Takashi Komai

Preparation of carboxymethyl-gellan

Abstract In order to obtain a water-soluble gellan derivative, carboxymethyl-gellan was prepared. The carboxymethyl side group was introduced by adding the sodium monochloroacetic acid to gellan under alkaline conditions. The degree of substitution was determined by two different methods; potentiometric titration and infrared spectroscopy. A linear relationship was found between results from the two methods. It was found that the degree of substitution can be controlled by changing the reaction conditions. The solubility of gellan in water is found to increase by the carboxymethylation. The carboxymethyl gellan obtained here dissolved in water at concentrations up to 10% (wt/wt) at 25 °C. It is was also found that the solution of the carboxymethyl-gellan does not form a gel when it was cooled to 0 °C.

Preparation of gellan sulfate as an artificial ligand for removal of extra domain A containing fibronectin.

The extra domain A containing fibronectin (EDA(+)FN) concentration in plasma of rheumatoid arthritis (RA) is abnormally higher than the normal level. We synthesized various gellan-sulfate (GS) candidates as artificial ligands for removing EDA(+)FN from plasma. The interaction between these artificial ligands and EDA(+)FN was evaluated using affinity constants (KA), which were determined by surface plasmon resonance measurement. The KA (3.6 x 10(8) per M) of GS-25 [degree of substitution for sulfonation (DS) = 25%] with EDA(+)FN was higher than those of other molecules: GS-16 (DS=16%) at 8.3 x 10(7) per M, and GS-35 (DS = 35%) at 1.7 x 10(8) per M. Furthermore, GSs displayed selectivity of EDA(+)FN for binding with plasma FN (KAEDA(+)FN)/KA(plasma FN)>2). The removal ratio in plasma was measured by using GS-immobilized gel. Removals of 66, 11, 7.7, 6.2, 6.9, and 12% for EDA(+)FN, plasma FN, fibrinogen, albumin, immunoglobulin G (IgG) and antithrombin III from the patient-model plasma were, respectively, achieved with GS-25-immobilized gel. These results suggest that GS may be used as a selective artificial ligand for EDA(+)FN removal from plasma in RA treatment.

Polymer network dynamics in shrinking patterns of gels

The pattern formation process in the shrinking gel is studied using a confocal laser scanning microscope. The time evolution of the spatial distribution of polymer network within the gel, as well as that of the shape of the gel, is clarified. These results indicate that the pattern formation process of shrinking gel consists of four stages. In the early stages, the diffusion of solvent plays essential roles to form the membrane on the surface portion of the gel. The mechanical properties of the membrane thus formed prevent the uniform shrinking of the gel and finally pattern is formed in the gel.

Confocal Laser Scanning Microscope Imaging of a Pattern in Shrinking Gel.

Pattern formation in a shrinking gel is studied using the confocal laser scanning microscope to reveal the time evolution of the spatial distribution of the polymer network in the gel. The polymer network is distributed uniformly in the gel at the early stage of bubble pattern formation. The distribution of the polymer network in the gel, however, rapidly changes with time. Finally, hollow bubbles are built up in the gel. This process occurs much faster than the cooperative diffusion of the gel.

Multiple volume phase transition of nonionic thermosensitive gel

The volume phase transition is studied in the ternary system of the nonionic poly (N‐normalpropylacrylamide) gel, water, and various alcohol. The gel shows a reentrant volume phase transition in all mixed solvent systems studied here. It is found that the phase transition is multiple in the mixed solvent systems of water‐1‐propanol and water‐2‐propanol. These results are discussed by the mean field theory in which the effects of the chain stiffening due to the adsorption of the solvent molecules are taken into account. These results strongly suggest that the adsorption and/or the desorption of the solvent molecules promote the multiple phase transition of the nonionic poly (N‐normalpropylacrylamide) gel.

Novel plasma-separation dilayer gellan–gellan-sulfate adsorber for direct removal of extra domain A containing fibronectin from the blood of rheumatoid arthritis patients

Rheumatoid arthritis (RA) patients, in whom cryogelation occurs in the presence of heparin, exhibit abnormally high concentrations of extra domain A containing fibronectin [EDA(+)FN] in their plasma. The selective removal of EDA(+)FN from patient blood is therefore of potential therapeutic benefit. Gellan-sulfate is a candidate ligand for the removal of EDA(+)FN due to its high affinity for FN. In this study, we prepare a novel adsorber for the direct removal of EDA(+)FN from patient blood. The adsorber has both a plasma separation function and EDA(+)FN trapping zones, and is prepared by cross-linking gellan-sulfate with epichlorohydrine. The ratio of gellan-sulfate to gellan in the adsorber is 48%. The surface and internal structure of gellan beads were observed by a range of microscopic techniques, and the beads were found to have a dilayer structure, consisting of a porous outer layer and an underlying gellan-sulfate phase as the adsorber. The affinity constants of the gellan-sulfate beads for EDA(+)FN were almost the same in blood as in buffer because the porous gellan coating acts to separate plasma from the cellular fraction of the blood. The removal rate of plasma proteins and blood cells from mock RA blood was measured for coated and uncoated gellan-sulfate beads. Removal rates were 30-32% for EDA(+)FN, 6-10% for fibrinogen, 10-14% for antithrombin III, 8% for C3, 4-7% for C4, and 0% for albumin. The removal rates of uncoated beads were 11% for white blood cells, 0% for red blood cells and 33% for platelets, whereas removal rates of 0% for white blood cells, 0% for red blood cells and 20% for platelets were achieved for coated beads. The coating effectively inhibits the adsorption of white blood cells and platelets. Existing problems with direct adsorbers, including selectivity and plasma separation, have been solved by this material.

Development of the selective adsorbent for EDA containing fibronectin using heparin immobilized cellulose.

We have noticed that extra domain A containing fibronectin (EDA(+)FN) is closely related to inflammatory diseases and accordingly investigated a selective adsorbent to remove EDA(+)FN, which may increase the severity of the symptoms, from the plasma of rheumatoid arthritis (RA) patients. Three types of heparin immobilized cellulose (HC) were prepared and their adsorptive properties were evaluated by batchwise adsorption tests. The results showed that EDA(+)FN was adsorbed more efficiently than total FN on HC. In particular HC in which heparin was immobilized to amino residue had an excellent capacity. The adsorption rates of EDA(+)FN, total FN and antithrombin-III were 89, 15 and 17% respectively. This adsorbent is extremely useful in selectively removing EDA(+)FN from plasma.

Cryogelation in vitro

Cryogel is a physical gel formed by the heterophilic aggregation of extra domain A containing fibronectin (EDA(+)FN), plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep). Cryogelation is controlled by the interactions between each aggregate and the amount of aggregates. Therefore, the present study attempted to elucidate these properties by studying turbidity (tau). Although only Fbg formed a self-aggregate under low temperatures, from the temperature dependence of tau, the amount of aggregate in three-element (pFN/Fbg/Hep) solution surpassed that of the EDA(+)FN/Fbg/Hep system. The optimal condition for cryogelation was afforded by a solution with Fbg/EDA(+)FN/pFN/Hep expressed in the molar ratio of 12:0.04:0.79:1. This cryogel structure in solution was probably formed via structural changes induced by pFN in Fbg. The structural change in Fbg was examined by circular dichroism under optimal conditions. This concept was based on observations of the direct transmission scanning electron microscopy of a cryogel. The EDA(+)FN/pFN/Fbg/Hep aggregates displayed a network structure that manifested particulate crosslinkage. Cryogelation, a phenomenon related to induction of rheumatoid arthritis in humans, was facilitated by both the EDA(+)FN-Hep interaction and the structural changes of Fbg induced by pFN.

Adhesion of 3-carboxymethyl-cellulose-6-sulfate to extra domain A-containing fibronectin: development of ligands for cryogel removal

Abstract Rheumatoid arthritis (RA) patients, in whom cryogelation occurs in the presence of heparin (HP), ex-hibit abnormally high concentrations of extra domain A-containing fibronectin [EDA(+)FN] in their plasma. RA-associated cryogel is a complex of EDA(+)FN, plasma FN, fibrinogen, and HP in patient plasma. We believe that removal of EDA(+)FN will result in effective inhibition of cryogelation. Although HP has a high affinity for FN and selectivity for EDA(+)FN over plasma FN, HP also binds antithrombin III (which is a normal plasma protein). In this study, we synthesized the following three artificial ligands, which do not bind antithrombin III, for the purpose of removing EDA(+)FN from plasma: cellulose-6-sulfate (C-6S), 3-carboxymethyl cellulose-6-sulfate (3CM-C-6S), and 2-carboxymethyl cellulose-6-sulfate (2CM-C-6S). We evaluated interaction between these artificial ligands and EDA(+)FN by determining affinity constants (KAs). The KA of 3CM-C-6S for binding to EDA(+)FN (7.86 × 108 M−1) was higher than that of other molecules: C-6S, 3.03 × 108 M−1; 2CM-C-6S, 3.57 × 108 M−1. Furthermore, selectivity of adsorption (S = KAEDA(+)FN/KAEDA(−)FN) of 3CM-C-6S for EDA(+)FN over EDA(−)FN (S = 6.25) was higher than that of HP (S = 2.52). These results suggest 3CM-C-6S is a selective ligand suitable for removal of EDA(+)FN from plasma, as a method of inhibition of cryogelation.

Immobilized gellan sulfate surface for cell adhesion and multiplication: development of cell-hybrid biomaterials using self-produced fibronectin.

A new concept for cell-hybrid biomaterial is proposed in which human unbilical vein endothelial cells (HUVEC) are adhered to an immobilized gellan sulfate (GS) surface. Extra domain A containing fibronectin (EDA(+)FN) released from HUVEC is necessary for cell adhesion and multiplication. The material design in this study is based on these self-released cell adhesion proteins. The interaction between GS and EDA(+)FN was evaluated using the affinity constant (KA); the value obtained was 1.03x10(8) (M(-1)). These results suggest that the adhesion of HUVEC to GS may be supported by the adhesion of EDA(+)FN to GS. We also found that this new material adheres to HUVEC, allowing the reintroduction of EDA(+)FN, which is self-produced by the cell. This material is relatively easy to produce, not requiring the usual coating of adhesion proteins in pretreatment.