Effect of cyclic topology versus linear terpolymers on antibacterial activity and biocompatibility: antimicrobial peptide avatars

Abstract

Host-defense peptides (HDPs) and their analogs hold significant potential for combating multidrug-resistant (MDR) bacterial infections. However, their clinical use has been hindered by susceptibility to proteases, high production costs, and cytotoxicity towards mammalian cells. Synthetic polymers with diverse topologies and compositions, designed to mimic HDPs, show promise for treating bacterial infections. In this study, we explored the antibacterial activity and biocompatibility of synthetic amphiphilic linear (LPs) and cyclic terpolymers (CPs) containing hydrophobic groups 2-ethylhexyl (E) and 2-phenylethyl (P) at 20% and 30% content. LPs were synthesized via RAFT polymerization and then cyclized into CPs through a hetero-Diels-Alder click reaction. The bioactivity of these terpolymers was correlated with their topology (LPs vs. CPs) and hydrophobic composition. LPs demonstrated superior antibacterial efficacy compared to CPs against four Gram-negative bacterial strains, with terpolymers containing (P) outperforming those with (E). Increasing the hydrophobicity from 20% to 30% in the terpolymers increased toxicity to both bacterial and mammalian cells. Notably, our terpolymers inhibited the MDR Gram-negative bacterial strain PA37 more effectively than gentamicin and ciprofloxacin. Furthermore, our terpolymers were able to disrupt cell membranes and rapidly eliminate Gram-negative bacteria (99.99% within 15 minutes). Interestingly, CPs exhibited higher hemocompatibility and biocompatibility with mammalian macrophage cells compared to LPs, showcasing a better safety profile (CPs > LPs). These findings underscore the importance of tailoring polymer architectures and optimizing the hydrophilic/hydrophobic balance to address challenges related to toxicity and selectivity in developing antimicrobial polymers.

Fig. 1. (A) Synthesis scheme of RAFT agent. (B) Chemical structures of amphiphilic LPE, CPE, LPP, and CPP (X = percentage of cationic groups, Y = percentage of hydrophilic groups, and Z = percentage of hydrophobic groups.

Fig. 2. (A) UV-vis spectra of LPs (LPE-20 and LPP-30) in DMSO. (B) UV-vis spectra of Boc-protected LP (LPP-30) and its corresponding CP (CPP-30) in DMSO. (C) MWDs of the Boc-protected LPE-20 and CPE-20 terpolymers obtained by SEC.

Fig. 3. Proposed mechanism of interaction of cationic linear polymers (LPs) and cyclic polymers (CPs) with Gram-negative bacterial cell membranes. (A) Due to their high flexibility and mobility, LPs can maximize contact with the bacterial membrane, promoting stronger electrostatic interactions and efficient membrane disruption. (B) Conversely, the constrained conformation of CPs limits their surface contact and interaction with the bacterial membrane, potentially reducing their antibacterial activity.

Fig. 4. The red fluorescence (emitting at 670 nm) intensity of Gram-negative, E. coli in the presence of different concentrations of LPP-30 and CPP-30 terpolymers.

Fig. 5. Time-kill kinetics of LPP-30 and CPP-30 against P. aeruginosa PA 27853 cells, determined via CFU assay, using an initial concentration of PA 27853 cells of 4.7 × 10⁵ CFU mL⁻¹. The total PA 27853 bacterial cells killed after incubation with different concentration of the terpolymers at 37 °C in PBS (pH 7.4) was plotted against time. Figures (A and B) and (C and D) display the time-kill curves for PA 27853 cells treated with LPP-30 and CPP-30 terpolymers, respectively, at 1×MIC and 4×MIC concentrations for 15, 30, and 60 minutes (n = 3). The corresponding MIC₉₀ values for LPP-30 and CPP-30 against PA 27853 are provided in Table 2.