Oligonucleotide therapeutics have attracted great attention in recent years as a new modality of molecular–targeted drugs. To stabilize fragile oligonucleotides in the bloodstream and deliver them to target tissue, numerous oligonucleotide nanomedicines have been proposed. Although some of them are clinically approved, the current oligonucleotide nanomedicines can target only the liver. Here, we constructed a series of nano–assemblies (polyion complexes (PICs)) from anionic oligonucleotides and cationic polymers. Fine–tuning of the polymer/oligonucleotide structures provides well–organized PICs having controlled structures, physicochemical properties, and biological properties. These PIC systems successfully delivered the oligonucleotides to various tissues, including tumors. Our approach could provide a promising platform for oligonucleotides delivery.

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The share of biopharmaceuticals is expected to further increase in the future. Biopharmaceuticals, which consist of peptides and proteins, are anticipated to exhibit greater specificity to target molecules within the body compared to small molecule drugs. However, most biopharmaceuticals are administered by injection because they are broken down by enzymes when taken orally. We have been exploring the development of transdermal formulations (e.g., patches or ointments) as an alternative to injectable biopharmaceuticals. In this review, we introduce the transdermal delivery of insulin, a representative peptide drug, and present a transdermal vaccine for malaria as an alternative to injection. Finally, we discuss cutting–edge research on transdermal formulations for the latest nucleic acid drugs.

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Membrane curvature plays an important role in the functions of biological membranes. Here, I focus on synthetic molecular probes based on membrane curvature sensing peptides for the development of useful molecular tools toward extracellular vesicles. I introduce several examples of such probes, for fluorescent sensing , analysis and isolation of extracellular vesicles.

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Biological membranes, composed of lipids and proteins, exhibit dynamic curvature that influences cellular processes such as division, transport, and signaling. Curvature–sensing proteins, like BAR domain proteins, are known in eukaryotes for recognizing membrane shapes and inducing structural changes. Recent studies show that prokaryotes also possess such proteins, like SpoVM in Bacillus subtilis and MamY in Magnetospirillum sp., stabilizing curved membranes. However, understanding the mechanisms of membrane structure regulation in both prokaryotes and eukaryotes remains unclear, and many more curvature–sensing proteins likely exist within cells. To address this, we developed a novel method using spherical supported lipid bilayers (SSLBs) for proteomic analysis to comprehensively explore these proteins. This method successfully identifies curvature-sensing proteins from various samples, including cancer, normal human cells, and Escherichia coli. This review emphasizes our novel techniques and discusses research outcomes, providing insights into the identification of these proteins and their roles in membrane dynamics.

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All Gram–negative and Gram–positive bacteria isolated to date secrete membrane–embedded nanoparticles called extracellular membrane vesicles (MVs) or outer membrane vesicles (OMVs). These vesicles play important roles in bacterial survival, such as infection to host cells and biofilm formation. MVs are used as a tool for inter– or intra–communication in these bacterial processes. Membrane vesicle production by bacteria is important for understanding bacterial survival strategies, and the development of vaccines, drug delivery, and heterologous protein production using them has attracted much attention. Shewanella vesiculosa HM13, a Gram–negative bacterium, was isolated from fish intestines as a promising host strain for the heterologous protein secretion system. This strain secretes a large number of EMVs that uniquely carry a single major cargo, namely P49. This review summarizes a unique feature of this non–model bacterium and the unique protein cargo loading mechanism.

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Extracellular vesicles (EVs) carry various informative components, including signaling proteins, transcriptional regulators, lipids, and nucleic acids. EVs have shown great promise as pharmaceutical–targeting vesicles and have attracted the attention of researchers in the fields of biological and medical science because of their importance as diagnostic and prognostic markers. However, the detection and isolation of EVs in cell–cultured media remain challenging. In the present study, we used curvature–sensing peptides to develop a simple and rapid method for in–situ vesicle detection in cultured media without EV–purification steps and applied this method for rapid screening and identification of genes involved in bacterial extracellular vesicle production. Also we developed a novel methodology, i.e., an EV catch–and–release isolation system (EV–CaRiS) using a net–charge invertible curvature–sensing peptide (NIC). NIC was newly designed to reversibly capture and release EVs in a pH–dependent manner. NIC allowed us to achieve reproducible EV isolation from three human cell lines and single–particle imaging of EVs containing the ubiquitous exosome markers CD63 and CD81. EV–CaRiS was demonstrated as a simple and convenient methodology for EV isolation.

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As momentum for the utilization of cultured cells increases, cell manipulation technology using light is being investigated as a promising means to meet the diversifying needs for bio–manipulation. In particular, the control of crosslinking structure by light has been actively investigated as a new technology to create an unprecedented cell culture environment. This article focuses on polymer systems in which cross–linking is controlled on–demand by light, and introduces trends in related research at home and abroad, with particular emphasis on the application to optical control of biosystems, including the work of the authors.

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In vitro 3D and organoid culture methods that emulate the intricate complexity of cell populations and extracellular matrix (ECM) components In vitro 3D significantly contribute to advancing our understanding of various biological phenomena. However, achieving precise control over the complex shapes, architectures, and interactions among different tissues within cultured organoids remains a challenge. Current organoid development heavily relies on cellular self–organization, yet the uniform culture conditions In vitro 3D fall short of providing accurate spatial cues to cells. Conversely, leveraging engineering principles offers a promising avenue to customize the design, composition, and construction of organoids based on specific research objectives. We have developed an in vitro experimental platform for the organoid culture to design and control microenvironment. The simple cube device, which comprises a polycarbonate frame with rigid agarose walls and an inner ECM hydrogel, can be used as a carrier of organoid to (i) control the spatial distribution of cells by employing 3D–printed carbohydrate moulds to create cell seeding pockets in the ECM hydrogel, (ii) design tissues with localized ECM by isolating ECM hydrogels of varying the composition or stiffness in separate compartments, (iii) facilitate integration with microfluidics to generate the concentration gradient of morphogens to direct cell growth and differentiation, (iv) assemble multi–CUBE with organoids or tissues to express tissue-tissue interactions. By employing above technologies, we were able to replicate the notochord signal during the development of the neural tube. The Shh gradient, facilitated through a 100 μm slit on a CUBE device, was applied to the neural plate, resulting in the generation of a localized expression pattern on the neural tube organoid.

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We have developed a substrate surface that enables the photo–patterning of cells with single–cell precision and have been conducting research to acquire image data on molecular and cellular behaviors that were previously unattainable due to technical challenges. In these studies, we employed polyethylene glycol–lipids (PEG–lipids), a polymer material that interacts with cell membranes. We succeeded in converting PEG–lipids into photoresponsive materials and developed three types of photoresponsive cell–adhesive surfaces: photocleavable, photoactivatable, and photoswitchable. In this paper, we introduce the development of these surfaces and their applications in membrane protein localization analysis on non–adherent single cells and microscopic analysis of intracellular faces of intact cell membranes.

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Electrostatic interactions play an important role in various biological events. This article focuses on polyion complex (PIC) materials, which are formed by utilizing electrostatic interactions, and overviews the development of their functions as biomaterials and the new strategies for biomacromolecular assembly formation based on the rational control of electrostatic interactions. Particularly, PIC vesicles (PICsomes), which are membrane–based architectures, and coacervates, which are artificial biomolecular condensates, are highlighted. The utilization of their dynamic properties is discussed, as well as a novel method for preparing hierarchical structures utilizing self-assembled membranes for compartmentalization.

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Biointerfaces enable an active interaction between materials and biomolecules (including cell, biological tissue, or living organism). Herein, a systematic characterization method has been developed for self–assembled membranes (liposomes), which quantitatively provides surface properties such as fluidity, polarity, cation density, etc. Furthermore, the characterization method has been applied to investigate the properties of inorganic nanoparticles and polymer particles. The surface properties will become one of important factors to improve the activity of biointerfaces.

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Nanoparticle has attracted much attention for diagnostic applications. In these applications, nanoparticles need to reach their targets through various membrane barriers such as vessel walls, extracellular matrix, and cell membranes. Therefore, understanding the interaction between nanoparticles and membranes is important for their efficient delivery. Recent studies have revealed that physicochemical property of nanoparticles, including size, shape, and surface chemistry, significantly affect their interaction with biological environment. Based on our precise control of the physicochemical properties of nanoparticles, we aimed to elucidate the mechanisms of physicochemical property– dependent interaction of nanoparticles with membrane barriers. Our recent attempt to use nanoparticles for membrane analysis is also presented.

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Ionic liquids have been used in membrane separation for more than a decade due to their physical and chemical properties, such as non–volatility, thermal stability and unique solubility. In this paper, we present a new concept in chemically stabilized IL membranes using a silylated IL as a precursor chemical. The membranes showed selective permeability toward organic vapors against H₂ and CH₄. Their permeation and separation mechanism, and microstructure were discussed by gas and vapor permeation tests, Attenuated Total Reflection–IR, N₂ adsorption measurements, and nanopermporometry. These characterization revealed that the membranes depend on two permeation pathways, “only the dense ionic liquid regions” and “Si–O– network derived–micropores + dense ionic liquid regions”. The membranes contained about 1 nm–sized micropores, and the contributions of two permeation pathways to H₂ permeation were successfully evaluated. In addition, ionic liquid–immobilized siloxane membranes with a different Si–O– network structure were developed, and their CH₃OH permeation characteristics were discussed.

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In response to the challenge of treating water with high concentration of suspended solids (high SS water) through a costly multiple steps, we have developed a high permeability hollow fiber membrane module, named GL module, for treating high SS water. Integrating “hollow fiber membrane” and “module structure” technologies, the GL module features a high water permeability membrane and unique structural elements that are durable for high SS water treatment. The GL module enables a simplified membrane filtration process without pretreatment (i.e., coagulation, sedimentation, sand–filtration processes etc.), thereby reducing water treatment costs in high SS water treatment. Our technology holds promise for advancing membrane technology and fostering a sustainable society.

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