Raman scattering study of water clusters around polyrotaxane and pseudopolyrotaxane supramolecular assemblies.

We have measured Raman spectra of collective O-H stretching vibration of water clusters in polyrotaxane and pseudopolyrotaxane aqueous solutions and the aqueous solutions of their constituent molecules. The intensities of the collective bands of water clusters in the polyrotaxane and pseudopolyrotaxane solutions were approximately equal to that of their solvents. On the other hand, those in the solutions of linear polymeric chains and cyclic molecules were smaller. These results indicate that the water molecules in the solvents cannot approach to interact with the hydrophobic parts of the constituent molecules sterically when the constituent molecules form the inclusion complexes. Thus, the polyrotaxane and pseudopolyrotaxane molecules are observed as inert in terms of molecular interaction with water, although the constituent molecules have hydrophobic parts in their structure.

Enhanced accessibility of peptide substrate toward membrane-bound metalloexopeptidase by supramolecular structure of polyrotaxane.

A L-phenylalanlylglycylglycine- (H-L-PheGlyGly-) terminated polyrotaxane in which many alpha-cyclodextrins (alpha-CDs) are threaded onto poly(ethylene oxide) (PEO) was synthesized to evaluate the effect of alpha-CD threading on the degradation of the terminal H-L-PheGlyGly by a membrane-bound metalloexopeptidase (aminopeptidase M). The threading of alpha-CDs and introducing H-L-PheGlyGly to the terminals were confirmed by gel permeation chromatography and (1)H NMR spectroscopies. In vitro degradation and kinetic studies revealed that the supramolecular structure of the polyrotaxane enhanced the accessibility toward aminopeptidase M despite the higher molecular weight of the polyrotaxane (M(n): approximately 16,000). This finding provides a new design of biodegradable polymers for biomedical applications with controlled degradation profile.

Controllable erosion time and profile in poly(ethylene glycol) hydrogels by supramolecular structure of hydrolyzable polyrotaxane.

A series of poly(ethylene glycol) (PEG) hydrogels cross-linked by a hydrolyzable polyrotaxane was prepared and the hydrolytic erosion behavior was characterized. The hydrolyzable polyrotaxane consisting of many alpha-cyclodextrins (alpha-CDs) and a PEG chain capped with bulky end groups via ester linkages was used as a cross-linker in the PEG hydrogels, where alpha-CDs in the polyrotaxane were linked with another PEG chains to form hydrophilic PEG networks. From the result of the erosion study, the time to reach complete gel erosion was found to be prolonged by decreasing the polyrotaxane content and increasing the PEG/alpha-CD ratio. The PEG/alpha-CD ratio, indicating the number of PEG chains linked with one alpha-CD molecule, is considered to make the environment of the polyrotaxane more aqueous and lead to the hydrolysis of ester linkages in the polyrotaxane. However, the higher PEG/alpha-CD ratio prolonged the time of the hydrogel erosion. These results indicate the enhanced stability of ester hydrolysis in the hydrogels with highly water swollen state. Such an anomalous phenomenon may be due to the structural characteristic of the polyrotaxane: ester linkages may be included within the cavity of alpha-CDs, resulting in their enhanced stability. The erosion profile of the hydrogels was changeable by the M(n) of PEG-bisamine, independent of the polyrotaxane content. The hydrogels cross-linked by the polyrotaxane can be new candidates as long-term stable but actually hydrolyzable hydrogels for polymeric scaffolding in tissue engineering.

Modulatory factors on temperature-synchronized degradation of dextran grafted with thermoresponsive polymers and their hydrogels.

Several types of dextran grafted with poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide [dextran-g-poly(NIPAAm-co-DMAAm)] with different graft lengths and numbers were synthesized in a preciously controlled fashion, and their enzymatic degradation was examined by viscosity measurement and gel permeation chromatography as a function of temperature. Degradation of dextran-g-poly(NIPAAm-co-DMAAm)s decreased with increasing the graft length below their lower critical solution temperatures (LCSTs). Above the LCST, enzymatic degradation was independent of the graft length. A larger amount of the graft chain with increasing the graft length rather than the graft number was effective to modulate the temperature-synchronized degradation. Hydrogels were prepared by cross-linking the graft copolymers using 1,6-hexamethylenediamine. While all the hydrogels have water content of about 93-96% in a wide range of temperatures, their degradation behaviors show a significant dependence on a temperature change. Such a unique property is closely related to the structure of graft copolymers such as graft lengths. Consequently, introducing thermoresponsive grafts with longer length to dextran and its hydrogels is suggested to be an important factor for modulating enzymatic degradation of dextran in synchronization with temperature.

ATP-Sensitive potassium channels modulate glucose transport in cultured human skeletal muscle cells.

Several lines of evidence suggest that ATP-sensitive potassium (KATP) channels are involved in glucose uptake by insulin target tissues. The aim of the present study was to prove directly the effect of KATP channel activity on glucose transport into cultured human skeletal muscle cells. We used potassium channel openers PCO-400 and nicorandil alone or in combination with channel blockers glibenclamide and gliclazide to examine their effects on insulin- or high glucose concentration-induced glucose uptake using 2-deoxy-D-3H-glucose or 3-O-methyl-D-3H-glucose as tracer, respectively. PCO-400 inhibited the basal (non-stimulated) uptake of 2-DG or 3-OMG at the glucose concentration of 5 mM. PCO-400 and nicorandil dose-dependently inhibited insulin-stimulated glucose uptake, and their inhibitory effects were reversed by glibenclamide or gliclazide. In addition, PCO-400 inhibited high glucose concentration-facilitated glucose transport in the absence of insulin, and this effect was also antagonized by both sulfonylurea drugs. Regarding the mechanism by which KATP channels modulate glucose transport, we focused on protein kinase C (PKC), because PKC has been supposed to participate in both insulin- and high glucose concentration-stimulated glucose transport. PMA (phorbol 12-myristate 13-acetate) dose-dependently reversed the PCO-400-induced suppression of insulin-stimulated glucose uptake. On the other hand, PCO-400 at the concentration that inhibited glucose uptake caused no alteration of membrane-associated PKC activity in the presence of insulin or PMA. From these results we conclude that KATP channels modulate the basal and insulin-or high glucose level-stimulated glucose transport in skeletal muscle through a mechanism independent of PKC.

Transience in polyion complexation between nicotinamide-modified dextran and carboxymethyl dextran during enzymatic degradation of dextran.

A self-regulated degradation system using polyion complexation through oxidation reaction from degradation products was preliminarily studied. 1,4-Dihydronicotinamide-modified dextran (NAH-Dex) with different molecular weights was prepared, and NAH moiety in NAH-Dex was oxidized by H2O2 to the dehydrated form (NA+-Dex). The dependence of stoichiometry, concentration, and molecular weight on polyion complexation with carboxymethyl dextran (CMD) were examined. NA+-Dex with a molecular weight above 40000 formed an insoluble complex with CMD, and the complexation was found to proceed stoichiometrically. The extent of polyion complexation was dependent on the concentration of NA+-Dex and CMD, whereas the time to reach complexation was dependent on H2O2 concentration. When H2O2 and dextranase were added to the solution containing NAH-Dex, CMD, and dextran, transmittance dropped and then increased again. From these results, the addition of dextran into the system of H2O2, NAH-Dex, CMD, and dextranase can regulate formation and dissociation of the polyion complex between NA+-Dex and CMD. The antagonistical inhibition of the degradation of the polyion complex is a key parameter of the self-regulated degradation system.

Self-complex formation of nicotinamide-modified dextran with carboxymethyl dextran using their degradation products.

A pseudo-metabolic cycle as a self-degradation system was designed: enzymatic degradation products from a polysaccharide generate oxidants which introduce a cationic charge into the polysaccharide chains, and can form a polyion complex with an anionic polysaccharide. As a component of such a system, dextran, with various degrees of nicotinamide substitution, was prepared. Its degradation by dextranase, redox reaction via glucose oxidase-catalysis, and polyion complex formation with carboxymetyl dextran (CMD) were examined. Nicotinamide-modified dextran (NA-Dex) with nine nicotinamide moieties per 100 glucose units was soluble in PBS and completely oxidized by > 100 mM H2O2. The oxidized type of NA-Dex was found to form a 1:1 complex with CMD. By the addition of dextranase, isomaltase, and glucose oxidase (GOD) to phosphate buffer solution of the reduced type of NA-Dex and CMD, the transmittance of the solution dropped, suggesting polyion complex formation via the oxidation of 1,4-dihydronicotinamide in NA-Dex by H2O2 generated from GOD-catalytic reaction. These findings are of great importance for designing a self-complex formation system aimed at biodegradable and osillative drug release.

[Influence of the radioactive strontium (89Sr) using for nuclear medical radiation therapy upon radioactive draining-water system].

PURPOSE: Strontium-89 chloride (89Sr) is a new radiopharmaceutical that provides effective pain relief for metastatic bone lesions, and is expected to be available soon in the palliative management for metastatic bone pain in Japan. Because of relatively long physical half life (50.5 days), 89Sr may affect to the radioactive draining-water system by exceeding the limits of activity concentration for radioactive drain. In this article, the influence of 89Sr use on the radioactive drainage system was simulated. METHODS: The standard tank capacity of drainage and draining frequency was determined from the results of questionnaire carried out for the nationwide medical and research institutes where radioisotope treatment are performed. On the assumption that 89Sr of 148 MBq for one therapy was used twice a week and several common radionuclides were used as the same activity as used at Chiba Cancer Center, the influence of 89Sr was estimated. The calculation was performed using the activity contamination ration into the draining-water system of each radionuclide of 0.01, which was legally determined. RESULTS: The simulation revealed that the sum of the contamination ratios of individual radionuclides exceeded a legal value of 1.0 in standard drainage with the capacity of 5 m3 and 10 m3 and draining frequency of 7 times per year. The actual contamination ratios of common radiopharmaceuticals measured at Chiba Cancer Center ranged from 1/100 to 1/1000 of the legal values. CONCLUSION: It is necessary that the legal value of activity contamination ratios into the draining-water system should be reassessed before starting 89Sr therapy.

Preparation and characterization of poly(ethylene glycol) hydrogels cross-linked by hydrolyzable polyrotaxane.

PEG hydrogels cross-linked by a hydrolyzable polyrotaxane were prepared and their hydrolytic erosion characterized in terms of supramolecular dissociation of the polyrotaxane. The hydrolyzable polyrotaxane, in which many alpha-cyclodextrins (alpha-CDs) are threaded onto a poly(ethylene glycol) (PEG) chain capped with L-phenylalanine via ester linkages, was used as a multifunctional cross-linker: the PEG network was covalently bound to hydroxyl groups of alpha-CDs in the polyrotaxane. The contact angle and water content of the hydrogels were varied with the polyrotaxane content in the feed. In vitro hydrolysis study revealed that the time to reach complete gel erosion was shortened by increasing the polyrotaxane content in the feed in relation to the decreased number of chemical cross-links between PEG and alpha-CDs in the polyrotaxane. The hydrogel degradation in a physiological condition was found to be followed by bulk mechanism. These findings suggest that changing the preparative conditions such as polyrotaxane content will make it possible to control programmed gel erosion for tissue engineering.

67Ga planar imaging with a low-energy collimator and scatter correction using the triple energy window method.

To improve the spatial resolution and contrast of 67Ga planar imaging, we used a low-energy collimator and two lower-energy windows with a triple-energy-window (TEW) scatter compensation method. The spatial resolution is better than with a medium-energy collimator, and the TEW method can correct for scattered photons and reduce the background counts. In a phantom study and a clinical study involving 44 patients, the images obtained by the proposed method were compared with the images obtained with a medium-energy collimator and three energy windows without scatter compensation (the conventional method). The spatial resolution and the counts were measured. Two nuclear medicine physicians interpreted the images and clinical usefulness was evaluated. The spatial resolution and contrast were improved by our proposed method. It enabled the detection of lesions in five locations in the clinical study. The counts were reduced but misreadings were not seen. We conclude that our proposed method shows a clinical advantage over the conventional method. It can be used easily and quickly with commercially available equipment and is useful in clinical practice.

[Tumor imaging using 201Tl chloride and 67Ga citrate in nucleomedical surveys].

Among nucleomedical examinations for cancer, tumor imaging using 201Tl chloride and 67Ga citrate can be easily performed at almost any hospital. However, the planar images usually obtained are not sufficient to detect small tumorous lesions. For more accurate detection, tomographic images particularly single photon emission CT (SPECT) is necessary. SPECT is also indispensable in cancer diagnosis. Whether 201Tl SPECT or 67Ga SPECT is chosen depends upon tumor localization, histological type, and the final purpose of the study. 201Tl accumulates in almost all tumors, but it is not suitable for detection of abdominal lesions, because there is much physiological accumulation in the small intestine and kidney. In contrast, 67Ga does not always accumulate in adenocarcinomas. 201Tl SPECT and 67Ga SPECT are more useful in the functional imaging of cancer than is morphological tumor diagnosis. Both methods are useful in monitoring treatment effectiveness, detecting recurrent lesions after surgery and radiotherapy, and predicting the grade of malignancy of the tumor. Tumor SPECT using 201Tl chloride and/or 67Ga citrate provides clinically useful information not obtained by morphological tumor diagnosis only.

Hyaluronic acid grafted with poly(ethylene glycol) as a novel peptide formulation.

Hyaluronic acids (HA) grafted with poly(ethylene glycol) (PEG) (PEG-g-HA) were synthesized. The materials characterization, enzymatic degradability and peptide (insulin) release from solutions of the copolymers were examined. Distribution of bioactive peptides within the polymer chain is well-known for combinations of PEG and polysaccharides as aqueous polymer two-phase systems. Insulin was preferentially partitioned into the PEG phase in a PEG/HA solution system. Enzymatic degradation of the copolymers was strongly dependent on the PEG content. Thermal analysis revealed that PEG-g-HA exhibited a variation in phase-separated structures depending on the PEG content. The solution of PEG-g-HA enabled insulin to remain in the PEG moieties dispersed in the HA matrix. Leakage of insulin from the copolymers was dependent upon the PEG content. Leakage rate of insulin from copolymer containing between 7 and 39% by weight of PEG were similar. A dramatic increase in leakage rate occurred when the PEG content was increased to greater than 39% by weight. It is considered that the loaded insulin was partitioned into the PEG moieties and became entangled with the PEG chains. The conformational change of insulin was effectively prevented in PEG-g-HA solutions, although insulin was denatured in storage of both phosphate buffered solution and HA solution. Such a heterogeneous-structured polymeric solution may be advantageous as an injectable therapeutic formulation for ophthalmic or arthritis treatment.

Synthesis of theophylline-polyrotaxane conjugates and their drug release via supramolecular dissociation.

Theophylline-polyrotaxane conjugates were synthesized by coupling theophylline with alpha-cyclodextrins (alpha-CDs) in the polyrotaxane. The polyrotaxane is a molecular assembly in which many alpha-CDs are threaded onto a poly(ethylene glycol) (PEG) chain capped with L-phenylalanine (L-Phe). Theophylline-7-acetic acid was activated by coupling with 4-nitrophenol, and then ethylenediamine was allowed to react with the active ester in order to obtain N-aminoethyl-theophylline-7-acetoamide. This derivative was coupled with a 4-nitrophenyl chloroformate-activated polyrotaxane to obtain the theophylline-polyrotaxane conjugates. The conjugates formed a specific association under physiological conditions, depending upon interactions between the theophylline molecules and/or the terminal l-Phe moiety in the conjugates. In vitro degradation of the conjugates revealed that theophylline-immobilized alpha-CDs were completely released by hydrolysis of the terminal peptide linkage in the polyrotaxane. This result indicates that the association of the conjugates does not induce the steric hindrance but rather enhances the accessibility of enzymes to the terminal peptide linkages. It is suggested that our designed drug-polyrotaxane conjugates can release the drugs via the dissociation of the supramolecular structure without steric hindrance of enzymatic accessibility to the terminal peptide linkages.

Pulsatile peptide release from multi-layered hydrogel formulations consisting of poly(ethylene glycol)-grafted and ungrafted dextrans.

Multi-layered hydrogel formulations consisting of poly(ethylene glycol)-grafted dextran (PEG-g-Dex) and ungrafted Dex were investigated as a model of pulsatile drug release. In these formulations, it is considered that the grafted PEG domains act as a drug reservoir dispersed in the Dex matrix based on aqueous polymer two-phase systems. The formulations exhibited surface-controlled degradation by dextranase, and insulin release was observed in a pulsatile manner because of the multi-layered structure: PEG-g-Dex hydrogel layers containing insulin and insulin-free Dex hydrogel layers. Thus, it is suggested that the multi-layered hydrogel formulations using PEG-g-Dex and Dex are feasible for chronopharmacological drug delivery systems.

Effect of acetylation of biodegradable polyrotaxanes on its supramolecular dissociation via terminal ester hydrolysis.

Acetylation of biodegradable polyrotaxanes was examined to estimate the effect on its supramolecular dissociation via terminal ester hydrolysis. The biodegradable polyrotaxanes, in which many alpha-cyclodextrins (alpha-CD) are threaded onto a poly(ethylene glycol) chain capped with L-phenylalanine via ester linkages, were acetylated using acetic anhydride; alpha-CD release behavior was then characterized by in vitro hydrolysis. The degree of acetylation was changed by the concentration of acetic anhydride and the reaction time. The results of the in vitro hydrolysis indicate that the critical degree of acetylation to prolong supramolecular dissociation lies at around 30%. The terminal hydrolysis proceeded completely even with 100% of acetylation. These findings suggest that the hydrophobization of alpha-CDs in the polyrotaxane makes it possible to delay the time to complete the supramolecular dissociation. The hydrophobization of the polyrotaxane is of great importance for designing implantable materials that maintain their supramolecular structure until tissue regeneration with complete terminal hydrolysis.

Polyrotaxanes: synthesis, structure, and potential in drug delivery.

This article reviews the potential of polyrotaxanes in drug delivery with the historical background of polyrotaxane syntheses. Pseudopolyrotaxanes and polyrotaxanes, including classifications, synthetic methods, structures and physical properties are discussed in the first section. The second section provides our concept of drug carriers using drug-polyrotaxane conjugates in comparison with conventional drug-polymer conjugates. The third and fourth sections describe the synthetic method for biodegradable polyrotaxanes, the conjugation with drugs, and their association under physiological conditions. The fifth section discusses other possibilities for the polyrotaxanes such as drug penetration enhancers. These studies suggest the potential of polyrotaxanes in pharmaceutical applications.

Dual-stimuli-responsive drug release from interpenetrating polymer network-structured hydrogels of gelatin and dextran.

Interpenetrating polymer network (IPN)-structured hydrogels of gelatin (Gtn) and dextran (Dex) were prepared with lipid microspheres (LMs) as a drug microreservoir, and LM release from these hydrogels was examined in relation to their dual-stimuli-responsive degradation. A phase morphology in the IPN-structured hydrogels was varied with the preparation temperature, i.e. above or below the sol-gel transition temperature (Ttrans) of Gtn. The IPN-structured hydrogel prepared below Ttrans exhibited a specific degradation-controlled LM release behavior: LM release from the hydrogel in the presence of either alpha-chymotrypsin or dextranase alone was completely hindered, whereas LM release was observed in the presence of both enzymes. It is concluded that dual-stimuli-responsive drug release can be achieved by specific degradation of a particular IPN-structured hydrogel.

Venous response after lidocaine administration in arm veins.

We previously reported that cubital venous pressure (Pv) tended to increase initially, but this was followed by a drop in a dose-dependent response after intravenous lidocaine administration in subjects with various diseases. In this study we examined whether Pv responses after small-dose intravenous lidocaine administrations are related to the stimulating effect of lidocaine on vascular smooth muscle (VSM). In 5 subjects free of cardiovascular disease, Pv increased slightly with decreased pulsations after a 10 mg dose (p<0.01) with no change in central venous pressure. In the cinephlebographic test performed on 2 healthy volunteers, Pv increased during recovery from proximal venoconstriction caused by an injection of contrast medium mixed with 10 mg lidocaine. In 9 subjects with cardiovascular disease, deltaPv spread in the same directions (+ or -) after 5 and 10 mg drug administrations. In 6 of those tested with both drug doses, deltaPv had positive means and no significant difference was observed. Thus, Pv responses after small-dose lidocaine administrations are consistent with neither the stimulating effect of lidocaine nor with a dose-dependent response. They could be attributed to the spasmolytic effect of lidocaine on the basal tone of VSM, which could be modulated by disease conditions.

Mechanistic aspects of blood-contacting properties of polypropylene surfaces--from the viewpoint of macromolecular entanglement and hydrophobic interaction via water molecules.

Polypropylene surfaces with a particular crystalline-amorphous microstructure have been demonstrated to reduce protein adsorption and platelet activation. Such blood-contacting properties may be affected by the crystalline-amorphous microstructure of the surfaces, although wettability such as dynamic contact angles and surface free energy components were almost constant, being independent from the variation in the microstructure. In order to clarify the mechanistic aspects on their blood-contacting properties, the physicochemical properties of the surfaces were evaluated for a series of compression-molded polypropylene sheets in terms of the work of adhesion and the structure of sorbed water. The work of adhesion of the compression-molded sheets increased with decreasing surface layer crystallinity, presumably due to macromolecular entanglement with a polymeric glue used. The work of adhesion involving macromolecular entanglement may occur between proteins and the surfaces. Thus, a decrease in the surface layer crystallinity is considered to cause an increase in the protein adsorption. The structure of water sorbed into the sheets changed--it was more gaseous (isolated) at the surfaces with a higher crystallinity. This suggests that the hydrophobic interaction via water molecules increased with surface layer crystallinity, resulting in increasing protein adsorption and denaturation. Thus, it is considered that both macromolecular entanglement and hydrophobic interaction are important on the mechanistic aspects of blood-contacting properties of polypropylene surfaces. In order to confirm this hypothesis, the evaluation of the physicochemical properties and blood-contacting properties was also performed on a series of uniaxially drawn polypropylene films. A decrease in the work of adhesion and the hydrophobic interaction at the surfaces was observed with increasing draw ratio, and the protein adsorption and platelet activation were effectively prevented with increasing draw ratio. This result supports our hypothesis. Therefore, it is concluded that the excellent blood-contacting properties of polypropylene surfaces can be achieved by reducing the macromolecular entanglement and the hydrophobic interaction with proteins.