Yoshikatsu Akiyama

Preparation of thermoresponsive cationic copolymer brush surfaces and application of the surface to separation of biomolecules.

We have prepared poly( N-isopropylacrylamide (IPAAm)- co-2-(dimethylamino)ethylmethacrylate (DMAEMA)) brush-grafted silica bead surfaces through surface-initiated atom transfer radical polymerization (ATRP) using the CuCl/CuCl 2/Me 6TREN catalytic system in 2-propanol at 25 degrees C for 16 h. The prepared temperature-responsive surfaces were characterized by chromatographic analysis using the modified silica beads as stationary phases. Chromatographic retention times for adenosine nucleotides in aqueous mobile phases were significantly increased compared to that previously reported for other cationic hydrogel surfaces, indicating that strong electrostatic cationic copolymer brush interactions occur between the surfaces and nucleotide analytes. Retention times for adenosine nucleotides significantly decreased with increasing column temperature, explained by the decreasing basicity in the copolymer with increasing temperature. Step-temperature gradients from 10 to 50 degrees C shorten ATP retention times. These results indicate that cationic copolymer brush surfaces prepared by ATRP can rapidly alter their electrostatic properties by changing aqueous temperature.

Temperature-responsive glass coverslips with an ultrathin poly(N-isopropylacrylamide) layer

A temperature-responsive cross-linked polymer gel was covalently grafted onto glass coverslips by electron beam irradiation. The grafted thickness and amount of polymer as well as the surface wettability increased with the initial monomer concentration. When the monomer concentration was 5 wt.%, the grafted polymer density was 0.84microgcm(-2), and cells adhered and spread on the surface at 37 degrees C, but detached at 20 degrees C. In contrast, when the monomer concentration was 35 wt.%, the polymer density was 1.28microgcm(-2), and the surfaces were cell repellent even at 37 degrees C. These results show a remarkable contrast to those obtained from temperature-responsive polymer-grafted tissue culture polystyrene dishes, since various types of cells showed temperature-dependent cell adhesion/detachment when the grafted density was around 2microgcm(-2) on these surfaces. We discuss the possible molecular mechanisms underlying this discrepancy.

Characterization of Ultra‐Thin Temperature‐Responsive Polymer Layer and Its Polymer Thickness Dependency on Cell Attachment/Detachment Properties

Ultra thin poly(N-isopropylacrylamide) (PIPAAm) modified glass coverslips (PIAPAm-CS) using electron beam irradiation exhibited a clear relationship between the polymer thickness and thermal cell adhesion/detachment behavior. The polymer thickness dependency and the characteristic of ultra thin PIPAAm layer, has been illustrated in terms of the molecular motion of the modified PIPAAm chains. PIPAAm-CSs surfaces with various area-polymer densities and thicknesses were characterized by AFM and protein adsorption assay. The newly obtained results gave a further insight into the illustration. Finally, the future application of intelligent surfaces was discussed for fabricating tissue and organ.

Thermo-responsive polymer brush-grafted porous polystyrene beads for all-aqueous chromatography

Poly(N-isopropylacrylamide) (PIPAAm) brush-grafted porous polystyrene beads with variable grafted polymer densities were prepared using surface-initiated atom transfer radical polymerization (ATRP) for applications in thermo-responsive chromatography. Utilization of these grafted beads as a stationary phase in aqueous chromatographic analysis of insulin provides a graft density-dependent analyte retention behavior. The separations calibration curve on PIPAAm-grafted polystyrene was obtained using pullulan standards and exhibited inflection points attributed to analyte diffusion into bead pores and partitioning into grafted PIPAAm brush surfaces. Presence of these inflection points supports a separation mechanism where insulin penetrates pores in polystyrene beads and hydrophobically interacts with PIPAAm brushes grafted within the pores. Control of PIPAAm brush graft density on polystyrene facilitates effective aqueous phase separation of peptides based on thermally modulated hydrophobic interactions with grafted PIPAAm within stationary phase pores. These results indicated that PIPAAm brush-grafted porous polystyrene beads prepared by surface-initiated ATRP was effective stationary phase of thermo-responsive chromatography for aqueous phase peptide separations.

Comb-type grafted poly(N-isopropylacrylamide) gel modified surfaces for rapid detachment of cell sheet

A comb-type grafted poly(N-isopropylacrylamide) (PIPAAm) gel modified surface was newly developed for providing a rapid cell sheet recovery for tissue engineering. PIPAAm macromonomer was prepared by the etherification reaction of the hydroxyl terminal moieties of PIPAAm with acryloyl chloride, followed by the radical telomerization reaction of N-isopropylacrylamide (IPAAm) monomer using 2-mercaptoethanol as a chain transfer agent. Solution containing IPAAm monomer and PIPAAm macromonomer was spread on the surface of tissue culture polystyrene (TCPS), and then the surface was subjected to electron beam irradiation for grafting the monomer and macromonomer on the surfaces, resulting in comb-type grafted PIPAAm gel modified TCPS (GG-TCPS). Besides the difference of the amount of the modified PIPAAm, no distinct difference was found between the properties of GG-TCPSs and normal-type PIPAAm gel modified TCPS (NG-TCPS) through XPS, AFM and a contact angle measurement. At 37 degrees C, bovine aortic endothelial cells (BAECs) were well adhered and spread on GG-TCPS as well as NG-TCPS regardless of the macromonomer concentration. By lowering temperature to 20 degrees C, BAECs detached themselves more rapidly from GG-TCPS compared with NG-TCPS. Upon lowering temperature, the grafted polymer was speculated to accelerate the hydration of modified PIPAAm gel, resulting in a rapid cell sheet detachment.

Thermally-modulated on/off-adsorption materials for pharmaceutical protein purification

For the development of temperature-responsive adsorption materials for pharmaceutical protein purification, poly(N-isopropylacrylamide-co-N,N-dimethylaminopropylacrylamide-co-N-tert-butylacrylamide) (P(IPAAm-co-DMAPAAm-co-tBAAm) brush grafted silica beads were prepared through a surface-initiated atom transfer radical polymerization (ATRP). The prepared silica beads as a chromatographic stationary phase were evaluated by observing their thermo-responsive elution profiles of plasma proteins including human serum albumin (HSA) and γ-globulin. Chromatograms of two proteins indicated that negatively-charged HSA was adsorbed on the cationic copolymer brush modified silica beads at higher temperatures with low concentration of phosphate buffer (PB) (pH 7.0) as a mobile phase. The HSA adsorption was attributed to (1) an enhanced electrostatic interaction with the cationic copolymer brush at low concentration of PB and (2) an increased hydrophobic interaction from the dehydrated copolymer at high temperature. Step-temperature gradient enabled HSA and γ-globulin to be separated by the modulation of HSA adsorption/desorption onto the copolymer brush grafted silica beads. These results suggested that the prepared copolymer brush grafted silica beads adsorbed negatively-charged proteins both through electrostatic and hydrophobic interactions by the modulation of column temperature and gave attractive adsorption materials for protein purification process.

Thermoresponsive Polymer Brush Surfaces with Hydrophobic Groups for All-Aqueous Chromatography

For developing thermoresponsive chromatographic matrices with a strong hydrophobicity, poly(N-isopropylacrylamide-co-n-butyl methacrylate) (poly(IPAAm-co-BMA)) brush grafted silica beads were prepared through a surface-initiated atom transfer radical polymerization (ATRP) with a CuCl/CuCl(2)/Me(6)TREN catalytic system in 2-propanol at 25 degrees C for 16 h. The prepared beads were characterized by chromatographic analysis. Chromatograms of the benzoic-acid family and phenol as model analytes were obtained with high-resolution peaks because of their strong hydrophobic interactions to the densely grafted hydrophobized copolymers on the beads. Retention times of the analytes increased with the increase in BMA composition ratio. Dehydration of grafted copolymer with large BMA composition was performed at low temperature. These results indicated that the copolymer-brush-grafted surface prepared by ATRP was an effective tool for separating hydrophilic analytes at low temperature through modulating the strong hydrophobic interaction.

Switching of cell growth/detachment on heparin-functionalized thermoresponsive surface for rapid cell sheet fabrication and manipulation.

Heparin-functionalized poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [P(IPAAm-co-CIPAAm)] grafted surface was designed for the switching of cell growth/detachment, achieved by the regulation of affinity binding between basic fibroblast growth factor (bFGF) and immobilized heparin through the temperature-dependent conformational change of grafted P(IPAAm-co-CIPAAm) chains. At 37 °C, bFGF-bound heparin-thermoresponsive surfaces were able to hold the two- to three-fold number of mouse fibroblast (NIH/3T3) cells than both bFGF-physisorbed surface and PIPAAm surface with soluble bFGF after a 3-day cultivation. Bound bFGF via heparin on shrunken grafted P(IPAAm-co-CIPAAm) chains at 37 °C was able to reinforce the formation and stabilization of bFGF-FGF receptor complex, although the activity of physisorbed bFGF on PIPAAm-grafted surfaces was decreased by non-specific and randomly oriented adsorption. At 20 °C, the cultured NIH/3T3 cell sheet with bFGF detached from heparin-functionalized thermoresponsive surface. The release of bFGF from the surfaces was induced by reducing the affinity binding between bFGF and immobilized-heparin due to increasing the mobility of the swollen grafted P(IPAAm-co-CIPAAm) chains. Therefore, heparin-functionalized thermoresponsive surface was able to enhance cell proliferation, and confluent cells detached themselves as a contiguous cell sheet due to switching cell growth by changing temperature. A cell culture system using this surface is useful for rapid cell sheet fabrication and manipulation.

Effect of malic acid on structure of silicon alkoxide derived silica

Abstract The effect of malic acid on specific surface area, pore size distribution and gel structure of sol-gel derived silica gels was investigated. Tetraethoxysilane (TEOS) used as starting material was modified with malic acid prior to hydrolysis. The specific surface area of calcined gels increased and the pore size distribution shifted to smaller values and became sharper with an increase in malic acid. Malic acid affected particle size and the extent of condensation due to its acidity and condensation hindrance. Under high malic acid concentrations particulate gels were formed. Malic acid chemically bonded or adsorbed to silica polymers prevented particle growth and condensation among particles. Gels obtained at above malic acid/TEOS = 1.17 (mol/mol) and calcined at 450°C for 3 h showed high specific surface areas about 1000 m 2 g −1 . The gel had uniform mesopores with around 2 nm radius.

Thermo-responsive protein adsorbing materials for purifying pharmaceutical protein on exposed charging surface

Thermo-responsive anionic terpolymer brushes with high selectivity for proteins were prepared though surface-initiated ATRP. Utilizing these terpolymer brushes, pure lysozyme was successfully separated from egg-white. Thus, the prepared thermo-responsive terpolymer brush is an effective separation tool for the pharmaceutical and biotechnological industries that produce valuable proteins.