Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Mechanism of Antibacterial Activity of Choline-Based Ionic Liquids (CAGE) Kelly N. Ibsen,†,‡ Huilin Ma,§ Amrita Banerjee,† Eden E. L. Tanner,‡ Shikha Nangia,§ and Samir Mitragotri*,‡ †
Department of Chemical Engineering, University of California, Santa Barbara, California 93106, United States School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, United States § Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, New York 13244, United States ‡
ABSTRACT: The continued emergence of antibiotic-resistant organisms has severely depleted our arsenal of effective antimicrobials. Ionic liquids (ILs) show great promise as antibacterial agents but understanding the mechanism of attack on bacterial cells is key to ensuring that design of IL-based biocides impart maximum efficacy with minimal toxicity, while also avoiding the potential for the target organisms to become resistant. Here we report the antibacterial attributes of a set of choline and geranate (CAGE)-based ILs and identify the mechanism by which they interact with the Gram-negative cell wall of Escherichia coli. CAGE is envisaged as an antimicrobial agent to treat topical infections in skin. Our earlier work has shown that CAGE is highly effective across a breadth of bacterial, fungal, and viral species and is benign to human cells. This combination makes CAGE an ideal antimicrobial for human use. Four CAGE variants with varying ratios of choline and geranic acid were synthesized and tested for their antibacterial activity (1:4, 1:2, 1:1, and 2:1 choline:geranic acid). The minimum bactericidal concentration required to kill E. coli correlated with the geranic acid content. Using molecular dynamics (MD) simulations, we identified the mechanism of CAGE action on the E. coli membrane, namely that choline is attracted to the negatively charged cell membrane and consequently inserts geranic acid into the lipid bilayer. The disruption of the cell membrane was confirmed with propidium iodide staining via flow cytometry and scanning electron microscopy. Fourier Transform infrared spectroscopic analysis of treated cells showed an altered lipid profile similar to phase transition, indicating the disruption of the lipid bilayer conformation. E. coli cells repeatedly exposed to CAGE did not exhibit resistance. This study provides the fundamental mechanism of the action of choline-based ILs on Gram-negative bacteria and demonstrates the promise of CAGE as a powerful antimicrobial agent to treat infections. KEYWORDS: antibacterial, ionic liquid, membrane disruption, molecular dynamics simulation
1. INTRODUCTION
particular interest to this study, several groups have reported on the antibacterial properties of ILs containing choline, another quaternary ammonium cation, with a variety of counterions or functionalizing the choline cation.2−6 Previously, through a systematic evaluation of several different cation/anion pairings, we developed a Choline And GEranate ionic liquid (CAGE) with superior antimicrobial activity and low cytotoxicity to human cells.4 CAGE was tested for potential application as an antimicrobial agent to treat skin infection by determining its efficacy against 47 different strains of Gramnegative and Gram-positive bacteria, fungi, and viruses. In all cases, low concentrations of CAGE provided complete pathogen neutralization, including against E. coli (5% CAGE), methicillin-
The development of robust, broad-spectrum antibiotics that do not trigger microbial resistance continues to be one of the major challenges facing the healthcare community today. Ionic liquids (ILs), with their intrinsic antimicrobial qualities and highly tunable nature, have the potential to address this need. ILs are a broad class of compounds most commonly described by their low melting points ( 2:1> choline bicarbonate > pure geranic acid > Na+1:4. The order corroborates with the experimentally observed CAGE toxicity. The results of MBC testing showed this same trend in bactericidal activity among the four CAGE variants, with increasing geranic acid/ geranate content resulting in lower minimum bactericidal concentrations. Although the MBCs of the variants are not significantly different from their nearest neighbors when ordered by geranic acid content, the extremes (1:4 and 2:1) are significantly different (p < 0.05). From the growth kinetics of E. coli in sublethal doses of 1:2 CAGE variant, we determined that 50% of the lethal concentration was immediately inhibitory, while 25% (6.5 mM) inhibited growth after 3−4 h. Flow cytometry confirmed that cells remained viable after being exposed to a sublethal, or bacteriostatic dose (8 mM) for 2 h. There are
number of ions in 1:4 CAGE with 1:4 sodium geranate (Table 1) shows that replacing choline with the harder Na+ in the system results in the decrease of geranate and geranic acid adsorption in the membrane; geranate decreases from 16 to
