Giant Unilamellar Vesicles Research Articles

Photoswitchable Molecular Motor Phospholipid: Synthesis, Characterization, and Integration into Lipid Vesicles.

Lipid membranes are essential for cellular function, acting as barriers and platforms for major cellular and biochemical activities. The integration of photoisomerizable units into lipid structures allows for tunable membrane properties, offering insights into major membrane-related processes. In this study, we present the first molecular-motor-conjugated phospholipid system. The synthesis of two phosphatidylcholine derivatives is reported, where one acyl chain is replaced with a light-responsive molecular rotary motor moiety. We explore the photochemical and thermodynamic behaviors of these compounds in solution and as self-assembled systems, demonstrating their rotation cycles under illumination and their dynamic properties in combination with lipid molecules. Additionally, giant unilamellar vesicles with these compounds are formed to investigate the mechanisms of the photoinduced responses in synthetic lipid membranes. Our findings show that molecular motor-based lipids can operate in aqueous solution and with natural phospholipids, maintaining photoisomerization properties and enabling oxidation-driven release within giant lipid vesicles.

Phase-separated cationic giant unilamellar vesicles as templates for the polymerization of tetraethyl orthosilicate (TEOS)

Unlike homogeneous liposomes, phase-separated liposomes have the potential to be attractive soft materials because they exhibit different properties for each phase. In this study, phase separation was observed when liposomes were prepared using 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol. The pH of the DOTAP-rich phase was evaluated using a coumarin derivative, and measurements showed that DOTAP molecules accumulated hydroxyl ions (OH-) in the DOTAP-rich phase. Such accumulation of OH- was not observed when homogeneous DSPC liposomes were used. The difference in local concentration of OH- in each phase was applied to prepare hollow silica particles with large pores. The OH- promotes the polymerization of tetraethyl orthosilicate (TEOS). Therefore, TEOS polymerized preferentially in the DOTAP-rich phase, whereas a silica membrane barely formed in the DSPC-rich phase.

Dithiocarbamate-Based Sequence-Defined Oligomers as Promising Membrane-Disrupting Antibacterial Agents: Design, Activity, and Mechanism.

The emerging prevalence of antimicrobial resistance demands cutting-edge therapeutic agents to treat bacterial infections. We present a synthetic strategy to construct sequence-defined oligomers (SDOs) by using dithiocarbamate (DTC). The antibacterial activity of the synthesized library of SDOs was studied using a Gram-positive B. subtilis and a Gram-negative E. coli. Among SDOs, Dec (with C10 aliphatic chain) was found to be the most promising antibacterial agent exhibiting a minimum inhibitory concentration (MIC) of 3 μg/mL against B. subtilis. Structure-activity relationship studies led to a 400-fold improvement in the MIC within the SDO library. The mode of action of the SDOs was elucidated on a model system, where bacterial membranes mimicking giant unilamellar vesicles (GUVs) were exposed to the SDOs. Membrane disruption and pore formation were found to be the key mechanisms through which SDOs act. In addition, scanning electron microscopy (SEM) and confocal laser scanning microscopy analysis of Dec-treated bacteria confirmed the loss of cell membrane integrity. Finally, the hemolysis assay with SDOs revealed their excellent selectivity toward bacterial cells. Taken together, we developed a modular platform for the synthesis of SDOs having promising antibacterial activity and superior selectivity toward bacteria, with the membrane disruption mode of action confirmed via studies on the model GUV system and SEM analysis.

Micrometer-sized liposomes self-aggregate by forming DNA duplexes on their surfaces more sensitively than metallic nanoparticles

Abstract We demonstrate that liposomes with diameters of more than 10 µm (giant unilamellar vesicles) self-aggregate in a noncrosslinking manner by forming DNA duplexes with blunt ends on their surfaces much more sensitively than gold nanoparticles by virtue of their unique features.

Metal-organic frameworks as anchors for giant unilamellar vesicle immobilization.

Giant unilamellar vesicles (GUVs) are ideal for studying cellular mechanisms due to their cell-mimicking morphology and size. The formation, stability, and immobilization of these vesicles are crucial for drug delivery and bioimaging studies. Separately, metal-organic frameworks (MOFs) are actively researched owing to their unique and varied properties, yet little is known about the interaction between MOFs and phospholipids. This study investigates the influence of the metal-phosphate interface on the formation, size distribution, and stability of GUVs with different lipid compositions. GUVs were electroformed in the presence of a series of MOFs. The results show Al, Zn, Cu, Fe, Zr, and Ca metal centers of MOFs can coordinate to phospholipids on the surface of GUVs, leading to the formation of functional GUV@MOF constructs, with stablilities over 12 hours. Macroscopically, society has seen biology (people, plants, microbes) interacting with inorganic materials regularly. We now explore how microscopic biological models behave in the presence of inorganic constructs. This research opens new avenues for advanced biomedical applications interacting tailored frameworks with liposomes.

Morphology remodelling and membrane channel formation in synthetic cells via reconfigurable DNA nanorafts

The shape of biological matter is central to cell function at different length scales and determines how cellular components recognize, interact and respond to one another. However, their shapes are often transient and hard to reprogramme. Here we construct a synthetic cell model composed of signal-responsive DNA nanorafts, biogenic pores and giant unilamellar vesicles (GUVs). We demonstrate that reshaping of DNA rafts at the nanoscale can be coupled to reshaping of GUVs at the microscale. The nanorafts collectively undergo reversible transitions between isotropic and short-range local order on the lipid membrane, programmably remodelling the GUV shape. Assisted by the biogenic pores, during GUV shape recovery the locally ordered DNA rafts perforate the membrane, forming sealable synthetic channels for large cargo transport. Our work outlines a versatile platform for interfacing reconfigurable DNA nanostructures with synthetic cells, expanding the potential of DNA nanotechnology in synthetic biology.

Controlled Lipid Domain Positioning and Polarization in Confined Minimal Cell Models

AbstractGiant unilamellar vesicles (GUVs) are widely used minimal cell models where essential biological features can be reproduced, isolated and studied. Although precise spatio‐temporal distribution of membrane domains is a process of crucial importance in living cells, it is still highly challenging to generate anisotropic GUVs with domains at user‐defined positions. Here we describe a novel and robust method to control the spatial position of lipid domains of liquid‐ordered (Lo)/liquid‐disordered (Ld) phase in giant unilamellar vesicles. Our strategy consists in confining Lo/Ld phase‐separating GUVs in microfluidic channels to define free curved regions where the minority‐phase domains localize and coalesce by decreasing the line energy through domain fusion. We show that this process is governed by the respective fraction of the two phases, and not by the chemical nature of the lipids involved. The spatial position and number of domains are controlled by the design of the confining microchannel and could result in polarized GUVs with a controllable number of poles. The developed method is versatile and user‐friendly, while allowing multiple single‐vesicle experiments in parallel.

Rapid Multi-Well Evaluation of Assorted Materials for Hydrogel-Assisted Giant Unilamellar Vesicle Production: Empowering Bottom-Up Synthetic Biology.

Giant unilamellar vesicles (GUVs) are versatile cell models in biomedical and environmental research. Of the various GUV production methods, hydrogel-assisted GUV production is most easily implemented in a typical biological laboratory. To date, agarose, polyvinyl alcohol, cross-linked dextran-PEG, polyacrylamide, and starch hydrogels have been used to produce GUVs. Some leach and contaminate the GUVs, while others require handling toxic material or specialised chemistry, thus limiting their use by novices. Alternative hydrogel materials could address these issues or even offer novel advantages. To facilitate discovery, we replaced the manual spreading of reagents with controlled drop-casting in glass Petri dishes and polystyrene multi-well plates, allowing us to rapidly screen up to 96 GUV-production formulations simultaneously. Exploiting this, we rapidly evaluated assorted biomedical hydrogels, including PEG-DA, cross-linked hyaluronic acid, Matrigel, and cross-linked DNA. All of these alternatives successfully produced GUVs. In the process, we also developed a treatment for recycling agarose and polyvinyl alcohol hydrogels for GUV production, and successfully encapsulated porcine liver esterase (PLE-GUVs). PLE-GUVs offer a novel method of GUV labelling and tracing, which emulates the calcein-AM staining behaviour of cells. Our results highlight the utility of our protocol for potentiating substrate material discovery, as well as protocol and product development.

Effect of Cholic Acid Salt and Its Mixed Micelles on the Morphology of Giant Unilamellar Vesicles (GUV)

Bile salts, present in the gastrointestinal tract as biosurfactants, play a crucial role in emulsifying and solubilizing fat-soluble nutrients and drugs, thereby facilitating their absorption. However, the cellular permeation of bile acid-mixed micelles solubilized with lipophilic substances remains inadequately explored. To comprehend the cell permeation behavior of bile salts and their mixed micelles, giant unilamellar vesicles (GUVs) were employed as a cell-mimetic system, prepared with dioleylphosphatidylcholine (DOPC). Confocal laser scanning microscopy, utilizing fluorescent dyes doped in the lipid membrane and solubilized substances, was employed to observe morphological changes in GUVs subsequent to the application of sodium cholate (NaC) alone and NaC-mixed micelles solubilized with lipophilic components. In the case of NaC alone, below the critical micelle concentration (CMC), the monomer interacts with the lipid membrane of the GUV, inducing endocytic morphological changes that result in the formation of small vesicles containing the bulk liquid inside the GUV. Conversely, when both monomers and micelles interacted with the lipid membrane beyond the CMC, lipid aggregates such as buds and threads protruded outward from the GUV. Contrastingly, upon application of three types of NaC mixed micelles-NaC-P solubilized with Palmitoyloleoylphosphocholine (POPC), NaC-P-F solubilized with oleic acid (OA) and monoolein (MO), and NaC-P-P solubilized with perylene, a liposoluble dye-to the GUV, the lipid membranes formed aggregates or vesicles and migrated into the interior of the GUV. In the case of NaC-P and NaC-P-P, the coexistence of drawn lipid aggregates and solubilized substances was scarcely observed. In contrast, for NaC-P-F, the coexistence of solubilized substances was observed in both lipid aggregates and small vesicles that migrated into the GUV. It is suggested that the partitioning of the solubilized substance from the mixed micelles adsorbed on the GUV to the lipid bilayer is implicated in the permeation of the solubilized substance through the cell membrane.

Effect of osmotic pressure on membrane permeation through antimicrobial peptide-induced pores.

Most antimicrobial peptides (AMPs) induce membrane damage such as pore formation in bacterial cells, resulting in rapid cell death. On the other hand, bacterial cells have a large intracellular turgor pressure, i.e., an osmotic pressure (Π) due to higher osmolarity inside bacterial cells, but the effects of Π on the membrane permeation of the internal contents of lipid vesicles and cells through AMP-induced pores are unknown. Here, we investigated the effect of Π on the membrane permeability of a water-soluble fluorescent probe, AlexaFluor 488 hydrazide (AF488), when passing through peptidyl-glycylleucine-carboxyamide (PGLa)- or magainin 2 (Mag)-induced nanopores in giant unilamellar vesicles (GUVs). For the interaction of PGLa with single GUVs under Π, the onset of pore formation was followed by a gradual increase in the membrane permeability coefficient, MP, until MP reached a steady value, Ps. On the other hand, for the interaction of Mag with single GUVs under Π, the onset of pore formation was rapidly followed by a change of MP to Ps. Small Π values enhanced the Ps values of AF488 passing through the PGLa- or Mag-induced nanopores. The mechanisms underlying the increase of Ps at small Π values were discussed. Based on these results and our previous results that the membrane tension (due to Π) enhances rate of AMP-induced pore formation, we consider the role of turgor pressure in AMP-induced damage in bacterial membranes and the efflux of internal contents.

Development and characterization of fluorescent cholesteryl probes with enhanced solvatochromic and pH-sensitive properties for live-cell imaging

We present novel fluorescent cholesteryl probes (CNDs) with a modular design based on the solvatochromic 1,8-phthalimide scaffold. We have explored how different modules—linkers and head groups—affect the ability of these probes to integrate into lipid membranes and how they distribute intracellularly in mouse astrocytes and fibroblasts targeting lysosomes and lipid droplets. Each compound was assessed for its solvatochromic behavior in organic solvents and model membranes. Molecular dynamics simulations and lipid partitioning using giant unilamellar vesicles showed how these analogs behave in model membranes compared to cholesterol. Live-cell imaging demonstrated distinct staining patterns and cellular uptake behaviors, further validating the utility of these probes in biological systems. We compared the empirical results with those of BODIPY-cholesterol, a well-regarded fluorescent cholesterol analog. The internalization efficiency of fluorescent CND probes varies in different cell types and is affected mainly by the head groups. Our results demonstrate that the modular design significantly simplifies the creation of fluorescent cholesteryl probes bearing distinct spectral, biophysical, and cellular targeting features. It is a valuable toolkit for imaging in live cells, measuring cellular membrane dynamics, and studying cholesterol-related processes.

Simultaneous Detection of Multiple Analytes at Ambient Temperature Using Eukaryotic Artificial Cells with Modular and Robust Synthetic Riboswitches.

Cell-free systems, which can express an easily detectable output (protein) with a DNA or mRNA template, are promising as foundations of biosensors devoid of cellular constraints. Moreover, by encasing them in membranes such as natural cells to create artificial cells, these systems can avoid the adverse effects of environmental inhibitory molecules. However, the bacterial systems generally used for this purpose do not function well at ambient temperatures. We here encapsulated a eukaryotic cell-free system consisting of wheat germ extract (WGE) and a DNA template encoding an analyte-responsive regulatory RNA (called a riboswitch) into giant unilamellar vesicles (GUVs) to create eukaryotic artificial cell-based sensors that function well at ambient temperature. First, we improved our previously reported eukaryotic synthetic riboswitches and WGE for use in GUVs by chimerizing two internal ribosome entry sites and optimizing magnesium concentrations, respectively, both of which increased the expression efficiency in GUVs several fold. Then, a DNA template encoding one of these riboswitches followed by a reporter protein was encapsulated with the optimized GUV-friendly WGE. Importantly, our previously established versatile method allowed for the rational design of highly efficient eukaryotic riboswitches that are responsive to a user-defined analyte. In fact, we utilized this method to successfully create three types of artificial cells, each of which responded to a specific, membrane-permeable analyte with wide-range, analyte-dose dependency and high sensitivity at ambient temperature. Finally, due to their orthogonality and robustness, we were able to mix a cocktail of these artificial cells to achieve simultaneous detection of the three analytes without significant barriers.

Biomolecular Condensates can Induce Local Membrane Potentials.

Biomolecular condensates are a ubiquitous component of cells, known for their ability to selectively partition and compartmentalize biomolecules without the need for a lipid membrane. Nevertheless, condensates have been shown to interact with lipid membranes in diverse biological processes, such as autophagy and T-cell activation. Since many condensates are known to have a net surface charge density and associated electric potential(s), we hypothesized that they can induce a local membrane potential. Using an electrochromic dye, we demonstrate that poly-lysine/ATP condensates induce a localized membrane potential in Giant Unilamellar Vesicles. This effect diminishes with increasing salt concentration and higher ATP-to-poly-lysine ratios, underscoring the key role of condensate charge. Numerical modeling of the condensate-membrane interface using an electro-thermodynamic framework supports our experimental findings and highlights parameters expected to play a key role in the effect. These results have broad implications for biological processes regulated by membrane potential, particularly in contexts such as neuronal signaling, where condensate interactions with membranes may play a previously unrecognized regulatory role.

Controlled Lipid Domain Positioning and Polarization in Confined Minimal Cell Models.

Giant unilamellar vesicles (GUVs) are widely used minimal cell models where essential biological features can be reproduced, isolated and studied. Although precise spatio-temporal distribution of membrane domains is a process of crucial importance in living cells, it is still highly challenging to generate anisotropic GUVs with domains at user-defined positions. Here we describe a novel and robust method to control the spatial position of lipid domains of liquid-ordered (Lo)/liquid-disordered (Ld) phase in giant unilamellar vesicles. Our strategy consists in confining Lo/Ld phase-separating GUVs in microfluidic channels to define free curved regions where the minority-phase domains localize and coalesce by decreasing the line energy through domain fusion. We show that this process is governed by the respective fraction of the two phases, and not by the chemical nature of the lipids involved. The spatial position and number of domains are controlled by the design of the confining microchannel and could result in polarized GUVs with a controllable number of poles. The developed method is versatile and user-friendly, while allowing multiple single-vesicle experiments in parallel.

Adaptive metal ion transport and metalloregulation-driven differentiation in pluripotent synthetic cells.

Pluripotent cells can yield different cell types determined by the specific sequence of differentiation signals that they encounter as the cell activates or deactivates functions and retains memory of previous inputs. Here, we achieved pluripotency in synthetic cells by incorporating three dormant apo-metalloenzymes such that they could differentiate towards distinct fates, depending on the sequence of specific metal ion transport with ionophores. In the first differentiation step, we selectively transported one of three extracellular metal ion cofactors into pluripotent giant unilamellar vesicles (GUVs), which resulted in elevation of intracellular pH, hydrogen peroxide production or GUV lysis. Previously added ionophores suppress transport with subsequent ionophores owing to interactions among them in the membrane, as corroborated by atomistic simulations. Consequently, the addition of a second ionophore elicits a dampened response in the multipotent GUV and a third ionophore results in no further response, reminiscent of a terminally differentiated GUV. The pluripotent GUV can differentiate into five final fates, depending on the sequence in which the three ionophores are added.

Flow Cytometric Analysis for Evaluating Protein Synthesis Efficiency in Giant Unilamellar Vesicles with Charged Lipids

Quantitative investigation of the relationship between endosomal translation reactions and phospholipid membrane composition is crucial for enhancing protein translation efficiency in artificial cells. In this study, we quantitatively compared the translation reactions within liposomes containing negatively and positively charged lipids using green fluorescent protein fluorescence as an indicator to investigate whether lipid membrane charge affects translation reaction efficiency in artificial cells. Thus, translation efficiency reduced in liposomes containing both negatively and positively charged lipids. Interestingly, flow cytometry analysis revealed that the percentage of liposomes undergoing translational reactions was reduced by the charged phospholipids. This translation reaction inhibition was alleviated by adding equal amounts of negatively and positively charged lipids, indicating that phospholipid membrane charges affected translation reaction efficiency. The relationship between membrane composition and translation reaction efficiency identified in this study is significant for the constructing complex artificial cells, particularly concerning membrane composition design

Photophysical behavior of meso-N-butylcarbazole-substituted BODIPY in different nano-scale organized media.

The present work focuses on the photophysical behavior of meso-N-butylcarbazole-substituted BODIPY (CBZ-BDP) in different organized media towards exploring the possible use of the dye as a molecular sensor and imaging agent. The molecule shows an appreciable change in absorption and emission spectra at 75% water-acetonitrile mixture compared to pure acetonitrile. In water-acetonitrile mixture, it displays aggregate-induced emission (AIE) bands. New emission peaks are observed at 560nm and 630nm, corresponding to LE (locally excited) and ICT (intramolecular charge transfer) states of CBZ-BDP aggregates. The fluorescence anisotropy studies of CBZ-BDP in glycerol medium show its better sensitivity towards the microenvironment. CBZ-BDP was used to probe various microheterogeneous systems like bile salts, pluronics, and lipid bilayer systems in aqueous medium. The dye displays sensitive variation in emission intensity and fluorescence anisotropy in sodium cholate (NaC) bile salt in aqueous medium as a function of the bile salt concentration. The molecule detects the temperature-induced phase transitions in pluronic P123 and F127, as well as 1,2-dimyristoylphosphatidylcholine (DMPC)and 1,2-dipalmitoylphosphatidylcholine (DPPC) lipid bilayer systems in aqueous medium. These studies strongly suggest that CBZ-BDP can be used as an efficient fluorescent probe in sensing the micro-environmental changes in bile salts, pluronics, and lipid bilayers in aqueous medium. The imaging studies of CBZ-BDP-embedded Giant Unilamellar Vesicles (GUVs) were carried out. The molecule stains the lipid bilayers and displays bright-green fluorescent images, suggesting its potential in lipid bilayer imaging.

Interaction of an Oomycete Nep1-like Cytolysin with Natural and Plant Cell-Mimicking Membranes.

Plants are attacked by various pathogens that secrete a variety of effectors to damage host cells and facilitate infection. One of the largest and so far understudied microbial protein families of effectors is necrosis- and ethylene-inducing peptide-1-like proteins (NLPs), which are involved in important plant diseases. Many NLPs act as cytolytic toxins that cause cell death and tissue necrosis by disrupting the plant's plasma membrane. Their mechanism of action is unique and leads to the formation of small, transient membrane ruptures. Here, we capture the interaction of the cytotoxic model NLP from the oomycete Pythium aphanidermatum, NLPPya, with plant cell-mimicking membranes of giant unilamellar vesicles (GUVs) and tobacco protoplasts using confocal fluorescence microscopy. We show that the permeabilization of GUVs by NLPPya is concentration- and time-dependent, confirm the small size of the pores by observing the inability of NLPPya monomers to pass through them, image the morphological changes of GUVs at higher concentrations of NLPPya and confirm its oligomerization on the membrane of GUVs. In addition, NLPPya bound to plasma membranes of protoplasts, which showed varying responses. Our results provide new insights into the interaction of NLPPya with model lipid membranes containing plant-derived sphingolipids.

Topological and Morphological Membrane Dynamics in Giant Lipid Vesicles Driven by Monoolein Cubosomes

AbstractLipid nanoparticles have important applications as biomedical delivery platforms and broader engineering biology applications in artificial cell technologies. These emerging technologies often require changes in the shape and topology of biological or biomimetic membranes. Here we show that topologically‐active lyotropic liquid crystal nanoparticles (LCNPs) can trigger such transformations in the membranes of giant unilamellar vesicles (GUVs). Monoolein (MO) LCNPs, cubosomes with an internal nanostructure of space group incorporate into 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) GUVs creating excess membrane area with stored curvature stress. Using time‐resolved fluorescence confocal and lattice light sheet microscopy, we observe and characterise various life‐like dynamic events in these GUVs, including growth, division, tubulation, membrane budding and fusion. Our results shed new light on the interactions of LCNPs with bilayer lipid membranes, providing insights relevant to how these nanoparticles might interact with cellular membranes during drug delivery and highlighting their potential as minimal triggers of topological transitions in artificial cells.

Mass-Guided Single-Cell MALDI Imaging of Low-Mass Metabolites Reveals Cellular Activation Markers.

Single-cell MALDI mass spectrometry imaging (MSI) of lipids and metabolites >200Da has recently come to the forefront of biomedical research and chemical biology. However, cell-targeting and metabolome-preserving methods for analysis of low mass, hydrophilic metabolites (<200Da) in large cell populations are lacking. Here, the PRISM-MS (PRescan Imaging for Small Molecule - Mass Spectrometry) mass-guided MSI workflow is presented, which enables space-efficient single cell lipid and metabolite analysis. In conjunction with giant unilamellar vesicles (GUVs) as MSI ground truth for cell-sized objects and Monte Carlo reference-based consensus clustering for data-dependent identification of cell subpopulations, PRISM-MS enables MSI and on-cell MS2-based identification of low-mass metabolites like amino acids or Krebs cycle intermediates involved in stimulus-dependent cell activation. The utility of PRISM-MS is demonstrated through the characterization of complex metabolome changes in lipopolysaccharide (LPS)-stimulated microglial cells and human-induced pluripotent stem cell-derived microglia. Translation of single cell results to endogenous microglia in organotypic hippocampal slice cultures indicates that LPS-activation involves changes of the itaconate-to-taurine ratio and alterations in neuron-to-glia glutamine-glutamate shuttling. The data suggests that PRISM-MS can serve as a standard method in single cell metabolomics, given its capability to characterize larger cell populations and low-mass metabolites.