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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, Chakravarthy S Srinandan, C. Uma Maheswari, and Subbiah Nagarajan ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01806 • Publication Date (Web): 07 Nov 2016 Downloaded from http://pubs.acs.org on November 9, 2016

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ACS Sustainable Chemistry & Engineering

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*† †

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. *E-mail: [email protected], [email protected]. KEYWORDS: Renewable resource, Assembled thin film, Cardanol, Antibacterial, Anti-biofilm, Linseed oil.

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

ACS Paragon Plus Environment

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phenolic compound having unsaturated hydrophobic tail isolated from cashew fruits, and linseed oil, a vegetable oil and an important bio-based 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 epoxidised 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 wide range of applications. 1H NMR and FT-IR analysis revealed the existence of strong interaction between ligand and metal, which pave a way to develop nonleachable metal based thin film coating. 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 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 hold promise to be used as metal enabled, non-leachable coating materials for public bacterial threats, food and biomedical applications. In particular, this material can be potentially used for developing urinary catheter tubes with anti-bacterial properties.

1. INTRODUCTION


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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, 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 to air form an excellent scratch free coating by simple air oxidation process via 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 non-leachable antibacterial thin film coating material for various applications discussed below. Bacterial infections are considered as a major health issue causing increased number of health risks especially in hospitals, public toilets, railway stations, schools, sanitary facilities, 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 but due to its overuse, bacteria has evolved resistance thereby reducing the efficacy of antibiotics.23 Moreover, bacteria predominantly attach to biotic or abiotic surfaces and display tolerance towards 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,


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antimicrobial treatment of such 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 non-leachable antimicrobial thin film coating material from readily available renewable raw material to overcome microbial threats in public, biomedical and industrial environments. Nano-biomedical thin film techniques are more attractive to overcome the limitations of existing tools.31,32 Zinc and copper plays vital role in various biological processes, and researchers have exponentially explored the antimicrobial activity of copper and zinc complexes.33-35 At commercial level, zinc and copper has been blended in consumer products and present as zinc pyrithione in shampoo,36 zinc lactate and zinc sulfate in toothpaste and mouthwash,37 zinc acetate and zinc gluconate in mineral supplements.38 Moreover, EPA has approved wide variety of copper based antimicrobial material with public health benefit such as antimicrobial copper-alloy touch surfaces, etc.39 Most of the previous work in this area concerned more on metal nanoparticles and metal complexes of copper, silver, gold and platinum.40-42. Metal incorporated in coating material could possibly inhibit biofilm formation of bacteria and could be employed as an antimicrobial and antifouling agents. There are several disadvantages on commercial use of metal blended coating such as leachability, stability of coated surface, availability of metal to the environment.43-47 However, in order to overcome the existing limitations on the development of antimicrobial thin film surface, in this report we discuss the synthesis and assembly of zinc and copper based Schiff base complexes derived from renewable resource, cardanol in Acrylated Epoxidised Linseed Oil


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(AELO) and subsequent use in the development of antimicrobial thin film coating material has been explored. 2. EXPERIMENTAL SECTION 2.1 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 pre-coated 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. 2.2 Characterization methods. 1H- and

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C-NMR spectra were recorded on a Bruker Avance

300 MHz instrument in either CDCl3 or CDCl3 with few drops of DMSO-d6 at room temperature. Chemical 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. Infrared spectrum was obtained using FTIR Shimadzu 8000 Spectrometer as KBr pellets in the spectral range of 4000 cm-1 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.


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2.3 Purification of Cardanol. The major constituent present in cashew nut shell liquid (CNSL) is cardanol 1a, a bio-based non isoprene lipid, comprising of phenolic lipid mixture: 5% of 3-npentadecylphenol (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 mm Hg to get cardanol.4851

Cardanol was obtained as pale yellow liquid, which darkens during storage. After a second

distillation, mixture of cardanol mono-, di- and tri-ene was obtained and were used for the preparation of antimicrobial thin film coating material. Hydrogenated cardanol, 1b can be easily obtained as pure compound by hydrogenation of the double bonds present in the side chain of distilled cardanol. 2.4 Synthesis 2.4.1 General procedure for the synthesis of 2-hydroxy-4-alkylbenzaldehydes 2a and 2b. To a mixture of 3-alkyl phenol (4 mmol), anhydrous magnesium chloride (6 mmol) and triethylamine (15 mmol) in acetonitrile (25 mL), dry paraformaldehyde (35 mmol) was added and 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 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);

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C NMR (75 MHz, CDCl3) δ = 195.8, 161.8, 153.8, 133.6, 130.0, 129.7,


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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 white crystalline solid; yield = 92%. 1H 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);

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C

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. 2.4.2 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), allylamine (15.0 mmol) was added 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 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), 5.955.80 (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);

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C NMR (CDCl3, 75

MHz) δ = 165.4, 161.4, 148.0, 135.1, 131.2, 130.3, 129.8, 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.


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Compound 3b. Obtained as 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).

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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. 2.3.3 General procedure for the synthesis of zinc complexs 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 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,


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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; observed = m/z 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);

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C 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; observed = m/z 805.0579. 2.3.4 General procedure for the synthesis of copper complexs 4c and 4d. To the Schiff base ligand 3a or 3b (10.0 mmol) dissolved in methanol (4.0 mL), triethylamine (10.0 mmol) and copper acetate (5.0 mmol) was added and refluxed for 2h. After monitoring the progress of the reaction using TLC, the crude product was extracted using dichloromethane and dried using anhydrous Na2SO4. Compound 4c. Obtained as green viscous liquid. HRMS (ESI): m/z calculated for C50H76N2O2Cu [M+MeOH]+ = 831.5454; observed = m/z 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). HRMS (ESI): Compound 4d. Obtained as green solid; HRMS (ESI): m/z calculated for C50H80N2O2Cu [M+H]+ = 804.5583; observed = m/z 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).


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2.3.5 General procedure for the synthesis of epoxidised linseed oil (ELO). Epoxidation of linseed oil was carried out in a three neck 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), hydrogen peroxide (12.24 g, 108 mmol) was added slowly and refluxed at 60 oC 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 solvent was removed under reduced pressure to get the epoxidised linseed oil. 1H NMR spectra of ELO is given in supporting information. 2.3.6 Acrylation of epoxidised Linseed oil. Epoxidised linseed oil was acrylated via ring opening of 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 epoxidised linseed oil (0.891 g, 1 mmol) and acrylic acid (0.4 mL, 6 mmol), triethyl amine (0.14 mL, 1 mmol) and hydroquinone (0.110, 1 mmol) were added. The mixture was then heated at 110 oC 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 epoxidised linseed oil (AELO). 1H NMR spectra of AELO is given in supporting information. 2.4 Preparation of metal based antimicrobial thin film coating. Different curable formulations 5%, 10% and 20% were prepared by consistently mixing 5 mg, 10 mg 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 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


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degree of unsaturation present in AELO, compound 4a and 4c makes them sensitive to autooxidation under atmospheric condition within 2 days, whereas precursor compound 3a took little long time to cure. Abbreviation and composition of auto-oxidizable thin film coating materials are given in Table 1. Table 1. Abbreviation and nature of thin film coating materials prepared. S.

Compounds

Abbreviations used

No.

Nature of thin film formation with AELO (Time duration for curing: 2448 h)

1 2 3 4 5 6

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

CAI

Scratch free thin film#

PAI

Viscous nature

ZnCAI

Zinc enabled CAI thin film

ZnPAI

Viscous nature

CuCAI

Copper enabled CAI thin film

CuPAI

Viscous nature

Note: 20% curable formulation was used for thin film formation studies.

#

Time duration for

curing: > 48 h. 2.5 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. 2.6 Morphological analysis. Morphological analysis of thin film coated on glass slide was studied using JEOL JSM-6701F ultrahigh resolution field emission scanning electron microscope


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coupled to an Energy Dispersive X-ray (EDX) detector. The coated slides were cut into small piece of size 1cm x 1cm and were coated with gold prior to imaging. 2.7 Mechanical stability test. 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 thin film coating on glass slide and then detached, this process was repeated up to 20 times. 2.8 Leaching Studies. Leaching of copper or zinc or compound 3a from ZnCAI and CuCAI thin film was studied by suspending individual thin film coated glass slide in a beaker containing 10 mL of tap water, distilled water and sea water and also at pH = 4 and pH = 10. Immediately after suspending glass plate in water, 2 mL of aliquot was withdrawn and scanned using UV-vis spectrophotometer, thereafter leaching was tracked at regular interval of once in a five days. After recording UV-vis spectra, aliquot was transferred back to the beaker. UV-vis spectral data obtained were then used for comparison of leaching behavior of thin films. 2.9 Rheological measurement. Flow behavior of 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.


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2.10 Contact angle measurement. Contact angle measurements for polymerized linseed oil, polymerized AELO, ZnCAI thin film and CuCAI thin film were performed using goniometer at room temperature. 2.11 Bacterial strains and culture conditions. Strains used in this work are Uropathogenic E.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 ELISA reader (Tecan Sunrise ELISA Reader). 2.12 Biofilm assay. The quantitative biofilm formation was done by the method described by Srinandan et al. in 96 well microtiter plate.53 Briefly, an overnight grown culture was diluted to 1:100 and inoculated in 200 µL LB medium containing different concentration 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 non-adherent cells and wells were 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 de-stain 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 1h. After 1 h, the slides were rinsed with PBS. In dark condition, the live dead backlight® stain was added according to the manufacturer’s instruction. Slides were then observed under the fluorescence microscope. The


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images were then processed with auto-thresholding technique and intensity measured in the ImageJ software. The number of images analyzed were >30 for each sample. Congo red (CR) plate was used to test the ability of the organism to produce exopolymeric components (EPS). YESCA media (0.5 gL-1 yeast extract, 10 gL-1 casamino acids) containing 40 µg mL-1 of CR and 20 µg mL-1 of coomassie brilliant blue (CBB) and 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 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. 2.13 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. Bacterial cell-suspension from the overnight grown culture (107 cfu mL-1) was inoculated into the YECAS 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 JSM6701J). 2.14 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


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Non-parametric tests were used to find the significance between two samples. The data with P < 0.05 was considered significant. 3 Results and discussion 3.1. 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 bio-based non-isoprene unit derived from CNSL comprised of a rich mixture of phenolic lipids: 5% of 3-(pentadecyl) phenol (3-PDP), 50% of 3-(8Z-pentadecenyl) phenol, 16% of 3-(8Z,11Zpentadecadienyl) phenol and 29% of 3-(8Z,11Z,14-pentadecatrienyl) phenol and unique in property by means of having unsaturated tail, which is prone to form a polymer under atmospheric conditions.58 3-n-Pentadecylphenol, 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 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 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 Crosslinking and mechanical behavior of films obtained from cardanol via thermal curing process were similar to those of a commercially available CNSL-formaldehyde resin.


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Lipophilic lanthanide bis-phthalocyanines derived from cardanol displayed discotic liquid crystalline

nature

and

reasonable

UV-photo-stability.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 non-leachable 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 presence of anhydrous MgCl2 and TEA produced 2-hydroxy-4alkylbenzaldehyde 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).


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Scheme 1 Synthesis of zinc and copper complexes used to derive ZnCAI and CuCAI thin films. 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 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.


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Figure 1. 1H NMR spectra of (a) compound 3a and (b) compound 4a in CDCl3 at 278 K. 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 vibration of ligand 3a and its complex 4a are 1626 cm-1 and 1614 cm-1 respectively, shift in stretching frequency to lower frequency indicate 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. Naturally linseed oil contains triglycerides consisting of a mixture of linolenic, linoleic and oleic acids prone to polymerize at room temperature. The cross-linking nature of the polymer formed


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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 epoxidised linseed oil (AELO) via epoxidation of linseed oil followed by the ring opening of oxirane group using acrylic acid (Scheme 2).

Scheme 2. Synthesis of ELO, AELO, ZnCAI and CuCAI thin films. Thin film coating material was prepared by consistently mixing metal complexes 4a-4d derived from cardanol with AELO. The material obtained by dispersing 4a in AELO (ZnCAI), 4b in AELO (ZnPAI), 4c in AELO (CuCAI) and 4d in AELO (CuPAI) were coated on glass surface and studied their curing behavior. After applying on 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. Based on the curing data, we have chosen ZnCAI and CuCAI for our further investigation.


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3.2 Leaching studies. Past few decades’ substantial methods have been developed in order to predict and quantify metal or antimicrobials that is being leached from coated surfaces. In order 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, sea water and acidic (pH = 4) and basic (pH = 10) buffer solution 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 peak 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. Absence of absorption band corresponding to Cu2+, Zn2+, 3a, 4a and 4c clearly suggest 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 coordinate bond.

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


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3.3. 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 nanoscale level. A large number of spherical nanoparticles with a diameter of 1012 nm appears in a hierarchical manner, and well dispersed in the coated surface. Uniformly distributed spherical structures assembled in the form of fiber-like 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 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 the presence of Zn, C, N, and O elements, and Cu, C, N and O elements in ZnCAI and CuCAI respectively (SEE ESI).

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


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3.4 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 phase then the stress and strain will be out of phase. Generally, most of the thin film coating materials exhibits 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 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 & 4b). 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. In order to study the effect of strain amplitude on the thin film coating material we have carried out strain sweep experiment on ZnCAI and CuCAI, which is in good agreement with the above discussion (Figure 4c & 4d).


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Figure 4 (a & b) Frequency sweep of thin film coating material (a) ZnCAI and (b) CuCAI; (c & d) amplitude sweep of (c) ZnCAI and (d) CuCAI respectively. Creep is an invaluable technique, which provides clear insight about the behavior of coating a material at small constant stress for 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 has been calculated from creep curves (Figure 5a and 5b). Zero shear viscosity (η0) and equilibrium compliance (Je0) for ZnCAI and CuCAI are 17.91 and 97.67 Pa.S, and 0.022097 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.


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Figure 5. Creep behavior of thin film coating material (a) ZnCAI; (b) CuCAI and (c) Flow characteristics of coating materials. 3.5 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 pattern of polymerized linseed oil,66 polymerized AELO,1ZnCAI and CuCAI thin films are displayed in Figure 6. Generally, the interlayer distance between alkyl chain in the amorphous cross-linked linseed oil and AELO thin film is observed around 0.44 nm (2θ = 20o). XRD diffractogram of linseed oil and AELO films displayed single broad reflection at 2θ = 20o indicates the amorphous nature of the thin film. In addition to the broad peak at 2θ = 20o, ZnCAI and CuCAI thin films displayed 2θ = 13.6o and 2θ = 12.2o respectively, which is attributed to the molecular assembly of ZnCAI and CuCAI in


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the polymerized linseed oil. WAXD of ZnCAI and CuCAI thin films also displayed peaks between 2θ = 12-15o and at 2θ = 20o 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 and 6f. Even though these compounds didn’t show any intra- or intermolecular hydrogen bonding, the existence of molecular assembly is due to the van der Waals interactions (See ESI).


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Figure 6 SAXD of (a) polymerized linseed oil; (b) polymerized ALEO; (c) polymerized ZnCAI thin film, (d) CuCAI thin film, (e) molecular assembly of compound 5 and (f) molecular assembly of compound 6. 3.6 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 oC, which is attributed to the degradation of the material (Figure 7a). DSC curves of ZnCAI and CuCAI thin films during the first heating cycle showed exothermic peaks at 159 and 259 oC, and 173 and 259 oC respectively (Figure 7b). The exothermic peak observed at 259 oC is for polymerized AELO and the peaks at 159 and 173 o

C are for assembled structures existed in the polymerized AELO. Thermal studies reveal the

presence of assembled structure in amorphous AELO thin film.

Figure 7. (a) TGA thermograms of ZnCAI and CuCAI thin films and (b) DSC curves of ZnCAI and CuCAI thin films 3.7 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 hydrogen


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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 65.8o and 79.4o respectively. The formation of assembled structure by compound 4a and 4c in AELO i.e. ZnCAI and CuCAI thin films further enhance the water repellent to 92 and 94.5o respectively. Contact angle measurement of ZnCAI and CuCAI thin films reveal the hydrophobic character and the surface developed out of these compounds were repulsive to water, suitable for biomedical applications.

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. 3.8 Antibacterial studies. The compounds 3a, 4a and 4c derived from renewable resource 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 concentration, however, the growth inhibition was clearly dose dependent (Figure 9a). The compounds 4a and 4c were consistently significant (Unpaired t test, P

Intrinsic Hydrophobic Antibacterial Thin Film from ... - ACS Publications

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