Antiviral screening of natural, anti-inflammatory compound library against African swine fever virus.

BACKGROUND: African swine fever virus (ASFV) is a major threat to pig production and the lack of effective vaccines underscores the need to develop robust antiviral countermeasures. Pathologically, a significant elevation in pro-inflammatory cytokine production is associated with ASFV infection in pigs and there is high interest in identifying dual-acting natural compounds that exhibit antiviral and anti-inflammatory activities. METHODS: Using the laboratory-adapted ASFV BA71V strain, we screened a library of 297 natural, anti-inflammatory compounds to identify promising candidates that protected Vero cells against virus-induced cytopathic effect (CPE). Virus yield reduction, virucidal, and cell cytotoxicity experiments were performed on positive hits and two lead compounds were further characterized in dose-dependent assays along with time-of-addition, time-of-removal, virus entry, and viral protein synthesis assays. The antiviral effects of the two lead compounds on mitigating virulent ASFV infection in porcine macrophages (PAMs) were also tested using similar methods, and the ability to inhibit pro-inflammatory cytokine production during virulent ASFV infection was assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The screen identified five compounds that inhibited ASFV-induced CPE by greater than 50% and virus yield reduction experiments showed that two of these compounds, tetrandrine and berbamine, exhibited particularly high levels of anti-ASFV activity. Mechanistic analysis confirmed that both compounds potently inhibited early stages of ASFV infection and that the compounds also inhibited infection of PAMs by the virulent ASFV Arm/07 isolate. Importantly, during ASFV infection in PAM cells, both compounds markedly reduced the production of pro-inflammatory cytokines involved in disease pathogenesis while tetrandrine had a greater and more sustained anti-inflammatory effect than berbamine. CONCLUSIONS: Together, these findings support that dual-acting natural compounds with antiviral and anti-inflammatory properties hold promise as preventative and therapeutic agents to combat ASFV infection by simultaneously inhibiting viral replication and reducing virus-induced cytokine production.

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.

Unraveling How Antimicrobial Lipid Mixtures Disrupt Virus-Mimicking Lipid Vesicles: A QCM-D Study.

Single-chain lipid amphiphiles such as fatty acids and monoglycerides are promising antimicrobial alternatives to replace industrial surfactants for membrane-enveloped pathogen inhibition. Biomimetic lipid membrane platforms in combination with label-free biosensing techniques offer a promising route to compare the membrane-disruptive properties of different fatty acids and monoglycerides individually and within mixtures. Until recently, most related studies have utilized planar model membrane platforms, and there is an outstanding need to investigate how antimicrobial lipid mixtures disrupt curved model membrane platforms such as intact vesicle adlayers that are within the size range of membrane-enveloped virus particles. This need is especially evident because certain surfactants that completely disrupt planar/low-curvature membranes are appreciably less active against high-curvature membranes. Herein, we conducted quartz crystal microbalance-dissipation (QCM-D) measurements to investigate the membrane-disruptive properties of glycerol monolaurate (GML) monoglyceride and lauric acid (LA) fatty acid mixtures to rupture high-curvature, ~75 nm diameter lipid vesicle adlayers. We identified that the vesicle rupture activity of GML/LA mixtures mainly occurred above the respective critical micelle concentration (CMC) of each mixture, and that 25/75 mol% GML/LA micelles exhibited the greatest degree of vesicle rupture activity with ~100% efficiency that exceeded the rupture activity of other tested mixtures, individual compounds, and past reported values with industrial surfactants. Importantly, 25/75 GML/LA micelles outperformed 50/50 GML/LA micelles, which were previously reported to have the greatest membrane-disruptive activity towards planar model membranes. We discuss the mechanistic principles behind how antimicrobial lipid engineering can influence membrane-disruptive activity in terms of optimizing the balance between competitive membrane remodeling processes and inducing anisotropic vs. isotropic spontaneous curvature in lipid membrane systems.

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.

Interindividual- and blood-correlated sweat phenylalanine multimodal analytical biochips for tracking exercise metabolism.

In situ monitoring of endogenous amino acid loss through sweat can provide physiological insights into health and metabolism. However, existing amino acid biosensors are unable to quantitatively assess metabolic status during exercise and are rarely used to establish blood-sweat correlations because they only detect a single concentration indicator and disregard sweat rate. Here, we present a wearable multimodal biochip integrated with advanced electrochemical electrodes and multipurpose microfluidic channels that enables simultaneous quantification of multiple sweat indicators, including phenylalanine and chloride, as well as sweat rate. This combined measurement approach reveals a negative correlation between sweat phenylalanine levels and sweat rates among individuals, which further enables identification of individuals at high metabolic risk. By tracking phenylalanine fluctuations induced by protein intake during exercise and normalizing the concentration indicator by sweat rates to reduce interindividual variability, we demonstrate a reliable method to correlate and analyze sweat-blood phenylalanine levels for personal health monitoring.

Cholesterol-Enriched Hybrid Lipid Bilayer Formation on Inverse Phosphocholine Lipid-Functionalized Titanium Oxide Surfaces.

Hybrid lipid bilayers (HLBs) are rugged biomimetic cell membrane interfaces that can form on inorganic surfaces and be designed to contain biologically important components like cholesterol. In general, HLBs are formed by depositing phospholipids on top of a hydrophobic self-assembled monolayer (SAM) composed of one-tail amphiphiles, while recent findings have shown that two-tail amphiphiles such as inverse phosphocholine (CP) lipids can have advantageous properties to promote zwitterionic HLB formation. Herein, we explored the feasibility of fabricating cholesterol-enriched HLBs on CP SAM-functionalized TiO2 surfaces with the solvent exchange and vesicle fusion methods. All stages of the HLB fabrication process were tracked by quartz crystal microbalance-dissipation (QCM-D) measurements and revealed important differences in fabrication outcome depending on the chosen method. With the solvent exchange method, it was possible to fabricate HLBs with well-controlled cholesterol fractions up to ~65 mol% in the upper leaflet as confirmed by a methyl-ß-cyclodextrin (MßCD) extraction assay. In marked contrast, the vesicle fusion method was only effective at forming HLBs from precursor vesicles containing up to ~35 mol% cholesterol, but this performance was still superior to past results on hydrophilic SiO2. We discuss the contributing factors to the different efficiencies of the two methods as well as the general utility of two-tail CP SAMs as favorable interfaces to incorporate cholesterol into HLBs. Accordingly, our findings support that the solvent exchange method is a versatile tool to fabricate cholesterol-enriched HLBs on CP SAM-functionalized TiO2 surfaces.

Glycerol Monolaurate Inhibits Wild-Type African Swine Fever Virus Infection in Porcine Macrophages.

Naturally abundant antimicrobial lipids, such as fatty acids and monoglycerides, that disrupt membrane-enveloped viruses are promising mitigants to inhibit African swine fever virus (ASFV). Among mitigant candidates in this class, glycerol monolaurate (GML) has demonstrated particularly high antiviral activity against laboratory-adapted ASFV strains. However, there is an outstanding need to further determine the effects of GML on wild-type ASFV strains, which can have different virulence levels and sensitivities to membrane-disrupting compounds as compared to laboratory-adapted strains. Herein, we investigated the antiviral effects of GML on a highly virulent strain of a wild-type ASFV isolate (Armenia/07) in an in vitro porcine macrophage model. GML treatment caused a concentration-dependent reduction in viral infectivity, and there was a sharp transition between inactive and active GML concentrations. Low GML concentrations had negligible effect on viral infectivity, whereas sufficiently high GML concentrations caused a >99% decrease in viral infectivity. The concentration onset of antiviral activity matched the critical micelle concentration (CMC) value of GML, reinforcing that GML micelles play a critical role in enabling anti-ASFV activity. These findings validate that GML can potently inhibit wild-type ASFV infection of porcine macrophages and support a biophysical explanation to guide antimicrobial lipid performance optimization for pathogen mitigation applications.

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.

pH-Modulated Nanoarchitectonics for Enhancement of Multivalency-Induced Vesicle Shape Deformation at Receptor-Presenting Lipid Membrane Interfaces.

Multivalent ligand-receptor interactions between receptor-presenting lipid membranes and ligand-modified biological and biomimetic nanoparticles influence cellular entry and fusion processes. Environmental pH changes can drive these membrane-related interactions by affecting membrane nanomechanical properties. Quantitatively, however, the corresponding effects on high-curvature, sub-100 nm lipid vesicles are scarcely understood, especially in the multivalent binding context. Herein, we employed the label-free localized surface plasmon resonance (LSPR) sensing technique to track the multivalent attachment kinetics, shape deformation, and surface coverage of biotin ligand-functionalized, zwitterionic lipid vesicles with different ligand densities on a streptavidin receptor-coated supported lipid bilayer under varying pH conditions (4.5, 6, 7.5). Our results demonstrate that more extensive multivalent interactions caused greater vesicle shape deformation across the tested pH conditions, which affected vesicle surface packing as well. Notably, there were also pH-specific differences, i.e., a higher degree of vesicle shape deformation was triggered at a lower multivalent binding energy in pH 4.5 than in pH 6 and 7.5 conditions. These findings support that the nanomechanical properties of high-curvature lipid membranes, especially the membrane bending energy and the corresponding responsiveness to multivalent binding interactions, are sensitive to solution pH, and indicate that multivalency-induced vesicle shape deformation occurs slightly more readily in acidic pH conditions relevant to biological environments.

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.

Curvature-sensing peptide inhibits tumour-derived exosomes for enhanced cancer immunotherapy.

Tumour-derived exosomes (T-EXOs) impede immune checkpoint blockade therapies, motivating pharmacological efforts to inhibit them. Inspired by how antiviral curvature-sensing peptides disrupt membrane-enveloped virus particles in the exosome size range, we devised a broadly useful strategy that repurposes an engineered antiviral peptide to disrupt membrane-enveloped T-EXOs for synergistic cancer immunotherapy. The membrane-targeting peptide inhibits T-EXOs from various cancer types and exhibits pH-enhanced membrane disruption relevant to the tumour microenvironment. The combination of T-EXO-disrupting peptide and programmed cell death protein-1 antibody-based immune checkpoint blockade therapy improves treatment outcomes in tumour-bearing mice. Peptide-mediated disruption of T-EXOs not only reduces levels of circulating exosomal programmed death-ligand 1, but also restores CD8+ T cell effector function, prevents premetastatic niche formation and reshapes the tumour microenvironment in vivo. Our findings demonstrate that peptide-induced T-EXO depletion can enhance cancer immunotherapy and support the potential of peptide engineering for exosome-targeting applications.

Unraveling How Cholesterol Affects Multivalency-Induced Membrane Deformation of Sub-100 nm Lipid Vesicles.

Cholesterol plays a critical role in modulating the lipid membrane properties of biological and biomimetic systems and recent attention has focused on its role in the functions of sub-100 nm lipid vesicles and lipid nanoparticles. These functions often rely on multivalent ligand-receptor interactions involving membrane attachment and dynamic shape transformations while the extent to which cholesterol can influence such interaction processes is largely unknown. To address this question, herein, we investigated the attachment of sub-100 nm lipid vesicles containing varying cholesterol fractions (0-45 mol %) to membrane-mimicking supported lipid bilayer (SLB) platforms. Biotinylated lipids and streptavidin proteins were used as model ligands and receptors, respectively, while the localized surface plasmon resonance sensing technique was employed to track vesicle attachment kinetics in combination with analytical modeling of vesicle shape changes. Across various conditions mimicking low and high multivalency, our findings revealed that cholesterol-containing vesicles could bind to receptor-functionalized membranes but underwent appreciably less multivalency-induced shape deformation than vesicles without cholesterol, which can be explained by a cholesterol-mediated increase in membrane bending rigidity. Interestingly, the extent of vesicle deformation that occurred in response to increasingly strong multivalent interactions was less pronounced for vesicles with greater cholesterol fraction. The latter trend was rationalized by taking into account the strong dependence of the membrane bending energy on the area of the vesicle-SLB contact region and such insights can aid the engineering of membrane-enveloped nanoparticles with tailored biophysical properties.

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.

Optimizing Plasmonic Gold Nanorod Deposition on Glass Surfaces for High-Sensitivity Refractometric Biosensing.

Owing to high surface sensitivity, gold nanorods (AuNRs) are widely used to construct surface-based nanoplasmonic biosensing platforms for label-free molecular diagnostic applications. A key fabrication step involves controlling AuNR deposition onto the target surface, which requires maximizing surface density while minimizing inter-particle aggregation, and is often achieved by surface functionalization with a self-assembled monolayer (SAM) prior to AuNR deposition. To date, existing studies have typically used a fixed concentration of SAM-forming organic molecules (0.2-10% v/v) while understanding how SAM density affects AuNR deposition and resulting sensing performance would be advantageous. Herein, we systematically investigated how controlling the (3-aminopropyl)triethoxysilane (APTES) concentration (1-30% v/v) during SAM preparation affects the fabrication of AuNR-coated glass surfaces for nanoplasmonic biosensing applications. Using scanning electron microscopy (SEM) and UV-visible spectroscopy, we identified an intermediate APTES concentration range that yielded the highest density of individually deposited AuNRs with minimal aggregation and also the highest peak wavelength in aqueous solution. Bulk refractive index sensitivity measurements indicated that the AuNR configuration had a strong effect on the sensing performance, and the corresponding wavelength-shift responses ranged from 125 to 290 nm per refractive index unit (RIU) depending on the APTES concentration used. Biosensing experiments involving protein detection and antigen-antibody interactions further demonstrated the high surface sensitivity of the optimized AuNR platform, especially in the low protein concentration range where the measurement shift was ~8-fold higher than that obtained with previously used sensing platforms.

Facile and scalable fabrication of exosome-mimicking nanovesicles through PEGylated lipid detergent-aided cell extrusion.

We report a scalable fabrication method to generate exosome-mimicking nanovesicles (ENVs) by using a biocompatible, cell-binding lipid detergent during cell extrusion. A PEGylated mannosylerythritol lipid (MELPEG) detergent was rationally engineered to strongly associate with phospholipid membranes to increase cell membrane deformability and the corresponding friction force during extrusion and to enhance the dispersibility of ENVs. Compared to cell extrusion without detergent, cell extrusion in the presence of MELPEG increased the ENV production yield by approximately 20 times and cellular protein content per MELPEG-functionalized ENV by approximately 2-fold relative to that of unmodified ENVs. We verified that MELPEG strongly binds to ENV membranes and increases membrane deformability via expansion/swelling while preserving the integrity of the phospholipid bilayer structure. The results highlight that the MELPEG-aided cell extrusion process broadly applies to various cell lines; hence, it could be helpful in the production of ENVs for tissue regeneration, drug delivery, and cancer nanomedicine.

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.

Distinct Binding Properties of Neutravidin and Streptavidin Proteins to Biotinylated Supported Lipid Bilayers: Implications for Sensor Functionalization.

The exceptional strength and stability of noncovalent avidin-biotin binding is widely utilized as an effective bioconjugation strategy in various biosensing applications, and neutravidin and streptavidin proteins are two commonly used avidin analogues. It is often regarded that the biotin-binding abilities of neutravidin and streptavidin are similar, and hence their use is interchangeable; however, a deeper examination of how these two proteins attach to sensor surfaces is needed to develop reliable surface functionalization options. Herein, we conducted quartz crystal microbalance-dissipation (QCM-D) biosensing experiments to investigate neutravidin and streptavidin binding to biotinylated supported lipid bilayers (SLBs) in different pH conditions. While streptavidin binding to biotinylated lipid receptors was stable and robust across the tested pH conditions, neutravidin binding strongly depended on the solution pH and was greater with increasingly acidic pH conditions. These findings led us to propose a two-step mechanistic model, whereby streptavidin and neutravidin binding to biotinylated sensing interfaces first involves nonspecific protein adsorption that is mainly influenced by electrostatic interactions, followed by structural rearrangement of adsorbed proteins to specifically bind to biotin functional groups. Practically, our findings demonstrate that streptavidin is preferable to neutravidin for constructing SLB-based sensing platforms and can improve sensing performance for detecting antibody-antigen interactions.

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.

Inkjet-Printed Phospholipid Bilayers on Titanium Oxide Surfaces: Towards Functional Membrane Biointerfaces.

Functional biointerfaces hold broad significance for designing cell-responsive medical implants and sensor devices. Solid-supported phospholipid bilayers are a promising class of biological materials to build bioinspired thin-film coatings, as they can facilitate interactions with cell membranes. However, it remains challenging to fabricate lipid bilayers on medically relevant materials such as titanium oxide surfaces. There are also limitations in existing bilayer printing capabilities since most approaches are restricted to either deposition alone or to fixed microarray patterning. By combining advances in lipid surface chemistry and on-demand inkjet printing, we demonstrate the direct deposition and patterning of covalently tethered lipid bilayer membranes on titanium oxide surfaces, in ambient conditions and without any surface pretreatment process. The deposition conditions were evaluated by quartz crystal microbalance-dissipation (QCM-D) measurements, with corresponding resonance frequency (Δf) and energy dissipation (ΔD) shifts of around −25 Hz and <1 × 10−6, respectively, that indicated successful bilayer printing. The resulting printed phospholipid bilayers are stable in air and do not collapse following dehydration; through rehydration, the bilayers regain their functional properties, such as lateral mobility (>1 µm2/s diffusion coefficient), according to fluorescence recovery after photobleaching (FRAP) measurements. By taking advantage of the lipid bilayer patterned architectures and the unique features of titanium oxide's photoactivity, we further show how patterned cell culture arrays can be fabricated. Looking forward, this work presents new capabilities to achieve stable lipid bilayer patterns that can potentially be translated into implantable biomedical devices.

Effect of Membrane Curvature Nanoarchitectonics on Membrane-Disruptive Interactions of Antimicrobial Lipids and Surfactants.

Single-chain lipid amphiphiles such as fatty acids and monoglycerides along with structurally related surfactants have received significant attention as membrane-disrupting antimicrobials to inhibit bacteria and viruses. Such promise has motivated deeper exploration of how these compounds disrupt phospholipid membranes, and the membrane-mimicking, supported lipid bilayer (SLB) platform has provided a useful model system to evaluate corresponding mechanisms of action and potency levels. Even so, it remains largely unknown how biologically relevant membrane properties, such as sub-100 nm membrane curvature, might affect these membrane-disruptive interactions, especially from a nanoarchitectonics perspective. Herein, using the quartz crystal microbalance-dissipation (QCM-D) technique, we fabricated intact vesicle adlayers composed of different-size vesicles (70 or 120 nm diameter) with varying degrees of membrane curvature on a titanium oxide surface and tracked changes in vesicle adlayer properties upon adding lauric acid (LA), glycerol monolaurate (GML), or sodium dodecyl sulfate (SDS). Above their critical micelle concentration (CMC) values, LA and GML caused QCM-D measurement shifts associated with tubule- and bud-like formation, respectively, and both compounds interacted similarly with small (high curvature) and large (low curvature) vesicles. In marked contrast, SDS exhibited distinct interactions with small and large vesicles. For large vesicles, SDS caused nearly complete membrane solubilization in a CMC-independent manner, whereas SDS was largely ineffective at solubilizing small vesicles at all tested concentrations. We rationalize these experimental observations by taking into account the interplay of the headgroup properties of LA, GML, and SDS and curvature-induced membrane geometry, and our findings demonstrate that membrane curvature nanoarchitectonics can strongly influence the membrane interaction profiles of antimicrobial lipids and surfactants.