyst and hydroquinone as a free radical inhibitor.1,52 To a mixture of epoxidized linseed oil (0.891 g, 1 mmol) and acrylic acid (0.4 mL, 6 mmol) were added triethyl amine (0.14 mL, 1 mmol) and hydroquinone (0.110, 1 mmol). The mixture was then heated at 110 °C with constant stirring for 36 h. After completion of reaction, as identified by TLC, the reaction mixture was diluted with hexane and unreacted acrylic acid and base were removed by extraction with water. The organic layer was separated, dried under anhydrous Na2SO4 to get pure acrylated epoxidized linseed oil (AELO). 1H NMR spectra of AELO is given in the Supporting Information. Preparation of Metal Based Antimicrobial Thin Film Coating. Different curable formulations, 5%, 10%, and 20%, were prepared by consistently mixing 5, 10, and 20 mg of synthesized compounds (3a and 3b), zinc based complexes (4a and 4b), and copper based complexes (4c and 4d) in 1.0 mL of AELO using a vortex mixer. The resultant mixture was loaded into the pistol type air brush with a nozzle of 0.15 mm and sprayed on the glass surface. The high degree of unsaturation present in AELO, compound 4a, and 4c makes them sensitive to auto-oxidation under atmospheric condition within 2 days, whereas precursor compound 3a took a little longer time to cure. Abbreviations and compositions of auto-oxidizable thin film coating materials are given in Table 1.

Table 1. Abbreviation and Nature of Thin Film Coating Materials Prepareda S. No. Compounds 1 2 3 4 5 6

3a 3b 4a 4b 4c 4d

Abbreviations used

Nature of thin film formation with AELO (Time duration for curing: 24−48 h)

CAI PAI ZnCAI ZnPAI CuCAI CuPAI

Scratch free thin filmb Viscous nature Zinc enabled CAI thin film Viscous nature Copper enabled CAI thin film Viscous nature

Note: 20% curable formulation was used for thin film formation studies. bTime duration for curing: >48 h. a

X-ray Diffraction Studies. A small portion of a thin film formed on glass surface was scratched off, and measurement was performed on a BRUKER-binary V3 diffractometer system. Morphological Analysis. Morphological analysis of thin film coated on a glass slide was studied using a JEOL JSM-6701F ultrahigh resolution field emission scanning electron microscope coupled to an energy dispersive X-ray (EDX) detector. The coated slides were cut into small piece of size 1 cm × 1 cm and were coated with gold prior to imaging. Mechanical Stability Test. A tape peeling test was performed to assess the surface resistance to the adhesive force of cello tape and Scotch 810 tape. The tape was pasted on the thin film coating on a glass slide and then detached; this process was repeated up to 20 times. Leaching Studies. Leaching of copper or zinc or compound 3a from ZnCAI and CuCAI thin film was studied by suspending an individual thin film coated glass slide in a beaker containing 10 mL of tap water, distilled water, and seawater and also at pH = 4 and pH = 10. Immediately after the glass plate was suspended in water, 2 mL of aliquot was withdrawn and scanned using a UV−vis spectrophotometer. Thereafter, leaching was tracked at a regular interval of once in 5 days. After UV−vis spectra were recorded, the aliquot was transferred back to the beaker. UV−vis spectral data obtained were then used for comparison of leaching behavior of thin films. 438


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ACS Sustainable Chemistry & Engineering Rheological Measurement. The flow behavior of the thin film coating mixture was identified using a stress controlled rheometer (Anton Paar 302 rheometer) implemented with a steel-coated 25 mm diameter parallel-plate geometry. The gap between two plates for rheological testing of thin film coating mixture was fixed as 1 mm and experiments were carried out at 23 °C. To study the viscoelastic behavior of coating mixture, oscillation measurement was performed at 23 °C. An amplitude sweep measurement was conducted first to identify the linear viscoelastic range, then the storage modulus, G′ and the loss modulus, G″ were monitored as functions of frequency from 0.1 to 100 rad s−1. Creep test was performed to examine the recovering ability of materials from the deformation that occurred during creep. The coating nature of the material was determined using flow studies. Contact Angle Measurement. Contact angle measurements for polymerized linseed oil, polymerized AELO, ZnCAI thin film, and CuCAI thin film were performed using a goniometer at room temperature. Bacterial Strains and Culture Conditions. Strains used in this work are uropathogenic Escherichia coli UTI89 (UPEC), Staphylococcus aureus ATCC 25923 (SA), Pseudomonas aeruginosa ATCC 27853 (PA), and Salmonella enterica Typhimurium 14028 (STM). Lysogeny broth (LB) medium was used to maintain and grow the bacteria for most of the bacteriological experiments unless otherwise specified. The bacterial planktonic growth was monitored by measuring the turbidity in the media at 600 nm in an ELISA reader (Tecan Sunrise ELISA Reader). Biofilm Assay. The quantitative biofilm formation was done by the method described by Srinandan et al. in a 96 well microtiter plate.53 Briefly, an overnight grown culture was diluted to 1:100 and inoculated in 200 μL LB medium containing different concentrations of the compounds in the microtiter well and was incubated for 24 h at 37 °C. The grown culture was aspirated, and wells were washed three times with 200 μL of phosphate-buffered saline (PBS) to remove the nonadherent cells. Wells were then dried in an inverted position. Adherent biofilm was stained with 200 μL of 1% crystal violet (CV) for 15 min. The unbound CV was removed by rinsing thrice with 200 μL of PBS. Further, 70% ethanol was added and incubated for 15 min to destain the CV and the absorbance of each well was measured at 600 nm using an ELISA reader as a proxy for the biomass. Live dead staining for the biofilm was done by treating the biofilm grown on glass slides with compound 4a and DMSO as control, followed by incubation for 1 h. After 1 h, the slides were rinsed with PBS. In dark conditions, the live/dead BacLight Bacterial Viability Kit was used. Slides were then observed under the fluorescence microscope. The images were then processed with autothresholding technique and intensity measured in the ImageJ software. The number of images analyzed were >30 for each sample. A Congo red (CR) plate was used to test the ability of the organism to produce exopolymeric components (EPS). YESCA media (0.5 g L−1 yeast extract, 10 g L−1 casamino acids) containing 40 μg mL−1 of CR and 20 μg mL−1 of coomassie brilliant blue (CBB). 1.5% agar was used to grow and analyze the colony morphology of UPEC. The colony morphology on the CR plate determines the ability of the organism to produce biofilm EPS. A red dry and rough (RDAR) morphology is expected if the colony produces cellulose and curli protein that is required for biofilm formation.54 A colony that does not produce cellulose or curli protein will form a smooth and white (SAW) colony, implying that it is not capable of biofilm formation. Field Emission Scanning Electron Microscopy of Biofilm. The medical catheter tubes (made up of PVC) were used for the biofilm studies. The catheter was thin film coated with AELO and the ZnCAI with a concentration of 200 μg mL−1. The bacterial cell-suspension from the overnight grown culture (107 cfu mL−1) was inoculated into the YESCA broth that was dispensed in a catheter tube and incubated for 24 h. The catheter tubes were then rinsed 3−4 times with sterile PBS (pH 7.2) and fixed with 2.5% glutaraldehyde for 24 h. The samples were dehydrated using an ethanol gradient, for 15 min each. The samples were mounted onto aluminum stubs, sputter-coated with gold− palladium (1.8 kV and 6 mA for 60 s), and desiccated. The electron micrographs were analyzed by digital field emission scanning electron microscope (JEOL JSM-6701J).

Statistical Data Analysis. All the bacteriological experiments were performed in five or more replicates. The data was analyzed using Graphpad Prism V5 software. Unpaired t tests or nonparametric tests were used to find the significance between two samples. The data with P < 0.05 was considered significant.

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RESULTS AND DISCUSSION Synthesis. Establishing and optimizing efficient materials from renewable resources has been a focus that substantially deals the need of the 21st century.55 Vegetable oils such as linseed, tung, soybean, rapeseed, castor oils, and cashew nut shell liquid (CNSL) have a wide variety of applications.56,57 Among these, we choose CNSL, an agricultural byproduct obtained from cashew tree, Anacardium occidentale L, abundantly available all over the world. Cardanol (1a), a biobased nonisoprene unit derived from CNSL composed of a rich mixture of phenolic lipids: 5% of 3-(pentadecyl) phenol (3-PDP), 50% of 3-(8Zpentadecenyl) phenol, 16% of 3-(8Z,11Z-pentadecadienyl)phenol, and 29% of 3-(8Z,11Z,14-pentadecatrienyl)phenol and unique in property by means of having an unsaturated tail, which is prone to form a polymer under atmospheric conditions.58 3-nPentadecylphenol, 1b, can be obtained by hydrogenation of distilled cardanol. Zafer and co-workers have developed Mn2+ and Co2+ based nanostructured transparent polymer film from cardanol by adopting a solid-state methodology. Antimicrobial and antibiofilm evaluation of amorphous, porous nanostructured coordination polymer against various pathogens displayed moderate activity.59 The lack of formation of the assembled structure and strong coordination unit pose several disadvantages on commercial use of such metal blended coating such as leachability, stability of coated surface, hydrophobicity balance, and availability of toxic metal to the environment. Kobayashi and co-workers have oxidatively polymerized cardanol at room temperature in the presence of various metal complexes as catalyst.60 Cross-linking and mechanical behavior of films obtained from cardanol via thermal curing process were similar to those of a commercially available CNSL−formaldehyde resin. Lipophilic lanthanide bis-phthalocyanines derived from cardanol displayed discotic liquid crystalline nature and reasonable UV-photostability.61 Electro-polymerized copper metalloporphyrin synthesized from cardanol was used for the detection of nitric oxide in aqueous solution.62 Cardanol based polymers display very important features of possible practical significance. Our interest in generating nonleachable metal based thin film coating led us to generate cardanol based Schiff base ligands 3a and 3b, and their corresponding zinc and copper complexes 4. The electrophilic aromatic substitution reaction in 3-alkylphenol 1 with paraformaldehyde in the presence of anhydrous MgCl2 and TEA produced 2-hydroxy-4-alkylbenzaldehyde 2. The allyl substituted Schiff base ligands 3a and 3b were prepared by refluxing allyl amine with compound 2 for 2 h using standard literature procedure.63 Treatment of two equivalents of Schiff base ligands 3a or 3b with zinc acetate and copper acetate in the presence of triethylamine resulted in the formation of corresponding zinc and copper complexes 4a−d, respectively (Scheme 1). After completion of complexation, the entire contents were poured into ice water and extracted using dichloromethane. Both ligands and zinc complexes were characterized using 1H and 13C NMR spectral techniques. The use of NMR for the characterization of copper complex is limited because of its paramagnetic character, whereas the product formation was confirmed by FT-IR and mass spectral analysis. The 1H NMR spectra of zinc 439


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ACS Sustainable Chemistry & Engineering Scheme 1. Synthesis of Zinc and Copper Complexes Used to Derive ZnCAI and CuCAI Thin Films

Figure 1. 1H NMR spectra of (a) compound 3a and (b) compound 4a in CDCl3 at 298 K.

ligand 3a and its complex 4a are 1626 and 1614 cm−1 respectively. The shift in stretching frequency to lower frequency indicates the coordination of Schiff base 3a via azomethine nitrogen to the metal (See ESI). Linseed oil is one of the vegetable oils extensively used as a medium for coating, due to its capacity to form a continuous thin film with good optical and mechanical properties. Natural linseed oil contains triglycerides consisting of a mixture of linolenic, linoleic, and oleic acids prone to polymerize at room temperature.

based complex 4a displayed very small upfield shift for imine signal in comparison with its ligand, which strongly depend upon the coordination metal. Moreover, the coordination of phenolic −OH to the metal shifts the aromatic signals with respect to those in free ligand (Figure 1). Formation of the complex was further confirmed by 13C NMR spectral studies. FT-IR was also used to investigate the binding of Schiff base ligand to metal in the complexes by comparing the free ligand with that of metal complexes. The CN stretching vibrations of 440


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ACS Sustainable Chemistry & Engineering Scheme 2. Synthesis of ELO, AELO, ZnCAI, and CuCAI Thin Films

Figure 2. UV−vis absorption spectra of (a) compound 3a, 4a, 4c, Zn(OAc)2, and Cu(OAc)2 dissolved in methanol (concentration: 1 × 10−5 M) and (b) aliquot withdrawn after 72 h and 60 days from the beaker in which coated slides were immersed in water.

solutions at room temperature for 60 days. In this study, a simple quantitative technique to determine the leaching of copper or zinc or any other composite from coated surface was developed by using UV−vis spectroscopy (Figure 2). UV−vis spectra of compounds 3a displayed absorbance peaks at 261 and 401 nm. Compound 3a on complexation with Zn2+ and Cu2+ showed absorbance maxima at 275 and 364 nm. The absorbance peak of Zn(OAc)2 and Cu(OAc)2 appears at 234 and 265 nm, respectively. Eluates obtained at different intervals of time were subjected to UV−vis spectral analysis. The absence of absorption bands corresponding to Cu2+, Zn2+, 3a, 4a, and 4c clearly suggests that there was no leaching of thin film into water. This result clearly suggests the highest degree of cross-linking of complexes with AELO, wherein metal was immobilized by a coordinate bond. Morphological Analysis. Morphologies of ZnCAI and CuCAI thin films were analyzed by FE-SEM as shown in Figure 3. The micrograph reveals the uniform coating without any visible cracks, even at the nanoscale level. A large number of spherical nanoparticles with a diameter of 10−12 nm appear in a hierarchical manner, and well dispersed in the coated surface. Uniformly distributed spherical structures assembled in the form of a fiberlike structure were observed, which is because of the interaction between hydroxyl group and Schiff base nitrogen with Cu2+ and Zn2+ ions. Moreover, the polar group of ZnCAI and CuCAI

The cross-linking nature of the polymer formed by linseed oil depends upon the degree of unsaturation present in it. Aiming at the development of antimicrobial thin film coating material, we have synthesized acrylated epoxidized linseed oil (AELO) via epoxidation of linseed oil followed by the ring opening of oxirane group using acrylic acid (Scheme 2). Thin film coating material was prepared by consistently mixing metal complexes 4a−d derived from cardanol with AELO. The materials obtained by dispersing 4a in AELO (ZnCAI), 4b in AELO (ZnPAI), 4c in AELO (CuCAI), and 4d in AELO (CuPAI) were coated on a glass surface and their curing behavior studied. After applied on a glass surface using pistol type air brush, ZnCAI and CuCAI were cured within 24 h because of oxygen mediated polymerization of 4a and 4c with AELO, whereas ZnPAI and CuPAI failed to polymerize even after 5 days because of the absence of unsaturation. On the basis of the curing data, we have chosen ZnCAI and CuCAI for our further investigation. Leaching Studies. In the past few decades, substantial methods have been developed in order to predict and quantify metal or antimicrobials that is being leached from coated surfaces. To investigate the leaching behavior of immobilized metal or compounds 3a, 4a, and 4c from the coated surface, ZnCAI and CuCAI coated glass slides were immersed in distilled water, tap water, seawater, and acidic (pH = 4) and basic (pH = 10) buffer 441


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ACS Sustainable Chemistry & Engineering

Figure 3. FESEM images of (a and b) ZnCAI thin film and (c and d) CuCAI thin film.

Figure 4. (a and b) Frequency sweep of thin film coating material (a) ZnCAI and (b) CuCAI; (c and d) amplitude sweep of (c) ZnCAI and (d) CuCAI, respectively.

the presence of Zn, C, N, and O elements, and Cu, C, N, and O elements in ZnCAI and CuCAI, respectively (see ESI). Rheological Analysis. The rheological behavior of thin film coating materials was evaluated using oscillation measurement.64 If thin film coating material is in elastic nature, then the stress and strain waves will be in phase, whereas the material is in viscous

prevents the formation of nanoparticle agglomerates. The presence of unsaturated long alkyl chain was responsible for the formation assembled thin film with AELO. Thus, successful uniform dispersion of zinc and copper nanoparticles was achieved by assembly process without the use of particle surface treatments and modifiers. EDAX spectrum of thin films confirms 442


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0.022 097 and 0.1424 Pa−1, respectively. The flow characteristics of ZnCAI and CuCAI were determined by measuring shear rate under controlled increasing rate of stress (Figure 5c). From Figure 5c, we could clearly observe the decrease in viscosity with increasing shear rate, which is more suitable for thin film coating applications. XRD Analysis. Both small-angle X-ray diffraction (SAXD) and wide-angle X-ray diffraction (WAXD) experiments was employed to acquire the information about the assembly of ZnCAI and CuCAI in AELO thin films. SAXD patterns of polymerized linseed oil,66 polymerized AELO,1 ZnCAI, and CuCAI thin films are displayed in Figure 6. Generally, the interlayer distance between alkyl chain in the amorphous crosslinked linseed oil and AELO thin film is observed around 0.44 nm (2θ = 20°). The XRD diffractogram of linseed oil and AELO films displayed a single broad reflection at 2θ = 20° , which indicates the amorphous nature of the thin film. In addition to the broad peak at 2θ = 20°, ZnCAI and CuCAI thin films displayed 2θ = 13.6° and 2θ = 12.2° respectively, which is attributed to the molecular assembly of ZnCAI and CuCAI in the polymerized linseed oil. WAXD of ZnCAI and CuCAI thin films also displayed peaks between 2θ = 12−15° and at 2θ = 20°, further confirming the existence of assembled structure in the amorphous linseed oil thin film (see ESI). Single crystal X-ray diffraction of compounds 5 and 6,63 which is structural analog of ZnCAI and CuCAI, is shown is Figure 6e,f. Even though these compounds did not show any intra- or intermolecular hydrogen bonding, the existence of molecular assembly is due to the van der Waals interactions (see ESI). Thermal Analysis of Thin Films. The thermal behavior of metal based thin films were investigated using TGA and DSC studies. The TGA thermograms of ZnCAI and CuCAI thin films displayed the weight loss at around 200−400 °C, which is attributed to the degradation of the material (Figure 7a). DSC curves of ZnCAI and CuCAI thin films during the first heating

phase then the stress and strain will be out of phase. Generally, most of the thin film coating materials exhibit a viscoelastic behavior, which lies between elastic and viscous character.65 In Figure 4, we have plotted storage modulus, G′ (elastic) and loss modulus, G″ (viscous) as a function of angular frequency for thin film coating material (ZnCAI and CuCAI). It has been observed that the elastic modulus is slightly greater than that of the viscous modulus and both displayed a significant increase as a function of frequency sweep. In the rheology experiment, tan δ is the ratio of viscous to elastic behavior, which provides an idea about the nature of the thin film coating material (Figure 4a,b). Generally, thin film coating materials with tan δ < 1 explains the highly ordered structure (i.e., molecules are highly associated via weak forces and materials with higher tan δ value (>10)) implies the fluid like character, wherein the molecules are in disassembled form. For assembled thin film coating material, intermediate tan δ is desirable for the formation of stable uniform thin film coating on the desired substrates. The tan δ value appears at 5.011 and 3.621 for ZnCAI and CuCAI clearly demonstrates the existence of assembled structure, which undergoes further polymerization in the presence of atmospheric oxygen. The loss factor, tan δ does not vary dramatically with increasing frequency, indicating the existence of storage stability. To study the effect of strain amplitude on the thin film coating material, we have carried out a strain sweep experiment on ZnCAI and CuCAI, which is in good agreement with the above discussion (Figure 4c,d). Creep is an invaluable technique, which provides clear insight about the behavior of coating a material at small constant stress for a long period of time. We have investigated the recovery of ZnCAI and CuCAI from the disassembly by removing the applied strain. The important parameters such as zero shear viscosity (η0) and equilibrium compliance (Je0), which is a measure of elastic recoil, have been calculated from creep curves (Figure 5a,b). Zero shear viscosity (η0) and equilibrium compliance (Je0) for ZnCAI and CuCAI are 17.91 and 97.67 Pa·s, and

Figure 5. Creep behavior of thin film coating material (a) ZnCAI; (b) CuCAI. (c) Flow characteristics of coating materials. 443


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Figure 6. SAXD of (a) polymerized linseed oil, (b) polymerized ALEO,(c) polymerized ZnCAI thin film, and (d) CuCAI thin film. (e) Molecular assembly of compound 5 and (f) molecular assembly of compound 6.

Figure 7. (a) TGA thermograms of ZnCAI and CuCAI thin films and (b) DSC curves of ZnCAI and CuCAI thin films.

cycle showed exothermic peaks at 159 and 259 °C, and 173 and 259 °C, respectively (Figure 7b). The exothermic peak observed at 259 °C is for polymerized AELO, and the peaks at 159 and 173 °C are for assembled structures that existed in the polymerized AELO. Thermal studies reveal the presence of assembled structure in amorphous AELO thin film.

Contact Angle and Surface Analysis of Thin Film. Contact angle measurement is a versatile method for the detection of molecular arrangement in a polymer or an assembled thin film surface. Hydrophobicity is a repulsive force exerted by the alkyl chain on surface of the film and hydrophilicity is the affinity of thin film surface to the water by means of forming 444


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Figure 8. Images displaying the static contact angle of (a) polymerized linseed oil, (b) AELO thin film, (c) ZnCAI thin film, and (d) CuCAI thin film.

Figure 9. Influence of varying concentrations of the compounds on the planktonic growth of the pathogens (a) uropathogenic E. coli, (b) Typhimurium S. aureus, (c) P. aeruginosa, and (d) S. enterica. Error bars represent the SEM.

concentration; however, the growth inhibition was clearly dose dependent (Figure 9a). The compounds 4a and 4c were consistently significant (Unpaired t test, P < 0.01, n = 5) at increasing concentrations than the precursor compound 3a in displaying the growth inhibitory effects of UPEC and S. aureus (SA) (Figure 9b). For P. aeruginosa (PA), the compounds 3a, 4a, and 4c displayed an increase in growth inhibition following a nearly arithmetic progression. Among these compounds, 4a showed a significant inhibition of planktonic growth of PA (Unpaired t test, P = < 0.0001, n = 5) at 500 μg concentration. The Cu(OAc)2 displayed 20%−40% inhibition in growth of PA at all concentrations used in this study. The compound 3a was showing the antimicrobial effect on S. enterica Typhimurium (STM) in a dose-dependent manner with 500 μg showing a complete growth inhibition (Figure 9d). However, the compounds 4a and 4c display substantial antimicrobial effect at high

hydrogen bond. To access the wetting properties of ZnCAI and CuCAI thin films, contact angle was measured. As shown in Figure 8, a double distilled water droplet placed on top of the surface of polymerized linseed oil and AELO thin films, which displayed a static contact angle of 65.8° and 79.4°, respectively. The formation of an assembled structure by compound 4a and 4c in AELO, i.e., ZnCAI and CuCAI thin films, further enhances the water repellent to 92 and 94.5°, respectively. Contact angle measurement of ZnCAI and CuCAI thin films reveals the hydrophobic character and the surface developed out of these compounds were repulsive to water, suitable for biomedical applications. Antibacterial Studies. The compounds 3a, 4a, and 4c derived from renewable resources were preliminarily tested for their influence on the growth of prominent pathogenic bacteria. It was observed that the compounds 4a and 4c inhibited the growth of uropathogenic E. coli (UPEC) to nearly 100% at 500 μg 445


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ACS Sustainable Chemistry & Engineering concentration of 500 μg. Altogether, it can be inferred that the compound 4a and 4c are more effective against UPEC planktonic growth and the compounds 3a, 4a, and 4c against SA at higher concentration. However, the compound 4a showed their inhibitory effects against the growth PA and 3a against STM in a dose-dependent fashion. The MIC of the compounds 4a and 4c on planktonic growth are shown in the Table 2.

Both the compounds had significant biofilm inhibiting activity, whereas 4a had a pronounced effect on UPEC and STM and 4c was effective on SA (Figure 10). The minimum concentration that was necessary to inhibit biofilm formation by 4a was 100 μg mL−1 for UPEC, SA, and PA, whereas the STM biofilm was inhibited at nearly 90% at 500 μg mL−1 as compared to the control (Figure 10 and Table 2). The minimum concentration required for complete biofilm inhibition by compound 4c was 350 μg mL−1 in UPEC, 100 μg mL−1 in SA and PA, and 500 μg mL−1 in STM (Figure 10 and Table 2). Further, we have performed the biofilm inhibition assay and bactericidal property of compound 4a on the grown biofilm. The biofilm biomass significantly reduced (nonparametric t test, P = < 0.001, n = 5) on treatment of compound 4a to the mature biofilm grown for 24 and 48 h (Figure S23). Nevertheless, the values suggest that the compound have a moderate bactericidal effect on the already formed biofilm, but effectively inhibited its formation. Indwelling catheter associated urinary tract infections (CAUTI) are predominantly caused by UPEC biofilm.69 The biofilm formation in E. coli is potentiated by the production of EPS components consisting predominantly of cellulose and curli proteins. The cells that produce these EPS components forms a red dry and rough (RDAR) colony morphology on Congo red (CR) agar.70 Cells that does not produce EPS forms a smooth and white (SAW) colony. The SAW morphotypes are incapable of biofilm formation. To investigate the influence of compounds 3a and 4a on the production of biofilm EPS components, we grew the UPEC in the CR agar media containing 3a and 4a. It was observed that the compounds 3a and 4a did influence the production of EPS (Figure 11). Smooth colonies were formed, and the periphery was made up of SAW cells, whereas the cores of the colony were red colored. This result implies that the secretion of the EPS is mainly targeted by the compounds 3a and 4a leading to a significant reduction in the biofilm formation (Figures 10 and 11). To investigate the potential application of 4a, we coated AELO

Table 2. Minimum Inhibitory Concentration on Planktonic Growth and Minimum Biofilm Inhibitory Concentration of the Compounds 4a and 4c Minimum Inhibitory Concentration (MIC) in (μg ml−1)

Minimum Biofilm Inhibitory Concentration (MBIC) in (μg ml−1)

Bacterial Species

Compound 4a

Compound 4c

Compound 4a

Compound 4c

uropathogenic E. coli S. enterica Typhimurium S. aureus P. aeruginosa

150

ND

100

350

450

500

ND

ND

100 100

100 100

100 100

100 100

Influence on Biofilm Formation. Biofilm is the predominant lifestyle of bacteria in its natural environment. Bacteria aggregate, stick to the surfaces, and enmesh themselves by producing Exo-Polymeric Substances (EPS). Generally, the biofilm bacteria are resilient to environmental stresses. For example, the biofilm bacteria are almost 2000 times more tolerant to antimicrobials than their planktonic counterparts. It is estimated by the Centers for Disease Control (CDC) that 65% of the bacterial infections are in the biofilm mode,67 but according to National Institutes of Health (NIH), the biofilm is responsible for 80% of bacterial infections (NIH 2002).68 We investigated the potential of the synthesized compounds 4a and 4c to limit the biofilm formation, as it is also the persistent state for the bacteria.

Figure 10. Influence of the compounds, 4a (white bar) and 4c (black bar) on biofilm formation of the pathogenic organisms. (a) uropathogenic E. coli, (b) S. aureus, (c) P. aeruginosa, (d) S. enterica Typhimurium. Error bars represent the SEM. 446


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Figure 11. Colony growth of UPEC on Congo red agar plates with (a) control plate (media containing no exogenous compounds), (b) media containing DMSO, (c) media containing 200 μg of 3a, and (d) media containing 200 μg of 4a.

Figure 12. Scanning electron micrographs of biofilm growth of UPEC on surface of catheter tubes (a) AELO coated surface and (b) ZnCAI coated surface.

thin film coating material inhibited effectively the formation of uropathogenic E. coli bacterial film. Overall, the derived thin film coating material could be potentially used to develop antibacterial urinary catheter tubes with intrinsic hydrophobicity. These thin films also hold promising applications in industrial and biomedical sectors.

(control) and ZnCAI (derived from compound 4a in AELO) in a urinary catheter made of PVC, and incubated the UPEC cells. Both the urinary catheter tubes were processed to test for the biofilm formation. The SEM images of the catheter coated with the ZnCAI thin film displayed no apparent biofilm microcolony formation (Figure 12). Further, the catheter was also incubated for 10 days in the culture broth, but no biofilm could be found, suggesting that the ZnCAI is potentially effective against biofilm formation in the indwelling urinary catheters (Figure S24).

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ASSOCIATED CONTENT

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b01806.

CONCLUSION In this study, we focused on the synthesis of cardanol based metal complexes having an unsaturated hydrophobic unit and AELO prepared via the epoxidation of the double bonds followed by acrylation. The metal complexes dispersed in AELO undergo air oxidation under atmospheric conditions to form an assembled thin film, even in the absence of initiators. The leaching behavior, morphology, thermal, mechanical, antibacterial, and antibiofilm properties of the resulting thin film were analyzed. UV−vis measurements corroborated the nonleaching behavior of thin film coating under various conditions. The mechanical properties of assembled thin film coating material were described using oscillatory rheology. The formation of the assembled thin film structure was identified by FE-SEM and SAXD analysis. The thin film showed intrinsic hydrophobicity and thermal stability. These compounds exhibited antimicrobial activity against both Grampositive and Gram-negative bacteria, and the effectiveness increased with the increase in concentration. Notably, biobased

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H and 13C NMR spectra, FT-IR spectra, FE-SEM images, WXRD of thin films, EDAX spectra of thin films, short contacts existing in compounds 5 and 6 (PDF)

AUTHOR INFORMATION

Corresponding Author

*S. Nagarajan. Fax: 04362264120. Tel: 04362304270. E-mail: [email protected]; [email protected]. Author Contributions

The paper was written through the contributions of all authors. M.S. and C.S.S. contributed toward the antibacterial and antibiofilm investigation. All authors have given approval to the final version of the paper. Notes

The authors declare no competing financial interest. 447


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ACKNOWLEDGMENTS This work was financially supported by the Department of Science and Technology (IFA-CH-04 and #SB/FT/CS-024/ 2013), India and Board of Research in Nuclear Science (#37(1)/ 20/47/2014), Department of Atomic Energy. Financial support to this work was also provided by the Science and Engineering Research Board under the Start-Up grant scheme to C.S.S. (SB/YS/LS-283/2013). The DST-FIST funded (SR/FST/ETI331/2013) fluorescence microscopy facility is acknowledged. S.N. sincerely thanks SASTRA University for financial support under DESH-VIDESH scheme.

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ABBREVIATIONS AELO, acrylated epoxidized linseed oil EPS, exopolymeric components CAI, cardanyl-N-allyl imine CNSL, cashew nut shell liquid CR, Congo red CuCAI, copper complex of CAI CuPAI, copper complex of PAI ELO, epoxidized linseed oil LB, Lysogeny broth PA, Pseudomonas aeruginosa PAI, n-pentadecyl-N-allyl imine RDAR, red dry and rough SA, Staphylococcus aureus STM, Salmonella enterica Typhimurium UPEC, uropathogenic Escherichia coli ZnCAI, zinc complex of CAI ZnPAI, zinc complex of PAI

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