Contagious Aggregation: Transmittable Protein Aggregation in Cellular Communities Initiated by Synthetic Cells.

Aggregation of amyloidogenic proteins causing neurodegenerative diseases is an uncontrollable and contagious process that is often associated with lipid membranes in a highly complex physiological environment. Although several approaches using natural cells and membrane models have been reported, systematic investigations focusing on the association with the membranes are highly challenging, mostly because of the lack of proper molecular tools. Here, we report a new supramolecular approach using a synthetic cell system capable of controlling the initiation of protein aggregation and mimicking various conditions of lipid membranes, thereby enabling systematic investigations of membrane-dependent effects on protein aggregation by visualization. Extending this strategy through concurrent use of synthetic cells and natural cells, we demonstrate the potential of this approach for systematic and in-depth studies on interrogating inter- and intracellularly transmittable protein aggregation. Thus, this new approach offers opportunities for gaining insights into the pathological implications of contagious protein aggregation associated with membranes for neurotoxicity.

A bioinspired and hierarchically structured shape-memory material.

Shape-memory polymeric materials lack long-range molecular order that enables more controlled and efficient actuation mechanisms. Here, we develop a hierarchical structured keratin-based system that has long-range molecular order and shape-memory properties in response to hydration. We explore the metastable reconfiguration of the keratin secondary structure, the transition from α-helix to ß-sheet, as an actuation mechanism to design a high-strength shape-memory material that is biocompatible and processable through fibre spinning and three-dimensional (3D) printing. We extract keratin protofibrils from animal hair and subject them to shear stress to induce their self-organization into a nematic phase, which recapitulates the native hierarchical organization of the protein. This self-assembly process can be tuned to create materials with desired anisotropic structuring and responsiveness. Our combination of bottom-up assembly and top-down manufacturing allows for the scalable fabrication of strong and hierarchically structured shape-memory fibres and 3D-printed scaffolds with potential applications in bioengineering and smart textiles.

Controlled metabolic cascades for protein synthesis in an artificial cell.

In recent years, researchers have been pursuing a method to design and to construct life forms from scratch - in other words, to create artificial cells. In many studies, artificial cellular membranes have been successfully fabricated, allowing the research field to grow by leaps and bounds. Moreover, in addition to lipid bilayer membranes, proteins are essential factors required to construct any cellular metabolic reaction; for that reason, different cell-free expression systems under various conditions to achieve the goal of controlling the synthetic cascades of proteins in a confined area have been reported. Thus, in this review, we will discuss recent issues and strategies, enabling to control protein synthesis cascades that are being used, particularly in research on artificial cells.

Cross-Linked Polymersomes with Reversible Deformability and Oxygen Transportability.

Polymersomes are of interest as nanocarriers due to their physical and chemical robustness, which arises from the macromolecular nature of their block copolymer components. However, the physical robustness of polymersomes impairs transmembrane diffusion and responsiveness to mechanical forces. Polymer nanocarriers that can reversibly deform under stress while maintaining structural integrity and transmembrane diffusivity are desired for development of gas transport vehicles. Here, we report polymersomes composed of amphiphilic block copolymers containing polydimethylsiloxane with side-chain pendant vinyl groups. A reversibly deformable polymersome compartmentalizing membrane was obtained by cross-linkage of PEG- b-poly(dimethyl- r-methylvinyl)silane in a self-assembled bilayer via photoradical generation in aqueous media. The covalently cross-linked polymersomes exhibited superior physical robustness compared to unlinked polymersomes while maintaining deformability under stress. Transmembrane oxygen diffusion was confirmed when lumen-encapsulated Zn-porphyrin generated singlet O2 under irradiation, and the anthracene-9,10-dipropionic acid O2 quencher was consumed. Polymersome-encapsulated hemoglobin bound oxygen reversibly, indicating the polymersomes could be used as O2 carriers that reversibly deform without sacrificing structural integrity or oxygen transportability.

The fabrication of patterned gold nanoparticle arrays via selective ion irradiation and plasma treatment.

A simple and facile method for the patterning of gold nanoparticles (GNPs) was described via selective ion irradiation and oxygen plasma etching. Thin Pluronic films containing HAuCI4 as the precursor of GNPs were selectively irradiated through a pattern mask with 200 keV proton ions to generate GNP-embedded Pluronic patterns. The Pluronic was then removed by an oxygen plasma etching process for the pattern formation of GNPs. Based on the results of the UV-Vis, FE-SEM, and EDX analyses, 50 µm negative-tone line patterns of the GNP-embedded Pluronic were successfully generated at a fluence of less than 1 x 10(16) ions/cm2. The changes in the morphology and elemental composition of the formed GNP-embedded Pluronic patterns with different time periods of oxygen plasma etching were investigated using an FE-SEM with an EDX. The experimental results demonstrated that the patterns of GNPs were effectively generated by the oxygen plasma etching of the formed GNP-embedded Pluronic patterns for 15 min. Furthermore, the XRD results revealed that GNPs in the patterns formed by ion irradiation were further grown during the subsequent oxygen plasma etching.

A Self-Regenerating Artificial Cell, that is One Step Closer to Living Cells: Challenges and Perspectives.

Controllable, self-regenerating artificial cells (SRACs) can be a vital advancement in the field of synthetic biology, which seeks to create living cells by recombining various biological molecules in the lab. This represents, more importantly, the first step on a long journey toward creating reproductive cells from rather fragmentary biochemical mimics. However, it is still a difficult task to replicate the complex processes involved in cell regeneration, such as genetic material replication and cell membrane division, in artificially created spaces. This review highlights recent advances in the field of controllable, SRACs and the strategies to achieve the goal of creating such cells. Self-regenerating cells start by replicating DNA and transferring it to a location where proteins can be synthesized. Functional but essential proteins must be synthesized for sustained energy generation and survival needs and function in the same liposomal space. Finally, self-division and repeated cycling lead to autonomous, self-regenerating cells. The pursuit of controllable, SRACs will enable authors to make bold advances in understanding life at the cellular level, ultimately providing an opportunity to use this knowledge to understand the nature of life.

An Extracellular Matrix-Liposome Composite, a Novel Extracellular Matrix Delivery System for Accelerated Tissue Regeneration.

The unfolded states of fibronectin (FN) subsequently induce the formation of an extracellular matrix (ECM) fibrillar network, which is necessary to generate new substitutive tissues. Here, the authors demonstrate that negatively charged small unilamellar vesicles (SUVs) qualify as candidates for FN delivery due to their remarkable effects on the autonomous binding and unfolding of FN, which leads to increased tissue regeneration. In vitro experiments revealed that the FN-SUV complex remarkably increased the attachment, differentiation, and migration of fibroblasts. The potential utilization of this complex in vivo to treat inflammatory colon diseases is also described based on results obtained for ameliorated conditions in rats with ulcerative colitis (UC) that had been treated with the FN-SUV complex. Their findings provide a new ECM-delivery platform for ECM-based therapeutic applications and suggest that properly designed SUVs may be an unprecedented FN-delivery system that is highly effective in treating UC and inflammatory bowel diseases.

Influence of charge densities of randomly sulfonated polystyrene surfaces on cell attachment and proliferation.

Attachment and proliferation of NIH-3T3 fibroblast cells on random polymer surfaces, polystyrene sulfonated acid (PSSAx) with five different degrees of sulfonation (x = 0%, 5%, 10%, 15% and 33%) and on a tissue culture polystyrene (TCPS) surface were studied. The surface properties, wettability and roughness were measured by water-contact angle and atomic force microscopy measurement. The wettability and surface roughness increased with increasing the content of sulfonic acid groups on the surfaces. The number of cells attached on the surface after seeding increased with increasing x and reached to the maximum value on PSSA15. The cell proliferation also increased with increasing x. However, cell proliferation was slow down on PSSA33 in comparison to PSSA10 and PSSA15 surfaces after 48 h culture.

Determining the effects of PEI adsorption on the permeability of 1,2-dipalmitoylphosphatidylcholine/bis(monoacylglycero)phosphate membranes under osmotic stress.

Polycations are used for a number of biological applications, including antibiotics and gene therapy. One aspect of the use of polycation gene carriers such as polyethylenemine (PEI) in gene therapy that is not well understood is their ability to escape from the vesicles they are internalized in. Here, in an attempt to gain a better understanding of PEI interaction with endosomal lipids under osmotic stress, we performed investigations using monolayers and vesicles derived from a mixture of neutral and negative lipids (1,2-dipalmitoylphosphatidylcholine (DPPC) and bis(monoacylglycero)phosphate (BMP), respectively). X-ray reflectivity (XR) and Langmuir trough measurements confirmed PEI adsorption to the negatively charged membrane. Confocal microscopy imaging indicated that PEI adsorption actually increases the overall integrity of the DPPC/BMP vesicle against osmotic stresses while also causing overall deformation and permeabilization of the lipid membrane, thus leading to leakage of contents from the interior of the vesicle. These confocal microscopy observations were also supported by data gathered by dynamic light scattering (DLS). STATEMENT OF SIGNIFICANCE: In recent decades, researchers have investigated polyamine-based gene delivery systems as useful alternatives to viral gene carriers. One step that is crucial to the performance of polyamine gene carriers such as polyethylenemine (PEI) is escape from late endosomal vesicles during intracellular delivery. However, the ability of polyamine/DNA polyplexes to effectively escape from endosomes is a little-understood part of the gene therapy techniques that use these polyplexes. Here, we performed investigations using monolayers and vesicles derived from a mixture of neutral and negative lipids (1,2-dipalmitoylphosphatidylcholine (DPPC) and bis(monoacylglycero)phosphate (BMP), respectively) as model systems for late endosomes in order to examine the interactions of PEI with the DPPC/BMP membranes and study the subsequent effects on the stability and permeability of these membranes.

Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system.

Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis. Independent optical activation of the two photoconverters allows dynamic control of ATP synthesis: red light facilitates and green light impedes ATP synthesis. We encapsulated the photosynthetic organelles in a giant vesicle to form a protocellular system and demonstrated optical control of two ATP-dependent reactions, carbon fixation and actin polymerization, with the latter altering outer vesicle morphology. Switchable photosynthetic organelles may enable the development of biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviors.

Surface modification for polystyrene colloidal particles with controlled charge densities.

A significant amount of polystyrene sulfonated acid (PSSA) and poly(styrene-ran-acrylic acid) (PSAA) random copolymer can be adsorbed by dispersion of PS particles via a swelling-quenching process. A THF-water mixed solvent was used in the swelling process and a large amount of pure water was used, to give a low concentration of THF% in quenching process. Our results showed that functional PSSA groups were randomly and tightly adsorbed to the PS particles. When the mol.% of charged segments was increased, the progressive adsorption of PSSA chains to the PS particles leads to an increase in the electrophoretic mobility and zeta-potential of aqueous dispersions. Thus, we were able to obtain well-distributed surface charge density on the PS particles.

Simple and Biocompatible Ion Beam Micropatterning of a Cell-Repellent Polymer on Cell-Adhesive Surfaces to Manipulate Cell Adhesion.

In this paper, the simple and biocompatible micropatterning of cell-repellent poly(N-isopropylacrylamide) (PNIPAAm) on a cell-adhesive substrate by ion beam micropatterning to control cell adhesion is described. Cell-repellent PNIPAAm films spin-coated on cell-adhesive tissue culture polystyrene (TCPS) substrates were selectively irradiated by energetic proton ions at various fluences through a pattern mask, and subsequently developed to create the micropatterns of PNIPAAm. Well-defined negative-type PNIPAAm micropatterns were successfully created on the TCPS substrates at fluences higher than 5 x 10¹4 ions/cm², and their chemical properties were dependent on the fluence. Moreover, based on the results of the protein adsorption and in-vitro cell culture tests, 200 µm well-defined micropatterns of mammalian cells were clearly formed on the PNIPAAm-micropatterned TCPS substrates though the preferential adsorption and growth of cells on the TCPS regions due to the strong cell-repellency of PNIPAAm.

Combined study of X-ray reflectivity and atomic force microscopy on a surface-grafted phospholipid monolayer on a solid.

We investigated the detailed structure of a surface-grafted phospholipid monolayer, which was polymerized in situ onto a methacryloyl-silanized solid surface. By the combined study of X-ray reflectivity and atomic force microscopy, the in situ polymerization step of the lipid molecules are sufficiently detailed to reveal the molecular structure of lipid molecules before and after in situ polymerization. From the data of the X-ray reflectivity, we confirmed that the in situ polymerization process produces a flat lipid monolayer structure and that the lipid monolayer is substantially grafted on a silanized surface by chemical bonding. After the polymerization and washing processes, the thickness of the head group was 9 angstroms and the thickness of the tail group was 21 angstroms. The surface morphology of the polymerized phospholipid monolayer obtained by the measurements of atomic force microscopy was consistent with the results of the X-ray reflectivity. The cross-sectional analysis shows that the surface coverage of lipid molecules, which are chemically grafted onto a silanized surface, is approximately 89%.

Generation of functionalized polymer nanolayer on implant surface via initiated chemical vapor deposition (iCVD).

Initiated chemical vapor deposition (iCVD) was utilized to generate a 200nm thick, uniform, functionalized polymer nanolayer comprised of glycidyl methacrylate (GMA) on the surface of titanium implants as a means to improve cellular attachment. Dot-patterned GMA-coated specimens were prepared as well as fully coated specimens. In vitro cellular responses, including cell morphology, protein adsorption, cell proliferation assays, alkaline phosphate activity (ALP) assays, and calcium deposition assays were studied using adipose derived stem cells. The mechanical stability of the thin film was investigated by XPS and FE-SEM analysis of the GMA-coated implant after implantation to an extracted bone from a pig. The GMA-coated specimens displayed increased protein adsorption, higher alkaline phosphatase activities, and higher calcium deposition as compared to control sample with no cytotoxicity. Additionally, no defect was observed in the test of mechanical stability. Notably, dot-patterned GMA-coated samples displayed higher alkaline phosphatase activities than others. Functionalized polymer nanolayer deposition via iCVD is a flexible and robust technique capable of mass production of biocompatible layers. These properties make this technique very suitable for implant applications in a variety of ways.

Engineering hybrid polymer-protein super-aligned nanofibers via rotary jet spinning.

Cellular microenvironments are important in coaxing cells to behave collectively as functional, structured tissues. Important cues in this microenvironment are the chemical, mechanical and spatial arrangement of the supporting matrix in the extracellular space. In engineered tissues, synthetic scaffolding provides many of these microenvironmental cues. Key requirements are that synthetic scaffolds should recapitulate the native three-dimensional (3D) hierarchical fibrillar structure, possess biomimetic surface properties and demonstrate mechanical integrity, and in some tissues, anisotropy. Electrospinning is a popular technique used to fabricate anisotropic nanofiber scaffolds. However, it suffers from relatively low production rates and poor control of fiber alignment without substantial modifications to the fiber collector mechanism. Additionally, many biomaterials are not amenable for fabrication via high-voltage electrospinning methods. Hence, we reasoned that we could utilize rotary jet spinning (RJS) to fabricate highly aligned hybrid protein-polymer with tunable chemical and physical properties. In this study, we engineered highly aligned nanofiber constructs with robust fiber alignment from blends of the proteins collagen and gelatin, and the polymer poly-ε-caprolactone via RJS and electrospinning. RJS-spun fibers retain greater protein content on the surface and are also fabricated at a higher production rate compared to those fabricated via electrospinning. We measured increased fiber diameter and viscosity, and decreasing fiber alignment as protein content increased in RJS hybrid fibers. RJS nanofiber constructs also demonstrate highly anisotropic mechanical properties mimicking several biological tissue types. We demonstrate the bio-functionality of RJS scaffold fibers by testing their ability to support cell growth and maturation with a variety of cell types. Our highly anisotropic RJS fibers are therefore able to support cellular alignment, maturation and self-organization. The hybrid nanofiber constructs fabricated by RJS therefore have the potential to be used as scaffold material for a wide variety of biological tissues and organs, as an alternative to electrospinning.

Hydrodynamic filtration in microfluidic channels as size-selection process for giant unilamellar vesicles.

We have developed hydrodynamic filtration method in microfluidic device for the efficient size-selection of polydisperse lipid vesicles for giant unilamellar vesicles (GUVs), in which vesicles were formed by electroformation method. Combining pinched flow channel design before hydrodynamic filtration, GUVs were flowed and guided to filtration channels, in which small lipid vesicles were further filtered and GUV were remained in main flow channels. For increasing the selectivity of GUV in outlets, length of slit section, or relative flow rate were controlled and drain channels were introduced for avoiding back flow. At higher flow rate in a pinched flow, the fraction of recovered GUVs (>10 microm) were increased, in which most of small vesicles were filtered.

Micropatterning of mammalian cells on indium tin oxide substrates using ion implantation.

In this study, a simple surface patterning method to create micropatterns of mammalian cells on indium tin oxide (ITO) substrates was developed using ion implantation. Thin polystyrene (PS) films spin-coated on an ITO glass was selectively implanted with accelerated proton ions through a pattern mask and then developed to generate PS micropatterns. Well-organized negative PS patterns were generated on the ITO glass. The results of the in vitro cell culture on the PS-patterned ITO glass with two types of cancer cell lines revealed the formation of well-defined cell patterns through a selective cell adhesion and proliferation only onto the ITO regions separated by PS regions. This facile method for cell patterning may be used to create a desired platform for cellular device applications, such as biosensors and cell microarrays.

Micropatterning of cells on electron-irradiated poly(dimethylsiloxane) surface.

In this study, a facile route to fabricate micropatterns of cells is presented on the basis of electron irradiation of poly(dimethylsiloxane) (PDMS). PDMS films were irradiated with electron beams through a pattern mask with micrometer-sized grids. After irradiation, the changes in the chemical composition, morphology, and wettability of the PDMS surface were investigated by using an X-ray photoelectron spectrometer, an atomic force microscope, and a contact anglometer. The results of the surface analysis revealed that the hydrophobic PDMS surface was changed into a hydrophilic one by the electron irradiation. Furthermore, on the basis of cell culturing on the selectively-irradiated PDMS, cells such as NIH3T3 and L929 were selectively adhered to and proliferated on the irradiated regions of the PDMS surface, resulting in the micropatterns of the cells on the PDMS surface.

Analysis of thickness of a hydrophobic fluoropolymer film based on electrowetting.

The electrowetting of water drops on a dielectric fluoropolymer film was studied experimentally. The dependence of the contact angles of the water drops on the applied voltage has been well explained in the low-voltage limit by using the classical Young-Lippmann theory. With this theory, the thicknesses of films coated on glass substrates by using a spin-coater were obtained indirectly by fitting the contact angle data and were confirmed by using X-ray reflectometry. The two sets of results showed a good agreement. In addition, we confirmed that the contact angle saturation at high voltage were consistent with Peykov's model.

Nanostructured metal/carbon hybrids for electrocatalysis by direct carbonization of inverse micelle multilayers.

A synthetic strategy for the fabrication of graphitic carbon nanomaterials containing highly dispersed arrays of metal nanoparticles is reported. This synthetic strategy involves successive deposition of inverse micelle monolayers containing a metal precursor and reduction of the latter, followed by direct carbonization of the obtained multilayer structure of inverse micelles containing metal nanoparticles. Thus, a "direct-carbonization" concept, in which the block copolymer simultaneously serves as soft template and as carbon source, was combined with a multilayer buildup protocol. The inner architecture of the multilayer structures consisting of carbon and metal nanoparticles was studied by X-ray reflectivity, grazing incidence small-angle X-ray scattering, and cross-sectional transmission electron microscopy imaging. The hexagonal near ordering of the metal nanoparticles in the block copolymer micelle multilayers was by and large conserved after carbonization. The resulting carbon structures containing multilayers of highly dispersed metal nanoparticles exhibit superior electrocatalytic activity in formic acid and methanol oxidation, suggesting that they are promising electrode materials for fuel cells.