Scalable Synthesis of Planar Macroscopic Lipid-Based Multi-Compartment Structures.

As life evolved, the path from simple single cell organisms to multicellular enabled increasingly complex functionalities. The spatial separation of reactions at the micron scale achieved by cellular structures allowed diverse and scalable implementation in biomolecular systems. Mimicking such spatially separated domains in a scalable approach could open a route to creating synthetic cell-like structured systems. Here, we report a facile and scalable method to create multicellular-like, multi-compartment (MC) structures. Aqueous droplet-based compartments ranging from 50 to 400 µm were stabilized and connected together by hydrophobic layers composed of phospholipids and an emulsifier. Planar centimeter-scale MC structures were formed by droplet deposition on a water interface. Further, the resulting macroscopic shapes were shown to be achieved by spatially controlled deposition. To demonstrate configurability and potential versatility, MC assemblies of both homogeneous and mixed compartment types were shown. Notably, magnetically heterogeneous systems were achieved by the inclusion of magnetic nanoparticles in defined sections. Such structures demonstrated actuated motion with structurally imparted directionality. These novel and functionalized structures exemplify a route toward future applications including compartmentally assembled "multicellular" molecular robots.

DNA cytoskeleton for stabilizing artificial cells.

Cell-sized liposomes and droplets coated with lipid layers have been used as platforms for understanding live cells, constructing artificial cells, and implementing functional biomedical tools such as biosensing platforms and drug delivery systems. However, these systems are very fragile, which results from the absence of cytoskeletons in these systems. Here, we construct an artificial cytoskeleton using DNA nanostructures. The designed DNA oligomers form a Y-shaped nanostructure and connect to each other with their complementary sticky ends to form networks. To undercoat lipid membranes with this DNA network, we used cationic lipids that attract negatively charged DNA. By encapsulating the DNA into the droplets, we successfully created a DNA shell underneath the membrane. The DNA shells increased interfacial tension, elastic modulus, and shear modulus of the droplet surface, consequently stabilizing the lipid droplets. Such drastic changes in stability were detected only when the DNA shell was in the gel phase. Furthermore, we demonstrate that liposomes with the DNA gel shell are substantially tolerant against outer osmotic shock. These results clearly show the DNA gel shell is a stabilizer of the lipid membrane akin to the cytoskeleton in live cells.

Large-Scale Preparation of Giant Vesicles by Squeezing a Lipid-Coated Marshmallow-like Silicone Gel in a Buffer.

Giant vesicles were efficiently produced by squeezing a lipid (l-α-phosphatidylcholine from egg yolk)-coated marshmallow-like flexible macroporous silicone monolith in a buffer. The mean diameter of the obtained vesicles was 2 µm, showing a wide distribution, up to tens of micrometers, which was similar to that of vesicles formed by a natural swelling method. It was possible to prepare vesicle dispersions on a scale from several microliters to several hundred milliliters. A protein synthesis system (PURE system) contained in vesicles prepared using this method functioned effectively. Our absorbing-squeezing method is expected to help in studies that use giant vesicles such as artificial cells and drug delivery systems.

Unzipping and shearing DNA with electrophoresed nanoparticles in hydrogels.

We show electric control of unzipping and shearing dehybridization of a DNA duplex anchored to a hydrogel. Tensile force is applied by electrophoresing (25 V cm-1) gold nanoparticles pulling the DNA duplex. The pulled DNA strand is gradually released from the hydrogel. The unzipping release rate is faster than shearing; for example, 3-fold for a 15 base pair duplex, which helps to design electrically driven DNA devices.

Synthesis and in situ insertion of a site-specific fluorescently labeled membrane protein into cell-sized liposomes.

The integral membrane protein bacteriorhodopsin, containing a fluorescent amino acid at a specific position, was synthesized in the presence of hydrated lipid films using an in vitro translation system expanded with a four-base codon/anticodon pair. Cell-sized liposomes with the labeled protein inserted into the liposome membranes were generated after the translation reaction. This study also demonstrated that this labeling method could be used to analyze the dynamic properties of membrane proteins in situ by fluorescence correlation spectroscopy.

A large, square-shaped, DNA origami nanopore with sealing function on a giant vesicle membrane.

Intaking molecular information from the external environment is essential for the normal functioning of artificial cells/molecular robots. Herein, we report the design and function of a membrane nanopore using a DNA origami square tube with a cross-section of 100 nm2. When the nanopore is added to a giant vesicle that mimics a cell membrane, the permeation of large external hydrophilic fluorescent molecules is observed. Furthermore, the addition of up to four ssDNA strands enables size-based selective transport of molecules. A controllable artificial nanopore should facilitate the communication between the vesicle components and their environment.

Degassing-assisted patterning of cell culture surfaces.

We developed an alternative patterning technique which is capable of producing both topographic and biochemical features for cell culture studies. This technique is based on microaspiration induced with a degassed poly (dimethylsiloxane) (PDMS) mold. After degassing in a rough vacuum chamber and placed on a sample surface, liquid solution can be aspired through channels and cavities created in the PDMS mold. Depending on the composition of the solution and the associated drying or incubation processes, a variety of surface patterns can be produced without applying external pressure. For demonstration, we fabricated agarose gel microwells and biomolecule patterns either on a glass plate or in a cell culture Petri dish, both applicable for cell culture studies.

Isothermal amplification of specific DNA molecules inside giant unilamellar vesicles.

An isothermal amplification circuit for specific DNA molecules was implemented in giant unilamellar vesicles. Using this circuit, over 5000-fold amplification of output DNAs was achieved, and the amplification behaviour depended on the concentration of input signal DNAs in a cell-sized compartment. Moreover, initiation of the amplification by photo-stimulation was demonstrated.

Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis.

We investigated the direct constitution of membrane proteins into giant liposomes in cell-free (in vitro) protein synthesis. Giant liposomes were present in a translation reaction cocktail of a wheat germ cell-free protein translation system. Apo cytochrome b(5) (b5) and its fusion proteins were synthesized and directly localized in the liposomes. After the translation reaction, the proteo-liposomes were isolated by simplified discontinuous density-gradient centrifugation. Apo cytochrome b(5) conjugated dihydrofolate reductase (DHFR) was synthesized in the same procedure and the protein was directly displayed on the liposome surface. b5 acts as a "hydrophobic tag" for recruitment to the liposome surface.

Changes in the morphology of cell-size liposomes in the presence of cholesterol: formation of neuron-like tubes and liposome networks.

Spontaneous changes in the morphology of cell-size liposomes (dioleoylphosphatidylcholine, DOPC and egg PC) as model cells were investigated in the presence of cholesterol. Tube structures and liposome networks connected by the tubes were observed in the presence of 5-30% cholesterol by dark-field and laser-scanning microscopy. Furthermore, in the presence of more than 40 mol% of cholesterol, the tubes disappeared and changed to small liposomes. Thus, cholesterol induced a morphological change in giant liposomes from tubes to small liposomes. These phenomena may be related to the role of cholesterol in the morphological changes in living cells such as neurons.

Trading polymeric microspheres: exchanging DNA molecules via microsphere interaction.

A new class of artificial molecular transport system is constructed by polymeric microspheres. The microspheres are prepared by self-assembly of poly(ethylene glycol)-block-poly(3-dimethyl(methacryloyloxyethyl)ammonium propane sulfonate), PEG-b-PDMAPS, by intermolecular dipole-dipole interaction of sulfobetaine side chains in water. Below the upper critical solution temperature (UCST) of PEG-b-PDMAPS, the microspheres (∼1µm) interact with other microspheres by partial and transit fusion. In order to apply the interaction between microspheres, a 3'-TAMRA-labeled single-stranded DNA oligomer (ssDNA) is encapsulated into a PEG-b-PDMAPS microsphere by thermal treatment. The exchange of ssDNA between microspheres is confirmed by fluorescence resonance energy transfer (FRET) quenching derived from double-stranded formation with complementary 5'-BHQ-2-labeled ssDNA encapsulated in PEG-b-PDMAPS microspheres. The exchange rate of ssDNA is controllable by tuning the composition of the polymer. The contact-dependent transport of molecules can be applied in the areas of microreactors, sensor devices, etc.

Induction of neuron-like tubes and liposome networks by cooperative effect of gangliosides and phospholipids.

Although there is a rather large abundance of gangliosides in neurons, their functional role is still unclear. We focused on a physicochemical role of gangliosides in the formation of tubular structures, such as axons or dendrites in neurons. When a ganglioside, GM3, was added to cell-size liposomes that consisted of dioleoylphosphatidyl-choline, tubular structures were induced and liposome networks connected by the tubes were observed by differential interference microscopy and fluorescence microscopy. The potential for various gangliosides to induce tubes was dependent on the structures of their hydrophilic head group. With a large excess of gangliosides, the tubes are destabilized and small fragments, or micelles, are generated. The phenomenon was suggested by physical model calculation. Gangliosides may play a role as building material in neural unique tubular structures.

Introducing micrometer-sized artificial objects into live cells: a method for cell-giant unilamellar vesicle electrofusion.

Here, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o) emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC) field to induce a linear cell-GUV alignment, and then a direct current (DC) pulse was applied to facilitate transient electrofusion. With uniformly sized fluorescent beads as size indexes, we successfully and efficiently introduced beads of 1 µm in diameter into living cells along with a plasmid mammalian expression vector. Our electrofusion did not affect cell viability. After the electrofusion, cells proliferated normally until confluence was reached, and the introduced fluorescent beads were inherited during cell division. Analysis by both confocal microscopy and flow cytometry supported these findings. As an alternative approach, we also introduced a designed nanostructure (DNA origami) into live cells. The results we report here represent a milestone for designing artificial symbiosis of functionally active objects (such as micro-machines) in living cells. Moreover, our technique can be used for drug delivery, tissue engineering, and cell manipulation.

Temperature-responsive telechelic dipalmitoylglyceryl poly(N-isopropylacrylamide) vesicles: real-time morphology observation in aqueous suspension and in the presence of giant liposomes.

Telechelic α,ω-di(twin-tailed poly(N-isopropylacrylamides)) form polymersomes in water that increase in size by fusion when the water temperature exceeds the polymers cloud point temperature. Hybrid vesicles form in mixed suspensions of giant phospholipid liposomes and polymersomes by adsorption/fusion, and undergo further transformations, such as fission.

Direct integration of cell-free-synthesized connexin-43 into liposomes and hemichannel formation.

Proteoliposomes were directly prepared by synthesizing membrane proteins with the use of minimal protein synthesis factors isolated from Escherichia coli (the PURE system) in the presence of liposomes. Connexin-43 (Cx43), which is a water-insoluble integral membrane protein that forms a hexameric complex in membranes, was cotranslationally integrated with an essentially uniform orientation in liposomes. The addition of liposomes following protein expression (post-translational presence of liposomes) did not lead to the integration of Cx43 into the liposome membranes. The amount of integrated Cx43 increased as the liposome concentration increased. The presence of liposomes did not influence the total amount of synthesized Cx43. The Cx43 integrated into the liposome membranes formed open membrane pores. These results indicate that the liposomes act in a chaperone-like manner by preventing Cx43 from aggregating in solution, because of integration into the bilayer, and also by functionalization of the integrated Cx43 in the membrane. This is the first report that cell-free-synthesized water-insoluble membrane protein is directly integrated with a uniform orientation as a functional oligomer into liposome membranes. This simple proteoliposome preparation procedure should be a valuable approach for structural and functional studies of membrane proteins.

Direct formation of proteo-liposomes by in vitro synthesis and cellular cytosolic delivery with connexin-expressing liposomes.

Liposomes are widely utilized in molecular biology and medicine as drug carriers. Here we report a new liposome-cell interaction through connexins. Connexin 43 (Cx43)-containing liposomes were prepared by using cell-free transcription/translation systems with plasmids encoding Cx43 in the presence of liposome. The expressed membrane protein, Cx43, was directly constituted to the liposome membrane upon in vitro synthesis, leading to pure membrane protein-containing liposomes. The hydrophilic dye calcein was efficiently transferred from Cx43-expressing liposomes to cultured cells (Cx43 expressing). The transfer is significantly blocked in the presence of gap junction inhibitor (18beta-glycyrrhetinic acid) and in the case of the other type of connexin (Cx32)-expressing cell. The results show that calcein entered the cell through connexin-mediated pathway. Cx43 liposomes containing a soluble NEMO-binding domain peptide suppressed the intracellular signaling cascade IL-1beta-induced NF-kappaB activation and cyclooxygenase-2 expression in Cx43-expressing cells, confirming effective peptide transfer into the cell. This is a new method for direct cytosolic delivery of hydrophilic molecules.

Protein synthesis in giant liposomes using the in vitro translation system of Thermococcus kodakaraensis.

An in vitro translation system, based on cell components of the hyperthermophilic archaeon, Thermococcus kodakaraensis, has previously been developed. The system has been optimized and applied for protein production at high temperatures (60-65 degrees C). In this paper, we have examined the possibilities to utilize this system at a lower temperature range using green fluorescence protein (GFP) as the reporter protein. By optimizing the composition of the reaction mixture, and adding chaperonins from the mesophilic Escherichia coli, the yield of protein production at 40 degrees C was increased by fivefold. For liposome encapsulation of the optimized system, water-in-oil cell-sized emulsions were prepared by adding the translation system/GFP mRNA mixture to mineral oil supplemented with 1,2-dioleoyl-sn -glycero-3-phosphatidylcholine (DOPC). Giant liposomes were formed when these emulsions passed across a water/oil interface occupied with DOPC. The liposomes were incubated at 40 degrees C for 90 min, and fluorescence was examined by laser confocal microscopy. A significant increase in average fluorescence intensity was observed in liposomes with GFP mRNA, but not in those without mRNA. Our results indicate that the T. kodakaraensis in vitro translation system is applicable for protein production within giant liposomes, and these artificial cell models should provide the methodology to reconstitute various cell functions from a constitutional biology approach.

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.