Flow-Driven Self-Propulsion of Oil Droplet on a Surfactant Solution Surface, as Observed by Time-Resolved Interfacial Tension and Surface Flow Speed Measurements.

The imbalanced force of the interfacial tension applied to an object has often been taken into account in the analysis of the motion mechanism of self-propelled systems. However, heterogeneous distributions of the interfacial tension also cause Marangoni flows, and these flows also contribute to the self-propulsion through the viscous force. The contribution of such flows has not been observed directly, while the interfacial tension difference has been measured in some systems. In this study, simultaneous measurements of the interfacial tension and surface flow speed of the unidirectional self-propelled motion of a butyl salicylate (BS) droplet in a circular channel on a sodium dodecyl sulfate (SDS) aqueous solution were achieved by the quasi-elastic laser scattering method. The droplet position was also recorded by observing its fluorescence excited by a UV light. The BS droplet speed dependence of the interfacial tension and surface flow speed were measured by varying the initial BS concentration codissolved in the SDS aqueous solution. As a result, a periodic decrease of the interfacial tension and a periodic increase of the speed of both forward and backward flows were observed when the droplet passed the sampling position of the time-resolved measurements. When they were converted to the distribution in space of the droplet position, no droplet speed dependence of the interfacial tension difference between the front and rear of the droplet was observed. On the other hand, the speed of both forward and backward flows increased as the droplet speed increased. By analysis of the above results with a simplified model, it was clarified that the forward flow driven by the interfacial tension gradient at the droplet front is actually important in the mechanism of the unidirectional self-propelled motion of a droplet.

Release of liposomally formulated near-infrared fluorescent probes included in giant cluster vesicles by ultrasound irradiation.

Detection of tumors and regional lymph nodes during surgery has been proposed in the diagnosis of lymphatic metastasis and the surgical treatment of malignant diseases. Giant cluster vesicles (GCVs), including liposomally formulated indocyanine green (LP-ICG) derivatives, are a possible candidate for agents to realize the two contradictory properties, i.e., retention in tissue for lesion-marking and trace for sentinel lymph nodes (SLNs) identification. We attempted to release the LP-ICG derivatives from GCVs using ultrasound contrast agents (UCAs) under ultrasound irradiation. An absorption spectrophotometer quantitatively evaluated the amounts of released LP-ICG derivatives. As a result, we demonstrated that it depended on conditions for sound pressure, burst length, and number density of UCAs, and had a sound pressure threshold independent of burst length and number density of UCAs. The results will aid to determine appropriate conditions to maximize the released amount of LP-ICG derivatives while keeping safety.

A Practical Guide to Preparation and Applications of Giant Unilamellar Vesicles Formed via Centrifugation of Water-in-Oil Emulsion Droplets.

Giant vesicles (GVs), which are closed lipid bilayer membranes with a diameter of more than 1 µm, have attracted attention not only as model cell membranes but also for the construction of artificial cells. For encapsulating water-soluble materials and/or water-dispersible particles or functionalizing membrane proteins and/or other synthesized amphiphiles, giant unilamellar vesicles (GUVs) have been applied in various fields, such as supramolecular chemistry, soft matter physics, life sciences, and bioengineering. In this review, we focus on a preparation technique for GUVs that encapsulate water-soluble materials and/or water-dispersible particles. It is based on the centrifugation of a water-in-oil emulsion layered on water and does not require special equipment other than a centrifuge, which makes it the first choice for laboratory use. Furthermore, we review recent studies on GUV-based artificial cells prepared using this technique and discuss their future applications.

Time-Resolved Measurements of Interfacial Tension and Flow Speed of the Inclined Water Surface around a Self-propelled Camphor Boat by the Quasi-elastic Laser Scattering Method.

An inclined liquid surface, such as a meniscus, plays an important role in advection and transport phenomena at a liquid's surface. However, there is no time-resolved measurement method for the interfacial tension of an inclined liquid-air interface. Here, a noninvasive method for simultaneous measurements of the interfacial tension and surface flow speed for an inclined water surface is described. This is an upgrade of the quasi-elastic laser scattering method with a closed-loop control system that introduces the dynamically tracked scattered and referential light into the detector. For the evaluation of the tilt compensation by dynamic tracking, the relationship between the apparent interfacial tension and surface inclination was examined for a water meniscus at 0-5° inclinations. It was also demonstrated that simultaneous measurements of the interfacial tension and surface flow speed around a self-propelled camphor boat on a pure water surface inclined by >3° at the back end of the boat are difficult to conduct accurately without dynamic tracking. Both the interfacial tension difference and the backward flow speed increased as the boat speed increased to 0.1 m/s; that had not been evaluated to date because of the high velocity of the boat and the surface inclination of the water around it. The direct experimental evaluation of the interfacial tension and the flow speed supported the model that the driving force of the camphor boat is the interfacial tension difference and the resistance force proportional to the boat velocity reduces its acceleration.

Stannous colloid mixed with indocyanine green as a tracer for sentinel lymph node navigation surgery.

The combined use of a vital dye and radioactive colloid reportedly performs better in detecting sentinel lymph nodes (SLNs) for cancers than the use of either of them alone. However, especially for gastric cancer, two endoscopic procedures are required to administer these two tracers, which burdens the patients and practitioners. Here we propose the use of stannous colloid (SnC) mixed with indocyanine green (ICG) as a new mixed tracer (SnC-ICG); its characteristics were investigated in vivo and in vitro to estimate its usefulness for SLN navigation. The tracers were administered to rats and the accumulation of radioactivity and/or near-infrared fluorescence were evaluated in the regional lymph nodes (LNs) using single positron emission computed tomography and near-infrared fluorescence imaging, respectively. SnC-ICG showed significantly better clearance from the injection site and better migration to primary LNs than the single administration of SnC or ICG aqueous solution. SnC-ICG demonstrated a wide particle size variability, stabilized to 1200-nm upon the addition of albumin in vitro; These properties could contribute to its behavior in vivo. The use of SnC-ICG could contribute better performance to detect SLNs for gastric cancer with less burden on both patients and medical practitioners.

Evolution of Proliferative Model Protocells Highly Responsive to the Environment.

In this review, we discuss various methods of reproducing life dynamics using a constructive approach. An increase in the structural complexity of a model protocell is accompanied by an increase in the stage of reproduction of a compartment (giant vesicle; GV) from simple reproduction to linked reproduction with the replication of information molecules (DNA), and eventually to recursive proliferation of a model protocell. An encounter between a plural protic catalyst (C) and DNA within a GV membrane containing a plural cationic lipid (V) spontaneously forms a supramolecular catalyst (C@DNA) that catalyzes the production of cationic membrane lipid V. The local formation of V causes budding deformation of the GV and equivolume divisions. The length of the DNA strand influences the frequency of proliferation, associated with the emergence of a primitive information flow that induces phenotypic plasticity in response to environmental conditions. A predominant protocell appears from the competitive proliferation of protocells containing DNA with different strand lengths, leading to an evolvable model protocell. Recently, peptides of amino acid thioesters have been used to construct peptide droplets through liquid-liquid phase separation. These droplets grew, owing to the supply of nutrients, and were divided repeatedly under a physical stimulus. This proposed chemical system demonstrates a new perspective of the origins of membraneless protocells, i.e., the "droplet world" hypothesis. Proliferative model protocells can be regarded as autonomous supramolecular machines. This concept of this review may open new horizons of "evolution" for intelligent supramolecular machines and robotics.

Identifying and Manipulating Giant Vesicles: Review of Recent Approaches.

Giant vesicles (GVs) are closed bilayer membranes that primarily comprise amphiphiles with diameters of more than 1 µm. Compared with regular vesicles (several tens of nanometers in size), GVs are of greater scientific interest as model cell membranes and protocells because of their structure and size, which are similar to those of biological systems. Biopolymers and nano-/microparticles can be encapsulated in GVs at high concentrations, and their application as artificial cell bodies has piqued interest. It is essential to develop methods for investigating and manipulating the properties of GVs toward engineering applications. In this review, we discuss current improvements in microscopy, micromanipulation, and microfabrication technologies for progress in GV identification and engineering tools. Combined with the advancement of GV preparation technologies, these technological advancements can aid the development of artificial cell systems such as alternative tissues and GV-based chemical signal processing systems.

Construction of Supramolecular Systems That Achieve Lifelike Functions.

The Nobel Prize in Chemistry was awarded in 1987 and 2016 for research in supramolecular chemistry on the "development and use of molecules with structure-specific interactions of high selectivity" and the "design and production of molecular machines", respectively. This confirmed the explosive development of supramolecular chemistry. In addition, attempts have been made in systems chemistry to embody the complex functions of living organisms as artificial non-equilibrium chemical systems, which have not received much attention in supramolecular chemistry. In this review, we explain recent developments in supramolecular chemistry through four categories: stimuli-responsiveness, time evolution, dissipative self-assembly, and hierarchical expression of functions. We discuss the development of non-equilibrium supramolecular systems, including the use of molecules with precisely designed properties, to achieve functions found in life as a hierarchical chemical system.

Role of Negatively Charged Lipids Achieving Rapid Accumulation of Water-Soluble Molecules and Macromolecules into Cell-Sized Liposomes against a Concentration Gradient.

Liposomes, molecular self-assemblies resembling biological membranes, are a promising scaffold to investigate the physicochemical logic behind the complexity of living cells. Despite elaborate synthetic studies constructing cell-like chemical systems using liposomes, less attention has been paid to the proactive role of the membrane emerging as dynamics of the molecular self-assembly. This study investigated the liposomes containing anionic phospholipids by exposing them to steady flow conditions using a newly constructed automatic microfluidic observation platform. We demonstrated that the liposomes accumulated even macromolecules under the microfluidic condition without pore formation. By investigating the effect of composition of liposomes and visualizing negatively charged phospholipids upon the flow, we presumed that the external flow caused a compositional asymmetry of anionic phospholipids between the inner/outer leaflets, and the asymmetry enabled a rapid accumulation of those molecules against the concentration gradient. The current study opens new research interests regarding the nature of biological membranes under steady flow conditions.

Molecular Transformation for Self-reproducing Vesicles and Underlying Analysis Methods.

Closed bilayer membranes of amphiphiles in water, termed vesicles, represent one of the promising models of primitive cellular compartments. Herein, we reviewed studies on the design and construction of vesicle-based cell models capable of sequential growth and division and their underlying analysis methods. We discussed the potential contribution of these studies to the universal understanding of the chemical/physical logics behind the steady reproduction of cellular membranes.

Perfusion Chamber for Observing a Liposome-Based Cell Model Prepared by a Water-in-Oil Emulsion Transfer Method.

For the construction of a chemical model of contemporary living cells, the so-called water-in-oil emulsion transfer (WOET) method has drawn much attention as one of the promising preparation protocols for cell-sized liposomes encapsulating macromolecules and even micrometer-sized colloidal particles in high yields. Combining the throughput and accuracy of the observation is the key to developing a synthetic approach based on the liposomes prepared by the WOET method. Recent advances in microfluidic technology can provide a solution. By means of surface modification of a poly(dimethylsiloxane)-type microfluidic device integrating size-sorting and trapping modules, here, we enabled a simultaneous direct observation of the liposomes with a narrow size distribution, which were prepared by the WOET method. As a demonstration, we evaluated the variance of encapsulation of polystyrene colloidal particles and water permeability of the cell-sized liposomes prepared by the WOET method in the device. Since the liposomes prepared by the WOET method are useful for constructing cell models with an easy protocol, the current system will lead to a critical development of not only supramolecular chemistry and soft matter physics but also synthetic biology.

Environment-Sensitive Intelligent Self-Reproducing Artificial Cell with a Modification-Active Lipo-Deoxyribozyme.

As a supramolecular micromachine with information flow, a giant vesicle (GV)-based artificial cell that exhibits a linked proliferation between GV reproduction and internal DNA amplification has been explored in this study. The linked proliferation is controlled by a complex consisting of GV membrane-intruded DNA with acidic amphiphilic catalysts, working overall as a lipo-deoxyribozyme. Here, we investigated how a GV-based artificial cell containing this lipo-deoxyribozyme responds to diverse external and internal environments, changing its proliferative dynamics. We observed morphological changes (phenotypic expression) in GVs induced by the addition of membrane precursors with different intervals of addition (starvation periods). First, we focused on a new phenotype, the "multiple tubulated" form, which emerged after a long starvation period. Compared to other forms, the multiple tubulated form is characterized by a larger membrane surface with a heavily cationic charge. A second consideration is the effect of the chain length of encapsulated DNA on competitive proliferation. The competitive proliferation among three different species of artificial cells containing different lengths of DNA was investigated. The results clearly showed a distinct intervention in the proliferation dynamics of the artificial cells with each other. In this sense, our GV-based artificial cell can be regarded as an intelligent supramolecular machine responding to external and internal environments, providing a new concept for developing molecular machines and robotics.

Hydrodynamic accumulation of small molecules and ions into cell-sized liposomes against a concentration gradient.

In investigations of the emergence of protocells at the origin of life, repeatable and continuous supply of molecules and ions into the closed lipid bilayer membrane (liposome) is one of the fundamental challenges. Demonstrating an abiotic process to accumulate substances into preformed liposomes against the concentration gradient can provide a clue. Here we show that, without proteins, cell-sized liposomes under hydrodynamic environment repeatedly permeate small molecules and ions, including an analogue of adenosine triphosphate, even against the concentration gradient. The mechanism underlying this accumulation of the molecules and ions is shown to involve their unique partitioning at the liposomal membrane under forced external flow in a constrained space. This abiotic mechanism to accumulate substances inside of the liposomal compartment without light could provide an energetically up-hill process for protocells as a critical step toward the contemporary cells.

Temperature-Dependent Dynamics of Giant Vesicles Composed of Hydrolysable Lipids Having an Amide Linkage.

Various amphiphiles including surfactants and lipids have been designed and synthesized to improve and create new functionalities. In particular, the emergence of cell-like behaviors of giant vesicles (GVs) composed of synthetic lipids has drawn much attention in the development of chemical models for cells. The aim of this study was to measure temperature-dependent morphological changes of GVs induced by fragmentation and subsequent growth using hydrolysable cationic lipids having an amide linkage. Results from differential scanning calorimetry, fluorescence spectroscopy using an environment-responsive probe, and confocal Raman microscopy showed that the dynamics observed were due to changes in the vesicle membrane, including variation in the lipid composition, induced by thermal stimulation.

DNA Length-dependent Division of a Giant Vesicle-based Model Protocell.

DNA is an essential carrier of sequence-based genetic information for all life today. However, the chemical and physical properties of DNA may also affect the structure and dynamics of a vesicle-based model protocell in which it is encapsulated. To test these effects, we constructed a polyethylene glycol-grafted giant vesicle system capable of undergoing growth and division. The system incorporates a specific interaction between DNA and lipophilic catalysts as well as components of PCR. We found that vesicle division depends on the length of the encapsulated DNA, and the self-assembly of an internal supramolecular catalyst possibly leads to the direct causal relationship between DNA length and the capacity of the vesicle to self-reproduce. These results may help elucidate how nucleic acids could have functioned in the division of prebiotic protocells.

A sustainable self-reproducing liposome consisting of a synthetic phospholipid.

A novel phosphoric membrane lipid (phospholipid) bearing an oleyl group as one of the hydrophobic chains formed a liposome with a thin lamella in water. Since the anionic membrane of pre-existing liposomes acted as a catalytic surface in phosphate buffer, membrane lipids could be generated from their precursor in an autocatalytic manner without the inclusion of catalytic amphiphiles in the liposome. The morphological changes of this anionic liposome were monitored both by flow cytometry and optical microscopy, and it was found that the liposomes deformed into a budding shape, followed by division, after the addition of a membrane precursor. Hence, this anionic monocomponent liposome could be regarded as a sustainable self-reproducing system. This liposome was also able to provide a reaction cavity for enzymatic reactions, such as DNA amplification by a polymerase chain reaction.

Budding and Division of Giant Vesicles Linked to Phospholipid Production.

The self-reproduction of supramolecular assemblies based on the synthesis and self-assembly of building blocks is a critical step towards the construction of chemical systems with autonomous, adaptive, and propagation properties. In this report, we demonstrate that giant vesicles can grow and produce daughter vesicles by synthesizing and incorporating phospholipids in situ from ad-hoc precursors. Our model involves acyl chain elongation via copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition reaction and the ensuing production of synthetic phospholipids to induce budding and division. In addition, the growth and budding of giant vesicles were compatible with the encapsulation and transfer of macromolecules as large as lambda phage DNA to the buds. This chemical system provides a useful model towards the implementation of cell-like compartments capable of propagation and transport of biological materials.

Topology-Reset Execution: Repeatable Postcyclization Recyclization of Cyclic Polymers.

Repeatable topological transformation of polymers for the modulation of material functions is a challenge. We have developed a method for repeatedly resetting a cyclic macromolecular architecture to a linear architecture by photostimulation, namely, topology-reset execution (T-rex) based on the photochemistry of hexaarylbiimidazoles (HABIs). We synthesized cyclic poly(dimethylsiloxane)s (PDMSs) of various ring sizes with HABIs linked in the chains. UV irradiation of the cyclic PDMSs produced telechelic linear PDMSs with triphenylimidazolyl radical (TPIR) end groups. After termination of UV irradiation, end-to-end recyclization occurred by the recoupling of TPIRs. The cyclic PDMSs also responded to ultrasound, which decreased their molecular weight (MW) by site-specific cleavage of in-chain HABI moieties, and we are able to reset the MWs by subsequent phototriggered T-rex. Furthermore, T-rex enabled solvent-free switching of the rheological properties of the materials while retaining the liquid character of PDMS.

Toward Experimental Evolution with Giant Vesicles.

Experimental evolution in chemical models of cells could reveal the fundamental mechanisms of cells today. Various chemical cell models, water-in-oil emulsions, oil-on-water droplets, and vesicles have been constructed in order to conduct research on experimental evolution. In this review, firstly, recent studies with these candidate models are introduced and discussed with regards to the two hierarchical directions of experimental evolution (chemical evolution and evolution of a molecular self-assembly). Secondly, we suggest giant vesicles (GVs), which have diameters larger than 1 µm, as promising chemical cell models for studying experimental evolution. Thirdly, since technical difficulties still exist in conventional GV experiments, recent developments of microfluidic devices to deal with GVs are reviewed with regards to the realization of open-ended evolution in GVs. Finally, as a future perspective, we link the concept of messy chemistry to the promising, unexplored direction of experimental evolution in GVs.

Irreversible aggregation of alternating tetra-block-like amphiphile in water.

As a frontier topic of soft condensed matter physics, irreversible aggregation has drawn attention for a better understanding of the complex behavior of biomaterials. In this study, we have described the synthesis of an artificial amphiphilic molecule, an alternating tetra-block-like amphiphile, which was able to diversify its aggregate structure in water. The aggregated state of its aqueous dispersion was obtained by slow evaporation of the organic solvent at room temperature, and it collapsed irreversibly at ~ 50°C. By using a cryo-transmission electron microscope and a differential scanning calorimeter, it was revealed that two types of molecular nanostructures were formed and developed into submicro- and micrometer-sized fibrils in the aggregated material.