Microplate-Based Cryopreservation of Adherent-Cultured Human Cell Lines Using Amino Acids and Proteins.

With the progression of regenerative medicine and cell therapy, the importance of cryopreservation techniques for cultured cells continues to rise. Traditional cryoprotectants, such as dimethyl sulfoxide and glycerol, are effective in cryopreserving suspended cells, but they do not demonstrate sufficient efficacy for two-dimensional (2D)-cultured cells. In the past decade, small molecules and polymers have been studied as cryoprotectants. Some L-amino acids have been reported to be natural and biocompatible cryoprotectants. However, the cryoprotective effects of D-amino acids have not been investigated for such organized cells. In the present study, the cryoprotective effects of D- and L-amino acids and previously reported cryoprotectants were assessed using HepG2 cells cultured on a microplate without suspending the cells. d-Proline had the highest cryoprotective effect on 2D-cultured cells. The composition of the cell-freezing solution and freezing conditions were then optimized. The d-proline-containing cell-freezing solution also effectively worked for other cell lines. To minimize the amount of animal-derived components, fetal bovine serum in the cell freezing solution was substituted with bovine serum albumin and StemFit (a commercial supplement for stem cell induction). Further investigations on the mechanism of cryopreservation suggested that d-proline protected enzymes essential for cell survival from freeze-induced damage. In conclusion, an effective and xeno-free cell-freezing solution was produced using d-proline combined with dimethyl sulfoxide and StemFit for 2D-cultured cells.

In Situ Synthesis of an Anticancer Peptide Amphiphile Using Tyrosine Kinase Overexpressed in Cancer Cells.

Cell-selective killing using molecular self-assemblies is an emerging concept for cancer therapy. Reported molecular self-assemblies are triggered by hydrolysis of well-designed molecules inside or outside cancer cells. This hydrolysis can occur in cancer and normal cells because of the abundance of water in living systems. Here, we report the in situ synthesis of a self-assembling molecule using a tyrosine kinase overexpressed in cancer cells. We designed a tyrosine-containing peptide amphiphile (C16-E4Y) that is transformed into a phosphorylated peptide amphiphile (C16-E4pY) by the overexpressed tyrosine kinase. Phosphorylation of C16-E4Y promoted self-assembly to form nanofibers in cancer cells. C16-E4Y exhibited selective cytotoxicity toward cancer cells overexpressing the tyrosine kinase. Self-assembled C16-E4pY induced endoplasmic reticulum stress that caused apoptotic cell death. Animal experiments revealed that C16-E4Y has antitumor activity. These results show that an enzyme overexpressed in cancer cells is available for intracellular synthesis of an antitumor self-assembling drug that is cell-selective.

Inhibition of Melittin Activity Using a Small Molecule with an Indole Ring.

We investigated d-amino acids as potential inhibitors targeting l-peptide toxins. Among the l- and d-amino acids tested, we found that d-tryptophan (d-Trp) acted as an inhibitor of melittin-induced hemolysis. We then evaluated various Trp derivatives and found that 5-chlorotryptamine (5CT) had the largest inhibitory effect on melittin. The indole ring, amino group, and steric hindrance of an inhibitor played important roles in the inhibition of melittin activity. Despite the small size and simple molecular structure of 5CT, its IC50 was approximately 13 µg/mL. Fluorescence quenching, circular dichroism measurements, and size-exclusion chromatography revealed that 5CT interacted with Trp19 in melittin and affected the formation of the melittin tetramer involved in hemolysis. Molecular dynamics simulation of melittin also indicated that the interaction of 5CT with Trp19 in melittin affected the formation of the tetramer.

Rewritable Surface on a Plastic Substrate Using Fluorous Affinity.

Fluorous chemistry has unique features and high potential applicability, which are distinct from those of nonfluorinated organic compounds. However, there are limited reports detailing the applications of fluorous-fluorous interactions (fluorophilicity or fluorous affinity), likely because these interactions are not found in nature. In the present study, we describe the rewritable surface functionalization of a plastic substrate based on fluorous affinity. Plastic substrates were dip-coated with a series of methacrylate-based fluoropolymers to generate fluorous surfaces. Fluorous-tagged small molecules [perfluoroalkyl (Rf) amines] were immobilized on the fluorous surfaces via fluorous-fluorous interactions, thereby introducing reactive functional groups (amino moieties) on the surface. The amino groups displayed on the surface (accessible by a reactant) were successfully quantified using a reactive fluorophore, which enabled quantitative analysis of the Rf-amines immobilized on the fluorous surface that were available for the subsequent reaction. The effects of the molecular structures of the fluoropolymers and Rf-amines on the surface immobilization of Rf-amines were also investigated quantitatively. The surface coated with a fluoropolymer containing -C8F17 most effectively immobilized an Rf-amine comprising two -C6F13 chains. The adhered Rf-amines were easily removed by washing the surface with methanol, and then, they could successfully be re-immobilized on the surface. Finally, the presented approach enabled the rewritable micropatterning of an Rf-tagged biomolecule on a plastic surface through microcontact printing.

Restoration of the defect in radial glial fiber migration and cortical plate organization in a brain organoid model of Fukuyama muscular dystrophy.

Fukuyama congenital muscular dystrophy (FCMD) is a severe, intractable genetic disease that affects the skeletal muscle, eyes, and brain and is attributed to a defect in alpha dystroglycan (αDG) O-mannosyl glycosylation. We previously established disease models of FCMD; however, they did not fully recapitulate the phenotypes observed in human patients. In this study, we generated induced pluripotent stem cells (iPSCs) from a human FCMD patient and differentiated these cells into three-dimensional brain organoids and skeletal muscle. The brain organoids successfully mimicked patient phenotypes not reliably reproduced by existing models, including decreased αDG glycosylation and abnormal radial glial (RG) fiber migration. The basic polycyclic compound Mannan-007 (Mn007) restored αDG glycosylation in the brain and muscle models tested and partially rescued the abnormal RG fiber migration observed in cortical organoids. Therefore, our study underscores the importance of αDG O-mannosyl glycans for normal RG fiber architecture and proper neuronal migration in corticogenesis.

Microenvironment pH-Induced Selective Cell Death for Potential Cancer Therapy Using Nanofibrous Self-Assembly of a Peptide Amphiphile.

Self-assembly of synthetic molecules has been drawing broad attention as a novel emerging approach in drug discovery. Here, we report selective cell death induced by a novel peptide amphiphile that self-assembles to form entangled nanofibers (hydrogel) based on intracellular pH (pHi). We found that a palmitoylated hexapeptide (C16-VVAEEE) formed a hydrogel below pH 7. The formation of the nanofibrous self-assembly was responsive to a small pH change around pH 7. The cytotoxicity of C16-VVAEEE was correlated with pHi of cells. Microscope observation demonstrated the self-assembly of C16-VVAEEE inside HEK293 cells. In vivo experiments revealed that the transcutaneous administration of C16-VVAEEE showed remarkable anti-tumor activity. This study proposes that distinct microenvironment inside living cells can be used as a trigger for the intracellular self-assembly of a peptide amphiphile, which provide a new clue to drug discovery.

Molecular Design of pH-Responsive Helix Peptides That Can Damage Tumor Cells Selectively.

It is known that tumor cells have a lower pH compared to normal cells. We have designed peptides that have no definite structure at neutral pH but at lower pH penetrate into the cell membrane by forming an α-helix structure and thus damage tumor cells selectively. These peptides were designed by combining a pH-responsive artificially designed α-helix structure and a flanking sequence that controls membrane insertion. In aqueous solution, some of these peptides were found to be unstructured at neutral pH and helical at acidic pH and showed destruction ability against negatively charged liposomes only at acidic pH. In living cells, one of the designed peptides induced 40% cell death against cervical cancer HeLa and breast cancer MCF-7 cells, while almost no cell death was observed against normal cells. The designed peptide can thus cause tumor cell death without side effects by responding to the peculiar environment of the cell membrane.

Covalent immobilization of gold nanoparticles on a plastic substrate and subsequent immobilization of biomolecules.

We propose a novel approach to stably immobilize gold nanoparticles (AuNPs) on a plastic substrate and demonstrate that the modified substrate is also capable of immobilizing biomolecules. To immobilize citrate-capped AuNPs, an acrylic substrate was simply dip-coated in a functional polymer solution to decorate the outermost surface with amino groups. Electrostatic interactions between AuNPs and the amino groups immobilized the AuNPs with a high density. The AuNP-modified acrylic substrate was transparent with a red tint. A heat treatment promoted the formation of amide bonds between carboxy groups on the AuNPs and amino groups on the substrate surface. These covalent bonds stabilized the immobilized AuNPs and the resulting substrate was resistant to washing with acid and thiol-containing solutions. The surface density of AuNPs was controlled by the surface density of amino groups on the substrate surface, which was in turn controlled by the dip-coating in the functional polymer solution. We attempted to immobilize functional biomolecules on the AuNPs-functionalized plastic surface by two different approaches. An enzyme (horseradish peroxidase) was successfully immobilized on the AuNPs through amide formation and 5'-thiolated DNA was also immobilized on the AuNPs through S-Au interactions. These chemistries allow for simultaneous immobilization of two different kinds of biomolecules on a plastic substrate without loss of their functional properties.

Comparative analyses of site-directed mutagenesis of human melatonin MTNR1A and MTNR1B receptors using a yeast fluorescent biosensor.

Melatonin is an indoleamine neurohormone made by the pineal gland. Its receptors, MTNR1A and MTNR1B, are members of the G-protein-coupled receptor (GPCR) family and are involved in sleep, circadian rhythm, and mood disorders, and in the inhibition of cancer growth. These receptors, therefore, represent significant molecular targets for insomnia, circadian sleep disorders, and cancer. The yeast Saccharomyces cerevisiae is an attractive host for assaying agonistic activity for human GPCR. We previously constructed a GPCR-based biosensor employing a high-sensitivity yeast strain that incorporated both a chimeric yeast-human Gα protein and a bright fluorescent reporter gene (ZsGreen). Similar approaches have been used for simple and convenient measurements of various GPCR activities. In the current study, we constructed a fluorescence-based yeast biosensor for monitoring the signaling activation of human melatonin receptors. We used this system to analyze point mutations, including previously unreported mutations of the consensus sequences of MTNR1A and MTNR1B melatonin receptors and compared their effects. Most mutations in the consensus sequences significantly affected the signaling capacities of both receptors, but several mutations showed differences between these subtype receptors. Thus, this yeast biosensor holds promise for revealing the functions of melatonin receptors.

Hydrogel formation by short D-peptide for cell-culture scaffolds.

The present study reports that a short oligopeptide D-P1, consisting of only five D-amino acids, self-assembled into entangled nanofibers to form a hydrogel that functioned as a scaffold for cell cultures. D-P1 (Ac-D-Phe-D-Phe-D-Phe-Gly-D-Lys) gelated aqueous buffer solution and water at a minimum gelation concentration of 0.5 wt%. The circular dichroism (CD) measurements demonstrated the formation of a ß-sheet structure in the self-assembly of D-P1. We investigated the gelation properties and CD spectra of both the D- and L-forms of the oligopeptide, and found only a minimal difference between them. The D-P1 hydrogel was resistant to a protease, whereas the L-P1 hydrogel was rapidly degraded. Both oligopeptides exhibited nontoxic properties to human cancer cells and embryoid bodies (EBs) derived from human-induced pluripotent stem cells. Additionally, we succeeded in forming spheroids of HeLa cells on the D-P1 hydrogel, which indicates the potential of this hydrogel for 3-dimensional cell culture.

One-Step Biotinylation of Cellulose Paper by Polymer Coating to Prepare a Paper-Based Analytical Device.

Cellulose paper has strong potential as an analytical platform owing to its unique characteristics. In the present study, we investigated a procedure for functionalizing the surface of cellulose paper by dip-coating a mixture of a functional polymer and a perfluoroalkylated surfactant (surfactant 1). The functional polymer comprised a mixture of methyl methacrylate and poly(ethylene glycol) methacrylate monomers. The monomer ratio in the functional polymer affected the hydrophilicity and water absorbance of the cellulose paper after dip-coating. Furthermore, the presence of surfactant 1 during dip-coating promoted the surface segregation of poly(ethylene glycol) (PEG) moieties in the polymer, which enhanced the hydrophilicity, prevented nonspecific protein adsorption, and maintained the water absorbance of the dip-coated cellulose paper. Dip-coating with another functional polymer containing biotin groups produced a cellulose paper with a biotin-decorated surface in a one-step procedure. The displayed biotin groups immobilized avidin on the surface, and the PEG moieties in the polymer prevented nonspecific protein adsorption. We then immobilized a thrombin-binding DNA aptamer on the avidin-immobilized cellulose paper to prepare a paper-based analytical device. It is possible to visualize thrombin in model solutions and serum using the paper-based analytical device.

Quantification of azide groups on a material surface and a biomolecule using a clickable and cleavable fluorescent compound.

Here, we propose a novel method for quantifying azide groups on a solid surface and a protein using a clickable and cleavable fluorescent compound. The clickable and cleavable fluorescent compound conjugates with the azide groups on the material surface and the fluorophore is then liberated into the solvent via a cleavage reaction, which can be simply quantified with a conventional fluorometer.

Short Oligopeptides for Biocompatible and Biodegradable Supramolecular Hydrogels.

Short Phe-rich oligopeptides, consisting of only four and five amino acids, worked as effective supramolecular hydrogelators for buffer solutions at low gelator concentrations (0.5-1.5 wt %). Among 10 different oligopeptides synthesized, peptide P1 (Ac-Phe-Phe-Phe-Gly-Lys) showed high gelation ability. Transmission electron microscopy observations suggested that the peptide molecules self-assembled into nanofibrous networks, which turned into gels. The hydrogel of peptide P1 showed reversible thermal gel-sol transition and viscoelastic properties typical of a gel. Circular dichroism spectra revealed that peptide P1 formed a ß-sheetlike structure, which decreased with increasing temperature. The self-assembly of peptide P1 occurred even in the presence of nutrients in culture media and common surfactants. Escherichia coli and yeast successfully grew on the hydrogel. The hydrogel exhibited low cytotoxicity to animal cells. Finally, we demonstrated that functional compounds can be released from the hydrogel in different manners based on the interaction between the compounds and the hydrogel.

Controlling Surface Segregation of a Polymer To Display Carboxy Groups on an Outermost Surface Using Perfluoroacyl Groups.

Controlling the surface properties of solid polymers is important for practical applications. We here succeeded in controlling the surface segregation of polymers to display carboxy groups on an outermost surface, which allowed the covalent immobilization of functional molecules via the carboxy groups on a substrate surface. Random methacrylate-based copolymers containing carboxy groups, which were protected with perfluoroacyl (Rf) groups, were dip-coated on acrylic substrate surfaces. X-ray photoelectron spectroscopy and contact-angle measurements revealed that the Rf groups were segregated to the outermost surface of the dip-coated substrates. The Rf groups were removed by hydrolysis of the Rf esters in the copolymers, resulting in the display of carboxy groups on the surface. The quantification of carboxy groups on a surface revealed that the carboxy groups were reactive to a water-soluble solute in an aqueous solution. The surface segregation was affected by the molecular structure of the copolymer used for dip-coating.

In Situ Synthesis of a Supramolecular Hydrogelator at an Oil/Water Interface for Stabilization and Stimuli-Induced Fusion of Microdroplets.

Supramolecular hydrogels are expected to have applications as novel soft materials in various fields owing to their designable functional properties. Herein, we developed an in situ synthesis of supramolecular hydrogelators, which can trigger gelation of an aqueous solution without the need for temperature change. This was achieved by mixing two precursors, which induced the synthesis of a supramolecular gelator and its instantaneous self-assembly into nanofibers. We then performed the in situ synthesis of this supramolecular gelator at an oil/water interface to produce nanofibers that covered the surfaces of the oil droplets (nanofiber-stabilized oil droplets). External stimuli induced fusion of the droplets owing to disassembly of the gelator molecules. Finally, we demonstrated that this stimuli-induced droplet fusion triggered a synthetic reaction within the droplets. This means that the confined nanofiber-stabilized droplets can be utilized as stimuli-responsive microreactors.

A DNA-gold nanoparticle hybrid hydrogel network prepared by enzymatic reaction.

We report a DNA-gold nanoparticle (AuNP) hybrid hydrogel in which the AuNPs crosslink enzymatically synthesized DNA to form a gel network. PCR-elongated DNA and AuNPs act as a one-dimensional polymer and cross-linkers, respectively. The DNA-AuNP hydrogel has the functional properties of both long DNA and the AuNPs.

Rational and practical exfoliation of graphite using well-defined poly(3-hexylthiophene) for the preparation of conductive polymer/graphene composite.

Processing and manipulation of highly conductive pristine graphene in large quantities are still major challenges in the practical application of graphene for electric device. In the present study, we report the liquid-phase exfoliation of graphite in toluene using well-defined poly(3-hexylthiophene) (P3HT) to produce a P3HT/graphene composite. We synthesize and use regioregular P3HT with controlled molecular weights as conductive dispersants for graphene. Simple ultrasonication of graphite flakes with the P3HT successfully produces single-layer and few-layer graphene sheets dispersed in toluene. The produced P3HT/graphene composite can be used as conductive graphene ink, indicating that the P3HT/graphene composite has high electrical conductivity owing to the high conductivity of P3HT and graphene. The P3HT/graphene composite also works as an oxidation-resistant and conductive film for a copper substrate, which is due to the high gas-barrier property of graphene.

Mapping of endoglucanases displayed on yeast cell surface using atomic force microscopy.

The surface of yeast cells has been an attractive interface for the effective use of cellulose. Surface enzymes, however, are difficult to visualize and evaluate. In this study, two kinds of unique anchoring regions were used to display the cellulase, endoglucanase (EG), on a yeast cell surface. Differences in the display level and the localization of EG were observed by atomic force microscopy. By surveying the yeast cell surface with a chemically modified cantilever, the interactive force between the cellulose and EG was measured. Force curve mapping revealed differences in the display levels and the localization of EG according to anchoring regions. The proposed methodology enables visualization of displayed enzymes such as EG on the yeast cell surface.

Liquid-liquid extraction of enzymatically synthesized functional RNA oligonucleotides using reverse micelles with a DNA-surfactant.

We successfully implemented solvent extraction of short, single-stranded RNA using reverse micelles (water-in-oil microemulsions) with a DNA-surfactant. A thrombin-binding RNA aptamer was enzymatically synthesized and purified by extraction using the reverse micellar system. The extracted RNA aptamer retained thrombin-binding activity after the extraction procedure.

Highly Conductive Ionic-Liquid Gels Prepared with Orthogonal Double Networks of a Low-Molecular-Weight Gelator and Cross-Linked Polymer.

We prepared a heterogeneous double-network (DN) ionogel containing a low-molecular-weight gelator network and a polymer network that can exhibit high ionic conductivity and high mechanical strength. An imidazolium-based ionic liquid was first gelated by the molecular self-assembly of a low-molecular-weight gelator (benzenetricarboxamide derivative), and methyl methacrylate was polymerized with a cross-linker to form a cross-linked poly(methyl methacrylate) (PMMA) network within the ionogel. Microscopic observation and calorimetric measurement revealed that the fibrous network of the low-molecular-weight gelator was maintained in the DN ionogel. The PMMA network strengthened the ionogel of the low-molecular-weight gelator and allowed us to handle the ionogel using tweezers. The orthogonal DNs produced ionogels with a broad range of storage elastic moduli. DN ionogels with low PMMA concentrations exhibited high ionic conductivity that was comparable to that of a neat ionic liquid. The present study demonstrates that the ionic conductivities of the DN and single-network, low-molecular-weight gelator or polymer ionogels strongly depended on their storage elastic moduli.