Bacterial Membrane Selective Antimicrobial Peptide-Mimetic Polyurethanes: Structure–Property Correlations and Mechanisms of Action

Abstract

The rise in prevalence of antibiotic resistant strains of bacteria is a very significant challenge for treating life-threatening infections worldwide. A source of novel therapeutics that has shown great promise is a class of biomolecules known as antimicrobial peptides. Previously, within our laboratories, we developed a new family of water-soluble antimicrobial polyurethanes that mimic antimicrobial peptides. Within this current investigation, studies were carried out to gain a greater understanding of the structure/property relationships of the polyurethanes. This was achieved by synthesizing a variety of pendant group functionalized polyurethanes and testing their effectiveness as an antimicrobial by carrying out minimum inhibitory concentration testing and determining their compatibility with blood cells. Additionally, insight into the mode of action of the polyurethanes was obtained through experiments using dye encapsulated phospholipids and assays of bacterial cells that indicated the ability of the polyurethanes to penetrate and disrupt membranes. Collectively, the results indicate that the addition of hydrophobic, uncharged polar, and anionic moieties do not have a strong influence on the antimicrobial activity; yet, the addition of hydrophobic groups enhances cytoplasmic membrane disruption, a larger proportion of cationic pendant groups promotes greater outer membrane disruption of Gram negative bacteria, and uncharged polar groups and anionic groups improve compatibility of the polyurethanes with mammalian cells.

Antimicrobial and Antibiofilm Peptides

“…Their mechanism of action has been widely studied. AMPs can lead to bacterial cell death through both membranolytic [33][34][35] and non-membranolytic mechanisms, interacting with intracellular targets, such as DNA, RNA, and proteins [36][37][38][39]. Both Gram-negative and Gram-positive bacteria have molecules on the outer membrane that confer a negative net charge, allowing the electrostatic interaction with cationic peptides [24].…”

Peptidomimetic polyurethanes disrupt surface established bacterial biofilms and prevent biofilm formation

“…The polyurethanes were synthesized using a simple, one-pot step-growth polymerization of N-functionalized diethanolamide monomers with hexamethylene diisocyanate using methods previously optimized in our lab, and detailed in the experimental section (Figure 1). [43][44] The monomers were synthesized as reported previously and were designed to be structural mimics of lysine (mLys), alanine (mAla), and phenylalanine (mPhe). 44,48 To mimic arginine (mArg), the mLys units were guanylated after the polymerization using 1H-Pyrazole-1-carboxamidine hydrochloride in the presence of triethylamine in DMF.…”

“…Table 1 shows that the molar masses (Mn)of the polyurethanes range between 9-10 kg/mol. Since the molar mass and hydrophobicity of the polymer directly influence its toxicity towards mammalian cells, [43][44] we minimized the cytotoxicity of the designed polymers by synthesizing polymers with lower molecular weights to compensate for the addition of hydrophobic pendant groups. We first tested the ability of these polymers to prevent biofilm formation on abiotic surfaces against three common, biofilm-forming opportunistic bacterial pathogens, viz., P. aeruginosa, S. aureus, and E. coli, in BM2 minimal medium supplemented with 0.4% glucose and 0.5% casamino acids (which supports biofilm formation of all three species studied).…”

“…Since these polymers share design features with antimicrobial polymers, we also evaluated their antimicrobial activity against planktonic bacteria using the modified broth microdilution method. [42][43][44] The MIC of the polymers was determined in MHB, LB, and BM2 minimal medium. As depicted in Table 2 the polymers showed no inhibitory activity against P. aeruginosa, S. aureus, or E. coli in MHB or LB until 250 μg/mL above which they could not be tested due to their poor solubility in LB and MHB.…”

“…25 We have previously reported a library of synthetic peptidomimetic antimicrobial polymers with tunable selectivity and low cytotoxicity. [42][43][44][45] In this study, we focused on variants of these polymers with activity against biofilms and systematically explored the mechanisms behind their antibiofilm characteristics through structureproperty relationships. Inspired by natural [31][32] and synthetic [33][34][35]46 antibiofilm peptides reported in the literature, we designed polyurethanes using our pendant-functionalized, amino acid mimetic monomer library to understand how individual amino acid mimetic units would influence the activity of the polyurethane against biofilms, and the surface interactions of bacteria preceding biofilm formation.…”