Comparison of facially amphiphilic versus segregated monomers in the design of antibacterial copolymers

Abstract

A direct comparison of two strategies for designing antimicrobial polymers is presented. Previously, we published several reports on the use of facially amphiphilic (FA) monomers which led to polynorbornenes with excellent antimicrobial activities and selectivities. Our polymers obtained by copolymerization of structurally similar segregated monomers, in which cationic and non-polar moieties reside on separate repeat units, led to polymers with less pronounced activities. A wide range of polymer amphiphilicities was surveyed by pairing a cationic oxanorbornene with eleven different non-polar monomers and varying the comonomer feed ratios. Their properties were tested using antimicrobial assays and copolymers possessing intermediate hydrophobicities were the most active. Polymer-induced leakage of dye-filled liposomes and microscopy of polymer-treated bacteria support a membrane-based mode of action. From these results there appears to be profound differences in how a polymer made from FA monomers interacts with the phospholipid bilayer compared with copolymers from segregated monomers. We conclude that a well-defined spatial relationship of the whole polymer is crucial to obtain synthetic mimics of antimicrobial peptides (SMAMPs): charged and non-polar moieties need to be balanced locally, for example, at the monomer level, and not just globally. We advocate the use of FA monomers for better control of biological properties. It is expected that this principle will be usefully applied to other backbones such as the polyacrylates, polystyrenes, and non-natural polyamides.

Figure 1

Two methods to endow polynorbornenes, based on polyamine oxanorbornene, PAON, with antibacterial properties. The center structure was previously reported.^[20] The right structure, A5′, synthesized for this study, shows a five-carbon branched chain as the non-polar moiety (one of eleven alkyl chains tested). Presumably, rotation about the single bonds (red curved arrows) in the polymer backbone allows for the orientation of these copolymers into a globally FA conformation.

Figure 2

50:50 random copolymers synthesized at two different molecular weights. PAON and several non-50:50 compositions were also synthesized for a total of thirty-two polymers. Notation examples: PAON = the homo-amine polymer, A5′=50:50 random copolymer of the amine and the 5′ non-polar monomer.

Figure 3

MICs for E. coli of selected polymers illustrating optimal hydrophobicity for the most active ones A4, A5 (low M_n) and A3 (high M_n).

Figure 4

Cartoon proposing that polymer interactions with the polar head group (black circles) and the non-polar lipid tails (squiggly lines) of the membrane are significantly different for polymers from segregated monomers (A) versus that of polymers from FA monomers (B).

Figure 5

Graph of polymer-induced dye leakage.

Figure 6

Fluorescence microscopy of stained E. coli in the absence and presence of polymer. Each sample consisted of 10⁸ bacteria cells incubated for 30 min with polymer (75 μg mL⁻¹ final concentration) or buffer. Left panels were visualized with a green filter to display SYTO9 emission and the right panels show the same fields visualized with a red filter to display propidium iodide emission.