Membrane-active molecules provide a promising strategy to target and kill pathogenic bacteria. Understanding how specific molecular features drive interactions with membrane components and subsequently cause disruption that leads to antimicrobial activity is a crucial step in designing next-generation treatments. Here, we test a library of lipid-like compounds (lipidoids) against Gram-negative bacteria Escherichia coli to garner in-depth structure–activity relationships using antimicrobial assays. Modular lipidoid molecules were synthesized in high-throughput, such that we could analyze 104 compounds with variable combinations of hydrophobic tails and cationic headgroups. Antibacterial activity was strongly correlated to specific structural features, including tail hydrophobicity and headgroup charge density, and also to the overall molecular shape and propensity for self-assembly into curved liquid crystalline phases. Dye permeabilization assays showed that E. coli membranes were permeabilized by lipidoids, confirming their membrane-active nature. The reduced permeabilization, as compared to Gram-positive Bacillus subtilis, alludes to the challenge of permeabilizing the additional outer membrane layer of E. coli. The effect of headgroup solubility in gemini-type lipidoids was also demonstrated, revealing that a headgroup with a more hydrophilic spacer between amine groups had enhanced activity against B. subtilis but not E. coli. This provides insight into features enabling outer membrane penetration and governing selectivity between bacterial species.

The search for next-generation antibacterial compounds that overcome the development of resistance can be facilitated by identifying how to target the cell membrane of bacteria. Understanding the key molecular features that enable interactions with lipids and lead to membrane disruption is therefore crucial. Here we employ a library of lipid-like compounds (lipidoids) comprising modular structures with tunable hydrophobic and hydrophilic architecture, to shed light on how the chemical functionality and molecular shape of synthetic amphiphilic compounds determine their activity against bacterial membranes. Synthesized from combinations of 8 different polyamines as headgroups and 13 acrylates as tails, 104 different lipidoids are tested for activity against a model Gram-positive bacterial strain (Bacillus subtilis). Results from the high-throughput antimicrobial screening assay show that lipidoids with the most potent antimicrobial properties (down to 2 μM) have intermediate tail hydrophobicity (i.e. logP values between 3 and 4), and lower headgroup charge density (i.e. longer spacer groups between charged amines). However, the most important factor appeared to be the ability of a lipidoid to self-assemble into an inverse hexagonal liquid crystalline phase, as observed by small angle X-ray scattering (SAXS) analysis. The lipidoids active at lowest concentrations, and which induced the most significant membrane damage during propidium iodide (PI) permeabilization assays, were those that aggregated into highly-curved inverse hexagonal liquid crystal phases. These observations suggest that the introduction of strong curvature stress into the membrane is one way to maximize membrane disruption and lipidoid antimicrobial activity. Lipidoids that demonstrated the ability to furnish this phase consisted of either i) branched or linear headgroups with shorter linear tails, or ii) cyclic headgroups with 4 bulky non-linear tails. On the contrary, lipidoids previously observed to adopt disc-like conformations that pack into bicontinuous cubic phases were significantly less effective against B. subtilis. The discovery of these structure-property relationships demonstrates that it is not simply a balance of hydrophobic and hydrophilic moieties that governs membrane-active antibacterial activity, but also their intrinsic curvature and collective behavior.