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The development of mRNA delivery systems utilizing lipid-based assemblies holds immense potential for precise control of gene expression and targeted therapeutic interventions. Despite advancements in lipid-based gene delivery systems, a critical knowledge gap remains in understanding how the biophysical characteristics of lipid assemblies and mRNA complexes influence these systems. Herein, we investigate the biophysical properties of cationic liposomes and their role in shaping mRNA lipoplexes by comparing various fabrication methods. Notably, an innovative fabrication technique called the liposome under cryo-assembly (LUCA) cycle, involving a precisely controlled freeze-thaw-vortex process, produces distinctive onion-like concentric multilamellar structures in cationic DOTAP/DOPE liposomes, in contrast to a conventional extrusion method that yields unilamellar liposomes. The inclusion of short-chain DHPC lipids further modulates the structure of cationic liposomes, transforming them from multilamellar to unilamellar structures during the LUCA cycle. Furthermore, the biophysical and biological evaluations of mRNA lipoplexes unveil that the optimal N/P charge ratio in the lipoplex can vary depending on the structure of initial cationic liposomes. Cryo-EM structural analysis demonstrates that multilamellar cationic liposomes induce two distinct interlamellar spacings in cationic lipoplexes, emphasizing the significant impact of the liposome structures on the final structure of mRNA lipoplexes. Taken together, our results provide an intriguing insight into the relationship between lipid assembly structures and the biophysical characteristics of the resulting lipoplexes. These relationships may open the door for advancing lipid-based mRNA delivery systems through more streamlined manufacturing processes.

The size of membrane-enveloped virus particles, exosomes, and lipid vesicles strongly impacts functional properties in biological and applied contexts. Multivalent ligand-receptor interactions involving nanoparticle shape deformation are critical to such functions, yet the corresponding effect of nanoparticle size remains largely elusive. Herein, using an indirect nanoplasmonic sensing approach, we investigated how the nanoscale size properties of ligand-modified lipid vesicles affect real-time binding interactions, especially vesicle deformation processes, with a receptor-modified, cell membrane-mimicking platform. Together with theoretical analyses, our findings reveal a pronounced, size-dependent transition in the membrane bending properties of nanoscale lipid vesicles between 60 and 180 nm in diameter. For smaller vesicles, a large membrane bending energy enhanced vesicle stiffness while the osmotic pressure energy was the dominant modulating factor for larger, less stiff vesicles. These findings advance our fundamental understanding of how nanoparticle size affects multivalency-induced nanoparticle shape deformation and can provide guidance for the design of biomimetic nanoparticles with tailored nanomechanical properties.

There is enormous interest in utilizing biologically active fatty acids and monoglycerides to treat phospholipid membrane-related medical diseases, especially with the global health importance of membrane-enveloped viruses and bacteria. However, it is difficult to practically deliver lipophilic fatty acids and monoglycerides for therapeutic applications, which has led to the emergence of lipid nanoparticle platforms that support molecular encapsulation and functional presentation. Herein, we introduce various classes of lipid nanoparticle technology and critically examine the latest progress in utilizing lipid nanoparticles to deliver fatty acids and monoglycerides in order to treat medical diseases related to infectious pathogens, cancer, and inflammation. Particular emphasis is placed on understanding how nanoparticle structure is related to biological function in terms of mechanism, potency, selectivity, and targeting. We also discuss translational opportunities and regulatory needs for utilizing lipid nanoparticles to deliver fatty acids and monoglycerides, including unmet clinical opportunities.

Co-assembly of binary systems driven by specific non-covalent interactions can greatly expand the structural and functional space of supramolecular nanostructures. We report here on the self-assembly of peptide amphiphiles and fatty acids driven primarily by anion-π interactions. The peptide sequences investigated were functionalized with a perfluorinated phenylalanine residue to promote anion-π interactions with carboxylate headgroups in fatty acids. These interactions were verified here by NMR and circular dichroism experiments as well as investigated using atomistic simulations. Positioning the aromatic units close to the N-terminus of the peptide backbone near the hydrophobic core of cylindrical nanofibers leads to strong anion-π interactions between both components. With a low content of dodecanoic acid in this position, the cylindrical morphology is preserved. However, as the aromatic units are moved along the peptide backbone away from the hydrophobic core, the interactions with dodecanoic acid transform the cylindrical supramolecular morphology into ribbon-like structures. Increasing the ratio of dodecanoic acid to PA leads to either the formation of large vesicles in the binary systems where the anion-π interactions are strong, or a heterogeneous mixture of assemblies when the peptide amphiphiles associate weakly with dodecanoic acid. Our findings reveal how co-assembly involving designed specific interactions can drastically change supramolecular morphology and even cross from nano to micro scales.

Pore formation by membrane-active antimicrobial peptides is a classic strategy of pathogen inactivation through disruption of membrane biochemical gradients. It remains unknown why some membrane-active peptides also inhibit enveloped viruses, which do not depend on biochemical gradients. Here, we employ a label-free biosensing approach based on simultaneous quartz crystal microbalance-dissipation and ellipsometry measurements in order to investigate how a pore-forming, virucidal peptide destabilizes lipid vesicles in a surface-based experimental configuration. A key advantage of the approach is that it enables direct kinetic measurement of the surface-bound peptide-to-lipid (P:L) ratio. Comprehensive experiments involving different bulk peptide concentrations and biologically relevant membrane compositions support a unified model that membrane lysis occurs at or above a critical P:L ratio, which is at least several-fold greater than the value corresponding to the onset of pore formation. That is consistent with peptide-induced pores causing additional membrane strain that leads to lysis of highly curved membranes. Collectively, the work presents a new model that describes how peptide-induced pores may destabilize lipid membranes through a membrane strain-related lytic process, and this knowledge has important implications for the design and application of membrane-active peptides.

Tunable nanoplasmonic biosensing for lipid and protein applications is reported based on controlling lipid membrane architecture on surfaces. The interaction of a peptide with lipid membranes is highly sensitive to the membrane architecture on top of plasmonic nanodisks, and the measurement response varies in a manner which is consistent with the surrounding lipid environment., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An indirect nanoplasmonic sensing platform is reported for investigating the kinetics of attachment and shape deformation associated with lipid vesicle adsorption onto a titanium oxide-coated substrate. The localized surface plasmon resonance (LSPR) originates from embedded gold nanodisks and is highly sensitive to the local lipid environment. To interpret the corresponding results, we have extended treatments of diffusion-limited adsorption kinetics and adsorbate-related LSPR physics, identified the expected scaling laws for the LSPR-tracked kinetics measured at different lipid concentrations and/or nanometer-scale vesicle sizes in the case when vesicle deformation is negligible, and scrutinized experimental deviations accordingly. After adsorption, the smallest 58 nm diameter vesicles were found to maintain shape on the time scale of adsorption at high lipid concentrations in solution, and shape deformation became more appreciable at lower lipid concentrations. Higher saturation coverage was observed with increasing lipid concentration, which is attributed to the difference in relative time scales of vesicle attachment and deformation. For larger vesicles between 80 and 160 nm diameter, deviations associated with their shape deformation and correlations with the location of gold nanodisks became more apparent at moderate and high coverages. Taken together, the results obtained support that the quantitative measurement capabilities of nanoplasmonic biosensing should be considered for applications demanding highly surface-sensitive characterization of soft matter adsorption and related phenomena at liquid-solid interfaces.

An amphipathic α-helical (AH) peptide was recently discovered that can rupture the lipid envelope of many viruses including HIV, hepatitis C, dengue, and herpes simplex. Despite its broad-spectrum activity, the AH peptide specifically targets small viruses only and does not affect large viruses. Indirect observations of virus size-specific targeting have been confirmed in a model system comprised of intact lipid vesicles on a gold substrate. Depending on vesicle size, AH peptide can promote vesicle rupture, but the mechanism by which vesicle size influences the rupture process remains to be elucidated. Herein, using the dynamic light scattering and quartz crystal microbalance with dissipation techniques, we have combined experiment and theory to understand the effects of vesicle size on the interaction between the AH peptide and vesicles. We identified that the AH peptide-binding interaction can induce a structural rearrangement of the vesicle's lipid bilayer, which occurs independently of vesicle size. Kinetic analysis also revealed that AH peptide-binding occurs cooperatively for small vesicles only. Binding cooperativity is consistent with pore formation leading to vesicle rupture. By contrast, for large vesicles, AH peptide-binding is noncooperative and does not cause vesicle rupture, suggesting that the binding interaction occurs via a different mechanism. Compared to previous estimates that AH peptide is most effective against viruses with a diameter of less than 70 nm, our evidence validates that AH peptide may target a wider size range of enveloped viruses up to 160 nm in diameter. Taken together, our findings provide a quantitative rationale to understand the targeting specificity of AH peptide as a broad-spectrum antiviral drug candidate.

Using tethered sub-100 nm lipid vesicles that mimic enveloped viruses with nanoscale membrane curvature, we have in this work designed a total internal reflection fluorescence microscopy-based single vesicle assay to investigate how an antiviral amphipathic α-helical (AH) peptide interacts with lipid membranes to induce membrane curvature-dependent pore formation and membrane destabilization. Based on a combination of statistics from single vesicle imaging, binding kinetics data, and theoretical analysis, we propose a mechanistic model that is consistent with the experimentally observed peptide association and pore formation kinetics at medically relevant peptide concentrations (10 nM to 1 μM) and unusually low peptide-to-lipid (P/L) ratio (~1/1000). Importantly, the preference of the AH peptide to selectively rupture virions with sub-100 nm diameters appears to be related to membrane strain-dependent pore formation rather than to previously observed nanoscale membrane curvature facilitated binding of AH peptides. Compared to other known proteins and peptides, the combination of low effective P/L ratio and high specificity for nm-sized membrane curvature lends this particular AH peptide great potential to serve as a framework for developing a highly specific and potent antiviral agent for prophylactic and therapeutic applications while avoiding toxic side effects against host cell membranes.

Supported lipid platforms are versatile cell membrane mimics whose structural properties can be tailored to suit the application of interest. By identifying parameters that control the self-assembly of these platforms, there is potential to develop advanced biomimetic systems that overcome the surface specificity of lipid vesicle interactions under physiological conditions. In this work, we investigated the adsorption kinetics of vesicles onto silicon and titanium oxides as a function of pH. On each substrate, a planar bilayer and a layer of intact vesicles could be self-assembled in a pH-dependent manner, demonstrating the role of surface charge density in the self-assembly process. Under acidic pH conditions where both zwitterionic lipid vesicles and the oxide films possess near-neutral electric surface charges, vesicle rupture could occur, demonstrating that the process is driven by nonelectrostatic interactions. However, we observed that the initial rupturing process is insufficient for propagating bilayer formation. The role of electrostatic interactions for propagating bilayer formation differs for the two substrates; electrostatic attraction between vesicles and the substrate is necessary for complete bilayer formation on titanium oxide but is not necessary on silicon oxide. Conversely, in the high pH regime, repulsive electrostatic interactions can result in the irreversible adsorption of intact vesicles on silicon oxide and even a reversibly adsorbed vesicle layer on titanium oxide. Together, the results show that pH is an effective tool to modulate vesicle-substrate interactions in order to create various self-assembled lipid platforms on hydrophilic substrates.

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-β-cyclodextrin (MβCD) 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. [ABSTRACT FROM AUTHOR]

Pharmaceutical drugs and vaccines require the use of material containers for protection, storage, and transportation. Glass and plastic materials are widely used for packaging, and a longstanding challenge in the field is the nonspecific adsorption of pharmaceutical drugs to container walls – the so‐called "sticky containers, vanishing drugs" problem – that effectively reduces the active drug concentration and can cause drug denaturation. This challenge has been frequently discussed in the case of the anticancer drug, paclitaxel, and the ongoing coronavirus disease 2019 (COVID‐19) pandemic has brought renewed attention to this material science challenge in light of the need to scale up COVID‐19 vaccine production and to secure sufficient quantities of packaging containers. To reduce nonspecific adsorption on inner container walls, various strategies based on siliconization and thin polymer films have been explored, while it would be advantageous to develop mass‐manufacturable, natural material solutions, especially ones involving pharmaceutical grade excipients. Inspired by how lipid nanoparticles have revolutionized the vaccine field, in this perspective, we discuss the prospects for developing lipid bilayer coatings to prevent nonspecific adsorption of pharmaceutical drugs and vaccines and how recent advances in lipid bilayer coating fabrication technologies are poised to accelerate progress in the field. We critically discuss recent examples of how lipid bilayer coatings can prevent nonspecific sticking of proteins and vaccines to relevant material surfaces and examine future translational prospects. [ABSTRACT FROM AUTHOR]

The solvent-assisted lipid bilayer (SALB) formation method provides a simple and efficient, microfluidic-based strategy to fabricate supported lipid bilayers (SLBs) with rich compositional diversity on a wide range of solid supports. While various studies have been performed to characterize SLBs formed using the SALB method, relatively limited work has been carried out to understand the underlying mechanisms of SALB formation under various experimental conditions. Through thermodynamic modeling, we studied the experimental parameters that affect the SALB formation process, including substrate surface properties, initial lipid concentration, and temperature. It was found that all the parameters are critically important to successfully form high-quality SLBs. The model also helps to identify the range of parameter space within which conformal, homogeneous SLBs can be fabricated, and provides mechanistic guidance to optimize experimental conditions for lipid membrane-related applications. [ABSTRACT FROM AUTHOR]

• Measurement stability is affected by dye leakage and imaging-related photobleaching. • Membrane composition of tethered lipid vesicles can be tuned to minimize leakage. • Imaging interval can be modulated to minimize photobleaching effects. • Optimal conditions improved accuracy when measuring the rate of peptide-induced pore formation. Highly parallel measurements on single, tethered lipid vesicles enable real-time monitoring of dynamic membrane interactions of relevance to medical, pharmaceutical, and biotechnological applications. Monitoring the time-dependent release of entrapped fluorescent dyes is a popular measurement approach, although it is often challenging to accurately extract quantitative biochemical parameters. Key issues include dye leakage and imaging-related photobleaching, and corrective measures are needed. Herein, we present an extended analytical framework to collect and interpret time-lapsed fluorescence microscopy imaging data, and demonstrate its utility for tracking membrane-peptide interactions. Our approach is focused on improving platform design and data analysis. First, we identified suitable membrane compositions to minimize dye leakage while enhancing the biomimetic character of lipid vesicles. Second, a data normalization procedure was developed to correct for experimental artifacts, namely dye leakage and photobleaching, and hence improve measurement accuracy. This analytical procedure was applied to experimentally determine the rate of peptide-induced pore formation in single lipid vesicles, and there was up to a nearly three-fold decrease in the measured rate, as compared to uncorrected data. Taken together, the results present a broadly applicable analytical framework to account for experimental artifacts and improve measurement accuracy in highly parallel, single lipid vesicle arrays. [ABSTRACT FROM AUTHOR]

The adhesion of lipid vesicles tosolid supports represents animportant step in the molecular self-assembly of model membrane platforms.A wide range of experimental parameters are involved in controllingthis process, including substrate material and topology, lipid composition,vesicle size, solution pH, ionic strength, and osmotic pressure. Atpresent, it is not well understood how the magnitude and directionof the osmotic pressure exerted on a vesicle influence the correspondingadsorption kinetics. In this work, using quartz crystal microbalancewith dissipation (QCM-D) monitoring, we have experimentally studiedthe role of osmotic pressure in the adsorption of zwitterionic vesiclesonto silicon oxide. The osmotic pressure was induced by changing theionic strength of the solvent across an appreciably wider range (from25 to 1000 mM NaCl outside of the vesicle, and 125 mM NaCl insideof the vesicle, unless otherwise noted) compared to that used in earlierworks. Our key finding is demonstration that, by changing osmoticpressure, all three generic types of the kinetics of vesicle adsorptionand rupture can be observed in one system, including (i) adsorptionof intact vesicles, (ii) adsorption and rupture after reaching a criticalvesicle coverage, and (iii) rupture just after adsorption. Furthermore,theoretical analysis of pressure-induced deformation of adsorbed vesiclesand a DLVO-type analysis of the vesicle–substrate interactionqualitatively support our observations. Taken together, the findingsin this work demonstrate that osmotic pressure can either promoteor impede the rupture of adsorbed vesicles on silicon oxide, and offerexperimental evidence to support adhesion energy-based models thatdescribe the adsorption and spontaneous rupture of vesicles on solidsupports. [ABSTRACT FROM AUTHOR]

We report the development of a real-time localized surface plasmon resonance (LSPR) biosensing strategy to detect three-dimensional morphological changes in a supported lipid bilayer (SLB) on a plasmonic substrate. The sensing concept advances on past efforts to detect subtle conformational changes in adsorbed biomacromolecules by demonstrating the capability to track large-scale, complex adsorbate shape changes and to classify different types of shape changes based on specific, multi-step measurement signatures. To validate this concept, we tested the addition of antimicrobial fatty acids, monoglycerides, and surfactants in micellar form to the SLB platform, which triggered specific three-dimensional membrane morphological changes such as tubule or bud formation along with solubilization. Experimentally, the different remodeling events were detected by distinct measurement signatures related to the shape and size of lipid protrusions that formed and evolved over time, which agreed well with a newly developed theoretical model. Our conceptual approach and formalism are applicable to various biosensing techniques, including not only LSPR but also surface plasmon resonance (SPR) and total internal reflection fluorescence (TIRF) microscopy. These sensing capabilities are advantageous for evaluating the mechanisms of antimicrobial drug candidates and other membrane-active compounds, and the measurement strategy is extendable to a wide range of biomimetic lipid compositions. Image 1 [ABSTRACT FROM AUTHOR]