Cell-sized Lipid Vesicles Research Articles

Measuring Vesicle Loading with Holographic Microscopy and Bulk Light Scattering.

We report efforts to quantify the loading of cell-sized lipid vesicles using in-line digital holographic microscopy. This method does not require fluorescent reporters, fluorescent tracers, or radioactive tracers. A single-color LED light source takes the place of conventional illumination to generate holograms rather than bright field images. By modeling the vesicle's scattering in a microscope with a Lorenz-Mie light scattering model and comparing the results to data holograms, we are able to measure the vesicle's refractive index and thus loading. Performing the same comparison for bulk light scattering measurements enables the retrieval of vesicle loading for nanoscale vesicles.

Artificial Methylotrophic Cells via Bottom-Up Integration of a Methanol-Utilizing Pathway.

Methanol has gained substantial attention as a substrate for biomanufacturing due to plentiful stocks and nonreliance on agriculture, and it can be sourced renewably. However, due to inevitable complexities in cell metabolism, microbial methanol conversion requires further improvement before industrial applicability. Here, we present a novel, parallel strategy using artificial cells to provide a simplified and well-defined environment for methanol utilization as artificial methylotrophic cells. We compartmentalized a methanol-utilizing enzyme cascade, including NAD-dependent methanol dehydrogenase (Mdh) and pyruvate-dependent aldolase (KHB aldolase), in cell-sized lipid vesicles using the inverted emulsion method. The reduction of cofactor NAD+ to NADH was used to quantify the conversion of methanol within individual artificial methylotrophic cells via flow cytometry. Compartmentalization of the reaction cascade in liposomes led to a 4-fold higher NADH production compared with bulk enzyme experiments, and the incorporation of KHB aldolase facilitated another 2-fold increase above the Mdh-only reaction. This methanol-utilizing platform can serve as an alternative route to speed up methanol biological conversion, eventually shifting sugar-based bioproduction toward a sustainable methanol bioeconomy.

Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements.

To develop artificial cell models that mimic living cells, cell-sized lipid vesicles encapsulating cell-free protein synthesis (CFPS) systems are useful for protein expressions or artificial gene circuits for vesicle-vesicle communications. Therefore, investigating the transcriptional and translational properties of CFPS systems in lipid vesicles is important for maximizing the synthesis and functions of proteins. Although transcription and translation using CFPS systems inside lipid vesicles are more important than that outside lipid vesicles, the former processes are not investigated by changing the lipid composition of lipid vesicles. Herein, we investigated changes in transcription and translation using CFPS systems inside giant lipid vesicles (approximately 5-20μm in diameter) caused by changing the lipid composition of lipid vesicles containing neutral, positively, and negatively charged lipids. After incubating for 30min, 1h, 2h, and 4h, the transcriptional and translational activities in these lipid vesicles were determined by detecting the fluorescence intensities of the fluorogenic RNA aptamer on the 3'-untranslated region of mRNA (transcription) and the fluorescent protein sfCherry (translation), respectively. The results revealed that transcriptional and translational activities in a lipid vesicle containing positively charged lipids were high when the protein was synthesized using the CFPS system inside the lipid vesicle. Thus, the present study provides an experimental basis for constructing complex artificial cell models using bottom-up approaches.

The Influence of Functional Materials on the Size of the Lipid Vesicles in Beverages

By investigating the hydrophobic properties and functional components including ethyl caproate (EC), caproic acid (CA), isoamyl acetate (IA), isoamyl alcohol (IAA), isovaleraldehyde (IVA), and procyanidin B2 (PB2) in beverages, one can incorporate them with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids to create cell-sized lipid vesicles. The aim of this study was to explore the correlation between the concentration of flavors or functional compounds and the size of the lipid vesicles. It was observed that EC, CA, IA, and IAA decreased the size of lipid vesicles. In contrast, IVA and PB2 increased their size. To comprehend this correlation, both the chemical structure of these compounds in relation to DOPC membranes and the fluidity of the membranes were considered. The size of the lipid vesicles was influenced by the molecular interactions between the compounds and DOPC. Those were caused by, in particular, the balance between hydrophobicity and hydrophilicity. Compounds with higher hydrophobicity tended to decrease the size of the lipid vesicles, whereas compounds with greater hydrophilicity had the opposite effect, leading to an increase in size. These findings suggest that the size of lipid vesicles can serve as a potential indicator for rapidly evaluating the concentration of these components in beverages.

Cell penetrating peptides facilitate protein transport into the cell-sized asymmetric lipid vesicles at arbitrary timing.

Cell penetrating peptides (CPP) play an important role of drug delivery system. CPP have a strategy to overcame the membrane impermeability. There are two pathways to across the lipid bilayer via CPPs such as direct translocation and endocytosis. The direct translocation of CPPs was investigated using an artificial lipid membrane, such as planar lipid bilayers and nano-sized lipid vesicles. The direct translocation via CPPs using the planar lipid bilayers suggests that the negative transmembrane potential generating between planar lipid bilayers plays an important role for the direct translocation of β-gal via pep-1. In this study, the interactions between CPPs and cell-sized lipid vesicles were investigated using two peptides (Pep-1, penetratin). Next, the interaction between the CPP-DNase I complexes and membrane were observed. In addition, we investigated the DNase I translocation of CPP-DNase I complexes into the GUVs. Finally, we demonstrated the formation of a cross-linked actin network in GUVs using CPP-mediated translocation.CPPs-DNase I complexes was easily interacted with the asymmetric GUVs containing DOPG or DOPS in the inner leaflet. Therefore, CPPs mediated direct translocation of DNase I into the asymmetric GUVs with negatively charged lipids on the inner leaflet. These results indicate that the CPPs-mediated direct translocation could be triggered by the transmembrane potential generating by asymmetric GUV membrane. We found that the penetratin-mediated translocation of DNase I was more efficient than the Pep-1-mediated translocation. Using this asymmetric membrane component and CPP condition, we could translocate streptavidin into GUVs. Thus, highly versatile of CPP-mediated translocation was shown. This system will contribute to creations of well-defined artificial cell models with the capacity to control the start of inside reaction.

Autonomous Vesicle/Sheet Transformation of Cell-Sized Lipid Bilayers by Hetero-Grafted Copolymers.

Lipid bilayer transformations are involved in biological phenomena including cell division, autophagy, virus infection, and vesicle transport. Artificial materials to manipulate membrane dynamics play a vital role in cellular engineering and drug delivery technology that accesses the membranes of cells or liposomes. Transformation from 3D lipid vesicles to 2D nanosheets is thermodynamically prohibited because the apolar/polar interfaces between the hydrophobic bilayer edges and water are energetically unfavorable. We recently reported that cell-sized lipid vesicles (or giant vesicles) can be thoroughly transformed to 2D nanosheets by the addition of the amphiphilic E5 peptide and a cationic graft copolymer. Here, to understand the mechanisms underlying the lipid nanosheet formation, we systematically investigated the structural effects of the cationic copolymers on nanosheet formation. We found that lipid nanosheet formation is controlled in an all-or-nothing manner when the graft content of the copolymer is increased from 5.7 mol % to 7.7 mol %. This finding prompted us to obtain autonomous 2D/3D transformation system. A newly designed hetero-grafted cationic copolymers with thermoresponsive poly(N-isopropylacrylamide) grafts enables spontaneous 3D vesicle/2D nanosheet transformation in response to temperature. These findings would enable us to obtain smart nanointerfaces that trigger cell-sized lipid membrane dynamics in response to diverse stimuli and to create 2D-3D convertible lipid-based biomaterials.

Confined Motion: Motility of Active Microparticles in Cell-Sized Lipid Vesicles.

Active materials can transduce external energy into kinetic energy at the nano and micron length scales. This unique feature has sparked much research, which ranges from achieving fundamental understanding of their motility to the assessment of potential applications. Traditionally, motility is studied as a function of internal features such as particle topology, while external parameters such as energy source are assessed mainly in bulk. However, in real-life applications, confinement plays a crucial role in determining the type of motion active particles can adapt. This feature has been however surprisingly underexplored experimentally. Here, we showcase a tunable experimental platform to gain an insight into the dynamics of active particles in environments with restricted 3D topology. Particularly, we examined the autonomous motion of coacervate micromotors confined in giant unilamellar vesicles (GUVs) spanning 10–50 μm in diameter and varied parameters including fuel and micromotor concentration. We observed anomalous diffusion upon confinement, leading to decreased motility, which was more pronounced in smaller compartments. The results indicate that the theoretically predicted hydrodynamic effect dominates the motion mechanism within this platform. Our study provides a versatile approach to understand the behavior of active matter under controlled, compartmentalized conditions.

Quantifying proton-induced membrane polarization in single biomimetic giant vesicles

Proton gradients are utilized by cells to power the transport activity of many membrane proteins. Synthetic cells, such as proteo-giant unilamellar vesicles, offer an advanced approach for studying the functionality of membrane proteins in isolation. However, understanding of protein-based transport in vitro requires accurate measurements of proton flux and its accompanying electrochemical gradient across the lipid bilayer. We present an approach to directly quantify the flux of protons across single cell-sized lipid vesicles under modulated electrochemical gradients. Our measurements reveal the corresponding association between proton permeation and transmembrane potential development and its relation to the chemical nature of the conjugated anion (base). In the case of formic acid, we showed that, out of the total amount of permeated protons, a fraction of ≈0.2 traverse the lipid bilayer as H+, with the rest (≈0.8) in the form of a neutral acid. For strong acids (HCl or HNO3), proton permeation was governed by translocation of H+. Accordingly, a larger proton motive force (pmf) was generated for strong acids (pmf=14.2 mV) relative to formic acid (pmf=1.3 mV). We anticipate that our approach will guide the development of protein-based transport driven by proton gradient in artificial cell models and enable a deeper understanding of how vital acids, such as fatty acids, amino acids, bile acids, and carboxylic acid-containing drugs, traverse the lipid bilayer.

Vesicle-Based Gel via Polyelectrolyte-Induced Adhesion: Structure, Rheology, and Response.

We describe an experimental study of soft solids composed of micron-scale lipid bilayer vesicles that adhere to one another through electrostatic attraction to an oppositely charged polymer (PDADMAC). As the polymer concentration was increased, we found a fluid phase, a solid gel phase, and a gel composed of internally reorganized vesicles. Optical microscopy images showed a nearly close-packed structure of adhered vesicles that retained their closed-cell morphology. Shear rheology measurements showed that the gel phase is a solid with a modulus at the Pa scale and with linear response up to 70% strain. We found that the modulus depends on the energy per area of membrane-membrane adhesion but does not depend on the vesicle size. We further found that the gels survived osmotic stress or dilution of the adhering polymer but could be rapidly disrupted in response to the addition of strongly binding silica nanoparticles. These results demonstrate the potential for cell-sized lipid vesicles to form a solid platform that maintains the responsive properties of the membranes. Such materials may find applications as triggerable, protective coatings of delicate surfaces.

Investigation of Fusion between Nanosized Lipid Vesicles and a Lipid Monolayer Toward Formation of Giant Lipid Vesicles with Various Kinds of Biomolecules.

We determined the properties of fusion between large unilamellar vesicles (LUVs) and the lipid monolayer by measuring the fluorescence intensity of rhodamine-conjugated phospholipids in cell-sized lipid vesicles. The charge of LUVs (containing cationic lipids) and lipid droplets (containing anionic lipids) promoted lipid membrane fusion. We also investigated the formation of cell-sized lipid vesicles with asymmetric lipid distribution using this fusion method. Moreover, cell-sized asymmetric ganglioside vesicles can be generated from the planar lipid bilayer formed at the interface between the lipid droplets with/without LUVs containing ganglioside. The flip-flop dynamics of ganglioside were observed on the asymmetric ganglioside vesicles. This fusion method can be used to form asymmetric lipid vesicles with poor solubility in n-decane or lipid vesicles containing various types of membrane proteins for the development of complex artificial cell models.

Size of Cells and Physicochemical Properties of Membranes are Related to Flavor Production during Sake Brewing in the Yeast Saccharomyces cerevisiae

Ethyl caproate (EC) and isoamyl acetate (IA) are key flavor components of sake. Recently, attempts have been made to increase the content of good flavor components, such as EC and IA, in sake brewing. However, the functions of EC and IA in yeast cells remain poorly understood. Therefore, we investigated the effects of EC and IA using cell-sized lipid vesicles. We also investigated lipid vesicles containing EC and/or caproic acid (CA) as well as IA and/or isoamyl alcohol (IAA). CA and IAA are precursors of EC and IA, respectively, and are important flavors in sake brewing. The size of a vesicle is influenced by flavor compounds and their precursors in a concentration-dependent manner. We aimed to establish the conditions in which the vesicles contained more flavors simultaneously and with different ratios. Interestingly, vesicles were largest in a mixture of 50% of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with 25% EC and 25% CA or a mixture of 50% DOPC with 25% IA and 25% IAA. The impact of flavor additives on membrane fluidity was also studied using Laurdan generalized polarization. During the production process, flavors may regulate the fluidity of lipid membranes.

Influence of Ethyl Caproate on the Size of Lipid Vesicles and Yeast Cells

Ethyl caproate (EC) is a key flavor component of sake. Recently, in sake brewing, an effort has been underway to increase the content of aromatic components such as EC. However, the function of EC in yeast cells remains poorly understood. Therefore, we investigated the effects of EC using cell-sized lipid vesicles. We found that vesicle size decreases in a concentration-dependent manner when EC is contained in lipid vesicles. Furthermore, yeast experiments showed that a strain producing high quantities of EC in its stationary phase decreased in size during EC production. Given caproic acid’s (CA) status as the esterification precursor of EC in yeast, we also compared lipid vesicles containing CA with those containing EC. We found that CA vesicles were smaller than EC vesicles of the same concentration. These results suggest that EC production may function apparently to maintain cell size.

Controlled division of cell-sized vesicles by low densities of membrane-bound proteins

The proliferation of life on earth is based on the ability of single cells to divide into two daughter cells. During cell division, the plasma membrane undergoes a series of morphological transformations which ultimately lead to membrane fission. Here, we show that analogous remodeling processes can be induced by low densities of proteins bound to the membranes of cell-sized lipid vesicles. Using His-tagged fluorescent proteins, we are able to precisely control the spontaneous curvature of the vesicle membranes. By fine-tuning this curvature, we obtain dumbbell-shaped vesicles with closed membrane necks as well as neck fission and complete vesicle division. Our results demonstrate that the spontaneous curvature generates constriction forces around the membrane necks and that these forces can easily cover the force range found in vivo. Our approach involves only one species of membrane-bound proteins at low densities, thereby providing a simple and extendible module for bottom-up synthetic biology.

Freeze-thaw cycles induce content exchange between cell-sized lipid vesicles

Early protocells are commonly assumed to consist of an amphiphilic membrane enclosing an RNA-based self-replicating genetic system and a primitive metabolism without protein enzymes. Thus, protocell evolution must have relied on simple physicochemical self-organization processes within and across such vesicular structures. We investigate freeze-thaw (FT) cycling as a potential environmental driver for the necessary content exchange between vesicles. To this end, we developed a conceptually simple yet statistically powerful high-throughput procedure based on nucleic acid-containing giant unilamellar vesicles (GUVs) as model protocells. GUVs are formed by emulsion transfer in glass bottom microtiter plates and hence can be manipulated and monitored by fluorescence microscopy without additional pipetting and sample handling steps. This new protocol greatly minimizes artefacts, such as unintended GUV rupture or fusion by shear forces. Using DNA-encapsulating phospholipid GUVs fabricated by this method, we quantified the extent of content mixing between GUVs under different FT conditions. We found evidence of nucleic acid exchange in all detected vesicles if fast freezing of GUVs at −80 °C is followed by slow thawing at room temperature. In contrast, slow freezing and fast thawing both adversely affected content mixing. Surprisingly, and in contrast to previous reports for FT-induced content mixing, we found that the content is not exchanged through vesicle fusion and fission, but that vesicles largely maintain their membrane identity and even large molecules are exchanged via diffusion across the membranes. Our approach supports efficient screening of prebiotically plausible molecules and environmental conditions, to yield universal mechanistic insights into how cellular life may have emerged.

Cell-sized lipid vesicles for cell-cell synaptic therapies.

Cell-sized lipid vesicles (CLVs) have shown great promise for therapeutic and artificial cell applications, but their fragility and short shelf life has hindered widespread adoption and commercial viability. We present a method to circumvent the storage limitations of CLVs such as giant unilamellar vesicles (GUVs) and single-compartment multisomes (SCMs) by storing them in a double emulsion precursor form. The double emulsions can be stored for at least 8 months and readily converted into either GUVs or SCMs at any time. In this study, we investigate the interfacial parameters responsible for this morphological change, and we also demonstrate the therapeutic potential of CLVs by utilizing them to present a transmembrane protein, neuroligin-2, to pancreatic β-cells, forming cell-cell synapses that stimulate insulin secretion and cellular growth.

Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4–5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases.

Ca-mediated electroformation of cell-sized lipid vesicles.

Cell-sized lipid giant unilamellar vesicles (GUVs) are formed when lipid molecules self-assemble to construct a single bilayer compartment with similar morphology to living cells. The physics of self-assembly process is only generally understood and the size distribution of GUVs tends to be very polydisperse. Herein we report a strategy for the production of controlled size distributions of GUVs by a novel mechanism dissecting the mediation ability of calcium (Ca) on the conventional electroformation of GUVs. We finely construct both of the calcium ion (Ca2+) and calcium carbonate (CaCO3) mineral adsorption layers on a lipid film surface respectively during the electroformation of GUVs. It is found that Ca2+ Slip plane polarized by alternating electric field could induce a pattern of electroosmotic flow across the surface, and thus confine the fusion and growth of GUVs to facilitate the formation of uniform GUVs. The model is further improved by directly using CaCO3 that is in situ formed on a lipid film surface, providing a GUV population with narrow polydispersity. The two models deciphers the new biological function of calcium on the birth of cell-like lipid vesicles, and thus might be potentially relevant to the construction of new model to elucidate the cellular development process.

Real-time observation of model membrane dynamics induced by Alzheimer's amyloid beta

Amyloid beta (Aβ) has been strongly implicated in inducing neurotoxicity in the pathology of Alzheimer's disease (AD). However, the underlying mechanisms remain unknown. In this study, we examined, in real-time, the spatio-temporal changes in individual model membranes induced by the presence of different Aβ-40 molecular assemblies (species). We used cell-sized lipid vesicles to enable the direct observation of these changes. We found three significantly different membrane-transformation pathways. We characterized the biophysical mechanisms behind these transformations in terms of the change in inner vesicle volume and surface area. Oligomeric Aβ exhibited the highest tendency to cause membrane fluctuation and transformations. Interestingly, mature fibrils, which are often considered inert species, also induced profound membrane changes. Furthermore, we imaged the localization of pre-fibrillar species on membranes. The real-time observation of these morphological transformations, which can be missed in a discretised analysis, may help to unlock the mechanisms of AD's Aβ-induced neuro-degeneration.

Construction of Asymmetric Cell-Sized Lipid Vesicles from Lipid-Coated Water-in-Oil Microdroplets

We present a simple, rapid, and robust method for preparing asymmetric cell-sized lipid bilayer vesicles using water-in-oil (W/O) microdroplets transferred through an oil-water interface. The efficiency for producing cell-sized model membranes is elucidated in relation to the vesicular size and the weight of contained water-soluble molecules. We demonstrate the biological asymmetric nature and the formation of lipid raft microdomain structures using fluorescence microscopy.

Gene expression within cell-sized lipid vesicles.

Functional protein synthesis was observed in cell-sized lipid vesicles following encapsulation of a gene-expression system. Expression of rsGFP (red-shifted green fluorescent protein) within individual vesicles was observed by fluorescence microscopy. Interestingly, at the early stage of the reaction, the expression efficiency inside the vesicle was remarkably higher than that in the solution outside. The synthesized rsGFP in individual vesicles is safe from attack by proteinase K added to the external aqueous solution. Studies on cell-sized vesicles expressing protein should contribute to a fundamental understanding of certain aspects of living systems and will be useful for practical applications, such as the construction of microreactors.