Langmuir

We report formation of coacervate-supported phospholipid membranes by hydrating a dried lipid film in the presence of coacervate droplets. In contrast to traditional giant lipid vesicles formed by gentle hydration in the absence of coacervates, the coacervate-templated membrane vesicles are more uniform in size, shape, and apparent lamellarity. Due to their fully-coacervate model cytoplasm, these simple artificial cells are macromolecularly crowded and can be easily pre-loaded with high concentrations of proteins or nucleic acids. Coacervate-supported membranes were characterized by fluorescence imaging, polarization, fluorescence recovery after photobleaching of labeled lipids, lipid quenching experiments, and solute uptake experiments. Our findings are consistent with the presence of lipid membranes around the coacervates, with many droplets fully coated with what appear to be continuous lipid bilayers. Within the same population, other coacervate droplets are coated with membranes having defects or pores that permit solute entry, and still others are coated with multilayered membranes. These membranes surrounding protein-based coacervate droplets provided protection from a protease added to the external solution. The simplicity of producing artificial cells having a coacervate model cytoplasm surrounded by a model membrane is at the same time interesting as a potential mechanism for prebiotic protocell formation and appealing for biotechnology. We anticipate that such structures could serve as a new type of model system for understanding interactions between intracellular phases and cell- or organelle membranes, which are implicated in a growing number of processes ranging from neurotransmission to signaling.

The delivery of therapeutics to cells enables both the treatment and the prevention of diseases. To protect therapeutics from degradation and enable cell-specific targeting, they are often encapsulated into drug delivery vehicles such as lipid nanoparticles, viral vectors or lipid vesicles. These delivery vehicles have been extremely successful in delivering small molecules, nucleic acids or proteins. However, there is no universal drug delivery vehicle that can deliver therapeutics irrespective of the choice of cargo. Here, we present a method to engineer lipid vesicles with asymmetric leaflets and show that they can deliver mRNA and proteins to cells. We also find that the leaflet asymmetry can increase the lipid vesicle uptake by cells. When we load asymmetric vesicles with mRNA, we observe a 5-fold increase in the transfection efficiency indicative of an improved uptake and release by asymmetric vesicles. Moreover, our findings extend beyond mRNA cargos by showcasing the effectiveness of asymmetric vesicles in delivering a wide range of proteins to cells, including the promising CRISPR/Cas9 gene editing system. Our method and findings expand the parameter space for engineering drug delivery vehicles and demonstrate the pivotal role of leaflet asymmetry in determining the performance of drug delivery vehicles. Consequently, our work leads to many applications, including the formation of more efficient universal drug carriers and the delivery of gene-editing proteins to cells.

Lipids, and cationic lipids in particular, are of interest as delivery vectors for hydrophobic drugs such as the cancer therapeutic paclitaxel, and the structures of lipid assemblies affect their efficacy. We investigated the effect of incorporating the multivalent cationic lipid MVL5 (+5e) and poly(ethylene glycol)-lipids (PEG-lipids), alone and in combination, on the structure of fluid-phase lipid assemblies of the charge-neutral lipid 1,2-dioleoyl-sn-glycero-phosphocholine (DOPC). This allowed us to elucidate lipid-liposome structure correlations in sonicated formulations with high charge density, which are not accessible with univalent lipids such as the well-studied DOTAP (+1e). Cryogenic TEM allowed us to determine the structure of the lipid assemblies, revealing diverse combinations of vesicles and disc-shaped, worm-like, and spherical micelles. Remarkably, MVL5 forms an essentially pure phase of disc micelles at 50 mol% MVL5. At higher (75 mol%) content of MVL5, short and intermediate-length worm-like micellar rods were observed and, in ternary mixtures with PEG-lipid, longer and highly flexible worm-like micelles formed. Independent of their length, the worm-like micelles coexisted with spherical micelles. In stark contrast, DOTAP forms mixtures of vesicles, disc micelles and spherical micelles at all studied compositions, even when combined with PEG-lipids. The observed similarities and differences in the effects of charge (multivalent versus univalent) and high curvature (multivalent charge versus PEG-lipid) on assembly structure provide insights into parameters that control the size of fluid lipid nanodiscs, relevant for future applications.

Liposomes are widely used as synthetic analogues of cell membranes and for drug delivery. Lipid-binding DNA nanostructures can modify the shape, porosity and reactivity of liposomes, mediated by cholesterol-modifications. DNA nanostructures can also be designed to switch conformations by DNA strand displacement. However, the optimal conditions to facilitate stable, high-yield DNA-lipid binding while allowing controlled switching by strand-displacement are not known. Here we characterised the effect of cholesterol arrangement, DNA structure, buffer and lipid composition on DNA-lipid binding and strand displacement. We observed that binding was inhibited below pH 4, and above 200 mM NaCl or 40 mM MgCl2, was independent of lipid type, and increased with membrane cholesterol content. For simple motifs, binding yield was slightly higher for double-stranded DNA than single-stranded. For larger DNA origami tiles, 4 - 8 cholesterol modifications were optimal, while edge positions and longer spacers increased yield of lipid-binding. Strand displacement achieved controlled removal of DNA tiles from membranes, but was inhibited by overhang domains, which are used to prevent cholesterol aggregation. These findings provide design guidelines for integrating strand-displacement switching with lipid-binding DNA nanostructures. This paves the way for achieving dynamic control of membrane morphology, enabling broader applications in nanomedicine and biophysics.

Biomolecular condensates driven by liquid-liquid phase separation (LLPS) have received attention as novel activity regulators of living organisms. In intracellular LLPS, an important question is what type of biomolecules form condensates under what conditions. In this regard, possible interactions between biomolecules have been investigated. Recently, LLPS condensates have been reported to regulate the membrane structure upon wetting. However, the possibility of membrane wetting, in which the membrane conversely regulates the LLPS, remains unexplored. Using droplets of short polyethylene glycol and long dextran blends encapsulated with a lipid membrane, we demonstrate that membrane wetting regulates LLPS in cell-size spaces and alters the equilibrium state. In smaller droplets, the two-phase region expands beyond the bulk system, and the fractionation degree increases, particularly during the separation between short PEG and long dextran. We explain the space-size dependent LLPS based on the competitive membrane wetting between the polymers. Smaller droplets promote the membrane wetting of short PEG, which enhances the depletion force between long dextran molecules and finally induces LLPS. This shows that competition for membrane wettability among various molecules can regulate LLPS in cell-size spaces, rendering this LLPS principle feasible in living cells.

The polymorphism of lipid aggregates has long attracted detailed study due to the myriad factors that determine the final mesophase observed. This study is driven by the need to understand mesophase behaviour for a number of applications, such as drug delivery and membrane protein crystallography. In the case of the latter, the role of the so-called sponge (L3) mesophase has been often noted, but not extensively studied by itself. The L3 mesophase can be formed in monoolein/water systems on the addition of butanediol to water, which partitions the headgroup region of the membrane, and decreases its elastic moduli. Like cubic mesophases, it is bicontinuous, but unlike them, has no long-range translational symmetry. In our present study, we show that the formation of the L3 phase can delicately depend on the addition of dopant lipids to the mesophase. While electrostatically neutral molecules similar in shape to monoolein (DOPE, cholesterol) have little effect on the general mesophase behaviour, others (DOPC, DDM) significantly reduce the region in which it can form. Additionally, we show that by combining cholesterol with the anionic lipid DOPG, it is possible to form the largest stable L3 mesophases observed to date, with correlation lengths over 220 [A].

Significant cellular processes, including protein sorting, signal transduction, and pathogen entry, amongst others, are associated with membrane microdomains, also known as lipid rafts. Lipid rafts, due to their unique biophysical properties compared to their surrounding environment, which stem from their distinct lipid and protein profiles, have garnered interest in methods and techniques that tune their coexisting liquid-ordered/liquid-disordered state, aiming to disrupt or destabilize them. Since cholesterol stabilizes the membrane domain, cholesterol-depleting compounds like cyclodextrin can be used to destabilize and disrupt the membrane rafts. Overall, given the membrane rafts importance in biological processes, it is crucial to understand the biophysical factors that influence its stability. In this study, we present a new method for disrupting and dissolving lipid rafts in a model system of phase-separated supported lipid bilayer (SLB) patches composed of DOPC, DPPC, and cholesterol. Using fluorescence microscopy to monitor the liquid ordered (Lo) and liquid disordered (Ld) phases of the SLB patches, we observed that adding DOPC liposomes causes a transformation of the co-existing Ld and Lo phases into a single-phase bilayer. On the other hand, adding liposomes that match the lipid content of the phase-separated SLB patch increase the areas of the existing Ld and Lo phases. This work also offers a new method for redistributing raft-localized molecules, confirmed by tracking the redistribution of cholera toxin bound to GM1 after domain dissolution with DOPC liposomes. The work describes an alternative method for dynamically altering membrane composition and dissolving domains via liposome addition, rather than lipid depletion or exchange.

The origin of life likely involved a complex interplay between organic molecules and mineral surfaces, yet the molecular details of these interactions remain poorly understood. Over recent decades, considerable research has focused on the individual roles of key biomolecules - such as RNA, lipids, and proteins - in early abiogenesis. However, this reductionist view offers only a partial picture because the emergence of life likely involved networks of molecular interactions that collectively shaped early functional assemblies. In this study, we examine the ability of peptides - arguably one of the most abundant early polymers - to interact with mineral surfaces and lipid vesicles, prebiotic interfaces and compartments. Using peptide libraries constructed from either prebiotically plausible or contemporary amino acids, we demonstrate that while acidic residues drive peptide binding to mineral surfaces (such as fluorapatite, studied here), the inclusion of arginine - a basic residue that may have been accessible in specific prebiotic environments - synergistically enhances the mobilization of bioavailable phosphate from geological reservoirs. Furthermore, we observe a functional divergence in vesicle interactions: while prebiotic alphabets promote dynamic membrane behaviours such as budding, libraries with late canonical amino acids can help preserve vesicle integrity against salt-induced collapse. Our finding supports the view that interactions with peptides can elicit changes in both prebiotic minerals and vesicles, underscoring the importance of studying these systems collectively.

As the central organelles of lipid and energy homeostasis in plant seeds, lipid droplets (LDs) consist of a neutral lipid core, decorated by phospholipids (prominently phosphatidylcholines, PCs) and surfactant-like proteins (mostly oleosins, OLs). So far, the dynamic interplay between PCs and OLs at the LD interface remains unclear. The presented work addresses this knowledge gap by reconstituting oil-in-water emulsions stabilized by OLs and PCs using microfluidic systems. Our results show that the resistance to droplet coalescence is primarily provided by OLs. We further reveal that the addition of PCs alters the assembly of OLs at the interface, reducing the OL network density and interfacial elasticity, thereby rendering a weaker interface. In conclusion, our study suggests complementary roles, with OLs acting as the primary stabilizers while PCs playing a destabilizing role. This contrast likely contributes to the observed metastability of LDs and can be exploited to design stimuli-responsive emulsions. HIGHLIGHTSO_LIOleosins form an interfacial network that stabilizes the oil-water interface. C_LIO_LIPhosphatidylcholines globally weaken the oleosin network and promote droplet coalescence. C_LIO_LIMicrofluidics enables controlled reconstitution and real-time analysis of lipid droplets. C_LIO_LIOleosin-phospholipid interplay explains lipid droplet metastability and can guide the design of bio-inspired emulsions. C_LI

Live cell plasma membranes show spatially heterogeneous liquid-ordered (Lo)-like and liquid-disordered (Ld)-like regions similar to the co-existing Lo/Ld phases observed in lipid vesicles. The Lo-like regions are relatively less hydrated and less polar due to tight packing of the membrane components compared to the Ld-like regions. The steady-state fluorescence spectra of Di-4-ANEPPDHQ (Di-4), a widely used polarity-sensitive probe, is blue or red shifted when solvated in less polar (Ld- like) or more polar (Lo-like) regions respectively. However, quantification of Di-4 fluorescence in blue and red channels for the evaluation of membrane phase state suffers from the lack of specific wavelength choice for these two channels and Di-4s relatively higher concentration in Ld phase (red channel) due to its partitioning preference. To address these issues, we employed fluorescence lifetime of Di-4, a concentration independent photophysical parameter, to understand membrane biophysical properties. The fluorescence lifetime of Di-4 in lipid vesicles exhibits Arrhenius-like temperature dependence. Centred around this energetic feature of Di-4 photophysics, we developed a novel analytical module, namely excited state relaxation activation energy (ESRAct), that serves as an intrinsic descriptor of the membrane nano-environment sensed by this probe. We show that the ESRAact value scales with increasing disorder in nanoscale phase separation (i.e., ESRAct of pure Ld > mixed Ld/Lo > pure Lo phase). We then extended its applications to giant plasma membrane vesicles (GPMVs) isolated from MCF-7 cells and found that these vesicles exhibit nanoscale Lo/Ld co-existing phase within 16-37{degrees}C. We envisage wide applications of ESRAct to delineate plasma membrane phase behavior as well as general photophysical studies on other newly designed polarity-sensitive probes.

Giant membrane vesicles (GUVs) and giant plasma membrane vesicles (GPMVs) are useful models for studying cellular membrane properties. Our research analyzed the reduced volume of vesicles made from phospholipid and 10% cholesterol to investigate transbilayer sugar asymmetries. We found that GPMVs have an average reduced volume of (0.88 {+/-} 0.06) with buffer asymmetry of 323 mM, lower than the (0.92 {+/-} 0.08) observed for DOPC: cholesterol vesicles with sucrose/glucose asymmetry of 390 mM. GUVs with different sugars inside and outside were more deflated, demonstrating a greater volume reduction than those with the same sugar inside and out. We applied the area-difference elasticity (ADE) model to map GPMVs and used the spontaneous curvature (SC) model to analyze DOPC: cholesterol GUVs, extracting spontaneous curvature based on their reduced volume. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/622000v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@178828forg.highwire.dtl.DTLVardef@c38f8eorg.highwire.dtl.DTLVardef@105c783org.highwire.dtl.DTLVardef@6da36d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Lipid nanoparticles (LNPs) are the most successful drug delivery carrier to date, but optimizing lipid formulations to improve membrane fusion capabilities for effective drug release has been challenging due to lack of a quantitative measure for fusogenicity. Here we introduce a new framework based on small angle X-ray scattering to experimentally measure [Formula] for lipids used in LNP formulations such as glycerol monooleate (GMO) and ionizable lipids (SM-102 and ALC-0315). Q intrinsically captures spontaneous curvature (J0), which is traditionally used to assess fusogenicity. The change of cubic lattice parameters with temperature was measured for GMO-containing lipid mixtures, and the Q extracted quantitatively correlated with LNP fusogenicity power validated by fluorescence-based fusion assays and cryogenic electron microscopy. Fusogenicity of SM-102 and ALC-0315 was quantified by adding them to host membranes and assessing change in Q. This framework provides researchers with the ability to optimize the fusogenicity of LNP formulations for potent drug release and enhances understanding of parameters governing fusion in all biomembranes.

Antipsychotics (APs) are used in the treatment of severe mental disorders. Their mechanism of action involves interaction with multiple brain targets, notably the dopamine D2 receptors (D2R), where they compete with dopamine. Due to their lipophilic nature, APs also partition and accumulate in lipid membranes, particularly around the D2R and in synaptic vesicles. When intercalated into brain membranes, APs slowly accumulate and act as a reservoir, allowing their rapid release on demand to modulate neurotransmitter signaling. They also modify the physicochemical and mechanical properties of the lipid bilayer. These modifications can subsequently affect the conformational changes of embedded membrane proteins like the D2R. The present study investigated two major APs with different pharmacological and clinical profiles: chlorpromazine, which exerts its clinical activity mainly through a strong antagonistic action at the D2R, and clozapine, the weakest D2R antagonist of all APs. Surprisingly, although D2R antagonism is usually associated with AP potency, clozapine has repeatedly demonstrated clinical superior efficacy to all APs and is therefore recommended for treatment-resistant schizophrenia. The current work aims to extend the classical AP receptor mediated paradigmatic mode of action to their potential and unique membrane remodeling properties by thoroughly comparing their partitioning and impact on the physicochemical properties of the lipid membrane. Lipid model membranes mimicking synaptic vesicles have been investigated using a combination of several biophysical methods. The study aims to determine how the partitioning of the two APs modifies membrane order, phase transition, thickness, elasticity, phase separation, membrane integrity and charge. Differences have been demonstrated between these two compounds, which may further differ both over time as they accumulate as well as depending on their pre- or post-synaptic location.

The spontaneous self-assembly of single chain amphiphiles (SCAs) would have resulted in multiple protocell species in an early-Earth niche. Considering the heterogeneity inherent in a prebiotic milieu, interactions between physicochemically distinct protocell populations was evaluated to discern if emergent properties occurred at a systems level. This study demonstrates that depending on the physicochemical properties of the membrane, interacting populations are endowed with varied emergent properties owing to their coexistence. In a multispecies paradigm involving a two-candidate protocell system, the fitter population acted as a predator and grew at the expense of the less-fit prey population. The observed growth could be attributed to the predator attaining a more robust membrane via chemical evolution. Importantly, the prey population also accrued emergent properties like molecular crowding, and coexist in balance with the predator population, without being completely outcompeted. When extrapolating these results to a three-candidate population, the outcomes were multipronged. These findings suggest a possible route for protocell membrane evolution that could have occurred even in the absence of any sophisticated protein machinery, benefitting coexisting populations, while also illustrating evolutionary trajectories that potentially resulted in functionally complex protocells.

Optimizing the production of lipid nanoparticle (LNP) therapeutics is necessary for drug delivery efficiency, stability, and scalability. A small but growing body of literature has begun to recognize that LNP properties (e.g., size, shape, and internal structure) depend on the flow conditions during mixing for antisolvent precipitation, in which LNPs are formulated. Here, we use different mixers, varying flow patterns (e.g., laminar or turbulent mixing) and flow rate ratios (FRR), i.e., 3:1 and 1:1, to prepare a standard LNP formulation. We then characterize the resulting formulations using small angle x-ray scattering (SAXS) to provide insights into particle shape/morphology, internal organization (L and HII phases) of yeast RNA (yRNA), and structural differences/similarities that arise from the different mixing methods. The effect of ethanol removal on the LNPs structure, formulated from each mixing technique, is also discussed. We observed the 3:1 FRR mixers outperform the 1:1 configurations in certain desired LNP physiochemical properties. The differences observed in the LNPs produced across the two configurations are discussed. Furthermore, we use computational fluid dynamics to explain the turbulent mixing schemes among the 3:1 and 1:1 mixers.

This study investigates pH-triggered fusion dynamics of lipid nanoparticles (LNPs) with an endosomal membrane mimic, addressing mechanistic aspects of a crucial yet elusive process for effective mRNA delivery. Utilizing time-resolved total internal reflection fluorescence (TIRF) imaging, we observed a delayed onset of LNP fusion upon pH drop, lasting seconds to minutes depending on pH and LNP size. Once initiated, LNP fusion and cargo escape occurred rapidly within tens to hundreds of milliseconds. While LNP disintegration is observed to lead to release of a significant portion of mRNA into the acidic environment, some mRNA molecules remained mobile on the endosomal membrane mimic due to deprotonation-resistant complex salt formation. Comparison of the fusion efficiency of two LNP formulations correlated with protein translation in human primary cell transfection data, emphasizing the importance of biophysical investigations in understanding ionizable-lipid-containing LNP-assisted mRNA delivery mechanisms and providing insights for optimizing mRNA-LNP design for enhanced endosomal escape.

Enveloped viruses can enter host cells by fusing their membrane with that of the host cell, a process known as membrane fusion. This process depends on specific fusion proteins located on the viral particle surface, which contain a short, relatively hydrophobic segment called "fusion peptide" that binds to the host membrane. To investigate the fusion efficiency of various fusion peptides, we create simplified non-infectious virus like particles decorated with different fusion peptides and fuse them with an artificial cell membrane. For this purpose, microfluidic devices are used to create supported lipid bilayers while the result of the fusion process is studied by fluorescence microscopy. Our study provides structural insights into the interactions between virus particles and cell membranes, which can facilitate the development of new therapeutic strategies and more effective viral vectors for therapeutic applications.

COVID-19 vaccines, such as Pfizer-BioNTechs BNT162b and Modernas mRNA-1273, have demonstrated robust efficacy. However, direct comparisons of their delivery vehicles remain limited. Notably, BNT162b requires storage at -80{degrees}C, while mRNA-1273 is stored at -20{degrees}C. This discrepancy in storage temperatures may be influenced by differences in the structure and stability of the lipid nanoparticles (LNPs) used in these vaccines. Ionizable lipids, such as SM-102 in Modernas vaccine and ALC-0315 in Pfizers vaccine, play a crucial role in LNP stability and function, affecting endosomal escape, cellular uptake, and drug release. Understanding these variations is essential for optimizing vaccine delivery systems. In our study, we use molecular dynamics simulations with the coarse-grained Martini forcefield to compare the LNPs in Moderna and Pfizers COVID-19 vaccines, providing insights at an experimental scale. Our findings indicate that the ionizable lipid tail of BNT162b (ALC-0315) exhibits a higher degree of branching, resulting in a more bifurcated appearance compared to the structure of the ionizable lipids in mRNA-1273 (SM-102).

Lipid nanoparticles (LNPs) are crucial in advancing the delivery of RNA-based therapeutics within the domain of gene therapy. A comprehensive understanding of their formation and stability is critical for optimizing the clinical efficacy of LNPs. This study systematically investigates the influence of concentration variations of positive and neutral ionizable lipids - specifically, 2-[2,2-bis[(9Z,12Z)-octadeca-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethanamine (DLinKC2-DMA) and 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) - along with cholesterol and polyethylene glycol, on the formation of LNPs and encapsulation of small interfering RNA (siRNA). Utilizing coarse-grained classical molecular dynamics (MD) simulations with a system size matching experimental range (approximately 0.6 million beads), we conduct a comparative analysis and offer mechanistic insights into siRNA formulation within LNPs containing positive and neutral DLinKC2-DMA. We found that the LNPs with positive ionizable lipids encapsulate more than twice the siRNA compared to the LNPs with neutral ionizable lipids. In addition to the formation of LNPs, our study extends to the forces governing siRNA escape from LNPs, employing steered molecular dynamics simulations. The force experienced by siRNA to cross the LNP lipid layer containing positive ionizable lipids was 400kJ/mol/nm more than that of neutral ionizable lipids, suggesting the encapsulation is more favorable with positive ionisable lipids.

Microplastics and nanoplastics (MNPs), originating from plastic degradation, have arisen to be a threat to ecology and human health. Alarmingly, the penetration of MNPs across the highly selective blood-brain barrier (BBB) poses an emerging and urgent risk, yet its molecular mechanism remains unexplored. In this work, using long-time-scale (over 27 s) all-atom explicit solvent steered molecular dynamics, we examine the free energy of the passive permeation of four polymer nanoparticles: polyethylene, polypropylene, polystyrene, and polyethylene terephthalate. Polyethylene and polypropylene nanoparticles exhibited a remarkable preference for entering the BBB, attributed to their high hydrophobicity. Our study reveals that polymers can enter the BBB as polymerized nanoplastics and exit as dispersed polymer chains as the nanoparticles dissolve within the BBB. Further, the crystalline structure of polyethylene nanoparticles is found to adopt varying orientations. Our work advances the knowledge about the mechanism of nanoplastic penetration across the BBB, which could aid in the rational design of therapeutics for nanoplastic penetration inhibitors. TOC Graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/675462v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@1b20796org.highwire.dtl.DTLVardef@1102985org.highwire.dtl.DTLVardef@15fc22org.highwire.dtl.DTLVardef@15c2260_HPS_FORMAT_FIGEXP M_FIG C_FIG