Makoto Onishi

Blood compatible aspects of poly(2-methoxyethylacrylate) (PMEA)--relationship between protein adsorption and platelet adhesion on PMEA surface.

Platelet adhesion and spreading is suppressed when a poly(2-methoxyethylacrylate) (PMEA) surface is used, compared with other polymer surfaces. To clarify the reason for this suppression, the relationship among the amount of the plasma protein adsorbed onto PMEA, its secondary structure and platelet adhesion was investigated. Poly(2-hydroxyethylmethacrylate) (PHEMA) and polyacrylate analogous were used as references. The amount of protein adsorbed onto PMEA was very low and similar to that absorbed onto PHEMA. Circular dichroism (CD) spectroscopy was applied to examine changes in the secondary structure of the proteins after adsorption onto the polymer surface. The conformation of the proteins adsorbed onto PHEMA changed considerably, but that of proteins adsorbed onto PMEA differed only a little from the native one. These results suggest that low platelet adhesion and spreading are closely related to the low degree of the denaturation of the protein adsorbed onto PMEA. PMEA could be developed as a promising material to produce a useful blood-contacting surface for medical devices.

Cold crystallization of water in hydrated poly(2-methoxyethyl acrylate) (PMEA)

The structure of water associated with poly(2-methoxyethyl acrylate) (PMEA), which is known to exhibit excellent blood compatibility, has been investigated using differential scanning calorimetry (DSC). The total equilibrium water content (EWC) of PMEA was 9.0 wt%. Water in the PMEA could be classified into three types: non-freezing, freezing-bound and free water. Cold crystallization of water was clearly observed at about −42 °C on heating when the water content was more than 3.0 wt%. Cold crystallization is attributed to the phase transition from the amorphous ice to the crystal ice in PMEA. The relative proportions of freezing-bound water at the EWC is 48 % of all the water in hydrated PMEA. © 2000 Society of Chemical Industry

In situ studies on protein adsorption onto a poly(2-methoxyethylacrylate) surface by a quartz crystal microbalance

Abstract We have reported that poly(2-methoxyethylacrylate) (PMEA) showed excellent blood compatibility though it had a simple chemical structure, and have been making efforts to clarify the reason for the blood compatibility. It is well-known that the adsorption behavior of the protein affects the compatibility. Therefore, the adsorption behaviors of bovine serum albumin (BSA) and human fibrinogen onto the surfaces of PMEA, poly(2-hydroxyethyl methacrylate) (PHEMA) and polypropylene (PP) were investigated by using a quartz crystal microbalance (QCM), where PHEMA and PP were selected as the representatives of hydrophilic and hydrophobic polymers, respectively. Both proteins were observed to adsorb onto all the polymer surfaces according to Langmuirs adsorption isotherm. The maximum adsorption amounts and the apparent association constants of the proteins for PMEA obtained from the isotherm were lower than those for PHEMA and PP. These results suggest that the interaction between PMEA and the proteins is weaker than the interaction with PP and PHEMA.

Study on kinetics of early stage protein adsorption on poly(2-methoxyethylacrylate) (PMEA) surface

Abstract We have already reported that poly(2-methoxyethylacrylate) (PMEA) showed excellent blood compatibility featured by the significantly low adsorption of plasma protein and the low platelet adhesion. In this study, we tried to analyze the adsorption behavior of the plasma proteins (albumin and fibrinogen) on PMEA surface in terms of kinetics in the early stage of the adsorption reaction by using dynamic quartz crystal microbalance (QCM) method. In addition, the conformational changes of the proteins on the polymer surfaces were investigated. It was concluded from the results that the QCM method could be applied to the analysis of the kinetics in such a polymer–protein system. The characteristic of PMEA is that its detachment rate constant k−1, was higher than those from poly(2-hydroxyethylmethacrylate) (PHEMA) and polypropylene (PP) which were used as references. The degree of the conformational changes of the proteins decreases in the following order; PP>PHEMA>>PMEA. This was strongly related to the difference of the detachment rate constant k−1.

Design of a new plasma separation membrane by graft copolymerization

Abstract A new type of hydrophilic membrane for blood plasma separation has been successfully developed by vapor-phase glow discharge-initiated graft copolymerization. After exposing microporous polypropylene (PP) membrane to argon glow discharge, it was allowed to react with 2-methoxyethylacrylate (MEA) vapor to produce graft polymers. The polyMEA-grafted PP (PP-g-PMEA) membrane has the novel property of not causing hemolysis when blood first comes into contact with it in a dry state. It is thought that hydrophilic microporous membranes in a dry state cause hemolysis when they are initially exposed to blood, because plasma immediately penetrates into their pores by capillary attraction and erythrocytes are trapped rapidly on the micropores and lysed. Since PP-g-PMEA membranes have a weakly hydrophilic character, plasma penetrates into the micropores only slowly and hemolysis does not occur. Therefore, priming with physiological saline prior to use is not required, and consequently plasma separation procedures are simplified and shortened. A disk-type plasma separator equipped with a PP-g-PMEA membrane had good hemo-compatibility and an excellent separation capacity, enabling high recovery of plasma components. A decreased adsorption of plasma proteins due to the PMEA-grafted layer may be the reason for the performance of the membrane.

Anti-HIV-1 activity of an ionically modified porous polypropylene membrane determined by filtration of a viral suspension

We describe here a unique anti‐HIV‐1 membrane, derived from a chemically modified porous polypropylene (PP) membrane, which lowers viral infectivity upon the filtration of HIV‐1 suspension. A cationic polymer, polyethyleneimine (PEI) was graft‐polymerized onto the PP filter membrane (PP‐PEI), and infectious HIV‐1HTLV‐IIIB derived from MOLT‐4/HIV‐1HTLV‐IIIB cells (HIV‐1HTLV‐IIIB(MOLT‐4)) was applied. When a viral suspension of high titer (103.93 TCID50 ml 1) was filtered, efficient reduction (>99%) of gag p24 antigen levels and infectious titer resulted. In a viral suspension of medium titer (102.37 TCID50 ml 1), a significant decrease in the p24 antigen did not occur, although the titer was markedly reduced (>95%). Electron microscopic observation suggested that PEI induced viral aggregations under high titer conditions, and under medium titer conditions, PEI deprived HIV‐1HTLV‐IIIB of its infectivity alone to avoid virus adsorption. In contrast, HIV‐1 propagated in human peripheral blood mononuclear cells (PBMC) such as HIV‐1HTLV‐IIIB(PBMC) was more efficiently trapped by PP‐PEI at lower titers as compared with HIV‐1HTLV‐IIIB(MOLT‐4) from MOLT‐4/HIV‐1HTLV‐IIIB cells. These data suggest host cell modification in the interactions between PP‐PEI and HIV‐1 strains. Since HIV‐1HTLV‐IIIB(MOLT‐4) and HIV‐1HTLV‐IIIB(PBMC) were almost electrically neutral and negative, respectively, we concluded that the divergent effect of PEI on each HIV‐1HTLV‐IIIB resulted from their different electrical characteristics.

Enhancement of Human Immunodeficiency Virus Type 1 (HIV‐1) Infection via Increased Membrane Fluidity by a Cationic Polymer

Cationic polymers are known to have potent activity against bacteria, but their effects on viral activity have been little studied. We investigated the effect of one such polymer, polyethyleneimine (PEI), on HIV‐1 infection. Although virus‐cell binding was significantly inhibited by PEI, HIV‐1 infection in human T‐cell lines such as MT‐4 and MOLT‐4 was accelerated conversely when the drug treatment was carried out, after the virus had attached to the cells or PEI was simultaneously added to the virus and cell culture system. This paradoxical effect of PEI on HIV‐1 infection was examined using HIV‐1 chronically infected cells (MOLT‐4/HIV‐1). Dissociation of the glycoprotein gp120 (as revealed by exposure of transmembrane protein gp41) from MOLT‐4/HIV‐1 cells and the resultant fusion of these cells was shown to be induced by the addition of PEI. Accordingly, it was suggested that the binding inhibition of HIV‐1 to CD4‐positive cells by PEI was due to the shedding of gp120 from HIV‐1 particles, and this PEI rather promoted membrane fusion between the virus and cells leading to the enhancement of HIV‐1 infection. Similarly, dissociation of gp120 from MOLT‐4/HIV‐1 was also induced by sCD4. The effect of these reagents on changes in membrane fluidity was evaluated by polarization (p) measurements, and it was observed that the acceleration of membrane fluidity occurred only in the PEI system. Therefore, it is likely that PEI accelerates HIV‐1 infection by facilitating virus entry into the host cells through an increase in membrane fluidity.

Anticoagulant and antiprotease activities of a heparinoid sulfated glucoside-bearing polymer

We studied anticoagulant and antiprotease activities of the poly(glucosyloxyethyl methacrylate) sulfate [poly(GEMA)-sulfate] in plasma and purified enzyme systems in order to evaluate the anticoagulant behavior of a heparin-like sulfated glucoside-bearing polymer. As a result, we found that poly(GEMA)-sulfate can inhibit some coagulation proteases, although its antiprotease behavior differed from those of heparin and dextran sulfate. Poly(GEMA)-sulfate could not enhance antithrombin activity; therefore, we did not observe any significant inhibition of Factor Xa via antithrombin. However, we found that poly(GEMA)-sulfate was able to inhibit thrombin through the activation of heparin cofactor II. In addition, poly(GEMA)-sulfate was able to inhibit Tenase. In our previous research. we found that the anticoagulant activity of poly(GEMA)-sulfate is due primarily to the formation of an insoluble complex with fibrinogen. This paper showed that the antiprotease activities of poly(GEMA)-sulfate contribute to some extent to its anticoagulant activity.

Preparation and properties of plasma-initiated graft copolymerized membranes for blood plasma separation

Abstract A hydrophilic composite membrane for blood plasma separation has been prepared by surface graft copolymerization initiated by low-temperature plasma (LTP). After short LTP pre-irradiation onto a microporous polypropylene (PP) membrane, N-N -dimethylacrylamide (DMAA) vapor was introduced for grafting. The PP membrane had a 0.45 μm effective pore size and a 130 μm thickness. The rate of DMAA grafting onto PP was very high, even in vapor-solid phase reaction under reduced pressure; DMAA 1 mm Hg (133Pa). The percentage of grafted poly-DMAA (PDMAA) reached 15% within 5 min post graft polymerization, and the membrane surface, including the interior surface of pores, became completely hydrophilic. There was no apparent change observed in the membrane morphology in the dry state after the PDMAA-grafted layer was formed. However, water flux significantly decreased, probably due to swelling of the PDMAA-grafted layer. With a grafting yield below 17%, the PDMAA-grafted PP (PP-g-PDMAA) membrane showed a good separation capability of plasma from whole blood. The PP-g-PDMAA membrane exhibited low complement activating potential, high sieving coefficient for plasma proteins and high blood compatibility. Decreases in adsorption of blood cells, plasma proteins, and other biomolecules may be the reason for the membrane performance.

—BIOCOMPATIBLE ASPECTS OF POLY(2-METHOXYETHYLACRYLATE) (PMEA)—THE RELATIONSHIP BETWEEN AMOUNT OF ADSORBED PROTEIN, ITS CONFORMATIONAL CHANGE, AND PLATELET ADHESION ON PMEA SURFACE

ABSTRACT Poly(2-methoxyethylacrylate) (PMEA) surface suppresses platelet adhesion and spreading when compared with other polymer surfaces. To clarify the reason, the relationship between the amount of the plasma protein adsorbed on PMEA, its secondary structure and platelet adhesion were investigated. Poly(2-hydroxyethylmethacrylate) (PHEMA) and polyacrylate analogous were used as references. The amount of the protein adsorbed on PMEA was very low and almost equal to that of PHEMA. Circular dichroism (CD) spectroscopy was applied to examine the changes in the secondary structure of the proteins, resulting from adsorption on the polymer surface. The conformation of the proteins adsorbed on PHEMA changed considerably, but that adsorbed on PMEA differed a little from the native one. These results suggest that low platelet adhesion and spreading have a close relation to the low degree of denaturation of the protein adsorbed on PMEA. PMEA is a promising material to produce for blood contacting surfaces for medical devices.