Mid-infrared spectroscopic investigation of the perfect vitrification of poly(ethylene glycol) aqueous solutions.

Crystallization/recrystallization behaviors of poly(ethylene glycol) (PEG) aqueous solutions with water contents (WC's) of ∼36-51 wt % were investigated by temperature-variable mid-infrared spectroscopy. At a WC of 43.2 wt %, crystallization and recrystallization of water and PEG were not observed. At this specific WC value (WCPV), perfect vitrification occurred. Below and above the WCPV value, crystallization/recrystallization behaviors changed drastically. The crystallization temperature below WCPV (237 K) was ∼10 K greater than that above WCPV (226 K). Recrystallization above and below WCPV occurred in one (213 K) and two (198 and 210 K) steps, respectively. These findings resulted from the difference in the (re)crystallization behaviors of water molecules associated with PEG chains with helical and random-coil conformations. These two types of water molecules might have limiting concentrations for their (re)crystallization, indicating that perfect vitrification might have occurred when the concentrations of the two types of water molecules were less than the limiting concentrations of their (re)crystallization.

Clinical Significance of Circulating Tumor Cells in Patients with Esophageal Cancer.

Objective: In recent years, circulating tumor cells (CTCs) have attracted attention for prediction of metastasis in breast, prostate, and colon cancers. This study aimed to investigate whether detection of CTCs could be prognostic factor in esophageal cancer. Methods: This study involved 38 patients treated at Juntendo University from May 2010 to April 2013 who provided consent. CTCs were measured using CellSearch® system in preoperative peripheral blood. Clinicopathological parameters and prognostic factors were retrieved from our medical records. Results: CTCs were detected in 6 of 38 patients (15.8%). Among patients' characteristics and clinicopathological features, CTC-positive group had higher serum SCC levels and tended to have more advanced cStages than the CTC-negative group. The CTC-negative group showed better survival curves than CTCs positive-group in both overall survival (OS) and disease-free survival (DFS) although the differences were not statistically significant. CTCs positivity has a possibility to be prognostic marker according to multivariable analysis of OS and DFS. Conclusion: Although this study has some limitations, our results suggest that CTCs in preoperative peripheral blood has potential to be a prognostic marker for esophageal cancer.

Molecular recognition at the exterior surface of a zwitterionic telomer brush.

3-Sulfo-N,N-dimethyl-N-(2'-methacryloyloxyethyl)propanaminium inner salt (SPB) was polymerized on a glass plate with a surface-confined initiator of atom transfer radical polymerization (ATRP) having a 2-bromoisobutyryl group. The glass plate modified with a brush of sulfobetaine telomer (PSPB) was highly hydrophilic and showed a strong resistance against nonspecific adsorption of proteins such as lysozyme and albumin. Through the polymerization from the free surface of PSPB chain by ATRP, furthermore, N-methacryloyloxysuccinimide (MAOSu) residues were introduced, and the incubation of the telomer (PSPB-b-PMAOSu)-modified glass chip with a lectin (concanavalin A, Con A) gave a glass chip covered with the Con-A-modified PSPB brush. The Con A fixed to the zwitterionic telomer brush pursued specific binding of mannose residues accumulated on the surface of Au colloidal particles, resulting in the increase in absorbance at 550 nm ascribable to localized surface plasmon resonance, while the nonspecific adsorption of proteins to the surface of the glass chip was still largely suppressed. The present results indicate usefulness of the zwitterionic telomer surface with antibiofouling properties as a scaffold for specific sensing devices.

Novel anti-biofouling and drug releasing materials for contact lenses.

Contact lens users very often become patients of allergic conjunctivitis, which is caused by protein and bacteria adsorption to the eye, because contact lenses easily adsorb proteins and bacteria. However, even if contact lens users develop eye diseases such as allergic conjunctivitis, most of them continue to use contact lenses to avoid interference to daily life or a decrease in their quality of life. If novel contact lenses able to prevent and additionally cure eye diseases can be manufactured, they could improve the quality of life of contact lens users worldwide. Thus, we aim to develop a novel material for contact lenses to prevent diseases by incorporating a zwitterionic polymer with the ability to suppress protein and bacteria adsorption. In addition, we also aim to effectively introduce and release a drug against allergic conjunctivitis from the contact lens material. Because the poorly water-soluble drug for allergic conjunctivitis (pranoprofen) forms a rigid crystal structure, we developed the novel "hot-melt press method" to construct a contact lens able to effectively release it. In the present study, polymer sheets containing carboxymethyl betaine (a kind of zwitterionic monomer), 2-hydroxyethyl methacrylate, and 1-vinyl-2-pyrrolidone were prepared using three different procedures. The sheets were hydrophilic and showed a strong resistance against protein and bacteria adsorption. The sheets prepared by the hot-melt press method were transparent and seemed to have potential as a material for contact lenses. In addition, the drug introduced into the sheets during preparation was observed to release at a practically appropriate dose. Therefore, it is expected that the sheets could possibly be used as a material for contact lenses which not only protect against the development of eye trouble due to protein and bacterial adsorption, but also heal allergic conjunctivitis.

Functional surfaces for efficient differentiation of neural stem/progenitor cells into dopaminergic neurons.

Various techniques and systems have been reported for the efficient differentiation of neural stem/progenitor cells into dopaminergic neurons. Although a comparatively high percentage of dopaminergic neurons can be obtained using these techniques, the differentiated cells display varied cellular phenotypes such as astrocytes and oligodendrocytes. Generation of highly pure dopaminergic neurons is important for cell-based therapy and in vitro evaluation of dopaminergic neuron function. In this study, we developed a culture surface anchored with several neurotrophic factors and a neuronal cell-adhesive protein for efficient differentiation of neural stem/progenitor cells into dopaminergic neurons. Oligohistidine-fused brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, synthesized using genetic engineering, were co-immobilized on the surface via metal chelation. To facilitate cell adhesion, a cell-adhesive chimeric protein derived from laminin (LN-G) was also immobilized on the surface. Approximately 40% of the cells cultured for 14 days with these protein-immobilized substrates expressed tyrosine hydroxylase, a marker of dopaminergic neurons, with a three-fold increase in differentiation efficiency than that reported previously. In addition, the number of tyrosine hydroxylase-positive cells increased to approximately 80% of the culture after 30 days. These cells secreted dopamine and expressed dopaminergic neuron-specific genes. Interestingly, cell types (glial cells and oligodendrocytes) other than neuronal cells (immature and mature dopaminergic neurons) were not detected on the protein-anchored surface. Our results demonstrate that highly pure dopaminergic neurons can be exclusively obtained using the novel substrate without extra purification steps such as cell sorting. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 860-871, 2019.

Drug assessment based on detection of L-glutamate released from C6 glioma cells using an enzyme-luminescence method.

Monitoring of excitation activity of nerve cells is very useful for not only brain research but also assessment of the effects of various chemicals, including drugs and toxins. We previously reported a novel enzyme-luminescence method for real-time monitoring of l-glutamate release from C6 glioma cells with high levels of sensitivity ( approximately 10 nM) and temporal resolution (<1 s) using a luminescence plate reader. In the present study, we tested the applicability of this novel system for assessment of effects of drugs in vitro. Several drugs (e.g., veratridine and 4-aminopyridine) were administered to C6 glioma cells for inducing glutamate release. Moreover, antagonists of voltage-dependent Ca (2+) channels (e.g., nifedipine, flunarizine, and NiCl 2) and Na (+) channels (e.g., carbamazepine and lidocaine) were applied separately for evaluating the effects of these chemicals on glutamate release from the cells. The combined effect of carbamazepine and lidocaine was also investigated by using our method, and the combined effect was found to be more potent than that of single drug administration. These results indicated that the glutamate release from C6 cells was modulated by these drugs in a way similar to that found by using several conventional analytical techniques. We therefore conclude that the developed monitoring system for real-time detection of dynamic l-glutamate release from cells could be very useful for application to assessment of drugs acting on the nervous system.

Recrystallization of water in a non-water-soluble polymer examined by Fourier transform infrared spectroscopy: poly(2-methoxyethylacrylate) with low water content.

Crystallization of water during heating, the so-called "recrystallization of water", in poly(2-methoxyethylacrylate) (PMEA) was investigated by temperature-variable Fourier transform infrared spectroscopy. Recrystallization in a polymer-water system is generally understood to be a phase transition from glassy water (condensed water) to crystalline water. However, infrared spectral changes of the PMEA-water system with low water content indicated that the formation of ice I h during heating occurred by a vapor deposition process rather than by a crystallization process.

Crystal growth of ice Ih by revapor-deposition and diffusion suppression of monomolecular water in a polymer solid: spectroscopic observation of phase transition of water sorbed into solid polystyrene.

Monomolecular water in a solid polymer, which has no effective hydrogen bonding sites, was revealed by temperature-variable Fourier transform infrared spectroscopy to be condensable and crystallizable. Ice Ih formed in the polymer matrix was grown by vapor deposition and was reduced by sublimation. Moreover, rapid cooling induced crystal growth by vapor deposition during heating (revapor-deposition). These results indicate the requirement of a change in the generally accepted understanding of the thermal responses of water in a polymer matrix and give rise to a problem for general interpretation of the category of water in a polymer matrix based on calorimetrical analysis at very low water contents.

Spectroscopic evidence of phase transition of monomolecular water in solid polystyrene.

Phase transition behavior of monomolecular water in solid polystyrene was examined by temperature variable Fourier transform infrared spectroscopy. Spectral changes showed for the first time that monomolecular water in a polymer matrix (in a closed system) could be condensed and then frozen and also that the ice formed could be grown and diminished by vapor deposition in cooling process and sublimation in heating process, respectively.

Structure of water incorporated in amphoteric polymer thin films as revealed by FT-IR spectroscopy.

The structure and hydrogen bonding of water incorporated in a thin film of amphoteric terpolymers composed of various ratios of MA, DMAPMA, and BMA were analyzed using the band shapes of the O--H stretching in the IR spectra. At an early stage of sorption of water, the IR spectrum for the water incorporated in the film with comparative contents of MA and DMAPMA residues was similar to that for free water. This is consistent with the tendency for zwitterionic polymers, but is in contrast with the drastic change in the IR spectrum of water incorporated in non-ionic polymer films such as polyBMA. These results suggest a correlation between the mildness of the charge-balanced polymers to the structure of incorporated water and their blood compatibilities.

Interaction of wheat germ agglutinin with an N-acetylglucosamine-carrying telomer brush accumulated on a colloidal gold monolayer.

A disulfide-carrying telomer with many pendent N-acetylglucosamine (GlcNAc) residues (Cys-PMHGlcNAc) was obtained by photo-polymerization of 1-(6'-methacryloylaminohexyl)-2-N-acetoamido-2-deoxy d-glucopyranoside) (MHGlcNAc) using a benzyl N,N-diethyldithiocarbamoyl (BDC) derivative that shows abilities of initiation, transfer, and termination (iniferter). The disulfide-carrying telomer was accumulated on a monolayer of colloidal Au on a glass substrate, and the interaction of wheat germ agglutinin (WGA) with GlcNAc residue at the polymer brush-solution interface was examined by using the localized surface plasmon resonance (LSPR) technique. For comparison, an amphiphile carrying many pendent GlcNAc residues was also prepared with MHGlcNAc and a lipophilic radical initiator and was incorporated in a phospholipid liposome to examine interaction of the GlcNAc residue with WGA on the liposome surface using turbidity measurements. Both the colloidal gold optical device and the liposome showed a concentration-dependent specific binding of WGA, and the GlcNAc-carrying liposome had a detection limit of 100 nM for WGA, whereas that of the colloidal gold device was 10nM. The sugar-carrying telomer-coated device examined here is not only useful as a simple biosensor chip but is also expected to expand our knowledge of bio-related phenomena at the liquid-telomer brush interfaces on a colloidal Au.

Binding of Ricinus communis agglutinin to a galactose-carrying polymer brush on a colloidal gold monolayer.

A polymer with many pendent galactose residues was prepared by atom-transfer radical polymerization (ATRP) of galactose-carrying vinyl monomer, 2-lactobionamidoethyl methacrylate (LAMA), with a disulfide-carrying ATRP initiator, 2-(2'-bromoisobutyroyl)ethyl disulfide (DT-Br). The galactose-carrying polymer obtained (DT-PLAMA) was accumulated as a polymer brush via Au-S bond on a colloidal gold monolayer deposited on a cover glass. For comparison, a disulfide which carried one galactose residue at both ends (2-lactobionamidoethyl disulfide, Cys-Lac) was accumulated as a self-assembled monolayer (SAM) on the colloidal gold monolayer, too. The association and dissociation processes of galactose residues on the colloidal gold with a lectin, Ricinus communis agglutinin (RCA(120)), were observed by the increase and decrease in absorbance at 550nm corresponding to localized surface plasmon resonance (LSPR) phenomena. The Cys-Lac SAM-carrying glass chip showed a strong non-specific adsorption of the lectin, whereas the DT-PLAMA brush-carrying one reversibly associated with the lectin, indicating reusability of the latter device. The apparent association constant of the lectin with the galactose residues in the DT-PLAMA brush was much larger than the association constant for free galactose, and the detection limit of RCA(120) by the glycopolymer brush-modified device was satisfactorily low. Furthermore, a microscopic observation clearly indicated that the DT-PLAMA brush could reversibly associate with a HepG2 cell having galactose receptors, though these processes could not be observed spectrophotometrically due to a gigantic size of the cell.

A novel approach for UV-patterning with binary polymer brushes.

A mixed self-assembled monolayer (SAM) of an initiator (3-(2-bromo-2-isobutyryloxy)propyl triethoxysilane) for atom transfer radical polymerization (ATRP) and an agent (6-(triethoxysilyl)hexyl 2-(((methylthio)carbonothioyl)thio)-2-phenylacetate) for reversible addition-fragmentation chain transfer (RAFT) polymerization was constructed on the surface of a silicon wafer or glass plate by a silane coupling reaction. When a UV light at 254nm was irradiated at the mixed SAM through a photomask, the surface density of the bromine atom at the end of BPE in the irradiated region was drastically reduced by UV-driven scission of the BrC bond, as observed by X-ray photoelectron spectroscopy. Consequently, the surface-initiated (SI)-ATRP of 2-ethylhexyl methacrylate (EHMA) was used to easily construct the poly(EHMA) (PEHMA) brush domain. Subsequently, SI-RAFT polymerization of a zwitterionic vinyl monomer, carboxymethyl betaine (CMB), was performed. Using the sequential polymerization, the PCMB and PEHMA brush domains on the solid substrate could be very easily patterned. Patterning proteins and cells with the binary polymer brush is expected because the PCMB brush indicated strong suppression of protein adsorption and cell adhesion, and the PEHMA brush had non-polar properties. This technique is very simple and useful for regulating the shape and size of bio-fouling and anti-biofouling domains on solid surfaces.

Breaking of the supercooled state of water by a nanocavity with disordered atomic configuration I: freezing behavior of sorbed water into polymethylmethacrylate film as examined by Fourier transform infrared spectroscopy.

A freezing behavior of sorbed water into an annealed atactic polymethylmethacrylate (PMMA) film was examined by Fourier transform infrared (IR) spectroscopy. Temperature dependence of IR spectra of the sorbed water indicated that nonfreezable water in the PMMA film formed associated waters on cooling and were not universally nonfreezable. Its freezing temperature was found to be 249 K, which is much higher than the homogeneous nucleation temperature of water, 236 K. This result suggests that a disordered atomic configuration of a nanocavity wall with hydrogen bond acceptors induces a breaking of the supercooled state of water. The former two findings were visibly clarified by the vibrational spectroscopic method for the first time, and the latest finding was different from the literature on supercooled water in small spaces.

Resistance of surface-confined telomers with pendent glucosylurea groups against non-specific adsorption of proteins.

A disulfide-carrying random telomer with pendent D-glucosylurea groups (Cys-poly(glucosylureaethyl methacrylate-r-ureaethyl methacrylate), Cys-Poly(GUMA-r-UMA)) was obtained by UV-irradiation of GUMA and UMA in the presence of benzyl N,N-diethyldithiocarbamoyl (BDC) derivative which shows the abilities of initiation, transfer, and termination (iniferter). The disulfide-carrying telomer was accumulated on a gold electrode and colloidal gold-immobilized glass substrate, and the binding of various proteins to the surface of the polymer brush was examined by both cyclic voltammetry (CV) with hydroquinone as a probe, and localized surface plasmon resonance (LSPR) absorption method. The Cys-Poly(GUMA-r-UMA) brush did not show a significant non-specific adsorption of proteins such as bovine serum albumin and egg white lysozyme. Furthermore, sugar binding proteins, concanavalin A (with an affinity for mannose and glucose) and wheat germ agglutinin (with an affinity for N-acetylglucosamine), were only slightly adsorbed to the GUMA-carrying brush, which is in contrast with the prompt and distinct binding of these proteins to the brushes composed of 2-methacryloyloxyethyl D-glucopyranoside and 1-(6'-methacrylamido)hexyl-2-N-acetoamido-2-deoxy-D-glucopyranoside, respectively. The glucosylurea group-carrying telomer brush prepared here might be useful to provide a "bio-inert" surface in bio-medical fields.

Recognition of sugars on surface-bound cap-shaped gold particles modified with a polymer brush.

A dithiolated random copolymer with pendent phenylboronic acid residues (Cys-Poly(3-acrylamidophenylboronic acid-co-2-dimethylaminopropyl methacrylamide), Cys-Poly(APBA-co-DMAPMA)) obtained by photo-iniferter method was accumulated as a polymer brush on a cap-shaped gold particles deposited on a vacuum-evaporated gold film, and the usefulness of the polymer brush as a sensing element for glycoprotein, ovalbumin (OVA), was examined by using UV-vis spectroscopy with a help of surface plasmon resonance. A similar system was constructed with a dithiolated mannose-carrying polymer, dithiolated-poly(2-methacryloyloxyethyl-D-mannopyranoside) (DT-PMEMan), prepared by the atom transfer radical polymerization (ATRP). The brush composed of this polymer was examined as a sensing element for lectin, concanavalin A (Con A). The sensor cells modified with Cys-Poly(APBA-co-DMAPMA) and DT-PMEMan showed a concentration-dependent binding of OVA and Con A, respectively, with a comparable detection limit to those with a monolayer of polymer brush-coated gold particle deposited on a glass substrate. Using this system, it can be expected to open a new perspective to various functional polymer brushes fixed to the cap-shaped gold particle on a solid substrate.

Functionalization of single-walled carbon nanotube by the covalent modification with polymer chains.

A single-walled carbon nanotube (SWNT), which had been oxidized by incubation with a mixture of nitric acid and sulfuric acid to afford carboxyl groups at its ends, was incubated with an azo-type radical initiator carrying poly(2-methacryloyloxyethyl D-glucopyranoside) blocks at both ends (PMEGlc-initiator). Due to its high radical trapping activity, the SWNT could be coated with glycopolymers corresponding to the cloven macro-initiator (PMEGlc-SWNT). The PMEGlc-SWNT indicated a lectin (concanavalin A, Con A)-induced aggregation, and a buckey sheet composed of PMEGlc-SWNT could be used for the recovery of Con A from its aqueous solution. Furthermore, the carboxylated SWNT was also incubated with a terminal-aminated poly(N-isopropyl acrylamide) (PIPA) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl salt (PIPA-SWNT). The PIPA-SWNT indicated a definite temperature-responsiveness in the turbidity of its dispersion. These methods would be promising to modify SWNT with various functional polymers.

Structure of water in the vicinity of amphoteric polymers as revealed by Raman spectroscopy.

The structure and hydrogen bonding of water in an aqueous solution of amphoteric copolymers (poly(MA-r-DMAPMA), 3x10(3)

Titanium alloy modified with anti-biofouling zwitterionic polymer to facilitate formation of bio-mineral layer.

The surface of a titanium (Ti) alloy was modified with a self-assembled monolayer of poly(ethylene glycol) methacrylate phosphate (Phosmer PE). A zwitterionic monomer (carboxymethyl betaine, CMB) could be copolymerized with the surface-bound Phosmer PE due to a flexible linker between the Ti alloy surface and a methacryloyl group of Phosmer PE. The poly(CMB) (PCMB)-modified Ti alloy plate exhibited strong suppression of protein adsorption and cell adhesion, and induced approximately twice the amount of calcium (Ca2+) deposition as compared to the unmodified Ti alloy plate. The zwitterionic polymer-modified surfaces not only showed enhanced mineralization clusters creation and growth, but they were also highly non-responsive to biologically derived materials such as proteins and cells. Therefore, it is possible to easily form highly pure and rigid hydroxyapatite layers on Ti alloy surfaces without the incorporation of organic molecules such as proteins. The present surface modification technique and strategy can be applied to implantable orthopedic materials as a means of encouraging integration with host tissues, such as the thigh bone.

Diffusion-Controlled Recrystallization of Water Sorbed into Poly(meth)acrylates Revealed by Variable-Temperature Mid-Infrared Spectroscopy and Molecular Dynamics Simulation.

Recrystallization behaviors of water sorbed into four poly(meth)acrylates, poly(2-methoxyethyl acrylate), poly(tetrahydrofurfuryl acrylate), poly(methyl acrylate), and poly(methyl methacrylate), are investigated by variable-temperature mid-infrared (VT-MIR) spectroscopy and molecular dynamics (MD) simulation. VT-MIR spectra demonstrate that recrystallization temperatures of water sorbed into the polymers are positively correlated with their glass-transition temperatures reported previously. The present MD simulation shows that a lower-limit temperature of the diffusion for the sorbed water and the glass-transition temperatures of the polymers also have a positive correlation, indicating that the recrystallization is controlled by diffusion mechanism rather than reorientation mechanism. Detailed molecular processes of not only recrystallization during rewarming but also crystallization during cooling and hydrogen-bonding states of water in the polymers are systematically analyzed and discussed.