Permanent, non-leaching antibacterial surfaces—2: How high density cationic surfaces kill bacterial cells

Abstract

Rational controlled synthesis of poly(quaternary ammonium) compounds has been used to prepare antimicrobial polymer brushes on inorganic surfaces. The systematic variation of several structural parameters of the polymeric brushes allowed us to elicit the minimum surface requirements and a probable mechanism of action for Escherichia coli cell kill. Polymeric brushes were prepared by surface-initiated atom transfer radical polymerization of 2-(dimethylamino)ethyl methacrylate (DMAEMA), a method that allows the molecular weight of the polymer chains to be precisely controlled as they grow from the target surface. The tertiary amino groups of the polyDMAEMA were then quaternized with alkyl bromides to provide a surface with antimicrobial activity. Dry layer thickness of the polymer brushes was controlled by polymerization time and/or initiator density on the surface. This tunability of surface structure allows the antimicrobial polymer brushes to be tailored rationally. A combinatorial screening tool was developed to elucidate the role of chain length and chain density on cell kill in a single experiment. The results indicate that surface charge density, is a critical element in designing a surface for maximum kill efficiency. The most biocidal surfaces had charge densities of greater than 1–5×10¹⁵ accessible quaternary amine units/cm². The relevance of this finding to the mechanism of action is discussed.

Introduction

Antimicrobial materials are used to prevent microbial infection in a wide variety of industrial, medical, community, and private settings. To obtain biocidal effect without releasing biocide into the environment, antimicrobial species can be irreversibly (covalently) coupled to material surfaces. Such materials also reduce the likelihood of generating resistance to the active agent. Recent studies have reported the successful covalent attachment of polymeric antimicrobial materials onto glass, [1], [2], [3], [4] polymer, [5], [6], [7], [8], [9], [10], [11], [12] paper, [4] and metal [13]. In many of these cases the biocidal polymer [14], [15] contained cationic groups, such as alkyl pyridinium [1], [2], [3], [5], [6], [7], [8] or quaternary ammonium [4], [9], [10], [11], [12], [13]. Cationic antimicrobials are especially well positioned to play a role in the development of self-disinfecting surfaces [16]. Recently, quaternized poly-2-(dimethylamino)ethyl methacrylate (polyDMAEMA) has been used as a cationic surfactant [17], within polymer microspheres, [18] in which they exhibit high levels of antibacterial activity.

The quaternary ammonium salts (QAS) are among the most commonly used of the cationic antimicrobials [16]. Within the large group of QAS, the polymeric quaternary amines show perhaps the greatest promise in the realm of surface-active compounds. Several reports have suggested that the mechanism of action of polycations involves disruption of the integrity of the cell membrane [1], [3], [6], [7], [8], [9], [10]. Two mechanistic hypotheses have been put forward to explain why a wide range of species are susceptible to polyquaternary amine-induced death. The most quoted theory hypothesizes that long cationic polymers penetrate cells and thereby disrupt the membrane, like a needle bursting a balloon [1], [3], [6], [12]. This concept has persisted in the literature for some time [14], [15] despite the fact that in one of the first demonstrations of a permanent antimicrobial surface, a monolayer of 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride bound to a surface, was known to be highly antimicrobial [19]. The mechanistically-driven design of even more effective surface active QAS-based antimicrobials naturally depends on whether this hypothesis fully explains the observation that all polymeric QAS can kill microbes, from viruses to fungi, even though the different microbes have dramatically different membrane properties and dimensions. An alternative mechanism that has been offered to explain the breadth of bioactivity posits that a highly charged surface can induce what is essentially an ion exchange between the positive charges on the surface and structurally critical mobile cations within the membrane [2]. Upon approaching a cationic surface, the structurally essential divalent cations of the membrane are relieved of their role in charge neutralization of the membrane components and are thus free to diffuse out of the membrane. The loss of these structural cations results in a loss of membrane integrity [2].

While soluble compounds may be able to penetrate the cell envelope, surface-bound molecules are constrained by their molecular length and could only penetrate the cell membrane if they extended far enough away from the surface. Most bacteria have non-deformable, rigid outer envelopes. For the Gram negative Escherichia coli, the thickness of the cell envelope (cytoplasmic membrane, periplasmic space, peptidoglycan, and outer membrane from inside to outside) has recently been shown to be 46 nm [20], [21], [22]. The measurement is about 45–55 nm for the Gram positive Bacillus subtilis [20] which lacks an outer membrane but has a thicker peptidoglycan layer. This would mean that the wet layer thickness of a polymer coating on a surface should be at least 75 nm in order to effectively penetrate the cytoplasmic membrane and kill the cells. QAS polymers have recently been shown to kill fungal mycelia which have cell walls wider than 80 nm [23]. We have begun to explore how high density polymer brushes exhibit such broad bioactivity. Some of the highest density polymer brushes can be formed by surface-initiated atom transfer radical polymerization (ATRP) [4], which is among the most efficient controlled/“living” radical polymerization systems [24], [25], [26], [27], [28], [29], [30], [31]. ATRP-synthesized covalently attached polymer brushes also yield precise molecular weights and/or grafting density. We show herein that short chains with high grafting density and long chains with low grafting density are equally effective against E. coli.