Tuning the biological activity profile of antibacterial polymers via subunit substitution pattern

Abstract

Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp(3) carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the hydrophobic subunits. The α,α,β,β-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be generally valuable in the development of synthetic polymers for biological applications.

Figure 1

(a) β-Lactams used in this study, (b) representative copolymer synthesis, (c) nylon-3 copolymers prepared from equimolar binary β-lactam mixtures and containing 50% DM and 50% of a hydrophobic subunit, and (d) PHMB. The DM and CH subunits are racemic. All polymers are heterochiral.

Figure 2

(a) Consumption of β-lactams as a function of reaction progress for the copolymerization of 1:1 DM:CH (×), 1:1 DM:TM (○), or 1:1 DM:βDE (red ▲). Reactions were conducted at rm temp with an initial concentration of 50 mM for each β-lactam and 5 mM for the co-initiator tBuBzCl (5 mol % relative to the total amount of β-lactam), to prepare copolymers with an average 20-mer length. Measurement of subunit incorporation is described in the Supporting Information. (b) Cartoons of copolymers showing differences in compositional drift along the polymer chains.

Figure 3

Binary hydrophobic-cationic nylon-3 copolymers containing TM, βDE, or βCP subunits and DM subunits. The DM precursor was racemic, so all copolymers are heterochiral. Polymers within each series have variable subunit proportion; x + y = 100, with x = 40–100.

Figure 4

Summary of biological activity profiles (antibacterial activities, 3T3 fibroblast toxicity, and hemolytic activities) as a function of cationic:hydrophobic subunit proportion for the three sets of binary nylon-3 copolymers shown in Figure 3. The lines drawn for 3T3 fibroblast toxicity and hemolysis merely connect data points. MIC is the minimum inhibitory concentration for bacterial growth; IC₁₀ is the polymer concentration required to induce 10% 3T3 fibroblast death; and HC₁₀ is the polymer concentration required to cause 10% lysis of human red blood cells. When the IC₁₀ or HC₁₀ value is >400 μg/mL, the plot shows a concentration at 400 μg/mL.

Figure 5

Antibacterial resistance tests for 1:1 DM:TM with E. coli and MRSA. MIC is the minimum inhibitory concentration for bacterial growth; MBC is the minimum bactericidal concentration for 99.9% killing of the bacteria.

Figure 6

Antibacterial activity of copolymer 1:1 DM:TM against four pathogenic bacteria.