Antimicrobial lipids such as fatty acids and monoglycerides are promising antibacterial agents that destabilize bacterial cell membranes, causing a wide range of direct and indirect inhibitory effects. The goal of this review is to introduce the latest experimental approaches for characterizing how antimicrobial lipids destabilize phospholipid membranes within the broader scope of introducing current knowledge about the biological activities of antimicrobial lipids, testing strategies, and applications for treating bacterial infections. To this end, a general background on antimicrobial lipids, including structural classification, is provided along with a detailed description of their targeting spectrum and currently understood antibacterial mechanisms. Building on this knowledge, different experimental approaches to characterize antimicrobial lipids are presented, including cell-based biological and model membrane-based biophysical measurement techniques. Particular emphasis is placed on drawing out how biological and biophysical approaches complement one another and can yield mechanistic insights into how the physicochemical properties of antimicrobial lipids influence molecular self-assembly and concentration-dependent interactions with model phospholipid and bacterial cell membranes. Examples of possible therapeutic applications are briefly introduced to highlight the potential significance of antimicrobial lipids for human health and medicine, and to motivate the importance of employing orthogonal measurement strategies to characterize the activity profile of antimicrobial lipids.

Medium-chain saturated fatty acids and related compounds (e.g., monoglycerides) represent one class of membrane-active surfactants with antimicrobial properties. Most related studies have been in vitro evaluations of bacterial growth inhibition, and there is limited knowledge about how the compounds in this class destabilize lipid bilayers, which are the purported target within the bacterial cell membrane. Herein, the interaction between three representative compounds in this class and a supported lipid bilayer platform was investigated using quartz crystal microbalance-dissipation and fluorescence microscopy in order to examine membrane destabilization. The three tested compounds were lauric acid, sodium dodecyl sulfate, and glycerol monolaurate. For each compound, we discovered striking differences in the resulting morphological changes of supported lipid bilayers. The experimental trends indicate that the compounds have membrane-disruptive behavior against supported lipid bilayers principally above the respective critical micelle concentration values. The growth inhibition properties of the compounds against standard and methicillin-resistant Staphylococcus aureus bacterial strains were also tested. Taken together, the findings in this work improve our knowledge about how saturated fatty acids and related compounds destabilize lipid bilayers, offering insight into the corresponding molecular mechanisms that lead to membrane morphological responses.

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Supported lipid bilayers (SLBs) are widely studied model membrane platforms that are compatible with various surface-sensitive measurement techniques. SLBs are typically formed on silica-based materials, and there are numerous possible fabrication routes involving either bottom-up molecular self-assembly or vesicle adsorption and rupture. In between these two classes of fabrication strategies lies an emerging approach based on depositing quasi-two-dimensional lamellar, bicellar disks composed of a mixture of long-chain and short-chain phospholipids to promote the formation of SLBs. This approach takes advantage of the thermodynamic preference of long-chain phospholipids to form planar SLBs, whereas short-chain phospholipids have brief residence times. Although a few studies have shown that SLBs can be formed on silica-based materials from bicellar mixtures, outstanding questions remain about the self-assembly mechanism as well as the influence of the total phospholipid concentration, ratio of the two phospholipids (termed the "q-ratio"), and process of sample preparation. Herein, we address these questions through comprehensive quartz crystal microbalance-dissipation, fluorescence microscopy, and fluorescence recovery after photobleaching experiments. Our findings identify that optimal SLB formation occurs at lower total concentrations of phospholipids than previously used as short-chain phospholipids behave like membrane-destabilizing detergents at higher concentrations. Using lower phospholipid concentrations, we also discovered that the formation of SLBs proceeds through a two-step mechanism involving a critical coverage of bicellar disks akin to vesicle fusion. In addition, the results indicate that at least one cycle of freeze-thaw-vortexing is useful during the sample preparation process to produce SLBs. Taken together, the findings in this work identify optimal routes for fabricating SLBs from bicellar mixtures and reveal mechanistic details about the bicelle-mediated SLB formation process, which will aid further exploration of bicellar mixtures as tools for model membrane fabrication.

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