With the recent emergence of reports on resistant Gram-negative 'superbugs', infections caused by multidrug-resistant (MDR) Gram-negative bacteria have been named as one of the most urgent global health threats due to the lack of effective and biocompatible drugs. Here, we show that a class of antimicrobial agents, termed 'structurally nanoengineered antimicrobial peptide polymers' (SNAPPs) exhibit sub-μM activity against all Gram-negative bacteria tested, including ESKAPE and colistin-resistant and MDR (CMDR) pathogens, while demonstrating low toxicity. SNAPPs are highly effective in combating CMDR Acinetobacter baumannii infections in vivo, the first example of a synthetic antimicrobial polymer with CMDR Gram-negative pathogen efficacy. Furthermore, we did not observe any resistance acquisition by A. baumannii (including the CMDR strain) to SNAPPs. Comprehensive analyses using a range of microscopy and (bio)assay techniques revealed that the antimicrobial activity of SNAPPs proceeds via a multimodal mechanism of bacterial cell death by outer membrane destabilization, unregulated ion movement across the cytoplasmic membrane and induction of the apoptotic-like death pathway, possibly accounting for why we did not observe resistance to SNAPPs in CMDR bacteria. Overall, SNAPPs show great promise as low-cost and effective antimicrobial agents and may represent a weapon in combating the growing threat of MDR Gram-negative bacteria.

Infections caused by multidrug-resistant bacteria are on the rise and, therefore, new antimicrobial agents are required to prevent the onset of a postantibiotic era. In this study, we develop new antimicrobial compounds in the form of single-chain polymeric nanoparticles (SCPNs) that exhibit excellent antimicrobial activity against Gram-negative bacteria (e.g., Pseudomonas aeruginosa) at micromolar concentrations (e.g., 1.4 μM) and remarkably kill ≥99.99% of both planktonic cells and biofilm within an hour. Linear random copolymers, which comprise oligoethylene glycol (OEG), hydrophobic, and amine groups, undergo self-folding in aqueous systems due to intramolecular hydrophobic interactions to yield these SCPNs. By systematically varying the hydrophobicity of the polymer, we can tune the extent of cell membrane wall disruption, which in turn governs the antimicrobial activity and rate of resistance acquisition in bacteria. We also show that the incorporation of OEG groups into the polymer design is essential in preventing complexation with proteins in biological medium, thereby maintaining the antimicrobial efficacy of the compound even in in vivo mimicking conditions. In comparison to the last-resort antibiotic colistin, our lead agents have a higher therapeutic index (by ca. 2-3 times) and hence better biocompatibility. We believe that the SCPNs developed here have potential for clinical applications and the information pertaining to their structure-activity relationship will be valuable toward the general design of synthetic antimicrobial (macro)molecules.

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'Structurally nanoengineered antimicrobial peptide polymers' (SNAPPs), in the form of star-shaped peptide polymer nanoparticles, have been recently demonstrated as a new class of antimicrobial agents with superior in vitro and in vivo efficacy against Gram-negative pathogens, including multidrug-resistant species. Herein, we present a detailed bionano interaction study on SNAPPs by assessing their antimicrobial activities against several Gram-negative bacteria in complex biological matrices. Simulated body fluid and animal serum were used as test media to reveal factors that influence the antimicrobial efficacy of SNAPPs. With the exception of Acinetobacter baumannii, the presence of divalent cations at physiological concentrations reduced the antimicrobial efficacy of SNAPPs from minimum inhibitory concentrations (MICs) within the nanomolar range (40-300 nM) against Escherichia coli, Pseudomanas aeruginosa, and Klebsiella pneumoniae to 0.6-4.7 μM. By using E. coli as a representative bacterial species, we demonstrated that the reduction in activity was due to a decrease in the ability of SNAPPs to cause outer and inner membrane disruption. This effect could be reversed through coadministration with a chelating agent. Interestingly, the potency of SNAPPs against A. baumannii was retained even under high salt concentrations. The presence of serum proteins was also found to affect the interaction of SNAPPs with bacterial membranes, possibly through intermolecular binding. Collectively, this study highlights the need to consider the possible interactions of (bio)molecules present in vivo with any new antimicrobial agent under development. We also demonstrate that outer membrane disruption/destabilization is an important but hitherto under-recognized target for the antimicrobial action of peptide-based agents, such as antimicrobial peptides (AMPs). Overall, the findings presented herein could aid in the design of more efficient peptide-based antimicrobial agents with uncompromised potency even under physiological conditions.

This study presents a novel approach to synthesize biocompatible single-chain polymeric nanoparticles (SCPN) under mild reaction conditions via organo-catalyzed ring-opening polymerization (ROP). Linear polymeric precursors containing pendent polymerizable caprolactone groups, made by reversible addition−fragmentation chain transfer (RAFT) polymerization, were intramolecularly crosslinked via ROP in the presence of benzyl alcohol (nucleophilic initiator) and methanesulfonic acid (organo catalyst) to form discrete, well-defined SCPN, as confirmed by GPC, DLS, 1 H NMR, and AFM analysis. The formed SCPN are tunable in size (2−5 nm), depending on the molecular weight of the parent linear macromolecule. Furthermore, cytotoxicity studies revealed that the SCPN, which were covalently cross-linked by biodegradable polyester linkages, were nontoxic toward human embryonic kidney (HEK293T) cells. This study demonstrates the efficiency and versatility of this approach to generate uniformly sized soft nanoparticles with tunable dimensions that are potentially useful for a range of targeted applications, including drug delivery systems and membranes for gas separation technologies.