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Since autonomic dysfunction is closely associated with autoimmune encephalitis (AE), the objective of this study was to determine the autonomic symptoms and the prevalence of anti-α3 subunit of the ganglionic-type nicotinic acetylcholine receptor (gAChRα3) antibodies in the patients with AE. We reviewed the clinical features of 19 AE patients, and specifically analyzed sera for anti-gAChRα3 antibodies using the luciferase immunoprecipitation system (LIPS) assay. Cardiovascular autonomic symptoms were found to be common in patients with AE, and hypersalivation was seen only in patients with NMDAR encephalitis. LIPS detected anti-gAChRα3 antibodies in the sera from patients with AE (5/29, 26%). This study is the first to demonstrate that clinical characteristics including autonomic symptoms of AE patients with seropositivity for gAChR autoantibodies. It will be important to verify the role of gAChR antibodies in autonomic dysfunction and brain symptoms to clarify the pathogenesis of AE.

Proteinase 3 anti-neutrophil cytoplasmic antibody (PR3-ANCA) is reported to be highly specific to vasculitis compared to myeloperoxidase (MPO)-ANCA. We report a case of a 19-year-old woman with chronic inflammatory demyelinating polyneuropathy (CIDP) with high PR3-ANCA positivity. The patient responded well to intravenous immunoglobulin plus oral steroid, and showed no signs of systemic vasculitis during the subsequent 10 months of follow-up. Our present case suggests that CIDP may accompany high PR3-ANCA levels, which should be differentiated from axonal neuropathy due to vasculitis.

Objective To determine the features of alexia or agraphia with a left angular or supramarginal gyrus lesion. Methods We assessed the reading and writing abilities of three patients using kanji (Japanese morphograms) and kana (Japanese syllabograms). Results Patient 1 showed kana alexia and kanji agraphia following a hemorrhage in the left angular gyrus and the adjacent lateral occipital gyri. Patient 2 presented with minimal pure agraphia for both kanji and kana after an infarction in the left angular gyrus involving part of the supramarginal gyrus. Patient 3 also showed moderate pure agraphia for both kanji and kana after an infarction in the left supramarginal and postcentral gyri. All three patients made transposition errors (changing of sequential order of kana characters) in reading. Patient 1 showed letter-by-letter reading and a word-length effect and made substitution errors (changing hiragana [one form of kana] characters in a word to katakana [another form of kana] characters and vice versa) in writing. Conclusion Alexia occurs as “angular” alexia only when the lesion involves the adjacent lateral occipital gyri. Transposition errors suggest disrupted sequential phonological processing from the angular and lateral occipital gyri to the supramarginal gyrus. Substitution errors suggest impaired allographic conversion between hiragana and katakana attributable to a dysfunction in the angular/lateral occipital gyri.

A patient with progressive anomia and alexia with agraphia for kanji (Japanese morphograms) is described. The patient showed a deficit in single-word comprehension and on-reading (a type of reading that conveys phonetic value) dominance in kanji reading, i.e. on-preceding (pronouncing first with on-reading, irrespective of its preferred reading) and kun-deletion (inability to recall and recognize kun-reading [another type of reading that conveys meaning]) when reading a single-character kanji. These features were due to loss of lexico-semantic information and thus the patient was regarded as having progressive Gogi (word-meaning) aphasia by Imura, a Japanese manifestation of semantic dementia. Macroscopically, neuropathological examination disclosed atrophy of the left frontotemporal lobe with accentuation in the anterior portion of the temporal lobe. Histologically, there was neuronal loss in the cerebral cortex, hippocampus, parahippocampal gyrus, amygdala, caudate nucleus, and putamen. Ubiquitin-immunoreactive neuronal inclusions were present in the hippocampal dentate granular cells. This case demonstrates that progressive Gogi aphasia is semiologically identical to semantic dementia, and our patient clinicopathologically resembled those of Rossor et al. [Rossor, M.N., Revesz, T., Lantos, P.L., Warrington, E.K. Semantic dementia with ubiquitin-positive tau-negative inclusion bodies. Brain 2000; 123: 267-76.] and Hodges et al. [Hodges, J.R., Davies, R.R., Xuereb, J.H., Casey, B., Broe, M., Bak, T.H., et al. Clinicopathological correlates in frontotemporal dementia. Ann Neurol 2004; 56: 399-406.].

Vitamin B12 deficiency mainly affects the dorsal column. In this study, we investigated which part of the sensory pathway is affected in vitamin B12 deficiency using NCS and SEPs. We retrospectively reviewed EMG database of Teikyo University and Mitsui Memorial Hospital from 2008 to 2016, and enrolled patients who presented with sensory symptoms and/or gait disturbance, and whose serum B12 level was below 170 ng/ml. Enrolled were 17 patients. Spinal MRI demonstrated abnormalities in 4/17. NCS was abnormal in 3/15 patients for the sural nerve and in 5/12 patients for the median nerve. When NCS and peripheral segments of the SEPs were combined, conduction delay was detected in 12/15 patients for the lower limb and 8/13 patients for the upper limb. Delay of the central conduction was rarer, 3/15 patients in tibial SEPs. N9o to P13/14o segment in median SEPs was abnormal in 4/8 patients, although this is a mixture of the peripheral and central conductions. Vitamin B12 deficiency patients showed abnormalities of the peripheral nerve conduction more frequently than those of the central conduction. SEPs are useful in localizing the lesion along the sensory pathway, not only for CCT but for peripheral conduction.

We show the result of pre-clinical study of NK911, a polymeric micelle carrier system for doxorubicin (DOX). The NK911 micelle carrier consists of polyethyleneglycol and conjugated doxorubicin-polyaspartic acid. It has high hydrophobic inner core, and therefore can entrap the sufficient amount of DOX. NK911 has a small particle size of about 40 nm in diameter that accumulates in tumor tissue by EPR effect showing much stronger activity than the free DOX. We plan to perform a clinical trial at National Cancer Center Hospital, Japan from 2001.

We evaluated the alexia and agraphia of three patients with different lesions using Japanese kanji (morphograms) and kana (phonograms) and made a lesion-to-symptom analysis. Patient 1 (pure alexia for both kanji and kana and minor agraphia for kanji after a fusiform lesion) made more paragraphic errors for kanji, whereas patient 2 (alexia with agraphia for kanji after a posterior inferior temporal lesion) showed severe reading and writing disturbances and more agraphic errors for kanji. Brodmann Area 37 was affected in both patients, but in patient 2 the lesion was located lateral to that in patient 1. Patient 3 showed agraphia without alexia after restricted lesion to the angular gyrus. We believe that pure alexia (patient 1) results from a disconnection between the medial fusiform gyrus and posterior inferior temporal area (the lateral fusiform and inferior temporal gyri), whereas alexia with agraphia for kanji (patient 2), corresponding to lexical agraphia in Western countries, results from damage to the posterior inferior temporal area, in which whole-word images of words are thought to be stored. Furthermore, restricted lesion in the angular gyrus (patient 3) does not produce alexia; the alexic symptom of “angular” alexia with agraphia may be the result of damage to the adjacent lateral occipital gyri.

Adriamycin (ADR) dimer was prepared and its antitumor activity was evaluated with mouse colon adenocarcinoma 26 (C 26). As compared with original ADR, the dimer did not show significant antitumor activity, either in vitro or in vivo. Furthermore, polymeric micelles containing varied ratios of the dimer to the original ADR were prepared. Polymeric micelles with a higher dimer/ADR ratio (9.7) showed significant antitumor activity, but the effective dose shifted higher. Effective doses were found to largely depend on the concentration of the original ADR, rather than that of the dimer at the tumor sites. Therefore, it was presumed that the original ADR played a major role in antitumor activity, and the dimer played a supplementary role to contribute selective delivery of ADR to the tumor sites.

To fabricate peptide delivery systems using polymeric drug carriers, an oligopeptide model drug, [Arg8]-vasopressin(AVP), was incorporated into nano-associates comprising poly(ethylene glycol)-( l -aspartic acid block copolymer (PEG-P(Asp)). Incorporation of the AVP was accomplished using a dialysis method. Static light scattering measurements revealed that the acid-type and mixture-type PEG-P(Asp)s formed nano-associates independently without AVP, while the salt form PEG-P(Asp) did not. High loading of AVP into acid-type PEG-P(Asp) was observed with loading levels controlled by changing the molar ratio of drug and block copolymer. Mixture-type PEG-P(Asp) did not show high loading and the salt-type PEG-P(Asp) form did not at all. Acid-type P(Asp) homopolymer formed associates including AVP, however, it was insoluble in aqueous medium. Dynamic light scattering measurements showed that the acid-type PEG-P(Asp) associates sizes narrowly clustered around 150 nm. This finding suggests that associates of acid-type PEG-P(Asp) effectively incorporates peptides possibly via a hydrogen bonding interaction between the block copolymer and the peptide.

To achieve a combination of spatial specificity in a passive manner with a stimuli-responsive targeting mechanism, a temperature-responsive polymeric micelle is prepared using block copolymers of (poly(N-isopropylacrylamide-b-butylmethacrylate) (PIPAAm-PBMA)). The micelle inner core formed by self-aggregates of PBMA segments successfully loaded with a drug (adriamycin), and the outer shell of PIPAAm chains played a role of stabilization and initiation of micellar thermo-response. Optimum conditions were investigated for the micelle formation and drug loading into the inner cores in a view of micellar stability and function as drug carriers. Outer shell hydrophilicity that prevents inner core interaction with biocomponents and other micelles can be suddenly switched to hydrophobic at a specific site by local temperature increase beyond the LCST (lower critical solution temperature) (32.5 degrees C). These micelles showed reversible structural changes allowing drug release upon heating/cooling thermal fluctuations through the LCST. Polymeric micelles incorporated with adriamycin showed a dramatic thermo-responsive on/off switching behavior for both drug release and in vitro cytotoxicity according to the temperature responsive structural changes of a micellar shell structure. The reversible and sensitive thermo-response of the micelle opens up opportunities to construct a novel drug delivery system in conjunction with localized hyperthermia.

Adriamycin (ADR)-loaded thermally responsive polymeric micelles composed of poly( N -isopropylacrylamide- co - N , N -dimethylacrylamide)- b -poly( d,l -lactide) were prepared by dialysis from its dimethylacetamide solution against water. Microfiltration was successfully applied to removal of block copolymer associates, which were smaller than micellar structures. By this microfiltration polymeric micelles showing a lower critical solution temperature (LCST) at 40°C in phosphate buffered saline was obtained with a monodispersed size distribution of 69.2 nm in cumulant average diameter. ADR-loaded micelles released more ADR at 42.5°C (above the LCST) than at 37°C (below the LCST). ADR-loaded micelles did not show much cytotoxic activity against bovine aorta endothelial cells at 37°C, in contrast to high cytotoxicity at 42.5°C. On the other hand, free ADR expressed high cytotoxicity at both the incubation temperatures. Thus, thermally responsive polymeric micelles showed distinct control of ADR cytotoxic activity by temperature, while free ADR did not. From these results, an effective target therapy against solid tumors is feasible for these polymeric micelles by a combination of selective delivery to tumor sites based on stable micellar structures at 37°C and enhanced cytotoxic activity of these drug-loaded micelles at 42.5°C by local heating at tumor sites.

Four types of hydrophilic gel microcapsules containing water have been prepared by an interfacial polymerization method. Each type of microcapsules has a membrane of different composition. Using three kinds of monomers, N,N-dimethylacrylamide (DMAAm), 4-(aminomethyl)styrene (AmSt), and N,N-dimethylaminopropylacrylamide (DMAPAA), one type of aqueous copolymer having primary and tertiary amino groups was obtained. By the polymerization of three kinds of monomers, DMAAm, AmSt, and 2-[(methacryloyloxy)ethyl] trimethylammoniumchloride (METAC), another type of aqueous copolymer having primary and quaternary ammonium groups was also obtained. Two more types of copolymers were synthesized by copolymerization of α-acryloxy-ω-methoxy-poly(ethylene glycol) (a-PEG) with the above two kinds of monomer mixture. These copolymers were polymerized with terephthaloyldichloride at the water/oil interface to prepare four types of microcapsules containing water, i.e., poly(DMAAm-co-DMAPAA-co-AmSt-alt-terephthalic acid) microcapsules, poly(DMAAm-co-DMAPAA-co-AmSt-co-PEG-alt-terephthalic acid) microcapsules, poly (DMAAm-co-METAC-co-AmSt-alt-terephthalic acid) microcapsules, and poly (DMAAm-co-METAC-co-AmSt-co-PEG-alt-terephthalic acid) microcapsules, which will be abbreviated to MC 1, MC 2, MC 3, and MC 4, respectively. It has been predicted that the microcapsule membranes are hydrophilic and soft and have two-sublayer structures from electrophoretic mobility measurements and from the analysis of the data with Ohshima’s electrokinetic theory for soft particles. The outer sublayers of MC 1 and MC 2 are negatively charged and those of MC 3 and 4 are slightly positively charged. Also, the surfaces of MC 1 and MC 2 are harder than those of MC 3 and 4. By PEGylation, the surface charge density in the membranes decreases and the surface becomes softer. It has been found that the membrane of red blood cells (RBC) is also soft and is composed of two-sublayers, the outer sublayer of which is negatively charged and the inner one is positively charged. The interaction of four types of microcapsules with RBC has been studied. It was found that microcapsules with soft surfaces (MC 3 and MC 4) do not interact with RBC, even though the microcapsule surfaces are positively charged and the surface of RBC is negatively charged. On the other hand, microcapsules with negatively charged but harder surfaces (MC 1) interact with RBC to introduce hemolysis. The membrane surface of MC 2, which is obtained by PEGylation of MC 1, becomes softer than that of MC 1 so that the interaction with RBC was weakly suppressed. From these, it was concluded that the dominant factor to control the interaction between synthetic polymer surfaces and biological cell surfaces is not the surface charges carried by the polymer surfaces but the softness of the polymer surfaces.

Interaction between protein and cell was discussed using bovine serum albumin (BSA) and yeast protoplast. Glutaraldehyde was used for crosslinking intracellular and intrinsic membrane proteins, which enabled experiments to be carried under various conditions such as pH, salt, and temperature. BSA bound to the surface of yeast protoplast mainly due to the electrostatic interaction, which was determined by investigating the surface potential of the protoplast and the binding isotherms at various pHs. The BSA binding demonstrated the modified Langmuir type and the binding constants and cooperativity parameters were calculated. At the isoelectric point of both BSA and protoplast, the amount of binding obtained the maximum, while at lower and higher pH the binding was significantly decreased. The binding was further discussed in terms of lipid fluidity of the protoplast.

Four types of hydrophilic gel microcapsules containing water have been prepared by an interfacial polymerization method. Each type of microcapsules has membranes of different compositions. By the combination of three kinds of monomers, N , N -dimethylacrylamide (DMAAm), 4-(aminomethyl)styrene (AmSt), and N , N -dimethylamino-propylacrylamide (DMAPAA), two types of water-soluble copolymers having tertiary amino groups were obtained. Two more types of copolymers were synthesized by copolymerization of above three monomers with α -acryloxy- ω -methoxy-poly(ethylene glycol) (a-PEG). These four types of copolymers were polymerized with terephthaloy-dichloride at the water/oil interface to prepare four types of microcapsules containing water. Effects of the stirring rate during the preparation of microcapsules upon the membrane properties were studied. The effects of relative permittivity of dispersing media on microcapsule sizes were studied. It was found that the microcapsule sizes are controlled by the composition of the membranes and by the relative permittivity of the solvents in which microcapsules are redispersed. Depending on the stirring rates during the preparation procedure, the different changes in microcapsule size were observed during the exchanges of dispersing media of various relative permittivities. From these results, it is concluded that different microcapsules in their membrane compositions and in their properties are obtained depending on the stirring rate during the preparation of microcapsules, even if other preparation conditions are kept constant.

Deswelling mechanism for comb-type grafted poly( N -isopropylacrylamide) (PIPAAm) hydrogel was investigated to make clear the effect of gel architecture on the gel deswelling kinetics. Deswelling rate and mechanism were compared with those for conventional crosslinked PIPAAm gel and poly(ethylene oxide) (PEO)-grafted PIPAAm gel in response to temperature increase from below to above the gel-phase transition temperatures. The deswelling rate for the conventional PIPAAm gel are reciprocal to the gel size square, indicative of collective diffusion mediated manner for crosslinked network into water. Although PEO graft chains formed water release channels within the network to produce fast gel deswelling changes, deswelling change of PEO-grafted gel also demonstrated collective diffusion mediated manner of crosslinked backbone chains. On the other hand, one order faster rate of deswelling change of PIPAAm-grafted gel was observed than that of PEO-grafted gel with identical gel dimension. The deswelling change of PIPAAm grafted gel did not obey collective diffusion mediated manner, demonstrating reciprocal to the gel size to 1.58th power. This deswelling mechanism was explained in terms of rapid hydrophobic aggregation of freely mobile PIPAAm graft chains and an operation of intrinsic elastic forces of polymer network. The operation of both elastic force and strong hydrophobic aggregation force between the dehydrated PIPAAm graft chains released water inside the gel, causing more rapid deswelling changes of the PIPAAm-grafted gels.

We describe a patient with progressive aphemia with agrammatism that was later overlaid with buccofacial apraxia and pseudobulbar palsy. Pathological findings were consistent with those of classic Pick's disease with argyrophilic inclusions and neuronal achromasia, except for restricted cortical atrophy in the frontal operculum posterior to the pars opercularis (Brodmann Area 44). In addition, major neuronal loss was confined to the premotor cortex and the anterior half of the precentral gyrus (Area 6), which apparently explained the aphemia. The present case demonstrated that classic Pick's disease can show quite limited cortical atrophy in a patient who clinically presents with progressive aphemia. Also, our patient differed from the progressive non-fluent aphasia patients reported as having Pick's disease, who were all Pick variants, revealing that classic Pick's disease can be included in the spectrum of progressive aphasia syndrome.

Interaction of rat lymphocyte subpopulations (B cells and T cells) with surfaces of poly ( N,N -dimethylacryl-amide- co -3-acrylamidophenylboronic acid) (DB copolymers) with varying composition was evaluated under a controlled shear stress by using a plane-parallel-type channel equipped with a polymer-coated bottom wail [field-flow fractionation/ adhesion chromatography (FFF/AC)]. This method enables to control the hydrodynamic shear stress at the channel wall resulting from the parabolic flow in the channel. The recovery profile of lymphocytes from DB channels was different among each subpopulation, and the selective recovery of T cells from the channel was achieved with most of B cells remaining in the channel. Phenylboronic acid residues in DB copolymers seem to work as binding sites for sugar residues, presumably sialic acid residues, existing on the membrane surface of lymphocytes. Yield and efficiency of the separation are in the practical range for the application in immunological field, allowing this system to be useful as a novel lymphocyte separator.

To evaluate the role of the sub-cortical white matter and cortical areas of the supramarginal gyrus in short-term memory impairment (shortened digit or letter span) and repetition difficulty, four patients with conduction aphasia and impaired short-term memory and two patients with only short-term memory impairment were given digit span, letter span, speech audiometry and dichotic listening tests. The results showed that in most of the patients letter span was inferior to digit span and that bilateral ear suppression in the dichotic listening test was observed in two patients with a lesion in the inferior part of the supramarginal gyrus, suggesting that what was affected was phonological information and that the supramarginal gyrus was the storage site. The overlapped lesion of conduction aphasia patients with short-term memory impairment was the periventricular white matter at the upper to middle part of the trigone, while patients with only short-term memory impairment had a lesion in the inferior supramarginal gyrus in common. Thus, damage to the periventricular white matter at the trigone may yield the phonemic paraphasia characteristic of conduction aphasia, while damage to the inferior part of the supramarginal gyrus may result in the impairment of short-term memory. We believe that as a part of the mechanisms of short-term memory and repetition, phonological information is processed in the primary auditory cortex and goes through the periventricular white matter to the inferior part of the supramarginal gyrus and is temporarily stored there.

An anticancer drug adriamycin (ADR) was incorporated into polymeric micelles forming from poly(ethylene glycol)-poly(aspartic acid) block copolymer by chemical conjugation and physical entrapment. Structural stability of the polymeric micelles was found to be dependent on both the contents of chemically conjugated and physically entrapped ADR. The polymeric micelle with high contents of the chemically conjugated ADR and the physically entrapped ADR expressed very high in vivo antitumor activity against murine C 26 tumor, while the polymeric micelle with only the chemically conjugated ADR showed negligible in vivo activity. This indicates that the physically entrapped ADR played a major role in antitumor activity in vivo. For the polymeric micelle with the high ADR contents, it was found that a dimer of adriamycin molecules formed and that this dimer was physically entrapped in the inner core of the micelle as well as intact ADR.

Micelles of poly(ethylene oxide)-block-poly(β-benzyl- l -aspartate) (PEO-PBLA) were loaded with doxorubicin (DOX) and were characterized in relation to their use as drug vehicles. First, an oil-in-water emulsion method was developed to load DOX in PEO-PBLA micelles. The level of DOX in PEO-PBLA micelles was 5–12% w/w. Whereas the mean diameter of unloaded, PEO-PBLA micelles was ca., 19 nm, the mean diameter of PEO-PBLA micelles loaded with DOX was ≈37 nm. Minimal chemical degradation of DOX occurred as a result of loading in PEO-PBLA micelles. In addition, DOX in PEO-PBLA micelles was less susceptible to chemical degradation than free DOX in aqueous solution. There was evidence for retention of DOX in PEO-PBLA micelles even after freeze-drying and reconstitution in water. Lastly, PEO-PBLA micelles served as drug depots, slowly releasing DOX (days), even in the presence of 10% w/v serum albumin. The results suggest a number of pharmaceutical advantages of PEO-PBLA micelles for the delivery of DOX.

The success of most advanced drug delivery strategies requires development of sophisticated new site-specific carriers. Several new targeting methods use physical and chemical signals such as magnetic fields or changes in pH or temperature as targeting and triggering tools. In addition to site-specificity, the carrier should achieve passive targeting to evade the body's reticulo-endothelial system (RES) and exhibit long blood circulation times in order to efficiently distribute active drug to the site of action (active targeting). To fulfil these requirements, thermo-responsive polymeric micelles have been prepared from amphiphilic block copolymers composed of N -isopropylacrylamide (IPAAm) (a thermo-responsive outer shell) and styrene (St) (hydrophobic inner core). The polymeric micelle which is very stable in aqueous media was formed by the dialyzed method from DMF solution against water. The micelles have a unimodal size distribution (24±4 nm) and CMC was around 10 mg/l (ml→l). These micelles have a small diameter with a low critical micelle concentration, providing a carrier that may have long blood circulation times and a low RES uptake. When the temperature is increased above the transition temperature of the thermo-responsive block chains (32°C), the outer shell chains dehydrate and collapse, allowing aggregation between micelles and favoring binding interactions with cell membrane surfaces. Moreover, these changes are reversible. Hydrophobic molecules are shown to be incorporated into the inner hydrophobic core of the thermo-responsive micelles. Consequently, these micelles are valuable for site-specific delivery of drugs using changes in temperature as a trigger.

Sodium alginate forms a hydrogel upon contact with calcium ions in aqueous solution due to the physical crosslinking (chelation) between the carboxylate anions of guluronate units in alginate and the calcium ions. Alginate disintegration (dissolution) in phosphate buffered saline solution (pH 7.4, Ca 2+ -, Mg 2+ -free) occurs completely in a short time period after a certain lag time. Calcium ion release from the alginate gels is believed to be an influential factor in alginate dissolution. Using this alginate dissolution mechanism, we tried to release macromolecular dextran (nominal molecular weight of 145 000) in a pulsatile manner. As was expected, dextran with molecular weight of 145 000 was released completely in a short time range after a certain lag time. The lag time could be controlled by either alginate molecular weight, alginate concentration in the preparation, or gel beads size. The larger the diameter of the alginate gel beads, the later the observed release time onset. Thereafter dextran was released without changing release rate. Furthermore, we have succeeded to release dextran in a pulsatile fashion using calcium-alginate gel beads by mixing different bead sizes. These results indicate that pulsatile release of macromolecular drugs such as peptides and proteins could be achieved using calcium-alginate gel beads.

Alkyl-terminated PIPAAm was obtained by covalent coupling of alkyl groups of different chain lenghts (C3, C6, C8, C12 and C18) to one end of poly(N-isopropylacrylamide) (PIPAAm). At the molecular level, incorporation of the alkyl chains with PIPAAm chains promoted aggregation at temperatures below the LCST (lower critical solution temperature) of pure PIPAAm, reflecting the hydrophobic contribution of the alkyl chains to the LCST. The LCST changes were greatest for stearoyl (C18)-terminated PIPAAm (PIPAAm-C18H35). Interestingly, this polymer above its critical micelle concentration (cmc, 80 mg/L) in aqueous solution had the same LCST as unmodified PIPAAm, owing to formation of a core-shell micellar structure shielding the hydrophobic inner alkyl core from interactions with water. Micelle formation and the micellar structure of the alkyl-terminated PIPAAm aqueous dispersions were analyzed by fluorescence, surface tension and DLS (dynamic light scattering) measurements. Micelle formation is facilitated by both an increase in hydrophobicity of the incorporated group and suppression of PIPAAm hydrophilicity upon heating, closely relating to intramolecular hydrophilic/hydrophobic balance. PIPAAm-C18H35 micelles exhibit two heterogeneous microdomains; a hydrophobic inner core highly capable of solubilizing hydrophobic pyrene molecules, plus a hydrated outer shell that stabilizes this micellar structure below its LCST and shows reversible thermoresponsive aggregation/dispersion in heating/cooling thermal cycles through the LCST.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

The swelling behaviour of interpenetrating polymer networks (IPN) composed of poly (acrylic acid) (PAAc) and poly (acrylamide(AAm)-co-butyl methacrylate (BMA)) in water was studied and compared to crosslinked poly(AAm-co-BMA) and crosslinked poly(AAm-co-AAc-co-BMA). The IPNs were prepared by a sequential IPN method in which crosslinked PAAc chains were formed inside of three-dimensional networks ofpoly(AAm-co-BMA) as initial gels. Crosslinked poly(AAcco-AAm-co-BMA) gels (random gels) were also synthesized with the same monomer compositions as the IPNs to elucidate the effect of gel first-order structure on swelling behaviour. Positive swelling changes, i.e. lower swelling ratio at lower temperature and higher swelling ratio at higher temperature, were observed with temperature changes for both the IPNs and the random gels, while no change in swelling was observed for crosslinked poly(AAm-co-BMA) without AAc. In particular, IPN swelling showed a transition point at a certain temperature as a function of temperature change, while random gels showed simple and dull swelling changes with temperature change. The transition temperature of the IPNs shifts to higher temperature with increasing BMA content in IPNs. The positive temperature dependence is suggested to be linked to the formation and dissociation of hydrogen-bond complexes between AAc and AAm with temperature changes in IPNs and random gels. The observed transition in swelling changes in the IPN series is hypothesized to result from concurrent dissociation of continuous ladder-like polymer complexes between PAAc and PAAm matrices in the IPN. The shift of transition temperature induced by incorporation of BMA was due to the stabilization of the complex at higher temperature by increasing hydrophobic interactions. Reversible swelling changes, i.e. higher swelling ratio at high temperature (20°C, 30 °C, 40 °C) and lower swelling ratio at 10°C, in response to step-wise temperature changes between low and high temperature, were observed only for BMA-incorporated IPNs, while irreversible swelling was observed for IPNs without BMA. This result suggests that the presence of hydrophobic BMA had an important role in reversible swelling change of IPNs in response to temperature change. To evaluate the feasibility of IPNs for drug delivery systems, the release of ketoprofen from IPNs was performed with fluctuation of temperature between 10°C and 30 °C. The release rate of drug was reduced at 10°C, while it was increased at 30 °C. The difference of release rate between 10°C and 30 °C was considered to be due to different drug diffusivities between the temperatures induced by swelling changes of IPNs.

Frontal chromatography was used as a technique to examine adsorption behavior of serum proteins on polymeric biomaterials. The effect of feeding conditions, such as flow rate and concentration of protein solution loaded into column, on the adsorption mechanism is described in the present paper. The amount of bovine serum albumin and bovine immunoglobulin G (IgG) adsorbed on polystyrene was observed to be less influenced by feeding conditions, suggesting a formation of stable protein layer even in an initial stage of the adsorption due to strong hydrophobic interaction. On the other hand, the amount of adsorbed IgG on poly(2-hydroxyethyl methacrylate) (PHEMA) was increased with increasing flow rate and concentration of IgG solution loaded. This result suggests that the feeding conditions change the adsorption process of IgG, and the increase in the adsorbed amount may be explained by considering a relative difference in the rate of two consecutive processes assumed as follows: (1) adsorption of IgG to PHEMA surface, and (2) subsequent stabilization of adsorbed IgG on the surface.