In JoVE (1)

Other Publications (200)

Articles by Kazuhiko Ishihara in JoVE

Other articles by Kazuhiko Ishihara on PubMed

Microfluidic Flow Control on Charged Phospholipid Polymer Interface

Lab on a Chip. Feb, 2007  |  Pubmed ID: 17268622

A type of charged phospholipid polymer biointerface was constructed on a quartz microfluidic chip to control the electroosmotic flow (EOF) and to suppress non-specific protein adsorption through one-step modification. A negatively charged phospholipid copolymer containing 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate (BMA), potassium 3-methacryloyloxypropyl sulfonate (PMPS) and 3-methacryloxypropyl trimethoxysilane (MPTMSi) moieties (referred to as PMBSSi) was synthesized to introduce such phosphorylcholine segments as well as surface charges onto the silica-based microchannels via chemical bonding. At neutral pH, the homogenous microchannel surface modified with 0.3 wt% PMBSSi in alcoholic solution, retained a significant cathodic EOF ((1.0 +/- 0.1) x 10(-4) cm(2) V(-1) s(-1)) with approximately one-half of the EOF of the unmodified microchannel ((1.9 +/- 0.1) x 10(-4) cm(2) V(-1) s(-1)). Along with another non-charged copolymer (poly(MPC-co-MPTMSi), PMSi), the regulation of the surface charge density can be realized by adjusting the concentration of PMBSSi or PMSi initial solutions for modification. Coincidently, the zeta-potential and the EOF mobility at neutral pH showed a monotonically descending trend with the decrease in the charge densities on the surfaces. This provides a simple but feasible approach to controlling the EOF, especially with regard to satisfying the requisites of miniaturized systems for biological applications requiring neutral buffer conditions. In addition, the EOF in microchannels modified with PMBSSi and PMSi could maintain stability for a long time at neutral pH. In contrast to the EOF in the unmodified microchannel, the EOF in the modified microchannel was only slightly affected by the change in pH (from 1 to 10). Most importantly, although PMBSSi possesses negative charges, the non-specific adsorptions of both anionic and cationic proteins (considering albumin and cytochrome c, respectively, as examples) were effectively suppressed to a level of 0.15 microg cm(-2) and lesser in the case of the 0.3 wt% PMBSSi modification. Consequently, the variation in the EOF mobility resulting from the protein adsorption was also suppressed simultaneously. To facilitate easy EOF control with compatibility to biomolecules delivered in the microfluidic devices, the charged interface described could provide a promising option.

Protective Effects of the Whisky Congeners on Ethanol-induced Gastric Mucosal Damage

Alcoholism, Clinical and Experimental Research. Mar, 2007  |  Pubmed ID: 17295722

The ingestion of both ethanol and whisky can induce acute gastrointestinal bleeding. The effects of the congeners, substances other than ethanol in whisky, on the ethanol-induced gastric mucosal damage were examined in the rat model.

Nanometer-scale Patterned Surfaces for Control of Cell Adhesion

Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry. Mar, 2007  |  Pubmed ID: 17372362

A novel cell-adhesion surface, controlled by nanometer-scale topography and chemical patterning, was developed using semiconductor fabrication methods and the formation of self-assembled monolayers. The patterned surface had a sharp contrast between the adsorption and non-adsorption of proteins and cells, and the contrast could be maintained for more than 10 days. The patterning method could easily realize a single cell array and control of the cell morphology. The nanometer-scale patterned surface could control cell adhesion and proliferation. Using the patterned surface will contribute to studies about cell-surface interactions.

A Monoclonal Antibody, PGM34, Against 6-sulfated Blood-group H Type 2 Antigen, on the Carbohydrate Moiety of Mucin

The FEBS Journal. Apr, 2007  |  Pubmed ID: 17381513

Mucin, a major component of mucus, is a highly O-glycosylated, high-molecular-mass glycoprotein extensively involved in the physiology of gastrointestinal mucosa. To detect and characterize mucins derived from site-specific mucous cells, we developed a monoclonal antibody, designated PGM34, by immunizing a mouse with purified pig gastric mucin. The reactivity of PGM34 with mucin was inhibited by periodate treatment of the mucin, but not by trypsin digestion. This suggests that PGM34 recognizes the carbohydrate portion of mucin. To determine the epitope, oligosaccharide-alditols obtained from pig gastric mucin were fractionated by successive gel-filtration, ion-exchange, and normal-phase HPLC, and tested for reactivity with PGM34. Two purified oligosaccharide-alditols that reacted with PGM34 were obtained. Their structures were determined by NMR spectroscopy as Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Fucalpha1-2Galbeta1-3)GalNAc-ol and Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Galbeta1-3)GalNAc-ol. None of the defucosylated or desulfated forms of these oligosaccharides reacted with PGM34. Thus, the epitope of PGM34 was determined as the Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta- sequence. Immunohistochemical examination of rat gastrointestinal tract showed that PGM34 stained surface mucous cells close to the generative cell zone in the gastric fundus and goblet cells in the small intestine, but only slightly stained antral mucous cells in the stomach. These data, taken together, show that PGM34 is a very useful tool for elucidating the role of mucins with characteristic sulfated oligosaccharides.

High Lubricious Surface of Cobalt-chromium-molybdenum Alloy Prepared by Grafting Poly(2-methacryloyloxyethyl Phosphorylcholine)

Biomaterials. Jul, 2007  |  Pubmed ID: 17416412

Osteolysis caused by wear particles from polyethylene in artificial hip joints is of great concern. Various bearing couple combinations, bearing material improvements, and surface modifications have been attempted to reduce such wear particles. With the aim of reducing the wear and developing a novel artificial hip-joint system, we created a highly lubricious metal-bearing material: A 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer was grafted onto the surface of the cobalt-chromium-molybdenum (Co-Cr-Mo) alloy. For ensuring the long-term retention of poly(MPC) on the Co-Cr-Mo alloy, we used a 4-methacryloxyethyl trimellitate anhydride (4-META) intermediate layer and photo-induced graft polymerization technique to create a strong bonding between the Co-Cr-Mo substrate and the poly(MPC) chain via the 4-META layer. The Co-Cr-Mo alloy was pretreated with nitric acid and O(2) plasma to facilitate efficient interaction between the 4-META carboxyl group and the surface hydroxyl group on the Cr oxide passive layer of the Co-Cr-Mo alloy. After MPC grafting, the MPC unit peaks were clearly observed in the Fourier-transform infrared spectroscopy with attenuated total reflection (FT-IR/ATR) and X-ray photoelectron spectroscopy (XPS) spectra of the Co-Cr-Mo surface. Tribological studies with a pin-on-plate machine revealed that surface MPC grafting markedly lowered the friction coefficient. We concluded that the grafted poly(MPC) layer successfully provided high lubricity to the Co-Cr-Mo surface.

Feasibility of a Tiny Centrifugal Blood Pump (TinyPump) for Pediatric Extracorporeal Circulatory Support

Artificial Organs. May, 2007  |  Pubmed ID: 17470213

In this study, the performances of the TinyPump (priming volume 5 mL) system including the pediatric cannulae (Stöckert Pediatric Arterial Cannulae 2.6, 3.0, and 4.0 mm, Stöckert Instruments GmbH, Munich, Germany; Polystan 20-Fr Venous Catheter, MAQUET GmbH, Rastatt, Germany) and an oxygenator (Terumo Capiox RX05 Baby-RX, Terumo Cardiovascular Systems Co., Tokyo, Japan) were studied in vitro followed with preliminary ex vivo studies in 20-kg piglets. In vitro results revealed that the TinyPump system met the requirements for pump speed, pump flow, and pressure drop as extracorporeal circulatory support during open heart surgery and extracorporeal membrane oxygenation (ECMO) in pediatric patients. In 2-h ex vivo studies using 20-kg piglets where the blood contacting surface of the TinyPump was coated with a biocompatible phospholipid polymer, the plasma-free hemoglobin levels remained less than 5.0 mg/dL and no thrombus formation was observed inside the pump. The TinyPump system including the oxygenator and connecting circuits resulted in an overall priming volume of 68 mL, the smallest ever reported. The TinyPump can be a safe option for pediatric circulatory support during open heart surgery and ECMO without requiring blood transfusion.

Effects of Photo-induced Graft Polymerization of 2-methacryloyloxyethyl Phosphorylcholine on Physical Properties of Cross-linked Polyethylene in Artificial Hip Joints

Journal of Materials Science. Materials in Medicine. Sep, 2007  |  Pubmed ID: 17483881

Osteolysis caused by wear particles from polyethylene in the artificial hip joints is a serious issue. We have used photo-induced radical graft polymerization to graft 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer onto the surface of cross-linked polyethylene (CLPE-g-MPC) in order to reduce friction and wear at the bearing surface of the joint. The physical and mechanical properties of CLPE and CLPE-g-MPC were not significantly different, expect that the friction coefficient of untreated CLPE cups was 0.0075, compared with 0.0009 for CLPE-g-MPC cup, an 88% reduction. After 3.0 x 10(6) cycles in the hip joint simulator test, we could not observe any wear of CLPE-g-MPC cups. We concluded that the advantage of photo-induced radical graft polymerization technique was that the grafted MPC polymer gave a high lubricity only on the surface and has no effect on the bulk properties of the CLPE substrate.

Tissue-compatible and Adhesive Polyion Complex Hydrogels Composed of Amphiphilic Phospholipid Polymers

Journal of Biomaterials Science. Polymer Edition. 2007  |  Pubmed ID: 17550663

To investigate the tissue adhesive function of a hydrogel composed of biocompatible amphiphilic polymers, polymers with various architectures were prepared from 2-methacryloyloxyethyl phosphorylcholine (MPC), electrolyte monomers and hydrophobic n-butyl methacrylate (BMA). A polyion complex (PIC) hydrogel was formed within a few minutes after aqueous solutions containing the cationic and anionic MPC polymers were mixed. Provided the electrical charge of the cationic and anionic MPC polymers was approximately balanced, the PIC hydrogel existed stably in a large amount of aqueous medium. The results of the fluorescence study of the MPC polymers suggested that dissociation was suppressed and that the electrostatic interaction was enhanced in the block and graft polymers compared to the random polymers. This is due to the strategically designed architectures and the hydrophobic BMA units. Based on the results of the cytotoxicity test, the cytotoxicity of the MPC polymers was lower than that of glutaraldehyde, a cross-linker contained in aldehyde-type tissue adhesives. The cationic MPC polymers demonstrated higher cytotoxicity compared to the anionic ones, which demonstrated no significant cytotoxicity at examined concentrations. The tissue adhesion of the PIC hydrogels was evaluated with a dura incision model. The results indicated that the tissue adhesion strength of the PIC hydrogel was lower than that of a commercially available fibrin glue. However, the tissue adhesion strength increased with an increase in the polymer concentration and could be controlled by the water content of the hydrogel. Although further investigation of the biocompatibility of the PIC hydrogels and control of the water content is crucial, it can be concluded that the PIC hydrogels formed by the amphiphilic MPC polymers can be promising tissue adhesives which demonstrate properties according to the architectures and chemical structures.

Efficacy of an MPC-BMA Co-polymer As a Nanotransporter for Paclitaxel

Anticancer Research. May-Jun, 2007  |  Pubmed ID: 17595758

Paclitaxel (PTX) is administered as a solution in polyoxyethylated castor oil (CO) due to its low water solubility, but solvent-induced side-effects may be severe.

Instantaneous Determination Via Bimolecular Recognition: Usefulness of FRET in Phosphorylcholine Group Enriched Nanoparticles

Bioconjugate Chemistry. Nov-Dec, 2007  |  Pubmed ID: 17874838

This paper deals with smart bimolecular recognition for instantaneous determination. In particular, we installed the fluorescence resonance energy transfer (FRET) system in phosphorylcholine (PC) group enriched nanoparticles (NPs). The most favorable characteristics were as follows: (i) the suppression of nonspecific protein adsorption by the PC group enriched surface and (ii) simple bioassay protocol relative to the conventional enzyme-linked immunosorbent assay (ELISA). In the case of immunoassays, nonspecific interaction and complex protocols are known dominant problems. To address these issues, we designed FRET-installed NPs. Agglutination of NPs is a fundamental immunoassay technique; however, it is not quantitative. By evaluating the degree of agglutination based on the fluorescence intensity, the resulting information can be used for diagnosis. Therefore, we installed the FRET system on the surface of the NPs. In this paper, C-reactive protein (CRP) and osteopontin (OPN) were the target biomarkers for instantaneous determination, and the resulting fluorescence intensity correlated well with changes in the concentrations of the target molecules. The immunoassay protocol was quite simple, involving only the mixing of FRET-installed NPs and target molecules, such as CRP and OPN antigens. We successfully evaluated the concentration of the target biomarkers, even when human serum albumin was present as an interference molecule.

Effects of a Novel Histamine H2-receptor Antagonist, Lafutidine, on the Mucus Barrier of Human Gastric Mucosa

Journal of Gastroenterology and Hepatology. Nov, 2007  |  Pubmed ID: 17914953

Lafutidine is a novel histamine H(2)-receptor antagonist used primarily as an antisecretory agent in Japan. Previous human studies have not assessed its gastroprotective effects. The purpose of the present study was to determine the effects of lafutidine on the human gastric mucus layer using both histological and biochemical methods.

Enhanced Wear Resistance of Orthopaedic Bearing Due to the Cross-linking of Poly(MPC) Graft Chains Induced by Gamma-ray Irradiation

Journal of Biomedical Materials Research. Part B, Applied Biomaterials. Feb, 2008  |  Pubmed ID: 17588248

We assumed that the extra energy supplied by gamma-ray irradiation produced cross-links in 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer grafted cross-linked polyethylene (CLPE-g-MPC) and investigated its effects on the tribological properties of CLPE-g-MPC. In this study, we found that the gamma-ray irradiation produced cross-links in three kinds of regions of CLPE-g-MPC: poly(MPC) layer, CLPE-MPC interface, and CLPE substrate. The dynamic coefficient of friction of CLPE-g-MPC slightly increased with increasing irradiation doses. After the simulator test, both the nonsterilized and gamma-ray sterilized CLPE-g-MPC cups exhibited lower wear than the untreated CLPE ones. In particular, the gamma-ray sterilized CLPE-g-MPC cups showed extremely low and stable wear. As for the nonsterilized CLPE-g-MPC cups, the weight change varied with each cup. When the CLPE surface is modified by poly(MPC) grafting, the MPC graft polymer leads to a significant reduction in the sliding friction between the surfaces that are grafted because water thin films formed can behave as extremely efficient lubricants. Such a cross-link of poly(MPC) slightly increases the friction of CLPE by gamma-ray irradiation but provides a stable wear resistant layer on the friction surface. The cross-links formed by gamma-ray irradiation would give further longevity to the CLPE-g-MPC cups.

Effect of 2-methacryloyloxyethyl Phosphorylcholine Concentration on Photo-induced Graft Polymerization of Polyethylene in Reducing the Wear of Orthopaedic Bearing Surface

Journal of Biomedical Materials Research. Part A. Aug, 2008  |  Pubmed ID: 17975818

Photo-induced graft polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) on cross-linked polyethylene (CLPE) has been developed as a novel technology for reducing wear of orthopaedic bearings. In this study, the effect of MPC concentration on graft polymerization and the resultant properties of the grafted poly (MPC) layer have been investigated. The grafted poly (MPC) layer thickness increased with the MPC concentration in feed. The hip simulator wear test confirmed that CLPE-g-MPC cups exhibited minimal wear compared with untreated CLPE cups. Since MPC is a highly hydrophilic methacrylate, the water-wettability of CLPE-g-MPC was greater than that of untreated CLPE due to the formation of a poly(MPC) nanometer-scale layer. The CLPE-g-MPC orthopaedic bearing surface exhibited high lubricity, because of the present of the poly(MPC) layer even at a thickness of 10 nm. This layer is considered responsible for the improved wear resistance. Nanometer-scale modification of CLPE with poly(MPC) is expected to significantly increase the durability of the orthopaedic bearings. Poly (MPC) layer thickness can be controlled by changing the MPC concentration in feed. In order to achieve nanometer-scale modification of poly(MPC) in this manner, it is necessary to use a long photo-irradiation time for the MPC graft polymerization system, which contains a high-concentration monomer without its gelation.

Bioconjugated Phospholipid Polymer Biointerface for Enzyme-linked Immunosorbent Assay

Biomacromolecules. Jan, 2008  |  Pubmed ID: 18092759

This study aimed to develop a sensitive and reliable immunoassay by applying a highly functional phospholipid polymer biointerface. We synthesized a phospholipid polymer--poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)] (PMBN). MEONP contains active ester groups on the side chains for immobilization of antibodies via oxyethylene. PMBN with different compositions and oxyethylene chain lengths were synthesized; their effects on nonspecific and specific values in the immunoassay were evaluated. MPC units reduce the background by preventing nonspecific protein adsorption. MEONP units could conjugate antibodies and enhance the specific signal. The specific signal was independent of the oxyethylene chain length, but long oxyethylene chains increased the background. Specific signals corresponding to the antigen were observed with the PMBN coating, and a liner standard curve was obtained. The PMBN-coated surface maintained residual activity after long-term storage. This surface affords a low background without requiring blocking treatment and is suitable for immobilized antibodies.

Surface Tethering of Phosphorylcholine Groups Onto Poly(dimethylsiloxane) Through Swelling--deswelling Methods with Phospholipids Moiety Containing ABA-type Block Copolymers

Biomaterials. Apr, 2008  |  Pubmed ID: 18155763

The surface modification of poly(dimethylsiloxane) (PDMS) substrates by using ABA-type block copolymers comprising poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) (PMPC) and PDMS segments was investigated. The hydrophobic interaction between the swelling-deswelling nature of PDMS and PDMS segments in block copolymers was the main mechanism for surface modification. Block copolymers with various compositions were synthesized by using the atom transfer radical polymerization (ATRP) method. The kinetic plots revealed that polymerization could be initiated by PDMS macroinitiators and it proceeds in a well-controlled manner; therefore, the compositions of the block copolymers were controllable. The obtained block copolymers were dissolved in a chloroform/ethanol mixed solvent. The surface of the PDMS substrate was modified using block copolymers by the swelling-deswelling method. Static contact angle and X-ray photoelectron spectroscopy (XPS) measurements revealed that the hydrophobic surface of the PDMS substrate was converted to a hydrophilic surface because of modification by surface-tethered PMPC segments. Protein adsorption test and L929 cell adhesion test were carried out for evaluating the biocompatibility. As observed, the amount of adsorbed proteins and cell adhesion were drastically reduced as compared to those in the non-treated PDMS substrate. We conclude that this procedure is effective in fabricating biocompatible surfaces on PDMS substrates.

Photografting of 2-methacryloyloxyethyl Phosphorylcholine from Polydimethylsiloxane: Tunable Protein Repellency and Lubrication Property

Colloids and Surfaces. B, Biointerfaces. May, 2008  |  Pubmed ID: 18226507

The phosphorylcholine group functional methacrylate monomer, 2-methacryloyloxyethyl phosphorylcholine (MPC), was graft polymerized from the polydimethylsiloxane (PDMS) substrate using ultraviolet irradiation and using benzophenone as a photoinitiator. The varying monomer concentrations and irradiation times were investigated in order to verify the relationships between graft density and protein resistance under specific biological conditions. The ellipsometry analysis revealed that the layer thickness of the grafted polymer depended on the monomer concentrations after the irradiation for 1 min, however, it stabilized thereafter in all the specified conditions. The curve fitting of the C1s spectrum obtained by X-ray photoelectron spectroscopy analysis showed that the amount of grafted polymer increased with an increase in both monomer concentration and irradiation time. Atomic force microscopic images revealed that the terminations among the graft chains became dominant due to magnified chain mobility followed by growth of their length. In vitro albumin and fibrinogen adsorption results indicated that the resistance to protein adsorption was easily tuned by the specified conditions due to the controlled graft density. Lubrication was dramatically enhanced by the grafting and it was further promoted by an increase in the graft density in good solvents, indicating that the interactions between the graft chains and the solvents resulted in the lubrication system. These basic findings regarding the grafted PDMS surface are important for versatile applications, including its use as a biomaterial and microfluidic device.

Polymer Nanoparticles Covered with Phosphorylcholine Groups and Immobilized with Antibody for High-affinity Separation of Proteins

Biomacromolecules. Mar, 2008  |  Pubmed ID: 18247529

Novel polymer nanoparticles were prepared for the selective capture of a specific protein from a mixture with high effectiveness. The nanoparticle surface was covered with hydrophilic phosphorylcholine groups and active ester groups for easy immobilization of antibodies. Phospholipid polymers (PMBN) composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate, were synthesized for the surface modification of poly( l-lactic acid) nanoparticles. Surface analysis of the nanoparticles using laser-Doppler electrophoresis and X-ray photoelectron spectroscopy revealed that the surface of nanoparticles was covered with PMBN. Protein adsorption was evaluated with regard to the nonspecific adsorption on the nanoparticles that was effectively suppressed by the phosphorylcholine groups. The immobilization of antibodies on nanoparticles was carried out under physiological conditions to ensure specific binding of antigens. The antibody immobilized on the nanoparticles exhibited high activity and strong affinity for the antigen similar to that exhibited by an antibody in a solution. The selective binding of a specific protein as an antigen from a protein mixture was relatively high compared to that observed with conventional antibody-immobilized polymer nanoparticles. In conclusion, nanoparticles having both phosphorylcholine and active ester groups for antibody immobilization have strong potential for use in highly selective separation based on the biological affinities between biomolecules.

Prevention of Biofilm Formation with a Coating of 2-methacryloyloxyethyl Phosphorylcholine Polymer

The Journal of Veterinary Medical Science. Feb, 2008  |  Pubmed ID: 18319577

Device-associated infections are serious complications, and their prevention is an issue of considerable importance. Since biofilms are responsible for these refractory infections, effective methods to inhibit biofilm formation are required. In this investigation, stainless steel plates with and without 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, i.e., poly (MPC-co-n-butyl methacrylate) (PMB) coating, were incubated in a medium containing bacteria. In the course of incubation, half of the specimens received antibiotics. The specimens were stained for nucleic acid and polysaccharides, and then examined with a confocal laser scanning microscope. The numbers of bacteria on the specimen surfaces were evaluated by an ATP assay. On the surfaces of the specimens without PMB coating, the formation of a biofilm enveloping bacteria was confirmed. The addition of antibiotics did not effectively decrease the number of bacteria. On the other hand, on the surfaces of the specimens with PMB coating, no biofilm formation was observed, and the number of bacteria was significantly decreased. The addition of potent antibiotics further decreased the number of bacteria by 1/100 to 1/1000 times. The PMB coating combined with the validated use of antibiotics might provide a method for the simultaneous achievement of biocompatible surfaces of devices and the prevention of device-associated infections.

Effect of Tea Catechins on Body Fat Accumulation in Rats Fed a Normal Diet

Biomedical Research (Tokyo, Japan). Feb, 2008  |  Pubmed ID: 18344595

Although it is known that tea catechins exert potent effects in obese subjects, there is scant information concerning these effects on body weight gain and body fat accumulation in the non-obese. We studied normal rats fed a normal diet and water containing either 0.1% or 0.5% tea catechins to examine the effects on body fat content and serum cholesterol levels, as well as evaluating whether the effect is related to bile acids, which in recent years have emerged as an inducer of energy expenditure. The administration of 0.5% catechins decreased the accumulation of body fat and the serum levels of cholesterol and bile acids. These results indicate that tea catechins modulate lipid metabolism not only in obese subjects, but also in the non-obese.

Effects of Acid Antisecretory Drugs on Mucus Barrier of the Rat Against 5-fluorouracil-induced Gastrointestinal Mucositis

Scandinavian Journal of Gastroenterology. 2008  |  Pubmed ID: 18415744

Acid antisecretory agents are used for the prophylaxis of cancer chemotherapy (CT)-induced gastrointestinal (GI) mucositis. Although these drugs seem to be clinically beneficial, data on their effects on the GI mucosal defense during CT treatment are scant. The objective of this study was to compare the effects of omeprazole, lansoprazole, and lafutidine on mucin, a major mucus component, during 5-fluorouracil (5-FU) treatment, as a CT regimen.

Antithrombogenic Properties of a Monopivot Magnetic Suspension Centrifugal Pump for Circulatory Assist

Artificial Organs. Jun, 2008  |  Pubmed ID: 18422795

The National Institute of Advanced Industrial Science and Technology (AIST) monopivot magnetic suspension centrifugal pump (MC105) was developed for open-heart surgery and several weeks of circulatory assist. The monopivot centrifugal pump has a closed impeller of 50 mm in diameter, supported by a single pivot bearing, and is driven through a magnetic coupling to widen the fluid gap. Design parameters such as pivot length and tongue radius were determined through flow visualization experiments, and the effectiveness was verified in preliminary animal experiments. The maximum overall pump efficiency reached 18%, and the normalized index of hemolysis tested with bovine blood was as low as 0.0013 g/100 L. Animal experiments with MC105 were conducted in sheep for 3, 15, 29, and 35 days in a configuration of left ventricle bypass. No thrombus was formed around the pivot bearing except when the pump speed was reduced by 20% of normal operational speed, which reduced the pump flow by 40% to avoid inlet suction. Subsequently, the antithrombogenic design was verified in animal experiments for 5 weeks at a minimum rotational speed of greater than 1500 rpm and a minimum pump flow greater than 1.0 L/min; no thrombus formation was observed under these conditions.

Establishing Ultimate Biointerfaces Covered with Phosphorylcholine Groups

Colloids and Surfaces. B, Biointerfaces. Sep, 2008  |  Pubmed ID: 18556178

The phospholipid molecule is a typical component of the cell membrane. In particular, the phosphorylcholine polar group is an electrically neutral head group. 2-Methacryloyloxyethyl phosphorylcholine (MPC) comprising a phosphorylcholine group side chain was designed with the cell membrane as an inspiration. Versatile polymers comprising MPC could be synthesized, and their specific biofunctions were evaluated. Establishing an ultimate biointerface with multiple functions is important from the viewpoint of biomaterials science. Nonspecific protein adsorption is essential for achieving versatile biomedical applications. Simultaneously, bioconjugation and retention of its biofunction are crucial for a high-performance biointerface. In this review article, a tunable biointerface comprising MPCs was introduced. In particular, the use of nanoparticles and polymer brushes as biointerfaces was described along with the perspective versatility of their biological applications.

Dimensions of a Free Linear Polymer and Polymer Immobilized on Silica Nanoparticles of a Zwitterionic Polymer in Aqueous Solutions with Various Ionic Strengths

Langmuir : the ACS Journal of Surfaces and Colloids. Aug, 2008  |  Pubmed ID: 18627181

The dimensions and intermolecular interactions of a surface-grafted and unbound free polyampholyte, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), were estimated in aqueous solutions with various ionic strengths. Free linear PMPC was synthesized by atom-transfer radical polymerization (ATRP), and static light scattering (SLS) and dynamic light scattering (DLS) were carried out for the PMPC solutions with various concentrations of NaCl, c s. The hydrodynamic radius R H and the second virial coefficient A 2 of PMPC were independent of c s (0-0.5 M), though both R H and A 2 of polyampholytes usually strongly depend on the ionic strength. PMPC-immobilized silica nanoparticles (PMPC-SiNP) were also synthesized by surface-initiated ATRP, and DLS was carried out as for the solutions of linear PMPC to investigate the dependence of the dimensions of PMPC immobilized on a solid surface on the ionic strength. The molecular weight and surface density of PMPC immobilized on SiNP were estimated from the results obtained by GPC, NMR, and thermogravimetric analysis. The independence of R H of PMPC-SiNP was also observed, but its magnitude was larger than that of linear PMPC, although the molecular weight of PMPC immobilized on SiNP was smaller than that of linear PMPC. The larger dimension of PMPC immobilized on SiNP was explained by the excluded volume effect between the immobilized polymer chains.

Rapid Development of Hydrophilicity and Protein Adsorption Resistance by Polymer Surfaces Bearing Phosphorylcholine and Naphthalene Groups

Langmuir : the ACS Journal of Surfaces and Colloids. Sep, 2008  |  Pubmed ID: 18698868

In order to provide a protein adsorption resistant surface even when the surface was in contact with a protein solution under completely dry conditions, a new phospholipid copolymer, poly (2-methacryloyloxyethyl phosphorylcholine (MPC)- co-2-vinylnaphthalene (vN)) (PMvN), was synthesized. Poly(ethylene terephthalate) (PET) could be readily coated with PMvN by a solvent evaporation method. Dynamic contact angle measurements with water revealed that the surface was wetted very rapidly and had strong hydrophilic characteristics; moreover, molecular mobility at the surface was extremely low. When the surface came in contact with a plasma protein solution containing bovine serum albumin (BSA), the amounts of the plasma protein adsorbed on the dry surface coated with PMvN and that adsorbed on a dry surface coated with poly(MPC-co-n-butyl methacrylate) (PMB) were compared. Substantially lower protein adsorption was observed with PMvN coating. This is due to the rapid hydration behavior of PMvN. We concluded that PMvN can be used as a functional coating material for medical devices without any wetting pretreatment.

Artificial Cell Membrane-covered Nanoparticles Embedding Quantum Dots As Stable and Highly Sensitive Fluorescence Bioimaging Probes

Biomacromolecules. Nov, 2008  |  Pubmed ID: 18842054

To obtain a stable and highly sensitive bioimaging fluorescence probe, polymer nanoparticles with embedded quantum dots were covered with an artificial cell membrane. These nanoparticles were designed by assembling phospholipid polar groups as a platform, and oligopeptide was immobilized as a bioaffinity moiety on the surface of the nanoparticles. The polymer nanoparticles showed resistance to cellular uptake from HeLa cells owing to the nature of the phosphorylcholine groups. When arginine octapeptide was immobilized at the surface of the nanoparticles, they were able to penetrate the membrane of HeLa cells effectively. Cytotoxicity of the nanoparticles was not observed even after immobilization of oligopeptide. Thus, we obtained stable fluorescent polymer nanoparticles covered with an artificial cell membrane, which are useful as an excellent bioimaging probe and as a novel evaluation tool for oligopeptide functions in the target cells.

Surface Immobilization of Biocompatible Phospholipid Polymer Multilayered Hydrogel on Titanium Alloy

Colloids and Surfaces. B, Biointerfaces. Dec, 2008  |  Pubmed ID: 18930384

The aim of this study is to improve the biocompatibility of titanium alloy (Ti) implants by immobilization of multilayered phospholipid polymer hydrogel able to reduce protein adsorption and cell adhesion. We fabricated and characterized a multilayered hydrogel on Ti substrate via a layer-by-layer self-assembly deposition method using a phospholipid polymer bearing a phenylboronic acid moiety and poly(vinyl alcohol) (PVA). The water-soluble phospholipid polymer (PMBV) was synthesized from 2-methacrylocyloxyethyl phosphorylcholine, n-butyl methacrylate, and 4-vinylphenylboronic acid (VPBA). The PMBV reacted with PVA and formed a hydrogel due to covalent linkage between the VPBA units and hydroxyl groups of PVA. The hydrogel layer growth on the Ti surface was initialized by the deposition of one layer of photoreactive PVA bonded by UV irradiation to the Ti surface, which was modified with an alkylsilane compound. The multilayered hydrogel was built up by alternating the deposition of the PMBV and PVA; this was monitored by several methods: static contact angle measurement, X-ray photoelectron spectroscopy, and attenuated Fourier-transform infrared spectroscopy. The results revealed clearly the progressive construction of the multilayered hydrogel on the Ti substrate. The PMBV/PVA multilayer prepared on the Ti substrate reduced the adhesion of L929 cells compared with that on an untreated Ti substrate. Thus, we concluded that the formation of the multilayered hydrogel is effective to improve the biocompatibility on Ti-based medical devices.

Changes in the Mucus Barrier of the Rat During 5-fluorouracil-induced Gastrointestinal Mucositis

Scandinavian Journal of Gastroenterology. Jan, 2008  |  Pubmed ID: 18938749

A frequent complication of antineoplastic chemotherapy (CT) is gastrointestinal (GI) mucositis. Although clinically this mucositis can be treated, data on the effect of CTon the mucosal defense mechanisms are scant, so the effects of 5-fluorouracil (5-FU) on mucin, one of the principal defense factors of the GI mucosa, were investigated.

Effects of Tea Catechins on the Gastrointestinal Mucosa in Rats

Journal of Agricultural and Food Chemistry. Dec, 2008  |  Pubmed ID: 19035783

Although tea catechins are known to exert a potent antiulcer effect on the alimentary tract, there is scant information concerning their effects on normal mucus cell functions. Using original anti-mucin monoclonal antibodies, we studied the influences of long-term administration of catechins on the quantity and quality of mucin in rat gastrointestinal mucosa. Administration of 0.5% tea catechins significantly increased the mucin content of the ileum, but not the stomach. An enzyme-linked immunosorbent assay (ELISA) showed no remarkable qualitative changes in gastric mucin, but a selective increase and decrease in sulfo- and sialomucins, respectively, in the ileum of rats administered catechins. The ELISA results were consistent with both the immunohistochemical findings and the high-iron diamine-alcian blue staining pattern. These findings indicate that tea catechins modulate ileal mucin metabolism in the ileal mucosa, suggesting that further studies focusing on the ileal epithelium will assist in further elucidation of the mechanism of catechin effects.

Effects of Mobility/immobility of Surface Modification by 2-methacryloyloxyethyl Phosphorylcholine Polymer on the Durability of Polyethylene for Artificial Joints

Journal of Biomedical Materials Research. Part A. Aug, 2009  |  Pubmed ID: 18521890

Surface modification is important for the improvement in medical device materials. 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers have attracted considerable attention as surface modifiable polymers for several medical devices. In this study, we hypothesize that the structure of the surface modification layers might affect the long-term stability, hydration kinetics, wear resistance, and so forth, of medical devices such as artificial joints, and the poly(MPC) (PMPC) grafted surface might assure the long-term performance of such devices. Therefore, we investigate the surface properties of various surface modifications by using dip coatings of MPC-co-n-butyl methacrylate (PMB30) and MPC-co-3-methacryloxypropyl trimethoxysilane (PMSi90) polymers, or photoinduced radical grafting of PMPC and also the effects of the surface properties on the durability of cross-linked polyethylene (CLPE) for artificial joints. The PMPC-grafted CLPE has an extremely low and stable coefficient of dynamic friction and volumetric wear as compared to the untreated CLPE, PMB30-coated CLPE, and PMSi90-coated CLPE. It is concluded that the photoinduced radical graft polymerization of MPC is the best method to retain the benefits of the MPC polymer used in artificial joints under variable and multidirectional loads for long periods with strong bonding between the MPC polymer and the CLPE surface, and also to retain the high mobility of the MPC polymer.

Protein Adsorption Resistance and Oxygen Permeability of Chemically Crosslinked Phospholipid Polymer Hydrogel for Ophthalmologic Biomaterials

Journal of Biomedical Materials Research. Part B, Applied Biomaterials. Apr, 2009  |  Pubmed ID: 18777582

The biomimetic structure of a polymer hydrogel bearing phosphorylcholine groups was obtained from 2-methacryloyloxyethylphosphorylcholline (MPC) and a novel crosslinker, 2-(methacryloyloxy)ethyl-N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC), to prepare biocompatible ocular materials. MMPC is a dimethacrylate with phosphorylcholine-analogous linkage. Previous reports clarified that the affinity of MMPC to MPC enables the water contents and mechanical properties of the poly(MPC) hydrogels to be varied without disturbing the bulk phases. In this study, we examined the protein adsorption resistance, water wettability, oxygen permeability, and electrolyte permeability of the mechanically enhanced poly(MPC) hydrogel crosslinked with MMPC. The amount of protein adsorbed on this hydrogel was 0.9 microg/cm(2), which accounted for 30% of Omafilcon A and 3% of Etafilcon A. Water contact angle experiments revealed the high wettability of the poly(MPC) hydrogels. The oxygen permeability and NaCl diffusion constant of the poly(MPC) hydrogels were 64 barrer and 48 x 10(-6) cm(2)/s, respectively. This high permeability resulted from the high water content, similar to the case of the human cornea. These results suggested that poly(MPC) hydrogels have good potential for use in ophthalmologic biomaterials.

Superlubricious Surface Mimicking Articular Cartilage by Grafting Poly(2-methacryloyloxyethyl Phosphorylcholine) on Orthopaedic Metal Bearings

Journal of Biomedical Materials Research. Part A. Dec, 2009  |  Pubmed ID: 19048637

Aseptic loosening of the artificial hip joint with osteolysis due to the wear particles from polyethylene cup has remained as a serious issue. To reduce this wear and develop a novel artificial hip joint system, we produced a superlubricious metal-bearing material: for this, we grafted a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer onto the surface of a cobalt-chromium-molybdenum (Co-Cr-Mo) alloy. For ensuring long-term benefit retention of poly(MPC) on the Co-Cr-Mo alloy for application as a novel artificial hip joint system, several issues must be considered: strong bonding between poly(MPC) and Co-Cr-Mo surface, high mobility of free end groups of the poly(MPC) layer, and high density of the introduced poly(MPC). Considering these issues, we introduced a 3-methacryloxypropyl trimethoxysilane (MPSi) intermediate layer and a photoinduced graft polymerization technique to create a strong covalent bond between the Co-Cr-Mo substrate and the poly(MPC) chain via the MPSi layer. The thickness and density of the poly(MPC) layer on the surface increased with the MPC concentration and photoirradiation time. The grafted poly(MPC) layer successfully provided super-lubricity to the Co-Cr-Mo surface. The poly(MPC)-grafted crosslinked polyethylene/poly(MPC)-grafted Co-Cr-Mo or cartilage/poly(MPC)-grafted Co-Cr-Mo bearing interface mimicking natural joints showed an extremely low friction coefficient of 0.01, which is as low as that of natural cartilage interface. A superlubricious metal-bearing surface would enable the development of a novel biocompatible artificial hip joint system-artificial femoral head for partial hemiarthroplasty and metal-on-polymer/metal type for total hip arthroplasty.

Surface Modification by 2-methacryloyloxyethyl Phosphorylcholine Coupled to a Photolabile Linker for Cell Micropatterning

Biomaterials. Mar, 2009  |  Pubmed ID: 19081624

This report describes a new surface-treatment technique for cell micropatterning. Cell attachment was selectively controlled on the glass surface using a photochemical reaction. This strategy is based on combining 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, which is known to reduce non-specific adsorption, and a photolabile linker (PL) for selective cell patterning. The MPC polymer was coated directly on the glass surface using a straightforward surface modification method, and was removed by ultraviolet (UV) light illumination. All the surface modification steps were evaluated using static water contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), measurements of non-specific protein adsorption, and the cell attachment test. After selective cleavage of the MPC polymer through the photomask, cells attached only to the UV-illuminated region where the MPC polymer was removed, which made the hydrophilic surface relatively hydrophobic. Furthermore, the size of the MC-3T3 E1 cell patterns could be controlled by single cell level. Stability of the cell micropatterns was demonstrated by culturing MC-3T3 E1 cell patterns for 5 weeks on glass slide. The micropatterns were stable during culturing; cell viability also was verified. This method can be a powerful tool for cell patterning research.

Suppression of Protein Adsorption on a Charged Phospholipid Polymer Interface

Biomacromolecules. Feb, 2009  |  Pubmed ID: 19090783

High capability of a charged interface to suppress adsorption of both anionic and cationic proteins was reported. The interface was covalently constructed on quartz by modifying with an anionic phospholipid copolymer, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-potassium 3-methacryloyloxypropyl sulfonate (PMPS)-co-3-methacryloxypropyl trimethoxysilane (MPTMSi)) (PMBSSi). The PMBSSi interfaces were very hydrophilic and homogeneous and could function effectively for a long time even under long-term fluidic working conditions. The PMBSSi density on the interface, which was controllable by adjusting the PMBSSi concentration of the modification solution, affected the surface properties, including the surface contact angle, the surface roughness, and the surface zeta-potential. When a PMBSSi modification was applied, the adsorption of various proteins (isoelectric point varying from 1.0 to 11.0) on quartz was reduced to at least 87% in amount, despite the various electrical natures these proteins have. The protein adsorption behavior on the PMBSSi interface depended more on the PMBSSi density than on the surface charge. The PMBSSi modification had a stable impact on the surface, not only at the physiologic ionic strength, but also over a range of the ionic strength, suggesting that electrostatic interactions do not dominate the behavior of protein adsorption to the PMBSSi surface.

Selective Targeting by PreS1 Domain of Hepatitis B Surface Antigen Conjugated with Phosphorylcholine-based Amphiphilic Block Copolymer Micelles As a Biocompatible, Drug Delivery Carrier for Treatment of Human Hepatocellular Carcinoma with Paclitaxel

International Journal of Cancer. May, 2009  |  Pubmed ID: 19173297

Using dithioester-capped 2-methacryloyloxyethyl phosphorylcholine (MPC) as a macro chain transfer agent, a diblock copolymer was synthesized with n-butyl methacrylate (BMA) as hydrophobic core-forming blocks. The MPC-BMA unit was copolymerized with an immobilizable unit, p-nitrophenylcarbonyloxyethyl methacrylate (NPMA). The NPMA moiety then was modified by the addition of preS1 domain of hepatitis B surface antigen (HBsAg). This micelle-forming nanoparticle, the poly (MPC-co-BMA-co-NPMA) (PMBN) conjugated with preS1 enables solubilization of paclitaxel (PTX) with increased hepatotropism. The 50% inhibitory concentration (IC(50)) values of PTX and PTX/PMBN-preS1 against the human hepatocellular carcinoma cell line, HepG2, were 1,008 and 131 nM, respectively (p < 0.05). Conjugation of preS1 to PMBN enhanced strongly the synergistic inhibitory effect of paclitaxel on HepG2 cells in vitro, whereas such a change in IC(50) was not detected against the human squamous cell carcinoma cell line, A431. Tumor growth rates of a HepG2 xenograft in Balb/c nude mice after intraperitoneal injection of PTX, PTX/PMBN and PTX/PMBN-preS1 were +97.9%, -74.3% and -96.2%*, respectively (*p < 0.05 versus PTX). The local paclitaxel levels after administration of the PMBN-preS1 conjugate were determined in the xenografts by high-performance liquid chromatography and were 8 times higher than that after administration of paclitaxel alone. No side effects attributable to PMBN-preS1 were observed histologically in vital organs, and body weight loss was significantly less in the PTX/PMBN-preS1 group. These studies demonstrate that PMBN-preS1 may be used as a human hepatocyte-specific drug delivery carrier without serious adverse effects.

Effects of Combination Treatment with Famotidine and Methylmethionine Sulfonium Chloride on the Mucus Barrier of Rat Gastric Mucosa

Journal of Gastroenterology and Hepatology. Mar, 2009  |  Pubmed ID: 19175830

In Japan, peptic ulcer disease (PUD) is treated clinically with a combination of a mucosal protectant and acid suppressants, but there is scant information regarding the effects of these drugs on normal gastric mucus cells. In the present study, the effects of co-administration of methylmethionine sulfonium chloride (MMSC) and famotidine on rat gastric mucus cells were investigated using both biochemical and histological methods.

Evaluation of Conditions for Release of Mucin-type Oligosaccharides from Glycoproteins by Hydrazine Gas Treatment

Journal of Biochemistry. Jun, 2009  |  Pubmed ID: 19237440

By using commercially available anhydrous hydrazine in the gas-phase, mucin-type oligosaccharides were released from porcine gastric mucin (PGM) and bovine fetuin. The data indicated that a certain amount of the oligosaccharides from PGM were further degraded. Despite this, the HPLC elution profile of the anthranilic acid (AA)-derivatized oligosaccharides obtained by the treatment with hydrazine at 65 degrees C for 6 h resembled those obtained from the alkaline-borohydride treatment, except for the additional disaccharide fractions derived from the core 1 side of the oligosaccharides by further degradation. The other degraded products derived from the core 2 side could not be derivatized by AA, therefore, not visible by fluorescence detection. Liberation of the oligosaccharides was incomplete by the hydrazine treatment for 6 h. Although almost complete liberation was achieved by extending the treatment to 18 h, the degraded products also increased. In this case, the addition of barium oxide to the reaction vessel decreased the degree of further degradation. Results similar to PGM were obtained from bovine fetuin, but with less degradation. Application of this method for the analysis of rat gastric mucin (RGM) obtained from the corpus and antral region revealed that RGM has a large oligosaccharide portion on the core 1 side.

RAFT Synthesis and Stimulus-induced Self-assembly in Water of Copolymers Based on the Biocompatible Monomer 2-(methacryloyloxy)ethyl Phosphorylcholine

Biomacromolecules. Apr, 2009  |  Pubmed ID: 19243090

Reversible addition-fragmentation chain transfer (RAFT) radical polymerization, mediated by 4-cyanopentanoic acid dithiobenzoate and 4,4'-azobis(4-cyanovaleric acid) (V-501) in water at 70 degrees C, of biocompatible 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) yields a macro-chain transfer agent (CTA) that was employed in the synthesis of a range of stimulus-responsive AB diblock copolymers in protic media. Well-defined block copolymers of varying molar composition, with narrow molecular weight distributions (M(w)/M(n) = 1.10-1.24) were prepared with N,N-diethylacrylamide (DEAm), 4-vinylbenzoic acid (VBZ), N-(3-sulfopropyl)-N-methacryloyloxyethyl-N,N-dimethylammonium betaine (DMAPS), and the newly synthesized N,N-di-n-propylbenzylvinylamine (DnPBVA) in either methanol, 2,2,2-trifluoroethanol, or aqueous media. When a combination of (1)H NMR spectroscopy and dynamic light scattering is used, it is shown that all block copolymers are capable of existing as molecularly dissolved chains in aqueous media with average hydrodynamic diameters of approximately 6-7 nm provided the aqueous environment is appropriately tuned. Similarly, these unimers can be induced to undergo self-assembly in the same aqueous environment provided the correct external stimulus (change in temperature, pH, or electrolyte concentration) is applied. In such instances, aggregates with average sizes in the range of approximately 22-180 nm are formed and are most likely due to the formation of polymeric micelles and vesicles. Such self-assembly is also completely reversible. Removal, or reversal, of the applied stimulus results in the reorganization to the unimeric state.

Wear Resistance of Artificial Hip Joints with Poly(2-methacryloyloxyethyl Phosphorylcholine) Grafted Polyethylene: Comparisons with the Effect of Polyethylene Cross-linking and Ceramic Femoral Heads

Biomaterials. Jun, 2009  |  Pubmed ID: 19269686

Aseptic loosening of artificial hip joints induced by wear particles from the polyethylene (PE) liner remains the ruinous problem limiting their longevity. We reported here that grafting with a polymer, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) (PMPC), on the PE liner surface dramatically decreased the wear production under a hip joint simulator condition. We examined that the effect of properties of both PE by cross-linking and femoral head by changing the materials on wearing properties of PE. The PMPC grafting on the liners increased hydrophilicity and decreased friction torque, regardless of the cross-linking of the PE liner or the difference in the femoral head materials. During the hip joint simulator experiments (5 x 10(6) cycles of loading), cross-linking caused a decrease of wear amount and a reduction of the particle size, while the femoral head materials did not affect it. The PMPC grafting abrogated the wear production, confirmed by almost no wear of the liner surface, independently of the liner cross-linking or the femoral head material. We concluded that the PMPC grafting on the PE liner surpasses the liner cross-linking or the change of femoral head materials for extending longevity of artificial hip joints.

Effects of Indomethacin on the Rat Small Intestinal Mucosa: Immunohistochemical and Biochemical Studies Using Anti-mucin Monoclonal Antibodies

Journal of Gastroenterology. 2009  |  Pubmed ID: 19280111

The luminal surface of the gastrointestinal tract is covered by a viscoelastic gel layer that acts as a protective barrier against the intraluminal environment. Because the situation of the small intestine has not been elucidated to the same degree as other sections, in this study, we investigated the effects of indomethacin on the rat small intestinal mucosa.

Neutral Cysteine Protease Bleomycin Hydrolase is Essential for the Breakdown of Deiminated Filaggrin into Amino Acids

The Journal of Biological Chemistry. May, 2009  |  Pubmed ID: 19286660

Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH(2) terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from beta-naphthylamide derivatives and hydrolyzed citrulline-beta-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin.

Bioinspired Interface for Nanobiodevices Based on Phospholipid Polymer Chemistry

Journal of the Royal Society, Interface. Jun, 2009  |  Pubmed ID: 19324688

This review paper describes novel biointerfaces for nanobiodevices. Biocompatible and non-biofouling surfaces are designed largely based on cell membrane structure, and the preparation and functioning of the bioinspired interface are evaluated and compared between living and artificial systems. A molecular assembly of polymers with a phospholipid polar group has been developed as the platform of the interface. At the surface, protein adsorption is effectively reduced and the subsequent bioreactions are suppressed. Through this platform, biomolecules with a high affinity to the specific molecules are introduced under mild conditions. The activity of the biomolecules is retained even after immobilization. This bioinspired interface is adapted to construct bionanodevices, that is, microfluidic chips and nanoparticles for capturing target molecules and cells. The interface functions well and has a very high efficiency for biorecognition. This bioinspired interface is a promising universal platform that integrates various fields of science and has useful applications.

Development of Targeted Therapy with Paclitaxel Incorporated into EGF-conjugated Nanoparticles

Anticancer Research. Apr, 2009  |  Pubmed ID: 19414339

2-Methacryloyloxyethyl phosphorylcholine (MPC) polymer is a suitable vehicle for paclitaxel (PTX) delivery. A new targeted therapy has been developed by conjugating epidermal growth factor (EGF) to MPC polymer and its growth inhibitory and antitumor effects on cancer cells overexpressing EGF receptors (EGFR) has been investigated.

Controllable Nanostructured Surface Modification on Quantum Dot for Biomedical Application in Aqueous Medium

Journal of Nanoscience and Nanotechnology. Jan, 2009  |  Pubmed ID: 19441319

To develop biocompatible polymer modification method on a quantum dot (QD) surface, we have studied poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)) grafting on trioctylphosphine oxide (TOPO)-coated QD (CdSe/ZnS) surface using new strategy with a double functional reversible addition-fragmentation chain transfer (RAFT) agent. Sodium 2-dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionate (DMP-Na) was focused and prepared as the double functional RAFT agent. As the first function, the DMP-Na possessed surface activity that forms micelle in aqueous solution and solubilize the TOPO-coated QD into the solution. The DMP-Na indicated critical micelle concentration (CMC) from surface tension measurement in an aqueous medium. Over CMC, the QD was able to be solubilized into DMP-Na micelle and showed multicolor fluorescence spectra with narrow full-width at half maximum (FWHM) corresponding to diameter of the QD. Additionally, as the second function, the DMP-Na possessed chain transfer agent ability that synthesizes a poly(MPC)-grafted QD in an aqueous medium. In this strategy, fluorescence properties with narrow FWHM and peak top did not change before and after solubilization into the DMP-Na micelle aqueous solution and even after polymerization of the MPC initiated from the DMP-Na. The poly(MPC)-modified QD showed good biocompatibility based on the poly(MPC) properties, and was able to suppress uptake by HeLa cells despite diameter of the poly(MPC)-modified QD was approximately 12 nm.

Fabrication of a Cell-adhesive Protein Imprinting Surface with an Artificial Cell Membrane Structure for Cell Capturing

Biosensors & Bioelectronics. Nov, 2009  |  Pubmed ID: 19443203

We proposed a new molecular imprinting procedure based on molecular integration for the purpose of cell capture. We selected the cell-adhesive protein fibronectin (FN) as the imprinting protein for preparing templates and evaluated selective cell adhesion on the FN imprinting substrate. Silica beads with a diameter of 15 microm were used as the stamp matrix and FN molecules were adsorbed as a monolayer. The FN recognition sites were constructed by integrating a surfactant as the ligand and immobilizing it with new biocompatible photoreactive phospholipid polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units. As control substrates, imprinting procedures were carried out using albumin (BSA imprinting substrate) and without imprinting protein (non-imprinting substrate). The binding of FN from the cell culture medium with the fetal calf serum was achieved on the FN imprinting substrate, and induced the cell adhesion. On the other hand, on the non-imprinted and BSA imprinting substrates, the FN scarcely bound from the cell culture medium, and subsequent cell adhesion could not be observed on the substrate. These results indicate that the FN binding sites were well constructed by arranging the ligand surfactant to a suitable position and immobilized by the photoreactive MPC polymer. The MPC polymer prevented the nonspecific adsorption of proteins from the cell culture medium. We concluded that this procedure is convenient and can be potentially used for the preparation of surfaces for cell engineering devices.

Second-generation Histamine H(2)-receptor Antagonists with Gastric Mucosal Defensive Properties

Mini Reviews in Medicinal Chemistry. May, 2009  |  Pubmed ID: 19456288

Unlike the earlier agents in this class, certain of the newer histamine H(2)-receptor antagonists (so-called second-generation HB(2B)-receptor antagonists) have recently been reported to promote gastric mucosal defenses. We review herein the structure, specificity, and mechanisms of these agents with a special focus on their cytoprotective/gastroprotective actions.

Bioabsorbable Material-containing Phosphorylcholine Group-rich Surfaces for Temporary Scaffolding of the Vessel Wall

Tissue Engineering. Part C, Methods. Jun, 2009  |  Pubmed ID: 19505178

Novel blends of poly(L-lactic acid) (PLLA) and a water-soluble amphiphilic poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB30W) were prepared as materials for temporary scaffolding to provide mechanical support to the vessel wall until adequate vascular healing is achieved, after which the scaffold disappears. The PLLA/PMB30W cast films demonstrated higher breaking strengths than PLLA cast films, and their Young's modulus was similar to that of PLLA under dry conditions. A high density of phosphorylcholine head groups on the inner surface of PLLA/PMB30W tubing was developed by repeated coatings with the PLLA/PMB30W blend polymer solutions. The PLLA/PMB30W tubing showed stable degradation behaviors similar to those of the PLLA tubing. This is the first report that demonstrates cell membrane-like materials that can be used in temporary scaffolding of the vessel wall; these materials are characterized by strong mechanical properties and stable degradation behaviors that are superior or similar to those of high-molecular-weight PLLA.

Biodegradable Polymer Films for Releasing Nanovehicles Containing Sirolimus

Drug Delivery. May, 2009  |  Pubmed ID: 19514979

To obtain novel biodegradable sirolimus-releasing polymer films that could release nanovehicles incorporating sirolimus, polyester, a water-soluble amphiphilic phospholipid polymer, and sirolimus were blended. The sirolimus-releasing polymer films were characterized by scanning electron microscopy and differential scanning calorimetry. Nanovehicles were formed with the phospholipid polymer chains and could be released from the sirolimus-releasing polymer films. The hydrodynamic diameter of the nanovehicles in phosphate-buffered saline was smaller than 20 nm. The nanovehicles substantially enhanced the carrier-mediated delivery of sirolimus and attenuated its degradation product. This study demonstrates that the delivery of sirolimus was enhanced by nanovehicles released from sirolimus-eluting materials.

Conformational Recovery and Preservation of Protein Nature from Heat-induced Denaturation by Water-soluble Phospholipid Polymer Conjugation

Biomaterials. Oct, 2009  |  Pubmed ID: 19545892

The effect of water-soluble phospholipid polymer conjugated to a protein on conformational change during heat-quenched stress was investigated in this study. Well-defined pyridine disulfide end-functional poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) was synthesized by well controlled atom transfer radical polymerization method. Synthesized PMPC was the site specifically conjugated to a protein, and the effect on conformational change during heat-quenched stress was estimated by circular dichroism and fluorescence study. As a result, a single phospholipid polymer chain conjugated to a protein give a great effect on conformational preservation in both secondary and tertiary structures even after the heat-quenched process. Moreover, even the conformational recovery which gave the completely reversible conformational preservation was observed in circular dichroism study. The resulting protein activity was also confirmed, and no significant decline was induced by heat-quenched stress.

Development of a Method to Evaluate Caspase-3 Activity in a Single Cell Using a Nanoneedle and a Fluorescent Probe

Biosensors & Bioelectronics. Sep, 2009  |  Pubmed ID: 19553098

A method to detect an enzymatic reaction in a single living cell using an atomic force microscope equipped with an ultra-thin needle (a nanoneedle) and a fluorescent probe molecule was developed. The nanoneedle enables the low-invasive delivery of molecules attached onto its surface directly into a single cell. We hypothesized that an enzymatic reaction in a cell could be profiled by monitoring a probe immobilized on a nanoneedle introduced into the cell. In this study, a new probe substrate (NHGcas546) for caspase-3 activity based on fluorescent resonance energy transfer (FRET) was constructed and fixed on a nanoneedle. The NHGcas546-modified nanoneedle was inserted into apoptotic cells, in which caspase-3 is activated after apoptosis induction, and a change in the emission spectrum of the immobilized probe could be observed on the surface of the nanoneedle. Thus, we have developed a successful practical method for detecting a biological phenomenon in a single cell. We call the method MOlecular MEter with Nanoneedle Technology (MOMENT).

Protein Adsorption and Cell Adhesion on Cationic, Neutral, and Anionic 2-methacryloyloxyethyl Phosphorylcholine Copolymer Surfaces

Biomaterials. Oct, 2009  |  Pubmed ID: 19560198

Protein adsorption and cell adhesion on cationic, neutral, and anionic water-soluble 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer surfaces were compared. These model MPC copolymers coated SiO(2) surfaces exhibited comparable surface zeta-potentials of 26.1 mV, near 0 mV, and -24.2 mV, respectively. X-ray photoelectron spectroscopy analyses indicated the similarities and the differences in the surface composition between the sample surfaces. Atomic force microscopy analyses revealed that the type of the charged moiety did not affect the surface roughness. Static contact angle measurements and dynamic contact angle analyses not only indicated that the surfaces were very hydrophilic in general, but also provided information on the surface mobility and the dominant role of MPC at the surface in aqueous conditions. Comparing with the SiO(2) substrates on which protein seriously adsorbed and cell heavily adhered, three MPC copolymers coated surfaces, despite their different charge properties, exhibited significantly low adsorbed amounts of different proteins having various electrical natures and totally no cell adhesion. This suggested that the incorporation of charged moieties in the MPC copolymers did not significantly inspire both the protein adsorption and cell adhesion. The MPC moieties were predominant at the surface when in contact with aqueous conditions and thereby dominated the bio-adsorptions, while the possible effect from electrostatic interactions would be too small and too limited to influence the overall situation. Therefore, these MPC copolymer surfaces can satisfy those biological applications requiring not only electrical but also non-biofouling properties.

Controlled Drug Release from Multilayered Phospholipid Polymer Hydrogel on Titanium Alloy Surface

Biomaterials. Oct, 2009  |  Pubmed ID: 19560818

Here we describe the functionalization of a multilayered hydrogel layer on a Ti alloy with an antineoplastic agent, paclitaxel (PTX). The multilayered hydrogel was synthesized via layer-by-layer self-assembly (LbL) using selective intermolecular reactions between two water-soluble polymers, phospholipid polymer (PMBV) containing a phenylboronic acid unit and poly(vinyl alcohol) (PVA). Reversible covalent bonding between phenylboronic acid and the polyol provided the driving force for self-assembly. Poorly water-soluble PTX dissolves in PMBV aqueous solutions because PMBV is amphiphilic. Therefore, our multilayered hydrogel could be loaded with PTX at different locations to control the release profile and act as a drug reservoir. The amount of PTX incorporated in the hydrogel samples increased with the number of layers but was not directly proportional to the number of layers. However, as the step for making layers was repeated, the concentration of PTX in the PMBV layers increased. The different solubilities of PTX in PMBV and PVA aqueous solutions allow for the production of multilayered hydrogels loaded with PTX at different locations. In vitro experiments demonstrated that the location of PTX in the multilayered hydrogel influences the start and profile of PTX release. We expect that this rapid and facile LbL synthesis of multilayered hydrogels and technique for in situ loading with PTX, where the location of loading controls the release pattern, will find applications in biomedicine and pharmaceutics as a promising new technique.

Cell Adhesion on Phase-separated Surface of Block Copolymer Composed of Poly(2-methacryloyloxyethyl Phosphorylcholine) and Poly(dimethylsiloxane)

Biomaterials. Oct, 2009  |  Pubmed ID: 19592090

We investigated the morphological effect of phase-separated block copolymer surfaces composed of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) (PMPC) and poly(dimethylsiloxane) (PDMS) on protein adsorption and cell adhesion behavior. We observed three different types of phase-separated surface morphologies by TEM and AFM. The elemental composition of phosphorus on the surface increases with the PMPC composition. Furthermore, the polymer surface formed by a block copolymer-containing a higher MPC unit composition shows a slightly lower static water contact angle. This result indicates that the elemental surface ratio of the surface depends on the MPC composition in the block copolymer. Protein adsorption tests revealed that only hydrophobic PDMS domains showed selective protein adsorption. Cell adhesion tests revealed that the number of adhered cells increased with increasing hydrophobic PDMS domain size of block copolymers in serum-containing media. In contrast, no cells adhered onto block copolymer surfaces in serum-free media, whereas a large amount of adhered cells were observed on the hydrophobic PDMS surface. This result indicates that segregated hydrophobic domains on a biocompatible PMPC surface strongly affect serum protein adsorption, thereby promoting considerable cell adhesion, although the surface is hydrophilic. Thus, both the composition of MPC units and the segregated hydrophobic surface morphology are important considerations in biomaterial surface design.

Intraperitoneal Administration of Paclitaxel Solubilized with Poly(2-methacryloxyethyl Phosphorylcholine-co N-butyl Methacrylate) for Peritoneal Dissemination of Gastric Cancer

Cancer Science. Oct, 2009  |  Pubmed ID: 19604244

Intraperitoneal (i.p.) administration of paclitaxel (PTX) is a hopeful therapeutic strategy for peritoneal malignancy. Intravenously (i.v.) injected nanoparticle anticancer drugs are known to be retained in the blood stream for a long time and favorably extravasated from vessels into the interstitium of tumor tissue. In this study, we evaluated the effect of i.p. injection of PTX (PTX-30W), which was prepared by solubulization with water-soluble amphiphilic polymer composed of PMB-30W, a co-polymer of 2-methacryloxyethyl phosphorylcholine and n-butyl methacrylate, for peritoneal dissemination of gastric cancer. In a peritoneal metastasis model with transfer of MKN45P in nude mice, the effect of i.p. administration of PTX-30W was compared with conventional PTX dissolved in Cremophor EL (PTX-Cre). The drug accumulation in peritoneal nodules was evaluated with intratumor PTX concentration and fluorescence microscopic observation. PTX-30W reduced the number of metastatic nodules and tumor volume significantly more than did conventional PTX dissolved in Cremophor EL (PTX-Cre), and prolonged the survival time (P < 0.05). PTX concentration in disseminated tumors measured by HPLC was higher in the PTX-30W than in the PTX-Cre group up to 24 h after i.p. injection. Oregon green-conjugated PTX-30W, i.p. administered, preferentially accumulated in relatively hypovascular areas in the peripheral part of disseminated nodules, which was significantly greater than the accumulation of PTX-Cre. I.p. administration of PTX-30W may be a promising strategy for peritoneal dissemination, due to its superior characteristics to accumulate in peritoneal lesions.

Surface Modification of a Titanium Alloy with a Phospholipid Polymer Prepared by a Plasma-induced Grafting Technique to Improve Surface Thromboresistance

Colloids and Surfaces. B, Biointerfaces. Nov, 2009  |  Pubmed ID: 19647420

To improve the thromboresistance of a titanium alloy (TiAl(6)V(4)) surface which is currently utilized in several ventricular assist devices (VADs), a plasma-induced graft polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) was carried out and poly(MPC) (PMPC) chains were covalently attached onto a TiAl(6)V(4) surface by a plasma induced technique. Cleaned TiAl(6)V(4) surfaces were pretreated with H(2)O-vapor-plasma and silanated with 3-methacryloylpropyltrimethoxysilane (MPS). Next, a plasma-induced graft polymerization with MPC was performed after the surfaces were pretreated with Ar plasma. Surface compositions were verified by X-ray photoelectron spectroscopy (XPS). In vitro blood biocompatibility was evaluated by contacting the modified surfaces with ovine blood under continuous mixing. Bulk phase platelet activation was quantified by flow cytometric analysis, and surfaces were observed with scanning electron microscopy after blood contact. XPS data demonstrated successful modification of the TiAl(6)V(4) surfaces with PMPC as evidenced by increased N and P on modified surfaces. Platelet deposition was markedly reduced on the PMPC grafted surfaces and platelet activation in blood that contacted the PMPC-grafted samples was significantly reduced relative to the unmodified TiAl(6)V(4) and polystyrene control surfaces. Durability studies under continuously mixed water suggested no change in surface modification over a 1-month period. This modification strategy shows promise for further investigation as a means to reduce the thromboembolic risk associated with the metallic blood-contacting surfaces of VADs and other cardiovascular devices under development.

Nippostrongylus Brasiliensis: Increase of Sialomucins Reacting with Anti-mucin Monoclonal Antibody HCM31 in Rat Small Intestinal Mucosa with Primary Infection and Reinfection

Experimental Parasitology. Dec, 2009  |  Pubmed ID: 19703448

Infections with the parasitic helminth, Nippostrongylus brasiliensis, cause changes in rat small intestinal goblet cell mucin, particularly in the peripheral sugar residues of oligosaccharide. These changes may correlate with expulsion. In this study, we examined changes in mucin oligosaccharides caused by primary infection and reinfection with N. brasiliensis, using two monoclonal antibodies, HCM31 and PGM34, that react with sialomucin and sulfomucin, respectively. Enzyme-linked immunosorbent assay of jejunal mucins showed that the relative reactivity of mucins with HCM31, but not PGM34, increased up to 16 days after primary infection and 6 days after reinfection, the times when the worms were expelled from the rats. Immunohistochemical studies confirmed that goblet cells stained with HCM31 greatly increased at the time of worm expulsion. These results indicate that the marked increase observed in HCM31-reactive sialomucins may be related to expulsion of the worms.

Nanoscale Evaluation of Lubricity on Well-defined Polymer Brush Surfaces Using QCM-D and AFM

Colloids and Surfaces. B, Biointerfaces. Nov, 2009  |  Pubmed ID: 19720506

For preparing a "highly lubricated biointerface", which has both excellent lubricity and biocompatibility, we investigated the factors responsible for resistance to friction during polymer grafting. We prepared poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), poly(2-hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate) (PMMA) brush layers with high graft density and well-controlled thickness using atom transfer radical polymerization (ATRP). We measured the water absorptivity in the polymer brush layers and the viscoelasticity of the polymer-hydrated layers using a quartz crystal microbalance with dissipation monitoring (QCM-D) measurements. The PMPC brush layer had the highest water absorptivity, while the PMPC-hydrated layer had the highest fluidity. The friction properties of the polymer brush layers were determined in air, water, and toluene by atomic force microscopy (AFM). The friction on each polymer brush decreased only when a good solvent was chosen for each polymer. In conclusion, the brush layer possessing high water absorptivity and fluidity in water contributes to reduce friction. PMPC grafting is an effective and promising method for obtaining highly lubricated biointerfaces.

[Progress of Research in Osteoarthritis. Invention of Longer Lasting Artificial Joints]

Clinical Calcium. Nov, 2009  |  Pubmed ID: 19880996

In the advent of the aging society, the lifetime of artificial joints is a matter of concern. The major cause of revision surgery is periprosthetic osteolysis caused by polyethylene wear particles. To prevent osteolysis, both the reduction of wear and the suppression of osteoclast induction are necessary. For these purposes, we developed a new technology for grafting 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer on the surface of polyethylene liners. On the basis of encouraging results of the preclinical studies, we have started a large-scale clinical trial of new artificial hip joints since 2007.

Graft Linker Immobilization for Spatial Control of Protein Immobilization Inside Fused Microchips

Electrophoresis. Dec, 2009  |  Pubmed ID: 20013908

Fused silica glass microchips have several attractive features for lab-on-a-chip applications; they can be machined with excellent precision down to nanospace; are stable; transparent and can be modified with a range of silanization agents to change channel surface properties. For immobilization, however, ligands must be added after bonding, since the harsh bonding conditions using heat or hydrofluoric acid would remove all prior immobilized ligands. For spatial control over immobilization, UV-mediated immobilization offers several advantages; spots can be created in parallel, the feature size can be made small, and spatial control over patterns and positions is excellent. However, UV sensitive groups are often based on hydrophobic chemical moieties, which unfortunately result in greater non-specific binding of biomolecules, especially proteins. Here, we present techniques in which any -CH(x) (x=1,2,3) containing surface coating can be used as foundation for grafting a hydrophilic linker with a chemical anchor, a carboxyl group, to which proteins and amine containing molecules can be covalently coupled. Hence, the attractive features of many well-known protein and biomolecule repelling polymer coatings can be utilized while achieving site-specific immobilization only to pre-determined areas within the bonded microchips.

Self-initiated Surface Graft Polymerization of 2-methacryloyloxyethyl Phosphorylcholine on Poly(ether Ether Ketone) by Photoirradiation

ACS Applied Materials & Interfaces. Mar, 2009  |  Pubmed ID: 20355972

In the present paper, we reported the fabrication of a highly hydrophilic nanometer-scale modified surface on a poly(ether ether ketone) (PEEK) substrate by photoinduced graft polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) in the absence of photoinitiators. Photoirradiation results in the generation of semibenzopinacol-containing radicals of benzophenone units in the PEEK molecular structure, which acts as a photoinitiator during graft polymerization. The poly(MPC)-grafted PEEK surface fabricated by a novel and simple polymerization system exhibited unique characteristics such as high wettability and high antiprotein adsorption, which makes it highly suitable for medical applications.

Feasibility Study of Introducing Redox Property by Modification of PMBN Polymer for Biofuel Cell Applications

Applied Biochemistry and Biotechnology. Feb, 2010  |  Pubmed ID: 19455430

In this study, the feasibility of introducing redox property to an amphiphilic phospholipid polymer (PMBN) was investigated. The active ester group in the side chain of the polymer was used to react with pyrroloquinoline quinine (PQQ). Redox peaks that corresponded to PQQ redox potentials were observed after the modification. Glucose oxidase was immobilized to the modified polymer. Electrochemical oxidation of glucose was carried out with the polymer electrode. The oxidation current increased with elevating glucose concentration indicating electron transfer established between the electrode and enzyme. It suggests that by modification, PMBN is possible to use for enzyme electrode for bioelectronics.

Lubricity and Stability of Poly(2-methacryloyloxyethyl Phosphorylcholine) Polymer Layer on Co-Cr-Mo Surface for Hemi-arthroplasty to Prevent Degeneration of Articular Cartilage

Biomaterials. Feb, 2010  |  Pubmed ID: 19819011

Migration of the artificial femoral head to the inside of the pelvis due to the degeneration of acetabular cartilage has emerged as a serious issue in resurfacing or bipolar hemi-arthroplasty. Surface modification of cobalt-chromium-molybdenum alloy (Co-Cr-Mo) is one of the promising means of improving lubrication for preventing the migration of the artificial femoral head. In this study, we systematically investigated the surface properties, such as lubricity, biocompatibility, and stability of the various modification layers formed on the Co-Cr-Mo with the biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer by dip coating or grafting. The cartilage/poly(MPC) (PMPC)-grafted Co-Cr-Mo interface, which mimicked a natural joint, showed an extremely low friction coefficient of <0.01, as low as that of a natural cartilage interface. Moreover, the long-term stability in water was confirmed for the PMPC-grafted layer; no hydrolysis of the siloxane bond was observed throughout soaking in phosphate-buffered saline for 12 weeks. The PMPC-grafted Co-Cr-Mo femoral head for hemi-arthroplasty is a promising option for preserving acetabular cartilage and extending the duration before total hip arthroplasty.

Self-initiated Surface Grafting with Poly(2-methacryloyloxyethyl Phosphorylcholine) on Poly(ether-ether-ketone)

Biomaterials. Feb, 2010  |  Pubmed ID: 19906420

Poly(ether-ether-ketone) (PEEK)s are a group of polymeric biomaterials with excellent mechanical properties and chemical stability. In the present study, we demonstrate the fabrication of an antibiofouling and highly hydrophilic high-density nanometer-scaled layer on the surface of PEEK by photo-induced graft polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) without using any photo-initiators, i.e., "self-initiated surface graft polymerization." Our results indicated that the diphenylketone moiety in the polymer backbone acted as a photo-initiator similar to benzophenone. The density and thickness of the poly(MPC) (PMPC)-grafted layer were controlled by the photo-irradiation time and monomer concentration during polymerization, respectively. Since MPC is a highly hydrophilic compound, the water wettability (contact angle <10 degrees) and lubricity (coefficient of dynamic friction <0.01) of the PMPC-grafted PEEK surface were considerably lower than those of the untreated PEEK surface (90 degrees and 0.20, respectively) due to the formation of a PMPC nanometer-scale layer. In addition, the amount (0.05 microg/cm(2)) of BSA adsorbed on the PMPC-grafted PEEK surface was considerably lower, that is more than 90% reduction, compared to that (0.55 microg/cm(2)) for untreated PEEK. This photo-induced polymerization process occurs only on the surface of the PEEK substrate; therefore, the desirable mechanical properties of PEEK would be maintained irrespective of the treatment used.

Cell-penetrating Macromolecules: Direct Penetration of Amphipathic Phospholipid Polymers Across Plasma Membrane of Living Cells

Biomaterials. Mar, 2010  |  Pubmed ID: 20004016

Nanoscaled materials are normally engulfed in endosomes by energy-dependent endocytosis and fail to access the cytosolic cell machinery. Although some biomolecules may penetrate non-endocytically or fuse with plasma membranes without overt membrane disruption, to date no synthetic macromolecule of comparable size has been shown to exhibit this property. Here, we discovered mechanism of direct cell membrane penetration using synthetic phospholipid polymers. These water-soluble amphiphilic phospholipid polymers enter the cytoplasm of living mammalian cells in vitro within a few minutes without overt bilayer disruption even under conditions where energy-dependent endocytic uptakes are blocked. Furthermore, targeted cytosolic distribution to cell organelles was achieved by selecting specific fluorescent tags to the polymers. Thus, the phospholipid polymers can provide a new way of thinking about access to the cellular interior, namely direct membrane penetration.

Super-hydrophilic Silicone Hydrogels with Interpenetrating Poly(2-methacryloyloxyethyl Phosphorylcholine) Networks

Biomaterials. Apr, 2010  |  Pubmed ID: 20117831

We synthesized silicone hydrogels from 2-methacryloyloxyethyl phosphorylcholine (MPC) and bis(trimethylsilyloxy)methylsilylpropyl glycerol methacrylate (SiMA) using two methods: random copolymerization with a small amount of cross-linker (P(SiMA-co-MPC)) and construction of an interpenetration network (IPN) structure composed of cross-linked poly(MPC)(PMPC) chains and cross-linked poly(SiMA)(PSiMA) chains (PSiMA-ipn-PMPC). The polymerization was carried out by photoreaction. The surface hydrophilicity and water absorbability of P(SiMA-co-MPC) increased with an increase in the MPC unit composition. On the other hand, in the case of PSiMA-ipn-PMPC, a super-hydrophilic surface was obtained by the surface enrichment of MPC units. The optical and mechanical properties of PSiMA-ipn-PMPC are suitable for use as a material for preparing contact lenses. In addition, the oxygen permeability of PSiMA-ipn-PMPC remains high because of the PSiMA chains. The MPC units at the surface of the hydrogels reduce protein adsorption effectively. From these results for PSiMA-ipn-PMPC, we confirmed that it has the potential for application to silicone hydrogel contact lenses.

The Prevention of Peritendinous Adhesions by a Phospholipid Polymer Hydrogel Formed in Situ by Spontaneous Intermolecular Interactions

Biomaterials. May, 2010  |  Pubmed ID: 20149434

Preventing peritendinous adhesions after surgical repair of tendon is difficult. In order to establish an ideal anti-adhesion material, we prepared a spontaneously forming hydrogel by mixing the aqueous solutions of two polymers, poly(MPC-co-methacrylic acid) (PMA) and amphiphilic poly(MPC-co-n-butyl methacrylate) (PMB), in the presence of Fe(3+). This PMA/PMB/Fe(3+) hydrogel (MPC polymer hydrogel) had a honeycomb microstructure with nanometer-scale pores, which resist cell invasion but allow the passage of cytokines and growth factors for tendon healing. The dissociation rate of the hydrogel could be controlled by changing Fe(3+) concentration, and by examining the viscoelasticity of the hydrogel, we determined the optimal Fe(3+) concentration to be 0.05 M. We then examined the effects of the in situ application of this MPC polymer hydrogel containing 0.05 M Fe(3+) by using two animal models: the rat Achilles tendon model and the chicken flexor digitorum profundus tendon model. In both models, macroscopic and histological observation revealed that peritendinous adhesions were significantly decreased by the hydrogel application. Mechanical analyses revealed that the hydrogel prevented peritendinous adhesions but did not affect the tendon healing. Because of its characteristic microstructure and excellent biocompatibility, we believe that the MPC polymer hydrogel will be ideal for preventing peritendinous adhesions.

Stabilization of Phospholipid Polymer Surface with Three-dimensional Nanometer-scaled Structure for Highly Sensitive Immunoassay

Colloids and Surfaces. B, Biointerfaces. Jun, 2010  |  Pubmed ID: 20197230

A phospholipid polymer platform and an antibody as a bioaffinity ligand were used to construct a biointerface for a highly sensitive immunoassay. The platform had a nanometer-scaled particle deposition surface and it was constructed with poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)] (PMBN) by an electrospray deposition (ESD) method. The PMBN surface could immobilize specific antibodies through covalent chemical bonding by the reaction between MEONP units and amino groups in the antibody. In addition, the PMBN could prevent nonspecific protein adsorption from an analyte. However, the nanometer-scaled structure of the PMBN lost its shape after immersion in an aqueous medium. To stabilize the nanometer-scaled structure in an aqueous medium, the PMBN was cross-linked with 1,4-butylenediamine and then heat-treated. These treatments effectively improved the stability of the nanometer-scaled structure, that is, the structure had a high porosity even after immersing in an aqueous medium. The stabilization affected the specific signal in the enzyme-linked immunosorbent assay (ELISA), that is, the specific signal in ELISA was enhanced.

Vulnerable Sites and Changes in Mucin in the Rat Small Intestine After Non-steroidal Anti-inflammatory Drugs Administration

Digestive Diseases and Sciences. Dec, 2010  |  Pubmed ID: 20300842

The location of mucosal damage and changes in mucin content in the rat small intestine following administration of non-steroidal anti-inflammatory drugs (NSAIDs) have not been well elucidated.

Phospholipid Polymer Biointerfaces for Lab-on-a-Chip Devices

Annals of Biomedical Engineering. Jun, 2010  |  Pubmed ID: 20358288

This review summarizes recent achievements and progress in the development of various functional 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer biointerfaces for lab-on-a-chip devices and applications. As phospholipid polymers, MPC polymers can form cell-membrane-like surfaces by surface chemistry and physics and thereby provide biointerfaces capable of suppressing protein adsorption and many subsequent biological responses. In order to enable application to microfluidic devices, a number of MPC polymers with diverse functions have been specially designed and synthesized by incorporating functional units such as charge and active ester for generating the microfluidic flow and conjugating biomolecules, respectively. Furthermore, these polymers were incorporated with silane or hydrophobic moiety to construct stable interfaces on various substrate materials such as glass, quartz, poly(methyl methacrylate), and poly(dimethylsiloxane), via a silane-coupling reaction or hydrophobic interactions. The basic interfacial properties of these interfaces have been characterized from multiple aspects of chemistry, physics, and biology, and the suppression of nonspecific bioadsorption and control of microfluidic flow have been successfully achieved using these biointerfaces on a chip. Further, many chip-based biomedical applications such as immunoassays and DNA separation have been accomplished by integrating these biointerfaces on a chip. Therefore, functional phospholipid polymer interfaces are promising and useful for application to lab-on-a-chip devices in biomedicine.

An Efficient Surface Modification Using 2-methacryloyloxyethyl Phosphorylcholine to Control Cell Attachment Via Photochemical Reaction in a Microchannel

Lab on a Chip. Aug, 2010  |  Pubmed ID: 20498909

This report describes a direct approach for cell micropatterning in a closed glass microchannel. To control the cell adhesiveness inside the microchannel, the application of an external stimulus such as ultraviolet (UV) was indispensible. This technique focused on the use of a modified 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, which is known to be a non-biofouling compound that is a photocleavable linker (PL), to localize cells via connection to an amino-terminated silanized surface. Using UV light illumination, the MPC polymer was selectively eliminated by photochemical reaction that controlled the cell attachment inside the microchannel. For suitable cell micropatterning in a microchannel, the optimal UV illumination time and concentration for cell suspension were investigated. After selective removal of the MPC polymer through the photomask, MC-3T3 E1 cells and vascular endothelial cells (ECs) were localized only to the UV-exposed area. In addition, the stability of patterned ECs was also confirmed by culturing for 2 weeks in a microchannel under flow conditions. Furthermore, we employed two different types of cells inside the same microchannel through multiple removal of the MPC polymer. ECs and Piccells were localized in both the upper and down streams of the microchannel, respectively. When the ECs were stimulated by adenosine triphosphate (ATP), NO was secreted from the ECs and could be detected by fluorescence resonance energy transfer (FRET) in Piccells, which is a cell-based NO indicator. This technique can be a powerful tool for analyzing cell interaction research.

A Microfluidic Hydrogel Capable of Cell Preservation Without Perfusion Culture Under Cell-based Assay Conditions

Advanced Materials (Deerfield Beach, Fla.). Jul, 2010  |  Pubmed ID: 20503209

2-Methacryloyloxyethyl Phosphorylcholine Polymer (MPC)-coating Improves the Transfection Activity of GALA-modified Lipid Nanoparticles by Assisting the Cellular Uptake and Intracellular Dissociation of Plasmid DNA in Primary Hepatocytes

Biomaterials. Aug, 2010  |  Pubmed ID: 20537380

We previously reported that modification of GALA peptide on the surface of liposomes enhanced fusion with endosomal membrane, and cytoplasmic release of encapsulated macromolecules. We report herein that an additional coating of GALA-modified liposomes with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer resulted in a two order of magnitude enhancement in the transfection activity of encapsulating plasmid DNA (pDNA). Quantification of the delivered gene copies in whole cells and isolated nuclei revealed that the increase of transfection activity can be attributed to improved efficiencies in cellular uptake and post-nuclear delivery processes. Imaging studies revealed that the intracellular dissociation of pDNA from the lipid envelope is enhanced by GALA modification and further coating with MPC polymer in a stepwise manner. The MPC polymer-coating decreased the zeta-potential of GALA-modified liposomes, suggesting that it assisted in the functional display of negatively charged GALA on the cationic liposomes by providing shielding from mutual electrostatic interactions. Collectively, these data indicate that MPC polymer-coating induced the fusogenic activity of the GALA-modified envelope with endosomes, leading to a more effective cytoplasmic release pDNA. The extensive fusion of the lipid envelope may also reduce electrostatic interactions between mRNA and cationic lipid components, thereby resulting in an enhancement in the translation process.

Simple Surface Modification of a Titanium Alloy with Silanated Zwitterionic Phosphorylcholine or Sulfobetaine Modifiers to Reduce Thrombogenicity

Colloids and Surfaces. B, Biointerfaces. Sep, 2010  |  Pubmed ID: 20547042

Thrombosis and thromboembolism remain problematic for a large number of blood contacting medical devices and limit broader application of some technologies due to this surface bioincompatibility. In this study we focused on the covalent attachment of zwitterionic phosphorylcholine (PC) or sulfobetaine (SB) moieties onto a TiAl(6)V(4) surface with a single step modification method to obtain a stable blood compatible interface. Silanated PC or SB modifiers (PCSi or SBSi) which contain an alkoxy silane group and either PC or SB groups were prepared respectively from trimethoxysilane and 2-methacryloyloxyethyl phosphorylcholine (MPC) or N-(3-sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium betaine (SMDAB) monomers by a hydrosilylation reaction. A cleaned and oxidized TiAl(6)V(4) surface was then modified with the PCSi or SBSi modifiers by a simple surface silanization reaction. The surface was assessed with X-ray photoelectron spectroscopy (XPS), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and contact angle goniometry. Platelet deposition and bulk phase activation were evaluated following contact with anticoagulated ovine blood. XPS results verified successful modification of the PCSi or SBSi modifiers onto TiAl(6)V(4) based on increases in surface phosphorous or sulfur respectively. Surface contact angles in water decreased with the addition of hydrophilic PC or SB moieties. Both the PCSi and SBSi modified TiAl(6)V(4) surfaces showed decreased platelet deposition and bulk phase platelet activation compared to unmodified TiAl(6)V(4) and control surfaces. This single step modification with PCSi or SBSi modifiers offers promise for improving the surface hemocompatibility of TiAl(6)V(4) and is attractive for its ease of application to geometrically complex metallic blood contacting devices.

Simple Synthesis of a Library of Zwitterionic Surfactants Via Michael-type Addition of Methacrylate and Alkane Thiol Compounds

Langmuir : the ACS Journal of Surfaces and Colloids. Aug, 2010  |  Pubmed ID: 20695536

A library of zwitterionic phosphorylcholine surfactants with various alkyl chain lengths and compositions was readily prepared from a combination of 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) and various alkanethiol compounds via Michael-type addition. The critical micelle concentration (CMC) was evaluated by surface tensiometry. At the CMC, the surface tension of these aqueous solutions decreased significantly to below 40 mN/m. For the zwitterionic surfactants composed of MPC, and fluoroalkane and alkanedithiol, the surface tension was approximately 24, and 50 mN/m, respectively. From the equation log (CMC) = A - Bn, where n is the number of carbon atoms in the alkyl chain, A and B were calculated as 1.09 and 0.44, respectively. The value of the slope B was between that of ionic (B = 0.30) and nonionic surfactants (B = 0.50). Despite the ionic nature of the surfactants, their behavior was closer to that of nonionic surfactants.

Reduction of Protein Adsorption on Well-characterized Polymer Brush Layers with Varying Chemical Structures

Colloids and Surfaces. B, Biointerfaces. Nov, 2010  |  Pubmed ID: 20705439

To clarify protein adsorption behavior on polymer brush layers, surface characteristics and protein adsorption repellency on polymer brush layers should be precisely determined. Here, we clearly delineated the chemical structure of the polymer brush layers containing various hydrophilic groups, namely, phosphorylcholine, sulfoxybetaine, carboxybetaine (zwitterionic), and hydroxyl group (nonionic) and examined the effects of the chemical structure on initial protein adsorption behavior. Kinetic analysis performed during surface-initiated atom transfer radical polymerization revealed that graft polymerization proceeded in a living manner. The graft density of each type of polymer chain and its surface coverage were high enough to form dense polymer brush structures. The hydroxyl group-bearing polymer brush structure exhibited the highest graft density. Among the zwitterionic polymer brush structures, the graft density and surface coverage of sulfoxybetaine- and carboxybetaine-bearing polymer chains were higher than those of the phosphorylcholine-bearing polymer chains. The amount of protein relative to 100% serum adsorbed on polymer brush layers was quantified using quartz crystal microbalance with dissipation (QCM-D). Protein adsorption on all zwitterionic polymer brush layers apparently decreased with increasing thickness of the grafted polymer layers. Protein adsorption was highly suppressed on thick polymer brush layers bearing phosphorylcholine or sulfoxybetaine groups. However, the amount of proteins adsorbed on thick polymer brush layers bearing hydroxyl groups was 10 times more than that adsorbed on polymer brush layers bearing phosphorylcholine groups. Thus, we concluded that the chemical structure of the polymer brush layer is a significant factor affecting resistance to protein adsorption even for dense polymer brush structures.

The Biological Performance of Cell-containing Phospholipid Polymer Hydrogels in Bulk and Microscale Form

Biomaterials. Dec, 2010  |  Pubmed ID: 20732713

The biological performances of a cell-containing phospholipid polymer hydrogel in bulk and miniaturized formats without an additional culture medium support were investigated and compared. The cell-containing hydrogel was formed spontaneously when solutions of commercial polyvinyl alcohol (PVA) and the phospholipid polymer poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-vinylphenylboronic acid (VPBA)] (PMBV) suspended with cells in a cell culture medium are mixed together. Bulk and miniaturized hydrogels, with approximate thicknesses of 3.1 mm and 400 μm, respectively, were prepared in a 96-well microplate and a glass microchip, respectively. In both cases, the hydrogels were homogeneous, and cells were spatially encapsulated. The long-term observation (4 and 8 days) of cell morphology suggested that cells were passively attached to the interface of the hydrogel but were unable to spread and flatten, which inhibited cell growth in both hydrogels. Viability evaluations revealed that cells in both hydrogel formats maintained the same high viability levels after long-term encapsulation. Cytotoxicity assays indicated that the cells in the miniaturized hydrogel maintained a high degree of correlation in cytotoxic sensitivity with the cells in the bulk hydrogel and a routine medium culture. The PMBV/PVA hydrogel not only provides a beneficial cytocompatible microenvironment for long-term cell survival without an additional culture medium support but also creates a static condition for cell sustainment in a microchip similar to that in bulk. The uniform long-term performances of PMBV/PVA hydrogels in bulk and miniaturized formats make them ideal for the development of long-term, flexible, three-dimensional, living cell-based tools for routine cell-based assays and applications on bulk to microscale levels.

Control of Cell Function on a Phospholipid Polymer Having Phenylboronic Acid Moiety

Biomedical Materials (Bristol, England). Oct, 2010  |  Pubmed ID: 20876952

We synthesized a water-insoluble phospholipid polymer bearing a phenylboronic acid moiety (PMBV), which induces cell adhesion through a specific interaction with the glycoprotein, fibronectin. Surface plasmon resonance analysis revealed that fibronectin was adsorbed on the PMBV surface. When fibroblasts were cultured on the PMBV surface, the cells adhered and proliferated normally while showing a spherical morphology. In addition, the adherent cells were able to detach after the addition of sugar molecules, which bound to phenylboronic acid through an exchange reaction. The cell cycle of adherent cells was evaluated with the embedded HeLa-Fucci cells by using a fluorescent ubiquitination-based cell cycle indicator. The cell-cycle analysis by fluorescence microscopy indicated that the adherent HeLa-Fucci cells tended to converge to the G1 phase. The differentiation of mesenchymal stem cells to chondrocytes was accelerated on PMBV in the presence of bone morphogenetic protein-2. We concluded that PMBV is a useful surface in experiments for assessing cellular function and differentiation.

Single-cell Attachment and Culture Method Using a Photochemical Reaction in a Closed Microfluidic System

Biomicrofluidics. Sep, 2010  |  Pubmed ID: 21045929

Recently, interest in single cell analysis has increased because of its potential for improving our understanding of cellular processes. Single cell operation and attachment is indispensable to realize this task. In this paper, we employed a simple and direct method for single-cell attachment and culture in a closed microchannel. The microchannel surface was modified by applying a nonbiofouling polymer, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, and a nitrobenzyl photocleavable linker. Using ultraviolet (UV) light irradiation, the MPC polymer was selectively removed by a photochemical reaction that adjusted the cell adherence inside the microchannel. To obtain the desired single endothelial cell patterning in the microchannel, cell-adhesive regions were controlled by use of round photomasks with diameters of 10, 20, 30, or 50 μm. Single-cell adherence patterns were formed after 12 h of incubation, only when 20 and 30 μm photomasks were used, and the proportions of adherent and nonadherent cells among the entire UV-illuminated areas were 21.3%±0.3% and 7.9%±0.3%, respectively. The frequency of single-cell adherence in the case of the 20 μm photomask was 2.7 times greater than that in the case of the 30 μm photomask. We found that the 20 μm photomask was optimal for the formation of single-cell adherence patterns in the microchannel. This technique can be a powerful tool for analyzing environmental factors like cell-surface and cell-extracellular matrix contact.

Novel Polymer Biomaterials and Interfaces Inspired from Cell Membrane Functions

Biochimica Et Biophysica Acta. Mar, 2011  |  Pubmed ID: 20435095

Materials with excellent biocompatibility on interfaces between artificial system and biological system are needed to develop any equipments and devices in bioscience, bioengineering and medicinal science. Suppression of unfavorable biological response on the interface is most important for understanding real functions of biomolecules on the surface. So, we should design and prepare such biomaterials. SCOOP OF REVIEW: One of the best ways to design the biomaterials is generated from mimicking a cell membrane structure. It is composed of a phospholipid bilayered membrane and embedded proteins and polysaccharides. The surface of the cell membrane-like structure is constructed artificially by molecular integration of phospholipid polymer as platform and conjugated biomolecules. Here, it is introduced as the effectiveness of biointerface with highly biological functions observed on artificial cell membrane structure.

Spontaneous Formation of a Hydrogel Composed of Water-Soluble Phospholipid Polymers Grafted with Enantiomeric Oligo(lactic Acid) Chains

Journal of Biomaterials Science. Polymer Edition. 2011  |  Pubmed ID: 20546676

We designed and synthesized water-soluble biocompatible and biodegradable polymers composed of 2-methacryroyloxyethyl phosphorylcholine and oligo(L- or D-lactic acid) macromonomers to develop an injectable hydrogel matrix. Aqueous solutions containing the polymers with oligo(L-lactic acid) (OLLA) and oligo(D-lactic acid) (ODLA) chains underwent spontaneous gelation when mixed together. This was due to the formation of a stereocomplex between the OLLA and ODLA side-chains, which act as cross-linking components in the hydrogel. Therefore, the hydrogel could be re-dissolved in a buffer solution by hydrolysis of the oligo(lactic acid) chains. We obtained an injectable, biocompatible and degradable hydrogel, and we anticipate that it will be used in applications involving the controlled release of bioactive molecules and cell-based tissue engineering.

Spatial Distribution of Intraperitoneally Administrated Paclitaxel Nanoparticles Solubilized with Poly (2-methacryloxyethyl Phosphorylcholine-co N-butyl Methacrylate) in Peritoneal Metastatic Nodules

Cancer Science. Jan, 2011  |  Pubmed ID: 20942868

Intraperitoneal (i.p.) administration of paclitaxel nanoparticles (PTX-30W) prepared by solubulization with the amphiphilic copolymer of 2-methacryloxyethyl phosphorylcholine and n-butyl methacrylate can efficiently suppress the growth of peritoneal metastasis. In this study, we characterized the drug distribution of i.p. injected PTX-30W in peritoneal tumor and liver in a mouse model using MKN45, human gastric cancer cells. Oregon green-conjugated PTX-30W showed perivascular accumulation in MKN45 tumor in the peritoneum at 24 h after intravenous (i.v.) injection; however, the amount of PTX in tumor was markedly less than that in liver. In contrast, a larger amount of PTX accumulated in the peripheral area of disseminated nodules at 1 h after i.p. injection and the area gradually enlarged. The depth of PTX infiltration reached 1 mm from the tumor surface at 48 h after i.p. injection, and the fluorescence intensity was markedly greater than that in liver. Interestingly, i.p. injected PTX preferentially accumulated in relatively hypovascular areas, and many tumor cells in the vicinity of PTX accumulation showed apoptosis. This unique accumulation pattern and lesser washout in hypovascular areas are thought to be attributable to the superior penetrating activity of PTX-30W, and thus, PTX-30W is considered to be highly suitable for i.p. chemotherapy for peritoneal dissemination.

Effect of Hydrophilic Polymer Conjugation on Heat-induced Conformational Changes in a Protein

Acta Biomaterialia. Apr, 2011  |  Pubmed ID: 21111070

End-functional 2-methacryloyloxyethyl phosphorylcholine (MPC) co-polymers containing two different monomer units, 2-hydroxyethyl methacrylate (HEMA) and n-butyl methacrylate (BMA), with varying hydrophilicities were synthesized to investigate the effect of the conjugated hydrophilic polymer on the heat-induced conformational changes of a protein. MPC co-polymer-conjugated proteins containing the HEMA unit (PMH) could withstand thermal conformational changes better than those containing the more hydrophobic BMA unit (PMB). The changes in protein tertiary structures were estimated via the excitation of tryptophan. PMH-conjugated proteins could withstand heat-induced intensity changes better than the PMB-conjugated proteins. Thus, hydrophilic units in the conjugated polymer are probably essential in suppressing the heat-induced conformational changes of a protein. The changes in secondary and tertiary structures of poly(MPC)-(PMPC) and poly(HEMA) (PHEMA)-conjugated proteins were compared to validate the effect of MPC units on heat-induced conformational change. Although the thermally induced conformational changes in the secondary and tertiary structures of PHEMA-conjugated proteins were partially suppressed, the effect on PMPC-conjugated proteins was much greater, with significant conformational preservation. This is due to the specific hydration state of the hydrophilic PMPC chain, which reduces interaction between the protein molecules.

Protective Effect of Geranylgeranylacetone Against Loxoprofen Sodium-induced Small Intestinal Lesions in Rats

European Journal of Pharmacology. Feb, 2011  |  Pubmed ID: 21130762

Nonsteroidal anti-inflammatory drugs induce small intestinal ulcers but the preventive measures against it remain unknown. So we evaluated the effect of geranylgeranylacetone (GGA), a mucosal protectant, on both the mucus content and loxoprofen sodium-induced lesions in the rat small intestine. Normal male Wistar rats were given GGA (200 or 400mg/kg p.o.) and euthanized 3h later for measurement of mucin content and immunoreactivity. Other Wistar rats were given loxoprofen sodium (30mg/kg s.c.) and euthanized 24h later. GGA (30-400mg/kg p.o.) was administered twice: 30min before and 6h after loxoprofen sodium. The total mucin content of the small intestinal mucosa increased, especially the ratio of sialomucin, which increased approximately 20% more than the control level after a single dose of GGA. Loxoprofen sodium provoked linear ulcers along the mesenteric margin of the distal jejunum, accompanied by an increase in enterobacterial translocation. Treatment of the animals with GGA dose-dependently prevented the development of intestinal lesions, and bacterial translocation following loxoprofen sodium was also significantly decreased. GGA protects the small intestine against loxoprofen sodium-induced lesions, probably by inhibiting enterobacterial invasion of the mucosa as a result of the increase in the mucosal barrier.

Cartilage-mimicking, High-density Brush Structure Improves Wear Resistance of Crosslinked Polyethylene: a Pilot Study

Clinical Orthopaedics and Related Research. Aug, 2011  |  Pubmed ID: 21132412

In natural synovial joints under physiologic conditions, fluid thin-film lubrication by a hydrated layer of the cartilage is essential for the smooth motion of the joints. The considerably less efficient lubrication of artificial joints of polyethylene is prone to wear, leading to osteolysis and aseptic loosening and limiting the longevity of THA. A nanometer-scale layer of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) with cartilage-mimicking brushlike structures on a crosslinked polyethylene (CLPE) surface may provide hydrophilicity and lubricity resembling the physiologic joint surface.

Tissue Response to Poly(L-lactic Acid)-based Blend with Phospholipid Polymer for Biodegradable Cardiovascular Stents

Biomaterials. Mar, 2011  |  Pubmed ID: 21185597

A temporary cardiovascular stent device by bioabsorbable materials might reduce late stent thrombosis. A water-soluble amphiphilic phospholipid polymer bearing phosphorylcholine groups (PMB30W) was blended with a high-molecular-weight poly(l-lactic acid) (PLLA) to reduce unfavorable tissue responses at the surface. The PLLA implants and the polymer blend (PLLA/PMB30W) implants were inserted into subcutaneous tissues of rats, the infrarenal aorta of rats, and the internal carotid arteries of rabbits. After 6 months subcutaneous implantation, the PLLA/PMB30W maintained high density of phosphorylcholine groups on the surface without a significant bioabsorption. After intravascular implantation, the cross-sectional areas of polymer tubing with diameters less than 1.6 mm were histomorphometrically measured. Compared to the PLLA tubing, the PLLA/PMB30W tubing significantly reduced the thrombus formation during 30 d of implantation. Human peripheral blood mononuclear cells were cultured on the PLLA and the PLLA/PMB30W to compare inflammatory reactions. Enzyme-linked immunosorbent assay quantified substantially decreased proinflammatory cytokines in the case of the PLLA/PMB30W. They were almost the same level as the negative controls. Thus, we conclude that the phosphorylcholine groups could reduce tissue responses significantly both in vivo and in vitro, and the PLLA/PMB30W is a promising material for preparing temporary cardiovascular stent devices.

Bleomycin Hydrolase is Regulated Biphasically in a Differentiation- and Cytokine-dependent Manner: Relevance to Atopic Dermatitis

The Journal of Biological Chemistry. Mar, 2011  |  Pubmed ID: 21190945

Loss-of-function mutation in the profilaggrin gene is a major risk factor for atopic dermatitis (AD). Previously, we showed that a neutral cysteine protease, bleomycin hydrolase (BH), has a role in generating natural moisturizing factors, and calpain I is an upstream protease in the filaggrin degradation pathway. Here, we investigated the transcriptional regulatory mechanisms of BH and the relevance of BH to AD. First, we cloned the 5'-flanking region of BH. Deletion analyses identified a critical region for BH promoter activity within -216 bp upstream. Electrophoretic mobility shift assay revealed that MZF-1, Sp-1, and interferon regulatory factor-1/2 could bind to this region in vitro. Moreover, site-directed mutagenesis of the MZF-1 and Sp-1 motifs markedly reduced BH promoter activity. These data indicate that BH expression is up-regulated via MZF-1 and Sp-1. Interestingly, a Th1 cytokine, IFN-γ, significantly reduced the expression of BH. Analyses with site-directed mutagenesis and small interference RNA supported the suppressing effect of IFN-γ on BH expression. On the other hand, a Th2 cytokine, IL-4, did not show any direct effect on BH expression. However, it down-regulated MZF-1 and Sp-1 in cultured keratinocytes, indicating that IL-4 could work as a suppressor in BH regulation. Lastly, we examined expression of BH in skins of patients with AD. BH activity and expression were markedly decreased in AD lesional skin, suggesting a defect of the filaggrin degradation pathway in AD. Our results suggest that BH transcription would be modulated during both differentiation and inflammation.

Reduction of Peritendinous Adhesions by Hydrogel Containing Biocompatible Phospholipid Polymer MPC for Tendon Repair

The Journal of Bone and Joint Surgery. American Volume. Jan, 2011  |  Pubmed ID: 21248211

Peritendinous adhesions are serious complications after surgical repair of tendons. As an anti-adhesion material, we focused on 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, our original biocompatible polymer, and prepared an aqueous solution of MPC-containing polymer called poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid) (PMBV), which can be formed into hydrogel properties by mixture with another aqueous polymer, poly(vinyl alcohol) (PVA). The objective of the present study was to examine the possible application of the MPC hydrogel for the reduction of peritendinous adhesions.

Significance of Antibody Orientation Unraveled: Well-oriented Antibodies Recorded High Binding Affinity

Analytical Chemistry. Mar, 2011  |  Pubmed ID: 21338074

To investigate the effect of antibody orientation on its immunological activities, we developed a novel and versatile platform consisting of a well-defined phospholipid polymer surface on which staphylococcal protein A (SpA) was site-selectively immobilized. The application of a biocompatible phospholipid-based platform ensured minimal denaturation of immobilized antibodies, and the site-selective immobilization of SpA clarified the effect of antibody orientation on immunological activities. The phospholipid polymer platform was prepared on silicon substrates using the surface-initiated atom transfer radical polymerization (SI-ATRP) technique. An enzymatic reaction was performed for orientation-selective coupling of SpA molecules to the polymer brush surface. Orientation-controlled antibodies were achieved using enzymatic reactions, and these antibodies captured 1.8 ± 0.1 antigens on average, implying that at least 80% of immobilized antibodies reacted with two antigens. Theoretical multivalent binding analysis further revealed that orientation-controlled antibodies had antigen-antibody reaction equilibrium dissociation constants (K(d)) as low as 8.6 × 10(-10) mol/L, whereas randomly oriented and partially oriented antibodies showed K(d) values of 2.0 × 10(-7) and 1.2 × 10(-7) mol/L, respectively. Strict control of antibody orientation not only formed an approximately 100-fold stronger antigen-antibody complex than the controls but also sustained the native antibody K(d) (10(-10)-10(-9) mol/L). These findings support the significance of antibody orientation because controlling the orientation resulted in high reactivity and theoretical binding capacity.

Well-controlled Cationic Water-soluble Phospholipid Polymer-DNA Nanocomplexes for Gene Delivery

Bioconjugate Chemistry. Jun, 2011  |  Pubmed ID: 21539389

The facile synthesis of biocompatible and nontoxic gene delivery vectors has been the focus of research in recent years due to the high potential in treating genetic diseases. 2-Methacryloxyethyl phosphorylcholine (MPC) copolymers were recently studied for their ability to produce nontoxic and biocompatible materials. The synthesis of well-defined and water-soluble MPC polymer based cationic vectors for gene delivery purposes was therefore attractive, due to the potential excellent biocompatibility of the resulting copolymers. Herein, cationic MPC copolymers of varying architectures (block versus random) were produced by the reversible addition--fragmentation chain transfer (RAFT) polymerization technique. The copolymers produced were evaluated for their gene delivery efficacy in the presence and absence of serum. It was found that copolymer architectures and molecular weights do affect their gene delivery efficacy. The statistical copolymers produced larger particles, and showed poor gene transfection efficiency as compared to the diblock copolymers. The diblock copolymers served as efficient gene delivery vectors, in both the presence and absence of serum in vitro. To the best of our knowledge, this is the first report where the effect of architecture of MPC based copolymer on gene delivery efficacy has been studied.

In Vivo Evaluation of the "TinyPump" As a Pediatric Left Ventricular Assist Device

Artificial Organs. May, 2011  |  Pubmed ID: 21595723

Pediatric patients with end-stage heart failure require mechanical circulatory support (MCS) just as adults do. In order to meet the special requirements for neonates' and infants' MCS, pediatric circulatory support devices must be compact with low priming volume, easily controllable with low flow, less traumatic for blood cells and tissues, and biocompatible with minimum anticoagulation. We have designed and developed a miniature rotary centrifugal blood pump, "TinyPump," with a priming volume of 5 mL, which has already demonstrated its controllable performance for low flow and durability in vitro. To evaluate the feasibility of the TinyPump as a left ventricular assist device (LVAD) suitable for neonates and infants, we have examined the biocompatibility and hemodynamic performance of the TinyPump in a pediatric animal model using Shiba goats. The TinyPump is a miniaturized centrifugal pump weighing 150 g comprising a disposable pump head with a 30-mm diameter impeller having six straight-vanes and a reusable motor driver. The impeller in the pump head is supported by a hydrodynamic bearing at its center and is driven by radial magnetic force coupled to the motor driver. TinyPump implantations were performed in 22 Shiba goats (17 female and 5 male), with body weights ranging from 8.4 to 27.2 kg. Under gas anesthesia, via left lateral thoracotomy, a 22 Fr inflow cannula was inserted through the left ventricular apex, while a 6-mm outflow graft was anastomosed to the descending aorta, which were then connected to a TinyPump mounted on the animal's back. Postoperative hemodynamic monitoring included heart rate, arterial and central venous pressure, pump flow, and rotation speed. Target pump flow in all animals was maintained at 0.9 ± 0.1 L/min, which is approximately half the normal pulmonary artery flow measured in control animals. Blood samples were collected to evaluate peripheral organ functions, hemolysis, and thrombosis. Goats were divided into three groups-acute phase (6 h; n = 4), subchronic phase (6 h 2 postoperative days [POD]; n = 11), and chronic phase (3 POD-16 POD; n = 8)-based on their survival duration. In the early experiments, hemolysis and thrombi formation at the impeller bearing resulted in termination of the study. Subsequent modifications of the bearing design, pump housing design, and magnetic coupling force helped to minimize the hemolysis and thrombi formation, prolonging the survival duration of the Shiba goats to 2 weeks with minimum adverse effects on the blood components and organ functions. With further experiments and improvements in pump durability and hemocompatibility, the TinyPump can serve as a suitable circulatory support device for neonates and infants bridging to heart transplantation as well as to heart recovery.

Hydrolyzed Eggshell Membrane Immobilized on Phosphorylcholine Polymer Supplies Extracellular Matrix Environment for Human Dermal Fibroblasts

Cell and Tissue Research. Jul, 2011  |  Pubmed ID: 21597915

We have found that a water-soluble alkaline-digested form of eggshell membrane (ASESM) can provide an extracellular matrix (ECM) environment for human dermal fibroblast cells (HDF) in vitro. Avian eggshell membrane (ESM) has a fibrous-meshwork structure and has long been utilized as a Chinese medicine for recovery from burn injuries and wounds in Asian countries. Therefore, ESM is expected to provide an excellent natural material for biomedical use. However, such applications have been hampered by the insolubility of ESM proteins. We have used a recently developed artificial cell membrane biointerface, 2-methacryloyloxyethyl phosphorylcholine polymer (PMBN) to immobilize ASESM proteins. The surface shows a fibrous structure under the atomic force microscope, and adhesion of HDF to ASESM is ASESM-dose-dependent. Quantitative mRNA analysis has revealed that the expression of type III collagen, matrix metalloproteinase-2, and decorin mRNAs is more than two-fold higher when HDF come into contact with a lower dose ASESM proteins immobilized on PMBN surface. A particle-exclusion assay with fixed erythrocytes has visualized secreted water-binding molecules around the cells. Thus, HDF seems to possess an ECM environment on the newly designed PMBN-ASESM surface, and future applications of the ASESM-PMBN system for biomedical use should be of great interest.

Plaunotol Induces a Comparative Increase of Acidic Mucin Fraction in Gastric Juice

Hepato-gastroenterology. Mar-Apr, 2011  |  Pubmed ID: 21661449

Gastric mucus protects the gastric mucosa. Plaunotol, a gastroprotective agent, has been shown to increase mucus production in animal models. However, it is unclear whether plaunotol benefits human gastric mucus secretion.

A Bioconjugated Phospholipid Polymer Biointerface with Nanometer-scaled Structure for Highly Sensitive Immunoassays

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21674351

This method relates to the preparation of a phospholipid polymer platform and the immobilization of an antibody as a bioaffinity ligand onto the platform to construct a biointerface for highly sensitive immunoassays. The specific phospholipid polymer used in this work is poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)] (PMBN). The PMBN surface could immobilize specific antibodies through covalent chemical bonding by the reaction between MEONP units and amino groups in the antibody. In addition, the PMBN surface could prevent nonspecific protein adsorption from an analyte sample without the use of blocking reagents based on the fundamental properties of the MPC units. Furthermore, a nanometer-scaled particle deposition surface is constructed with PMBN by an electrospray deposition method to enhance the sensitivity by increasing the overall surface area of the biointerface.

Methacrylate Polymer Layers Bearing Poly(ethylene Oxide) and Phosphorylcholine Side Chains As Non-fouling Surfaces: in Vitro Interactions with Plasma Proteins and Platelets

Acta Biomaterialia. Oct, 2011  |  Pubmed ID: 21693202

Two methacrylate monomers, oligo(ethylene glycol) methyl ether methacrylate (OEGMA; MW=300 g mol(-1), poly(ethylene glycol) (PEG) side chains of average length n=4.5) and 2-methacryloyloxyethyl phosphorylcholine (MPC; MW=295 g mol(-1)), were grafted from silicon wafer surfaces via surface-initiated atom transfer radical polymerization. The grafted surfaces were used as model PEG and phosphorylcholine surface systems to allow comparison of the effectiveness of these two motifs in the prevention of plasma protein adsorption and platelet adhesion. It was found that at high graft density fibrinogen adsorption from plasma on the poly(MPC) and poly(OEGMA) surfaces for a given graft chain length was comparable and extremely low. At low graft density, poly(OEGMA) was slightly more effective than poly(MPC) in resisting fibrinogen adsorption from plasma. Flowing whole blood experiments showed that at low graft density the poly(OEGMA) surfaces were more resistant to fibrinogen adsorption and platelet adhesion than the poly(MPC) surfaces. At high graft density, both the poly(MPC) and poly(OEGMA) surfaces were highly resistant to fibrinogen and platelets. Immunoblots of proteins eluted from the surfaces after contact with human plasma were probed with antibodies against a range of proteins, including the contact phase clotting factors, fibrinogen, albumin, complement C3, IgG, vitronectin and apolipoprotein A-I. The blot responses were weak on the poly(MPC) and poly(OEGMA) surfaces at low graft density and zero at high graft density, again indicating strongly protein resistant properties for these surfaces. Since the side chains of the poly(OEGMA) are about 50% greater in size than those of poly(MPC), the difference in protein resistance between the poly(MPC) and poly(OEGMA) surfaces at low graft density may be due to the difference in surface coverage of the two graft types.

Effects of 3,4-dihydrophenyl Groups in Water-soluble Phospholipid Polymer on Stable Surface Modification of Titanium Alloy

Colloids and Surfaces. B, Biointerfaces. Nov, 2011  |  Pubmed ID: 21807481

The surface of a titanium (Ti) alloy substrate was modified by a simple and quick process using a water-soluble polymer, and the effects of 3,4-dihydroxyphenyl (DHP) groups in the polymer side chain on the modification process were examined. The polymers (PMDP) composed of both 2-methacryloyloxyethyl phosphorylcholine (MPC) unit and 3,4-dihydroxyphenyl methacrylate unit were synthesized for surface anchoring. The Ti alloy substrate was coated with PMDP using an aqueous solution of the polymer. A PMDP layer with a thickness of 20 nm was formed on the Ti alloy substrate simply by dip coating for 10s without drying. Even when the Ti alloy substrate with PMDP coating was immersed in the aqueous medium for 1 week, no change in the thickness was observed, i.e., the PMDP layer was bound to the surface very stably. Oxidation of the DHP groups reduced the stability of the polymer layer significantly. Thus, the DHP groups play a significant role in achieving stable binding. Protein was adsorbed on the Ti alloy substrate; however, this was not observed for the PMDP-coated Ti alloy substrate. In conclusion, we confirmed the effects of DHP groups in PMDP on the stability of the coating on the Ti alloy substrate. Moreover, we found that surface treatment using PMDP was simple, quick, and reliable, and thus, it has great potential for improving biofouling of Ti alloy substrates used in medical devices.

Photodynamic Therapy Using an Anti-EGF Receptor Antibody Complexed with Verteporfin Nanoparticles: a Proof of Concept Study

Cancer Biotherapy & Radiopharmaceuticals. Dec, 2011  |  Pubmed ID: 21861705

Photodynamic therapy (PDT) is a noninvasive optical treatment method in which the topical or systemic delivery of photosensitizing drugs is followed by irradiation with broadband red light. Coupling photosensitizers with a specific antibody may allow this approach to target specific cancers. This study determines the antitumor efficacy of coupling verteporfin (Visudyne(®)), a hydrophobic polyporphryin oligomer, with an antiepidermal growth factor receptor (anti-EGFR) antibody. Poly[2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-nitrophenylcarbonyloxyethyl methacrylate] (PMBN) was conjugated with an anti-EGFR antibody and mixed with verteporfin (verteporfin-PMBN-antibody complex). Tumor-bearing mice were intravenously injected with the verteporfin-PMBN-antibody complex or verteporfin plus PMBN without the antibody. Irradiation was conducted at 640 nm with a dose of 75 J/cm(2). The fluorescence intensity in A431 cells in vitro was threefold higher after exposure to verteporfin-PMBN-antibody complex than after exposure to verteporfin-PMBN. In A431 tumor-bearing mice, the intratumor concentration of verteporfin was 9.4 times higher than that of the skin, following administration of the verteporfin-PMBN-antibody complex. Tumor size significantly decreased within 8 days in mice treated with verteporfin-PMBN-antibody complex compared with those treated with verteporfin-PMBN. PDT using a PMBN-verteporfin-antibody complex offers a promising anticancer therapy.

Preparation and Surface Properties of Polyrotaxane-containing Tri-block Copolymers As a Design for Dynamic Biomaterials Surfaces

Colloids and Surfaces. B, Biointerfaces. Jan, 2012  |  Pubmed ID: 21974908

A tri-block copolymer series containing hydrophilic polyrotaxane and hydrophobic poly(iso-butylmethacrylate) (PiBMA) segments was prepared by atom transfer radical polymerization (ATRP), starting from a pseudopolyrotaxane consisting of 2-bromoisobutyryl end-capped poly(ethylene glycol) (PEG) and α-cyclodextrin (α-CD) and followed by methylation. The dynamic wettability and molecular mobility of the copolymer surfaces were evaluated by dynamic contact angle (DCA) and quartz crystal microbalance with dissipation (QCM-D) measurements, respectively. The polyrotaxane tri-block copolymer surfaces were found to show pronounced dynamic wettability and molecular mobility compared to the control surfaces-a tri-block polymer consisting of PEG and PiBMA, and a PiBMA homopolymer-suggesting that a polyrotaxane loop-like structure exists at the outermost surface in an aqueous environment and exhibits dynamic properties attributable to the possible mobile nature of hydrated α-CD molecules along the PEG backbone. Finally, excellent protein adsorption repellency was achieved on the polyrotaxane tri-block copolymer surface, presumably due to the mobile nature of the supramolecular architecture on the surface.

Cell Adhesion Control on Photoreactive Phospholipid Polymer Surfaces

Colloids and Surfaces. B, Biointerfaces. Nov, 2012  |  Pubmed ID: 21982212

Non-invasive and effective cell recovery from culture substrates is important for the passage and characterization of cells. In this study, a photoreactive polymer surface, which uses UV-irradiation to control substrate cell adhesion, was prepared. The photoreactive phospholipid polymer (PMB-PL) reported herein, was composed of a both 2-methacryloyloxyethyl phosphorylcholine (MPC) unit as a cytocompatible unit and methacrylate bearing a photolabile nitrobenzyl group. The PMB-PL polymer was used to coat a cell culture substrate thus affording a photoreactive surface. Surface analysis of the PMB-PL coating indicated a strong photoresponse owing to the sensitivity of the PL unit. Before light exposure, the PMB-PL surface provided cell adhesion. Following UV-irradiation, the PMB-PL coating was converted to a neutral ζ-potential and hydrophilic surface. The photoreactive surface conversion process allowed for the detachment of adhered cells from the PMB-PL surface while maintaining cell viability. This study demonstrates the promise and significance of the PMB-PL photoreactive surface as a method to control cell attachment and detachment for cell function investigation.

Regulation of Cell Proliferation by Multi-layered Phospholipid Polymer Hydrogel Coatings Through Controlled Release of Paclitaxel

Biomaterials. Jan, 2012  |  Pubmed ID: 22036102

We fabricated multi-layered hydrogels on titanium alloy (Ti) surfaces by applying alternating layers of a water-soluble phospholipid polymer (PMBV) and polyvinyl alcohol (PVA). This was accomplished by a layer-by-layer (LbL) process that is based on the formation of reversible covalent bonds between the boronic acid subunits in the PMBV and the hydroxyl groups in the PVA. When placed in an aqueous medium, PMBV acquires a polymeric aggregate structure with hydrophobic domains that can effectively solubilize hydrophobic molecules such as the anticancer drug paclitaxel (PTX) used in this study. The PTX-containing PMBV layer acted as a reservoir in the multi-layered hydrogels. To obtain diverse release profiles, the PTX was loaded in either the top layer (top-type) or the bottom layer (bottom-type) of the hydrogels; additional layers of PMBV and PVA, without PTX, functioned as a diffusion-barrier. In cell culture experiments, top-type hydrogels demonstrated excessive suppression of human epidermal carcinoma A431 cell proliferation over 5 days due to the initial high concentration of released PTX. However, bottom-type hydrogels were able to maintain a constant cell number profile. The release of PTX from multi-layered hydrogels was governed by both diffusion through the diffusion-barrier and dissociation of the hydrogel through an exchange reaction of phenylboronic acid subunits with the low-molecular weight D-glucose in the cell culture medium. In the cell culture experiments, the cell cycle was arrested in S and G2/M phases, as expected following PTX-mediated growth inhibition; control hydrogels did not demonstrate any appreciable cell cycle arrest. We concluded that cell proliferation could be controlled by the concentration of PTX released from the multi-layered hydrogels prepared through the LbL process. This system when used to solubilize bioactive agents at an appropriate layer within the hydrogel has potential for localized and surface-mediated delivery of bioactive molecules from biomedical devices.

Expression of Bleomycin Hydrolase in Keratinization Disorders

Archives of Dermatological Research. Jan, 2012  |  Pubmed ID: 22037625

A neutral cysteine protease, bleomycin hydrolase (BH), is widely expressed in mammalian tissues, with the skin seeming to contain the highest level. Our previous study revealed that BH transcription is modulated both during differentiation and by cytokines. However, BH involvement in keratinization disorder is not well known. In the present study, we performed immunohistochemical studies of BH and other serine/cysteine proteases in human normal skin and lesional skin with keratinization disorders. BH-positive cells were detected in granular layers of orthokeratotic and hyperkeratotic skin diseases, such as erythrokeratoderma and lichen planus. In parakeratotic skin diseases with porokeratosis, pityriasis rubra pilaris and psoriasis, BH staining was decreased in lesional skins compared to that in normal skin. Similar results were obtained for cysteine proteases, caspase-14 and calpain I. On the other hand, cells positive for serine proteases kallikrein 5 and 7 were increased in parakeratotic and inflammatory skin diseases, such as psoriasis. Semi-quantification analysis revealed that BH- and caspase-14-positive staining had higher intensity than those of the other proteases in normal epidermis. As BH is the major citrulline aminopeptidase in normal granular layer, the alternation would have a significant effect on terminal differentiation processes, such as aberrant processing of deiminated peptides. Therefore, BH may play an important role during the late stage of epidermal differentiation.

Association of Gastric Acid and Mucus Secretion Level with Low-dose Aspirin-induced Gastropathy

Journal of Gastroenterology. Feb, 2012  |  Pubmed ID: 22038552

Low-dose aspirin is known to cause upper gastrointestinal complications. The mechanism by which the aspirin disrupts gastric mucosal integrity remains to be clarified. In this study we investigated the temporal association of gastric secretory parameters (acid and mucus) with aspirin-induced gastropathy.

Mechanical Force-based Probing of Intracellular Proteins from Living Cells Using Antibody-immobilized Nanoneedles

Biosensors & Bioelectronics. Jan, 2012  |  Pubmed ID: 22093769

We developed a method combining atomic force microscopy (AFM) and antibody-immobilized nanoneedles to discriminate living cells by probing intracellular cytoskeletal proteins without the need for cell labeling. The nanoneedles are ultra-thin AFM probes sharpened to 200 nm in diameter. While retracting a nanoneedle inserted into a cell, we measured the mechanical force needed to unbind the antibody-target protein complex. Using this method, the intermediate filament protein, nestin and neurofilament were successfully detected in mouse embryonic carcinoma P19 cells and rat primary hippocampal cells within a minute for a single cell and cell differentiation states could be determined. Additionally, the measured magnitude of the force detecting nestin was indicative of the malignancy of breast cancer cells. This method was shown to affect neither the doubling time of cells nor does it leave extrinsic antibodies within the examined cells, allowing to be used in subsequent analyses in their native state.

Degradable Thermoresponsive Nanogels for Protein Encapsulation and Controlled Release

Bioconjugate Chemistry. Jan, 2012  |  Pubmed ID: 22171688

Reversible addition-fragmentation chain transfer (RAFT) polymerization technique was used for the fabrication of stable core cross-linked micelles (CCL) with thermoresponsive and degradable cores. Well-defined poly(2-methacryloyloxyethyl phosphorylcholine), poly(MPC) macroRAFT agent, was first synthesized with narrow molecular weight distribution via the RAFT process. These CCL micelles (termed as nanogels) with hydrophilic poly(MPC) shell and thermoresponsive core consisting of poly(methoxydiethylene glycol methacrylate) (poly(MeODEGM) and poly(2-aminoethyl methacrylamide hydrochloride) (poly(AEMA) were then obtained in a one-pot process by RAFT polymerization in the presence of an acid degradable cross-linker. These acid degradable nanogels were efficiently synthesized with tunable sizes and low polydispersities. The encapsulation efficiencies of the nanogels with different proteins such as insulin, BSA, and β-galactosidase were studied and found to be dependent of the cross-linker concentration, size of protein, and the cationic character of the nanogels imparted by the presence of AEMA in the core. The thermoresponsive nature of the synthesized nanogels plays a vital role in protein encapsulation: the hydrophilic core and shell of the nanogels at low temperature allow easy diffusion of the proteins inside out and, with an increase in temperature, the core becomes hydrophobic and the nanogels are easily separated out with entrapped protein. The release profile of insulin from nanogels at low pH was studied and results were analyzed using bicinchoninic assay (BCA). Controlled release of protein was observed over 48 h.

Spherical Phospholipid Polymer Hydrogels for Cell Encapsulation Prepared with a Flow-focusing Microfluidic Channel Device

Langmuir : the ACS Journal of Surfaces and Colloids. Jan, 2012  |  Pubmed ID: 22176809

To prepare spherical polymer hydrogels, we used a flow-focusing microfluidic channel device for mixing aqueous solutions of two water-soluble polymers. Continuous encapsulation of cells in the hydrogels was also examined. The polymers were bioinspired 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenyl boronic acid groups (PMBV) and poly(vinyl alcohol) (PVA), which spontaneously form a hydrogel in aqueous medium via specific molecular complexation upon mixing, even when they were in cell culture medium. The microfluidic device was prepared with polydimethylsiloxan, and the surface of the channel was treated with fluoroalkyl compound to prevent sticking of the polymers on the surface. The microfluidic channel process could control the diameter of the spherical hydrogels in the range of 30-90 μm and generated highly monodispersed diameter spherical hydrogels. We found that the polymer distribution in the hydrogel was influenced by the PVA concentration and that the hydrogel could be dissociated by the addition of d-sorbitol to the suspension. The single cells could be encapsulated and remain viable in the hydrogels. The localized distribution of polymers in the hydrogel may provide an environment for modulating cell function. It is concluded that the spontaneous hydrogel formation between PMBV and PVA in the flow-focusing microfluidic channel device is applicable for continuous preparation of a spherical hydrogel-encapsulating living cell.

Rapid and Specific Alterations of Goblet Cell Mucin in Rat Airway and Small Intestine Associated with Resistance Against Nippostrongylus Brasiliensis Reinfection

Experimental Parasitology. Mar, 2012  |  Pubmed ID: 22269441

The intestinal parasitic nematode Nippostrongylus brasiliensis is expelled rapidly from the rat in reinfection challenge compared with that of the primary infection owing to the host defense mechanisms raised against the pre-intestinal- and intestinal-stage larvae. We examined the relationship between the mucin alterations in airway and jejunal mucosae and the worm expulsion after third-stage larva reinfection. When rats had been inoculated with fourth-stage larvae and immunized with only the intestinal-stage worms for more than 8 days, the challenge larvae were expelled during the intestinal stage along with a rapid increase of the specific sialomucin in jejunal mucosa, without any effect on the bronchial mucus. When rats had been infected with third-stage larvae and immunized with only the pre-intestinal stage larvae by killing with antihelminthic, the challenge larvae were rejected during the pre-intestinal stage along with marked goblet cell hyperplasia and Muc5AC mucin hyperproduction on the bronchial mucosa, but not as a result of jejunal mucin alteration. Taking these finding together, immunization with pre-intestinal- and intestinal-stage worms independently increases the airway and intestinal goblet cell mucins, respectively, and in both cases, the mucin alterations may contribute to rapid worm expulsion upon reinfection.

Essential Role of Gastric Gland Mucin in Preventing Gastric Cancer in Mice

The Journal of Clinical Investigation. Mar, 2012  |  Pubmed ID: 22307328

Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides (O-glycans) having terminal α1,4-linked N-acetylglucosamine residues (αGlcNAc). Previously, we identified human α1,4-N-acetylglucosaminyltransferase (α4GnT), which is responsible for the O-glycan biosynthesis and characterized αGlcNAc function in suppressing Helicobacter pylori in vitro. In the present study, we engineered A4gnt(-/-) mice to better understand its role in vivo. A4gnt(-/-) mice showed complete lack of αGlcNAc expression in gastric gland mucin. Surprisingly, all the mutant mice developed gastric adenocarcinoma through a hyperplasia-dysplasia-carcinoma sequence in the absence of H. pylori infection. Microarray and quantitative RT-PCR analysis revealed upregulation of genes encoding inflammatory chemokine ligands, proinflammatory cytokines, and growth factors, such as Ccl2, Il-11, and Hgf in the gastric mucosa of A4gnt(-/-) mice. Further supporting an important role for this O-glycan in cancer progression, we also observed significantly reduced αGlcNAc in human gastric adenocarcinoma and adenoma. Our results demonstrate that the absence of αGlcNAc triggers gastric tumorigenesis through inflammation-associated pathways in vivo. Thus, αGlcNAc-terminated gastric mucin plays dual roles in preventing gastric cancer by inhibiting H. pylori infection and also suppressing tumor-promoting inflammation.

Different Effects of Two Types of H2-receptor Antagonists, Famotidine and Roxatidine, on the Mucus Barrier of Rat Gastric Mucosa

Biomedical Research (Tokyo, Japan). Feb, 2012  |  Pubmed ID: 22361886

Compared with the aggressive factors, little attention has been paid to the mucosal defensive factors in ulcer therapy, and the role of the H2-receptor antagonists in gastric mucosal protection has not been well characterized. In the present study, the effects of different types of H2-receptor antagonists (famotidine and roxatidine) on rat gastric mucus cells were investigated using both biochemical and histological methods. Each drug (famotidine, 3 mg/kg; roxatidine, 100 mg/kg) was orally administered to rats by gavage once daily for 7 days. The biosynthesis and tissue content of mucin were compared in the gastric mucosa treated with each drug. Using anti-mucin monoclonal antibodies, the mucin content and immunohistochemical localization were also compared. Both the biosynthesis and the accumulation of gastric mucin were significantly decreased in the famotidine-treated rats, but not in the roxatidine. Both the content and the immunoreactivity of surface mucus cell-derived mucin were reduced by famotidine, while they were maintained in roxatidine-treated rat stomachs. There was no difference between the groups in the content and immunoreactivity of mucous neck cell-derived mucin. H2-receptor antagonists should be classified in relation to gastric surface mucus cell function, raising the possibility of more effective ulcer therapy.

Fabrication of Polymeric Electron-transfer Mediator/enzyme Hydrogel Multilayer on an Au Electrode in a Layer-by-layer Process

Biosensors & Bioelectronics. Apr, 2012  |  Pubmed ID: 22365365

The layer-by-layer (LBL) construction of an enzyme electrode covered with a multilayer structure alternately composed of a polymeric electron transfer mediator and a polymer-modified enzyme was examined. Poly(2-methacryloyloxyethyl phosphorylcholine-co-p-vinylphenylboronic acid-co-vinylferrocene) (PMVF) was synthesized and used as a polymeric electron transfer mediator. Glucose oxidase (GOx) was selected as a model enzyme and poly(vinyl alcohol) (PVA) chains were bound to the GOx (GOx-PVA) under mild conditions. The PMVF and PVA formed a gel spontaneously through a selective reaction between phenylboronic acid units and hydroxyl groups in both polymers. Using the spin coating technique, a repeating PMVF/GOx-PVA multilayer was fabricated on the surface of an Au electrode. The thickness of each PMVF/GOx-PVA layer was around 5.8 nm, corresponding to the dimensions of GOx. The electrochemical performance of the electrode was evaluated in glucose concentration measurement. The oxidation current of glucose by GOx was measured at 0.38 V (vs. Ag/AgCl), verifying that ferrocene units in the PMVF of the hydrogel electrically wired the immobilized GOx. Moreover, the current increased with the number of PMVF/GOx-PVA layers. That is, both intermolecular electron transfer between each individual layer and the presence of a freely diffusing substrate in the hydrogel were achieved. We conclude that a LBL structure constructed from PMVF and a PVA-modified enzyme is effective for use in developing bioelectronic devices that employ enzyme molecules.

In Vivo Kinetics of Indoxyl Sulfate in Humans and Its Renal Interaction with Angiotensin-converting Enzyme Inhibitor Quinapril in Rats

The Journal of Pharmacology and Experimental Therapeutics. Jun, 2012  |  Pubmed ID: 22389425

Indoxyl sulfate (IS) is an organic anion uremic toxin that accumulates in patients with chronic kidney disease (CKD). The aims of this study were to examine the kinetic profiles of IS in humans at a steady state after multiple doses of L-Trp, a precursor of IS, and the in vivo interaction of IS with the angiotensin-converting enzyme inhibitor quinapril, whose active metabolite is a substrate of organic anion transporter 3 (OAT3) in rats. First, 12-h kinetics after single doses of Trp (2, 4, and 8 g) were examined in two healthy volunteers. Second, 24-h kinetics after a single dose of 2 g of Trp was studied in six volunteers. Third, 35-h kinetics after single and multiple doses of 2 g of Trp were examined in five volunteers. In anesthetized rats, quinapril or probenecid, an inhibitor of OATs, was given intravenously before IS, and blood and urine samples were taken until 90 min. Trp and IS concentrations were determined by high-performance liquid chromatography. Ultrafiltration was used to measure serum unbound IS concentrations. Renal tubular secretion of IS accounted for more than 90% of its renal clearance in the steady state of serum IS levels after multiple doses in humans. In animals, the serum area under the curve of IS increased in conjunction with a decrease in renal clearances after coadministration of IS with quinapril or probenecid. It is concluded that quinapril may inhibit the urine excretion of IS via OAT3-mediated renal tubular transport in patients with CKD.

Biomimetic Hydrogels Gate Transport of Calcium Ions Across Cell Culture Inserts

Biomedical Microdevices. Jun, 2012  |  Pubmed ID: 22426887

Control of the in vitro spatiotemporal availability of calcium ions is one means by which the microenvironments of hematopoietic stem cells grown in culture may be reproduced. The effects of cross-linking density on the diffusivity of calcium ions through cell culture compatible poly(2-hydroxyethyl methacrylate) [poly(HEMA)]-based bioactive hydrogels possessing 1.0 mol% 2-methacryloyloxyethyl phosphorylcholine (MPC), 5 mol% N,N-(dimethylamino)ethylmethacrylate (DMAEMA) and ca. 17 mol% n-butyl acrylate (n-BA) have been investigated to determine if varying cross-link density is a viable approach to controlling transport of calcium across hydrogel membranes. Cross-linking density was varied by changing the composition of cross-linker, tetraethyleneglycol diacrylate (TEGDA). The hydrogel membranes were formed by sandwich casting onto the external surface of track-etched polycarbonate membranes (T = 10 μm, φ = 0.4 μm pores) of cell culture inserts, polymerized in place by UV light irradiation and immersed in buffered (0.025 HEPES, pH 7.4) 0.10 M calcium chloride solution. The transport of calcium ions across the hydrogel membrane was monitored using a calcium ion selective electrode set within the insert. Degree of hydration (21.6 ± 1.0%) and void fraction were found to be constant across all cross-linking densities. Diffusion coefficients, determined using time-lag analysis, were shown to be strongly dependent on and to exponentially decrease with increasing cross-linking density. Compared to that found in buffer (2.0-2.5 × 10⁻⁶ cm²/s), diffusion coefficients ranged from 1.40 × 10⁻⁶ cm²/s to 1.80 × 10⁻⁷ cm²/s and tortuosity values ranged from 1.7 to 10.0 for the 1 and 12 mol% TEGDA cross-linked hydrogels respectively. Changes in tortuosity arising from variations in cross-link density were found to be the primary modality for controlling diffusivity through novel n-BA containing poly(HEMA)-based bioactive hydrogels.

Biomimetic Hydration Lubrication with Various Polyelectrolyte Layers on Cross-linked Polyethylene Orthopedic Bearing Materials

Biomaterials. Jun, 2012  |  Pubmed ID: 22465336

Natural joints rely on fluid thin-film lubrication by the hydrated polyelectrolyte layer of cartilage. However, current artificial joints with polyethylene (PE) surfaces have considerably less efficient lubrication and thus much greater wear, leading to osteolysis and aseptic loosening. This is considered a common factor limiting prosthetic longevity in total hip arthroplasty (THA). However, such wear could be mitigated by surface modification to mimic the role of cartilage. Here we report the development of nanometer-scale hydrophilic layers with varying charge (nonionic, cationic, anionic, or zwitterionic) on cross-linked PE (CLPE) surfaces, which could fully mimic the hydrophilicity and lubricity of the natural joint surface. We present evidence to support two lubrication mechanisms: the primary mechanism is due to the high level of hydration in the grafted layer, where water molecules act as very efficient lubricants; and the secondary mechanism is repulsion of protein molecules and positively charged inorganic ions by the grafted polyelectrolyte layer. Thus, such nanometer-scaled hydrophilic polymers or polyelectrolyte layers on the CLPE surface of acetabular cup bearings could confer high durability to THA prosthetics.

Wettability and Antifouling Behavior on the Surfaces of Superhydrophilic Polymer Brushes

Langmuir : the ACS Journal of Surfaces and Colloids. May, 2012  |  Pubmed ID: 22500465

The surface wettabilities of polymer brushes with hydrophobic and hydrophilic functional groups were discussed on the basis of conventional static and dynamic contact angle measurements of water and hexadecane in air and captive bubble measurements in water. Various types of high-density polymer brushes with nonionic and ionic functional groups were prepared on a silicon wafer by surface-initiated atom-transfer radical polymerization. The surface free energies of the brushes were estimated by Owens-Wendt equation using the contact angles of various probe liquids with different polarities. The decrease in the water contact angle corresponded to the polarity of fluoroalkyl, hydroxy, ethylene oxide, amino, carboxylic acid, ammonium salt, sulfonate, carboxybetaine, sulfobetaine, and phosphobetaine functional groups. The poly(2-perfluorooctylethyl acrylate) brush had a low surface free energy of approximately 8.7 mN/m, but the polyelectrolyte brushes revealed much higher surface free energies of 70-74 mN/m, close to the value for water. Polyelectrolyte brushes repelled both air bubbles and hexadecane in water. Even when the silicone oil was spread on the polyelectrolyte brush surfaces in air, once they were immersed in water, the oil quickly rolled up and detached from the brush surface. The oil detachment behavior observed on the superhydrophilic polyelectrolyte brush in water was explained by the low adhesion force between the brush and the oil, which could contribute to its excellent antifouling and self-cleaning properties.

Simple Surface Treatment Using Amphiphilic Phospholipid Polymers to Obtain Wetting and Lubricity on Polydimethylsiloxane-based Substrates

Colloids and Surfaces. B, Biointerfaces. Sep, 2012  |  Pubmed ID: 22609584

Simple surface treatment of polydimethylsiloxane (PDMS) substrates was performed using an aqueous-ethanolic solution of amphiphilic phospholipid polymers to reduce the hydrophobic and high friction characteristics of PDMS. The phospholipid polymers, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-2-ethylhexyl methacrylate (EHMA)-co-2-(N,N-dimethylamino)ethyl methacrylate) (PMED) and poly(MPC-co-EHMA) (PMEH) were synthesized, and the effects of the electric charge of the polymer chain on the stability of the attachment to the PDMS surface was investigated. The polymers were dissolved in a mixed solvent of ethanol and water, and the PDMS samples were treated by a simple dipping method using the polymer solution. Pure ethanol as the solvent was ineffective for the attachment of the polymers to the PDMS surface. It was considered that the hydrophobic interactions and electrostatic attraction forces between the polymer chains and the PDMS surface were too weak for efficient interaction in this solvent. On the other hand, the surface wettability and lubricity of PDMS could be improved by treatment with an aqueous-ethanolic solution of PMED. The static contact angle was decreased from 90° to 20° by this treatment, and the dynamic friction coefficient against a Co-Cr ball was decreased by nearly 80% compared with that of the untreated PDMS. The hydrophobic interactions and electrostatic attraction forces generated by PMED were both essential for the stable adsorption of the polymer layer on PDMS. Furthermore, the solubilized state of the polymers affected the adsorption of the polymer. We concluded that the surface of PDMS could be stably modified using aqueous-ethanolic solutions of PMED without the need for pretreatments.

Electrospun Phospholipid Polymer Substrate for Enhanced Performance in Immunoassay System

Biosensors & Bioelectronics. Oct-Dec, 2012  |  Pubmed ID: 22705405

A functional polymer bearing both phosphorylcholine and active ester groups, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-N-succinimidyloxycarbonyl di(ethylene glycol) methacrylate (PENHS)] (PMBS), provided a highly biomolecule-friendly platform for immunoassays. The nonbiofouling property of the PMBS remarkably reduces the background noise from nonspecific adsorption of proteins in the analyte in an enzyme-linked immunosorbent assay (ELISA), which improves the specificity and signal-to-noise ratio. Electrospinning deposition, a versatile and cost-effective technique, was employed to fabricate nanofibrous PMBS. This nanostructure increased the surface area of the polymer, allowing more antibodies to bind to the polymer interface and enhancing the sensitivity of the biosensing system. The electrospun PMBS fibers were stable and retained their unique morphologies after contact with an aqueous solution for 4.0 h. The ability of PMBS to reduce background noise without blocking by protein-based reagents was verified by comparison with an immunoassay conducted on a polystyrene substrate. The ELISA of human immunoglobulin-G with the electrospun PMBS substrate showed a good sigmoidal relationship with a linear detection range from 1.0 to 100 ng/mL. The detection time was 25% shorter than the conventional assay as the blocking step was omitted. The immobilized primary antibodies exhibited high stability on the electrospun PMBS; 60% and 25% of the residual bioactivity remained after storage in dry conditions for 2 and 4 weeks, respectively. Thus, for further development of biosensors, nanostructured PMBS can improve lifetime of immobilized biomolecules, and also contribute to an enhanced reliability and signal-to-noise ratio of immunoassay.

Impact of the Nature, Size and Chain Topologies of Carbohydrate-phosphorylcholine Polymeric Gene Delivery Systems

Biomaterials. Nov, 2012  |  Pubmed ID: 22818654

With the recent significant advances in the field polymer chemistry, it is now possible to produce well-defined and non-toxic cationic polymers with advanced molecular structures of desired molecular weights and compositions. Carefully engineered polymer architectures are found to impact significantly their DNA condensation and gene delivery efficacies. In a previous study, the statistical carbohydrates based copolymers were found to show high gene expression and low toxicity, however there aggregation in the presence of serum proteins was a major drawback. In this study, carbohydrate and phosphorylcholine based cationic polymers having a different architecture, compositions and varying molecular weights are produced and are termed as cationic 'block-statistical' copolymers. These cationic copolymers are evaluated for their gene delivery efficacies, interactions with serum protein, cellular uptake and nuclear localization ability. As compared to the statistical analogue, 'block-statistical' copolymers showed high gene expression, low interactions with serum proteins, as well as low toxicity in hepatocytes and human dermal fibroblasts. In addition, 2- methacryloyloxyethyl phosphorylcholine (MPC) based 'block-statistical' copolymers and their sugar incorporated analogues were prepared and were found to serve as improved gene delivery vectors than their statistical analogues.

The Helical Flow Pump with a Hydrodynamic Levitation Impeller

Journal of Artificial Organs : the Official Journal of the Japanese Society for Artificial Organs. Dec, 2012  |  Pubmed ID: 22926404

The helical flow pump (HFP) is a novel rotary blood pump invented for developing a total artificial heart (TAH). The HFP with a hydrodynamic levitation impeller, which consists of a multi-vane impeller involving rotor magnets, stator coils at the core position, and double helical-volute pump housing, was developed. Between the stator and impeller, a hydrodynamic bearing is formed. Since the helical volutes are formed at both sides of the impeller, blood flows with a helical flow pattern inside the pump. The developed HFP showed maximum output of 19 l/min against 100 mmHg of pressure head and 11 % maximum efficiency. The profile of the H-Q (pressure head vs. flow) curve was similar to that of the undulation pump. Hydrodynamic levitation of the impeller was possible with higher than 1,000 rpm rotation speed. The normalized index of the hemolysis ratio of the HFP to centrifugal pump (BPX-80) was from 2.61 to 8.07 depending on the design of the bearing. The HFP was implanted in two goats with a left ventricular bypass method. After surgery, hemolysis occurred in both goats. The hemolysis ceased on postoperative days 14 and 9, respectively. In the first experiment, no thrombus was found in the pump after 203 days of pumping. In the second experiment, a white thrombus was found in the pump after 23 days of pumping. While further research and development are necessary, we are expecting to develop an excellent TAH with the HFP.

The Effect of the Encapsulation of Bacteria in Redox Phospholipid Polymer Hydrogels on Electron Transfer Efficiency in Living Cell-based Devices

Biomaterials. Nov, 2012  |  Pubmed ID: 22938764

Development of living cell-based devices holds great promise in many biomedical and industrial applications. To increase our understanding of the process, we investigated the biological and electrochemical properties of a redox phospholipid polymer hydrogel containing an electron-generating bacteria (Shewanella oneidensis MR-1). A water-soluble and amphiphilic phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid-co-vinylferrocene) (PMBVF), was our choice for incorporation into a hydrogel matrix that promotes encapsulation of bacteria and acts as an electron transfer mediator. This hydrogel formed spontaneously and encapsulated Shewanella in three-dimensional structures. Visual analysis showed that the encapsulated Shewanella maintained viability and metabolic activity even after long-term storage. Cyclic voltammetry measurement indicated that the PMBVF/poly(vinyl alcohol) (PMBVF/PVA) hydrogel had stable and high electron transfer efficiency. Amperometric measurement showed that the hydrogel could maintain the electron transfer efficiency even when Shewanella was encapsulated. Thus, the PMBVF/PVA hydrogel not only provides a mild environment for long-term bacterial survival but also maintains electron transfer efficiency from the bacteria to the electrode. We conclude that hydrogel/bacteria hybrid biomaterials, such as PMBVF/PVA/Shewanella, may find application in the fabrication of living cell-based devices.

The Monoclonal Antibody HCM31 Specifically Recognises the Sd(a) Tetrasaccharide in Goblet Cell Mucin

FEBS Open Bio. 2012  |  Pubmed ID: 23650604

Rat small intestinal goblet cell mucins reacting with monoclonal antibody HCM31 increase significantly during regeneration from experimental mucosal damage and at the period of expulsion of parasitic nematode, Nippostrongylus brasiliensis (N.b). The reduction in reactivity of HCM31 with mucin upon neuraminidase treatment, suggested that HCM31 recognizes sialylated oligosaccharide on mucin. HCM31-reactive sialomucins are therefore considered to play an important role in the physiological and pathological changes in the gastrointestinal mucosa. To determine the epitope for HCM31, oligosaccharide-alditols reacted with HCM31 were obtained from the small intestinal mucins of N.b-infected rats and purified by ion-exchange chromatography followed by normal-phase HPLC. Two HCM31-reactive oligosaccharide-alditols were obtained. Analyses using tandem mass spectrometry and NMR spectroscopy showed that these oligosaccharides were core 4 mucin-type oligosaccharides having a common tetrasaccharide sequence, NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ- (Sd(a) blood group antigen). These structures were not found in the small intestinal mucin oligosaccharides from uninfected rats. This epitope specificity of HCM31 was also confirmed using previously established anti-GM2 and anti-Sd(a) antibodies. Taken together, these results strongly suggest that HCM31 specifically recognizes mucin-type oligosaccharides with the Sd(a) tetrasaccharide sequence. Immunohistochemical examination of human gastrointestinal tracts showed that HCM31 site-specifically stained the goblet cells in normal sigmoid colon and normal rectum, but the goblet cells stained with HCM31 were reduced in the corresponding cancer tissues. HCM31 seems to be useful for diagnosis of colonic cancer and for examining the function of secretory-type mucin with Sd(a) antigen.

Cell Membrane-inspired Phospholipid Polymers for Developing Medical Devices with Excellent Biointerfaces

Science and Technology of Advanced Materials. Dec, 2012  |  Pubmed ID: 27877525

This review article describes fundamental aspects of cell membrane-inspired phospholipid polymers and their usefulness in the development of medical devices. Since the early 1990s, polymers composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units have been considered in the preparation of biomaterials. MPC polymers can provide an artificial cell membrane structure at the surface and serve as excellent biointerfaces between artificial and biological systems. They have also been applied in the surface modification of some medical devices including long-term implantable artificial organs. An MPC polymer biointerface can suppress unfavorable biological reactions such as protein adsorption and cell adhesion - in other words, specific biomolecules immobilized on an MPC polymer surface retain their original functions. MPC polymers are also being increasingly used for creating biointerfaces with artificial cell membrane structures.

Evaluation of the Actin Cytoskeleton State Using an Antibody-functionalized Nanoneedle and an AFM

Biosensors & Bioelectronics. Feb, 2013  |  Pubmed ID: 22784496

A cell diagnosis technique was developed, which uses an Atomic Force Microscope (AFM) and an ultra-thin AFM probe sharpened to a diameter of 200 nm (nanoneedle). Due to the high aspect ratio of the nanoneedle, it was successfully inserted into a living cell without affecting its viability. Furthermore, by functionalizing the nanoneedle with specific antibodies and measuring the unbinding forces ('fishing forces') during evacuation of the nanoneedle from the cell, it was possible to measure specific mechanical interactions between the antibody-functionalized nanoneedle and the intracellular contents of the cell. In this study, an anti-actin-antibody-functionalized nanoneedle was used to evaluate the actin cytoskeleton state in living cells. To examine the effect of cytoskeleton condition on the measured fishing forces, the cytoskeleton-disrupting drugs cytochalasin D (cytD) and Y-27632 were used, showing a marked decrease in the measured fishing forces following incubation with either of the drugs. Furthermore, the technique was used to measure the time course changes in a single cell during incubation with cytD, showing a gradual time-dependent decrease in fishing forces. Even minute doses of the drugs, the effects of which were hardly evident by optical and fluorescence methods, could be clearly detected by the measurement of nanoneedle-protein fishing forces, pointing to the high sensitivity of this detection method. This technique may prove beneficial for the evaluation of cytoskeleton conditions in health and disease, and for the selection of specific cells according to their intracellular protein contents, without the need for introduction of marker proteins into the cell.

Direct Observation of Selective Protein Capturing on Molecular Imprinting Substrates

Biosensors & Bioelectronics. Feb, 2013  |  Pubmed ID: 22784498

A sensing interface for specific protein capture was fabricated using a novel molecular imprinting (MIP) process. Bovine serum albumin (BSA) and ovalbumin (OVA) were imprinted on a quartz substrate with modified alkyl groups, and target protein capture was detected using a deep-UV fluorescence image microscope (UVFLIM). The imprinted protein was immobilized to silica beads (diameter: 15 μm) using a phospholipid polymer containing both active ester groups and silane coupling groups, which were used as protein stamps to prepare the imprinting surface. Protein recognition sites were constructed by integrating sodium dodecyl sulfate (SDS) as the ligand, which was immobilized with a biocompatible photoreactive phospholipid polymer. When BSA solution was added to the BSA-based MIP substrate, strong fluorescence was observed from the tryptophan residue of BSA. In contrast, for the OVA-based MIP substrate and non-MIP substrate, no fluorescence was observed. The surface showed good selectivity of BSA against OVA. The phospholipid polymer layer prevented non-specific protein adsorption, resulting in highly selective protein recognition. Further, when the protein-imprinted substrate was constructed without ligands, neither protein was captured on the substrate. We demonstrated the importance of ligand integration for capturing target proteins at specific positions. UVFLIM can be used to detect biomolecules at the single-molecule level by using intrinsic fluorescence without molecular labeling. Our new protein-imprinted surface used with UVFLIM is a versatile tool for capturing biomolecules.

Direct Electron Transfer with Enzymes on Nanofiliform Titanium Oxide Films with Electron-transport Ability

Biosensors & Bioelectronics. Mar, 2013  |  Pubmed ID: 23040371

Direct electron transfer (DET) from biomolecules to electrode is a process without electron-mediators, thus superior selectivity and sensitivity is expected in order to monitor electron transfer between electrode and biomolecules without any mediator interference. However, DET is difficult because a redox center which is an electron active center of proteins such as enzymes is buried deep. So, a unique electrode nanostructure to reach the redox center is a critical factor. Here we have systematically investigated terms for DET using various nanofiliformed electrode morphologies and enzyme concentrations. It is pointed out that the reaction site is below 100 nm, the ration amounts of adsorbed enzyme per surface area are below 1.0 are contributed to the DET. As a great application, we have developed a biosensor monitoring the hydrogen peroxide (H(2)O(2)) detecting capability from peroxidase directly. For the fabricated HRP/nTOF/Ti-electrodes observed the catalytic current value was linear according to the increase in the concentration of H(2)O(2) up to 100 μM, which indicates a good potential for an H(2)O(2) biosensor.

Redox Phospholipid Polymer Microparticles As Doubly Functional Polymer Support for Immobilization of Enzyme Oxidase

Colloids and Surfaces. B, Biointerfaces. Feb, 2013  |  Pubmed ID: 23107964

We prepared redox phospholipid polymer microparticles for immobilizing an enzyme in order to maintain activity for a long time and obtain highly effective electron transfer to a gold substrate as an electrode. To achieve these double functions, an amphiphilic redox phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-nitrophenyloxycarbonyl oligo(ethylene glycol) methacrylate (MEONP)-co-vinylferrocene (VFc)) (PMBNF) was synthesized. The polystyrene (PS) microparticles were modified by employing a simple solution dip-coating technique to form the PMBNF layer on the surface. As one of the model enzyme oxidases, a glucose oxidase (GO(x)) was immobilized on the PMBNF/PS microparticles by the reaction between the MEONP units in the PMBNF layer and the amino group in the GO(x). The activity of immobilized GO(x) is maintained well; for example, activity of more than 80% of the initial activity was observed even after storage at both 4°C and 25°C (ionic strength: 0.10 mol/L, phosphate buffer solution, pH 7.0) for at least one month. The GO(x)/PMBNF/PS microparticles were arrayed on a gold substrate in a monolayer, and then, crosslinked to each other with a polymeric diamine compound. The PMBNF/PS microparticles demonstrated an efficient electron transfer from immobilized GO(x) to the gold surface. From these results, we concluded that the PMBNF layer on the PS microparticles possessed double functions such as stable enzyme immobilization ability and efficient electron transfer ability.

Detachment of Cells Adhered on the Photoreactive Phospholipid Polymer Surface by Photoirradiation and Their Functionality

Colloids and Surfaces. B, Biointerfaces. Mar, 2013  |  Pubmed ID: 23261571

Regulating the detachment of adhered living cells from a surface is a key technological requirement to obtain specific cells in the field of cellular engineering. Here, we describe dynamic control of cell adhesion and detachment at a photoreactive and cytocompatible phospholipid polymer surface. The surface was prepared using the amphiphilic and water-insoluble substance poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) bearing 4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid (PL) groups in its side chain (PMB-PL). On this prepared surface, the photoinduced control of human epidermoid carcinoma cancer cells (A431 cells) and primary murine embryonic fibroblast (PMEF) cells was examined. The PMB-PL surface allowed successful control of living cells adhesion with photoreactivity. The efficiency of cell detachment obtained was approximately 50% of the initial number of adhered cells. The PL groups at the surface provide adhesion points for cells, as evidenced by the fact that after photoreaction of PL groups by photoirradiation, the number of adhered cells on the surface considerably decreased. Additionally, when the polymer surface was re-used after the first photoirradiation, cells did not adhere to it, and low detachment was observed. The functionalities of the cells detached by photoreaction were evaluated. The proliferation rate and morphological changes of cells were as the same as those of cells detached by conventional enzymatic digestion. Moreover, the extracellular expression levels of transmembrane proteins on cells detached by photoirradiation showed no significant difference with those seen on normal cells. From these results, we conclude that the PMB-PL surface is a suitable platform to regulate cell adhesion and detachment via photochemical reaction.

The Significance of Hydrated Surface Molecular Mobility in the Control of the Morphology of Adhering Fibroblasts

Biomaterials. Apr, 2013  |  Pubmed ID: 23410683

The effects of the hydrated molecular mobility and the surface free energy of polymer surfaces on fibronectin adsorption and fibroblast adhesion were investigated. ABA-type block copolymers composed of polyrotaxane (PRX) with different number of threaded α-cyclodextrin (α-CD), random copolymers with similar chemical composition to the PRX block copolymers, and conventional polymers were prepared to determine a wide range of hydrated molecular mobility (Mf) values estimated by quartz crystal microbalance-dissipation (QCM-D) measurements. Fibronectin adsorption was highly dependent on surface free energy, and high surface fibronectin density resulted in a large projected cell area on the polymer surfaces. However, the morphology of adhering fibroblasts was not explained by the surface free energy, but it was found to be strongly dependent on the Mf values of the polymer surfaces in aqueous media. These results emphasize the importance of Mf in the discussion of the elongated morphology of adhering fibroblasts on various polymer surfaces.

Inducing Rapid Cellular Response on RGD-binding Threaded Macromolecular Surfaces

Journal of the American Chemical Society. Apr, 2013  |  Pubmed ID: 23544620

The rapid response of integrin β1 molecules to an RGD peptide on a dynamic polyrotaxane surface was successfully induced. As a result, RGD peptides introduced on a highly dynamic cyclodextrin molecule enhanced the frequency of contact with specific integrin molecules on the cell membrane at the early stage of material-cell interactions.

Hybridization of Poly(2-methacryloyloxyethyl Phosphorylcholine-block-2-ethylhexyl Methacrylate) with Segmented Polyurethane for Reducing Thrombogenicity

Colloids and Surfaces. B, Biointerfaces. Aug, 2013  |  Pubmed ID: 23563289

Segmented polyurethanes (SPUs) are widely used in biomedical devices owing to their superior mechanical properties. However, their applicability in blood-contacting devices such as small-diameter vascular prostheses is severely limited owing to their thrombogenicity. Thus, it is necessary to develop an SPU material that has a surface with low thrombogenicity. In this study, poly(2-methacryloyloxyethyl phosphorylcholine-block-2-ethylhexyl methacrylate) (B-PMEH) was synthesized as a well-defined diblock copolymer by reversible addition-fragmentation chain transfer (RAFT) polymerization. B-PMEH was hybridized with SPU by the integration of the polymer layers to prepare an SPU/B-PMEH polymer alloy membrane (SB membrane). The MPC units in the B-PMEH phase separated on the immersion of the SB membrane in water, producing a surface that drastically lowered the amount of the absorbed fibrinogen and platelet adhesion as compared to the SPU. Importantly, it was demonstrated that the bulk mechanical properties of the SPU were not affected by the addition of B-PMEH.

A Simple Procedure for the Preparation of Precise Spatial Multicellular Phospholipid Polymer Hydrogels

Colloids and Surfaces. B, Biointerfaces. Aug, 2013  |  Pubmed ID: 23587764

A precise spatial multicellular polymer hydrogel matrix was prepared by successive assembly of cell-laden hydrogel layers alternated by hydrogel layers without cells based on the spontaneous hydrogel formation between 2 aqueous polymer solutions. The polymers used were a water-soluble 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenylboronic acid groups (PMBV) and poly(vinyl alcohol) (PVA). Each cell-laden layer was deposited as a cell-laden PMBV solution on a PMBV/PVA precursor film. PMBV/PVA multilayer hydrogel was stacked on the top of a cell-laden layer by sequential coating with spinning of the PMBV and PVA solutions. This process allowed the formation of the PMBV/PVA multilayer hydrogel with finely controlled thickness. Finally, we succeeded in cell patterning by using a multilayer hydrogel matrix, forming a sandwich of 2 cell-laden layers separated by a PMBV/PVA multilayer hydrogel. The cells remained alive during the spinning process and maintained their metabolism for at least 24h. This precise spatial multicellular PMBV/PVA hydrogel can be used to examine interactions between many different cells and construct customized microenvironments for multicellular co-cultures.

Extracellular Electron Transfer Across Bacterial Cell Membranes Via a Cytocompatible Redox-active Polymer

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry. Jul, 2013  |  Pubmed ID: 23630181

A redox-active phospholipid polymer with a phospholipid-mimicking structure (2-methacryloyloxyethyl phosphorylcholine; MPC) was synthesized to construct a biocompatible electron mediator between bacteria and an electrode. In this study, a copolymer of MPC and vinylferrocene [VF; poly(MPC-co-VF)] (PMF) is synthesized. When PMF is added to cultures of the bacterial species Escherichia coli (Gram negative) and Lactobacillus plantarum (Gram positive), which have different cell wall structures, a catalytic current mediated by PMF is observed. In addition, growth curves and live/dead assays indicate that PMF does not decrease metabolic activity or cell viability. These results indicate that PMF mediates extracellular electron transfer across bacterial cell membranes without associated cytotoxicity.

The Use of the Mechanical Microenvironment of Phospholipid Polymer Hydrogels to Control Cell Behavior

Biomaterials. Aug, 2013  |  Pubmed ID: 23676454

We considered that properties of the microenvironment surrounding cells are important for the control of the cell functions. Cytocompatible polymer hydrogels are good candidates to study such microenvironment. Here, we prepared spontaneously forming hydrogels composed of two polymer systems, namely poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid) (PMBV) and poly(vinyl alcohol) (PVA). The PMBV/PVA hydrogels could be reversibly dissociated by the addition of d-sorbitol. The storage modulus was measured for evaluating the mechanical properties of the PMBV/PVA hydrogels. The storage modulus could be controlled in the range 0.30-2.5 kPa by changing the cross-linking density of the hydrogels. After pluripotent stem cells were encapsulated within the PMBV/PVA hydrogels during the preparation of the hydrogel under normal cell-culturing conditions, the proliferation rate and the cell cycle of the encapsulated cells were observed. Cells lived for more than three days in every PMBV/PVA hydrogel. However, the proliferation significantly depended on the storage modulus of the hydrogels. Although the cell cycle of the initial cells was heterogenous, it developed uniformity toward the G1 phase when the cells were encapsulated within the PMBV/PVA hydrogel with a storage modulus of 1.1 kPa for three days. That is, the mechanical properties of the PMBV/PVA environment influenced the biological functions of the cells encapsulated in the hydrogels. From these results, we conclude that PMBV/PVA hydrogels are useful for adjusting cell cycles and proliferation, thus providing uniform cells for applications in the field of cell engineering.

Surface Modification of a Biodegradable Magnesium Alloy with Phosphorylcholine (PC) and Sulfobetaine (SB) Functional Macromolecules for Reduced Thrombogenicity and Acute Corrosion Resistance

Langmuir : the ACS Journal of Surfaces and Colloids. Jul, 2013  |  Pubmed ID: 23705967

Siloxane functionalized phosphorylcholine (PC) or sulfobetaine (SB) macromolecules (PCSSi or SBSSi) were synthesized to act as surface modifying agents for degradable metallic surfaces to improve acute blood compatibility and slow initial corrosion rates. The macromolecules were synthesized using a thiol-ene radical photopolymerization technique and then utilized to modify magnesium (Mg) alloy (AZ31) surfaces via an anhydrous phase deposition of the silane functional groups. X-ray photoelectron spectroscopy surface analysis results indicated successful surface modification based on increased nitrogen and phosphorus or sulfur composition on the modified surfaces relative to unmodified AZ31. In vitro acute thrombogenicity assessment after ovine blood contact with the PCSSi and SBSSi modified surfaces showed a significant decrease in platelet deposition and bulk phase platelet activation compared with the control alloy surfaces. Potentiodynamic polarization and electrochemical impedance spectroscopy data obtained from electrochemical corrosion testing demonstrated increased corrosion resistance for PCSSi- and SBSSi-modified AZ31 versus unmodified surfaces. The developed coating technique using PCSSi or SBSSi showed promise in acutely reducing both the corrosion and thrombotic processes, which would be attractive for application to blood contacting devices, such as vascular stents, made from degradable Mg alloys.

A Large Mobility of Hydrophilic Molecules at the Outmost Layer Controls the Protein Adsorption and Adhering Behavior with the Actin Fiber Orientation of Human Umbilical Vein Endothelial Cells (HUVEC)

Journal of Biomaterials Science. Polymer Edition. 2013  |  Pubmed ID: 23796033

Adhesion behaviors of human umbilical vein endothelial cells (HUVECs) are interestingly affected by the mobility of hydrophilic chains on the material surfaces. Surfaces with different molecular mobilities were prepared using ABA-type block copolymers consisting polyrotaxane (PRX) or poly(ethylene glycol) (PEG) central block (A block), and amphiphilic anchoring B blocks of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB). Two different molecular mobilities of the PRX chains were designed by using normal α-cyclodextrin (α-CD) or α-CD whose hydroxyl groups were converted to methoxy groups in a given ratio to improve its molecular mobility (PRX-PMB and OMe-PRX-PMB). The surface mobility of these materials was assessed as the mobility factor (Mf), which is measured by quartz crystal microbalance with dissipation monitoring system. HUVECs adhered on OMe-PRX-PMB surface much more than PRX-PMB and PMB-block-PEG-block-PMB (PEG-PMB) surfaces. These different HUVEC adhesions were correlated with the density of cell-binding site of adsorbed fibronectin. In addition, the alignment of the actin cytoskeleton of adhered HUVECs was strongly suppressed on the PEG-PMB, PRX-PMB, and OMe-PRX-PMB in response to the increased Mf value. Remarkably, the HUVECs adhered on the OMe-PRX-PMB surface with much less actin organization. We concluded that not only the cell adhesion but also the cellular function are regulated by the molecular mobility of the outmost material surfaces.

Preparation and Characterization of Polyion Complex Micelles with Phosphobetaine Shells

Langmuir : the ACS Journal of Surfaces and Colloids. Aug, 2013  |  Pubmed ID: 23845059

A pair of oppositely charged diblock copolymers, poly(2-(methacryloyloxy)ethyl phosphorylcholine)-block-poly((3-(methacryloylamino)propyl)trimethylammonium chloride) (PMPC-b-PMAPTAC) and poly(2-(methacryloyloxy)ethyl phosphorylcholine)-block-poly(sodium 2-(acrylamido)-2-methylpropanesulfonate) (PMPC-b-PAMPS), was prepared via reversible addition-fragmentation chain transfer radical polymerization using a PMPC-based macro chain transfer agent. The pendant phosphorylcholine group in the hydrophilic PMPC block has anionic phosphate and cationic quaternary amino groups, which are neutralized within the pendant group. Therefore, the mixing of aqueous solutions of PMPC-b-PMAPTAC and PMPC-b-PAMPS leads to the spontaneous formation of simple core-shell spherical polyion complex (PIC) micelles comprising of a segregated PIC core and PMPC shells. The PIC micelles were characterized using (1)H NMR spin-spin (T2) and spin-lattice relaxation times (T1), diffusion-ordered NMR spectroscopy, static light scattering, dynamic light scattering (DLS), and transmission electron microscopy techniques. The hydrodynamic size of the PIC micelle depended on the mixing ratio of PMPC-b-PMAPTAC and PMPC-b-PAMPS; the maximum size occurred at the mixing ratio yielding stoichiometric charge neutralization. The PIC micelles disintegrated to become unimers with the addition of salts.

Poly(ether-ether-ketone) Orthopedic Bearing Surface Modified by Self-initiated Surface Grafting of Poly(2-methacryloyloxyethyl Phosphorylcholine)

Biomaterials. Oct, 2013  |  Pubmed ID: 23891520

We investigated the production of free radicals on a poly(ether-ether-ketone) (PEEK) substrate under ultraviolet (UV) irradiation. The amount of the ketyl radicals produced from the benzophenone (BP) units in the PEEK molecular structure initially increased rapidly and then became almost constant. Our observations revealed that the BP units in PEEK acted as photoinitiators, and that it was possible to use them to control the graft polymerization of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC). This "self-initiated surface graft polymerization" method is very convenient in the absence of external photoinitiator. We also investigated the effects of the monomer concentration and UV irradiation time on the extent of the grafted PMPC layer. Furthermore, as an application to improving the durability of artificial hips, we demonstrated the nanometer-scale photoinduced grafting of PMPC onto PEEK and carbon fiber-reinforced PEEK (CFR-PEEK) orthopedic bearing surfaces and interfaces. A variety of test revealed significant improvements in the water wettability, frictional properties, and wear resistance of the surfaces and interfaces.

Elastic Repulsion from Polymer Brush Layers Exhibiting High Protein Repellency

Langmuir : the ACS Journal of Surfaces and Colloids. Aug, 2013  |  Pubmed ID: 23898820

Hydrophilic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly(2-hydroxyethyl methacrylate) (PHEMA) brush layers with different thicknesses and graft densities were prepared to construct a model surface to elucidate protein-surface interactions. In particular, we focused on the steric repulsion of hydrophilic polymer layers as one of the surface properties that strongly influence protein adsorption and employed force-versus-distance (f-d) curve measurements obtained via atomic force microscopy to quantitatively evaluate the steric repulsion force, which is also referred to as the "elastic repulsion energy." We also analyzed direct interactions between the surface and proteins via the f-d curve, because these interactions trigger the protein-adsorption phenomenon. Protein-surface interactions were extremely suppressed at surfaces with high elastic repulsion energies and highly dense polymer brush structures, which is in contrast to those at surfaces with low elastic repulsion energies and low density of the grafted polymer layers. These results indicate that the elastic repulsion from the grafted polymer layer at the surface is an important parameter for controlling protein-surface interactions and protein adsorption phenomenon.

Synthesis of Photoreactive Phospholipid Polymers for Use in Versatile Surface Modification of Various Materials to Obtain Extreme Wettability

ACS Applied Materials & Interfaces. Aug, 2013  |  Pubmed ID: 23905848

We synthesized photoreactive phospholipid polymers for use in the versatile surface modification of various materials. The photoreactive methacrylate derivative 2-methacryloyloxyethyl-4-azidobenzoate (MPAz) was synthesized. MPAz was copolymerized with 2-methacryloyloxyethyl phosphorylcholine (MPC) to obtain poly(MPC-co-MPAz) (PMPAz) and with both MPC and n-butyl methacrylate (BMA) to obtain poly(MPC-co-BMA-co-MPAz) (PMBPAz). PMPAz and PMBPAz were bound to the surfaces of various materials, including polymers, glass, and metals, by exposure to ultraviolet irradiation. The azide groups in the MPAz units played a role in the surface anchoring; the polymer was bound covalently to the substrate. After the photoreaction, the surfaces were converted from hydrophobic to superhydrophilic, and their cell adhesion was effectively suppressed.

Increased Gastric Mucus Secretion Alleviates Non-steroidal Anti-inflammatory Drug-induced Abdominal Pain

The Tohoku Journal of Experimental Medicine. 2013  |  Pubmed ID: 24005244

Non-steroidal anti-inflammatory drugs (NSAIDs) can cause dyspeptic symptoms, including abdominal pain. Gastric mucus is important as the first line of defense against luminal irritants. In the present study, we investigated whether gastric mucus secretion could influence the severity of gastric mucosal injuries or NSAID-induced dyspeptic symptoms. Fifteen Helicobacter pylori-negative, healthy males were administered two types of NSAIDs, a non-selective cyclooxygenase inhibitor, naproxen (300 mg, twice a day), or a cyclooxygenase-2-selective inhibitor, etodolac (200 mg, twice a day), for 1 week in a crossover study, with an interval of ≥ 4 weeks. Study participants underwent endoscopic examinations before and after treatment. Pentagastrin-stimulated gastric secretions were collected for 10 min during endoscopic examinations, and were analyzed for gastric acid levels (mEq/10 min) and mucus output (mg hexose/10 min). The grade of gastric mucosal injury was assessed endoscopically. Among 29 subjects who completed the crossover study, 11 individuals reported abdominal pain following the administration of naproxen or etodolac for 1 week, as judged by elevated pain scores, while 18 individuals did not report abdominal pain. The occurrence of symptoms was not associated with the type of NSAIDs administered or the occurrence of erosive injury visualized by endoscopy. Gastric mucus secretion was significantly increased in subjects without drug-induced abdominal pain (P < 0.05), whereas it was significantly reduced in those with drug-induced abdominal pain (P < 0.05). In conclusion, the occurrence of NSAID-induced abdominal pain is associated with reduced levels of gastric mucus secretion rather than the occurrence of endoscopic mucosal injury.

The Helical Flow Total Artificial Heart: Implantation in Goats

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference. 2013  |  Pubmed ID: 24110289

To realize a total artificial heart (TAH) with high performance, high durability, good anatomical fitting, and good blood compatibility, the helical flow TAH (HFTAH) has been developed with two helical flow pumps having hydrodynamic levitation impeller. The HFTAH was implanted in goats to investigate its anatomical fitting, blood compatibility, mechanical stability, control stability, and so on. The size of the HFTAH was designed to be 80 mm in diameter and 84 mm wide. The maximum output was 19 L/min against 100 mmHg of pressure head. Eight adult female goats weighting from 45 to 56.3 kg (average 49.7 kg) were used. Under the extracorporeal circulation, natural heart was removed at the atrioventricular groove and the HFTAH was implanted. The HFTAH was driven with a pulsatile mode. The 1/R control was applied when the right atrial pressure recovered. The HFTAH could be implanted with good anatomical fitting in all goats. Two goats survived for more than a week. One goat is ongoing. Other goats did not survive for more than two days with various reasons. In the goats that survived for more than a week, the hydrodynamic bearing was worn and broken, which indicated that the bearing touched to the shaft. The cause was supposed to be the influence of the sucking effect. The potential of the HFTAH could be demonstrated with this study. The stability of the hydrodynamic bearing in a living body, especially the influence of the sucking effect, was considered to be very important and a further study should be necessary.

Identification and Characterization of Sulfated Carbohydrate-binding Protein from Lactobacillus Reuteri

PloS One. 2013  |  Pubmed ID: 24391811

We previously purified a putative sulfated-galactosylceramide (sulfatide)-binding protein with a molecular weight of 47 kDa from the cell surface of Lactobacillus reuteri JCM1081. The aim of this study was to identify the 47-kDa protein, examine its binding to sulfated glycolipids and mucins, and evaluate its role in bacterial adhesion to mucosal surfaces. By cloning and sequencing analysis, the 47-kDa protein was identified as elongation factor-Tu (EF-Tu). Adhesion properties were examined using 6 × Histidine-fused EF-Tu (His6-EF-Tu). Surface plasmon resonance analysis demonstrated pH-dependent binding of His6-EF-Tu to sulfated glycolipids, but not to neutral or sialylated glycolipids, suggesting that a sulfated galactose residue was responsible for EF-Tu binding. Furthermore, His6-EF-Tu was found to bind to porcine gastric mucin (PGM) by enzyme-linked immunosorbent assay. Binding was markedly reduced by sulfatase treatment of PGM and in the presence of acidic and desialylated oligosaccharide fractions containing sulfated carbohydrate residues prepared from PGM, demonstrating that sulfated carbohydrate moieties mediated binding. Histochemical staining revealed similar localization of His6-EF-Tu and high iron diamine staining in porcine mucosa. These results indicated that EF-Tu bound PGM via sulfated carbohydrate moieties. To characterize the contribution of EF-Tu to the interaction between bacterial cells and PGM, we tested whether anti-EF-Tu antibodies could inhibit the interaction. Binding of L. reuteri JCM1081 to PGM was significantly blocked in a concentration-dependent matter, demonstrating the involvement of EF-Tu in bacterial adhesion. In conclusion, the present results demonstrated, for the first time, that EF-Tu bound sulfated carbohydrate moieties of sulfated glycolipids and sulfomucin, thereby promoting adhesion of L. reuteri to mucosal surfaces.

Changes in the Mucus Barrier During Cisplatin-induced Intestinal Mucositis in Rats

BioMed Research International. 2013  |  Pubmed ID: 24455680

Gastrointestinal mucositis is a frequent complication of antineoplastic chemotherapy, but the effects of chemotherapy on mucosal defense mechanisms remain poorly understood. We studied the effects of cisplatin on mucin, one of the principal defense factors of the gastrointestinal mucosa, and evaluated the efficacy of two different types of H2-receptor antagonists against cisplatin-induced mucositis.

Grafting of Poly(2-methacryloyloxyethyl Phosphorylcholine) on Polyethylene Liner in Artificial Hip Joints Reduces Production of Wear Particles

Journal of the Mechanical Behavior of Biomedical Materials. Mar, 2014  |  Pubmed ID: 23651567

Despite improvements in the techniques, materials, and fixation of total hip arthroplasty, periprosthetic osteolysis, a complication that arises from this clinical procedure and causes aseptic loosening, is considered to be a major clinical problem associated with total hip arthroplasty. With the objective of reducing the production of wear particles and eliminating periprosthetic osteolysis, we prepared a novel hip polyethylene (PE) liner whose surface graft was made of a biocompatible phospholipid polymer-poly(2-methacryloyloxyethyl phosphorylcholine (MPC)). This study investigated the wear resistance of the poly(MPC)-grafted cross-linked PE (CLPE; MPC-CLPE) liner during 15×10(6) cycles of loading in a hip joint simulator. The gravimetric analysis showed that the wear of the acetabular liner was dramatically suppressed in the MPC-CLPE liner, as compared to that in the non-treated CLPE liner. Analyses of the MPC-CLPE liner surface revealed that it suffered from no or very little wear even after the simulator test, whereas the CLPE liners suffered from substantial wears. The scanning electron microscope (SEM) analysis of the wear particles isolated from the lubricants showed that poly(MPC) grafting dramatically decreased the total number, area, and volume of the wear particles. However, there was no significant difference in the particle size distributions, and, in particular, from the SEM image, it was observed that particles with diameters less than 0.50μm were present in the range of the highest frequency. In addition, there were no significant differences in the particle size descriptors and particle shape descriptors. The results obtained in this study show that poly(MPC) grafting markedly reduces the production of wear particles from CLPE liners, without affecting the size of the particles. These results suggest that poly(MPC) grafting is a promising technique for increasing the longevity of artificial hip joints.

Reduced Platelets and Bacteria Adhesion on Poly(ether Ether Ketone) by Photoinduced and Self-initiated Graft Polymerization of 2-methacryloyloxyethyl Phosphorylcholine

Journal of Biomedical Materials Research. Part A. May, 2014  |  Pubmed ID: 23720384

Aromatic poly(ether ether ketone) (PEEK) is a super engineering plastic, which has good mechanical properties and is resistant to physical and chemical stimuli. We have, therefore, attempted to use PEEK in cardiovascular devices. Synthetic cardiovascular devices require both high hemocompatibility and anti-inflammatory activity in addition to the mechanical properties. We modified the PEEK surface by photoinduced and self-initiated graft polymerization with 2-methacryloyloxyethyl phosphorylcholine (MPC; PMPC-grafted PEEK) for obtaining good antithrombogenicity. Polymerization was carried out on the surface of PEEK under radiation of ultraviolet (UV) light during which we controlled monomer concentrations, temperatures, and UV intensities. The biological performance of the PMPC-grafted PEEK was examined and compared with that of unmodified PEEK. With increase in the thickness of the PMPC layer, the amount of fibrinogen adsorption decreased significantly in comparison to that in the case of unmodified PEEK. When placed in contact with human platelet-rich plasma, surface of the PMPC-grafted PEEK clearly showed inhibition of platelet adhesion and activation. Also, bacterial adhesion was reduced dramatically on the PMPC-grafted PEEK. Thus, the PMPC grafting on PEEK improved the antithrombogenicity.

Detection of Microtubules in Vivo Using Antibody-immobilized Nanoneedles

Journal of Bioscience and Bioengineering. Jan, 2014  |  Pubmed ID: 23896017

We present here an alternative, force-based measurement method for the detection of intracellular cytoskeletal proteins in the live cell. High aspect ratio nanoneedles of 200 nm in diameter were functionalized with anti-tubulin antibodies and inserted, using an atomic force microscope (AFM), into live NIH3T3 cells, without affecting cell viability. Force curves were recorded during insertion and evacuation of nanoneedles from the cells, and used to analyse intracellular interactions of the nanoneedles with the microtubule cytoskeleton during evacuation from the cell. Disruption of microtubules led to a correlated time-dependent decrease in the measured intracellular binding forces, pointing to the high-sensitivity and high-specificity of this detection method. This analytical technique allows for real-time evaluation of the microtubule network in the live cell, without the need to use potentially harmful molecular markers as do conventional detection methods, and may prove beneficial in the diagnosis and investigation of cytoskeleton-associated diseases.

Effect of UV-irradiation Intensity on Graft Polymerization of 2-methacryloyloxyethyl Phosphorylcholine on Orthopedic Bearing Substrate

Journal of Biomedical Materials Research. Part A. Sep, 2014  |  Pubmed ID: 24124003

Photoinduced grafting of 2-methacryloyloxyethyl phosphorylcholine (MPC) onto cross-linked polyethylene (CLPE) was investigated for its ability to reduce the wear of orthopedic bearings. We investigated the effect of UV-irradiation intensity on the extent of poly(MPC) (PMPC) grafting, and found that it increased with increasing intensity up to 7.5 mW/cm(2), and the remained fairly constant. It was found to be extremely important to carefully control the UV intensity, as at higher values, a PMPC gel formed via homopolymerization of the MPC, resulting in the formation of cracks at the interface of the PMPC layer and the CLPE substrate. When the CLPE was exposed to UV-irradiation during the graft polymerization process, some of its physical and mechanical properties were slightly changed due to cross-linking and scission effects in the surface region; however, the results of all of the tests exceed the lower limits of the ASTM standards. Modification of the CLPE surface with the hydrophilic PMPC layer increased lubrication to levels that match articular cartilage. The highly hydrated thin PMPC films mimicked the native cartilage extracellular matrix that covers synovial joint surface, acting as an extremely efficient lubricant, and providing high-wear resistance.

Long-term Hip Simulator Testing of the Artificial Hip Joint Bearing Surface Grafted with Biocompatible Phospholipid Polymer

Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. Mar, 2014  |  Pubmed ID: 24249706

To prevent periprosthetic osteolysis and subsequent aseptic loosening of artificial hip joints, we recently developed a novel acetabular highly cross-linked polyethylene (CLPE) liner with graft polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) on its surface. We investigated the wear resistance of the poly(MPC) (PMPC)-grafted CLPE liner during 20 million cycles in a hip joint simulator. We extended the simulator test of one liner to 70 million cycles to investigate the long-term durability of the grafting. Gravimetric, surface, and wear particle analyses revealed that PMPC grafting onto the CLPE liner surface markedly decreased the production of wear particles and showed that the effect of PMPC grafting was maintained through 70 million cycles. We believe that PMPC grafting can significantly improve the wear resistance of artificial hip joints.

Cell-membrane-permeable and Cytocompatible Phospholipid Polymer Nanoprobes Conjugated with Molecular Beacons

Biomacromolecules. Jan, 2014  |  Pubmed ID: 24308501

To enable the visualization of the distribution and dynamics of intracellular biomolecules and thereby understand the mechanisms of intracellular bioreactions, we developed a specific functional nanoprobe through the combination of a well-designed, cytocompatible phospholipid polymer and molecular beacons (MBs). A water-soluble, amphiphilic phospholipid polymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-N-succinimidyloxycarbonyl tetra(ethylene glycol) methacrylate] (PMBS), was synthesized and conjugated with MBs to form nanoprobes via a chemical reaction between the ester group of N-hydroxysuccinimide and the amine group of the MBs. Surface tension measurements indicated that the polymeric nanoprobes had different conformations in aqueous solution, specifically at a concentration of 1.0 mg/mL. The PMBS, containing the large, hydrophobic BMA, formed polymer aggregates. The carcinoma cells used to test the probes remained 100% viable after incubation with PMBS-MB probes. The polymeric nanoprobes demonstrated not only a high target specificity but also resistance to nonspecific adsorption of proteins compared with unconjugated MBs and were able to penetrate the cytoplasm of the cells, allowing the live imaging of mRNA. In summary, MPC polymer-MB nanoprobes have great potential for practical application for the noninvasive monitoring of intracellular biomolecules and bioreactions in real time.

Quantitating Distance-dependent, Indirect Cell-cell Interactions with a Multilayered Phospholipid Polymer Hydrogel

Biomaterials. Feb, 2014  |  Pubmed ID: 24333029

Multilayered polymer hydrogels containing living cells were assembled for assessing the distance-dependent effects of soluble factors secreted by stroma cells on tumor cell cycle progression in vitro. A layer of tumor cells and a layer of stroma cells were separated with finely controlled spacing in a multilayered sandwich composed of a 2-methacryloyloxyethyl phosphorylcholine polymer and poly(vinyl alcohol) hydrogel. We demonstrated the utility of this tool for investigating intercellular communication between human cervical cancer HeLa cells and supportive stromal L929 fibroblast cells in co-culture. Time-lapse microscopic analyses of HeLa cells showed short distances (15 μm) between tumor cells and stroma cells induced a continuous increase in the percentage of HeLa cells in the S/G2/M phases of the cell cycle, while longer distances (70 μm) between the cells caused a slower increase followed by a sharp increase in the percentage of cells in S/G2/M phases. One possible explanation is gradient formation in the diffusion-dominant multilayer hydrogels by water-soluble factors such as those inducing growth, differentiation, and proliferation. This study provides insights into the potential effects of diffusion of soluble factors and related distance-dependent effects on cell behavior, which may contribute to the design of future co-culture systems.

Effects of Molecular Architecture of Phospholipid Polymers on Surface Modification of Segmented Polyurethanes

Journal of Biomaterials Science. Polymer Edition. 2014  |  Pubmed ID: 24417469

To modify the surface properties of segmented polyurethane (SPU), effects of the molecular architecture of the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers on the performance of the SPU/MPC polymer membrane were investigated. We combined the random-type, block-type, and graft-type of the MPC polymers with a typical SPU, Tecoflex(®) using double solution casting procedure. The graft-type MPC polymers composed of a poly(MPC) main chain and poly(2-ethylhexyl methacrylate (EHMA)) side chains were synthesized through the combination of two different living radical polymerization techniques to regulate the density and chain length of the side chains. The SPU membranes modified with the MPC polymers were characterized using X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The results revealed that the MPC units were located on the SPU surface. Although the breaking strength of the SPU membranes modified with block-type poly(MPC-block-EHMA) and graft-type poly(MPC-graft-EHMA) was lower than that of SPU membranes modified with random-type poly(MPC-random-EHMA), their breaking strengths were adequate for manufacturing medical devices. On the other hand, better stability was observed in the MPC polymer layer on the SPU membrane after immersion in an aqueous medium, wherein the SPU membrane had been modified with the poly(MPC-graft-EHMA). This was because of the intermixing of the hydrophobic poly(EHMA) segments in the domain of the hard segments in the SPU membrane. After this modification, each SPU/MPC polymer membrane showed hydrophilic nature based on the MPC polymers and a dramatic suppression of protein adsorption. From these results, we concluded that the SPU membrane modified with the poly(MPC-graft-EHMA) was one of the promising polymeric biomaterials for making blood-contacting medical devices.

Comprehensive Genetic Analysis of Early Host Body Reactions to the Bioactive and Bio-inert Porous Scaffolds

PloS One. 2014  |  Pubmed ID: 24454803

To design scaffolds for tissue regeneration, details of the host body reaction to the scaffolds must be studied. Host body reactions have been investigated mainly by immunohistological observations for a long time. Despite of recent dramatic development in genetic analysis technologies, genetically comprehensive changes in host body reactions are hardly studied. There is no information about host body reactions that can predict successful tissue regeneration in the future. In the present study, porous polyethylene scaffolds were coated with bioactive collagen or bio-inert poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) and were implanted subcutaneously and compared the host body reaction to those substrates by normalizing the result using control non-coat polyethylene scaffold. The comprehensive analyses of early host body reactions to the scaffolds were carried out using a DNA microarray assay. Within numerous genes which were expressed differently among these scaffolds, particular genes related to inflammation, wound healing, and angiogenesis were focused upon. Interleukin (IL)-1β and IL-10 are important cytokines in tissue responses to biomaterials because IL-1β promotes both inflammation and wound healing and IL-10 suppresses both of them. IL-1β was up-regulated in the collagen-coated scaffold. Collagen-specifically up-regulated genes contained both M1- and M2-macrophage-related genes. Marked vessel formation in the collagen-coated scaffold was occurred in accordance with the up-regulation of many angiogenesis-inducible factors. The DNA microarray assay provided global information regarding the host body reaction. Interestingly, several up-regulated genes were detected even on the very bio-inert PMB-coated surfaces and those genes include inflammation-suppressive and wound healing-suppressive IL-10, suggesting that not only active tissue response but also the inert response may relates to these genetic regulations.

Evaluation of the Durability and Antiadhesive Action of 2-methacryloyloxyethyl Phosphorylcholine Grafting on an Acrylic Resin Denture Base Material

The Journal of Prosthetic Dentistry. Aug, 2014  |  Pubmed ID: 24461942

The polymer 2-methacryloyloxyethyl phosphorylcholine is currently used on medical devices to prevent infection. Denture plaque-associated infection is regarded as a source of serious dental and medical complications in the elderly population, and denture hygiene, therefore, is an issue of considerable importance for denture wearers. Furthermore, because denture bases are exposed to mechanical stresses, for example, denture brushing, the durability of the coating is important for retaining the antiadhesive function of 2-methacryloyloxyethyl phosphorylcholine.

Calcium Mediated Formation of Phosphorylcholine-based Polyplexes for Efficient Knockdown of Epidermal Growth Factor Receptors (EGFR) in HeLa Cells

Chemical Communications (Cambridge, England). Mar, 2014  |  Pubmed ID: 24504362

2-Methacryloxyethyl phosphorylcholine (MPC) materials are well studied due to their excellent biocompatibility and are currently being used in many clinical applications. In this study, MPC based homopolymers and copolymers are prepared and are subsequently evaluated for their charge inversion properties in the presence of cations and subsequent DNA binding efficacies. These polymers are then studied for their epidermal growth factor receptor (EGFR) specific siRNA delivery in HeLa cells. The homopolymers of MPC and their copolymers show efficient EGFR knockdown efficacies in HeLa cells both in the presence and absence of serum proteins.

Quantitative Evaluation of Interaction Force Between Functional Groups in Protein and Polymer Brush Surfaces

Langmuir : the ACS Journal of Surfaces and Colloids. Mar, 2014  |  Pubmed ID: 24564418

To understand interactions between polymer surfaces and different functional groups in proteins, interaction forces were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Various polymer brush surfaces were systematically prepared by surface-initiated atom transfer radical polymerization as well-defined model surfaces to understand protein adsorption behavior. The polymer brush layers consisted of phosphorylcholine groups (zwitterionic/hydrophilic), trimethylammonium groups (cationic/hydrophilic), sulfonate groups (anionic/hydrophilic), hydroxyl groups (nonionic/hydrophilic), and n-butyl groups (nonionic/hydrophobic) in their side chains. The interaction forces between these polymer brush surfaces and different functional groups (carboxyl groups, amino groups, and methyl groups, which are typical functional groups existing in proteins) were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Furthermore, the amount of adsorbed protein on the polymer brush surfaces was quantified by surface plasmon resonance using albumin with a negative net charge and lysozyme with a positive net charge under physiological conditions. The amount of proteins adsorbed on the polymer brush surfaces corresponded to the interaction forces generated between the functional groups on the cantilever and the polymer brush surfaces. The weakest interaction force and least amount of protein adsorbed were observed in the case of the polymer brush surface with phosphorylcholine groups in the side chain. On the other hand, positive and negative surfaces generated strong forces against the oppositely charged functional groups. In addition, they showed significant adsorption with albumin and lysozyme, respectively. These results indicated that the interaction force at the functional group level might be a suitable parameter for understanding protein adsorption.

Regulation of the Cyanobacterial Circadian Clock by Electrochemically Controlled Extracellular Electron Transfer

Angewandte Chemie (International Ed. in English). Feb, 2014  |  Pubmed ID: 24573996

There is growing awareness that circadian clocks are closely related to the intracellular redox state across a range of species. As the redox state is determined by the exchange of the redox species, electrochemically controlled extracellular electron transfer (EC-EET), a process in which intracellular electrons are exchanged with extracellular electrodes, is a promising approach for the external regulation of circadian clocks. Herein, we discuss whether the circadian clock can be regulated by EC-EET using the cyanobacterium Synechococcus elongatus PCC7942 as a model system. In vivo monitoring of chlorophyll fluorescence revealed that the redox state of the plastoquionone pool could be controlled with EC-EET by simply changing the electrode potential. As a result, the endogenous circadian clock of S. elongatus cells was successfully entrained through periodically modulated EC-EET by emulating the natural light/dark cycle, even under constant illumination conditions. This is the first example of regulating the biological clock by electrochemistry.

Biomimetic Interfaces Reveal Activation Dynamics of C-reactive Protein in Local Microenvironments

Advanced Healthcare Materials. Nov, 2014  |  Pubmed ID: 24700816

Poly(2-methacryloyloxyethyl Phosphorylcholine) Grafting and Vitamin E Blending for High Wear Resistance and Oxidative Stability of Orthopedic Bearings

Biomaterials. Aug, 2014  |  Pubmed ID: 24836953

The ultimate goal in manipulating the surface and substrate of a cross-linked polyethylene (CLPE) liner is to obtain not only high wear resistance but also high oxidative stability and high-mechanical properties for life-long orthopedic bearings. We have demonstrated the fabrication of highly hydrophilic and lubricious poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) grafting layer onto the antioxidant vitamin E-blended CLPE (HD-CLPE(VE)) surface. The PMPC grafting layer with a thickness of 100 nm was successfully fabricated on the vitamin E-blended CLPE surface by using photoinduced-radical graft polymerization. Since PMPC has a highly hydrophilic nature, the water wettability and lubricity of the PMPC-grafted CLPE and HD-CLPE(VE) surfaces were greater than that of the untreated CLPE surface. The PMPC grafting contributed significantly to wear reduction in a hip-joint simulator wear test. Despite high-dose gamma-ray irradiation for cross-linking and further UV irradiation for PMPC grafting, the substrate modified by vitamin E blending maintained high-oxidative stability because vitamin E is an extremely efficient radical scavenger. Furthermore, the mechanical properties of the substrate remained almost unchanged even after PMPC grafting or vitamin E blending, or both PMPC grafting and vitamin E blending. In conclusion, the PMPC-grafted HD-CLPE(VE) provided simultaneously high-wear resistance, oxidative stability, and mechanical properties.

Durable Modification of Segmented Polyurethane for Elastic Blood-contacting Devices by Graft-type 2-methacryloyloxyethyl Phosphorylcholine Copolymer

Journal of Biomaterials Science. Polymer Edition. 2014  |  Pubmed ID: 24894706

We propose a novel application of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers for enhancing the performance of modified segmented polyurethane (SPU) surfaces for the development of a small-diameter vascular prosthesis. The SPU membranes were modified by random-type, block-type, and graft-type MPC polymers that were prepared using a double-solution casting procedure on stainless steel substrates. Among these MPC polymers, the graft-type poly(MPC-graft-2-ethylhexyl methacrylate [EHMA]), which is composed of a poly(MPC) segment as the main chain and poly(EHMA) segments as side chains, indicated a higher stability on the SPU membrane after being peeled off from the stainless steel substrate, as well as after immersion in an aqueous medium. This stability was caused by the intermiscibility in the domain of the poly(EHMA) segments and the soft segments of the SPU membrane. Each SPU/MPC polymer membrane exhibited a dramatic suppression of protein adsorption from human plasma and endothelium cell adhesion. Based on these results, the performance of SPU/poly(MPC-graft-EHMA) tubings 2 mm in diameter as vascular prostheses was investigated. Even after blood was passed through the tubings for 2 min, the graft-type MPC polymers effectively protected the blood-contacting surfaces from thrombus formation. In summary, SPU modified by graft-type MPC polymers has the potential for practical application in the form of a non-endothelium, small-diameter vascular prosthesis.

Water-soluble Polymers Bearing Phosphorylcholine Group and Other Zwitterionic Groups for Carrying DNA Derivatives

Journal of Biomaterials Science. Polymer Edition. 2014  |  Pubmed ID: 25010135

Water-soluble polymers with equal positive and negative charges in the same monomer unit, such as the phosphorylcholine group and other zwitterionic groups, exhibit promising potential in gene delivery with appreciable transfection efficiency, compared with the traditional poly(ethylene glycol)-based polycation-gene complexes. These zwitterionic polymers with various architectural structures and properties have been synthesized by various polymerization methods, such as conventional radical polymerization, atom-transfer radical-polymerization, reversible addition-fragmentation chain-transfer polymerization, and nitroxide-mediated radical polymerization. These techniques have been used to efficiently facilitate gene therapy by fabrication of non-viral vectors with high cytocompatibility, large gene-carrying capacity, effective cell-membrane permeability, and in vivo gene-loading/releasing functionality. Zwitterionic polymer-based gene delivery vectors systems can be categorized into soluble-polymer/gene mixing, molecular self-assembly, and polymer-gene conjugation systems. This review describes the preparation and characterization of various zwitterionic polymer-based gene delivery vectors, specifically water-soluble phospholipid polymers for carrying gene derivatives.

Quantitative Evaluation of Interaction Force of Fibrinogen at Well-defined Surfaces with Various Structures

Journal of Biomaterials Science. Polymer Edition. 2014  |  Pubmed ID: 25025547

The effects of functional groups and structures at the surface of biomaterials on protein adsorption were examined using direct interaction force measurements. Three kinds of surface structures were evaluated: polymer brushes, self-assembled monolayers with low molecular weight compounds, and surfaces with conventional polymer coatings. These surfaces had various functional groups including phosphorylcholine (PC) group. The surface characterization demonstrated that surface wettability and flexibility depended on both the structure of the surface and the functional groups at the surface. The interactions of protein with these surfaces were evaluated by a force vs. distance curve using an atomic force microscope (AFM). We used fibrinogen as the protein, and the fibrinogen was immobilized on the surface of the AFM cantilever by a conventional technique. It was observed that the interaction force of fibrinogen was strongly related to surface hydrophobic nature and flexibility. That is, the interaction force increased with the increasing hydrophobic nature of the surface. The relationship between the amount of fibrinogen adsorbed on the surface and the interaction force showed good correlation in the range of fibrinogen adsorption from 0 to 250 ng/cm(2), that is, in a monolayered adsorption region. The interaction force decreased with increasing surface viscoelasticity. The most effective surface for preventing fibrinogen adsorption was the polymer brush surface with phosphorylcholine (PC) groups, that is, poly(2-methacryloyloxyethyl phosphorylcholine) brush. The interaction force of this sample was less than 0.1 nN and the amount of fibrinogen adsorbed on the surface was minimal. It was found that the evaluation of protein adsorption based on the interaction force measurement is useful for low-protein adsorption surfaces. It was demonstrated that an extremely hydrophilic and flexible surface could weaken the protein interactions at the surface, resulting in greater resistance to protein adsorption.

Phospholipid Polymer-based Antibody Immobilization for Cell Rolling Surfaces in Stem Cell Purification System

Journal of Biomaterials Science. Polymer Edition. 2014  |  Pubmed ID: 25036279

We previously developed an antibody-conjugated cell rolling column that successfully separates stem cell subpopulations depending on the cell surface marker density, but a large amount of the injected cells were retained in the column because of non-specific interactions. In this study, an amphiphilic copolymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (nBMA)-co-N-vinyl formamide (NVf)], with phospholipid polar side groups was designed as a novel antibody-immobilizing modifier. The formamide groups in NVf units were converted to active maleimide groups. A plastic flow microfluidic chamber was coated with the copolymers, and a reduced anti-CD90 antibody was immobilized. The adipose tissue-derived stem cells isolated from the rat were injected into the flow chamber, and their rolling behavior was observed under a microscope with a high-speed camera. Non-specific cell adhesion was reduced strongly by means of this immobilization method because of the MPC unit, resulting in a high percentage of rolling cells. These results demonstrate that a surface coated with phospholipid polar groups can be used in an effective stem cell separation system based on the cell rolling process.

Gene Chip/PCR-array Analysis of Tissue Response to 2-methacryloyloxyethyl Phosphorylcholine (MPC) Polymer Surfaces in a Mouse Subcutaneous Transplantation System

Journal of Biomaterials Science. Polymer Edition. 2014  |  Pubmed ID: 25075735

To evaluate the in vivo foreign body reaction to bio-inert 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers, MPC polymer-coated porous substrates, with large surface area, were implanted subcutaneously in mice for 7 and 28 days, and the surrounding tissue response and cells infiltrating into the porous structure were evaluated. The MPC polymer surface induced low angiogenesis and thin encapsulation around the porous substrate, and slightly suppressed cell infiltration into the porous substrate. M1-type macrophage specific gene (CCR7) expression was suppressed by the MPC polymer surface after 7 days, resulting in the suppression of inflammatory cytokine/chemokine gene expression. However, the expression of these genes on the MPC polymer surface was higher than on the non-coated surface after 28 days. These findings suggest that MPC polymer surfaces successfully inhibit inflammatory responses during the early stage of tissue response, and seem to retard its occurrence over time.

Zwitterion Biomaterials. Foreword

Journal of Biomaterials Science. Polymer Edition. 2014  |  Pubmed ID: 25104314

Evaluation of Bifidobacterial Adhesion to Acidic Sugar Chains of Porcine Colonic Mucins

Bioscience, Biotechnology, and Biochemistry. 2014  |  Pubmed ID: 25130751

The aim of this study was to assess the adhesion of Bifidobacterium strains to acidic carbohydrate moieties of porcine colonic mucin. Mucins were extracted and purified via gel filtration chromatography followed by density-gradient ultracentrifugation. The presence of sulfated and sialylated carbohydrates in mucins was shown by enzyme-linked immunosorbent assays using PGM34 and HMC31 monoclonal antibodies (mAbs), respectively. Adhesion of Bifidobacterium strains to mucin preparations was markedly affected by the degree of purification. In eight of 22 strains, we observed increased adhesion to mucin preparations purified by ultracentrifugation. Moreover, in some of these eight strains, adhesion to mucin was reduced by pretreatment with sulfatase and/or sialidase, and competitively inhibited by pretreatment with PGM34 and/or HCM31 mAbs. Our results showed that some Bifidobacterium strains adhered to sulfo- and/or sialomucin and were able to recognize carbohydrate structures of the mAbs epitopes.

Neutron Reflectivity Study of the Swollen Structure of Polyzwitterion and Polyeletrolyte Brushes in Aqueous Solution

Journal of Biomaterials Science. Polymer Edition. 2014  |  Pubmed ID: 25178564

The swollen brush structures of polycation and zwitterionic polymer brushes, such as poly(2-methacryloyloxyethyltrimethylammonium chloride) (PMTAC), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), and poly[3-(N-2-methacryloyloxyethyl-N,N-dimethyl)ammonatopropanesulfonate] (PMAPS), in aqueous solutions of various ionic strengths were characterized by neutron reflectivity (NR) measurements. A series of the polyelectrolyte brushes were prepared by surface-initiated controlled radical polymerization on silicon substrates. A high-graft-density PMTAC brush in salt-free water (D2O) adopted a two-region step-like structure consisting of a shrunk region near the Si substrate surface and a diffuse brush region with a relatively stretched chain structure at the solution interface. The diffuse region of PMTAC was reduced with increase in salt (NaCl) concentration. The PMAPS brush in D2O formed a collapsed structure due to the strong molecular interaction between betaine groups, while significant increase in the swollen thickness was observed in salt aqueous solution. In contrast, no change was observed in the depth profile of the swollen PMPC brush in D2O with various salt concentrations. The unique solution behaviors of zwitterionic polymer brushes were described.

Clinical and Radiographic Outcomes of Total Hip Replacement with Poly(2-methacryloyloxyethyl Phosphorylcholine)-grafted Highly Cross-linked Polyethylene Liners: Three-year Results of a Prospective Consecutive Series

Modern Rheumatology. Mar, 2015  |  Pubmed ID: 25109744

This study aimed to evaluate the clinical safety and wear-resistance of the novel highly cross-linked polyethylene (HXLPE) acetabular liner with surface grafting of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) at 3 years after total hip replacement (THR).

Multidirectional Wear and Impact-to-wear Tests of Phospholipid-polymer-grafted and Vitamin E-blended Crosslinked Polyethylene: a Pilot Study

Clinical Orthopaedics and Related Research. Mar, 2015  |  Pubmed ID: 25342007

Modifying the surface and substrate of a crosslinked polyethylene (CLPE) liner may be beneficial for high wear resistance as well as high oxidative stability and excellent mechanical properties, which would be useful in contributing to the long-term performance of orthopaedic bearings. A grafted poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) layer on a vitamin E-blended crosslinked PE (HD-CLPE[VE]) surface may provide hydrophilicity and lubricity without compromising the oxidative stability or mechanical properties.

Mobility of the Arg-Gly-Asp Ligand on the Outermost Surface of Biomaterials Suppresses Integrin-mediated Mechanotransduction and Subsequent Cell Functions

Acta Biomaterialia. Feb, 2015  |  Pubmed ID: 25463493

Mechanotransduction in the regulation of cellular responses has been previously studied using elastic hydrogels. Because cells interact only with the surface of biomaterials, we are focusing on the molecular mobility at the outermost surface of biomaterials. In this study, surfaces with the mobile Arg-Gly-Asp-Ser (RGDS) peptide have been constructed. Cell culture substrates were coated with ABA-type block copolymers composed of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) segments (A) and a polyrotaxane (PRX) unit with RGDS bound to α-cyclodextrin (B). Adhesion, morphological changes and actin filament formation of human umbilical vein endothelial cells were reduced on the surfaces containing mobile PRX-RGDS in comparison to the immobile RGDS surfaces constructed from random copolymers with RGDS side groups (Prop-andom-RGDS). In the neurite outgrowth assay using rat adrenal pheochromocytoma cells (PC12), only ∼20% of adherent PC12 cells had neurites on PRX-RGDS surfaces, but more than 50% did on the Random-RGDS surface. The beating colony of dimethyl-sulfoxide-treated mouse embryonic carcinoma cells (P19CL6) were found 10 and 14 days after induction on PRX-RGDS and Random-RGDS surfaces, respectively. After 22 days, the beating colony disappeared on PRX-RGDS surfaces, but many colonies remained on Random-RGDS surfaces. These data suggest that the molecular mobility of the cell-binding ligand on the outermost surface of materials effectively suppresses the actin filament formation and differentiation of these functional cell lines, and may be used as a culture substrate for immature stem cells or progenitor cells.

Concentration-dependent Effects of Fibronectin Adsorbed on Hydroxyapatite Surfaces on Osteoblast Adhesion

Materials Science & Engineering. C, Materials for Biological Applications. Mar, 2015  |  Pubmed ID: 25579937

Fibronectin (Fn) is an essential protein that is involved in cell attachment, migration, and differentiation. It is known that the conformation of Fn changes depending on its surrounding environment or on the surface properties of the substrate to which it is adsorbed. Here, we show that Fn adopts different conformations in physiological solutions and can play different roles in osteoblast adhesion on hydroxyapatite (HAp) depending on its concentration. The results of in situ observations showed that the adsorption configuration of Fn was an oblate ellipsoidal structure at low concentrations and a fibrillar structure at high concentrations, suggesting that individual Fn molecules attach to HAp with a side-on or end-on orientation, under low and high concentrations, respectively. Osteoblasts adhered more to the HAp surface under low concentrations of adsorbed Fn than under high concentrations. These observations suggest that the Fn concentration gradient is important for regulating biomaterial-cell interactions in regenerative medicine.

Hollow Fiber Membrane Modification with Functional Zwitterionic Macromolecules for Improved Thromboresistance in Artificial Lungs

Langmuir : the ACS Journal of Surfaces and Colloids. Mar, 2015  |  Pubmed ID: 25669307

Respiratory assist devices seek optimized performance in terms of gas transfer efficiency and thromboresistance to minimize device size and reduce complications associated with inadequate blood biocompatibility. The exchange of gas with blood occurs at the surface of the hollow fiber membranes (HFMs) used in these devices. In this study, three zwitterionic macromolecules were attached to HFM surfaces to putatively improve thromboresistance: (1) carboxyl-functionalized zwitterionic phosphorylcholine (PC) and (2) sulfobetaine (SB) macromolecules (mPC or mSB-COOH) prepared by a simple thiol-ene radical polymerization and (3) a low-molecular weight sulfobetaine (SB)-co-methacrylic acid (MA) block copolymer (SBMAb-COOH) prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. Each macromolecule type was covalently immobilized on an aminated commercial HFM (Celg-A) by a condensation reaction, and HFM surface composition changes were analyzed by X-ray photoelectron spectroscopy. Thrombotic deposition on the HFMs was investigated after contact with ovine blood in vitro. The removal of CO2 by the HFMs was also evaluated using a model respiratory assistance device. The HFMs conjugated with zwitterionic macromolecules (Celg-mPC, Celg-mSB, and Celg-SBMAb) showed expected increases in phosphorus or sulfur surface content. Celg-mPC and Celg-SBMAb experienced rates of platelet deposition significantly lower than those of unmodified (Celg-A, >95% reduction) and heparin-coated (>88% reduction) control HFMs. Smaller reductions were seen with Celg-mSB. The CO2 removal rate for Celg-SBMAb HFMs remained comparable to that of Celg-A. In contrast, the rate of removal of CO2 for heparin-coated HFMs was significantly reduced. The results demonstrate a promising approach to modifying HFMs using zwitterionic macromolecules for artificial lung devices with improved thromboresistance without degradation of gas transfer.

Safety, Reliability, and Operability of Cochlear Implant Electrode Arrays Coated with Biocompatible Polymer

Acta Oto-laryngologica. Apr, 2015  |  Pubmed ID: 25719221

Polymer-coated electrodes can reduce surgically-induced trauma associated with the insertion of a cochlear implant (CI) electrode array.

Molecular Interaction Forces Generated During Protein Adsorption to Well-defined Polymer Brush Surfaces

Langmuir : the ACS Journal of Surfaces and Colloids. Mar, 2015  |  Pubmed ID: 25719761

The molecular interaction forces generated during the adsorption of proteins to surfaces were examined by the force-versus-distance (f-d) curve measurements of atomic force microscopy using probes modified with appropriate molecules. Various substrates with polymer brush layers bearing zwitterionic, cationic, anionic, and hydrophobic groups were systematically prepared by surface-initiated atom transfer radical polymerization. Surface interaction forces on these substrates were analyzed by the f-d curve measurements using probes with the same polymer brush layer as the substrate. Repulsive forces, which decreased depending on the ionic strength, were generated between cationic or anionic polyelectrolyte brush layers; these were considered to be electrostatic interaction forces. A strong adhesive force was detected between hydrophobic polymer brush layers during retraction; this corresponded to the hydrophobic interaction between two hydrophobic polymer layers. In contrast, no significant interaction forces were detected between zwitterionic polymer brush layers. Direct interaction forces between proteins and polymer brush layers were then quantitatively evaluated by the f-d curve measurements using protein-immobilized probes consisting of negatively charged albumin and positively charged lysozyme under physiological conditions. In addition, the amount of protein adsorbed on the polymer brush layer was quantified by surface plasmon resonance measurements. Relatively large amounts of protein adsorbed to the polyelectrolyte brush layers with opposite charges. It was considered that the detachment of the protein after contact with the polymer brush layer hardly occurred due to salt formation at the interface. Both proteins adsorbed significantly on the hydrophobic polymer brush layer, which was due to hydrophobic interactions at the interface. In contrast, the zwitterionic polymer brush layer exhibited no significant interaction force with proteins and suppressed protein adsorption. Taken together, our results suggest that to obtain the protein-repellent surfaces, the surface should not induce direct interaction forces with proteins after contact with them.

Wear Resistance of the Biocompatible Phospholipid Polymer-grafted Highly Cross-linked Polyethylene Liner Against Larger Femoral Head

Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. Jul, 2015  |  Pubmed ID: 25764495

The use of larger femoral heads to prevent the dislocation of artificial hip joints has recently become more common. However, concerns about the subsequent use of thinner polyethylene liners and their effects on wear rate have arisen. Previously, we prepared and evaluated the biological and mechanical effects of a novel highly cross-linked polyethylene (CLPE) liner with a nanometer-scaled graft layer of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC). Our findings showed that the PMPC-grafted particles were biologically inert and caused no subsequent bone resorptive responses and that the PMPC-grafting markedly decreased wear in a hip joint simulator. However, the metal or ceramic femoral heads used in this previous study had a diameter of 26 mm. Here, we investigated the wear-resistance of the PMPC-grafted CLPE liner with a 40-mm femoral head during 10 × 10(6) cycles of loading in the hip joint simulator. The results provide preliminary evidence that the grafting markedly decreased gravimetric wear rate and the volume of wear particles, even when coupled with larger femoral heads. Thus, we believe the PMPC-grafting will prolong artificial hip joint longevity both by preventing aseptic loosening and by improving the stability of articular surface.

Synthesis of Grafted Phosphorylcholine Polymer Layers As Specific Recognition Ligands for C-reactive Protein Focused on Grafting Density and Thickness to Achieve Highly Sensitive Detection

Physical Chemistry Chemical Physics : PCCP. Apr, 2015  |  Pubmed ID: 25783194

We studied the effects of layer thickness and grafting density of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) thin layers as specific ligands for the highly sensitive binding of C-reactive protein (CRP). PMPC layer thickness was controlled by surface-initiated activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP). PMPC grafting density was controlled by utilizing mixed self-assembled monolayers with different incorporation ratios of the bis[2-(2-bromoisobutyryloxy)undecyl] disulfide ATRP initiator, as modulated by altering the feed molar ratio with (11-mercaptoundecyl)tetra(ethylene glycol). X-ray photoelectron spectroscopy and ellipsometry measurements were used to characterize the modified surfaces. PMPC grafting densities were estimated from polymer thickness and the molecular weight obtained from sacrificial initiator during surface-initiated AGET ATRP. The effects of thickness and grafting density of the obtained PMPC layers on CRP binding performance were investigated using surface plasmon resonance employing a 10 mM Tris-HCl running buffer containing 140 mM NaCl and 2 mM CaCl2 (pH 7.4). Furthermore, the non-specific binding properties of the obtained layers were investigated using human serum albumin (HSA) as a reference protein. The PMPC layer which has 4.6 nm of thickness and 1.27 chains per nm(2) of grafting density showed highly sensitive CRP detection (limit of detection: 4.4 ng mL(-1)) with low non-specific HSA adsorption, which was improved 10 times than our previous report of 50 ng mL(-1).

Induction of Sd(a)-sialomucin and Sulfated H-sulfomucin in Mouse Small Intestinal Mucosa by Infection with Parasitic Helminth

Experimental Parasitology. Jun, 2015  |  Pubmed ID: 25819298

Mucin is a major component of mucus on gastrointestinal mucosa. Mucin alteration in the host is considered to be the principal event for expulsion of intestinal helminths. However, it is unclear what mucin alterations are induced by various helminth infections. In this study, the alterations of mouse small intestinal mucin after infection with two nematodes, Nippostrongylus brasiliensis and Heligmosomoides polygyrus, which parasitize the jejunal epithelium, and a cestode, Vampirolepis nana, which parasitizes the ileal epithelium, were examined biochemically and histologically using two anti-mucin monoclonal antibodies (mAbs), HCM31 and PGM34, which recognize Sd(a) antigen, NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ-, and sulphated H type 2 antigen, Fucα1-2Galβ1-4GlcNAc(6SO₃H)β-, respectively. The goblet cell mucins that reacted with HCM31 increased conspicuously on the jejunal mucosa concurrently with expulsion of N. brasiliensis. Increased levels of HCM31-reactive mucins were observed in the jejunal mucosa after H. polygyrus infection, despite the ongoing parasitism. Goblet cell mucins that reacted with PGM34 increased on the ileal mucosa during V. nana parasitism. Small intestinal goblet cells reacting with the two mAbs were not observed in non-infected mice, although sialomucins and sulfomucins were abundantly present. Additionally, the number of ileal goblet cells that reacted with the two mAbs was increased at the time of expulsion of heterophyid trematode. These results indicate that the type of specific acidic mucins expressed after infection varies among species of intestinal helminth, and, furthermore, that the relationship with worm expulsion is also different.

Therapeutic Effect of Intravesical Administration of Paclitaxel Solubilized with Poly(2-methacryloyloxyethyl Phosphorylcholine-co-n-butyl Methacrylate) in an Orthotopic Bladder Cancer Model

BMC Cancer. Apr, 2015  |  Pubmed ID: 25928041

To evaluate the effects of intravesical administration of paclitaxel (PTX-30W), which was prepared by solubilization with a water-soluble amphiphilic polymer composed of PMB30W, a copolymer of 2-methacryloyloxyethyl phosphorylcholine and n-butyl methacrylate, in an orthotopic bladder cancer model.

Efficient Differentiation of Stem Cells Encapsulated in a Cytocompatible Phospholipid Polymer Hydrogel with Tunable Physical Properties

Biomaterials. Jul, 2015  |  Pubmed ID: 25934282

A large number of lineage-committed progenitor cells are required for advanced regenerative medicine based on cell engineering. Due to their ability to differentiate into multiple cells lines, multipotent stem cells have emerged as a vital source for generating transplantable cells for use in regenerative medicine. Increment in differentiation efficiency of the mesenchymal stem cell was obtained by using hydrogel to adjust the proliferation cycle of encapsulated cells to signal sensitive phase. Three dimensional (3-D) polymer networks composed of poly(2-methacyloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-vinylphenylboronic acid (VPBA)) (PMBV) and poly(vinyl alcohol) (PVA) were prepared as a hydrogel. The proliferation of cells encapsulated in the PMBV/PVA hydrogel was highly sensitive to the storage modulus (G') of the hydrogel. That is, when the G' value of the hydrogel was higher than 1.0 kPa, the cell proliferation was ceased and the proliferation cycle of cells was converged to G1 phase, whereas when the G' value was below 1.0 kPa, cell proliferation proceeded. By changing the G' value of hydrogels under encapsulation the cells, proliferation cycle of encapsulated mesenchymal stem cells was regulated to G1 phase and thus signal sensitivity were increased. 3-D polymer networks as hydrogels with tunable physical properties can be effectively used to control proliferation and lineage-restricted differentiation of stem cells.

Preparation of Upper Critical Solution Temperature (UCST) Responsive Diblock Copolymers Bearing Pendant Ureido Groups and Their Micelle Formation Behavior in Water

Soft Matter. Jul, 2015  |  Pubmed ID: 25971855

Poly(2-ureidoethyl methacrylate) (PUEM) was prepared via reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization and a post-modification reaction. PUEM shows upper critical solution temperature (UCST) behavior in aqueous solution. Although PUEM can dissolve in water above the UCST, it cannot dissolve in water below the UCST. Diblock copolymers (MmUn) composed of a biocompatible hydrophilic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) block and a PUEM block with different compositions were prepared via RAFT radical polymerization and a post-modification reaction. "M" and "U" represent PMPC and PUEM blocks, respectively, and the subscripts represent the degree of polymerization of each block. M95U149 and M20U163 formed polymer micelles comprising a PUEM core and a PMPC shell below the critical aggregation temperature (Tc) in aqueous solution. Polymer micelles were formed from M20U163 below 32 °C, which can incorporate guest molecules into the core.

Influences of Dehydration and Rehydration on the Lubrication Properties of Phospholipid Polymer-grafted Cross-linked Polyethylene

Proceedings of the Institution of Mechanical Engineers. Part H, Journal of Engineering in Medicine. Jul, 2015  |  Pubmed ID: 26036469

Surface modification by grafting of biocompatible phospholipid polymer onto the surface of artificial joint material has been proposed to reduce the risk of aseptic loosening and improve the durability. Poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-grafted cross-linked polyethylene (CLPE) has shown promising results for reducing wear of CLPE. The main lubrication mechanism for the PMPC layer is considered to be the hydration lubrication. In this study, the lubrication properties of PMPC-grafted CLPE were evaluated in reciprocating friction test with rehydration process by unloading in various lubricants. The start-up friction of PMPC-grafted CLPE was reduced, and the damage of PMPC layer was suppressed by rehydration in water or hyaluronic acid solutions. In contrast, the start-up friction of PMPC-grafted CLPE increased in fetal bovine serum solution, and the damage for PMPC layer was quite noticeable. Interestingly, the start-up friction of PMPC-grafted CLPE was reduced in fetal bovine serum solution containing hyaluronic acid, and the damage of the PMPC layer was suppressed. These results indicate that the rehydration by unloading and hyaluronic acid are elemental in maximizing the lubrication effect of hydrated PMPC layer.

Prevention of Bacterial Adhesion and Biofilm Formation on a Vitamin E-blended, Cross-linked Polyethylene Surface with a Poly(2-methacryloyloxyethyl Phosphorylcholine) Layer

Acta Biomaterialia. Sep, 2015  |  Pubmed ID: 26050636

In the construction of artificial hip joint replacements, the surface and substrate of a cross-linked polyethylene (CLPE) liner are designed to achieve high wear resistance and prevent infection by bacteria. In this study, we fabricated a highly hydrophilic and antibiofouling poly(2-methacryloyloxyethyl phosphorylcholine [MPC]) (PMPC)-graft layer on the vitamin E-blended CLPE (HD-CLPE(VE)) surface. The 100-nm-thick, smooth, and electrically neutral PMPC layer was successfully fabricated on the HD-CLPE(VE) surface using photoinduced graft polymerization. The PMPC-grafted HD-CLPE(VE) was found to prevent bacterial adherence and biofilm formation on the surface because of the formation of a highly hydrophilic polyzwitterionic layer on the surface of HD-CLPE(VE), which can serve as an extremely efficient antibiofouling layer. The number of bacterial adhered on the PMPC-grafted HD-CLPE(VE) surface was reduced by 100-fold or more by PMPC grafting, regardless of the biofilm-production characteristics of the strains. In contrast, vitamin E blending did not affect bacterial adhesion. Moreover, the number of planktonic bacteria did not differ significantly, regardless of PMPC grafting and vitamin E blending. In conclusion, the PMPC-grafted HD-CLPE(VE) provided bacteriostatic effects associated with smooth, highly hydrophilic surfaces with a neutral electrostatic charge owing to the zwitterionic structure of the MPC unit. Thus, this modification may prove useful for the production of artificial hip joint replacement materials.

Photoreactive Polymers Bearing a Zwitterionic Phosphorylcholine Group for Surface Modification of Biomaterials

ACS Applied Materials & Interfaces. Aug, 2015  |  Pubmed ID: 26202385

Photoreactive polymers bearing zwitterionic phosphorylcholine and benzophenone groups on the side chain were synthesized and used as surface modification reagents for biomaterials. A photoreactive methacrylate containing the benzophenone group, 3-methacryloyloxy-2-hydroxypropyl-4-oxybenzophenone (MHPBP), was synthesized via a ring-opening and addition reaction between glycidyl methacrylate and 4-hydroxybenzophenone. Then, water-soluble, amphiphilic polymers poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-MHPBP) (PMH) and poly(MPC-co-n-butyl methacrylate-co-MHPBP), with different monomer unit compositions, were synthesized through radical polymerization. Ultraviolet-visible (UV/vis) absorption spectra of these polymer solutions showed that these polymers have maximum absorption peaks at 254 and 289 nm that can be attributed to the benzophenone unit. The intensity of UV adsorption at 289 nm was decreased with increased UV irradiation time, and it was saturated within a few minutes, indicating that the polymers are highly sensitive to UV irradiation. A commercial material (i.e., cyclic polyolefin) was simply modified by a UV irradiation for 1.0 min. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy analysis results indicated that the stability of the polymer on the surface was dramatically enhanced because of the photochemical reaction of the benzophenone moiety. The air contact angles of PMH surfaces measured in water were up to 160°. Thus, highly hydrophilic surfaces were obtained. The critical surface tension of the PMH-modified surface was 45.7 mN/m. By evaluating the biological reactivity of the treated surface, protein adsorption and cell adhesion were completely inhibited on the surface, which was prepared using a photopatterning procedure using PMH. In conclusion, photoreactive MPC polymers with a benzophenone moiety could be used as a novel and effective surface modifier.

Animal Experiments of the Helical Flow Total Artificial Heart

Artificial Organs. Aug, 2015  |  Pubmed ID: 26234448

Severe cardiac failure patients require a total artificial heart (TAH) to save life. To realize a TAH that can fit a body of small stature and has high performance, high durability, good anatomical fitting, good blood compatibility, and physiological control, we have been developing the helical flow TAH (HFTAH) with two helical flow pumps with hydrodynamic levitation impeller. Animal experiments of the HFTAH were conducted to perform in vivo studies. The HFTAH was implanted in 13 adult female goats weighing 45.0-64.0 kg. After surgery, neither anti-coagulant nor anti-platelet medication was given systemically. The HFTAH was usually driven with a quasi-pulsatile mode. The 1/R control or ΔP control was applied to control the circulation. The ΔP control is a new method using simplified equation of the 1/R control. The HFTAH could be implanted in all goats with good anatomical fitting. Two goats survived for a long time (100 and 68 days). Major causes of termination were device failure and surgical complications. In the device failure, trouble with hydrodynamic bearing was conspicuous. In the two long-term survived goats, experiments were terminated with bearing instability that was probably caused by the suction effect. In these goats, hemolysis occurred on postoperative day 88 and 44, which was considered to be relevant to the bearing trouble. Thrombus was found at the broken right bearing of the 100-day survived goat. However, antithrombogenicity of the pump is expected to be good unless bearing trouble occurs. In two long-term survived goats, the 1/R control or ΔP control worked appropriately to prevent the elevation of right atrial pressure. In both goats, hemodynamic parameters changed with the condition of the animals, liver and kidney functions remained almost normal except when recovering from surgery and during hemolysis, and total protein recovered 2 weeks after surgery. Although instability of the hydrodynamic bearing should be improved, performance of the HFTAH with physiological control could be demonstrated.

Preparation of Photoreactive Phospholipid Polymer Nanoparticles to Immobilize and Release Protein by Photoirradiation

Colloids and Surfaces. B, Biointerfaces. Nov, 2015  |  Pubmed ID: 26263222

Photoreactive and cytocompatible polymer nanoparticles for immobilizing and releasing proteins were prepared. A water-soluble and amphiphilic phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-4-(4-(1-methacryloyloxyethyl)-2-methoxy-5-nitrophenoxy) butyric acid (PL)) (PMB-PL) was synthesized. The PMB-PL underwent a cleavage reaction at the PL unit with photoirradiation at a wavelength of 365 nm. Additionally, the PMB-PL took polymer aggregate in aqueous medium and was used to modify the surface of biodegradable poly(L-lactic acid) (PLA) nanoparticle as an emulsifier. The morphology of the PMB-PL/PLA nanoparticle was spherical and approximately 130 nm in diameter. The carboxylic acid group in the PL unit could immobilize proteins by covalent bonding. The bound proteins were released by a photoinduced cleavage reaction. Within 60s, up to 90% of the immobilized proteins was released by photoirradiation. From these results and with an understanding of the fundamental properties of MPC polymers, we concluded that PMB-PL/PLA nanoparticles have the potential to be used as smart carriers to deliver proteins to biological systems, such as the inside of living cells.

Surface Functionalization of Quantum Dots with Fine-structured PH-sensitive Phospholipid Polymer Chains

Colloids and Surfaces. B, Biointerfaces. Nov, 2015  |  Pubmed ID: 26283498

To add novel functionality to quantum dots (QDs), we synthesized water-soluble and pH-responsive block-type polymers by reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymers were composed of cytocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer segments, which contain a small fraction of active ester groups and can be used to conjugate biologically active compounds to the polymer, and pH-responsive poly(2-(N,N-diethylamino) ethyl methacrylate (DEAEMA)) segments. One terminal of the polymer chain had a hydrophobic alkyl group that originated from the RAFT initiator. This hydrophobic group can bind to the hydrophobic layer on the QD surface. A fluorescent dye was conjugated to the polymer chains via the active ester group. The block-type polymers have an amphiphilic nature in aqueous medium. The polymers were thus easily bound to the QD surface upon evaporation of the solvent from a solution containing the block-type polymer and QDs, yielding QD/fluorescence dye-conjugated polymer hybrid nanoparticles. Fluorescence resonance energy transfer (FRET) between the QDs (donors) and the fluorescent dye molecules (acceptors) was used to obtain information on the conformational dynamics of the immobilized polymers. Higher FRET efficiency of the QD/fluorescent dye-conjugated polymer hybrid nanoparticles was observed at pH 7.4 as compared to pH 5.0 due to a stretching-shrinking conformational motion of the poly(DEAEMA) segments in response to changes in pH. We concluded that the block-type MPC polymer-modified nanoparticles could be used to evaluate the pH of cells via FRET fluorescence based on the cytocompatibility of the MPC polymer.

Fabrication of a Live Cell-containing Multilayered Polymer Hydrogel Membrane with Micrometer-scale Thickness to Evaluate Pharmaceutical Activity

Journal of Biomaterials Science. Polymer Edition. 2015  |  Pubmed ID: 26374190

We propose a spinning-assisted layer-by-layer method for simple fabrication of a multilayered polymer hydrogel membrane that contains living cells. Hydrogel formation occurred based on the spontaneous cross-linking reaction between two polymers in aqueous solution. A water-soluble 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenylboronic acid groups (PMBV) and poly(vinyl alcohol) (PVA) were used as polymers for hydrogel membrane formation. Changing the number of hydrogel membrane layers, polymer concentration, spinning rate, and processing time for diffusion-dependent gelation of PMBV and PVA facilitated the regulation of the multilayered polymer hydrogel membrane thickness and morphology. We concluded that a multilayered polymer hydrogel membrane prepared using 5.0 wt% PMBV and 5.0 wt% PVA at a spinning rate of 2000 rpm was suitable for precise spatial control of cells in single layers. This multilayered polymer hydrogel membrane was used to prepare a single cell-laden layer to minimize barriers to the diffusion of bioactive compounds while preserving the three-dimensional (3-D) context. The pharmaceutical effects of one of the anticancer agents, paclitaxel, on a human cervical cancer line, HeLa cells, were evaluated in vitro, and the usability of this culture model was demonstrated.

Spontaneous Packaging and Hypothermic Storage of Mammalian Cells with a Cell-Membrane-Mimetic Polymer Hydrogel in a Microchip

ACS Applied Materials & Interfaces. Oct, 2015  |  Pubmed ID: 26436637

Currently, continuous culture/passage and cryopreservation are two major, well-established methods to provide cultivated mammalian cells for experiments in laboratories. Due to the lack of flexibility, however, both laboratory-oriented methods are unable to meet the need for rapidly growing cell-based applications, which require cell supply in a variety of occasions outside of laboratories. Herein, we report spontaneous packaging and hypothermic storage of mammalian cells under refrigerated (4 °C) and ambient conditions (25 °C) using a cell-membrane-mimetic methacryloyloxyethyl phosphorylcholine (MPC) polymer hydrogel incorporated within a glass microchip. Its capability for hypothermic storage of cells was comparatively evaluated over 16 days. The results reveal that the cytocompatible MPC polymer hydrogel, in combination with the microchip structure, enabled hypothermic storage of cells with quite high viability, high intracellular esterase activity, maintained cell membrane integrity, and small morphological change for more than 1 week at 4 °C and at least 4 days at 25 °C. Furthermore, the stored cells could be released from the hydrogel and exhibited the ability to adhere to a surface and achieve confluence under standard cell culture conditions. Both hypothermic storage conditions are ordinary flexible conditions which can be easily established in places outside of laboratories. Therefore, cell packaging and storage using the hydrogel incorporated within the microchip would be a promising miniature and portable solution for flexible supply and delivery of small amounts of cells from bench to bedside.

Effects of Surface Modification and Bulk Geometry on the Biotribological Behavior of Cross-Linked Polyethylene: Wear Testing and Finite Element Analysis

BioMed Research International. 2015  |  Pubmed ID: 26583106

The wear and creep deformation resistances of polymeric orthopedic bearing materials are both important for extending their longevity. In this study, we evaluated the wear and creep deformation resistances, including backside damage, of different polyethylene (PE) materials, namely, conventional PE, cross-linked PE (CLPE), and poly(2-methacryloyloxyethyl phosphorylcholine)- (PMPC-) grafted CLPE, through wear tests and finite element analysis. The gravimetric and volumetric degrees of wear of disks (3 or 6 mm in thickness) of these materials against a cobalt-chromium-molybdenum alloy pin were examined using a multidirectional pin-on-disk tester. Cross-linking and PMPC grafting decreased the gravimetric wear of the PE disks significantly. The volumetric wear at the bearing surface and the volumetric penetration in the backside of the 3-mm thick PE disk were higher than those of the 6-mm thick PE disk, regardless of the bearing material. The geometrical changes induced in the PE disks consisted of creep, because the calculated internal von Mises stress at the bearing side of all disks and that at the backside of the 3-mm thick disks exceeded their actual yield strengths. A highly hydrated bearing surface layer, formed by PMPC grafting, and a cross-linking-strengthened substrate of adequate thickness are essential for increasing the wear and creep deformation resistances.

Effects of Extra Irradiation on Surface and Bulk Properties of PMPC-grafted Cross-linked Polyethylene

Journal of Biomedical Materials Research. Part A. Jan, 2016  |  Pubmed ID: 26148654

Sterilization using high-energy irradiation is an important aspect of implementing an ultra-high molecular weight polyethylene acetabular liner in total hip arthroplasty (THA). In this study, we evaluate the effects of extra irradiations such as gamma-ray or plasma irradiation during sterilization of the poly(2-methacryloyloxyethyl phosphorylcholine [MPC]) (PMPC) surface and cross-linked polyethylene (CLPE) substrate of a PMPC-grafted CLPE acetabular liner. The PMPC-grafted surface yielded high wettability and low friction properties regardless of the extra irradiations as compared with untreated CLPE. During a hip simulator test, wear resistance of the PMPC-grafted CLPE liner was maintained after extra irradiation, which is due to the high wettability characteristics of the PMPC surface. In particular, the PMPC-grafted CLPE liner treated with plasma irradiation showed greater wettability and wear resistance than that with gamma-ray irradiation. However, we could not clearly observe the changes in chemical properties and morphology of the PMPC surface after both extra irradiations. The physical and mechanical properties attributed to CLPE substrate performance were also unchanged. In contrast, PMPC-grafted CLPE treated with plasma irradiation showed improved oxidation resistance as compared to that treated with gamma-ray irradiation after accelerated aging. Thus, we conclude that PMPC-grafted CLPE with plasma irradiation has promise as a lifelong solution for bearing in THA.

Preparation of a Thick Polymer Brush Layer Composed of Poly(2-methacryloyloxyethyl Phosphorylcholine) by Surface-initiated Atom Transfer Radical Polymerization and Analysis of Protein Adsorption Resistance

Colloids and Surfaces. B, Biointerfaces. May, 2016  |  Pubmed ID: 26896657

The purpose of this study was to prepare a thick polymer brush layer composed of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) and assess its resistance to protein adsorption from the dissolved state of poly(MPC) chains in an aqueous condition. The thick poly(MPC) brush layer was prepared through the surface-initiated atom transfer radical polymerization (SI-ATRP) of MPC with a free initiator from an initiator-immobilized substrate at given [Monomer]/[Free initiator] ratios. The ellipsometric thickness of the poly(MPC) brush layers could be controlled by the polymerization degree of the poly(MPC) chains. The thickness of the poly(MPC) brush layer in an aqueous medium was larger than that in air, and this tendency became clearer when the polymerization degree of the poly(MPC) increased. The maximum thickness of the poly(MPC) brush layer in an aqueous medium was around 110 nm. The static air contact angle of the poly(MPC) brush layer in water indicated a reasonably hydrophilic nature, which was independent of the thickness of the poly(MPC) brush layer at the surface. This result occurred because the hydrated state of the poly(MPC) chains is not influenced by the environment surrounding them. Finally, as measured with a quartz crystal microbalance, the amount of protein adsorbed from a fetal bovine serum solution (10% in phosphate-buffered saline) on the original substrate was 420 ng/cm(2). However, the poly(MPC) brush layer reduced this value dramatically to less than 50 ng/cm(2). This effect was independent of the thickness of the poly(MPC) brush layer for thicknesses between 20 nm and about 110 nm. These results indicated that the surface covered with a poly(MPC) brush layer is a promising platform to avoid biofouling and could also be applied to analyze the reactions of biological molecules with a high signal/noise ratio.

Photoinduced Inhibition of DNA Unwinding in Vitro with Water-soluble Polymers Containing Both Phosphorylcholine and Photoreactive Groups

Acta Biomaterialia. Aug, 2016  |  Pubmed ID: 27045692

Nile blue (NB)-tagged DNA helix-targeting amphiphilic photoreactive 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, poly(MPC-co-3-methacryloyloxy-2-hydroxypropyl-4-oxybenzophenone-co-2-trimethylammonium ethyl methacrylate chloride) (PMHT-NB), containing a cationic group to facilitate cell membrane penetration and a benzophenone (BP) group to promote photoinduced conjugation with DNA helix was synthesized using radical polymerization method. Ultraviolet light (UV)-visible light absorption spectra of PMHT-NB showed absorption peaks at wavelengths 254, 289, and 600nm, suggesting successful incorporation of BP and NB groups. PMHT-NB was highly sensitive to photoirradiation with UV irradiation at the second level, as confirmed based on the degradation spectra of UV absorption peaks for the BP group in PBS (pH=7.4). PMHT-NB showed good solubility in both aqueous solution and in ethanol. In a cell culture medium containing 10mg/mL PMHT-NB, the NB group showed fluorescence peaks at an emission wavelength of 650nm and excitation wavelength of 633nm. PMHT-NB also showed low cytotoxicity and good cell membrane permeability toward cancerous HeLa cells. Further, photoinduced PMHT-NB effectively inhibited the unwinding of a molecular beacon with a hairpin structure, indicating that synthetic photoreactive MPC polymers photoregulated the unwinding of DNA.

Formation of Polyion Complex (PIC) Micelles and Vesicles with Anionic PH-Responsive Unimer Micelles and Cationic Diblock Copolymers in Water

Langmuir : the ACS Journal of Surfaces and Colloids. Apr, 2016  |  Pubmed ID: 27048989

A random copolymer (p(A/MaU)) of sodium 2-(acrylamido)-2-methylpropanesulfonate (AMPS) and sodium 11-methacrylamidoundecanate (MaU) was prepared via conventional radical polymerization, which formed a unimer micelle under acidic conditions due to intramolecular hydrophobic interactions between the pendant undecanoic acid groups. Under basic conditions, unimer micelles were opened up to an expanded chain conformation by electrostatic repulsion between the pendant sulfonate and undecanoate anions. A cationic diblock copolymer (P163M99) consisting of poly(3-(methacrylamido)propyl)trimethylammonium chloride (PMAPTAC) and hydrophilic polybetaine, 2-(methacryloyloxy)ethylphosphorylcholine (MPC), blocks was prepared via controlled radical polymerization. Mixing of p(A/MaU) and P163M99 in 0.1 M aqueous NaCl under acidic conditions resulted in the formation of spherical polyion complex (PIC) micelles and vesicles, depending on polymer concentration before mixing. Shapes of the PIC micelles and vesicles changed under basic conditions due to collapse of the charge balance between p(A/MaU) and P163M99. The PIC vesicles can incorporate nonionic hydrophilic guest molecules, and the PIC micelles and vesicles can accept hydrophobic guest molecules in the hydrophobic core formed from p(A/MaU).

High-efficiency Preparation of Poly(2-methacryloyloxyethyl Phosphorylcholine) Grafting Layer on Poly(ether Ether Ketone) by Photoinduced and Self-initiated Graft Polymerization in an Aqueous Solution in the Presence of Inorganic Salt Additives

Acta Biomaterialia. Aug, 2016  |  Pubmed ID: 27154499

A highly efficient methodology for preparing a poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) layer on the surface of poly(ether ether ketone) (PEEK) was examined by photoinduced and self-initiated graft polymerization. To enhance the polymerization rate, we demonstrated the effects of inorganic salt additives in the feed monomer solution on thickness of grafted PMPC layer. Photoinduced polymerization occurred and the PMPC graft layer was successfully formed on the PEEK surface, regardless of inorganic salt additives. Moreover, it was clearly observed that the addition of inorganic salt enhanced the grafting thickness of PMPC layer on the surface even when the photoirradiation time was shortened. The addition of inorganic salt additives in the feed monomer solution enhanced the polymerization rate of MPC and resulted in thicker PMPC layers. In particular, we evaluated the effect of NaCl concentration and how this affected the polymerization rate and layer thickness. We considered that this phenomenon was due to the hydration of ions in the feed monomer solution and subsequent apparent increase in the MPC concentration. A PMPC layer with over 100-nm-thick, which was prepared by 5-min photoirradiation in 2.5mol/L inorganic salt aqueous solution, showed good wettability and protein adsorption resistance compared to that of untreated PEEK. Hence, we concluded that the addition of NaCl into the MPC feed solution would be a convenient and efficient method for preparing a graft layer on PEEK.

Special Issue on Zwitterionic Materials

Acta Biomaterialia. Aug, 2016  |  Pubmed ID: 27450195

Discrimination of Rat Brunner's Gland Carbohydrate Antigens by Site-specific Monoclonal Antibodies

Carbohydrate Research. Sep, 2016  |  Pubmed ID: 27454489

Mucus produced and secreted by gastrointestinal mucosa contains various types of mucins equipped with unique sugar chains considered to play critical roles in protecting mucous membranes; therefore, the identification and verification of mucin sugar chains is important for understanding the underlying mechanisms. In our previous work, we generated three monoclonal antibodies (mAbs), RGM22, RGM26, and RGM42, which recognize sugar chains in rat gastric mucin. Here, we immunohistochemically analyzed the rat gastrointestinal mucosa and found that the antigens recognized by RGM22 and RGM42 were expressed in the rat antrum and Brunner's glands, whereas that recognized by RGM26 was detected in the antrum, but rarely in Brunner's glands. We found that these antibodies reacted with porcine gastric mucin-derived oligosaccharides bearing a common structure: GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-6GalNAc-ol. Moreover, epitope analysis revealed that RGM42 and RGM22 recognized α-linked GalNAc and GalNAcα1-3Gal, respectively, on the GalNAcα1-3(Fucα1-2)Gal structure, whereas RGM26 was specific for GalNAcα1-3(Fucα1-2)Gal. These results indicate that rat Brunner's glands express specific antigens bearing GalNAcα1-3Gal that are recognized by RGM22 and RGM42. Thus, RGM22, RGM26, and RGM42 with their unique antigen specificities could be useful tools for investigation of oligosaccharide diversity among mucins.

Diffusion-Induced Hydrophilic Conversion of Polydimethylsiloxane/Block-Type Phospholipid Polymer Hybrid Substrate for Temporal Cell-Adhesive Surface

ACS Applied Materials & Interfaces. Aug, 2016  |  Pubmed ID: 27488537

In this study, diffusion-induced hydrophobic-hydrophilic conversion of the surface of the cross-linked polydimethylsiloxane (PDMS) substrate was realized by employing a simple swelling-deswelling process of PDMS substrate in a block-type polymer solution with the aim of development of a temporal cell-adhesive substrate. The ABA block-type polymer composed of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) segment and PDMS segment with over 70% of dimethylsiloxane unit composition could be successfully incorporated in the PDMS substrate during the swelling-deswelling process to prepare the PDMS/phospholipid block copolymer hybrid substrates. During the aging process of the PDMS substrate for 4 days in aqueous medium, its surface property changed gradually from hydrophobic to hydrophilic. X-ray photoelectron spectroscopy and atomic force microscopy data provided strong evidence that the time-dependent hydrophilic conversion of the PDMS/block-type phospholipid polymer hybrid substrate was induced by the diffusion of the hydrophilic PMPC segment in the block-type polymer to be tethered on the substrate. During the hydrophilic conversion process, surface-adsorbed fibronectin was gradually desorbed from the substrate surface, and this resulted in successful detachment of two-dimensional connected cell crowds.

Precise Control of Surface Electrostatic Forces on Polymer Brush Layers with Opposite Charges for Resistance to Protein Adsorption

Biomaterials. Oct, 2016  |  Pubmed ID: 27512944

Various molecular interaction forces are generated during protein adsorption process on material surfaces. Thus, it is necessary to control them to suppress protein adsorption and the subsequent cell and tissue responses. A series of binary copolymer brush layers were prepared via surface-initiated atom transfer radical polymerization, by mixing the cationic monomer unit and anionic monomer unit randomly in various ratios. Surface characterization revealed that the constructed copolymer brush layers exhibited an uniform super-hydrophilic nature and different surface potentials. The strength of the electrostatic interaction forces operating on these mixed-charge copolymer brush surfaces was evaluated quantitatively using force-versus-distance (f-d) curve measurements by atomic force microscopy (AFM) and probes modified by negatively charged carboxyl groups or positively charged amino groups. The electrostatic interaction forces were determined based on the charge ratios of the copolymer brush layers. Notably, the surface containing equivalent cationic/anionic monomer units hardly interacted with both the charged groups. Furthermore, the protein adsorption force and the protein adsorption mass on these surfaces were examined by AFM f-d curve measurement and surface plasmon resonance measurement, respectively. To clarify the influence of the electrostatic interaction on the protein adsorption behavior on the surface, three kinds of proteins having negative, positive, and relatively neutral net charges under physiological conditions were used in this study. We quantitatively demonstrated that the amount of adsorbed proteins on the surfaces would have a strong correlation with the strength of surface-protein interaction forces, and that the strength of surface-protein interaction forces would be determined from the combination between the properties of the electrostatic interaction forces on the surfaces and the charge properties of the proteins. Especially, the copolymer brush surface composed of equivalent cationic/anionic monomer units exhibited no significant interaction forces, and dramatically suppressed the adsorption of proteins regardless of their charge properties. We conclude that the established methodology could elucidate relationship between the protein adsorption behavior and molecular interaction, especially the electrostatic interaction forces, and demonstrated that the suppression of the electrostatic interactions with the ionic functional groups would be important for the development of new polymeric biomaterials with a high repellency of protein adsorption.

Photoreactive Initiator for Surface-Initiated ATRP on Versatile Polymeric Substrates

ACS Applied Materials & Interfaces. Sep, 2016  |  Pubmed ID: 27623483

We synthesized 4-azidophenylcarbonyloxyethyl-2-bromoisobutyrate (AzEBI) for construction of a polymer brush layer on a desired area on various polymeric substrates. After 3.0 min of exposure to UV irradiation, the phenylazide groups of AzEBI decomposed and formed covalent bonds with the polymeric substrate surfaces to introduce an initiator of atom transfer radical polymerization (ATRP). The reaction area of AzEBI was regulated using a photomask during photoreaction and surface initiated ATRP of 2-methacryloyloxyethyl phosphorylcholine (MPC) occurred on the desired part of the surface. In the area with poly(MPC), the surface was superhydrophilic and the adhesion of HeLa cell was effectively suppressed. The AzEBI allows the construction of polymer brush layer in anywhere and would expand the potential application of ATRP to prepare polymer brush layer on polymeric substrates.

Movement of a Quantum Dot Covered with Cytocompatible and PH-Responsible Phospholipid Polymer Chains Under a Cellular Environment

Biomacromolecules. Dec, 2016  |  Pubmed ID: 27791358

Quantum dots (QDs) were functionalized with well-defined polymer chains having both cytocompatibility and pH-responsiveness to monitor the movement of nanoparticles in a cellular environment with changing local pH. We used a triblock-type water-soluble polymer composed of three segments: (1) a pH-responsive poly[2-(N,N-diethylamino)ethyl methacrylate; DEAEMA] segment, (2) a poly[ω-(p-nitrophenyloxycarbonyl oligo(ethylene glycol)) methacrylate; MEONP] segment bearing an active ester group to react with an amino compound, and (3) a cytocompatible poly[(2-methacryloyloxyethyl phosphorylcholine; MPC) segment. Moreover, hydrophobic and carboxyl groups were attached as terminals of the polymer chain. The triblock-type polymer was attached to the QD surface through a hydrophobic layer, which was covered with the QD by hydrophobic interaction. This produced hybrid QD particles (QD/MPC polymer nanoparticles). The QD/MPC polymer nanoparticles had good water-dispersion ability after the modification. A fluorescence resonance energy transfer (FRET) phenomenon between QD and fluorescence dye (Alexa) was clearly observed at pH 7.4 and 9.0 when a fluorescence dye was reacted with the poly(MEONP) segment of the polymer. However, the efficiency decreased at pH 5.0. This was due to a change in the distance between the QD and the fluorescence dye in response to the protonation degree of the poly(DEAEMA) segment. The permeability of QD/MPC polymer nanoparticles through the cell membrane was enhanced by reacting the cell-penetrating peptide, octaarginine (R8), to the carboxyl group at the end of the polymer. The R8-QD/MPC polymer/Alexa nanoparticles attached onto the HeLa cell membrane surface within 15 min after they were added to the cell culture. This attachment initiated nanoparticle penetration of the cell membrane by endocytosis. The nanoparticles could be followed continuously as they moved in the cell culture. The change in the FRET index was determined during this process. Use of the R8-QD/MPC polymer/Alexa nanoparticle enabled us to determine nanoparticle location, based on the surrounding local pH. We concluded that QDs, modified with a cytocompatible and pH-responsible MPC polymer, provide a new imaging and transport tool in cell-based science and engineering.

Reduced Blood Cell Adhesion on Polypropylene Substrates by a Simple Surface Zwitterionization

Langmuir : the ACS Journal of Surfaces and Colloids. Nov, 2016  |  Pubmed ID: 27802598

To conquer thrombogenic reactions of hydrocarbon-based biomaterials in clinical blood treatment, we introduce a model study of a surface zwitterionization of polypropylene (PP) substrate using a set of well-defined copolymers for controlling the adhesion of blood cells in vitro. Random and block copolymers containing zwitterionic units of 2-methacryloyloxyethyl phosphorylcholine (MPC), [3-(methacryloylamino)propyl] dimethyl(3-sulfopropyl)ammonium hydroxide inner salt (SBAA), or non-ionic units of 2-hydroxyethyl methacrylate (HEMA) with a controlled hydrophobic segment of 70% n-butyl methacrylate (BMA) units in these polymers were synthesized by reversible addition-fragmentation chain transfer polymerization. A systematic study of how zwitterionic and non-ionic copolymer architectures associated with chain orientation control via hydration processes affect blood compatibility is reported. The surface wettability of PP substrates coated with the block copolymer with poly(MPC) (PMPC) segments was higher than that of the random copolymer, poly(MPC-random-BMA). However, the copolymers with SBAA units showed the opposite tendency. The PP substrate coated with non-ionic copolymers containing HEMA units showed relative lower hydration capability associated with higher protein adsorption, platelet adhesion, and leukocyte attachment than those with zwitterionic copolymers. The random copolymer of poly(SBAA-random-BMA) coated on PP substrates exhibited resistance to cell adhesion in human whole blood at a level comparable to that of the MPC copolymers. An ideal zwitterionic PP substrate could be obtained by coating it with block copolymer composed of PMPC and poly(BMA) (PBMA) segments, PMPC-block-PBMA. The water contact angle decreased dramatically from ca. 100° on the original PP to 11° within 30 s. The number of blood cells attached on the PMPC-block-PBMA decreased significantly to less than 2.5% that on original PP. These results supported that the rational design of zwitterionic polymers incorporated with a hydrophobic anchoring portion provides a promising approach to reduce blood cell adhesion and protein adsorption of hydrocarbon-based biomaterials applied in directly contacting with human whole blood.

Clinical Safety and Wear Resistance of the Phospholipid Polymer-grafted Highly Cross-linked Polyethylene Liner

Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. Nov, 2016  |  Pubmed ID: 27813260

To reduce the production of wear particles and subsequent aseptic loosening, we created a human articular cartilage-mimicked surface for a highly cross-linked polyethylene liner, whose surface grafted layer consisted of a biocompatible phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine). Although our previous in vitro findings showed that poly(2-methacryloyloxyethyl phosphorylcholine)-grafted particles were biologically inert and caused no subsequent bone resorptive responses, and poly(2-methacryloyloxyethyl phosphorylcholine) grafting markedly decreased wear in hip joint simulator tests, the clinical safety, and in vivo wear resistance of poly(2-methacryloyloxyethyl phosphorylcholine)-grafted highly cross-linked polyethylene liners remained open to question. Therefore, in the present study, we evaluated clinical and radiographic outcomes of poly(2-methacryloyloxyethyl phosphorylcholine)-grafted highly cross-linked polyethylene liners 5 years subsequent to total hip replacement in 68 consecutive patients. No reoperation was required for any reason, and no adverse events were associated with the implanted liners. The average Harris Hip Score increased from 38.6 preoperatively to 96.5 5 years postoperatively, and health-related quality of life, as indicated by the Short Form 36 Health Survey, improved. Radiographic analyses showed no periprosthetic osteolysis or implant migration. Between 1 and 5 years postoperatively, the mean steady-state wear rate was 0.002 mm/year, which represented a marked reduction relative to other highly cross-linked polyethylene liners, and appeared to be unaffected by patient-related or surgical factors. Although longer follow up is required, poly(2-methacryloyloxyethyl phosphorylcholine)-grafted highly cross-linked polyethylene liners improved mid-term clinical outcomes. The clinical safety and wear-resistance results are encouraging with respect to the improvement of long-term clinical outcomes with poly(2-methacryloyloxyethyl phosphorylcholine)-grafted highly cross-linked polyethylene liners. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Focus on Nanomedicine Molecular Science

Science and Technology of Advanced Materials. 2016  |  Pubmed ID: 27877875

Cytocompatible and Multifunctional Polymeric Nanoparticles for Transportation of Bioactive Molecules into and Within Cells

Science and Technology of Advanced Materials. 2016  |  Pubmed ID: 27877883

Multifunctional polymeric nanoparticles are materials with great potential for a wide range of biomedical applications. For progression in this area of research, unfavorable interactions of these nanoparticles with proteins and cells must be avoided in biological environments, for example, through treatment of the nanoparticle surfaces. Construction of an artificial cell membrane structure based on polymers bearing the zwitterionic phosphorylcholine group can prevent biological reactions at the surface effectively. In addition, certain bioactive molecules can be immobilized on the surface of the polymer to generate enough affinity to capture target biomolecules. Furthermore, entrapment of inorganic nanoparticles inside polymeric matrices enhances the nanoparticle functionality significantly. This review summarizes the preparation and characterization of cytocompatible and multifunctional polymeric nanoparticles; it analyzes the efficiency of their fluorescence function, the nature of the artificial cell membrane structure, and their performance as in-cell devices; and finally, it evaluates both their chemical reactivity and effects in cells.

A Hydrated Phospholipid Polymer-grafted Layer Prevents Lipid-related Oxidative Degradation of Cross-linked Polyethylene

Biomaterials. Jan, 2017  |  Pubmed ID: 27760396

The surface and substrate of a cross-linked polyethylene (CLPE) liner are designed to achieve resistance against oxidative degradation in the construction of hip joint replacements. In this study, we aimed to evaluate the oxidative degradation caused by lipid absorption of a highly hydrophilic nanometer-scaled thickness layer prepared by grafting a poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) layer and a high-dose gamma-ray irradiated CLPE with vitamin E blending (HD-CLPE[VE]). The HD-CLPE(VE) and PMPC-grafted HD-CLPE(VE) exhibited extremely high oxidation resistance regardless of lipid absorption, even though residual-free radical levels were detectable. The water wettability of the PMPC-grafted CLPE and PMPC-grafted HD-CLPE(VE) surfaces was considerably greater than that of untreated surfaces. The hydrated PMPC-grafted layer also exhibited extremely low solubility for squalene. Lipids such as squalene and cholesterol esters diminished the oxidation resistance of CLPE despite the vitamin E improvement. Notably, the PMPC-grafted surface was resistant to lipid absorption and diffusion as well as subsequent lipid-related oxidative degradation, likely because of the presence of the hydrated PMPC-grafted layer. Together, these results provide preliminary evidence that the resistance against lipid absorption and diffusion of a hydrated PMPC-grafted layer might positively affect the extent of resistance to the in vivo oxidation of orthopedic implants.

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