Nanoparticles Based on Natural Lipids Reveal Extent of Impacts of Designed Physical Characteristics on Biological Functions.

Nanoparticles based on lipids (LNPs) are essential in pharmaceuticals and intercellular communication, and their design parameters span a diverse range of molecules and assemblies. In bridging the gap in insight between extracellular vesicles (EVs) and synthetic LNPs, one challenge is understanding their in-cell/in-body behavior when simultaneously assessing more than one physical characteristic. Herein, we demonstrate comprehensive evaluation of LNP behavior by using LNPs based on natural lipids (N-LNPs) with designed physical characteristics: size tuned using microfluidic methods, surface fluidity designed based on EV components, and stiffness tuned using biomolecules. We produce 12 types of N-LNPs having different physical characteristics─two sizes, three membrane fluidities, and two stiffnesses for in vitro evaluation─and evaluate cellular uptake vitality and endocytic pathways of N-LNPs based on the physical characteristics of N-LNPs. To reveal the extent of the impact of the predesigned physical characteristics of N-LNPs on cellular uptakes in vivo, we also carried out animal experiments with four types of N-LNPs having different sizes and fluidities. The use of N-LNPs has helped to clarify the extent of the impact of inextricably related, designed physical characteristics on transportation and provided a bidirectional guidepost for the streamlined design and understanding of the biological functions of LNPs.

Enhancing suspended cell transfection by inducing localized distribution of the membrane actin cortex before exposure to electromechanical stimulation.

OBJECTIVES: During physical transfection, an electrical field or mechanical force is used to induce cell transfection. We tested if the disruption of a dense actin layer underneath the membrane of a suspended cell enhances cell transfection. RESULTS: A bubble generator was used to electromechanically stimulate suspended cells. To clarify the influence of the actin layer (the actin cortex) on cell transfection efficiency, we used an actin polymerization inhibitor (cytochalasin D) to disrupt the actin cortex before electromechanical stimulation. Without cytochalasin D treatment, signals from the overall actin cortex decreased after electromechanical stimulation. With cytochalasin D treatment, there was localized F-actin aggregation under static conditions. After electromechanical stimulation, there was a partial loss (localized disruption), but no overall disruption, of the actin cortex. With the pretreatment with cytochalasin D, the transfection efficiency of plasmids (4.7, 8.3, or 11 kbp) into NIH/3T3 or UMR-106 cells increased significantly after exposure to electromechanical stimulation. CONCLUSIONS: Localized distribution of the actin cortex before exposure to electromechanical stimulation is crucial for inducing a partial loss of the cortex, which improves transfection efficiency and large plasmid delivery.

Designing artificial circadian environments with multisensory cares for supporting preterm infants' growth in NICUs.

Previous studies suggest the importance of stable circadian environments for fetuses to achieve sound physiology and intrauterine development. This idea is also supported by epidemiological and animal studies, in which pregnant females exposed to repeated shifting of light-dark cycles had increased rates of reproductive abnormalities and adverse pregnancy outcomes. In response to such findings, artificial circadian environments with light-dark (LD) cycles have been introduced to NICUs to promote better physical development of preterm infants. Such LD cycles, however, may not be fully effective for preterm infants who are less than 30 weeks gestational age (WGA) since they are too premature to be adequately responsive to light. Instead, circadian rhythmicity of incubated preterm infants less than 30 WGA may be able to be developed through stimulation of the non-visual senses such as touch and sound.

Viscosity-aided electromechanical poration of cells for transfecting molecules.

Cell poration technologies offer opportunities not only to understand the activities of biological molecules but also to investigate genetic manipulation possibilities. Unfortunately, transferring large molecules that can carry huge genomic information is challenging. Here, we demonstrate electromechanical poration using a core-shell-structured microbubble generator, consisting of a fine microelectrode covered with a dielectric material. By introducing a microcavity at its tip, we could concentrate the electrical field with the application of electric pulses and generate microbubbles for electromechanical stimulation of cells. Specifically, the technology enables transfection with molecules that are thousands of kDa even into osteoblasts and Chlamydomonas, which are generally considered to be difficult to inject. Notably, we found that the transfection efficiency can be enhanced by adjusting the viscosity of the cell suspension, which was presumably achieved by remodeling of the membrane cytoskeleton. The applicability of the approach to a variety of cell types opens up numerous emerging gene engineering applications.

On-Demand Metallization System Using Micro-Plasma Bubbles.

3D wiring technology is required for the integration of micro-nano devices on various 3D surfaces. However, current wiring technologies cannot be adapted to a variety of materials and surfaces. Here, we propose a new metal deposition method using only a micro-plasma bubble injector and a metal ion solution. Micro-plasma bubbles were generated on demand using pulses, and the localized reaction field enables metal deposition independent of the substrate. Three different modes of micro-plasma bubble generation were created depending on the power supply conditions and mode suitable for metal deposition. Furthermore, using a mode in which one bubble was generated for all pulses among the three modes, copper deposition on dry/wet materials, such as chicken tissue and glass substrates, was achieved. In addition, metal deposition of copper, nickel, chromium, cobalt, and zinc was achieved by simply changing the metal ion solution. Finally, patterning on glass and epoxy resin was performed. Notably, the proposed metal deposition method is conductivity independent. The proposed method is a starting point for 3D wiring of wet materials, which is difficult with existing technologies. Our complete system makes it possible to directly attach sensors and actuators to living organisms and robots, for example, and contribute to soft robotics and biomimetics.

Edge current and pairing order transition in chiral bacterial vortices.

Bacterial suspensions show turbulence-like spatiotemporal dynamics and vortices moving irregularly inside the suspensions. Understanding these ordered vortices is an ongoing challenge in active matter physics, and their application to the control of autonomous material transport will provide significant development in microfluidics. Despite the extensive studies, one of the key aspects of bacterial propulsion has remained elusive: The motion of bacteria is chiral, i.e., it breaks mirror symmetry. Therefore, the mechanism of control of macroscopic active turbulence by microscopic chirality is still poorly understood. Here, we report the selective stabilization of chiral rotational direction of bacterial vortices in achiral circular microwells sealed by an oil/water interface. The intrinsic chirality of bacterial swimming near the top and bottom interfaces generates chiral collective motions of bacteria at the lateral boundary of the microwell that are opposite in directions. These edge currents grow stronger as bacterial density increases, and, within different top and bottom interfaces, their competition leads to a global rotation of the bacterial suspension in a favored direction, breaking the mirror symmetry of the system. We further demonstrate that chiral edge current favors corotational configurations of interacting vortices, enhancing their ordering. The intrinsic chirality of bacteria is a key feature of the pairing order transition from active turbulence, and the geometric rule of pairing order transition may shed light on the strategy for designing chiral active matter.

Design of Electrohydrodynamic Devices with Consideration of Electrostatic Energy.

The importance of actuators that can be integrated with flexible robot structures and mechanisms has increased in recent years with the advance of soft robotics. In particular, electrohydrodynamic (EHD) actuators, which have expandable integrability to adapt to the flexible motion of soft robots, have received much attention in the field of soft robotics. Studies have deepened the understanding of steady states of EHD phenomena but nonsteady states are not well understood. We herein observe the development process of fluid in a microchannel adopting a Schlieren technique with the aid of a high-speed camera. In addition, we analyze the behavior of fluid flow in a microchannel that is designed to have pairs of parallel plate electrodes adopting a computational fluid dynamics technique. Results indicate the importance of considering flow generated by electrostatic energy, which tends to be ignored in constructing and evaluating EHD devices, and by the body force generated by the ion-drag force. By considering these effects, we estimate the development process of EHD flow and confirm the importance of considering the generation of vortices and their interactions inside the microchannel during the development of EHD devices.

Autonomous oil flow generated by self-oscillating polymer gels.

The previously reported gel and polymer actuators require external inputs, such as batteries, circuits, electronic circuits, etc., compared with autonomous motions produced by the living organisms. To realize the spontaneous motions, here, we propose to integrate a power supply, actuators, and control into a single-component self-oscillating hydrogel. We demonstrate self-actuating gel pumps driven by the oscillatory Belousov-Zhabotinsky (BZ) reaction without electronic components. We have developed the volume oscillation of gels synchronized with the BZ reaction (BZ gel). Since the self-actuating gel pumps are driven by chemo-mechanical energy from BZ gels, the self-actuating gel pumps don't require complex wiring designs, energy supply, and assembling. The mechanical work generated by BZ gels is extremely small. We formulated the thermodynamic cycle of BZ gels and maximized mechanical work. We found that pre-stretched BZ gel shows larger mechanical works. We physically separated the BZ gels and working fluid to create practical pumps. By using optimizing mechanical generated by BZ gels, we demonstrated the self-actuating gel pumps that transfer mechanical work through a stretchable elastomer membrane.

Identification of Aquatic Organisms Using a Magneto-Optical Element.

In recent advanced information society, it is important to individually identify products or living organisms automatically and quickly. However, with the current identifying technology such as RFID tag or biometrics, it is difficult to apply to amphibians such as frogs or newts because of its size, stability, weakness under a wet environment and so on. Thus, this research aims to establish a system that can trace small amphibians easily even in a wet environment and keep stable sensing for a long time. The magnetism was employed for identification because it was less influenced by water for a long time. Here, a novel magnetization-free micro-magnetic tag is proposed and fabricated with low cost for installation to a living target sensed by Magneto-Optical sensor for high throughput sensing. The sensing ability of the proposed method, which was evaluated by image analysis, indicated that it was less than half of the target value (1 mm) both in the water and air. The FEM analysis showed that it is approximately twice the actual identification ability under ideal conditions, which suggests that the actual sensing ability can be extended by further improvement of the sensing system. The developed magnetization-free micro-magnetic tag can contribute to keep up the increasing demand to identify a number of samples under a wet environment especially with the development of gene technology.

Plasma-Induced Bubble Microjet Metallization of Elastomer.

As the development of flexible materials and advanced materials progresses, innovative wiring methods for these materials are attracting attention. In this study, we investigated a new wiring technology using plasma-induced microbubbles for elastomer without any surface treatment. Our technology includes three main points. (1) Unlike electroless plating and other conventional methods, it does not require complicated pre-surface treatment processes before wiring. (2) A wiring resolution of 500 micro meter can be reached quickly and economically. (3) Robust metallic adhesion on a wide range of materials can be successfully carried out with precise positioning. Here, by applying our method, we adhered nickel nanoparticles to a latex rubber substrate and demonstrated the electrical conductivity of the created line. The result suggests that our method has potential as an innovative wiring technology to precisely, robustly, and simply fabricate an electric circuit without any complicated procedures or pre-treatment. Our method can contribute to microfabrication technologies.

Relationship between parenting stress and children's behavioral characteristics in Japan.

BACKGROUND: The evaluation of a child tends to differ between the mother and father regardless of whether the child has a disability or not, although parents have key information about the behavioral characteristics of the child. The number of reports in Japan, however, is limited. We, therefore, investigated the relationship between the parenting stress experienced by parents of non-clinical preschool children and the children's behavioral characteristics. METHOD: The subjects were 83 pairs of mothers and fathers with non-clinical children in kindergarten and nursery school (average age, 59.1 ± 13.0 months; 36 boys, 47 girls). The study was conducted using the Parenting Stress Index-Short Form (PSI-SF) and the Strengths and Difficulties Questionnaire (SDQ). RESULTS: There was no difference in the PSI-SF scores between mothers and fathers, but hyperactivity/inattention, peer relationship problems, and the total score of the SDQ were significantly higher in fathers. On multiple regression analysis, parenting stress experienced by fathers was significantly related to hyperactivity/inattention, while parenting stress experienced by mothers was significantly related to peer relationship problems and emotional symptoms. CONCLUSION: Children's behavioral characteristics related to parenting stress differ between mothers and fathers.

On-chip magnetically actuated robot with ultrasonic vibration for single cell manipulations.

This paper presents an innovative driving method for an on-chip robot actuated by permanent magnets in a microfluidic chip. A piezoelectric ceramic is applied to induce ultrasonic vibration to the microfluidic chip and the high-frequency vibration reduces the effective friction on the MMT significantly. As a result, we achieved 1.1 micrometre positioning accuracy of the microrobot, which is 100 times higher accuracy than without vibration. The response speed is also improved and the microrobot can be actuated with a speed of 5.5 mm s(-1) in 3 degrees of freedom. The novelty of the ultrasonic vibration appears in the output force as well. Contrary to the reduction of friction on the microrobot, the output force increased twice as much by the ultrasonic vibration. Using this high accuracy, high speed, and high power microrobot, swine oocyte manipulations are presented in a microfluidic chip.

Single live-bacterial cell assay of promoter activity and regulation.

Laboratory cultures of a single species of bacteria harboring the same genetic background include heterogeneous cell populations, each differing in apparent morphology and physiology, as found in natural environments. To get insights into difference in the genome expression between individual cells, we constructed various types of the cell chip for monitoring the growth and fate of individual bacterial cells. Immobilization of portions of Escherichia coli culture within these cell chips was established after raising the local temperature in the presence of poly-(N-isopropylacrylamide) (PNIPAAm). The newly developed cell-chip system allows the investigation of activity and regulation of green fluorescent protein (GFP)-fused promoter in single live-bacterial cells for prolonged time under controlled culture conditions. Using this single-cell observation system, we succeeded, for the first time, the real-time single-cell assay of promoter activity of the E. coli gcl gene encoding glyoxylate carboligase as a model system, and the kinetics of gcl induction by an effector glyoxylate. Marked heterogeneity was found in the expression level of the gcl promoter. The heterogeneity in gcl promoter activity was, however, confirmed by Flow cytometry of suspension cultures. Our success provides an experimental system for the increased demand of single-cell biology in bacterial studies.

Powerful actuation of magnetized microtools by focused magnetic field for particle sorting in a chip.

This paper describes a novel powerful noncontact actuation of a magnetically driven microtool (MMT), achieved by magnetization of the MMT and focusing of the magnetic field in a microfluidic chip for particle sorting. The following are the highlights of this study: (1) an MMT was successfully fabricated from a mixture of neodymium powder and polydimethylsiloxane; the MMT was magnetized such that it acted as an elastic micromagnet with a magnetic flux density that increased by about 100 times after magnetization, and (2) a pair of sharp magnetic needles was fabricated adjacent to a microchannel in a chip by electroplating, in order to focus the magnetic flux density generated by the electromagnetic coils below the biochip; these needles contribute to miniaturization of an actuation module that would enable the integration of multiple functions in the limited area of a chip. FEM analysis of the magnetic flux density around the MMT showed that the magnetic flux density in the setup with the magnetic needles was around 8 times better than that in the setup without the needles. By magnetization, the drive frequency of the MMT improved by about 10 times--from 18 Hz to 180 Hz. We successfully demonstrated the separation of copolymer beads of a particular size in a chip by image sensing.

On-chip cell immobilization and monitoring system using thermosensitive gel controlled by suspended polymeric microbridge.

In this paper, we describe the fabrication of a temperature-controlled microfluidic chip for cell immobilization using a thermosensitive hydrogel of poly (N-isopropylacrylamide) (PNIPAAm). A mixture of PNIPAAm solution, yeast cells, and Calcein-AM fluorescent dye is flowed in the microchannel, and the indium-tin-oxide (ITO) microheaters that were fabricated by micromachining technology heat a PNIPAAm gel. However, if the gel is very thick, it blocks the observation of the culturing cells and reduces the SNR. To address this problem, we fabricate a suspended microbridge above the microheater that limits the height of the gel, ensuring that it forms a thin and transparent layer above the heater. Microheaters and suspended biocompatible microbridge are integrated on a chip in which yeast-cell immobilization can be performed by gelation of a PNIPAAm solution.