Artificial cells for in vivo biomedical applications through red blood cell biomimicry.

Recent research in artificial cell production holds promise for the development of delivery agents with therapeutic effects akin to real cells. To succeed in these applications, these systems need to survive the circulatory conditions. In this review we present strategies that, inspired by the endurance of red blood cells, have enhanced the viability of large, cell-like vehicles for in vivo therapeutic use, particularly focusing on giant unilamellar vesicles. Insights from red blood cells can guide modifications that could transform these platforms into advanced drug delivery vehicles, showcasing biomimicry's potential in shaping the future of therapeutic applications.

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Melittin-Phospholipase A2 Synergism Is Mediated by Liquid-Liquid Miscibility Phase Transition in Giant Unilamellar Vesicles.

The primary constituents of honeybee venom, melittin and phospholipase A2 (PLA2), display toxin synergism in which the PLA2 activity is significantly enhanced by the presence of melittin. It has been shown previously that this is accomplished by the disruption in lipid packing, which allows PLA2 to become processive on the membrane surface. In this work, we show that melittin is capable of driving miscibility phase transition in giant unilamellar vesicles (GUVs) and that it raises the miscibility transition temperature (Tmisc) in a concentration-dependent manner. The induced phase separation enhances the processivity of PLA2, particularly at its boundaries, where a substantial difference in domain thickness creates a membrane discontinuity. The catalytic action of PLA2, in response, induces changes in the membrane, rendering it more conducive to melittin binding. This, in turn, facilitates further lipid phase separation and eventual vesicle lysis. Overall, our results show that melittin has powerful membrane-altering capabilities that activate PLA2 in various membrane contexts. More broadly, they exemplify how this biochemical system actively modulates and capitalizes on the spatial distribution of membrane lipids to efficiently achieve its objectives.

Giant vesicles form in physiological saline and encapsulate pDNA by the modified electroformation method.

Giant vesicles (GVs) are used to study the structures and functions of cells and cell membranes. Electroformation is the most commonly used method for GV preparation. However, the electroformation of GVs is hindered in highly concentrated ionic solutions, limiting their application as cell models for research under physiological conditions. In this study, giant multilayer vesicles were successfully generated in physiological saline using a modified electroformation device by adding an insulating layer between the two electrode plates. The influence of the electric frequency and strength on the electroformation of GVs in physiological saline was explored, and a possible mechanism for this improvement was assessed. It has been shown that an insulating layer between the two electrodes can improve the electroformation of GVs in physiological saline by increasing the electrical impedance, which is weakened by the saline solution, thereby restoring the reduced effective electric field strength. Furthermore, macromolecular plasmid DNA (pDNA) was successfully encapsulated in the electroformed GVs of the modified device. This modified electroformation method may be useful for generating eukaryotic cell models under physiological conditions.

Effect of Phosphatidylethanolamine on Pore Formation Induced by the Antimicrobial Peptide PGLa.

Most antimicrobial peptides (AMPs) induce pore formation and a burst of lipid bilayers and plasma membranes. This causes severe leakage of the internal contents and cell death. The AMP PGLa forms nanopores in giant unilamellar vesicles (GUVs) comprising dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG). We here elucidated the effect of the line tension of a prepore rim on PGLa-induced nanopore formation by investigating the interaction of PGLa with single GUVs comprising dioleoylphosphatidylethanolamine (DOPE)/DOPG (6:4) in buffer using the single GUV method. We found that PGLa forms nanopores in the GUV membrane, which evolved into a local burst and burst of GUVs. The rate of pore formation in DOPE/DOPG-GUVs was smaller than that in DOPC/DOPG-GUVs. PGLa is located only in the outer leaflet of a GUV bilayer just before a fluorescent probe AF647 leakage from the inside, indicating that this asymmetric distribution induces nanopore formation. PGLa-induced local burst and burst of GUVs were observed at 10 ms-time resolution. After nanopore formation started, dense particles and small vesicles appeared in the GUVs, followed by a decrease in the GUV diameter. The GUV was finally converted into smaller GUV or lipid membrane aggregates. We discuss the mechanisms of PGLa-induced nanopore formation and its direct evolution to a GUV burst.

Negative lipid membranes enhance the adsorption of TAT-decorated elastin-like polypeptide micelles.

A cell-penetrating peptide (CPP) is a short amino-acid sequence capable of efficiently translocating across the cellular membrane of mammalian cells. However, the potential of CPPs as a delivery vector is hampered by the strong reduction of its translocation efficiency when it bears an attached molecular cargo. To overcome this problem, we used previously developed diblock copolymers of elastin-like polypeptides (ELPBCs), which we end functionalized with TAT (transactivator of transcription), an archetypal CPP built from a positively charged amino acid sequence of the HIV-1 virus. These ELPBCs self-assemble into micelles at a specific temperature and present the TAT peptide on their corona. These micelles can recover the lost membrane affinity of TAT and can trigger interactions with the membrane despite the presence of a molecular cargo. Herein, we study the influence of membrane surface charge on the adsorption of TAT-functionalized ELP micelles onto giant unilamellar vesicles (GUVs). We show that the TAT-ELPBC micelles show an increased binding constant toward negatively charged membranes compared to neutral membranes, but no translocation is observed. The affinity of the TAT-ELPBC micelles for the GUVs displays a stepwise dependence on the lipid charge of the GUV, which, to our knowledge, has not been reported previously for interactions between peptides and lipid membranes. By unveiling the key steps controlling the interaction of an archetypal CPP with lipid membranes, through regulation of the charge of the lipid bilayer, our results pave the way for a better design of delivery vectors based on CPPs.

Dynamic Light-Induced Protein Patterns at Model Membranes.

The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell polarization, migration, and division. Thus, methods to recruit proteins to model membranes with subcellular resolution and high temporal control are essential when reproducing and controlling such processes in synthetic cells. Here, a method is described for fabricating light-regulated reversible protein patterns at lipid membranes with high spatiotemporal precision. For this purpose, we immobilize the photoswitchable protein iLID (improved light-inducible dimer) on supported lipid bilayers (SLBs) and on the outer membrane of giant unilamellar vesicles (GUVs). Upon local blue light illumination, iLID binds to its partner Nano (wild-type SspB) and allows the recruitment of any protein of interest (POI) fused to Nano from the solution to the illuminated area on the membrane. This binding is reversible in the dark, which provides dynamic binding and release of the POI. Overall, this is a flexible and versatile method for regulating the localization of proteins with high precision in space and time using blue light.

Determination of pore edge tension from the kinetics of rupture of giant unilamellar vesicles using the Arrhenius equation: effects of sugar concentration, surface charge and cholesterol.

The pore edge tension (Γ) of a membrane closely intertwines with membrane stability and plays a vital role in the mechanisms that facilitate membrane resealing following pore formation caused by electrical and mechanical tensions. We have explored a straightforward procedure to determine Γ by fitting the inverse of the tension-dependent logarithm of the rate constant of rupture of giant unilamellar vesicles (GUVs) using the Arrhenius equation. The GUVs were prepared using a combination of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) and 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in a physiological environment. The effects of sugar concentration, membrane surface charge density, and membrane cholesterol concentration on Γ have been investigated. The values of Γ increase with sugar concentration in the physiological buffer, measuring 9.6 ± 0.3, 10.4 ± 0.1, and 16.2 ± 0.1 pN for 40, 100, and 300 mM, respectively. A higher concentration of anionic lipids (70 mol% of DOPG) significantly reduces Γ. An increasing trend of Γ with cholesterol content was observed; specifically, the values of Γ were 11.9 ± 0.9, 13.9 ± 0.7, and 16.2 ± 0.4 pN for 15, 29, and 40 mol% cholesterol, respectively. Thus, the presence of higher anionic lipids in the bilayer led to a decrease in membrane stability. In contrast, the presence of higher sugar concentrations in the buffer and increased cholesterol concentration in the membranes enhanced membrane stability.

Bilayer Charge Asymmetry and Oil Residues Destabilize Membranes upon Poration.

Transmembrane asymmetry is ubiquitous in cells, particularly with respect to lipids, where charged lipids are mainly restricted to one monolayer. We investigate the influence of anionic lipid asymmetry on the stability of giant unilamellar vesicles (GUVs), minimal plasma membrane models. To quantify asymmetry, we apply the fluorescence quenching assay, which is often difficult to reproduce, and caution in handling the quencher is generally underestimated. We first optimize this assay and then apply it to GUVs prepared with the inverted emulsion transfer protocol by using increasing fractions of anionic lipids restricted to one leaflet. This protocol is found to produce highly asymmetric bilayers but with ∼20% interleaflet mixing. To probe the stability of asymmetric versus symmetric membranes, we expose the GUVs to porating electric pulses and monitor the fraction of destabilized vesicles. The pulses open macropores, and the GUVs either completely recover or exhibit leakage or bursting/collapse. Residual oil destabilizes porated membranes, and destabilization is even more pronounced in asymmetrically charged membranes. This is corroborated by the measured pore edge tension, which is also found to decrease with increasing charge asymmetry. Using GUVs with imposed transmembrane pH asymmetry, we confirm that poration-triggered destabilization does not depend on the approach used to generate membrane asymmetry.

Nonspecific membrane-matrix interactions influence diffusivity of lipid vesicles in hydrogels.

The diffusion of extracellular vesicles and liposomes in vivo is affected by different tissue environmental conditions and is of great interest in the development of liposome-based therapeutics and drug-delivery systems. Here, we use a bottom-up biomimetic approach to better isolate and study steric and electrostatic interactions and their influence on the diffusivity of synthetic large unilamellar vesicles in hydrogel environments. Single-particle tracking of these extracellular vesicle-like particles in agarose hydrogels as an extracellular matrix model shows that membrane deformability and surface charge affect the hydrogel pore spaces that vesicles have access to, which determines overall diffusivity. Moreover, we show that passivation of vesicles with PEGylated lipids, as often used in drug-delivery systems, enhances diffusivity, but that this effect cannot be fully explained with electrostatic interactions alone. Finally, we compare our experimental findings with existing computational and theoretical work in the field to help explain the nonspecific interactions between diffusing particles and gel matrix environments.

Large Unilamellar Vesicles of Phosphatidic Acid Reduce the Toxicity of α-Synuclein Fibrils.

Parkinson's disease (PD) is a severe pathology that is caused by a progressive degeneration of dopaminergic neurons in substantia nigra pars compacta as well as other areas in the brain. These neurodegeneration processes are linked to the abrupt aggregation of α-synuclein (α-syn), a small protein that is abundant at presynaptic nerve termini, where it regulates cell vesicle trafficking. Due to the direct interactions of α-syn with cell membranes, a substantial amount of work was done over the past decade to understand the role of lipids in α-syn aggregation. However, the role of phosphatidic acid (PA), a negatively charged phospholipid with a small polar head, remains unclear. In this study, we examined the effect of PA large unilamellar vesicles (LUVs) on α-syn aggregation. We found that PA LUVs with 16:0, 18:0, and 18:1 FAs drastically reduced the toxicity of α-syn fibrils if were present in a 1:1 molar ratio with the protein. Our results also showed that the presence of these vehicles changed the rate of α-syn aggregation and altered the morphology and secondary structure of α-syn fibrils. These results indicate that PA LUVs can be used as a potential therapeutic strategy to reduce the toxicity of α-syn fibrils formed upon PD.

An Efficient Method for the Production of High-Purity Bioinspired Large Unilamellar Vesicles.

In order to recapitulate complex eukaryotic compartmentalization, synthetic biology aims to recreate cellular membrane-lined compartments from the bottom-up. Many important cellular organelles and cell-produced extracellular vesicles are in the size range of several hundreds of nanometers. Although attaining a fundamental characterization and mimicry of their cellular functions is a compelling goal, the lack of methods for controlled vesicle formation in this size range has hindered full understanding. Here, we show the optimization of a simple and efficient protocol for the production of large unilamellar vesicles (LUVs) with a median diameter in the range of 450-550 nm with high purity. Importantly, we rely on commercial reagents and common laboratory equipment. We thoroughly characterize the influence of different experimental parameters on the concentration and size of the resulting vesicles and assess changes in their lipid composition and surface charge. We provide guidance for researchers to optimize LUV production further to suit specific applications.

Exploring the synergistic effects of amoxicillin and methylene blue on unsaturated lipid structures: A study of Langmuir monolayers and giant unilamellar vesicles.

The potentially toxic effects of emerging pollutant mixtures often deviate from the individual compound effects, presenting additive, synergistic, or agonistic interactions. This study delves into the complex world of emerging pollutants' mixtures, with a particular focus on their potential impact on unsaturated lipid DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine) structured as both monolayers and bilayers, which are valuable tools for mimicking cell membranes. Specifically, we examine the effects of two common types of pollutants: antibiotics (amoxicillin) and dyes (methylene blue). Utilizing Langmuir monolayers, our research reveals a synergistic effect within the pollutant mixture, as evidenced by pressure-area isotherms and polarization-modulated infrared reflection absorption spectroscopy. We identify the specific chemical interactions contributing to this synergistic effect. Furthermore, through contrast phase microscopy experiments on giant unilamellar vesicles (bilayer system), we find that the individual pollutants and the mixture exhibit similar molecular effects on the bilayer, revealing that the molecular size is a key factor in the bilayer-mixture of pollutant interaction. This highlights the importance of considering molecular size in the interactions with bilayer systems. In summary, our research dissects the critical factors of chemical interactions and molecular size concerning the effects of pollutants on DOPC, serving as simplified models of cell membranes. This study underscores the significance of comprehending the molecular effects of emerging pollutants on human health and the development of models for exploring their intricate interactions with cell membranes.

Bottom-up approach to explore alpha-amylase assisted membrane remodelling.

Soluble alpha-amylases play an important role in the catabolism of polysaccharides. In this work, we show that the malt α -amylase can interact with the lipid membrane and further alter its mechanical properties. Vesicle fluctuation spectroscopy is used for quantitative measurement of the membrane bending rigidity of phosphatidylcholines lipid vesicles from the shape fluctuation based on the whole contour of Giant Unilamellar Vesicles (GUVs). The bending rigidity of the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid vesicles in water increases significantly with the presence of 0.14 micromolar alpha-amylase (AA) in the exterior solution. It appears that the enzyme present in the external solution interacts with the outer layer of the bilayer membrane, leading to an asymmetry of the solution on either side of the bilayer membrane and altering its elasticity. At AA concentration of 1.5 micromolars and above, changes in the morphology of the GUV membrane are observed. The interaction between AA in the external solution and the external leaflet causes the bilayer membrane to curve spontaneously, leading to the formation of outbuds, giving a positive spontaneous curvature of C0 ≤ 0.05 µm-1 at ≈ 1 mg / ml of the AA concentration. We validate and characterize its concentration-dependent role in stabilizing the membrane curvature. Our findings indicate that the involvement of the enzyme, depending on the concentration, can have a considerable effect on the mechanical characteristics of the membrane.

Protein-Rich Rafts in Hybrid Polymer/Lipid Giant Unilamellar Vesicles.

Considerable attention has been dedicated to lipid rafts due to their importance in numerous cell functions such as membrane trafficking, polarization, and signaling. Next to studies in living cells, artificial micrometer-sized vesicles with a minimal set of components are established as a major tool to understand the phase separation dynamics and their intimate interplay with membrane proteins. In parallel, mixtures of phospholipids and certain amphiphilic polymers simultaneously offer an interface for proteins and mimic this segregation behavior, presenting a tangible synthetic alternative for fundamental studies and bottom-up design of cellular mimics. However, the simultaneous insertion of complex and sensitive membrane proteins is experimentally challenging and thus far has been largely limited to natural lipids. Here, we present the co-reconstitution of the proton pump bo3 oxidase and the proton consumer ATP synthase in hybrid polymer/lipid giant unilamellar vesicles (GUVs) via fusion/electroformation. Variations of the current method allow for tailored reconstitution protocols and control of the vesicle size. In particular, mixing of protein-free and protein-functionalized nanosized vesicles in the electroformation film results in larger GUVs, while separate reconstitution of the respiratory enzymes enables higher ATP synthesis rates. Furthermore, protein labeling provides a synthetic mechanism for phase separation and protein sequestration, mimicking lipid- and protein-mediated domain formation in nature. The latter means opens further possibilities for re-enacting phenomena like supercomplex assembly or symmetry breaking and enriches the toolbox of bottom-up synthetic biology.

Intravesicular Solute Delivery and Surface Area Regulation in Giant Unilamellar Vesicles Driven by Cycles of Osmotic Stresses.

Phospholipid bilayers are dynamic cellular components that undergo constant changes in their topology, facilitating a broad diversity of physiological functions including endo- and exocytosis, cell division, and intracellular trafficking. These shape transformations consume energy, supplied invariably by the activity of proteins. Here, we show that cycles of oppositely directed osmotic stresses─unassisted by any protein activity─can induce well-defined remodeling of giant unilamellar vesicles, minimally recapitulating the phenomenologies of surface area homeostasis and macropinocytosis. We find that a stress cycle consisting of deflationary hypertonic stress followed by an inflationary hypotonic one prompts an elaborate sequence of membrane shape changes ultimately transporting molecular cargo from the outside into the intravesicular milieu. The initial osmotic deflation produces microscopic spherical invaginations. During the subsequent inflation, the first subpopulation contributes area to the swelling membrane, thereby providing a means for surface area regulation and tensional homeostasis. The second subpopulation vesiculates into the lumens of the mother vesicles, producing pinocytic vesicles. Remarkably, the gradients of solute concentrations between the GUV and the daughter pinocytic vesicles create cascades of water current, inducing pulsatory transient poration that enable solute exchange between the buds and the GUV interior. This results in an efficient water-flux-mediated delivery of molecular cargo across the membrane boundary. Our findings suggest a primitive physical mechanism for communication and transport across protocellular compartments driven only by osmotic stresses. They also suggest plausible physical routes for intravesicular, and possibly intracellular, delivery of ions, solutes, and molecular cargo stimulated simply by cycles of osmotic currents of water.

Estimation of negative membrane tension in lipid bilayers and its effect on antimicrobial peptide magainin 2-induced pore formation.

Positive membrane tension in the stretched plasma membrane of cells and in the stretched lipid bilayer of vesicles has been well analyzed quantitatively, whereas there is limited quantitative information on negative membrane tension in compressed plasma membranes and lipid bilayers. Here, we examined negative membrane tension quantitatively. First, we developed a theory to describe negative membrane tension by analyzing the free energy of lipid bilayers to obtain a theoretical equation for negative membrane tension. This allowed us to obtain an equation describing the negative membrane tension (σosm) for giant unilamellar vesicles (GUVs) in hypertonic solutions due to negative osmotic pressure (Π). Then, we experimentally estimated the negative membrane tension for GUVs in hypertonic solutions by measuring the rate constant (kr) of rupture of the GUVs induced by the constant tension (σex) due to an external force as a function of σex. We found that larger σex values were required to induce the rupture of GUVs under negative Π compared with GUVs in isotonic solution and quantitatively determined the negative membrane tension induced by Π (σosm) by the difference between these σex values. At small negative Π, the experimental values of negative σosm agree with their theoretical values within experimental error, but as negative Π increases, the deviation increases. Negative tension increased the stability of GUVs because higher tensions were required for GUV rupture, and the rate constant of antimicrobial peptide magainin 2-induced pore formation decreased.

Effect of membrane tension on antimicrobial peptide PGLa-induced pore formation in lipid bilayers.

The osmotic pressure (Π) method has recently been developed to quantitatively examine the effect of membrane tension (σ) on pore formation in giant unilamellar vesicles (GUVs) induced by antimicrobial peptides (AMPs). Here, we used the Π method to reveal the effect of σ on the interaction of an AMP, PGLa, with lipid bilayers comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6). PGLa induced leakage of fluorescent probes from single GUVs under Π, indicating nanopore formation. Membrane tension did not transform a PGLa-induced nanopore into a micropore nor cause GUV burst up to 3.4 mN/m, which is in contrast with the effect of σ on another AMP, magainin 2-induced pore formation, where lower σ resulted in GUV burst. The fraction of leaking GUVs at a specific time increased with increasing σ, indicating that the rate of PGLa-induced pore formation increases with increasing σ. The rate of transfer of fluorescent probe-labeled PGLa across the lipid bilayer without pore formation also increased with increasing σ. PGLa-induced pore formation requires a symmetric distribution of peptides in both leaflets of the GUV bilayer, and thus we infer that the increase in the rate of PGLa transfer from the outer leaflet to the inner leaflet underlies the increase in the rate of pore formation with increasing σ. On the basis of these results, we discuss the difference between the effect of σ on nanopore formation in GUV membranes induced by PGLa and that by magainin 2.

Effects of grafted polymers on the lipid membrane fluidity.

Since the membrane fluidity controls the cellular functions, it is important to identify the factors that determine the cell membrane viscosity. Cell membranes are composed of not only lipids and proteins but also polysaccharide chain-anchored molecules, such as glycolipids. To reveal the effects of grafted polymers on the membrane fluidity, in this study, we measured the membrane viscosity of polymer-grafted giant unilamellar vesicles (GUVs), which were prepared by introducing the poly (ethylene glycol) (PEG)-anchored lipids to the ternary GUVs composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol. The membrane viscosity was obtained from the velocity field on the GUV generated by applying a point force, based on the hydrodynamic model proposed by Henle and Levine. The velocity field was visualized by a motion of the circular liquid ordered (Lo) domains formed by a phase separation. With increasing PEG density, the membrane viscosity of PEG-grafted GUVs increased gradually in the mushroom region and significantly in the brush region. We propose a hydrodynamic model that includes the excluded volume effect of PEG chains to explain the increase in membrane viscosity in the mushroom region. This work provides a basic understanding of how grafted polymers affect the membrane fluidity.

Magnetic micromotors crossing lipid membranes.

Nano/micromotors are self-propelled particles that show enhanced motion upon being triggered by a stimulus. Their use in nanomedicine has been widely explored, with special focus on imaging or drug delivery. However, a thorough understanding of the requirements for more efficient locomotion is still lacking. In this paper, we assembled magnetically propelled motors of different sizes (i.e., 0.5, 1 and 4 µm) and surface chemistries (positive charge or PEGylated) and assessed their motion in the presence of giant unilamellar lipid vesicles (GUVs) of varying compositions (zwitterionic, negatively charged and saturated lipids). Unexpectedly, the size does not seem to be the dominating characteristics that governs the ability of the motors to cross lipid membranes. Specifically, the 0.5 µm PEGylated motors have very limited ability to cross the lipid membrane of GUVs due to their non-interacting nature compared to their equally sized positively charged counterparts. Furthermore, membranes made of saturated lipids and, in particular, in combination with a weak magnetic field facilitate motors' crossing, regardless of their size. The results were validated by in-house data-driven statistical analysis that employs experimental data to allow for the identification of individual motor motion in the ensemble when meeting the lipid membranes. Altogether, we provide insight into motor locomotion when they interact with a biological barrier considering both the entire ensemble and the individual motors, which has the potential to support considerations of future motor designs.