Unraveling the Biophysical Mechanisms of How Antiviral Detergents Disrupt Supported Lipid Membranes: Toward Replacing Triton X-100.

Triton X-100 (TX-100) is a membrane-disrupting detergent that is widely used to inactivate membrane-enveloped viral pathogens, yet is being phased out due to environmental safety concerns. Intense efforts are underway to discover regulatory acceptable detergents to replace TX-100, but there is scarce mechanistic understanding about how these other detergents disrupt phospholipid membranes and hence which ones are suitable to replace TX-100 from a biophysical interaction perspective. Herein, using the quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) techniques in combination with supported lipid membrane platforms, we characterized the membrane-disruptive properties of a panel of TX-100 replacement candidates with varying antiviral activities and identified two distinct classes of membrane-interacting detergents with different critical micelle concentration (CMC) dependencies and biophysical mechanisms. While all tested detergents formed micelles, only a subset of the detergents caused CMC-dependent membrane solubilization similarly to that of TX-100, whereas other detergents adsorbed irreversibly to lipid membrane interfaces in a CMC-independent manner. We compared these biophysical results to virus inactivation data, which led us to identify that certain membrane-interaction profiles contribute to greater antiviral activity and such insights can help with the discovery and validation of antiviral detergents to replace TX-100.

Membrane-Disruptive Effects of Fatty Acid and Monoglyceride Mitigants on E. coli Bacteria-Derived Tethered Lipid Bilayers.

We report electrochemical impedance spectroscopy measurements to characterize the membrane-disruptive properties of medium-chain fatty acid and monoglyceride mitigants interacting with tethered bilayer lipid membrane (tBLM) platforms composed of E. coli bacterial lipid extracts. The tested mitigants included capric acid (CA) and monocaprin (MC) with 10-carbon long hydrocarbon chains, and lauric acid (LA) and glycerol monolaurate (GML) with 12-carbon long hydrocarbon chains. All four mitigants disrupted E. coli tBLM platforms above their respective critical micelle concentration (CMC) values; however, there were marked differences in the extent of membrane disruption. In general, CA and MC caused larger changes in ionic permeability and structural damage, whereas the membrane-disruptive effects of LA and GML were appreciably smaller. Importantly, the distinct magnitudes of permeability changes agreed well with the known antibacterial activity levels of the different mitigants against E. coli, whereby CA and MC are inhibitory and LA and GML are non-inhibitory. Mechanistic insights obtained from the EIS data help to rationalize why CA and MC are more effective than LA and GML at disrupting E. coli membranes, and these measurement capabilities support the potential of utilizing bacterial lipid-derived tethered lipid bilayers for predictive assessment of antibacterial drug candidates and mitigants.

Unraveling Membrane-Disruptive Properties of Sodium Lauroyl Lactylate and Its Hydrolytic Products: A QCM-D and EIS Study.

Membrane-disrupting lactylates are an important class of surfactant molecules that are esterified adducts of fatty acid and lactic acid and possess industrially attractive properties, such as high antimicrobial potency and hydrophilicity. Compared with antimicrobial lipids such as free fatty acids and monoglycerides, the membrane-disruptive properties of lactylates have been scarcely investigated from a biophysical perspective, and addressing this gap is important to build a molecular-level understanding of how lactylates work. Herein, using the quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) techniques, we investigated the real-time, membrane-disruptive interactions between sodium lauroyl lactylate (SLL)-a promising lactylate with a 12-carbon-long, saturated hydrocarbon chain-and supported lipid bilayer (SLB) and tethered bilayer lipid membrane (tBLM) platforms. For comparison, hydrolytic products of SLL that may be generated in biological environments, i.e., lauric acid (LA) and lactic acid (LacA), were also tested individually and as a mixture, along with a structurally related surfactant (sodium dodecyl sulfate, SDS). While SLL, LA, and SDS all had equivalent chain properties and critical micelle concentration (CMC) values, our findings reveal that SLL exhibits distinct membrane-disruptive properties that lie in between the rapid, complete solubilizing activity of SDS and the more modest disruptive properties of LA. Interestingly, the hydrolytic products of SLL, i.e., the LA + LacA mixture, induced a greater degree of transient, reversible membrane morphological changes but ultimately less permanent membrane disruption than SLL. These molecular-level insights support that careful tuning of antimicrobial lipid headgroup properties can modulate the spectrum of membrane-disruptive interactions, offering a pathway to design surfactants with tailored biodegradation profiles and reinforcing that SLL has attractive biophysical merits as a membrane-disrupting antimicrobial drug candidate.

Tethered Bilayer Lipid Membrane Platform for Screening Triton X-100 Detergent Replacements by Electrochemical Impedance Spectroscopy.

In light of regulatory considerations, there are ongoing efforts to identify Triton X-100 (TX-100) detergent alternatives for use in the biological manufacturing industry to mitigate membrane-enveloped pathogen contamination. Until now, the efficacy of antimicrobial detergent candidates to replace TX-100 has been tested regarding pathogen inhibition in endpoint biological assays or probing lipid membrane disruption in real-time biophysical testing platforms. The latter approach has proven especially useful to test compound potency and mechanism of action, however, existing analytical approaches have been limited to studying indirect effects of lipid membrane disruption such as membrane morphological changes. A direct readout of lipid membrane disruption by TX-100 detergent alternatives would be more practical to obtain biologically relevant information to guide compound discovery and optimization. Herein, we report the use of electrochemical impedance spectroscopy (EIS) to investigate how TX-100 and selected replacement candidates-Simulsol SL 11W (Simulsol) and cetyltrimethyl ammonium bromide (CTAB)-affect the ionic permeability of tethered bilayer lipid membrane (tBLM) platforms. The EIS results revealed that all three detergents exhibited dose-dependent effects mainly above their respective critical micelle concentration (CMC) values while displaying distinct membrane-disruptive behaviors. TX-100 caused irreversible membrane disruption leading to complete solubilization, whereas Simulsol caused reversible membrane disruption and CTAB induced irreversible, partial membrane defect formation. These findings establish that the EIS technique is useful for screening the membrane-disruptive behaviors of TX-100 detergent alternatives with multiplex formatting possibilities, rapid response, and quantitative readouts relevant to antimicrobial functions.

Nanoarchitectonics-based model membrane platforms for probing membrane-disruptive interactions of odd-chain antimicrobial lipids.

The use of nanoscience tools to investigate how antimicrobial lipids disrupt phospholipid membranes has greatly advanced molecular-level biophysical understanding and opened the door to new application possibilities. Until now, relevant studies have focused on even-chain antimicrobial lipids while there remains an outstanding need to investigate the membrane-disruptive properties of odd-chain antimicrobial lipids that are known to be highly biologically active. Herein, using the quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) techniques, we investigated how an 11-carbon, saturated fatty acid and its corresponding monoglyceride-termed undecanoic acid and monoundecanoin, respectively-disrupt membrane-mimicking phospholipid bilayers with different nanoarchitectures. QCM-D tracking revealed that undecanoic acid and monoundecanoin caused membrane tubulation and budding from supported lipid bilayers, respectively, and were only active above their experimentally determined critical micelle concentration (CMC) values. Monoundecanoin was more potent due to a lower CMC and electrochemical impedance spectroscopy (EIS) characterization demonstrated that monoundecanoin caused irreversible membrane disruption of a tethered lipid bilayer platform at sufficiently high compound concentrations, whereas undecanoic acid only induced transient membrane disruption. This integrated biophysical approach also led us to identify that the tested 11-carbon antimicrobial lipids cause more extensive membrane disruption than their respective 12-carbon analogues at 2 × CMC, which suggests that they could be promising molecular components within next-generation antimicrobial nanomedicine strategies.

Biophysical Characterization of LTX-315 Anticancer Peptide Interactions with Model Membrane Platforms: Effect of Membrane Surface Charge.

LTX-315 is a clinical-stage, anticancer peptide therapeutic that disrupts cancer cell membranes. Existing mechanistic knowledge about LTX-315 has been obtained from cell-based biological assays, and there is an outstanding need to directly characterize the corresponding membrane-peptide interactions from a biophysical perspective. Herein, we investigated the membrane-disruptive properties of the LTX-315 peptide using three cell-membrane-mimicking membrane platforms on solid supports, namely the supported lipid bilayer, intact vesicle adlayer, and tethered lipid bilayer, in combination with quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) measurements. The results showed that the cationic LTX-315 peptide selectively disrupted negatively charged phospholipid membranes to a greater extent than zwitterionic or positively charged phospholipid membranes, whereby electrostatic interactions were the main factor to influence peptide attachment and membrane curvature was a secondary factor. Of note, the EIS measurements showed that the LTX-315 peptide extensively and irreversibly permeabilized negatively charged, tethered lipid bilayers that contained high phosphatidylserine lipid levels representative of the outer leaflet of cancer cell membranes, while circular dichroism (CD) spectroscopy experiments indicated that the LTX-315 peptide was structureless and the corresponding membrane-disruptive interactions did not involve peptide conformational changes. Dynamic light scattering (DLS) measurements further verified that the LTX-315 peptide selectively caused irreversible disruption of negatively charged lipid vesicles. Together, our findings demonstrate that the LTX-315 peptide preferentially disrupts negatively charged phospholipid membranes in an irreversible manner, which reinforces its potential as an emerging cancer immunotherapy and offers a biophysical framework to guide future peptide engineering efforts.

Mechanistic Evaluation of Antimicrobial Lipid Interactions with Tethered Lipid Bilayers by Electrochemical Impedance Spectroscopy.

There is extensive interest in developing real-time biosensing strategies to characterize the membrane-disruptive properties of antimicrobial lipids and surfactants. Currently used biosensing strategies mainly focus on tracking membrane morphological changes such as budding and tubule formation, while there is an outstanding need to develop a label-free biosensing strategy to directly evaluate the molecular-level mechanistic details by which antimicrobial lipids and surfactants disrupt lipid membranes. Herein, using electrochemical impedance spectroscopy (EIS), we conducted label-free biosensing measurements to track the real-time interactions between three representative compounds-glycerol monolaurate (GML), lauric acid (LA), and sodium dodecyl sulfate (SDS)-and a tethered bilayer lipid membrane (tBLM) platform. The EIS measurements verified that all three compounds are mainly active above their respective critical micelle concentration (CMC) values, while also revealing that GML induces irreversible membrane damage whereas the membrane-disruptive effects of LA are largely reversible. In addition, SDS micelles caused membrane solubilization, while SDS monomers still caused membrane defect formation, shedding light on how antimicrobial lipids and surfactants can be active in, not only micellar form, but also as monomers in some cases. These findings expand our mechanistic knowledge of how antimicrobial lipids and surfactants disrupt lipid membranes and demonstrate the analytical merits of utilizing the EIS sensing approach to comparatively evaluate membrane-disruptive antimicrobial compounds.

Streamlined Fabrication of Hybrid Lipid Bilayer Membranes on Titanium Oxide Surfaces: A Comparison of One- and Two-Tail SAM Molecules.

There is broad interest in fabricating cell-membrane-mimicking, hybrid lipid bilayer (HLB) coatings on titanium oxide surfaces for medical implant and drug delivery applications. However, existing fabrication strategies are complex, and there is an outstanding need to develop a streamlined method that can be performed quickly at room temperature. Towards this goal, herein, we characterized the room-temperature deposition kinetics and adlayer properties of one- and two-tail phosphonic acid-functionalized molecules on titanium oxide surfaces in various solvent systems and identified optimal conditions to prepare self-assembled monolayers (SAMs), upon which HLBs could be formed in select cases. Among the molecular candidates, we identified a two-tail molecule that formed a rigidly attached SAM to enable HLB fabrication via vesicle fusion for membrane-based biosensing applications. By contrast, vesicles adsorbed but did not rupture on SAMs composed of one-tail molecules. Our findings support that two-tail phosphonic acid SAMs offer superior capabilities for rapid HLB coating fabrication at room temperature, and these streamlined capabilities could be useful to prepare durable lipid bilayer coatings on titanium-based materials.