In this study, we report that 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 increasing the number of cationic groups of copolymers to be more than the 30 mole % 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). On the other hand, the MIC values of copolymers with > 30 mole % of AEMA depend on only the mole % of EMA, indicating that the hydrophobic interactions of 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.

Antimicrobial peptides (AMPs) in free solution can kill bacteria by disrupting bacterial cell membranes. Their modes of action have been extensively studied, and various models ranging from pore formation to carpet-like mechanisms were proposed. Surface-immobilized AMPs have been used as coatings to kill bacteria and as sensors to capture bacteria, but the interaction mechanisms of surface-immobilized AMPs and bacteria are not fully understood. In this research, an analytical platform, sum frequency generation (SFG) microscope, which is composed of an SFG vibrational spectrometer and a fluorescence microscope, was used to probe molecular interactions between surface-immobilized AMPs and bacteria in situ in real time at the solid/liquid interface. SFG probed the molecular structure of surface-immobilized AMPs while interacting with bacteria, and fluorescence images of dead bacteria were monitored as a function of time during the peptide-bacteria interaction. It was believed that upon bacteria contact, the surface-immobilized peptides changed their orientation and killed bacteria. This research demonstrated that the SFG microscope platform can examine the structure and function (bacterial killing) at the same time in the same sample environment, providing in-depth understanding on the structure-activity relationships of surface-immobilized AMPs.

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