Comparative evaluation of antimicrobial activity of different types of ionic liquids

Comparative evaluation of antimicrobial activity of different types of ionic liquids

Materials Science & Engineering C 104 (2019) 109907 Contents lists available at ScienceDirect Materials Science & Engineering C journal homepage: ww...

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Materials Science & Engineering C 104 (2019) 109907

Contents lists available at ScienceDirect

Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec

Comparative evaluation of antimicrobial activity of different types of ionic liquids ⁎

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Walter Florioa, Stefano Becherinib, Felicia D'Andreab, , Antonella Lupettia, , Cinzia Chiappeb, Lorenzo Guazzellib a b

Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, Università di Pisa, Pisa, Italy Dipartimento di Farmacia, Università di Pisa, Pisa, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords: Ionic liquids Antimicrobial activity Hemolysis Biofilm Bactericidal activity

In order to identify most suitable ionic liquids (ILs) for potential applications in infection prevention and control, in the present study we comparatively evaluated the antimicrobial potency and hemolytic activity of 15 ILs, including 11 previously described and four newly synthesized ILs, using standard microbiological procedures against Gram-positive and Gram-negative bacteria. ILs showing the lowest minimum inhibitory concentration (MIC) were tested for their hemolytic activity. Three ILs characterized by low MIC values and low hemolytic activity, namely 1-methyl-3-dodecylimidazolium bromide, 1-dodecyl-1-methylpyrrolidinium bromide, and 1dodecyl-1-methylpiperidinium bromide were further investigated to determine their minimum bactericidal concentration (MBC), and their ability to inhibit biofilm formation by Staphylococcus aureus or Pseudomonas aeruginosa. Killing kinetics results revealed that both Gram-positive and Gram-negative bacteria are rapidly killed after exposure to MBC of the selected ILs. Furthermore, the selected ILs efficiently inhibited biofilm formation by S. aureus or P. aeruginosa. To our knowledge, this is the first systematic study investigating the antimicrobial potential of different types of ionic liquids using standard microbiological procedures. In the overall, the selected ILs showed low hemolytic and powerful antimicrobial activity, and efficient inhibition of biofilm formation, especially against S. aureus, suggesting their possible application as anti-biofilm agents.

1. Introduction Ionic liquids (ILs) are salts composed by an organic cation and either an organic or an inorganic anion, which are in the liquid state at temperatures below 100 °C. ILs are characterized by unique physicochemical properties such as high thermal stability, low combustibility, negligible vapor pressure, and favorable solvating properties for a range of polar and non-polar compounds [1–5]. An additional feature of ILs, possibly the most interesting one, is their tunability, which refers to the possibility to tailor their properties for addressing a specific problem by selecting proper constituting ions [6]. Due to these aspects, several applications have been proposed for ILs in many areas of science including synthesis and catalysis [7–9], analytical chemistry [10], biomass processing [11,12], extraction [13,14], electrochemistry [15], biotechnology and medical science [16,17]. The antimicrobial and cytotoxic properties of ILs have attracted significant attention from medical scientists in view of possible applications in drug synthesis and drug delivery systems [17]. Antimicrobial activity has been reported for several ILs following a variety of different procedures to assess the ⁎

antimicrobial susceptibility of a wide range of microorganisms [18–26]. In many instances, antimicrobial activity has been assessed using agar disk diffusion methods and expressed as diameter of microbial growth inhibition [23,24,26,27]. In other cases, various broth microdilutionbased protocols, using different culture media and inoculum size, have been applied to determine the minimum inhibitory concentration (MIC) of one or a few selected IL types against bacterial and/or fungal organisms [18–20,22,25]. In some studies, antimicrobial activity was evaluated after applying test microorganisms onto polymeric membranes with incorporated ILs [21] or made up of poly(ionic liquid) homopolymers [22,25]. In addition, toxicity for human cells has been evaluated only for some ILs, and in a limited number of studies [22,25,28–34]. Consequently, a comparison of results from the various studies on the potential of different types of ILs as antimicrobial agents results difficult. In general, although the use of ILs for systemic administration appears problematic, as in animal models they resulted of moderate toxicity and low bioavailability [35], some ILs might be employed in topical formulations and/or as disinfectants/antiseptics. Although the mechanisms of antimicrobial activity of ILs have not

Corresponding authors. E-mail addresses: [email protected] (F. D'Andrea), [email protected] (A. Lupetti).

https://doi.org/10.1016/j.msec.2019.109907 Received 15 February 2019; Received in revised form 18 June 2019; Accepted 20 June 2019 Available online 22 June 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Chemical structure of ionic liquids (ILs) and dicationic liquids (DILs) synthesized and used in this study.

recorded with the spectrometers operating at 62.9 MHz. The assignments were made, when possible, with the aid of COSY and HSQC experiments. The first order proton chemical shifts δ are referenced to either residual D2O (δH 4.70), CD3CN (δH 1.94, δC 1.28) or residual CD3OD (δH 3.31, δC 49.0) and J-values are given in Hz. All reactions were followed by TLC on Kieselgel 60 F254 with detection by UV light and/or with ethanolic 10% phosphomolybdic or sulfuric acid, and heating. Kieselgel 60 (Merck, 70–230 and 230–400 mesh, respectively) was used for column and flash chromatography. Some flash chromatography purifications were conducted by using the automated system Isolera Four SVTM (Biotage®), equipped with an UV detector with variable wavelength (200–400 nm). The reactions performed with the aid of ultrasound were performed with a SONICS Ultrasonic Processor Sonicator, model CV334, power 750 W. β-D-Glucopyranosyl azide, 2,3,4,6-tetraacetate (22) 2-Propynyl-tetra-O-acetyl-β-D-glucopyranoside (18), dry MeCN, dry DMF, dry MgSO4, Et2O, CH2Cl2, EtOAc, MeOH, NH3-MeOH 7N, acetone, hexane, toluene, were purchased from Sigma Aldrich and used as received. 1-methyl imidazole was also obtained from Sigma Aldrich, but it was distilled before use. 1-bromododecane, 1,6-dibromohexane and 1-methylpyrrolidine were purchased from ACROS Organics, which is part of Thermo Fisher Scientific. Sodium ascorbate, CuSO4 pentahydrate, 4-methyl-2-pentanone, were obtained from Alfa Aesar (Thermo Fisher Scientific). Ammonium Chloride was purchased from Merk and Copper Iodide from Riedel-de Haën. All reactions involving air- or moisture-sensitive reagents were performed under an argon atmosphere using commercial anhydrous solvents. MgSO4 or Na2SO4 were used as the drying agents for solutions. The chemical structures of the ionic liquids used in the tests are described in Fig. 1.

been fully elucidated yet, there is evidence that their alkyl chain plays a major role, most probably by affecting the integrity of biological membranes, with the alkyl chain length strongly influencing antimicrobial activity [19–22,25]. In particular, ILs with an alkyl chain length of 12 or 14 carbon atoms showed the highest antimicrobial activity, whereas for aliphatic chains of more than 16 or less than 10 carbon atoms a marked reduction of antimicrobial activity was observed [19,25]. In addition, the chemical structure and composition of polar groups in the hydrophilic moiety of these ILs may also affect their antimicrobial activity significantly, an aspect that has been studied in depth mainly with imidazolium, pyridinium and pyrrolidinium type ionic liquids [18,22,36,37]. Of interest, the presence of certain functional groups (e.g., amide, ester, carboxyl, hydroxyl) in the cation substituents has been shown to reduce the toxicity of ILs and enhance their biodegradability [38,39]. Terpene based ILs, originally proposed for a variety of other biological applications, have also shown potential as antimicrobials [40–42]. Dicationic ILs (DILs) represent another class of ILs that has attracted an increasing interest in the last few years, since lower toxicity of some DILs compared to mono cationic ILs has been described [43]. The potential of this class of ILs as antimicrobials, or lack thereof, may deserve to be explored and reported. In this study, a number of previously described ILs as well as some ILs not previously tested as antimicrobials have been comparatively evaluated, using standardized procedures for their antimicrobial potency and hemolytic activity for human red blood cells. The main aim of the study was to identify IL types showing strong selective toxicity for bacteria, hence the most promising ILs for possible applications in infection prevention and control, and to gain further insights into the effects of ILs structure on their properties. On the basis of the above considerations, four subsets of ILs were selected: ILs presenting the same long alkyl chain, albeit different cationic head groups (Group A); functionalized ILs, having additional hydroxyl groups linked to the imidazolium cation (Group B); terpene-based ILs (Group C); dicationic ILs (Group D).

2.1.1. Preparation of ILs 1-5 ILs 1–4 (Scheme 1) were synthesized by N-alkylation of commercial 1-methyl-imidazole, N-methyl-pyrrolidine, N-methyl-piperidine and Nmethyl-morpholine with 1-bromododecane 16 (CH3CN at 80 °C) following previously reported procedures [44–47]. Ionic liquid 5 (Scheme 1) was prepared in good yield (75%) by a reaction between 1-bromododecane 16 and the known imidazole derivative 17 [48]. A solution of commercial 1-bromododecane (16, 4.14 g, 16.6 mmol) in dry MeCN (40 mL) was treated with a solution of 3-(1H-imidazol-1yl)propane-1,2-diol (17, 2.36 g, 16.6 mmol) in dry MeCN (10 mL). The reaction mixture was stirred at 80 °C until the starting material was

2. Materials and methods 2.1. Chemistry 1

H NMR spectra were recorded in appropriate solvents with a Bruker Advance II operating at 250.12 MHz. 13C NMR spectra were 2

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Scheme 1. Synthesis of ionic liquid 1–5.

2.1.2.1. Preparation of triazolic derivative 20. Commercial alkyne 18 (386 mg, 1.0 mmol) and 1-azido-6-bromohexane 19 (226 mg, 1.1 mmol, 1.1 eq), CuSO4·5H2O (374 mg, 1.50 mmol, 1.5 eq), sodium ascorbate (594 mg, 3.00 mmol, 3 eq) were dissolved in a 4:1 DMF-H2O mixture (10 mL) and the solution was stirred at room temperature. After 2 h, TLC analysis (25:75 hexane-EtOAc) showed the complete disappearance of the starting material (Rf 0.66) and the formation of a new compound at Rf 0.30. The solvent was removed under diminished pressure and the residue was treated with saturated aqueous NH4Cl (15 mL) and CH2Cl2 (15 mL), the organic phase was separated, and the aqueous layer extracted with CH2Cl2 (3 × 15 mL). The combined organic phases were dried (MgSO4), filtered, and concentrated under diminished pressure. Flash chromatographic purification over silica gel (25:75 hexane-EtOAc) of the crude product gave pure triazolic derivative 20 (448 mg, 76% yield) as a light yellow syrup; Rf 0.30 (25:75 hexane-EtOAc); 1H NMR (CD3CN): δ 7.73 (s, 1H, H-triazole), 5.21 (dd, 1H, J2,3 = 9.6 Hz, J3,4 = 9.5 Hz, H-3), 5.02 (dd, 1H, J4,5 = 9.9 Hz, H-4), 4.87 (dd, 1H, J1,2 = 8.0 Hz, H-2), 4.83, 4.69 (AB system, 2H, JA,B = 12.0 Hz, CH2O), 4.73 (d, 1H, H-1), 4.34 (t, 2H, Jvic = 7.1 Hz, CH2N), 4.25 (dd, 1H, J5,6 = 4.9 Hz, J6a,6b = 12.3 Hz, H6b), 4.09 (dd, 1H, J5,6a = 2.5 Hz, H-6a), 3.83 (ddd, 1H, H-5), 3.45 (t, 2H, Jvic = 6.8 Hz, CH2Br), 2.03, 1.97, 1.92, 1.91 (4 s, each 3H, 4 × MeCO), 1.85 (m, 2H, CH2CH2N), 1.74 (m, 2H, CH2CH2Br), 1.45 [m, 2H, CH2(CH2)2N], 1.32 [m, 2H, CH2(CH2)2Br]; 13C NMR (CD3CN): δ 172.3, 170.8, 170.5, 170.3 (4 × C=O), 144.2 (C-triazole), 124.6 (CHtriazole), 100.2 (C-1), 73.2 (C-3), 72.4 (C-2), 71.9 (C-5), 69.3 (C-4), 62.7 (C-6), 63.2 (CH2O), 50.7 (CH2N), 35.1 (CH2Br), 33.2 (CH2CH2Br), 30.6 (CH2CH2N), 28.1 [CH2(CH2)2N], 26.1 [CH2(CH2)2Br], 20.9–20.8 (4 × MeCO).

completely reacted (TLC, EtOAc). After 4 days the solution was evaporated under diminished pressure and the trituration of the crude product with Et2O (3 × 10 mL) afforded pure bromide salt 5 (4.74 g, 75% yield) as a yellowish hygroscopic semisolid; 1H NMR (CDCl3): δ 9.5 (s, 1H, Im-H2), 7.78, 7.38 (2bs, each 1H, Im-H4, Im-H5), 4.99 (bs, 1H, OH), 4.61–4.31 (m, 3H, CH-OH, NCH2CH), 4.24 (t, 2H, J = 7.6 Hz, NCH2CH2), 4.05 (bs, 1H, OH), 1.87 (bt, 2H, NCH2CH2), 1.40–1.22 (m, 18 H, 9 × CH2), 0.85 (t, 3H, J = 6.9 Hz, CH3); 13C NMR (CDCl3): δ 136.6 (Im-C2), 124.2, 121.5 (Im-C4, Im-C5), 70.1 (CH-OH), 62.8 (CH2OH), 52.5, 50.2 (2 × CH2N), 32.0 (CH2CH2N), 30.3, 29.7, 29.7, 29.5, 29.4, 29.3, 29.2, 26.4 (8 × CH2), 22.8 (CH2CH3), 14.2 (CH3). 2.1.2. Preparation of IL 6 and IL 7 Known 1-azido-6-bromohexane (compound 19, Scheme 2) [49] was conjugated to the commercial alkyne 18 (Scheme 2) by CuAAC click chemistry according to reported conditions [50]. The reaction was performed in a mixture of DMF-H2O (4:1) with copper(II) sulfate and sodium ascorbate as catalytic system at 80 °C for 2 h. The 1,2,3-triazole derivative 20 (Scheme 2) was isolated after flash chromatographic purification with complete regiospecificity (75% yield). The bromo derivative 20 was subjected to a SN2 displacement with commercial 1methylimidazole in dry MeCN at 80 °C affording the ionic liquid derivative 21 in an excellent yield (96%). The reaction between β-glycosyl azide (compound 22, Scheme 2) [51] and ionic liquid 23 [52] was performed in a 1:1 MeOH-H2O mixture, in the presence of catalytic amount of CuI, under sonication at room temperature for 50 min (Scheme 2, bottom). The 1,2,3-triazole derivative 24 was isolated, after trituration of the crude product with Et2O, with complete regiospecificity (98% yield). Finally, the de-O-acetylation of compounds 21 and 24 by treatment with NH3-MeOH 3.5N afforded the deprotected derivatives 6 and 7 in nearly quantitative yields (98–99%), after trituration of crude products with Et2O.

2.1.2.2. Preparation of imidazolium salt derivative 21. To a solution of triazolic compound 20 (254 mg, 0.43 mmol) in dry MeCN (12 mL), 3

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Scheme 2. Synthesis of 1,2,3-triazole derivatives 6 and 7. i: CuSO4·5H2O, Sodium Ascorbate, DMF/H2O 4:1, rt., 2 h, 75%; ii: dry MeCN, 80 °C, 3 days, 96%; iii: NH3/ MeOH 3.5N, rt., 12 h, 96–99%; iv: CuI, MeOH/H2O 1:1, sonication for 50 min, 98%.

MeOH 7N (1 mL) and the mixture was stirred at room temperature until the starting compound was completely reacted (inverse phase TLC, 6:4 H2O-MeCN, 8–12 h). The solution was evaporated under diminished pressure and trituration of crude product with Et2O afforded pure deprotected ionic liquid 6 (99% yield) or 7 (96% yield).

commercial 1-methylimidazole (35 mg, 0.43 mmol) was added. The reaction was stirred at 80 °C and after 3 days TLC analysis (2:8 hexaneEtOAc) showed the complete disappearance of the bromo derivatives 20 (Rf 0.32). The solution was concentrated under diminished pressure and the crude product was triturated with Et2O (3 × 5 mL) affording pure imidazolium salt 21 (279 mg, 96% yield) as a white very hygroscopic solid; 1H NMR (CDCl3): δ 10.3 (s, 1H, Im-H2), 7.72 (s, 1H, H-triazolic), 7.36, 7.33 (2 s, each 1H, Im-H4, Im-H5), 5.13 (dd, 1H, J2,3 = 9.2 Hz, J3,4 = 9.5 Hz, H-3), 4.99 (dd, 1H, J4,5 = 9.6 Hz, H-4), 4.89 (dd, 1H, J1,2 = 8.0 Hz, H-2), 4.88–4.67 (m, 2H, H-6a, H-6b), 4.66 (d, 1H, H-1), 4.38–4.21 (m, 4H, 2 × CH2N), 4.20, 4.18 (2 m, each 1H, CH2O), 4.02 (s, 3H, CH3N), 3.73 (m, 1H, H-5), 2.01–1.72 (m, 4H, 2 × CH2CH2N), 2.02, 1.95, 1.92, 1.91 (4 s, each 3H, 4 × MeCO); 1.45–1.20 (m, 4H, CH2CH2); 13C NMR (CDCl3): δ 170.7, 170.2, 169.5, 169.4 (4 × C=O), 145.1 (C-triazolic), 137.5 (Im-C2), 123.5, 123.2 (ImC4, Im-C5), 121.9 (CH-triazolic), 99.7 (C-1), 72.6 (C-3), 71.7 (C-2), 71.7 (C-5), 68.2 (C-4), 62.8 (CH2O), 61.7 (C-6), 49.9, 49.6 (2 × CH2N), 36.6 (CH3N), 29.7, 29.6 (2 × CH2CH2N), 25.3, 25.0 (CH2CH2), 20.8–20.6 (4 × MeCO).

2.1.2.5. Imidazolium salt derivative 6. Colourless hygroscopic syrup; 1H NMR (CD3OD/D2O): δ 9.00 (s, 1H, Im-H2), 8.07 (s, 1H, H-triazolic), 7.64, 7.58 (2 s, each 1H, Im-H4, Im-H5), 4.96, 4.78 (AB system, 2H, JA,B = 12.4 Hz, CH2O), 4.41 (t, 2H, Jvic = 6.9 Hz, CH2N), 4.31 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.21 (t, 2H, Jvic = 7.2 Hz, CH2N), 3.94 (s, 3H, CH3N), 3.89 (dd, 1H, J5,6b = 1.4 Hz, J6a,6b = 11.8 Hz, H-6b), 3.68 (dd, 1H, J5,6a = 5.4 Hz, H-6a), 3.40–3.28 (m, 3H, H-3, H-4, H-5), 3.20 (dd, 1H, J2,3 = 8.7 Hz, H-2), 1.92–1.82 (m, 4H, 2 × CH2CH2N), 1.36–1.20 (m, 4H, CH2CH2); 13C NMR (CD3OD/D2O): δ 145.6 (C-triazolic), 137.3 (Im-C2), 125.5, 124.9 (Im-C4, Im-C5), 123.6 (CH-triazolic), 103.5 (C-1), 72.6 (C-3), 78.0 (C-2), 77.9 (C-4), 71.6 (C-5), 62.9 (CH2O), 62.7 (C-6), 51.1, 50.6 (2 × CH2N), 36.6 (CH3N), 30.8, 30.7 (2 × CH2CH2N), 26.7, 26.4 (CH2CH2).

2.1.2.3. Preparation of triazolic derivative 24. A solution of known alkyne 23 (74 mg, 0.37 mmol), β-glycosyl azide 22 (137.4 mg, 0.37 mmol) and CuI (18 mg, 0.092 mmol, 0.25 eq) in MeOH-H2O 1:1 (4 mL) was sonicated for 50 min (microtip probe, amplitude 30%, temp max 50 °C, pulse (sec) 30 on 10 off, power 1,000,000 J, 10 min for cycle). TLC analysis (EtOAc) showed the disappearance of the azide derivative 22 (Rf 0.69); the reaction mixture was filtered through alternate pads of Celite and cotton and washed with MeOH (4 × 10 mL). The combined organic phases were concentrated under diminished pressure and trituration of crude product with Et2O (3 × 10 mL) afforded pure 24 (208 mg, 98% yield) as a light brown hygroscopic solid; 1H NMR (CD3OD): δ 8.56 (s, 1H, Im-H2), 7.66–7.62 (m, 3H, Im-H4, Im-H5, H-triazolic), 6.22 (d, 1H, J1,2 = 9.0 Hz, H-1), 5.65 (s, 2H, CH2N), 5.62–5.55 (m, 2H, H-3, H-4), 5.28 (m, 1H, H-2), 4.36–4.20 (m, 3H, H-5, H-6a, H-6b), 3.96 (s, 3H, CH3N), 2.06, 2.04, 2.00, 1.83 (4 s, each 3H, 4 × MeCO); 13C NMR (CD3OD): δ 172.2, 171.4, 171.2, 170.6 (4 × C=O), 142.5 (C-triazolic), 137.8 (Im-C2), 125.2, 123.6 (Im-C4, Im-C5), 125.4 (CH-triazolic), 86.6 (C-1), 75.9 (C3), 73.8 (C-2), 72.2 (C-5), 69.1 (C-4), 62.9 (C-6), 45.0 (CH2N), 39.7 (CH3N), 20.7–20.2 (4 × MeCO).

2.1.2.6. Imidazolium salt derivative 7. Brown hygroscopic light solid; 1H NMR (CD3OD): δ 8.50 (s, 1H, Im-H2), 7.73–7.61 (m, 3H, Im-H4, Im-H5, H-triazolic), 5.77 (d, 1H, J1,2 = 9.1 Hz, H-1), 5.63 (s, 2H, CH2N), 3.96 (s, 3H, CH3N), 3.95–3.83 (m, 5H, H-6a, H-6b, 3 × OH), 3.78–3.45 (m, 5H, H-2, H-3, H-4, H-5, OH); 13C NMR (CD3OD): δ 141.8 (C-triazolic), 138.4 (Im-C2), 125.4, 123.7 (Im-C4, Im-C5), 123.4 (CH-triazolic), 89.5 (C-1), 81.0 (C-3), 78.2 (C-4), 74.0 (C-2), 70.8 (C-5), 62.2 (C-6), 45.0 (CH2N), 36.8 (CH3N).

2.1.2.4. General procedure for the transformation of acetyl derivative 21 and 24 into deprotected ionic liquids 6 and 7. A solution of protected ionic liquid 21 or 24 (0.1 mmol) in MeOH (1 mL) was treated with NH3-

2.1.5. Preparation of IL 14 and IL 15 Commercial 1,6-dibromohexane (compound 25, Scheme 5) and the dibromo-PEG 26 [56], were subjected to a SN2 displacement (Scheme

2.1.3. Preparation of IL 8 and IL 9 Ionic liquids 8 [53] and 9 [54] were synthesized according to previously established procedures starting from myrtenol and nopol, respectively (Scheme 3). 2.1.4. Preparation of dicationic ionic liquids 10-13 Dicationic ionic liquids 10–13 varying both in the alkyl linker length (C3 and C6) and in the length of the substituent on the imidazolium ring (1-methyl or 1-butyl), were synthesized by reacting selected 1-alkylimidazoles with the proper 1,n-dibromoalkane (Scheme 4), as recently reported [55].

4

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Scheme 3. Synthesis of ionic liquids 8 and 9

Staphylococcus epidermidis (ATCC 35984), Staphylococcus aureus (ATCC 6538), and Enterococcus faecalis (ATCC 29212). Stock cultures were prepared by growing bacterial strains in Mueller-Hinton broth (Oxoid, Thermo Fisher Scientific Inc., Hampshire, UK) until mid-log phase; then, cultures were centrifuged, resuspended in 1/10 volume of fresh medium added of 20% glycerol, and stored in aliquots at −80 °C. To assess the number of colony forming units (CFU), serial two-fold dilutions of microbial cultures were plated onto Mueller-Hinton agar (S. epidermidis, S. aureus, E. faecalis) or Luria-Bertani agar (E. coli, P. aeruginosa) and incubated for 24 h at 37 °C.

5) with commercial 1-methylimidazole (in dry toluene at 80 °C) or commercial 1-methylpyrrolidine (in 4-methyl-2-pentanone at 80 °C) to give dicationic liquids 14 and 15 in good yield (93% and 88% respectively). (See Scheme 5.) 2.1.5.1. Preparation of 1,1′-(hexane-1,6-diyl)bis(1-methylpyrrolidinium) bromide (IL 14). To a solution of commercial 1,6-dibromohexane (25, 58 mmol) in 4-methyl-2-pentanone (MIBK, 20 mL) cooled to 0 °C, a solution of 1-methylpyrrolidine (122 mmol) in MIBK (10 mL) was added dropwise. The mixture was warmed to 80 °C and stirred for 12 h (white solid precipitation was observed after 30 min). The precipitate was filtered under vacuum, washed with MIBK (3 × 50 mL), EtOAc (3 × 50 mL), and acetone (3 × 50 mL) and dried under reduced pressure, to afford pure 14 (93% yield) as a yellowish hygroscopic solid; 1H NMR (D2O): δ 3.49 (m, 8H, 4 × CH2N), 32 (m, 4H, 2 × CH2N), 3.02 (s, 6H, 2 × CH3N), 2.19 (m, 8H, 4 × CH2CH2N), 1.83 (m, 4H, 2 × CH2CH2N), 1.42 (m, 4H, CH2CH2); 13C NMR (D2O): δ 63.8, 63.5 (6 × CH2N), 47.5 (2 × CH3N), 24.8, 22.5 (6 × CH2CH2N), 20.78 CH2CH2).

2.3. MIC determination The MIC of ILs against S. epidermidis, S. aureus, E. faecalis, E. coli, and P. aeruginosa was evaluated by the microdilution method in roundbottom polystyrene 96-well microtiter plates (Corning Costar, Sigma) according to the guidelines of the Clinical Laboratory Standards Institute [57]. In a first series of experiments, ILs were tested in a high concentration range: 156.2 μg/mL-5 mg/mL for IL 2, 3, 4, 6 and 7; 312.5 μg/mL-10 mg/mL for ionic liquids 1, 5, 8, 9, 10, 11, 12, 13, 14 and 15. ILs that were still inhibitory at the lowest concentration were tested in a lower concentration range (625 ng/mL-320 μg/mL). The MIC of ILs was evaluated in 200 μL of Mueller-Hinton broth containing approximately 105 CFU (5 × 105 CFU/mL) of bacteria harvested from mid-log phase culture, diluted to the appropriate density in fresh Mueller-Hinton broth, and mixed with ILs previously diluted in the same medium. The MIC was defined as the lowest concentration of ILs for which no visible microbial growth was observed.

2.1.5.2. Preparation of 3,3′-(tetraethyleneglycol-1,11-diyl)bis(1-methyl1H-imidazolium) bromide (IL 15). A solution of commercial 1methylimidazole (122 mmol) in toluene (10 mL) was added dropwise to a solution of 1-bromo-2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethane 26 (58 mmol) in toluene (20 mL) cooled to 0 °C. The mixture was warmed to 80 °C and stirred for 12 h. The liquid products were decanted, washed with toluene, and dried under reduced pressure, to afford pure IL 15 (88% yield) as a brown highly viscous liquid; 1H NMR (D2O): δ 8.80 (s, 1H, Im-H2), 7.56, 7.48 (2bt, each 1H, Im-H4, Im-H5), 4.43 (t, 4H, Jvic = 5.1 Hz, 2 × OCH2CH2N), 3.92 (m, 10H, 2 × CH3N, 2 × CH2N), 3.69 (m, 8H, 4 × OCH2); 13C NMR (D2O): δ 136.5 (Im-C2), 123.5, 122.7 (Im-C4, Im-C5), 69.7, 69.5, 68.5 (6 × OCH2), 49.1 (2 × CH2N), 36.0 (2 × CH3N).

2.4. Minimum bactericidal concentration and killing kinetics of selected ILs The minimum bactericidal concentration (MBC) of ILs 1, 2 and 3 against S. aureus, E. faecalis, E. coli, and P. aeruginosa was evaluated by the microdilution method in 10 mM sodium-phosphate buffer, pH 7.4 (NaPB) or NaPB containing 0.5% (w/v) bovine serum albumin (BSA) (Sigma). Microbial strains were grown in Mueller-Hinton broth until mid-log phase, centrifuged 3 min at 13,000 rpm, washed once in NaPB, and suspended in NaPB or NaPB/0.5% BSA at a density of 2 × 106 CFU/

2.2. Bacterial strains and culture conditions The following reference laboratory strains were used for this study: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853),

Scheme 4. Synthesis of dicationic liquids 10–13 5

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Scheme 5. Synthesis of dicationic ionic liquids 14 and 15. i: 4-methyl-2-pentanone (MIBK), 80 °C, 12 h, 93%; ii: dry toluene, 80 °C, 12 h, 88%.

mL; then, 100 μL of bacterial suspension was added to 100 μL of NaPB or NaPB/0.5% BSA, respectively, containing different concentrations of ILs: 2.5–320 μg/mL for IL 1, and 5–640 μg/mL for IL 2 and 3. Microbial suspensions in NaPB or NaPB/0.5% BSA, respectively, at the same density of the assay were used as cell viability control. Samples were incubated at 37 °C with orbital shaking for 90 min; after incubation, serial 10-fold dilutions of microbial suspensions in Mueller-Hinton broth were plated onto Mueller-Hinton agar and incubated for 24 h at 37 °C to determine the number of CFU. The MBC was defined as the lowest concentration of ILs killing ≥99.9% of viable microorganisms after 90 min incubation [58]. For killing kinetics studies, microbial suspensions were prepared in NaPB as described above and incubated with ILs at concentrations corresponding to the MBC for the different microbial strains or in NaPB (positive control) for 10, 30, 60 or 90 min. The number of CFU at the different time points was assessed as described above.

microtiter plates (Corning Costar), and incubated at 37 °C for 24 h in the presence of different concentrations of each IL (20 μg/mL-640 μg/mL for P. aeruginosa; 312 ng/mL-10 μg/mL for S. aureus) or in BPM (positive control). As negative control, BPM was incubated without adding bacterial cells. Biofilm biomass was evaluated by a crystal violet (CV) staining assay. To this purpose, biofilms were washed three times with PBS, airdried for 15 min, and incubated with 0.1% (w/v) CV (Sigma) for 15 min. After incubation, the CV solution was removed and plates were washed with PBS; then, the biofilm associated CV was extracted with 99.8% ethanol (Fluka, Honeywell International Inc., Bucharest, Romania), and quantified by measuring the optical density at 570 nm with a microplate reader. For each experiment, the biofilm mass formed by untreated bacterial culture, evaluated by the crystal violet staining assay, was considered as 100% biofilm formation. The inhibition of biofilm formation was quantified according to the following formula: biofilm formation (%) = [(ODIL-ODnegative control)/(ODpositive control − ODnegative control)] × 100.

2.5. Hemolysis assay To test the hemolytic activity of ILs 1–5 against human red blood cells (RBCs), peripheral blood from three healthy donors was collected in a sterile Vacutest tube, containing 5.24 mg of K3EDTA, centrifuged for 5 min at 800 ×g, 25 °C, and washed twice with phosphate buffer saline, pH 7.4 (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), added of 0.25% (v/v) dimethyl-sulfoxide (DMSO). After washing, RBCs were suspended at a density of 8% (v/v) in PBS/ 0.25% DMSO, and mixed 1:1 with ILs in PBS/0.25% DMSO at different concentrations in round-bottom polystyrene 96-well microplates (Corning Costar). After 1 h of incubation at 37 °C, the cell suspension was centrifuged at 800 ×g for 5 min, 25 °C. RBCs incubated in PBS/ 0.25% DMSO alone were used as negative control (0% hemolysis), while cells exposed to 2% Triton X-100 in PBS/0.25% DMSO were taken as positive control (100% hemolysis). One hundred μL of each supernatant were transferred into a microtiter plate and the optical density (OD) at 450 nm was measured by means of a microplate reader. The hemolytic activity was quantified according to the following formula: hemolysis (%) = [(ODIL − ODnegative control)/(ODpositive control − ODnegative control)] × 100. The three blood samples were withdrawn from healthy volunteers, casually chosen among the authors of this manuscript, to perform the hemolysis assay. The local ethical committee ruled out that no notification was necessary in this case.

In order to evaluate their antimicrobial activity against different bacterial species, the MIC of 15 ILs was determined against three Grampositive cocci, namely S. epidermidis, S. aureus, and E. faecalis, and two Gram-negative bacteria, i.e. E. coli and P. aeruginosa. Overall, ILs were tested in a wide range of concentrations (625 ng/mL–10 mg/mL). In general, MIC values were lower for Gram-positive than for Gram-negative bacteria, with the lowest MIC values (2.5 μg/mL) observed for IL 1 against S. aureus and S. epidermidis (Table 1). IL 2, 3, 4 and 5 also showed MIC values ≤ 20 μg/mL, at least for some of the tested microbial species. The five ILs showing the lowest MIC values were also evaluated for their degree of toxicity for human red blood cells by a hemolysis assay. The percentage of hemolysis caused by the selected ILs at concentrations corresponding to their MIC and 10× MIC for S. aureus is reported in Fig. 2. The results show that three out of the five ILs tested, namely IL 1, 2 and 3 did not exhibit significant hemolytic activity at up to 10× MIC for S. aureus (Fig. 2). Ionic liquids 4 and 5, instead, showed > 2% hemolysis at 10× MIC (Fig. 2). All the tested ILs showed a high degree (> 30%) of hemolysis at 50× MIC (data not shown).

2.6. Biofilm inhibition assay

3.2. Bactericidal activity and killing kinetics of the selected ILs

The ability of IL 1-3 to inhibit biofilm formation was evaluated against S. aureus and P. aeruginosa. Bacteria were grown overnight at 37 °C in Tryptone Soya Broth (TSB) (Oxoid) supplemented with 0.25% (w/v) glucose. Stationary phase cultures were inoculated 1:1000 in Biofilm Promoting Medium (BPM), consisting of TSB diluted 1:1 with NaPB, pH 7.4, and added of 0.25% glucose. Bacterial suspensions were dispensed in 200 μL aliquots into flat-bottom polystyrene 96-well

To investigate the mechanisms by which the selected ILs exert their antimicrobial activity, the MBC of IL 1-3 was determined after 90 min incubation in sodium phosphate buffer with S. aureus, E. faecalis, E. coli, or P. aeruginosa. Surprisingly, in preliminary experiments, the MBC values for E. coli and P. aeruginosa resulted lower than the corresponding MICs for all the three ILs tested. To test the possibility that protein components contained in the liquid medium used for MIC

3. Results 3.1. MIC values and hemolytic activity of ILs

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Table 1 Minimum inhibitory concentration of ionic liquids 1–15 for tested microorganisms. Ionic liquid

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a

MICa value for Staphylococcus epidermidis

Staphylococcus aureus

Enterococcus faecalis

Escherichia coli

Pseudomonas aeruginosa

2.5 μg/mL 10 μg/mL 5 μg/mL 20 μg/mL 10 μg/mL ˃5 mg/mL 5 mg/mL 312.5 μg/mL 160 μg/mL ˃10 mg/mL 5 mg/mL 10 mg/mL 2.5 mg/mL 10 mg/mL ˃10 mg/mL

2.5 μg/mL 10 μg/mL 5 μg/mL 20 μg/mL 10 μg/mL ˃5 mg/mL ˃5 mg/mL 625 μg/mL 160 μg/mL ˃10 mg/mL ˃10 mg/mL 10 mg/mL 5 mg/mL ˃10 mg/mL ˃10 mg/mL

5 μg/mL 20 μg/mL 10 μg/mL 40 μg/mL 20 μg/mL 5 mg/mL ˃5 mg/mL 1.25 mg/mL 625 μg/mL ˃10 mg/mL 10 mg/mL ˃10 mg/mL 10 mg/mL ˃10 mg/mL ˃10 mg/mL

20 μg/mL 80 μg/mL 40 μg/mL 156.2 μg/mL 80 μg/mL ˃5 mg/mL ˃5 mg/mL 1.25 mg/mL 625 μg/mL 10 mg/mL ˃10 mg/mL ˃10 mg/mL 2.5 mg/mL ˃10 mg/mL ˃10 mg/mL

160 μg/mL 312.5 μg/mL 312.5 μg/mL 312.5 μg/mL 160 μg/mL ˃5 mg/mL ˃5 mg/mL 2.5 mg/mL 1.25 mg/mL ˃10 mg/mL ˃10 mg/mL ˃10 mg/mL ˃10 mg/mL ˃10 mg/mL ˃10 mg/mL

MIC: minimum inhibitory concentration.

decreased below the detection limit after 30–90 min exposure, depending on the microbial species and IL tested. For E. coli, the killing kinetics of IL 1–3 were less rapid than for the other tested microorganisms (Fig. 3C). 3.3. Inhibition of bacterial biofilm formation The ability of IL 1–3 to prevent bacterial biofilm formation upon an abiotic surface (polystyrene) was investigated by testing the inhibitory effect of IL 1–3 at different concentrations on S. aureus or P. aeruginosa biofilm formation at 24 h. The results reported in Fig. 4 show that all three ILs effectively inhibited S. aureus biofilm formation even at 0.5× the corresponding MICs (Fig. 4A) whereas for P. aeruginosa effective inhibition of biofilm formation was observed at MIC for all the tested ILs (Fig. 4B).

Fig. 2. Hemolytic activity of ionic liquids 1–5 at concentrations corresponding to their MIC and 10× MIC for S. aureus. Results are reported as mean percent of hemolysis ( ± S.E.M.) of three independent experiments.

determination might partly neutralize the antimicrobial activity of these ILs, MBCs were evaluated, in parallel, in the presence or in the absence of 0.5% BSA. The results showed that MBCs were two- to fourfold higher for the Gram-positive and four- to eight-fold higher for the Gram-negative bacteria in the presence of BSA (Table 2), thus supporting our hypothesis. In order to gain further insights into the mechanisms by which ILs exert their antimicrobial activity, time-killing curves of S. aureus, E. faecalis, E. coli, and P. aeruginosa exposed to MBCs of the selected ILs were determined by counting the number of CFU after 10, 30, 60, and 90 min exposure. The results reported in Fig. 3 show that the killing kinetics were quite rapid for all three ILs tested. A ˃3 Log CFU reduction was observed for S. aureus, and P. aeruginosa, soon after 10 min exposure with the three ILs tested, and at 30 min for E. faecalis, and E. coli. For all the tested microorganisms, but E. coli, the number of CFU

4. Discussion Effective antimicrobial agents are strongly needed for the prevention and control of infectious diseases, especially for hospital-acquired multidrug-resistant infections [59–61]. Several ILs have been shown to possess antimicrobial and/or antimalarial activities [20,25,26,35,62,63]. However, a comparison of their potential as antimicrobial agents is hampered by the different methods and procedures used to perform antimicrobial susceptibility testing as highlighted in a recent review on the topic [64]. In addition, for some of the ILs tested in the present study, no evaluation of their antimicrobial activity has been previously reported. In the present study, a panel of ILs was evaluated for their potential use as antimicrobial agents. The 15 different ILs tested in this study were chosen as representatives of different ILs classes which attracted a

Table 2 Influence of bovine serum albumin on the minimum bactericidal concentration of ionic liquids 1–3. Ionic liquid

MBCa value for Staphylococcus aureus b

1 2 3 a b c

c

Enterococcus faecalis

Escherichia coli

Pseudomonas aeruginosa

NaPB

NaPB, 0.5% BSA

NaPB

NaPB, 0.5% BSA

NaPB

NaPB, 0.5% BSA

NaPB

NaPB, 0.5% BSA

40 μg/mL 80 μg/mL 80 μg/mL

80 μg/mL 160 μg/mL 160 μg/mL

40 μg/mL 40 μg/mL 40 μg/mL

160 μg/mL 160 μg/mL 160 μg/mL

10 μg/mL 20 μg/mL 20 μg/mL

80 μg/mL 80 μg/mL 80 μg/mL

20 μg/mL 80 μg/mL 80 μg/mL

160 μg/mL 320 μg/mL 320 μg/mL

MBC: minimum bactericidal concentration. NaPB: 10 mM sodium phosphate buffer, pH 7.4. BSA: bovine serum albumin. 7

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Fig. 3. Time-killing curves of S. aureus (A), E. faecalis (B), E. coli (C) and P. aeruginosa (D) either exposed to minimum bactericidal concentration of ionic liquids 1, 2 or 3 (IL 1–3) or incubated in 10 mM sodium phosphate buffer, pH 7.4 (NaPB). Each value represents the mean CFU/mL ( ± S.E.M.) of three independent experiments.

and 3 exert high antimicrobial activity, especially against Gram-positive bacteria, and low degree of hemolysis at up to 10× MIC for S. aureus. Notably, biofilm formation by S. aureus was inhibited even at 0.5× MIC of IL 1-3. The results obtained confirm that a long, linear alkyl chain is a mandatory structural requirement for a potent antimicrobial activity. In fact, ILs of group A are characterized by the highest antimicrobial activity. A clear effect of the nature of the cation was also observed for S. epidermidis, S. aureus, E. faecalis and E. coli with the antimicrobial activity which decreased in the following order: imidazolium (IL 1) > piperidinium (IL 3) > pyrrolidinium (IL 2) > morpholinium (IL 4). In the case of P. aeruginosa a lower effect of the cation moiety was instead observed and only imidazolium IL 1 displayed a different and higher antimicrobial activity than ILs 2–4. A moderate decrease of the antimicrobial activity, against all bacterial species tested but P. aeruginosa, was observed for IL 5 (group B) as compared to IL 1, which confirmed that the presence of hydroxyl groups did not markedly impair antimicrobial potency when bound to the other nitrogen of the imidazolium cation. ILs 6 and 7 (group B), whose structures are remarkably different from IL 5 due to the presence of the glucose moiety and the triazole fragment, also showed a drastic decrease of the antimicrobial potency in spite of the different linker between the triazole and the imidazolium cation. Instead, an effect of the different number of methylene groups on the antimicrobial activity was observed when comparing the MIC values of IL 8 and 9 (group C). Terpenoid-based IL 9, which has a total of 10 carbon atoms on the imidazolium substituent and an additional methylene group, displayed a two-fold higher potency than IL 8 against all the tested bacteria but S. aureus. Possibly, the size of the hydrophobic imidazolium substituent approaches the aforementioned

growing interest in recent years. Group A is comprised of different cations (pyrrolidinium, imidazolium, piperidinium, and morpholinium) bearing the same alkyl substituent. The length of the alkyl chain of the tested ILs was established according to previous studies showing that alkyl chain length influences both their toxicity and antimicrobial activity [18,19,24,27,29,65–69], though further studies are needed to thoroughly understand the optimal structural requirements for selective toxicity. As mentioned previously, we investigated other ILs with selected structural variations: ILs with additional hydrophilic groups, namely either a diol on the other nitrogen of the imidazolium cation (IL 5), or a glucose at the end of the alkyl chain (IL 6 and 7); ILs with a comparable non polar frame albeit enclosed in natural terpenoid rings (IL 8 and 9); dicationic ILs with a linker of different length and nature between the two cationic imidazolium heads (IL 10, 11, 12, 13, 14 and 15). For this latter group, solubility issues did not allow for testing longer alkyl side chains (data not shown). All these ILs were evaluated following a sequential step analysis. First, the MIC of ILs was determined using standardized reference procedures against S. aureus, S. epidermidis, E. faecalis, E. coli, and P. aeruginosa [57]. Based on these results, ILs showing the lowest MIC values were evaluated for their hemolytic activity, which allowed further selection of IL 1-3. Noteworthy, the results of hemolysis assays provide a measure of the relative selective toxicity of the different ILs tested, rather than their absolute toxicity for red blood cells, since the concentrations of ILs tested were related to their MIC values against S. aureus. Next, the bactericidal activity of IL 1-3 against S. aureus, E. faecalis, E. coli and P. aeruginosa, and their ability to inhibit biofilm formation by S. aureus and P. aeruginosa were tested. We focused on the inhibition of biofilm formation as this could prevent the development of bacterial biofilms on medical devices, such as catheters, artificial heart valves, and prostheses [70,71]. Overall, the results obtained show that IL 1, 2

Fig. 4. Ionic liquids 1, 2 and 3 inhibit biofilm formation by S. aureus (A) and P. aeruginosa (B). Each value represents the mean percent of biofilm mass ( ± S.E.M.) of four independent experiments.

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Acknowledgements

optimal range when moving from IL 8 to IL 9. However, further studies are required to verify this hypothesis. Finally, among the DILs tested (group D), IL 13, characterized by the longest alkyl chain, showed the highest antimicrobial activity, at least against S. epidermidis, S. aureus, and E. coli. Interesting enough, IL 12 and 14, which differ only for the cationic head groups (imidazolium versus pyrrolidinium), exhibited similar antimicrobial profile in the conditions tested. Low activity was also found for IL 15 as compared to 1, in spite of the presence of a long chain between the cationic head groups. The antimicrobial activity of the selected ILs against Gram-negative bacteria was less potent, especially for P. aeruginosa, suggesting that the outer membrane of Gram-negative bacteria might represent a possible barrier to their activity. Our results are consistent with those of Venkata Nancharaiah et al. [72], who reported stronger antimicrobial and antibiofilm activity of 1-dodecyl-3-methylimiazolium iodide against S. aureus compared to P. aeruginosa, and more pronounced difference between MIC and MBC for the former compared to the latter microorganism. Further studies will be needed to evaluate the possibility to potentiate the antimicrobial activity of ILs against Gram-negative bacteria, e.g. by combining them with compounds able to act synergistically against Gram-negative bacteria. Notably, the MBCs of IL 1-3 against E. coli and P. aeruginosa in sodium phosphate buffer were lower than the corresponding MIC values. As it was previously reported that ILs are able to bind to proteins [26,72], we hypothesized that the lower MBC values could be related to the presence of proteins in the medium used for MIC determination. Indeed, the addition of BSA into the sodium phosphate buffer used for MBC determination decreased the bactericidal activity of ILs of up to 8 folds. In this context, fluorescence spectra and circular dichroism analyses have previously shown that imidazolium-based IL surfactants may cause BSA unfolding, mainly by interacting with tryptophan residues of BSA [73]. Moreover, the interaction between imidazolium-based ILs and human serum albumin (HSA) has been shown to alter the secondary structure of HSA [26]. Finally, the rapid killing of bacteria after exposure to IL 1-3 at MBC indicate a probable direct action of these ILs on the integrity of bacterial membranes, thus leading to bacterial cell lysis, though additional mechanisms for their antimicrobial activity cannot be excluded.

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5. Conclusions In the present study, a comparative evaluation of different types of ionic liquids as antimicrobial agents has been carried out. To our knowledge, this is the first systematic study investigating the antimicrobial potential of different types of ionic liquids using standard procedures for antimicrobial susceptibility testing. The high antimicrobial activity and relatively low hemolytic activity of IL 1-3 suggest their possible application as antimicrobials, such as anti-biofilm agents. Further studies will be needed to better understanding the mechanisms involved in their antimicrobial activity, and other variables regarding toxicity and, in general, exploitability for their possible application into clinical practice.

Contributors WF, SB, FD, AL, CC and LG conceived the study and contributed to the experimental design; WF, SB, FD and LG carried out the experiments; WF and FD wrote a draft of the manuscript; AL and CC critically revised the manuscript.

Declaration of Competing Interest None. 9

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