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Poly ( α -hydroxy ethylene oxide-co- β -benzyl l -aspartate), α -hydroxy PEO/PBLA, block copolymers were used for the formulation of new functional polymeric micelles. They have a small diameter (about 30 nm) and a very low critical micellar concentration (cmc) in water, ca. 4 mg l −1 . Doxorubicin (DOX) was physically entrapped into the hydrophobic inner core of these functional micelles. The diameter of DOX-loaded α -hydroxy PEO/PBLA micelles was determined to be approximatively the same as the diameter of the corresponding empty micelles, ca. 25 nm. Moreover, the DOX-loaded functional micelles, as the corresponding empty micelles, were shown to be stable in 0.1 M phosphate buffered solution (PBS) pH 7.4 even in presence of proteins. The cytotoxicity of DOX-loaded functional micelles against P388D1 leukemia cells was studied and compared to the one of DOX-loaded α -methoxy PEO/PBLA micelles and to the cytotoxicity of both α -hydroxy and α -methoxy PEO/PBLA micelles. From this study it was concluded that the DOX-loaded functional micelles seem to have a slightly higher cytotoxicity than the DOX-loaded α -methoxy PEO/PBLA micelles, while both empty micelles were shown to be non-cytotoxic against P388D1 leukemia cells.

Purpose. To design glucose-responsive gels based on the complexation between polymers having phenylboronic acid groups and poly (vinyl alcohol). Specifically, high-glucose sensitivity at physiological pH was achieved through the interaction of phenylborate with amino groups.

The synthesis and characterization of comb-type grafted thermo-sensitive hydrogels is presented. These hydrogels were synthesized by polymerization of N-isopropylacrylamide (IPAAm) with a PIPAAm macromonomer. This process leads to a crosslinked IPAAm backbone polymer, copolymerized with highly mobile comb-type PIPAAm chains. These new thermo-responsive copolymers displayed higher equilibrium swellings at lower temperatures and rapid deswelling kinetics at elevated temperatures. The swelling/deswelling for comb-type gels is dependent on the graft chain lengths, in contrast to normal PIPAAm gel lacking the graft chains. As the temperature is increased above the critical temperature, the dehydrated graft chains aggregated due to hydrophobic attraction. Rapid and reversible kinetics of the graft-type gel were observed in response to stepwise temperature changes within short time cycles: phenomena not observed in normal crosslinked thermo-sensitive gels. The influence of freely mobile graft chains on both the equilibrium and dynamic properties of comb-type PIPAAm gel is demonstrated. Possible application of graft-type gel is discussed for actuator systems.

Cisplatin, an anticancer drug, was bound to aspartic acid residues of poly(ethylene glycol)-poly(aspartic acid) block copolymer (PEG-P(Asp)) by ligand substitution reaction at platinum atoms of cisplatin. At a molar ratio of cisplatin and the aspartic acid residue of 1:1, polymeric micelles were formed with an average diameter of 16 nm. A polymeric micelle fraction was easily purified by ultrafiltration, and a micellar structure of this fraction was stable in distilled water and NaCl solution at 37°C for 24 h. The polymeric micelle showed 1 8 to 1 5 cytotoxicity of intact cisplatin against murine B 16 melanoma cells during 24–72 h incubation. This suggests release of platinum complexes from the micelle.

Glucose-Sensitive Lower Critical Solution Temperature Changes of Copolymers Composed of N -Isopropylacrylamide and Phenylboronic Acid Moieties

We have prepared a copolymer containing both phenylboronic acid and tertiary amine moieties. The copolymer forms a stable complex with poly(vinyl alcohol) (PVA) since boronate moieties interact with PVA hydroxyl groups. The polymer-polymer complex changes its swelling degree with glucose concentration in Dulbecco's phosphate-buffered saline solution (PBS) at pH 7.4, due to the higher complex formation of boronic acid moieties with glucose hydroxyl groups over those in PVA. Glucose-responsive swelling changes of a membrane complex were then utilized to control glucose-responsive current changes with a membrane-coated platinum electrode. Glucose addition to PBS induces swelling of the cast gel membrane, leading to increased diffusion of ion species and thus increased measurable current changes. Since the addition of methyl α-d-glucoside has little influence on the current changes, the current change by the addition of glucose is indicative of the high selectivity of this system for glucose and its cis-hydroxyl groups in glucose. It is observed that current changes are proportional to glucose concentration in the range 0-300 mg/dL. This range corresponds well to physiological blood glucose levels. Current change rates determined from the slope of the time course immediately after glucose addition are also proportional to glucose concentration within this range, yielding even higher sensitivity to the change in glucose concentration. Reproducible signal output is also demonstrated by repetitive, stepwise glucose concentration changes. These results support the applicability of the platinum electrode coated with the gel membrane complex comprising a phenylboronic acid-containing polymer and PVA for a novel glucose-sensing device.

Changes in cytoplasmic free calcium levels and membrane fluidity of platelets in contact with poly(acrylamide-co-methacrylic acid) (PAAmMAc) particles were examined to analyze the mechanistic aspect of regulating platelet function. Our previous studies demonstrated interesting features of PAAmMAc particles during interaction with platelets: (1) PAAmMAc particles induce no calcium increase but enhance membrane fluidity of platelets: (2) thrombin induces no calcium increase in platelets when the platelets were mixed previously with PAAmMAc particles; and (3) PAAmMAc particles induce a calcium increase in platelets when they were treated previously with sodium azide (NaN3). These results suggest the possibility that PAAmMAc surfaces may regulate the calcium level by influencing platelet metabolism. In this study, non-cross-linked PAAmMAc solution with the same chemical composition as the particles showed a suppressive effect on thrombin-induced calcium increase, but, no influence on membrane fluidity. This result indicates that aggregated macromolecular surface assemblies of PAAmMAc may dominate the increase in membrane fluidity of platelets although the calcium change is induced by discrete molecular level interaction between the PAAmMAc and platelet membranes. It was also revealed that the suppression of thrombin-induced calcium increase and the membrane fluidity increase in platelets by PAAmMAc particles were reduced by albumin-treatment of the particles. This result suggests that such phenomena may be due to a decrease in any physicochemical interaction of PAAmMAc surfaces with albumin, rather than platelet metabolic change. PAAmMAc particle surfaces with higher carboxyl groups exhibited a more suppressive effect on thrombin-induced calcium increase, whereas those with lower carboxyl groups derived a higher calcium increase when the platelets were treated previously with NaN3. These results suggest the importance of electrostatic and any other physicochemical interaction of PAAmMAc chains on regulating cytoplasmic calcium levels.

Polymer-enzyme hybrid conjugates modified by a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), have been synthesized. We have investigated the molecular architecture of PIPAAm-enzyme conjugates by preparing two types of PIPAAm-trypsin conjugates, wherein PIPAAm chains are attached by either single-end or multipoint chemistry. A semitelechelic co-oligomer (IDc) was attached to trypsin by single-point conjugation (IDc-trypsin). A copolymer (PIDAAc) consisting of acrylic acid and IPAAm randomly linked in polymer chains was attached to trypsin using multipoint conjugation (PIDAAc-trypsin). Both conjugates exhibited reversible temperature-responsive phase separation. The IDc-trypsin conjugate exhibited phase separation at the same temperature as pure IDc, due to the highly mobile free polymer end group which remains sensitive to small temperature changes. The PIDAAc-trypsin conjugate precipitated at higher temperatures than pure PIDAAc, whose movement was restricted by multiple binding points. Enzyme stability in solution was improved after introduction of PIPAAm chains, which prevented autolysis attributed to conjugate steric hindrance. Stability under repeated temperature cycling was also dependent on the architecture of conjugates; the IDc-trypsin conjugate was more stable than the PIDAAc-trypsin. As a consequence, single-end conjugation of polymer to enzyme provides novel bioconjugate with novel functionality attributed to attached polymer while retaining native biological function with high stability.

An advanced insulin delivery system using a glucose-responsive polymer comprising both phenylboronic acid (PBA) and amino groups was developed and investigated for its efficacy at physiological pH. Stability of complexation of amine-containing PBA gel beads with gluconated insulin (G-Ins) was evaluated using a liquid chromatography system. The amino group presented in the PBA gel beads strongly affected G-Ins release from PBA groups at physiological pH due to an enhanced stability of the boronic acid complex with polyol in the presence of amine. Amine-containing PBA gel showed less rapid decrease of G-Ins in the column under buffer rinse over PBA gel. The released amount of G-Ins from amine-containing PBA gel is much greater than for the PBA gel, responding to the addition of glucose at physiological pH. The results of long-term release studies demonstrate that the amine-containing PBA gel has the capability to respond to glucose challenge over 120 h. Furthermore, a linearity of released G-Ins was observed with glucose concentration. These results demonstrate the feasibility of a glucose-responsive system to achieve insulin release in response to glucose under physiological conditions.

Amino semitelechelic poly(N-isopropylacrylamide)s (PIPAAm) with three different molecular weights were synthesized by telomerization of IPAAm monomer with 2-aminoethanethiol as a chain transfer agent, changing the molar ratio of monomer to chain transfer agent. Macromonomers of thermosensitive PIPAAm were synthesized by condensation reaction of amino semitelechelic PIPAAm with N-acryloxysuccinimide. The molecular weights of macromonomers determined by titration of the terminal amino groups were 2900, 4000, and 9000, respectively. The comb-type grafted PIPAAm hydrogels having different lengths of graft chains were synthesized by radical copolymerization of IPAAm monomer with PIPAAm macromonomer in the presence of N,N'-methylenebisacrylamide as a cross-linker. An important aspect of the graft-type gels is the construction of a molecular architecture different from PIPAAm normal type ofgel even though the composition is same. Higher equilibrium swellings at lower temperatures were observed in graft-type gels in contrast to the normal-type gel, and longer graft chains resulted in higher equilibrium swelling due to the freely mobile grafted chains. Both the normal-type and the graft-type gels exhibited reversible swelling-deswelling changes in aqueous milieu in response to an alteration of temperature. The deswelling kinetics at 40 °C changed from equilibrium swelling states at 10 °C, however, exhibited remarkable differences, and rapid responses were observed for graft-type gels. The rapid dehydration of graft chains during gel shrinking was confirmed by analysis of DSC measurements. These dehydrated graft chains strongly aggregated with hydrophobic intermolecular forces, inducing the rapid deswelling of the gels. The attractive forces operating between dehydrated chains were larger in the gel having longer grafted chains, resulting in faster deswelling. A deswelling mechanism distinct from polymer network collective diffusion was demonstrated with these comb-type grafted hydrogels having freely mobile grafted chains in the network.

This is a review of our recent electrokinetic studies of the surface of poly(N-isopropylacrylamide) hydrogel-coated particles and plates. Electrophoresis measurements were used for particles, while electroosmosis measurements were used for plate samples. It was found that the phase transition of a poly(N-isopropylacrylamide) layer is well reflected in the electrokinetic data. We found excellent correlation between the results obtained via these two different methods. The obtained data were found to be well described by an electrokinetic theory for “soft” surfaces.

The electro-osmotic velocity has been measured on a poly( N -isopropylacrylamide) hydrogel plate at various temperatures. The results are analyzed on the basis of a theory for the electro-osmosis on a "soft" surface (i.e., a polyelectrolyte-coated surface) to determine the charge density and the "softness" of the hydrogel layer. It is found that as the temperature is raised, the charge density increases and the softness of poly( N -isopropylacrylamide) hydrogel decreases. These changes are most remarkable around 33°C. This is probably because poly( N -isopropylacrylamide) hydrogel shrinks above its LCST (33°C) and swells below it. This behavior is in good agreement with our previous results obtained from electrophoretic mobility measurements for poly ( N -isopropylacrylamide)-coated latex particles. The obtained values of the charge density in the hydrogel layer are compared with those that are estimated by potentiometric titration and the swelling ratio measurement of the hydrogel plate. Agreement is excellent, suggesting that the electro-osmosis measurements should be appropriate for analyzing the physicochemical properties of a soft surface.

Our research is directed toward development and fundamental studies of biomedically relevant modulation systems using the temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) functionalized with a carboxyl end group, as the switching element. This material has been attached to both biomolecules and solid surfaces to create new, modified bioconjugates and grafted surfaces, respectively. PIPAAm was used to introduce a reversible switching function correlated to hydration-dehydration changes of polymer chains in response to changes in temperature. The introduction of PIPAAm chains onto polymer surfaces is intended to impart temperature-responsive surface property changes. These surfaces were prepared using either PIPAAm with a terminal carboxyl end group or random copolymers of PIPAAm with acrylic acid. The effects of graft conformation on the dynamics of grafted PIPAAm species were investigated by means of aqueous dynamic contact angle measurement. Each PIPAAm-grafted surface showed completely hydrophilic nature below 20°C, however, these surfaces become hydrophobic above their critical temperature. The extent of hydrophobic property for multipoint-grafted surfaces was small compared to that of the terminally grafted surface. These hydrophilic/hydrophobic surface property changes affect the platelet contact activation. The increase in cytoplasmic [Ca2+]i in platelets was observed only when PIPAAm grafted surface become hydrophobic. The feasibility of cell activation/inactivation control by temperature-modulated surface changes is attractive as modulator for biomedical systems and this strategy may lead to development of new ‘intelligent materials' with specific temperature-modulated surface properties.

Temperature-responsive poly( N -isopropylacrylamide) and its copolymer gels with the hydrophilic comonomer, acrylamide and the hydrophobic comonomer, butyl methacrylate, all exhibit swelling-deswelling changes in response to external temperature changes. These hydrogels show negative swelling thermosensitivities, particularly swelling at lower temperature and complete deswelling over specific phase transition temperatures ( T p ). Shrinking kinetics of these gels from swollen to deswollen states at several different temperature changes have been investigated. When temperature changes are performed entirely below T p , the shrinking process is dominated by polymer network diffusion. On the other hand, shrinking kinetics for temperature changes from below to above T p , are dramatically influenced by gel surface structural changes and formation of a collapsed polymer skin layer. This surface skin formation prompts the accumulation of internal hydrodynamic pressure inside the gels upon shrinking by blocking the outflux of entrapped water. Both rate and magnitude of internal hydrodynamic pressure are modulated by the gel volume, the hydrophobicity of the gel polymer chains and the degree of external temperature changes. This internal pressure eventually causes convective outflow of water from the gel interior. Hydrodynamic internal pressure affects the drug release on-off pattern during gel shrinking.

Electrochemical control of drug release was demonstrated by redox-active micelles of non-ionic surfactants having a ferrocenyl moiety (FPEG). The surfactants formed micelles at the concentrations above the critical micelle concentration (CMC = 0.1 mM) in normal saline solutions when they existed as the reduced form (FPEG), whereas the micelles were disassembled into monomers when they existed as the oxidized form (FPEG + ). This change was reversible and electrochemically controlled. The electroactivity of FPEG determined by cyclic voltammetry was quite sensitive to the aggregation states. Perylene, which was used as a model of hydrophobic drugs, could be solubilized in a 2 mM FPEG + normal saline solution, however, it was hardly solubilized in a 2 mM FPEG + solution that was made by oxidation of the former solution. Quick and discrete response for releasing perylene from the redox active micelles was achieved by using controlled potential bulk electrolysis method.

The entrapment of Adriamycin (ADR) in micelles composed of AB block copolymers (poly(ethylene oxide-co-β-benzyl L-aspartate) (PEO-PBLA)) was investigated. The loading process involved transfer of ADR and PEO-PBLA into an aqueous milieu from dimethyl-formamide (DMF) through a dialysis procedure. Evidence for the physical entrapment of ADR in the polymeric micelles was derived from fluorescence spectroscopy and gel permeation chromatography (GPC). The total fluorescence intensity of ADR was low, suggesting that the drug was self-associated in the micelles. In addition, quenching experiments, using a water-soluble quencher (iodide (I–)), showed that the fluorescence of ADR present in micellar solutions was largely unaffected by I–, whereas the fluorescence of free ADR was readily quenched. From Stern-Volmer plots, quenching constants (KSV) of 2.2 and 17 M−l were determined for ADR in micellar solutions and free ADR, respectively. As a result of the entrapment of ADR in the micelles, ADR binds only slightly serum albumin as evidenced by GPC. In contrast, ADR readily binds serum albumin in aqueous solutions. The findings suggest that ADR is stably entrapped in PEO-PBLA micelles. ADR entrapment in polymeric micelles is expected to affect markedly the pharmacokinetics of ADR.

Temperature-responsive semitelechelic poly(N-isopropylacrylamide) (PIPAAm) bearing a carboxyl end group has been chemically immobilized on aminated polystyrene particle surfaces via condensation reaction. PIPAAm-grafted particles were uniformly suspended in aqueous media at lower temperatures. With increasing temperature, PIPAAm-grafted particles aggregated and precipitated. Such reversible changes in particle colloidal behaviour was correlated to temperature-modulated hydrophilic/hydrophobic changes of particle surfaces modified by PIPAAm hydration/dehydration with temperature changes. Interactions between platelets and PIPAAm-grafted surfaces were studied by monitoring cytoplasmic free Ca2+ concentration ([Ca2+]i) changes in platelets using intracellularly-trapped Ca2+ indicator dye, Fura 2, at various temperatures. Although changes in [Ca2+]i in platelets in contact with PIPAAm-grafted particles were not observed below the critical temperature of PIPAAm, significant changes in [Ca2+]i in platelets were induced by contact with particles above this critical temperature. Furthermore, temperature-modulated cell adsorption/desorption control by PIPAAm-grafted particles was investigated using a particle aggregation assay in the presence of lymphocytes. Below the critical temperature of PIPAAm, mixed suspensions were completely homogeneous due to minimal interaction between lymphocytes and hydrated particles. In contrast, aggregated precipitates were observed by increasing the suspension temperature above the critical temperature of PIPAAm resulting from strong hydrophobic interactions between particles with lymphocytes. These precipitates are reversibly resuspended in cold buffer. The feasibility of cell activation/inactivation or cell attachment/detachment control by temperature-modulated surface changes is attractive for suspension cell culture and drug delivery at targeted sites in vivo.

The influence of adsorbed plasma proteins on preventing contact-induced activation of platelets on poly(propylene oxide) (PPO)-segmented nylon-610 surfaces was investigated by monitoring changes in cytoplasmic free Ca2+ concentrations in platelets and adsorption/desorption of albumin and fibrinogen on these copolymer surfaces. Direct measurement of cytoplasmic free Ca2+ concentration in platelets in contact with copolymer surfaces was achieved by monitoring spectral changes of a fluorescent indicator dye, Fura 2. These copolymers were characterized by a surface microstructure composed of coexisting crystalline and amorphous phases. An increase in cytoplasmic free Ca2+ concentration in platelets interacting with polymer surfaces was observed, and this increase was found to be strongly reduced both by the adsorption of plasma proteins into the polymer surface and by modifying the surface microstructure of the polymer itself. Transient changes in cytoplasmic free Ca2+ concentration were observed in platelets in contact with the surface of copolymer 61P3-25, which exhibited excellent nonthrombogenicity in our previous studies, depending on the residence time of plasma and plasma concentration. Additionally, adsorption/desorption of albumin and fibrinogen on copolymer surfaces was estimated using 125I-labeled proteins. Exchange of the adsorbed albumin with fibrinogen and minimum fibrinogen adsorption were observed particularly on the 61P3-25 surface. Exchange of adsorbed fibrinogen with plasma proteins and/or increased fibrinogen adsorption were also observed on all other polymer surfaces examined. Finally, we conclude that controlled formation of a defined protein adsorption layer on the 61P3-25 surface via the transient exchange of adsorbed albumin with fibrinogen from plasma, can be a dominant factor in preventing platelet adhesion and activation on this surface.

The conjugation reaction of adriamycin (ADR) to poly (ethylene oxide)-poly (aspartic acid) block copolymer (PEO-P(Asp)) was optimized in order to inhibit side reactions, which damage the ADR residues. By diminishing alkaline components and lowering temperature, ADR was successfully conjugated to PEO-P(Asp) without side reactions. The obtained ADR-conjugated poly(ethylene oxide)-poly(aspartic acid) block copolymer [PEO-P(Asp(ADR))] was observed to form a micellar structure with a narrower distribution in size than the PEO-P(Asp(ADR)) synthesized by the previously reported method. Unconjugated ADR remained in the inner core of the PEO-P(Asp(ADR)) micelles was removed by dialysis in organic solvents to obtain the micelle without free ADR. Regulated amount of free ADR was then successfully entrapped into the inner core of these purified micelles. In vitro cytotoxicity against P388D1 cells of PEO-P(Asp(ADR)) micelle without free ADR was assayed to estimate the contribution of physically entrapped ADR on anti-tumor activity of the micelle-forming polymeric drug, PEO-P(Asp(ADR)).

We describe a clinico-pathological variant of a degenerative disorder involving Broca's, Wernicke's, and supplementary motor areas, which presented as primary progressive aphasia, dysarthria, bucco-facial apraxia, and hearing loss as initial symptoms, followed by organic personality changes. Postmortem examination revealed severe focal atrophy of the cerebral convolutions in the frontal operculum, superior frontal gyrus, and superior and transverse temporal gyri in addition to diffuse atrophy of the frontal and temporal lobes in both hemispheres. Microscopical examination revealed argyrophilic neuronal inclusions (ANIs) in the neuronal perikarya and presynaptic terminal throughout the central nervous system, as well as neuronal loss and swollen chromatolytic neurons in the affected cortices. Neocortical ANIs showed a positive immunoreaction with an anti-tau antibody but only a weak reaction with an anti-ubiquitin antibody immunohistochemically. Ultrastructurally, neocortical ANIs consisted of 15-nm thick smooth-surfaced tubules and tubules with constrictions at 120–150-nm intervals; thus they were different from the typical paired helical filaments of the 80-nm interval constrictions observed in the subiculum. ANIs were also found in the basal ganglia, brain stem nuclei, and cervical cord. Accordingly, ANIs appear distinct from neurofibrillary tangles (NFTs) of progressive supranuclear palsy, NFTs of Alzheimer-type dementia, and Pick bodies. The authors consider that this case fits the histopathological criteria of corticonigral degeneration with neuronal achromasia except for the unusual extension to the temporal lobes.

We have investigated temperature modulation of surface properties for hydrophilic/hydrophobic changes using poly(N-isopropylacrylamide) (PIPAAm). Two types of PIPAAm were used as surface modifiers: an end-functionalized PIPAAm with a carboxyl end group and a poly(IPAAm-co-acrylic acid) copolymer. By means of dynamic contact angle measurements in water, the wettability of terminally polymer grafted surfaces using end-functionalized PIPAAm with a carboxyl end group were compared with that of multipoint polymer grafted surfaces using PIPAAm copolymers containing carboxyl groups along the polymer chain. Each PIPAAm grafted surface showed completely hydrophilic properties under 20 o C

When designing a turbo-type blood pump as an artificial heart, the gap between a rotating shaft and a pump housing should be perfectly sealed to prevent any leakage or contamination through a seal. In addition, blood coagulation in a blood chamber must be avoided. To overcome these problems, we proposed five different nonrotating-type turbo pumps: a caudal-fin-type axial-flow pump, a caudal-fin-type centrifugal pump, a nutating-column-type centrifugal pump, a nutating-collapsible-tube-type centrifugal pump, and an oscillating-disk-type centrifugal pump. We selected and developed the oscillating-disk-type centrifugal pump that consists of a disk, a driving rod, a seal, an oscillation mechanism, and a pump housing. The disk is mounted on the end of the rod, which is connected to a high-speed DC motor through an oscillation mechanism. The rod and the disk do not rotate, but they oscillate in the pump housing. This movement of the disk generates forward fluid flow around the axis (i.e., the rotational fluid flow). Centrifugal force due to fluid rotation supports the pressure difference between the outlet and the inlet. The diameter of the disk is 39 mm, the maximum inner diameter of the pump housing is 40 mm, and the volume of the blood chamber for 25 degrees' oscillation is 16.9 ml. The performance of the pump was tested in a mock circulatory system.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical modification of proteins by use of functional polymers is expected to endow them with new properties without destroying their native functions, thus providing useful materials for application in different fields. We have synthesized poly(N-isopropylacrylamide) [poly(IPAAm)] co-oligomer with N,N-dimethylacrylamide (DMAAm) and reactive end groups by telomerization of IPAAm. This co-oligomer exhibits a lower critical solution temperature (LCST) at 37 degrees C. Using this temperature-responsive semitelechelic co-oligomer, we prepared polymer-enzyme conjugates of lipase by covalent coupling via carboxyl end-groups. This bioconjugate exhibits a LCST at 37 degrees C, having rapid, reversible hydration-dehydration changes due to highly mobile free polymer end groups. The conjugate retained its native enzymatic activity below this critical temperature, above which it precipitated and its catalytic function was shut off. This conjugate can be readily separated from reaction mixtures as a precipitate by simple temperature changes after reaction and reused in cycles without denaturation. Such a modulated system is attractive for application as a novel bioreactor system.

Well-defined poly(ethylene oxide)s with a primary amino group at one end and a hydroxyl group at the other terminus were synthesized with new sila-protected amino functionality initiator, potassium N-[2-(2,2,5,5-tetramethyl-1-aza-2,5-disilacyclopentyl)-ethyl]methyl amide [1b]. 1b initiated an anionic polymerization of ethylene oxide (EO) to form a polymer (PEO) without any side reactions such as a cleavage reaction of protective group and a chain transfer reaction. The molecular weights of the PEO determined from GPC and MALDI TOF-MS spectrometry agreed well with those from end group analysis using 1H and 13C NMR and TLC and also with the expected value from EO/initiator ratio. From these results, it was concluded that the polymers thus obtained had a primary amino group at one end and a hydroxy group at the other end and can be regarded as hetrobifunctional PEO.

A novel polymer system sensitive to glucose concentration has been studied as a can didate material for chemically regulated insulin release system. Phenylboronic acid is able to form reversible binding to cis-diol substances. A glucose responsive insulin release system has been studied with utilization of phenylboronic acid polymers for the key material in the exchange reaction between gluconic acid modified insulin (G-Ins) and glucose. The released concentration of G-Ins from the polymer was pulsatile in response to the repeated stepwise concentration changes of glu cose. The linearity between glucose concentration and peak height of released G-Ins and the release response demonstrated no lag time to changes in glucose concentration were other important finites. The phenylboronic acid polymer shows considerable promise for use in a self-regulating insulin delivery system.

Temperature-responsive poly(N-isopropylacrylamide-co-butyl methacrylate) gels exhibit “on—off” regulation of drug release in response to temperature. In a previous study, swelling kinetics of these gels from deswollen to swollen states at several temperature were investigated. It was demonstrated that the swelling behavior of the gel changed at various temperatures, yielding several patterns of drug release profiles. At 20°C, gel swelling increased with time, which was explained using a Case-II transport mechanism. In this mechanism, the glassy polymer matrix core acts to suppress the swelling of the outer region when swelling forces dominate. By reducing the experimental temperature to 10°C and utilizing the greatly enhanced hydration of polymer chains after disappearance of the glassy core, a sigmodal swelling pattern gives rise to novel drug release profiles. In this study, these swelling mechanisms have been verified in detail by theoretical analysis. The existence of a swelling front was confirmed by observation of the colored gel using a dye. When the thickness of gel was changed, the acceleration of swelling was delayed with increasing thickness, and the acceleration times agreed with theoretical values predicted from the model. The observed changes in diameter and thickness of the gel also supported the model. This results demonstrate validity of the model presented with the previous paper.

Complex formation between PDMAAm as a hydrogen-bonding acceptor and PAAc as a hydrogen-bonding donor, the temperature dependence of the equilibrium swelling for IPN hydrogels composed of PDMAAm and PAAc, and changes in ketoprofen release of these IPN hydrogels were investigated. Interpolymer complexes between PDMAAm and PAAc were very stable at 70°C in aqueous solution. Dissociation temperatures of the complex between poly(DMAAm-co-AAm) and PAAc shifted to higher values with increasing DMAAm content. These IPNs composed of poly(DMAAm-co-AAm) and PAAc showed limited swelling ratios between their swelling transition temperatures and lower swelling ratios above these transition temperatures. Transition temperatures shift to higher values with increasing DMAAm content

A new glucose-responsive insulin delivery system composed of phenylboronic acid (PBA) groups was prepared and investigated. Complexation of various diol-containing molecules with PBA gel beads was evaluated using frontal chromatography. The structural features of the diol-containing molecules strongly influenced their binding to PBA gel beads. In particular, open-chain monosaccharides demonstrated higher association constants (ca 9.5 x 10(2) to 5.1 x 10(3) l/mol) than glucose (ca 6.3 x 10(2) l/mol). Furthermore, a model system utilizing a fluorescent derivative of tris(hydroxymethyl)aminomethane was synthesized and bound to PBA gel beads. The molecules were released in a pulsatile manner in response to glucose. In addition, gluconic acids were chemically attached to insulin molecules. The modified insulin, containing two gluconic acid units per insulin molecule, was isolated using ion-exchange chromatography. This gluconic acid-modified insulin (G-Ins) was bound onto a PBA gel column, and the G-Ins release profile in response to varying glucose concentrations was investigated. The results demonstrate that the PBA gel beads release G-Ins in response to glucose concentration. Thus, this new system may be applied for self-regulated insulin delivery.

The ground state of the two-dimensional electron systems in Bernal bilayer and ABC-stacked multilayer graphenes in the presence of a strong magnetic field is investigated with the Hartree-Fock approximation. Phase diagrams of the systems are obtained, focusing on charge density wave states including states with vortices of valley pseudospins (called a Skyrme crystal). The single-electron states in these stacked graphenes are given by two-component wave functions. That of the first excited Landau level has the same component as the lowest Landau level of the ordinary two-dimensional electrons. Because of this localized wave functions, the Skyrme crystal has low energy in this first excited level up to four layers of graphene, when the inter-layer distance is assumed to be infinitesimal. At the same time, bubble crystals are suppressed, so the phase diagram is different from that of a single-layer graphene., Comment: 8 pages, 8 figures

Changes in platelet cytoplasmic free calcium levels were investigated in contact with cast film surfaces of a block copolymer containing 2-hydroxyethyl methacrylate (HEMA) and styrene (St) (0.5 mole fraction HEMA). These copolymer surfaces demonstrate microdomain alternating lamellae structures composed of hydrophilic HEMA domains (5 nm width) and hydrophobic St domains (20 nm width). The results were compared with those obtained from platelets contacting a random copolymer of HEMA-St (0.5 mole fraction HEMA) and from homopolymers of polystyrene (PSt) and poly(2-hydroxyethyl methacrylate) (HEMA). Cytoplasmic free calcium levels in platelets contacting the microdomain structured surfaces of the HEMA-St block copolymer remained relatively constant in contrast to the significant increases observed for the radically prepared HEMA-St copolymer, PSt, and PHEMA surfaces. Adhering platelets were stimulated by exogenously introduced thrombin and calcium ionophore A23187 20 min after platelet adherence to the polymer surfaces. Only platelets on the block copolymer surfaces showed active metabolic responses. These results suggest that adhering platelets on the microdomain structured surfaces maintain high sensitivities to external stimulation due to an intrinsic strong inhibition of platelet functional changes induced by surface contact. © 1993 John Wiley & Sons, Inc.

Poly(N-isopropyl acrylamide) (PIPAAm) demonstrated a fully expanded chain conformation below 32 degrees C and a collapsed, compact conformation at high temperatures. This unique temperature responsive polymer was grafted onto surfaces of commercial polystyrene dishes and used as temperature switches for creating hydrophilic surfaces below 32 degrees C and hydrophobic surfaces above 32 degrees C. Cell attachment and the growth of bovine endothelial cells and rat hepatocytes on PIPAAm-grafted surfaces at 37 degrees C demonstrated similar behavior to the commercialized culture dishes. Both cell types were observed to detach from the PIPAAm-grafted surface simply by reducing the temperature below the polymer transition temperature (collapse). Cells recovered by this method maintained substrate adhesivity, growth, and secretion activities nearly identical to those found in primary cultured cells in contrast to the compromised function found in cultured cells damaged by trypsinization. These results provide strong evidence that PIPAAm-grafted surfaces, as thermal switches are very effective for reversing cell attachment and detachment without cell damage. Properties of cell culture surfaces can be readily transformed by this technique reversibly into hydrophilic and hydrophobic coatings of PIPAAm-grafted polymers.

3-Acetamido-6-heptafluoropropylphenylboronic acid was synthesized by the perfluoroalkylation of 3-acetamidophenylboronic acid (pKa = 8.3) with bis(heptafluorobutyryl)peroxide. The perfluoroalkylated phenylboronic acid thus prepared was determined to have a low pKa as 6.6 by potentiometric titration. Lowered pKa suggests that the product has an advantage for binding with polyols under physiological pH, opening a wide application in the field of biological as well as material sciences.

A new concept for inflammation responsive drug delivery systems using biodegradable hydrogels of crosslinked hyaluronic acid (HA) has been proposed in our recent studies [ l-3 1. HA is well known to be degraded specifically b;r the presence of hydroxyl radicals, which are produced by phagocytic cells such as leukocytes and macrophages, locally at inflammatory sites [ 41. Our system was established by constructing a heterogeneous-structured device of drug microreservoirs (lipid microspheres) dispersed into degradable matrices of crosslinked HA gels. The most attractive characteristics of this device are: (1) to release lipid microspheres (LM) in response to surface-controlled degradation of crosslinked HA gels and (2) to be quantitatively inflammation-responsive and degradable. These unique features of the device are favorable to regulate drug delivery in several inflammatoryrelated diseases, where LM are released in response to inflammation-induced degradation. The heterogeneous structured device of our design has much potential in drug delivery for severe inflammation. It is well recognized that dysfunction induced by free radicals may be a

In recent years, temporal control of drug delivery has been of interest to achieve improved drug therapies. Intelligent drug delivery systems (DDS) are one expected result, demonstrating an ability to sense external environmental changes, judge the degree of external signal, and release appropriate amounts of drug. Intelligent DDS may be achieved using stimuli-responsive polymeric hydrogels which alter their structure and physical properties in response to external stimuli. Pulsatile drug release has the advantages of avoiding drug tolerance or matching the body's release of specific peptides or hormones. In this review, recent studies for pulsatile drug delivery in response to stimuli such as chemical agents, pH, electric fields, and temperature are discussed. Achievement of pulsatile drug release from stimuli-responsive polymeric hydrogels as on-off switches and its mechanism are reviewed in terms of control for stimuli-responsive swelling.

Micelle-forming block copolymer–drug conjugates, Adriamycin-conjugated polyethylene glycol–poly(aspartic acid) block copolymers (PEG-P[Asp(ADR)]), were synthesized in eight compositions with varying chain lengths of the segments constituting the block co-polymer. The antitumor activity of this micelle-forming polymeric anticancer drug against murine colon adenocarcinoma 26 (C 26) was evaluated by injection into the tail vein. The in vivo activity of the micelle-forming polymeric anticancer drug was revealed to be strongly dependent on the composition, while the in vitro cytotoxic activity was found to be in the same range regard-less of the composition. One composition of PEG–P[Asp(ADR)] was observed to significantly suppress tumor growth and to prolong survival of the treated mice in a wide dose range. These results indicated that selective delivery of the micelle-forming polymeric anti-cancer drug to the tumor was achieved by the appropriate composition of the block copolymer–drug conjugates.

The micelle-forming behavior of a drug–block copolymer conjugate adriamycm-conjugated poly(ethylene glycol)–poly(aspartic acid) block copolymer; PEG-P[Asp(ADR)] was analyzed by gel permeation chromatography (GPC). Four compositions of the conjugates were observed to form micellar structures in aqueous media, and their micelle-forming behavior was found to be dependent on the composition and media. These micelles did not reach equilibrium within short time periods like low molecular weight surfactants. One composition formed stable micelles in the presence of serum.

Changes in cytoplasmic free calcium levels ([Ca2+]i) in platelets in contact with polystyrene (PSt) and poly(acrylamide-co-methacrylic acid) (PAAmMAc) particles were evaluated and results were compared with those from two representative biological calcium agonists; thrombin and calcium ionophore A23187. PSt particles stimulated a steep increase in cytoplasmic calcium levels in platelets as much as thrombin and A23187. Serratia protease-treated platelets showed a steep increase in [Ca2+]i by PSt particles, suggesting that PSt surfaces can initiate platelet activation independent of a glycoprotein Ib (GPIb)-mediated pathway. By contrast, dibucaine-treated platelets showed little increase in [Ca2+]i by PSt particles, indicating that microfilament assembly, including binding of GPIb with actin binding protein, should be required for platelet activation in contact with PSt surfaces. PAAmMAc particles induced little increase in cytoplasmic calcium levels in platelets. However, PAAmMAc particle-treated platelets demonstrated little response to thrombin in terms of an increase in [Ca2+]i and ATP release, suggesting the possibility that PAAmMAc surfaces may regulate [Ca2+]i by influencing platelet metabolism. Furthermore, sodium azide-treated platelets showed an increase in [Ca2+]i in platelets when contacting PAAmMAc particles, supporting the suggestion that PAAmMAc surfaces could regulate platelet functions. Fluorescence polarization measurements using 1,6-diphenyl-1,3,5-hexatriene-loaded platelets revealed that PAAmMAc particles increased membrane fluidity in platelets, which may be due to physicochemical interaction with PAAmMAc surfaces.

3-Acetamido-6-heptafluoropropylphenylboronic acid was synthesized by the perfluoroalkylation of 3-acetamidophenylboronic acid (pKa = 8.3) with bis(heptafluorobutyryl)peroxide. The perfluoroalkylated phenylboronic acid thus prepared was determined to have a low pKa as 6.6 by potentiometric titration. Lowered pKa suggests that the product has an advantage for binding with polyols under physiological pH, opening a wide application in the field of biological as well as material sciences.

Thermoresponsive poly(N-isopropylacrylamide-co-alkyl methacrylate) gels are capable of on-off regulation of drug release in response to external temperature changes because a gel surface skin formed with increasing temperature stops drug release from the gel interior. In this gel shrinking process, observation of bubble formation on the surface indicates that pressure is induced within the gel. This pressure may result in an outward convection of water

Adriamycin, a hydrophobic anticancer drug, was conjugated with poly(ethylene oxide)-poly(aspartic acid) block copolymers composed of various lengths of each block copolymer segment ranging from 1000 to 12,000 in molecular weight and from 10 to 80 units, respectively. Conjugation was achieved without precipitation by adjusting the ratio of adriamycin to aspartic acid residues of the block copolymer and the quantity of DMF used for the reaction. Thus obtained conjugates showed high water solubility irrespective of a large amount of the conjugated adriamycin. Furthermore, these conjugates were found to form micellar structures with a hydrophobic inner core and a hydrophilic outer shell. This micellar architecture may be utilized for effective drug targeting.

A novel polymer complex system sensitive to glucose was studied as a candidate material for formulating a chemically regulated insulin delivery system. A phenylboronic acid (PBA) moiety was incorporated in poly(N-vinyl-2-pyrrolidone) [poly(NVP-co-PBA) ] as a glucose sensor molecule by the radical copolymerization of N-vinyl-2-pyrrolidone with m-acrylamidophenylboronic acid. Complex formation as well as dissociation was estimated by viscosity changes in the complex solution. A positive viscosity change was observed for the poly (vinyl alcohol) (PVA)/poly(NVP-co-PBA) complex system due to complex formation between PVA diol units and poly(NVP-co-PBA) boronate units; minimal or no change in viscosity was observed for either PVA/boric acid or PVA/poly(NVP). The viscosity of the PVA/poly(NVP-co-PBA) system is able to be controlled through changes in polymer molecular weight and/or polymer concentration. The maximum point in viscosity was observed at a molar ratio of PVA to poly (NVP-co-PBA) of 4:1 in this system. The diol-boronate complex interaction was further investigated by following decreased complex viscosity with the addition of a competitive polyol: glucose, tris(hydroxymethyl)aminomethane (TRIS) or (±)-3-amino-1,2-propanediol (APD). The viscosity decreased steeply when glucose was added while minimal change in viscosity was observed when either TRIS or APD was added. This suggested that the chemical structure of the diol compound had an important role in exchange with the diol-boronate complex. These results point to the promising use of PVA/poly(NVP-co-PBA) complex systems in the development of a novel glucose responsive insulin release system.

A structural change in the stratum corneum of hairless rat skin by subcutaneous enhancers and its enhancing effect on indomethacin permeation through the full-thickness skin was examind. The stratum corneum was characterized in terms of a bound water content and a extraction by an enhancer. Enhanced permeation of indomethacin was observed by particular treatments, which lowered a bound water content and showed some extraction. This result suggests that the exchange of bound water in polar lipids by enhancer molecules showed the solubilization and extraction of some lipids from the stratum corneum to cause a reconstructed structure of lipids with the enhancer molecules for accerelating the indomethacin permeation. On the contrary, some treatments which showed an increase in bound water content in the stratum cornuem exhibited a suppressive effect on the permeation. This result suggests that such natural moisturing factors penetrate the hydrated layers of intracellular lipid layers to increase the barrier property of the stratum cormeum. Thus, it is concluded that such a characterization of the stratum corneum can be a useful method on evaluating structural changes in skin and predicting the enhancing properties of several subcutaneous enhancers on drug permeation.

Thermosensitive co-polymers of isopropyl acrylamide (IPAAm) with butyl methacrylate (BMA) are capable of 'on-off' regulation of drug release in response to external temperature changes due to skin formation with increasing temperature. To clarify the role of the surface-modulated skin and controlled pulsatile drug release patterns, the surface shrinking process was regulated by changing the length of the methacrylate alkyl side-chain. Release of indomethacin in response to stepwise temperature changes between 20 and 30 degrees C from co-polymers of IPAAm with BMA, hexyl methacrylate (HMA), and lauryl methacrylate (LMA) was studied. The drug release rate during the 'on' state (20 degrees C) remained constant before and after the 'off' state (30 degrees C) when the period of the 'off' state was increased. These results suggest that the drug in the polymeric matrices diffused from the inside to the surface during the 'off' state even when no drug release was seen. The length of alkyl side-chain was found to be an important parameter in controlling the thickness and density of the surface skin layer.

New concept of inflammation responsive drug delivery system by biodegradable hydrogels of hyaluronic acid (HA) is reviewed. HA has been well known to be degraded specifically by the presence of hydroxyl radicals, which are produced locally at an inflammatory site. Our system is established by constructing a heterogeneous-structured device of drug microreservoirs (lipid microspheres) dispersed into the degradable matrices of crosslinked HA gel. The most attractive characteristics of this device are : (1) to performe the release of lipid microspherers in proportion to surface-controlled degradation of crosslinked HA gels, and (2) to be quantitatively inflammation-responsive degradable. These unique features of the device are favorable to regulated drug delivery to several inflammatory related diseases, where lipid microspheres can be released in response to inflammation-induced degradation although being stable under normal health conditions.

We report a 51-year-old woman with polymyositis accompanied by a high titer of antiacetylcholine receptor antibody. The patient presented with weakness of grip strength followed by rapidly progressive dyspnea, which required mechanical ventilation. She was treated with a glucocorticoid and came off the respirator one week later. Antiacetylcholine receptor antibody activity was elevated in the acute phase and decreased during recovery, although other signs of myasthenia gravis were negative. This patient suggested that in cases of rapidly progressive bulbar palsy and limb muscle weakness, it is necessary to include polymyositis associated with elevated antiacetylcholine receptor antibody activity in the differential diagnosis.

A 58-year-old man presented with an intramedullary spinal cord abscess (ISCA) manifesting as posterior neck pain, gait disturbance, and urinary retention, and transverse myelopathy 1 week later. Magnetic resonance imaging showed the ISCA at the C7 to T1 levels. He was treated under a diagnosis of cryptogenic ISCA with high-dose ampicillin and third- or fourth-generation cephalosporins, which resulted in complete recovery after 2 months. Review of the literature between January 1998 and August 2007 identified 26 cases of ISCA, including our patient. We also identified two additional nonsurgically treated ISCA patients reported between 1977 and 2007. The most common presentation was motor deficits in all patients, followed by fever, pain, and bladder dysfunction. The mortality rate was 1 of 26 patients, and neurological sequelae were observed in 15 of the 25 surviving patients. There was no significant difference in the frequency of neurological sequelae between surgically and nonsurgically treated patients. Mean length of the abscess in the surgically treated group was significantly larger than that in the medically treated group (5.8 vs. 2.2 vertebral bodies). All three nonsurgically treated patients with neurological sequelae had anaerobic infections and received antibiotic therapy later and for shorter periods than those with complete neurological recovery. Antibiotic treatment is comparable to surgery plus antibiotic treatment. Early broad-spectrum high-dose ampicillin and third-generation cephalosporin, covering Gram-positive, Gram-negative, and anaerobic organisms, should be the first choice of management for patients with ISCA.