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An insulin delivery system comprising phenylboronic acid (PBA) hydrogel was investigated for efficacy at physiological pH by an addition of amino moiety. Release of gluconated insulin from hydrogel beads having PBA group responded to glucose concentration was strongly affected by the incorporation of amine group in the beads under physiological pH. Released G-Ins indicates 17600 mIU/cm3·day, that value is practical for in vivo application. These results indicate the feasibility of insulin delivery system consisting of PBA and amino groups at physiological conditions.

Micelle forming polymeric drug(MFPD) containing chemically bound and physically incorporated adriamycin(ADR) was designed. Three MFPD samples containing various contents of physically incorporated ADR were prepared and their antitumor activities were evaluated with murine tumor cell C26 in vivo. The activities seemed to depend on a dose of physically incorporated ADR rather than a dose of total ADR. Therefore, it is presumed that the major part of the activity of MFPD depends on physically incorporated ADR. This MFPD with both chemically bound and physically incorporated ADR is promissing for a highly active antitumor agent against solid tumors.

We were already reported that the amphiphilic block copolymer composed from 2-hydroxyethylmathacryrate and styrene(HSB) which formed micro-domain structure show excellent nonthombogenicity. In this study, we applied HSB coated small vascular graft for vain. The grafts were implanted into the rabbits inferior vena cave. After 1 week implantation, the round shape HSB coated graft tube shows 100% patency after 1 week implantation. No thrombus was observed on the anastomotic site and the grafts surfaces. When the edges were rounded, the damage of natural blood tube and turbulent flow were perverted, the HSB coat graft tubes keep patency more than 1 week in low flow rabbit vain. The cross-section of graft was observer with TEM. After staining with OsO4, only monolayer-like protein layer(about 200A) were observed on surface. The monolayre-like absorbed protein layer improved blood compatibility without persudointima formation. Thus the HSB coated graft could be a promising small vessel prosthesis even in vaines.

We investigate the possible applicability of alginate gel beads for controlled release system of water-soluble macromolecular drugs, using Fluorescein Isothiocyanate Dextran (FITC-dex) as the model compound. Lower molecular weight dextran was released via the diffusion through alginate matrix, while alginate erosion strongly affected dex release with increasing molecular weight. For FITC-dex (MW: 145,000), pulsatile release was observed after a lag time of 90 min. The lag time could be controlled not only by molecular weight and concentration of alginate but also by particle size of beads. We have also succeeded to alter the release profile of FITC-dex (MW: 145,000) from pulsatile to sustained release by adding styrene-maleic anhydride copolymer (SMA) to alginate. These results suggest that alginate gel beads are utilized as vehicle for a delivery of bioactive compounds.

We report a patient with primary progressive aphasia who first presented with amnesic aphasia that developed over the course of 3 years into nonfluent aphasia with buccofacial apraxia, followed in the next year by cognitive impairment and parkinsonism. Pathological findings were typical for corticobasal degeneration except for the distribution of cortical atrophy. This case suggests that corticobasal degeneration should be included in the differential diagnosis of primary progressive aphasia, especially in association with parkinsonism.

Heterobifunctional poly(ethylene oxide-co-s-benzyl-L-aspartate) -PEO/PBLA- block copolymers having methoxy groups or hydroxy functions at the free end of the PEO chains were synthesized and characterized. They show a stable left-handed α-helix conformation in chloroform, whereas in dimethylsulfoxide (DMSO) and in dimethylacetamide (DMAc), their conformation is random-coil. a-hydroxy and a-methoxy PEO/PBLA polymeric micelles were obtained by dialysis against water of the corresponding block copolymers solution in DMAc. Both polymeric micelles have a similar diameter (≤ 50nm), a very low critical micellar concentration (cmc ≤ 10 mg/L in water) and a good stability in PBS and in PBS/Serum. About 18% of Adriamycin (ADR) was loaded in the inner-core of both polymeric micelles. The diameter of the ADR-loaded micelles was similar to the diameter of the corresponding empty micelles, and they were found to be stable in PBS and in PBS/Serum. The first results on the cytotoxicity of entraped ADR against P388D1 leukemia cells show quite high cytotoxicity level for both carriers.

We have synthesized poly (m-acrylamidophenylboronic acid-co-N, N-dimethylaminopropyl acrylamide-co-N,N-dimetylacryl amide); DBA and investigated use for insulin delivery. And polymer complex formation between DBA and poly (vinyl alcohol) (PVA) as well as its dissociation responding to glucose concentration were studied. It was found that amino groups of DMAPAA were important to improve the response to glucose at pH 7.4. It is considered that amino groups may coordinate to boronic acid group. This coordination was observed with 11B-NMR.

Poly(N-sopropylacrylamide) (PIPAAm)-grafted surfaces show hydrophilic/hydrophobic surface property alteration upon temperature changes. Utilizing this property changes, we can cultivate cells on its surface at 37°C and detach cultured cells by lowering temperature. Cellular metabolisms are revealed to be involved in cell detachment from PIPAAm-grafted surfaces by temperture changes. We have also succeeded to recover viable confluent culture of hepatocytes as well as endothelial cells from PIPAAm grafted surfaces by temperature treatment.

Electrochemical reaction of FPEG is coupled with the preceding disassembled reaction of micelles in aqueous solution. The change in diffusion species fromthe micelles to the monomers at cmc in the concentration gradient in diffusion layers appeared in case of bulk electrolysis of FPEG. The drug releasing mechanism from the redox active micelles were clarified.

Chemical modification of biomolecules with synthetic and natural polymers has developed new applications of proteins in the fields of medicine, bio-engineering and bioreactor. We have been investigating polymer-protein hybrid modified by a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) and reported the possibility of regulation and recycling system of PIPAAm-enzyme by changing temperature with PIPAAm-lipase. We have also discussed that the molecular architecture of PIPAAm-enzyme conjugate affects their response towards temperature and stability in solution and demonstrated the necessity of molecular designing in protein modification by functional polymers. In this study, we prepared PIPAAm-conjugated BSA with semitelechelic oligomer containing carboxyl group(IDc) and amino group(IDa), in which different number of polymer chains attach to BSA by single-end. Their response towards temperature change was reversible and dependent on concentration and number of polymer chains, which indicated phase separation of conjugates was enhanced by hydrophobic interaction of dehydrated PIPAAm chains.

To estimate a feasibility of novel containers for drugs, poly(ethylene oxide)-poly(s-benzyl L-aspartate) (PEO-PBLA) micelles were prepared by dialysis into water from different solvents (e.g., DMF, acetonitrile, THF, DMSO, DMAc, and ethyl alcohol), determining the critical micelle concentration (cmc) of the prepared micelles in distilled water by a fluorescence probe technique using pyrene. Then, indomethacin (IMC) as a model drug was incorporated into the micelles by dialysis and O/W emulsion method. Characteristics of PEO-PBLA micelle without and with the physically trapped IMC in the inner core of the micelles (IMC/PEO-PBLA) were studied by dynamic light scattering and GPC/HPLC as well as in vitro release test of IMC from the micelle was conducted.

The ternary copolymers composed of m-acrylamidophenylboronic acid, N,N-dimethylaminopropylmethacrylamide and N-isopropylacrylamide were synthesized. Bovine aortic endothelial cells (BAECs) on the copolymer substrata demonstrated adhesion and spontaneously developed into capillary networks. The interactions between phenylboronic acid groups in copolymer and glycoconjugates on endothelial cell plasma membranes are proposed to regulate the induction of tissue formation, since phenylboronic acid groups are known to specifically form reversible complexes with cis-diol compounds such as glucose. The copolymers having phenylboronic acid moieties are novel materials capable of mediating specific signals analogous to extracellular matrix to induce endothelial cells into capillary structures.

A new "intelligent' polymer system was developed utilizing the binding and exchange of phenylboronic acid (PBA) with polyols and/or glucose. In this improved system, an amine component was incorporated into the polymer chain along with PBA, to enhance binding between PBA and glucose under physiological conditions. The PBA-based polymer was formed by free-radical copolymerization of 3-methacrylamidophenylboronic acid (MAPB) with comonomers, N,N-dimethylaminopropylacrylamide (DMAPAA) and acrylamide (AAm) in the presence of N,N'-methylenebis(acrylamide) (Bis-AAm) as a cross-linker. The proportion of the amount of PBA groups complexed with glucose vs total amount of PBA groups was determined by the batch method. Compared to PBA copolymers synthesized without amine component, the proportion increased as a function of the amine content as well as the pH of the buffer. These results confirm that the interaction of neighboring amines (unprotonated) to PBA strengthens the binding with glucose, especially at pH 7.4 and above. This new PBA-amine copolymer is promising as a material useful for polyol separation, protection of polyols, and possibly, as an insulin delivery device.

From the standpoints of long-term stability and simple handling, a glucose sensor composed of totally synthetic sensing moieties should be of great advantage. This study was performed to demonstrate the feasibility of a novel glucose sensing system in an aqueous milieu based on a change in the fluorescence on competitive binding between a fluorescent diol compound and glucose toward a phenylborate compound. 6,7-Dihydroxy-4-methyl-coumarin (DHMC) was selected as a fluorophore, and 3-propionamidophenylboronic acid (PAPBA) as a phenylborate compound. The relationship between the glucose concentration and fluorescence intensity was linear in the range of 0 to 500 mg/dl of glucose. The availability of various derivatives of DHMC and phenylborate compounds allows the incorporation of these functionalities on the tip of an optical fiber to construct an opto-sensing system for glucose.

Poly(N-isopropylacrylamide) (PIPAAm), exhibiting a lower critical solution temperature (LCST) at 25 degrees C in physiological phosphate buffered saline solution (pH 7.4) and at 32 degrees C in pure water, was grafted onto the surfaces of commercial polystyrene cell culture dishes. This PIPAAm-grafted surface exhibited hydrophobic surface properties at temperatures over the LCST and hydrophilic surface properties below the LCST. Endothelial cells and hepatocytes attached and proliferated on PIPAAm-grafted surfaces at 37 degrees C, above the LCST. The cultured cells were readily detached from these surfaces by lowering the incubation temperature without the usual damage associated with trypsinization. In this case, the optimum temperature for cell detachment was 10 degrees C for hepatocytes and 20 degrees C for endothelial cells. Cell detachment was partially inhibited by sodium azide treatment, suggesting that cell metabolism directly affects cell detachment. Morphological changes of the adherent cells during cell detachment experiments indicated further involvement of active cellular metabolic processes. Cells detached from hydrophobic-hydrophilic PIPAAm surfaces not only via reduced cell-surface interactions caused by the spontaneous hydration of grafted PIPAAm chains, but also by active cell morphological changes which were a function of cell metabolism.

A ternary copolymer composed of m-acrylamidophenylboronic acid, N,N-dimethylaminopropylmethacrylamide and N-isopropylacrylamide was synthesized. Long-term culture of bovine aortic endothelial cells on this copolymer substrate demonstrated adhesion and proliferation of the cells. After 26 days in culture, endothelial cells spontaneously developed into capillary networks. The interactions between phenylboronic acid groups in copolymer and glycoconjugates on endothelial cell plasma membranes are proposed to regulate the induction of tissue formation, since phenylboronic acid groups are known to specifically form reversible complexes with cis-diol compounds such as glucose. This copolymer is a novel material capable of mediating specific signals analogous to extracellular matrix to promote proliferation of endothelial cells, inducing capillary structures and prompt angiogenesis.

Immunoglobulin G (IgG) has been modified by poly(N-isopropylacrylamide) (PIPAAm) to create a novel bioconjugate which exhibits reversible phase transition behavior at 32 degrees C in aqueous media. A terminal carboxyl group introduced into PIPAAm molecule by polymerization of IPAAm with 3-mercaptopropionic acid was used for conjugation to IgG via coupling reaction of activated ester with protein amino group. These conjugates exhibited rapid response to changes in solution temperature and significant phase separation above a critical solution temperature corresponding to that for the original PIPAAm. These conjugates bound to antigen quantitatively in aqueous system, and antigen-bound complex also demonstrated phase separation and precipitation above a critical temperature. Precipitate was reversibly redissolved in cold buffer. Though particular conjugate which includes 12 molecules of PIPAAm with 6,100 molecular weight suppressed more than 95% of Fc-dependent binding with protein A, it retained approximately 60% of original specific antigen binding activity. It was manifested that polymer content of conjugate was 20-30 wt% for the case of 6,100 molecular weight of PIPAAm to demonstrate specific antigen binding activity most effectively and to reduce Fc-dependent binding with protein A. IgG-PIPAAm conjugates were soluble in water and formed antigen-bound complex in homogeneous solution system below a critical temperature. These conjugates were separated from solution and other solutes corresponding to PIPAAm nature and scarcely bound to antigen above a critical temperature. It is revealed that temperature-responsive PIPAAm conjugated to biomolecule operated as a switching molecule. These phenomena are attractive for not only reversible bioreactors and protein separations but also carrier substrate to localize biomolecules such as drugs, peptides and hormones in a living body.

The case is described of a patient with alexia and agraphia for kanji, and severe anomia after a subcortical haemorrhage in the left posterior inferior temporal area. Magnetic resonance imaging at four months after onset showed a lesion in the inferior temporal and fusiform gyri, extending from the temporo-occipital junction toward the anterior third of the temporal lobe. Comparison with other reported cases of alexia with agraphia and anomia made it clear that when accompanied by severe anomia, the lesions extended either forward to the anterior part of the middle temporal gyrus or medially to the parahippocampal gyrus. It is suggested that the disconnection of association fibres between the parahippocampal, fusiform, middle, and inferior temporal gyri, especially between the parahippocampal gyrus and the other temporal gyri, or the cortical damage to the posterior part of these gyri is essential for the production of anomia.

Temperature-responsive polymer gels have been developed using N-isopropylacrylamide (IPAAm) and applied to intelligent drug delivery systems. “On-off regulation of drug release in response to temperature changes was achieved by using these gels. Phase transition temperature and thermosensitivity of the gel can be controlled by changing comonomers. From the analysis of dynamic process of gel shrinking, the role of the surface modulated skin layer as an on-off switch and the utility of changing the comonomer was elucidated. Shrinking and release stopping mechanisms for IPAAm copolymer gels were clarified.

Primary culture of the chromaffin cells on polypyrrole-coated indium-tin oxide was performed and the influence of polypyrroles having some kinds of dopants on cell function was investigated. While the cells cultured on indium-tin oxide showed decrease of catecholamine release, those on polypyrrole-coated indium-tin oxide maintained the cell function of release of catecholamines. Furthermore, no influence of various dopants on the secretory function from the cultured cells was observed. To prepare polypyrrole on electrode as a cell-culture substrate was considered to be available for supporting the cell function.

Temperature-responsive copolymer (or ternary copolymer) gels of N-isopropylacrylamide (IPAAm) were synthesized with hydrophobic alkyl methacrylate (RMA), hydrophilic acrylamide (AAm), N,N'-dimethylacrylamide (DMAAm), and N-acryloylpyrrolidine (APy) as comonomers. The effects of these comonomers on the phase transition temperature (LCST) and the thermosensitivity have been discussed. The LCST of poly(IPAAm) gel in phosphate buffered saline (PBS) was lowered by the introduction of hydrophobic RMA, and the change in equilibrium swelling ratio with temperature change became smaller with an increase in RMA content. However, a stable skin layer to achieve complete 'on-off' regulation of drug release was formed at a higher temperature by RMA due to hydrophobic interaction of alkyl chains. The LCST of poly(IPAAm-co-AAm) gel increased with an increase in AAm content. However, the thermosensitivity of the gel became smaller. It was suggested that hydrophilic AAm prevented the formation of a dense skin layer at a higher temperature. It was difficult to obtain a complete 'off' state due to an insufficiently dense skin layer in order to stop the drug release. The LCST was raised and great thermosensitivity was possible by the introduction of DMAAm or APy. Poly(IPAAm-co-DMAAm) enabled 'on-off' drug release in response to smaller temperature changes around the body temperature. The molecular design to control transition temperature and thermosensitivity of gel was established.

We have synthesized carboxyl semitelechelic oligo(N-isopropylacrylamide) (OIPAAm) using radical telomerization with 3-mercaptopropionic acid. This telomerization is also effective for the synthesis of carboxyl semitelechelic co-oligomers of IPAAm with butyl methacrylate (BMA) as hydrophobic or N,N-dimethylacrylamide (DMAAm) as hydrophilic comonomers. All co-oligomers are highly water-soluble at lower temperatures and exhibit phase separation with increasing temperature. Pure OIPAAm exhibits a lower critical solution temperature (LCST) at 32 degrees C, and the LCST for co-oligomers can be controlled to increase over 32 degrees C with increasing DMAAm composition and to decrease below 32 degrees C with increasing BMA composition. OIPAAm was grafted to bovine serum albumin (BSA) and bovine plasma fibrinogen (BPF) by activated ester-amine coupling. These OIPAAm-biomolecule conjugates maintain their temperature responses, are soluble in cold water, and precipitate over a range of temperatures related to oligomer content. Conjugates could be selectively precipitated and independently separated from conjugate solution mixtures with increasing temperature. In this case, the number of OIPAAm molecules attached to a conjugate affects the aggregate sizes of precipitated conjugates in mixtures. Both conjugate mixture ratios and solution concentrations influence the contamination of oligo(IPAAm-co-DMAAm)-BSA conjugates in precipitated oligo(IPAAm-co-BMA)-BPF conjugates. Furthermore, precipitated conjugates separated using centrifugation and filtration redissolve in water and maintain their biofunctionality, indicating the potential of strategy in reversible bioreactors and protein separations.

Thermo-responsive hydrogels of poly(N-isopropylacrylamide-co-butyl methacrylate) (poly-(IPAAm-co-BMA)) are capable of swelling-deswelling changes in response to external temperature. As poly(IPAAm-co-BMA) gels swell larger at a lower temperature, the degree and rate of the swelling could be controlled by temperature without altering the chemical structure. Therefore, drug release profiles were remarkably changed by alternation of temperature. The release profiles of indomethacin from poly(IPAAm-co-BMA) were observed to be zero-order at 20 degrees C. This release profile was explained in terms of a Case-II diffusion mechanism; which indicates relaxation of polymer chains with swelling was rate-determining. In the case of 10 degrees C, release demonstrated a sigmoidal profile. The acceleration of drug release was due to a rapid increase in swelling with disappearance of the glassy core which had constrained swelling. The regulation of the water-uptake process by changing external temperature remarkably affected drug release and resulted in several different release profiles.

Using 3-mercaptopropionic acid (MPA) as a chain-transfer agent, the radical oligomerization of N-isopropylacrylamie (IPAAm) was carried out with varying molar ratios of MPA to IPAAm and DMF. The molecular weight of oligo-IPAAm (OIPAAm) could be controlled by the ratio of MPA to IPAAm. The OIPAAm was confirmed to average one carboxyl end group per chain. All OIPAAms samples were highly water-soluble at lower temperatures and exhibited phase separation near 32 degrees C. The optical transmittance of the OIPAAms aqueous solutions changed drastically at 32 degrees C which was independent of OIPAAms molecular weight. In aqueous solutions of OIPAAm having concentrations higher than 1 wt% and molecular weight of 6100, the oligomers were precipitated and recovered in 85 wt% yield of their original content. Further, OIPAAm was grafted to atelo collagen by activated ester-amine coupling. The OIPAAm-collagen conjugates were able to dissolved in cold water and precipitated at 34 degrees C. Temperature-responsive OIPAAm-collagen conjugates are expected to maintain native collagen functionality in the solution state, react at lower temperatures, and be easily removed from the system with small temperature increases.

The metabolism of platelets in contact with poly(acrylamide-co-methacrylic acid) (PAAmMAc) particles was evaluated in terms of changes in cytoplasmic free calcium levels ([Ca2+]i), aderosine triphosphate (ATP) release, and membrane fluidity. PAAmMAc particles induced little increase in [Ca2+]i in platelets. However, PAAmMAc particle-treated platelets demonstrated a smaller response to thrombin, to calcium ionophore A23187, and to polystyrene (PSt) particles, suggesting that PAAmMAc surfaces may regulate [Ca2+]i and ATP release by altering platelet metabolism. Sodium azide-treated platelets showed an increase in [Ca2+]i when contacting PAAmMAc particles, supporting the idea that PAAmMAc surfaces could regulate platelet function. Fluorescence polarization measurements revealed that PAAmMAc particles increased membrane fluidity in platelets, which effect may be due to physicochemical interaction with PAAmMAc surfaces.

Positron emission tomography, involving the H2(15)O injection technique, was used to study changes in regional cerebral blood flow during Japanese kanji word reading. Three-times intrasubject averaging analysis revealed that the main cortical responses occurred in the bilateral (left-side dominant) posterior inferior temporal area for visual input. We believe the left posterior inferior temporal area processes highly complex morphological features of kanji. For language output, left-side dominant activation of the posterior inferior frontal gyrus and the basal ganglia was found.

Poly(N-isopropyl acrylamide)(IPAAm) has characteristics to exhibit a remarkable swelling change in aqueous system in response to step-wise temperature change. Random copolymers of IPAAm with alkyl methacrylate (RMA) having different length of alkyl side chain were synthesized to achieve complete on-off drug permeation and release in response to step-wise temperature. On-off release and permeation of indomethacin were found to be due to surface shrinking layer with increasing temperature. The formation process of these surface shrinking layers were confirmed to be controlled by alkyl side chain length of RMA. From these results, the effects of chemical structure (length of alkyl side chain) on temperature modulated drug permeation and palsatile drug release pattern have been discussed.

We have already reported that block copolymers composed of hydroxyethylmethacrylate (HEMA) and styrene (St), having hydrophilic/hydrophobic microdomain structures, demonstrated excellent nonthrombogenicity. In this study, we discuss the relationship between surface structure and activation of platelets by using well-defined HEMA-St block copolymers.