Decoupling the Functional Roles of Cationic and Hydrophobic Groups in the Antimicrobial and Hemolytic Activities of Methacrylate Random Copolymers

Abstract

In this study, we report the antimicrobial and hemolytic activities of ternary statistical methacrylate copolymers consisting of cationic ammonium (amino-ethyl methacrylate: AEMA), hydrophobic alkyl (ethyl methacrylate: EMA), and neutral hydroxyl (hydroxyethyl methacrylate: HEMA) side chain monomers. The cationic and hydrophobic functionalities of copolymers mimic the cationic amphiphilicity of naturally occurring antimicrobial peptides (AMPs). The HEMA monomer units were used to separately modulate the compositions of cationic and hydrophobic monomers, and we investigated the effect of each component on the antimicrobial and hemolytic activities of copolymers. Our data indicated that increasing the number of cationic groups of the copolymers to be more than 30 mol % did not increase their antimicrobial activity against Escherichia coli. The number of cationic side chains in a polymer chain at this threshold is 5.5-7.7, which is comparable to those of natural antimicrobial peptides such as maginin (+6). The MIC values of copolymers with >30 mol % of AEMA depend on only the mol % of EMA, indicating that the hydrophobic interactions of the copolymers with E. coli cell membranes determine the antimicrobial activity of copolymers. These results suggest that the roles of cationic and hydrophobic groups can be controlled independently by design in the ternary copolymers studied here.

Figure 1.

Synthesis of ternary statistical copolymers. The statistical copolymers were prepared by free-radical polymerization in the presence of thiol chain transfer agent methyl 3-mercaptopropionate. The boc and TMS groups were removed using trifluoroacetic acid at room temperature.

Figure 2.

Antimicrobial activity of copolymers against E. coli. The MIC values were plotted as a function of the mole percentage of (A) HEMA or (B) AEMA for each series of copolymers with different EMA compositions (20–50 mole %).

Figure 3.

Hemolytic activity of copolymers against human red blood cells. The HC50 values were plotted as a function of (A) HEMA or (B) AEMA for the copolymers with 50 mole % of EMA.

Figure 4.

Hemolytic activities of ternary copolymers with ~30 mole % AEMA.

Figure 5.

Proposed antimicrobial mechanism of cationic amphiphilic copolymers, (i) Cationic polymer chains are attracted to the anionic E. coli cell membrane surface by electrostatic interactions, and (ii) associated with the membrane surface, (iii) The hydrophobic groups were inserted into the bacterial cell membranes, followed by the formation of membrane-active conformation and (iv) membrane disruption (MIC-determining step).