Giant Unilamellar Lipid Vesicles Research Articles

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.

Artificial cells with viscoadaptive behavior based on hydrogel-loaded giant unilamellar vesicles.

Viscoadaptation is an essential process in natural cells, where supramolecular interactions between cytosolic components drive adaptation of the cellular mechanical features to regulate metabolic function. This important relationship between mechanical properties and function has until now been underexplored in artificial cell research. Here, we have created an artificial cell platform that exploits internal supramolecular interactions to display viscoadaptive behavior. As supramolecular material to mimic the cytosolic component of these artificial cells, we employed a pH-switchable hydrogelator based on poly(ethylene glycol) coupled to ureido-pyrimidinone units. The hydrogelator was membranized in its sol state in giant unilamellar lipid vesicles to include a cell-membrane mimetic component. The resulting hydrogelator-loaded giant unilamellar vesicles (designated as HL-GUVs) displayed reversible pH-switchable sol-gel behavior through multiple cycles. Furthermore, incorporation of the regulatory enzyme urease enabled us to increase the cytosolic pH upon conversion of its substrate urea. The system was able to switch between a high viscosity (at neutral pH) and a low viscosity (at basic pH) state upon addition of substrate. Finally, viscoadaptation was achieved via the incorporation of a second enzyme of which the activity was governed by the viscosity of the artificial cell. This work represents a new approach to install functional self-regulation in artificial cells, and opens new possibilities for the creation of complex artificial cells that mimic the structural and functional interplay found in biological systems.

Extracellular Cues Govern Shape and Cytoskeletal Organization in Giant Unilamellar Lipid Vesicles.

Spontaneous and induced front-rear polarization and a subsequent asymmetric actin cytoskeleton is a crucial event leading to cell migration, a key process involved in a variety of physiological and pathological conditions such as tissue development, wound healing, and cancer. Migration of adherent cells relies on the balance between adhesion to the underlying matrix and cytoskeleton-driven front protrusion and rear retraction. A current challenge is to uncouple the effect of adhesion and shape from the contribution of the cytoskeleton in regulating the onset of front-rear polarization. Here, we present a minimal model system that introduces an asymmetric actin cytoskeleton in synthetic cells, which are resembled by giant unilamellar lipid vesicles (GUVs) adhering onto symmetric and asymmetric micropatterned surfaces. Surface micropatterning of streptavidin-coated regions with varying adhesion shape and area was achieved by maskless UV photopatterning. To further study the effects of GUV shape on the cytoskeletal organization, actin filaments were polymerized together with bundling proteins inside the GUVs. The micropatterns induce synthetic cell deformation upon adhesion to the surface, with the cell shape adapting to the pattern shape and size. As expected, asymmetric patterns induce an asymmetric deformation in adherent synthetic cells. Actin filaments orient along the long axis of the deformed GUV, when having a length similar to the size of the major axis, whereas short filaments exhibit random orientation. With this bottom-up approach we have laid the first steps to identify the relationship between cell front-rear polarization and cytoskeleton organization in the future. Such a minimal system will allow us to further study the major components needed to create a polarized cytoskeleton at the onset of migration.

Curvature preference of cubic CsPbBr3 quantum dots embedded onto phospholipid bilayer membranes.

Curvature-mediated lipid-protein interactions are important determinants of numerous vital cellular reactions and mechanisms. Biomimetic lipid bilayer membranes, such as giant unilamellar vesicles (GUVs), coupled with quantum dot (QD) fluorescent probes, provide an avenue to elucidate the mechanisms and geometry of induced protein aggregation. However, essentially all QDs used in QD-lipid membrane studies encountered in the literature are of the cadmium selenide (CdSe) or CdSe core/ZnS shell type, which are quasispherically shaped. We report here the membrane curvature partitioning of cube-shaped CsPbBr3 QDs embedded within deformed GUV lipid bilayers versus that of a conventional small fluorophore (ATTO-488) and quasispherical CdSe core/ZnS shell QDs. In alignment with basic packing theory regarding cubes packed in curved confined spaces, the local relative concentration of CsPbBr3 is highest in areas of lowest relative curvature in the plane of observation; this partitioning behavior is significantly different from that of ATTO-488 (p = 0.0051) and CdSe (p = 1.10 × 10-11). In addition, when presented with only one principal radius of curvature in the observation plane, no significant difference (p = 0.172) was observed in the bilayer distribution of CsPbBr3versus that of ATTO-488, suggesting that both QD and lipid membrane geometry greatly impact the curvature preferences of the QDs. These results highlight a fully-synthetic analog to curvature-induced protein aggregation, and lay a framework for the structural and biophysical analysis of complexes between lipid membranes and the shape of intercalating particles.

A DNA Segregation Module for Synthetic Cells.

The bottom-up construction of an artificial cell requires the realization of synthetic cell division. Significant progress has been made toward reliable compartment division, yet mechanisms to segregate the DNA-encoded informational content are still in their infancy. Herein, droplets of DNA Y-motifs are formed by liquid-liquid phase separation. DNA droplet segregation is obtained by cleaving the linking component between two populations of DNA Y-motifs. In addition to enzymatic cleavage, photolabile sites are introduced for spatio-temporally controlled DNA segregation in bulk as well as in cell-sized water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). Notably, the segregation process is slower in confinement than in bulk. The ionic strength of the solution and the nucleobase sequences are employed to regulate the segregation dynamics. The experimental results are corroborated in a lattice-based theoretical model which mimics the interactions between the DNA Y-motif populations. Altogether, engineered DNA droplets, reconstituted in GUVs, can represent a strategy toward a DNA segregation module within bottom-up assembled syntheticcells.

Use of giant unilamellar lipid vesicles as antioxidant carriers in in vitro culture medium of bovine embryos

Giant unilamellar vesicles (GUVs) are composed of lipophilic layers and are sensitive to the action of reactive oxygen species (ROS). The use of GUVs as microcarriers of biological macromolecules is particularly interesting since ROS produced by gametes or embryos during in vitro culture can induce the opening of pores in the membrane of these vesicles and cause the release of their content. This study investigated the behavior of GUVs [composed of 2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)] in co-culture with in vitro produced bovine embryos, as well as their embryotoxicity and effectiveness as cysteine carriers in culture medium. Embryonic developmental rates were unaffected, demonstrating the absence of toxicity of GUVs co-cultured with the embryos. No increase of intracellular ROS levels was observed in the embryos co-cultured with GUVs, indicating that the higher lipid content of the culture environment resulting from the lipid composition of the GUV membrane itself did not increase oxidative stress. Variations in the diameter and number of GUVs demonstrated their sensitivity to ROS produced by embryos cultured under conditions that generate oxidative stress. Encapsulation of cysteine in GUVs was found to be more effective in controlling the production of ROS in embryonic cells than direct dilution of this antioxidant in the medium. In conclusion, the use of GUVs in in vitro culture was found to be safe since these vesicles did not promote toxic effects nor did they increase intracellular ROS concentrations in the embryos. GUVs were sensitive to oxidative stress, which resulted in structural changes in response to the action of ROS. The possible slow release of cysteine into the culture medium by GUV rupture would therefore favor the gradual supply of cysteine, prolonging its presence in the medium. Thus, the main implication of the use of GUVs as cysteine microcarriers is the greater effectiveness in preventing the intracytoplasmic increase of ROS in in vitro produced bovine embryos.

Dynamics of spontaneous wrapping of microparticles by floppy lipid membranes

Lipid membranes form the barrier between the inside and outside of cells and many of their subcompartments. As such, they bind to a wide variety of nano- and micrometer sized objects and, in the presence of strong adhesive forces, strongly deform and envelop particles. This wrapping plays a key role in many healthy and disease-related processes. So far, little work has focused on the dynamics of wrapping. Here, using a model system of micron-sized colloidal particles and giant unilamellar lipid vesicles with tunable adhesive forces, we measure the velocity of the particle during wrapping as well as the forces exerted on it by the lipid membrane. Dissipation near the contact line appears to be the main factor determining the wrapping velocity and time to wrap an object.

Bottom-Up Assembly of Synthetic Cells with a DNA Cytoskeleton.

Cytoskeletal elements, like actin and myosin, have been reconstituted inside lipid vesicles toward the vision to reconstruct cells from the bottom up. Here, we realize the de novo assembly of entirely artificial DNA-based cytoskeletons with programmed multifunctionality inside synthetic cells. Giant unilamellar lipid vesicles (GUVs) serve as cell-like compartments, in which the DNA cytoskeletons are repeatedly and reversibly assembled and disassembled with light using the cis–trans isomerization of an azobenzene moiety positioned in the DNA tiles. Importantly, we induced ordered bundling of hundreds of DNA filaments into more rigid structures with molecular crowders. We quantify and tune the persistence length of the bundled filaments to achieve the formation of ring-like cortical structures inside GUVs, resembling actin rings that form during cell division. Additionally, we show that DNA filaments can be programmably linked to the compartment periphery using cholesterol-tagged DNA as a linker. The linker concentration determines the degree of the cortex-like network formation, and we demonstrate that the DNA cortex-like network can deform GUVs from within. All in all, this showcases the potential of DNA nanotechnology to mimic the diverse functions of a cytoskeleton in synthetic cells.

Printing and Erasing of DNA‐Based Photoresists Inside Synthetic Cells

AbstractIn the pursuit of producing functioning synthetic cells from the bottom‐up, DNA nanotechnology has proven to be a powerful tool. However, the crowded yet highly organized arrangement in living cells, bridging from the nano‐ to the micron‐scale, remains challenging to recreate with DNA‐based architectures. Here, laser microprinting is established to print and erase shape‐controlled DNA hydrogels inside the confinement of water‐in‐oil droplets and giant unilamellar lipid vesicles (GUVs). The DNA‐based photoresist consists of a photocleavable inactive DNA linker, which interconnects Y‐shaped DNA motifs when activated by local irradiation with a 405 nm laser. An alternative linker design allows to erase custom features from a preformed DNA hydrogel with feature sizes down to 1.38 μm. The present work demonstrates that the DNA hydrogels can serve as an internal support to stabilize non‐spherical GUV shapes. Overall, DNA‐based photoresists for laser printing in confinement allow to build up architectures on the interior of synthetic cells with light, which diversifies the toolbox of bottom‐up synthetic biology.

Two-Photon 3D Laser Printing Inside Synthetic Cells.

Toward the ambitious goal of manufacturing synthetic cells from the bottom up, various cellular components have already been reconstituted inside lipid vesicles. However, the deterministic positioning of these components inside the compartment has remained elusive. Here, by using two-photon 3Dlaser printing, 2D and 3D hydrogel architectures are manufactured with high precision and nearly arbitrary shape inside preformed giant unilamellar lipid vesicles (GUVs). The required water-soluble photoresist is brought into the GUVs by diffusion in a single mixing step. Crucially, femtosecond two-photon printing inside the compartment does not destroy the GUVs. Beyond this proof-of-principle demonstration, early functional architectures are realized. In particular, a transmembrane structure acting as a pore is 3Dprinted, thereby allowing for the transport of biological cargo, including DNA, into the synthetic compartment. These experiments show that two-photon 3D laser microprinting can be an important addition to the existing toolbox of syntheticbiology.

Proton gradients from light-harvesting E. coli control DNA assemblies for synthetic cells

Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness living systems. Here, we realize a strategic merger of both approaches to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. When DNA origami plates are modified with the pH-sensitive triplex motif, the proton-pumping E. coli can trigger their attachment to giant unilamellar lipid vesicles (GUVs) upon illumination. A DNA cortex is formed upon DNA origami polymerization, which sculpts and deforms the GUVs. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells.

Division and Regrowth of Phase‐Separated Giant Unilamellar Vesicles**

AbstractSuccess in the bottom‐up assembly of synthetic cells will depend on strategies for the division of protocellular compartments. Here, we describe the controlled division of phase‐separated giant unilamellar lipid vesicles (GUVs). We derive an analytical model based on the vesicle geometry, which makes four quantitative predictions that we verify experimentally. We find that the osmolarity ratio required for division is , independent of the GUV size, while asymmetric division happens at lower osmolarity ratios. Remarkably, we show that a suitable osmolarity change can be triggered by water evaporation, enzymatic decomposition of sucrose or light‐triggered uncaging of CMNB‐fluorescein. The latter provides full spatiotemporal control, such that a target GUV undergoes division whereas the surrounding GUVs remain unaffected. Finally, we grow phase‐separated vesicles from single‐phased vesicles by targeted fusion of the opposite lipid type with programmable DNA tags to enable subsequent division cycles.

Division and Regrowth of Phase-Separated Giant Unilamellar Vesicles*.

Success in the bottom‐up assembly of synthetic cells will depend on strategies for the division of protocellular compartments. Here, we describe the controlled division of phase‐separated giant unilamellar lipid vesicles (GUVs). We derive an analytical model based on the vesicle geometry, which makes four quantitative predictions that we verify experimentally. We find that the osmolarity ratio required for division is 2 , independent of the GUV size, while asymmetric division happens at lower osmolarity ratios. Remarkably, we show that a suitable osmolarity change can be triggered by water evaporation, enzymatic decomposition of sucrose or light‐triggered uncaging of CMNB‐fluorescein. The latter provides full spatiotemporal control, such that a target GUV undergoes division whereas the surrounding GUVs remain unaffected. Finally, we grow phase‐separated vesicles from single‐phased vesicles by targeted fusion of the opposite lipid type with programmable DNA tags to enable subsequent division cycles.

Wrapping of Microparticles by Floppy Lipid Vesicles.

Lipid membranes, the barrier defining living cells and many of their subcompartments, bind to a wide variety of nano- and micrometer sized objects. In the presence of strong adhesive forces, membranes can strongly deform and wrap the particles, an essential step in crossing the membrane for a variety of healthy and disease-related processes. A large body of theoretical and numerical work has focused on identifying the physical properties that underly wrapping. Using a model system of micron-sized colloidal particles and giant unilamellar lipid vesicles with tunable adhesive forces, we measure a wrapping phase diagram and make quantitative comparisons to theoretical models. Our data are consistent with a model of membrane-particle interactions accounting for the adhesive energy per unit area, membrane bending rigidity, particle size, and vesicle radius.

Calcium-Mediated Liposome Fusion to Engineer Giant Lipid Vesicles with Cytosolic Proteins and Reconstituted Mammalian Proteins.

Giant unilamellar lipid vesicles (GUVs) are widely used as model membrane systems and provide an excellent basis to construct artificial cells. To construct more sophisticated artificial cells, proteins-in particular membrane proteins-need to be incorporated in GUVs. However, current methods for protein reconstitution have limited throughput or are not generally applicable for all proteins because they depend on detergent solubilization. This limitation is addressed here by introducing calcium-mediated membrane fusion to transfer proteins between negatively charged GUVs and cell-derived plasma membrane vesicles (CDVs), derived from HEK293T cells overexpressing a membrane receptor protein. Fusion conditions are optimized using large unilamellar vesicles and GUVs containing phosphatidylserines and fusogenic lipids. The approach is then applied to induce lipid mixing and subsequent transfer of the overexpressed membrane receptor from CDVs into GUVs. The membrane receptor is detected by immunofluorescence on GUVs that underwent lipid mixing with CDVs. Those GUVs also exhibit esterase activity because cytosolic esterases entrapped in the CDVs are transferred during membrane fusion. Thus, content mixing is demonstrated. Using CDVs circumvents the need to purify or solubilize proteins. Moreover, calcium-mediated fusion allows transfer of lipids, water-soluble and membrane bound proteins in one step, resulting in a semi-synthetic cell.

Switchable Lipid Provides pH-Sensitive Properties to Lipid and Hybrid Polymer/Lipid Membranes.

Blending amphiphilic copolymers and lipids constitutes a novel approach to combine the advantages of polymersomes and liposomes into a new single hybrid membrane. Efforts have been made to design stimuli-responsive vesicles, in which the membrane’s dynamic is modulated by specific triggers. In this investigation, we proposed the design of pH-responsive hybrid vesicles formulated with poly(dimethylsiloxane)-block-poly(ethylene oxide) backbone (PDMS36-b-PEO23) and cationic switchable lipid (CSL). The latter undergoes a pH-triggered conformational change and induces membrane destabilization. Using confocal imaging and DLS measurements, we interrogated the structural changes in CSL-doped lipid and hybrid polymer/lipid unilamellar vesicles at the micro- and nanometric scale, respectively. Both switchable giant unilamellar lipid vesicles (GUV) and hybrid polymer/lipid unilamellar vesicles (GHUV) presented dynamic morphological changes, including protrusions and fission upon acidification. At the submicron scale, scattered intensity decreased for both switchable large unilamellar vesicles (LUV) and hybrid vesicles (LHUV) under acidic pH. Finally, monitoring the fluorescence leakage of encapsulated calcein, we attested that CSL increased the permeability of GUV and GHUV in a pH-specific fashion. Altogether, these results show that switchable lipids provide a pH-sensitive behavior to hybrid polymer/lipid vesicles that could be exploited for the triggered release of drugs, cell biomimicry studies, or as bioinspired micro/nanoreactors.

Enhancing membrane modulus of giant unilamellar lipid vesicles by lateral co-assembly of amphiphilic triblock copolymers

We report a facile, but robust approach to fabricate structurally stable giant unilamellar vesicles (GUVs), on which a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer membrane was made rigid by introducing amphiphilic block polymers. Particularly, we found that lateral co-assembly of an amphiphilic triblock copolymer (ATC) structured with a hydrophobic middle block and long molecular weight (20 K g/mol) hydrophilic end blocks remarkably enhanced the stretching modulus (k) of GUVs. When the membrane composition was optimized, the k value of ATC-hybridized GUVs increased to 6.2 × 108 Pa, which was approximately 10-fold higher than that of DPPC GUVs, thus leading to a much longer half-life. Moreover, we demonstrated that our ATC-hybridized GUVs enabled development of a fascinating vesicular model, which shows great potential as a structurally stable cell membrane mimic.

“Basicles”: Microbial Growth and Production Monitoring in Giant Lipid Vesicles

We present an optimized protocol to encapsulate bacteria inside giant unilamellar lipid vesicles combined with a microfluidic platform for real-time monitoring of microbial growth and production. The microfluidic device allows us to immobilize the lipid vesicles and record bacterial growth and production using automated microscopy. Moreover, the lipid vesicles retain hydrophilic molecules and therefore can be used to accumulate products of microbial biosynthesis, which we demonstrate here for a riboflavin-producing bacterial strain. We show that stimulation as well as inhibition of bacterial production can be performed through the liposomal membrane simply by passive diffusion of inducing or antibiotic compounds, respectively. The possibility to introduce as well as accumulate compounds in liposomal cultivation compartments represents great advantage over the current state of the art systems, emulsion droplets, and gel beads. Additionally, the encapsulation of bacteria and monitoring of individual lipid vesicles have been accomplished on a single microfluidic device. The presented system paves the way toward highly parallel microbial cultivation and monitoring as required in biotechnology, basic research, or drug discovery.

Sucrose solutions alter the electric capacitance and dielectric permittivity of lipid bilayers

Understanding sugar-membrane interactions is of fundamental and technological relevance considering the role of sugars in drought-protection mechanisms of plants as well as the cryo- and bio-preserving effect of carbohydrates in many industrial and medical applications. In this work, we investigated the effect of sucrose on the electrical properties of membranes. In particular, we measured the specific capacitance of palmitoyl-oleoyl phosphatidylcholine membranes in aqueous solutions of sodium chloride. Different concentrations of sucrose were examined. The capacitance was assessed from the frequency-dependent deformation of giant unilamellar lipid vesicles in alternating electric field. Our measurements on giant vesicles in sugar-free aqueous solutions yield lower specific capacitance compared to values obtained for suspended and supported bilayers. This might be a result of the higher membrane tension in the latter systems, which is coupled to smaller thickness of the bilayer. We also report an increase of the bilayer capacitance upon increasing the sugar content in water. This finding is consistent with the sugar-induced thinning of membranes reported in the literature. However, the thinning is not sufficient to explain the observed capacitance increase with rising sugar concentration. We interpret the trend as resulting from an increase in the membrane dielectric permittivity.

Extended photo-induced endosome-like structures in giant vesicles promoted by block-copolymer nanocarriers.

Upon irradiation, the photosensitizer pheophorbide-a causes dramatic morphological transitions in giant unilamellar lipid vesicles. This endocytosis-like process occurs only when the photoactive species are encapsulated in a copolymer nanocarrier and strictly depends on the chemical nature of the copolymer. Altogether, these results open up new perspectives in the field of photo-chemical internalization mediated by nanoassemblies.