Responsive Quaternized PDMAEMA Copolymers with Antimicrobial Action

Abstract

In this work, the antimicrobial action of partially quaternized poly(2-(dimethylamino)ethyl methacrylate) (PQDMAEMA) copolymers using different alkyl halides is presented. The poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) homopolymer was synthesized by group transfer polymerization, followed by the modification of its tertiary amine groups, using bromoethane, iodoethane, bromohexane and bromoethanol, to introduce permanent cationic, quaternary ammonium salt moieties, randomly distributed along the polymer chains. In all cases, the degree of quaternization was low, at ~10 mol%, as verified by proton nuclear magnetic resonance spectroscopy to preserve the thermo-responsive character of the PDMAEMA precursor polymer. The biocidal activity of the lightly quaternized PQDMAEMA copolymers against Escherichia coli and Staphylococcus aureus was evaluated by calculating the minimum inhibitory concentration (MIC) as well as the minimum bactericidal concentration (MBC) of the polymers and by comparing them to the respective values of the precursor non-quaternized PDMAEMA homopolymer. The antibacterial mechanism of action in the solution was studied by zeta potential measurements, scanning electron microscopy and protein leakage tests signifying the disruption of the outer membrane of the bacterial cells to release their periplasmic proteins.

Keywords: E. coli; MBC; MIC; PDMAEMA; S. aureus; antimicrobial polymers; minimum bactericidal concentration; minimum inhibitory concentration; quaternization.

Scheme 1

Synthesis of PDMAEMA homopolymer and quaternization reaction using different alkyl halides to obtain the PQDMAEMA copolymers.

Figure 1

Absorbance vs. temperature curves for 1 wt% aqueous solutions of the PDMAEMA, PQDMAEMA-EtI, PQDMAEMA-EtBr, PQDMAEMA-HexBr and PQDMAEMA-EtBrOH copolymers at pH 9.5.

Figure 2

Representative scanning electron microscopy images of E. coli (left) and S. aureus (right) following incubation with 5 mg/mL cationic polymers for 5 and 24 h. The control represents the morphology of cells cultured in the absence of the polymers. Magnification is 10,000× and the scale bars represent 1 μm.

Figure 3

Growth kinetics of E. coli in the presence of polymer concentrations ranging from 0.16 to 5 mg/mL. The optical densities were measured for 30 h incubation at 37 °C using a multi-detection microplate reader at 600 nm and were automatically recorded for each well every 15 min. In all experiments, the bacterial suspension in pure LB and in the presence of amoxicillin/clavulanic acid were measured as the negative (C−) and positive (C+) control, respectively.

Figure 4

Growth kinetics of S. aureus in the presence of polymer concentrations ranging from 0.16 to 5 mg/mL. The optical densities were measured for 30 h incubation at 37 °C using a multi-detection microplate reader at 600 nm and were automatically recorded for each well every 15 min. In all experiments, the bacterial suspension in pure LB and in the presence of amoxicillin/clavulanic acid were measured as the negative (C−) and positive (C+) control, respectively.

Figure 5

Protein leakage determined by the Bradford assay for E. coli and S. aureus incubated in the presence of the polymers at a concentration of 5 mg/mL, or in the absence of the polymers (negative control, C−) for 1, 2 or 4 h. The asterisk (*) designates significant differences in the presence of the different polymers compared to the E. coli and S. aureus control at each experimental time point (p < 0.001).

Figure 6

Zeta potential values for 5 mg/mL polymer solutions in LB (blue bars), and E. coli (green bars) and S. aureus (pink bars) suspensions incubated with 5 mg/mL cationic polymers in LB (blue bars).