Combinatorial Screening of Cationic Lipidoids Reveals How Molecular Conformation Affects Membrane-Targeting Antimicrobial Activity

The search for next-generation antibacterial compoundsthat overcomethe development of resistance can be facilitated by identifying howto target the cell membrane of bacteria. Understanding the key molecularfeatures that enable interactions with lipids and lead to membranedisruption is therefore crucial. Here, we employ a library of lipid-likecompounds (lipidoids) comprising modular structures with tunable hydrophobicand hydrophilic architecture to shed light on how the chemical functionalityand molecular shape of synthetic amphiphilic compounds determine theiractivity against bacterial membranes. Synthesized from combinationsof 8 different polyamines as headgroups and 13 acrylates as tails,104 different lipidoids are tested for activity against a model Gram-positivebacterial strain (Bacillus subtilis). Results from the combinatorial screening assay show that lipidoidswith the most potent antimicrobial properties (down to 2 & mu;M)have intermediate tail hydrophobicity (i.e., c log P values between 3 and 4) and lower headgroup charge density(i.e., longer spacers between charged amines). However, the most importantfactor appeared to be the ability of a lipidoid to self-assemble intoan inverse hexagonal liquid crystalline phase, as observed by small-angleX-ray scattering (SAXS) analysis. The lipidoids active at lowest concentrations,which induced the most significant membrane damage during propidiumiodide (PI) permeabilization assays, were those that aggregated intohighly curved inverse hexagonal liquid crystal phases. These observationssuggest that the introduction of strong curvature stress into themembrane is one way to maximize membrane disruption and lipidoid antimicrobialactivity. Lipidoids that demonstrated the ability to furnish thisphase consisted of either (i) branched or linear headgroups with shorterlinear tails or (ii) cyclic headgroups with 4 bulky nonlinear tails.On the contrary, lipidoids previously observed to adopt disc-likeconformations that pack into bicontinuous cubic phases were significantlyless effective against B. subtilis.The discovery of these structure-property relationships demonstratesthat it is not simply a balance of hydrophobic and hydrophilic moietiesthat govern membrane-active antibacterial activity, but also theirintrinsic curvature and collective behavior.