Hemolysis induced by antimicrobial polymers was examined to gain an understanding of the mechanism of polymer toxicity to human cells. A series of cationic amphiphilic methacrylate random copolymers containing primary ammonium groups as the cationic functionality and either butyl or methyl groups as hydrophobic side chains have been prepared by radical copolymerization. Polymers with 0−47 mol % methyl groups in the side chains, relative to the total number of monomeric units, showed antimicrobial activity but no hemolysis. The polymers with 65 mol % methyl groups or 27 mol % butyl groups displayed both antimicrobial and hemolytic activity. These polymers induced leakage of the fluorescent dye calcein trapped in human red blood cells (RBCs), exhibiting the same dose−response curves as for hemoglobin leakage. The percentage of disappeared RBCs after hemolysis increased in direct proportion to the hemolysis percentage, indicating complete release of hemoglobin from fractions of RBCs (all-or-none leakage) rather than partial release from all cells (graded leakage). An osmoprotection assay using poly(ethylene glycol)s (PEGs) as osmolytes indicated that the PEGs with MW > 600 provided protection against hemolysis while low molecular weight PEGs and sucrose had no significant effect on the hemolytic activity of polymers. Accordingly, we propose the mechanism of polymer-induced hemolysis is that the polymers produce nanosized pores in the cell membranes of RBCs, causing an influx of small solutes into the cells and leading to colloid-osmotic lysis.

Antimicrobial and hemolytic activities of amphiphilic random copolymers were modulated by the structure of the cationic side chain spacer arms, including 2-aminoethylene, 4-aminobutylene, and 6-aminohexylene groups. Cationic amphiphilic random copolymers with ethyl methacrylate (EMA) comonomer were prepared with a range of comonomer fractions, and the library of copolymers was screened for antimicrobial and hemolytic activities. Copolymers with 4-aminobutylene cationic side chains showed an order of magnitude enhancement in their antimicrobial activity relative to those with 2-aminoethylene spacer arms, without causing adverse hemolysis. When the spacer arms were further elongated to hexylene, the copolymers displayed potent antimicrobial and hemolytic activities. The 4-aminobutylene side chain appears to be the optimal spacer arm length for maximal antimicrobial potency and minimal hemolysis, when combined with hydrophobic ethylmethacrylate in a roughly 70/30 ratio. The copolymers displayed relatively rapid bactericidal kinetics and broad-spectrum activity against a panel of Gram-positive and Gram-negative bacteria. The effect of the spacer arms on the polymer conformation in the membrane-bound state was investigated by molecular dynamics simulations. The polymer backbones adopt an extended chain conformation, parallel to the membrane surface. A facially amphiphilic conformation at the membrane surface was observed, with the primary ammonium groups localized at the lipid phoshophate region and the nonpolar side chains of EMA comonomers buried in the hydrophobic membrane environment. This study demonstrates that the antimicrobial activity and molecular conformation of amphiphilic methacrylate random copolymers can be modulated by adjustment of cationic side chain spacer arms.