Cerium oxide nanoparticles (nanoceria), well known for their pro-and antioxidant features, have been recently proposed for the treatment of several pathologies, including cancer and neurodegenerative diseases. However, interaction between nanoceria and biological molecules such as proteins and lipids, short blood circulation time, and the need of a targeted delivery to desired sites are some aspects that require strong attention for further progresses in the clinical application of these nanoparticles. The aim of this work is the encapsulation of nanoceria into a liposomal formulation in order to improve their therapeutic potentialities. After the preparation through a reverse- phase evaporation method, size, Z-potential, morphology, and loading efficiency of nanoceria-loaded liposomes were investigated. Finally, preliminary in vitro studies were performed to test cell uptake efficiency and preserved antioxidant activity. Nanoceria-loaded liposomes showed a good colloidal stability, an excellent biocompatibility, and strong antioxidant properties due to the unaltered activity of the entrapped nanoceria. With these results, the possibility of exploiting liposomes as carriers for cerium oxide nanoparticles is demonstrated here for the first time, thus opening exciting new opportunities for in vivo applications.

Two lipid-based nanoformulations have been used to date in clinical studies: lipoplexes and lipid nanoparticles (LNPs). In this study, we prepared small interfering RNA (siRNA)-loaded carriers using lipid components of the same composition to form molecular assemblies of differing structures, and evaluated the impact of structure on cellular uptake and immune stimulation. Lipoplexes are electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic siRNA in an aqueous environment, whereas LNPs are nanoparticles embedding siRNA prepared by mixing an alcoholic lipid solution with an aqueous siRNA solution in one step. Although the physicochemical properties of lipoplexes and LNPs were similar except for small increases in apparent size of lipoplexes and zeta potential of LNPs, siRNA uptake efficiency of LNPs was significantly higher than that of lipoplexes. Furthermore, in the case of LNPs, both siRNA and lipid were effectively incorporated into cells in a co-assembled state
however, in the case of lipoplexes, the amount of siRNA internalized into cells was small in comparison with lipid. siRNAs in lipoplexes were thought to be more likely to localize on the particle surface and thereby undergo dissociation into the medium. Inflammatory cytokine responses also appeared to differ between lipoplexes and LNPs. For tumor necrosis factor-a, release was mainly caused by siRNA. On the other hand, the release of interleukin-1β was mainly due to the cationic nature of particles. LNPs released lower amounts of tumor necrosis factor-a and interleukin-1β than lipoplexes and were thus considered to be better tolerated with respect to cytokine release. In conclusion, siRNA-loaded nanoformulations effect their cellular uptake and immune stimulation in a manner that depends on the structure of the molecular assembly
therefore, nanoformulations should be optimized before extending studies into the in vivo environment.

1,5-Dihexadecyl N, N-diglutamyl-lysyl-L-glutamate (GGLG) liposomes were previously developed to enhance drug delivery efficiency in tumor cells owing to its pH-responsive properties. Herein, we report the modification of GGLG liposomes by conjugating a Fab' fragment of an ErbB2 antibody to the terminus of PEG (polyethylene glycol)-lipid (Fab'-GGLGliposomes). The conjugation of Fab' fragments did not affect the antibody activity, drug (doxorubicin, DOX) encapsulation efficiency, stability during storage or pH-sensitivity. However, the binding affinity of Fab'-GGLG liposomes was enhanced to ErbB2-overexpressing HCC1954 cells specifically, and the cell association increased 10-fold in comparison to GGLG liposomes. Consequently, intracellular DOX delivery was enhanced, with an increased cytotoxicity in HCC1954 cells (i.e., IC50 of 1.17 and 3.08 mu g/mL for Fab'-GGLG-DOX and GGLG-DOX liposomes, respectively). Further, a significantly enhanced tumor growth inhibition was obtained in an ErbB2-overexpressing breast cancer-bearing mouse model. Therefore, a potent anticancer drug delivery system was constructed by the immunological modification of pH-sensitive liposomes. (C) 2016 Elsevier Inc. All rights reserved.

In this paper, a microthermograph, temperature mapping with high spatial resolution, was established using luminescent molecules embedded ultrathin polymeric films (nanosheets), and demonstrated in a living small animal to map out and visualize temperature shift due to animal's muscular activity. Herein, we report super flexible and self-adhesive (no need of glue) nanothermosensor consisting of stacked two different polymeric nanosheets with thermosensitive (Eu-tris (dinaphthoylmethane)-bis-trioctylphosphine oxide: EuDT) and insensitive (Rhodamine 800) dyes being embedded. Such stacked nanosheets allow for the ratiometric thermometry, with which the undesired luminescence intensity shift due to focal drift or animal's z-axis displacement is eliminated and the desired intensity shift solely due to the temperature shift of the sample (living muscle) can be acquired. With the stacked luminescent nanosheets, we achieved the first-ever demonstration of video filming of chronologically changing temperature-shift distribution from the rest state to the active state of the muscles in the living animal. The polymer nanosheet engineering and in vivo microthermography presented in the paper are promising technologies to microscopically explore the heat production and heat transfer in living cells, tissues, and organisms with high spatial resolution beyond what existing thermometric technologies such as infrared thermography have ever achieved.

We present a facile method to fabricate polymer thin films with tens of nanometers thickness and several micrometers size (also called "microdiscs" herein) by applying phase separation of polymer blend. A water-soluble supporting layer is employed to obtain a freestanding microdisc suspension. Owing to their miniaturized size, microdiscs can be injected through a syringe needle. Herein, poly(D,L-lactic acid) microdiscs were fabricated with various thicknesses and sizes, in the range from ca. 10 to 60 nm and from ca. 1.0 to 10.0 mu m, respectively. Magnetic nanoparticles were deposited on polymer microdiscs with a surface coating method. The magnetic manipulation of microdiscs in a liquid environment under an external magnetic field was achieved with controllable velocity by adjusting the microdisc dimensions and the loading amount of magnetic components. Such biocompatible polymer microdiscs are expected to serve as injectable vehicles for targeted drug delivery.

Ultra-thin polymer films (nanosheets) fabricated by a layer-by-layer (LbL) method possess unique properties such as high flexibility, adhesive strength, and transparency, and can be peeled off from a substrate and attached to various surfaces via a water-soluble supporting film. Therefore, flexible and transferrable LbL nanosheets are convenient tools as coating materials. Here, we fabricated a novel antimicrobial coating material by embedding silver nanoparticles (AgNPs) in an LbL nanosheet composed of layers of chitosan and sodium alginate (Ag-LbL nanosheet) by means of a photo-reduction method. Optimizing the amount of irradiated energy applied led to robust antimicrobial efficacy against methicillin-resistant Staphylococcus aureus (MRSA), sufficient to meet ISO standards (ISO 22196), while maintaining the flexibility and adhesive potency of the LbL nanosheet. Thus, the Ag-LbL nanosheet is a promising coating material that can provide antimicrobial efficacy to various surfaces. (c) 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 585-593, 2016.

Thermoplastic elastomers are attractive materials because of the drastic changes in their physical properties above and below the glass transition temperature (T-g). In this paper, we report that free-standing polystyrene (PS, T-g: 100 degrees C) and polystyrene-polybutadiene-polystyrene triblock copolymer (SBS, T-g: -70 degrees C) thin films with a thickness of hundreds of nanometers were prepared by a gravure coating method. Among the mechanical properties of these thin films determined by bulge testing and tensile testing, the SBS thin films exhibited a much lower elastic modulus (ca. 0.045 GPa, 212 nm thickness) in comparison with the PS thin films (ca. 1.19 GPa, 217 nm thickness). The lower elastic modulus and lower thickness of the SBS thin films resulted in higher conformability and thus higher strength of adhesion to an uneven surface such as an artificial skin model with roughness (R-a = 10.6 mu m), even though they both have similar surface energies. By analyzing the mechanical properties of the SBS thin films, the elastic modulus and thickness of the thin films were strongly correlated with their conformability to a rough surface, which thus led to a high adhesive strength. Therefore, the SBS thin films will be useful as coating layers for a variety of materials.

Ca2+ distribution is spatially and temporally non-uniform inside cells due to cellular compartmentalization. However, Ca2+ sensing with small organic dyes, such as fura-2 and fluo-4, has been practically applied at a single cell level where the averaged signal from freely diffusing dye molecules is acquired. In this study, we aimed to target azide-functionalized fura-2 (N-3-fura-2) to a specific site of subcellular compartments to realize focal Ca2+ sensing. Using scAVD (single-chain avidin)-biotin interaction and a copper-free click reaction system, we linked N-3-fura-2 to specifically-targeted scAVD protein fused with a red fluorescent protein mCherry, so that Ca2+ sensors conjugated with four N-3-fura-2 dyes with dibenzocyclooctyne (DBCO)-PEG4-biotin as a linker were generated at subcellular compartments in living cells. In cytoplasm, N-3-fura-2 showed a prolonged retention period after binding to scAVD. Furthermore, the reacted N-3-fura-2 was retained inside cells even after free dyes were washed out by methanol fixation. When scAVD was overexpressed on endoplasmic reticulum (ER) membranes, N-3-fura-2 was accumulated on ER membranes. Upon histamine stimulation, which increases cytosolic Ca2+ concentration, ER-localized N-3-fura-2 successfully sensed the Ca2+ level changes at the cytosolic side of ER membrane. Our study demonstrated specific targeting of N-3-fura-2 to subcellular compartments and the ability of sensing focal Ca2+ level changes with the specifically targeted Ca2+ sensors.
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A preclinical study of dodecapeptide ((400)HHLGGAKQAGDV(411)) (H12)-(adenosine diphosphate, ADP)-liposomes for use as a synthetic platelet (PLT) substitute under conditions of red blood cell (RBC) transfusion-induced dilutional thrombocytopenia is limited to pharmacological effect. In this study, the pharmacokinetics of H12-(ADP)-liposomes in RBC transfusion-induced dilutional thrombocytopenic rats were evaluated. As evidenced by the use of C-14, H-3 double-radiolabeled H12-(ADP)-liposomes in which the encapsulated ADP and liposomal membrane were labeled with C-14 and H-3, respectively, the H12-(ADP)-liposomes remained intact in the blood circulation for up to 3 h after injection, and were mainly distributed to the liver and spleen. The encapsulated ADP was mainly eliminated in the urine, whereas the outer membrane was mainly eliminated in the feces. These successive pharmacokinetic properties of the H12-(ADP)-liposomes in RBC transfusion-induced dilutional thrombocytopenic rats were similar to those in healthy rats, except for the shorter retention time in the circulation. When H12-(ADP)-liposomes were repeatedly injected into RBC transfusion-induced dilutional thrombocytopenic rats at intervals of 5 days at a dose of 10 mg lipids/kg, the second dose of injected H12-(ADP)-liposomes were rapidly cleared from the circulation, namely, via the accelerated blood clearance phenomenon. These novel pharmacokinetic findings provide useful information for the further development of H12-(ADP)-liposomes as a PLT substitute. (c) 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3968-3976, 2015

We have proposed free-standing centimeter-sized ultra-thin films (nanosheets) for biomedical applications. Such nanosheets exhibit unique properties such as transparency, flexibility, and good adhesiveness. However, they are only easily adhered to broad and flat surfaces due to their dimensions. To this end, we recently proposed an innovative nanomaterial: the nanosheets fragmented into submillimeter-size pieces. Intriguingly, such fragmented nanosheets could be adhered to uneven and irregular surfaces in addition to flat surfaces in a spread-out patchwork manner. We herein review the fabrication procedure and characterization of fragmented nanosheets composed of biodegradable polyesters and thermostable bio-friendly polymers, and their biomedical applications in burn therapy and antithrombotic coating using a patchwork coating.

Micro/nano-bubbles are practical nanomaterials designed to increase the gas content in liquids. We attempted to use oxygen micro/nano-bubble dispersions as an oxygen-rich liquid as a means for total liquid ventilation. To determine the oxygen content in the bubble dispersion, a new method based on a spectrophotometric change between oxy- and deoxy-hemoglobin was established. The oxygen micro/nano-bubble dispersion was supplied to an experimental total ventilation liquid in anesthetic rats. Though the amount of dissolving oxygen was as low as 6 mg/L in physiological saline, the oxygen content in the oxygen micro/nano-bubble dispersion was increased to 45 mg/L. The positive correlation between the oxygen content and the life-saving time under liquid ventilation clearly indicates that the life-saving time is prolonged by increasing the oxygen content in the oxygen micro/nano-bubble dispersion. This is the first report indicating that the oxygen micro/nano-bubbles containing a sufficient amount of oxygen are useful in producing oxygen-rich liquid for the process of liquid ventilation.

Partial-thickness burn injury has the potential for reepithelialization and heals within 3 weeks. If the wound is infected by bacteria before reepithelization, however, the depth of disruption increases and the lesion easily progresses to the full-thickness dermal layers. In the treatment of partial-thickness burn injury, it is important to prevent the wound area from bacterial infection with an antimicrobial dressing. Here, we have tested the antimicrobial properties of polymeric ultra-thin films composed of poly(lactic acid) (termed "PLA nanosheets"), which have high flexibility, adhesive strength and transparency, and silver sulfadiazine (AgSD), which exhibits antimicrobial efficacy. The AgSD-loaded nanosheet released Ag+ for more than 3 days, and exerted antimicrobial efficacy against methicillin-resistant Staphylococcus aureus (MRSA) in an in vitro Kirby-Bauer test. By contrast, a cell viability assay indicated that the dose of AgSD used in the PLA nanosheets did not show significant cytotoxicity toward fibroblasts. In vivo evaluation using a mouse model of infection in a partial-thickness burn wound demonstrated that the nanosheet significantly reduced the number of MRSA bacteria on the lesion (more than 10(5)-fold) and suppressed the inflammatory reaction, thereby preventing a protracted wound healing process. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Mitochondrial thermodynamics is the key to understand cellular activities related to homeostasis and energy balance. Here, we report the first ratiometric fluorescent molecular probe (Mito-RTP) that is selectively localized in the mitochondria and visualize the temperature. We confirmed that Mito-RTP could work as a ratiometric thermometer in a cuvette and living cells.

We synthesized a novel cyclooctyne-based clickable fluorescent probe with versatile properties such as high cell-membrane permeability and free diffusibility in the cell. Our probe "FC-DBCO'' was conjugated to an azide-modified mannose via a Cu-free click reaction in living HeLa cells and displayed intracellular specific fluorescence imaging with low background signals.

We propose an instant micro-thermography method using a fluorescent-nanoparticle thermosensor capable of reporting temperature as the fluorescence intensity ratio of the temperature-sensitive dye to the reference. We demonstrate "temperature mapping" inside a fruit fly larva that was orally dosed with nanoparticle thermosensors.

A 1:3 molar complex of the fluoroalkyl side chain-substituted 2,6,10-tris-carboxymethoxy-3,7,11-tris(4,4,5,5,6,6,7,7,7-nonafluoroheptyloxy)triphenylene (TPF4) with the second generation dendron 3,5-bis(3,4-bis-dodecyloxybenzyloxy)-N-pyridin-4-yl-benzamide (DN) assembled through complementary hydrogen bonding to form a supramolecular columnar liquid crystal, which exhibited homeotropic alignment when sandwiched between octadecyltrichlorosilane (OTS)-coated or indium tin oxide (ITO)-coated glass plates due to specific interactions between the fluoroalkyl side chains and the substrates.

With a small amount of maleimide modification on the liposome surface, enhanced cellular uptake of liposomes and drug-delivery efficiency can be obtained both in vitro and in vivo. Herein, we describe the mechanisms underlying this enhanced cellular uptake. Suppression of the cellular uptake of maleimide-modified liposomes (M-GGLG, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate [GGLG]/cholesterol/poly(ethylene glycol) - 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [PEG5000-DSPE]/maleimide [M]-PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03) caused by temperature block and addition of serum was alleviated compared with that of liposomes without maleimide modification (GGLG liposomes, composed of GGLG/cholesterol/PEG5000-DSPE/PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03). When 0.01 nM N-ethylmaleimide was used to pre-block cellular thiols, the cellular uptake of M-GGLG liposomes was decreased to approximately 70% in HeLa, HCC1954, MDA-MB-468, and COS-7 cell lines. Moreover, inhibition of a thiol-related reductase such as protein disulfide isomerase resulted in a 15%-45% inhibition of the cellular uptake of M-GGLG liposomes, whereas GGLG liposomes were not influenced. Further, single and mixed inhibitors of clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis did not efficiently inhibit the cellular uptake of M-GGLG liposomes. Using confocal microscopy, we verified that M-GGLG liposomes were localized partially in lysosomes after inhibition of the mentioned conventional endocytic pathways. Therefore, it was hypothesized that the mechanisms underlying the enhanced cellular uptake of liposomes by maleimide modification was thiol-mediated membrane trafficking, including endocytosis and energy-independent transport. © 2014 Li and Takeoka.

A "sticker-like" PLGA nanosheet with microgrooved patterns is developed through a facile combination of spin coating and micropatterning techniques. The resulting microgrooved PLGA nanosheets can be physically adhered on flat or porous surfaces with excellent stability in aqueous environments and can harness the spatial arrangements of cells, which make it a promising candidate for generating biomimic periosteum for bone regenerative applications.

An amino acid-based cationic lipid having a TFA counterion (trifluoroacetic acid counterion) in the lysine headgroup was used to deliver functional proteins into human cervical cancer cells, HeLa, in the presence of serum. Proteins used in the study were fluorescein isothiocyanate (FITC) labeled bovine serum albumin, mouse anti-F actin antibody [NH3], and goat anti mouse IgG conjugated with FITC. The formation of liposome/protein complexes was confirmed using native polyacrylamide gel electrophoresis. Furthermore, the complexes were characterized in terms of their size and zeta potential at different pH values and found to be responsive to changes in pH. The highest delivery efficiency of the liposome/albumin complexes was 99% at 37 °C. The liposomes effectively delivered albumin and antibodies as confirmed by confocal laser scanning microscopy (CLSM). Inhibition studies showed that the cellular uptake mechanism of the complexes was via caveolae-mediated endocytosis, and the proteins were subsequently released from either the early endosomes or the caveosomes as suggested by CLSM. Thus, lysine-based cationic liposomes can be a useful tool for intracellular protein delivery. © 2013 American Chemical Society.

The homeostasis of body temperature and energy balance is one of the major principles in biology. Nanoscale thermometry of aqueous solutions is a challenging but crucial technique to understand the molecular basis of this essential process. Here, we developed a ratiometric nanothermometer (RNT) for intracellular temperature measurement in real time. Both the thermosensitive fluorophore, beta-diketonate chelate europium(III) thenoyltrifluoroacetonate, and the thermoinsensitive fluorophore, rhodamine 101, which was used as a self-reference, are embedded in a polymeric particle that protects the fluorophores from intracellular conditions. The ratiometric measurement of single RNT spots is independent of the displacement of the RNT along the z-axis. The temperature is therefore determined at the location of each RNT under an optical microscope regardless of the dynamic movement of living cells. As a demonstration of the spot-by-spot intracellular thermometry, we successfully followed the temperature change in individual RNT spots in a single cell together with the Ca2+ burst induced by the Ca2+ ionophore ionomycin. The temperature increases differently among different spots, implying heterogeneous heat production in the cell. We then show that, in some spots, the temperature gradually decreases, while in others it remains high. The average temperature elevation within a cell is positively correlated to the increase in Ca2+, suggesting that the activity and/or number of heat sources are dependent on the Ca2+ concentration.

The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C3-Glu2C14, and a lysine-based lipid, Lys-C3-Glu2C14, with two different types of counterion, chloride ion (Cl-) and trifluoroacetic acid (TFA -), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C3-Glu2C14 with TFA- induced a rate of transfection in primary cultured neurons higher than that of Lys-C 3-Glu2C14 using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C 3-Glu2C14 with TFA- showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C3-Glu2C14 with TFA- is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity. © 2013 American Chemical Society.

Maleimide is a stable and easy-to-handle moiety that rapidly and covalently conjugates thiol groups of cysteine residues in proteins or peptides. Herein, we use maleimide to modify the surface of liposomes in order to obtain an advanced drug delivery system. Employing a small amount (0.3 mol%) of maleimide-polyethylene glycol (PEG) to modify the surface of the liposomes M-GGLG-liposomes, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (GGLG)/cholesterol/poly(ethylene glycol) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG5000-DSPE)/maleimide-PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03, drug delivery efficiency was remarkably improved both in vitro and in vivo compared to unmodified liposomes (GGLG-liposomes, composed of GGLG/cholesterol/PEG5000-DSPE/PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03). Moreover, this modification did not elicit any detectable increase in cytotoxicity. The maleimide-modification did not alter the physical characteristics of the liposomes such as size, zeta potential, pH sensitivity, dispersibility and drug encapsulation efficiency. However, M-GGLG-liposomes were more rapidly (≥2-fold) internalized into HeLa, HCC1954, and MDA-MB-468 cells compared to GGLG-liposomes. In vivo, M-GGLG-liposomes encapsulating doxorubicin (M-GGLG-DOX-liposomes) also showed a more potent antitumor effect than GGLG-DOX-liposomes and the widely used 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-DOX-liposomes after two subcutaneous injections around breast cancer tissue in mice. The biodistribution of liposomes in this model was observed using an in vivo imaging system, which showed that M-GGLG-liposomes were present for significantly longer at the injection site compared to GGLG-liposomes. The outstanding biological functions of the maleimide-modified liposomes as a novel drug delivery system make them ideally suited to a wide range of applications. © 2013 Li and Takeoka.

Postoperative adhesion often causes serious adverse effects such as bowl obstruction, chronic abdominal pain, pelvic pain, and infertility. We previously reported that a poly-L-lactic acid (PLLA) nanosheet can efficiently seal a surgical incision without scarring. In this report, we examined whether the PLLA nanosheet can form an effective anti-adhesion barrier in partial hepatectomy accompanied by severe hemorrhaging in rats. To evaluate the anti-adhesive property of the nanosheet, the liver wound surface was covered with TachoComb((R)), a well-known hemostat material used in clinical procedures, and then with the PLLA nanosheet. Dressing the wound surface with TachoComb((R)) alone caused severe adhesion with omentum and/or residual parts of the liver. By contrast, combinational usage of TachoComb((R)) and the PLLA nanosheet significantly reduced such adhesion, presumably by inhibiting the permeation of oozing blood cells and the infiltration of fibroblastic cells. Moreover, the nanosheet displayed low permeability against serum proteins as well as cells in vitro, supporting the notion that the PLLA nanosheet has anti-adhesive properties in vivo. These results strongly suggested that the PLLA nanosheet is a promising material for reducing unwanted postoperative adhesion. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 101B: 1251-1258, 2013.

PURPOSE. We investigated the IOP reduction and safety of latanoprost-loaded biodegradable nanosheet (LBNS) as a new antiglaucoma drug delivery system (DDS).
METHODS. We fabricated a 40 nm thick multilayered biodegradable nanosheet that is composed of chitosan and sodium alginate by means of the layer-by-layer method. Latanoprost isopropyl ester was loaded on the nanosheet to prepare 25, 2.5, and 0.25 mu g/cm(2) LBNSs. A nanosheet without latanoprost isopropyl ester (NS) and 0.005% latanoprost ophthalmic solution were prepared as controls. LBNSs or NS was applied to rat cornea, and IOP was monitored for 9 days. Local adverse effects and eye scratching movement also were investigated. The amount of latanoprost acid in aqueous humor and was measured in rabbits.
RESULTS. The 0.25 mu g/cm(2) LBNS and 0.005% latanoprost ophthalmic solution showed significant IOP reduction only for 1 day after application, whereas the IOP reduction rates of 2.5 mu g/cm(2) LBNS at 1, 2, 4, 7, and 9 days after application were -27.0% +/- 14.8%, -22.0% +/- 16.7%, -25.8% +/- 18.0%, -22.7% +/- 20.9%, and -6.6% +/- 17.0%, respectively. The 25 mu g/cm(2) LBNS reduced IOP in a similar manner. The 25 mu g/cm(2) LBNS induced transient hyperemia, whereas the 0.25 and 2.5 mu g/cm(2) LBNSs did not exert any local adverse effects. The eye scratching movement test showed that application of 25 mu g/cm(2) LBNS did not cause any irritation of the eye. Latanoprost acid was detected in aqueous humor up to 6 days after application of 2.5 mu g/cm(2) LBNS.
CONCLUSIONS. LBNS may be used as a novel antiglaucoma DDS.

OBJECTIVE: To investigate the efficacy of multioverlapping therapy using a polysaccharide nanosheet having 75-nm thickness for sealing and stopping massive venous hemorrhage. METHODS: The hydrostatic durability of the polysaccharide nanosheet was evaluated in vitro when secured to an incised silicon tube. For in vivo studies, the inferior vena cava (IVC) of rabbits was cut longitudinally, and multiple polysaccharide nanosheets were overlapped onto the injured IVC. RESULTS: The mechanical hydrostatic durability of the nanosheets was gradually augmented by an increasing number of multilayered nanosheets in vitro. This durability was saturated at 80 ± 6 mm Hg by four layers of nanosheets, which was robust enough to seal injured vessel walls of the large IVC. Multioverlapping therapy using nanosheets effectively sealed and stopped bleeding from the injured IVC in vivo. One month later, no inflammatory tissue response was observed around the nanosheet attachment sites of the IVC, while conventional suturing repair in control rabbits showed a severe inflammatory response around the sutured area. CONCLUSIONS: The multioverlapping therapy using the polysaccharide nanosheets will effectively stop massive venous bleeding without adverse effects in the immediate or chronic postoperative setting.

Background: Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group. Methods: Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000. Results: We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular. Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity. Conclusion: The gene delivery efficiency of amino acid-based cationic assemblies is influenced by the amino acids (ie, arginine or lysine) present as the hydrophilic head group and their associated counterions. © 2013 Sarker et al, publisher and licensee Dove Medical Press Ltd.

The morphological stability of polymer films is critically important to their application as functional materials. The deformation of polymer surfaces on the nanoscale may be significantly influenced by geometrical confinement. Herein, we constructed a mechanically heterogeneous polymer surface by phase separation in a thin polymer film and investigated the deformation behavior of its nanostructure (∼30 nm thickness and ∼100 nm average diameter) with tapping-mode atomic force microscopy. By changing different scan parameters, we could induce deformation localized to the nanostructure in a controllable manner. A quantity called the deformation index is defined and shown to be correlated to energy dissipation by tip-sample interaction. We clarified that the plastic deformation of a polymer on the nanoscale is energy-dependent and is related to the glass-to-rubber transition. The mobility of polymer chains beneath the tapping tip is enhanced, and in the corresponding region a rubberlike deformation with the lateral motion of the tip is performed. The method we developed can provide insight into the geometrical confinement effects on polymer behavior. © 2013 American Chemical Society.

The authors evaluated the efficacy of an ultrathin nanosheet consisting of poly-L-lactic acid (75 nm thick) as a wound dressing material. A full-thickness skin defect was made on the backs of mice and overlapped with or without the poly-L-lactic acid nanosheet. Wound healing was more rapidly improved by overlapping with the nanosheet, especially in the early healing period (at 4 to 6 days). The remaining wound area in the treatment group was significantly smaller at 4 days than in the control group. Histologically, a clear layer was observed over the granulation layer by the nanosheet therapy at 4 days. Thus, overlapping therapy with the poly-L-lactic acid nanosheet accelerated wound healing and formed a clear layer just above the granulation tissue. The poly-L-lactic acid nanosheet may have potential as a novel wound dressing to promote wound healing. Copyright © 2013 by the American Society of Plastic Surgeons.

Freestanding poly(L-lactic acid) (PLLA) nanosheets are mass-produced by a simple combination of a spin-coating-assisted multi-layering process and a peeling technique. The resulting PLLA nanosheets are fragmented by homogenization and then reconstructed into a "patchwork" sheet on various surfaces without any adhesive reagents. The patchwork is shown to offer excellent protection against burn wound infection with Pseudomonas aeruginosa, and may therefore be an alternative to conventional burn therapy for prevention of infection.

Most polymers solidify below a glass transition temperature (;), which is important for the fabrication of polymeric materials. The glass transition dynamics (GTD) of polymers alters their physical properties and therefore the range of applications suitable for the particular materials. In this regard, most GTD studies were oriented to the thermodynamics of amorphous polymer systems, while little studies were known for semicrystalline polymers. Here, we focus on the glassy and crystalline properties of semicrystalline polymers such as poly(L-lactic acid) (PLLA) and envisage to control the nanostructure of free-standing PLLA ultrathin films (referred as "PLLA nanosheets"), via thermodynamic rearrangement of polymer chains entangled in a quasi-two-dimensional interface during the GTD process. The annealing process on the PLLA nanosheets (<100 nm thick) resulted in the formation of semicrystalline domains and microscopic apertures with polymer chains (similar to 100 nm in size). Such nanostructure surprisingly induced selective molecular permeability, which was controlled as a function of film thickness and inherent crystallinity. The present methodology demonstrates the direct conversion of thermodynamic properties of semicrystalline polymers into the functional nanostructured polymeric materials.

The powerful strategy of "intracellular click reaction" was used to retain a chemical Ca2+ indicator in the cytosol. Specifically, a novel clickable Ca2+ indicator "N-3-fura-2 AM" was coupled with dibenzylcyclooctyl-modified biomacromolecules via copper-free click reaction in living cells and Ca2+ oscillation was observed for an extended period of time.

In this paper, we report the development of nanoporous polymer which demonstrates the coordination property toward zinc porphyrin. A hydrogen-bonded columnar liquid crystalline precursor composed of a triphenylene template and three equivalent of the surrounding dendric amphiphile bearing a pyridyl head group and a polymerizable aliphatic chain, was covalently fixed by photo-polymerization, and then the subsequent selective removal of the template successively resulted in a nanoporous polymer in which the pore wall is modified with pyridyl groups. The nanoporous polymer reflected the conformation of template, and displayed considerable coordination ability of the pyridyl groups towards zinc porphyrin. The coordinative nanoporous polymer is promising as a nano-scaled scaffold for the organization of dyes into functional supramolecular architectures.

Recently, biomaterials have been widely used in a variety of medical applications. We previously reported that a poly-l-lactic acid (PLLA) nanosheet shows anti-adhesive properties and constitutes a useful biomaterial for preventing unwanted wound adhesion in surgical operations. In this article, we examine whether the PLLA nanosheet can be specifically modified with biomacromolecules on one surface only. Such an approach would endow each side of the nanosheet with discrete functions, that is anti-adhesive and pro-healing properties. We fabricated two distinct PLLA nanosheets: (i) collagen cast on the surface of a PLLA nanosheet (Col-Cast-PLLA) and (ii) collagen spin-coated on the nanosheet (Col-Spin-PLLA). In the Col-Spin-PLLA nanosheet, the collagen layer had a thickness of 5-10 nm on the PLLA surface and displayed increased hydrophilicity compared to both PLLA and Col-Cast-PLLA nanosheets. In addition, atomic force microscopy showed disorganized collagen fibril formation on the PLLA layer when covered using the spin-coating method, while apparent bundle formations of collagen were formed in the Col-Cast-PLLA nanosheet. The Col-Spin-PLLA nanosheet provided a microenvironment for cells to adhere and spread. By contrast, the Col-Cast-PLLA nanosheet displayed reduced cell adhesion compared to the Col-Spin-PLLA nanosheet. Consistent with these findings, immunocytochemical analysis clearly showed fine networks of actin filaments in cells cultured on the Col-Spin-PLLA, but not the Col-Cast-PLLA nanosheet. Therefore, the Col-Spin-PLLA nanosheet was shown to be more suitable for acting as a scaffold. In conclusion, we have succeeded in developing a heterofunctional nanosheet comprising a collagen modified side, which has the ability to rapidly adhere cells, and an unmodified side, which acts as an adhesion barrier, by using a spin-coating technique.

Polymeric ultra-thin films (nanosheets) possess unique properties that make them suitable materials for various biomedical applications. In our previous study, we assessed the use of an antibiotic (tetracycline, TC)-loaded nanosheet (or "TC-nanosheet") for the treatment of gastrointestinal tissue defects. The nanosheet consisted of three functional layers: layer-by-layer nanosheet as a stable platform, TC as an antimicrobial agent with autofluorescence for tracing, and a poly(vinyl acetate) nanosheet to act as a protecting layer. The TC-nanosheet has high flexibility, adhesive strength and transparency. Here, we evaluated the effectiveness of the TC-nanosheet in preventing full thickness burn-wound infections. In an in vivo study, murine dorsal skin was injured by full-thickness burns and then infected with Pseudomonas aeruginosa (P. aeruginosa), a common bacterium causing burn-associated infections. The wound site was treated either with a TC-nanosheet, TC-unloaded nanosheet or left untreated. Wound management was facilitated by the high transparency of the TC-nanosheet. The TC-nanosheet significantly improved burn-wound infection by P. aeruginosa in mice. Indeed, all mice treated with the TC-nanosheet survived, whereas the other treatment groups displayed increased rates of mortality due to bacterial infection. According to histological analyses and viable bacterial counting in the liver (bacterial translocation), the TC-nanosheets were able to prevent not only the local inflammation but also systemic inflammation. We conclude that the TC-nanosheet can act as an effective treatment for full-thickness burn-wound infection. Hence, the TC-nanosheet is a promising therapeutic tool for burn-wound management in severely burn-injured patients. (c) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

This paper describes a convenient screening method using ion trap electrospray ionization mass spectrometry to classify ligands to a target molecule in terms of kinetic parameters. We demonstrate this method in the screening of ligands to a hexahistidine tag from a pooled library synthesized by click chemistry. The ion trap mass spectrometry analysis revealed that higher stabilities of ligand-target complexes in; the gas phase were related to lower dissociation rate constants, i.e., off-rates in solution. Finally, we prepared a fluorescent probe utilizing the ligand with lowest off-rate and succeeded in performing single molecule observations of hexahistidine-tagged myosin V walking on actin filaments.

Burn wounds are highly susceptible to bacterial infection due to impairment of the skin's integrity. Therefore, prevention of bacterial colonization/infection in the wound is crucial for the management of burns, including partial-thickness burn injuries. Although partial-thickness burn injuries still retain the potential for reepithelialization, the complication of wound infection severely impairs the reepithelialization even in such superficial burn injuries. We recently developed a biocompatible nanosheet consisting of poly(l-lactic acid) (PLLA). The PLLA nanosheets have many useful and advantageous biological properties for their application as a wound dressing, such as sufficient flexibility, transparency, and adhesiveness. We herein investigated the suitability of the PLLA nanosheets as a wound dressing for partial-thickness burn wounds in mice. The PLLA nanosheets tightly adhered to the wound without any adhesive agents. Although wound infection with Pseudomonas aeruginosa in the controls significantly impaired reepithelialization of burn wounds, dressing with the PLLA nanosheet markedly protected against bacterial wound infection, thereby improving wound healing in the mice receiving partial-thickness burn injuries. The PLLA nanosheet also showed a potent barrier ability for protecting against bacterial penetration in vitro. The ultrathin PLLA nanosheet may be applied as a protective dressing to reduce environmental contamination of bacteria in a partial-thickness burn wound.

Polymer phase separation has established a series of bottom-up nanofabrication methods. Owing to the intrinsic immiscibility of most polymer blends, phase separation is typically produced by rapid quenching during spin-casting. However, the full sequence of events that occur during this process is still unclear, especially in the case of ultrathin polymer blend films. Herein, a freestanding method is first introduced to obtain morphological information from the bottom side of the film. We demonstrate that when the thickness of ultrathin film is comparable to the dimensional scale of the phase separation domains, it is feasible to prepare perforated films with uniform nanopores via selective solvent etching. Our results also provide direct evidence that the spinodal decomposition mechanism plays an important role in determining the final morphology within the ultrathin polymer blend films. These findings are of practical value in the fabrication of desired nanostructures by polymer phase separation.

The influence of both the ionization states and the hydrocarbon chain spacer of a series of amino acid-based cationic lipids was evaluated in terms of gene delivery efficiency and cytotoxicity to the COS-7 cell line and compared with that of Lipofectamine™ 2000. We synthesized a series of amino acid-based cationic lipids with different ionization states (i.e., -NH 2, -NH 3+Cl - or -NH 3+TFA -) in the lysine head group and different hydrocarbon chain spacers (i.e., 0, 3, 5 or 7 carbon atoms) between the hydrophilic head group and hydrophobic moieties. In the 3-carbon series, the cationic assemblies formed a micellar structure in the presence of -NH 3+Cl - and a vesicular structure both in the presence of -NH 2 and -NH 3+TFA -. Differential scanning calorimetry (DSC) data revealed a significantly lower (8.1°C) gel-to-liquid crystalline phase transition temperature for cationic assemblies bearing -NH 3+TFA - when compared to their -NH 2 counterparts. Furthermore, the zeta potential of cationic assemblies having -NH 3+TFA - in the hydrophilic head group was maximum followed by -NH 3+Cl - and -NH 2 irrespective of their hydrocarbon chain spacer length. The gene delivery efficiency in relation to the ionization states of the hydrophilic head group was as follows: -NH 3+TFA - > -NH 3+Cl - > -NH 2. © 2011 Elsevier B.V.

We fabricated fluorescent nanoparticles which monitor temperature changes without sensitivity to pH (4-10) and ionic strength (0-500 mM). The nanothermometers spontaneously enter living HeLa cells via endocytosis, enclosed in acidic organelles, i.e., endosome/lysosome, and then transported along microtubules in a temperature-dependent manner, working as "walking nanothermometers".

We report a mitochondrial targeted redox probe (MitoRP) that comprises a nitroxide radical (TEMPO) moiety and coumarin 343. Using isolated mitochondria in the presence/absence of substrates and inhibitors of oxidative phosphorylation, we demonstrated that MitoRP is a useful probe to monitor the electron flow associated with complex I.

Background One problem with polypropylene mesh (PPM) used to repair abdominal wall hernias is dense adhesions to the visceral surface. The authors developed the biocompatible poly-l-lactic acid (PLLA) nanosheet (thickness < 100 nm), which has the unique ability to adhere tightly to tissues but not to opposing tissues. This study investigated the antiadhesive and fixative characteristics of the PLLA nanosheet after placement of intraperitoneal onlay PPM (IPOM) overlaid with a PLLA nanosheet on intact peritoneum.
Methods The PLLA nanosheet was fabricated by the spin-coating method and peeling technique with polyvinyl alcohol (PVA) as a supporting film. Two 1.5-cm-square pieces of mesh were implanted on each peritoneal side of the midline incision. The mesh was fixed to the peritoneum with a suture and then overlaid with a 4-cm-square piece of Seprafilm or nanosheet. To examine the fixative property, mesh was overlaid with Seprafilm or nanosheet without a fixed suture. After 4 weeks, mesh adhesion, inflammatory reaction, fixation, and dislocation of mesh were evaluated.
Results Nanosheet-overlaid meshes were flexible and fit over the peritoneum. Adhesion was observed in 10% of the nanosheet-overlaid meshes and in 50% of the Seprafilm-overlaid meshes. The adhesion tenacity grade was significantly lower with the nanosheet-overlaid meshes (0.1 +/- A 0.1) than with the Seprafilm-overlaid meshes (1.0 +/- A 0.4) (p = 0.029), and the percentage of the adhesion area also was lower with the nanosheet-overlaid meshes (1.0 +/- A 1.0% vs 8.5 +/- A 3.2%; p = 0.037). The mean inflammatory cell counts were lower with the nanosheet-overlaid meshes (p = 0.0023). Regarding the fixative property, 37.5% of the nanosheet-overlaid meshes were fixated on the peritoneum, but no Seprafilm-overlaid mesh was fixated.
Conclusions Overlaying of a PLLA nanosheet was effective for adhesion prophylaxis of intraperitoneal mesh. It also may have a possible beneficial effect on fixation of mesh.

Ceramides play a crucial role in divergent signaling events, including differentiation, senescence, proliferation, and apoptosis. Ceramides are a minor lipid component in terms of content; thus, highly sensitive detection is required for accurate quantification. The recently developed isobaric tags for relative and absolute quantitation (iTRAQ) method enables a precise comparison of both protein and aminophospholipids. However, iTRAQ tagging had not been applied to the determination of sphingolipids. Here we report a method for the simultaneous measurement of multiple ceramide and monohexosylceramide samples using iTRAQ tags. Samples were hydrolyzed with sphingolipid ceramide N-deacylase (SCDase) to expose the free amino group of the sphingolipids, to which the N-hydroxysuccinimide group of iTRAQ reagent was conjugated. The reaction was performed in the presence of a cleavable detergent, 3-[3-(1,1-bisalkyloxyethyl)pyridine-1-yl]propane-1-sulfonate (PPS) to both improve the hydrolysis and ensure the accuracy of the mass spectrometry analysis performed after iTRAQ labeling. This method was successfully applied to the profiling of ceramides and monohexosylceramides in sphingomyelinase-treated Madin Darby canine kidney (MDCK) cells and apoptotic Jurkat cells.-Nabetani, T., A. Makino, F. Hullin-Matsuda, T. Hirakawa, S. Takeoka, N. Okino, M. Ito, T. Kobayashi, and Y. Hirabayashi. Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ). J. Lipid Res. 2011. 52: 1294-1302.

Aqueous colloidal mesoporous nanoparticles with ethenylene-bridged silsesquioxane frameworks with a uniform diameter of similar to 20 nm were prepared from bis(triethoxysilyl)ethenylene in a basic aqueous solution containing cationic surfactants. The nanopartides, which had higher hydrolysis resistance under aqueous conditions, showed lower hemolytic activity toward bovine red blood cells than colloidal mesoporous silica nanoparticles.

Freely suspended nanocomposite thin films based on soft polymers and functional nanostructures have been widely investigated for their potential application as active elements in microdevices. However, most studies are focused on the preparation of nanofilms composed of polyelectrolytes and charged colloidal particles. Here, a new technique for the preparation of poly(L-lactic acid) free-standing nanofilms embeddidng superparamagnetic iron oxide nanoparticles is presented. The fabrication process, based on a spin-coating deposition approach, is described, and the influence of each production parameter on the morphology and magnetic properties of the final structure is investigated. Superparamagnetic free-standing nanofilms were obtained, as evidenced by a magnetization hysteresis measurement performed with a superconducting quantum interference device (SQUID). Nanofilm surface morphology and thickness were evaluated by atomic force microscopy (AFM), and the nanoparticle dispersion inside the composites was investigated by transmission electron microscopy (TEM). These nanofilms, composed of a biodegradable polyester and remotely controllable by external magnetic fields, are promising candidates for many potential applications in the biomedical field.

Eukaryotic cells respond to signaling molecules with picomolar to nanomolar sensitivities. However, molar concentrations give no suggestion of the sufficient number of molecules per cell and are confusing when referring to physiological situations in which signaling molecules act in an immobilized state. Here, we studied platelet adhesion by thrombin, a key step in normal hemostasis and pathological arterial thrombosis. We generated a biofunctional nanosheet surface to mimic the in vivo solid-state interaction between platelets and thrombin at sites of injured tissues. We observed that <10 molecules readily activate platelets with high specificity, resulting in platelet adhesion and spreading. This number is much lower than expected from previous experiments in solution, in which the sole activation of platelets required a >1000-fold stoichiometric excess of thrombin. We conclude that immobilizing thrombin apposed to the membrane receptor allows platelets to respond with very high sensitivity. Moreover, we propose that irreversible cell activation may require several ligands to avoid activation by single, mislocalized signaling molecules.

Increased fluid viscosity of a solution of hemoglobin-based oxygen carriers (HBOCs) reduces vasoconstrictive effects because increased shear stress on the vascular wall enhances the production of vasorelaxation factors such as NO. Nevertheless, on a microcirculatory level, it remains unclear how viscosity affects the reaction of HBOCs and NO. In this study, different HBOCs were perfused through narrow gas-permeable tubes (25 mu m inner diameter at 1 mm/s centerline velocity; hemoglobin concentration [Hb] = 5 g/dL). The reaction was examined microscopically based on the Hb visible-light absorption spectrum. When immersed in a NO atmosphere, the NO-binding of deoxygenated Hb solution (viscosity, 1.1 cP at 1000 s(-1)) in the tube occurred about twice as rapidly as that of red blood cells (RBCs): 1.6 cP. Binding was reduced by PEGylation (PEG-Hb, 7.7 cP), by addition of a high molecular weight hydroxyethyl starch (HES) (2.8 cP), and by encapsulation to form Hb-vesicles (HbVs, 1.5 cP; particle size 279 nm). However, the reduction was not as great as that shown for RBCs. A mixture of HbVs and HES (6.2 cP) showed almost identical NO-binding to that of RBCs. Higher viscosity and particle size might reduce lateral diffusion when particles are flowing. The HbVs with HES showed the slowest NO-binding. Furthermore, Hb encapsulation and PEGylation, but not HES-addition, tended to retard CO-binding. Increased viscosity reportedly enhances production of endothelium NO. In addition, our results show that the increased viscosity also inhibits the reaction with NO. Each effect might mitigate vasoconstriction. (C) 2010 Elsevier Inc. All rights reserved.

A specific protein fluorescent labeling method has been used as a tool for bio-imaging in living cells. We developed a novel system of switching "fluorescent turn on" by the recognition of a fluorescent probe to a hexahistidine-tagged (His-tag) protein. The tetramethyl rhodamine bearing three nitrilotriacetic acids, which was used as a fluorescent probe to target a His-tagged protein, formed a reversible complex with the quencher, (Dabcyl)-conjugated oligohistidines, in the homogeneous solution, causing fluorescence of the fluorophore to be quenched. The complex when applied to living cells (COS-7) expressing His-tagged proteins on the cell surface caused the quencher-conjugated oligohistidines to be dissociated from the complex by specific binding of the fluorescent probe to the tagged protein, resulting in the fluorescent emission. The complex that did not participate in the binding event remained in the quenched state to maintain a low level of background fluorescence. (C) 2010 Elsevier Inc. All rights reserved.

A convenient method for surface modification is crucially important for the preparation of a functional interface. The precise control of surface properties by chemical and physical approaches is expected to enhance the potential application of innovative nanomaterials. In this study, we propose a methodology for convenient surface modification using a freestanding anti-biofouling ultra-thin film (nanosheet) bearing poly(2-methacryloyloxyethyl phosphorylcholine: MPC) (i.e., pMPC-nanosheets). The physicochemical properties of the pMPC-nanosheet such as physiological stability, surface wettability and anti-biofouling were precisely controlled by means of thermal crosslinking of the nanosheet, the grafting amount of MPC and tuning the thickness of pMPC brushes. These approaches revealed physicochemical insight into the pMPC-nanosheet and its remarkable biological response. The pMPC-nanosheet was prepared via a spin-coating assisted layer-by-layer method in combination with atom transfer radical polymerization of MPC. The freestanding pMPC-nanosheet, comprising an 11 nm-thick pMPC brushed layer with a hydrophilic surface, was easily transferred by tweezers, cut by scissors, patterned by a needle and patched onto various interfaces with the aid of a water-soluble supporting film. The pMPC-nanosheets patched on cell culture substrates displayed anti-biofouling properties such as anti-coagulant activity against human whole blood as well as the potential to microscopically pattern murine fibroblast cells. The present study not only reveals physicochemical properties of the surface-functionalized nanosheet for biological application, but also constitutes an alternative approach to conventional lithographic techniques using photoresists and micro-patterned molds; thereby providing a new tool in the field of nanobiotechnology.

Free-standing conductive ultra-thin films based on poly(3,4-ethylenedioxythiophene)/poly (styrenesulfonate) (PEDOT/PSS) are realized. A fabrication process based on a modified Supporting Layer technique is proposed that provides for the easy production of conductive nanofilms having a very large surface area with typical thickness of tens of nanometres. The proposed free-standing nanofilms can be manipulated, folded and unfolded in water many times without suffering from cracks, disaggregation or from loss of conductive properties. After collecting them onto rigid or soft substrates, they retain their functionality. Structural and functional properties of the nanofilms are described by means of their thickness, topography, conductivity and Young's modulus. Strong dependences of these properties on residual water, post-deposition treatments and environmental moisture are clearly evidenced. Possible applications are foreseen in the field of sensing and actuation, as well as in the biomedical field, e. g. as smart substrates for cell culturing and stimulation.

We have constructed phospholipid vesicles with hemostatic activity as a platelet substitute. The vesicles were conjugated with a dodecapeptide (HHLGGAKQAGDV, H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411). We have recently exploited these vesicles as a potential drug delivery system by encapsulation of adenosine 5'-diphosphate (ADP) (H12-(ADP)-vesicles).
Here we explore the relationship between the ADP release from H12-(ADP)-vesicles with different membrane properties and their hemostatic effects. In total, we prepared five kinds of H12-(ADP)-vesicles with different lamellarities and membrane flexibilities. By radioisotope-labeling, we directly show that H12-(ADP)-vesicles were capable of augmenting platelet aggregation by releasing ADP in an aggregation-dependent manner. The amount of ADP released from the vesicles was dependent on their membrane properties. Specifically, the amount of ADP released increased with decreasing lamellarity and tended to increase with increasing membrane flexibility. Our in vivo results clearly demonstrated that H12-(ADP)-vesicles with the ability to release ADP exert considerable hemostatic action in terms of correcting prolonged bleeding time in a busulphan-induced thrombocytopenic rat model.
We propose a recipe to control the hemostatic abilities of H12-(ADP)-vesicles by modulating ADP release based on membrane properties. We believe that this concept will be invaluable to the development of platelet substitutes and other drug carriers. (C) 2010 Elsevier B.V. All rights reserved.

Hexahistidine ((His)(6)) tags are used to purify genetically engineered proteins. Herein, we describe the construction of a 'turn-on' fluorescent probe system that consists of the fluorescence quencher, Dabcyl, conjugated to (His)(6), and fluorescent tetramethylrhodamine conjugated to nitrilotriacetic acid, which, in the presence of Ni2+, can bind (His)(6). The system is turned off when Dabcyl-(His)(6) is bound to the fluorescent nitrilotriacetic acid derivative. The binding strength of this system was assessed using electrospray ionization mass spectrometry, fluorescence correlation spectroscopy, and fluorescence intensity distribution analysis-polarization. Although there was no significant enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence increased from 14% to 40% of its initial intensity when an equimolar amount of (His)(6)-ubiquitin was added. Therefore, this system should be able to specifically recognize (His)(6)-proteins with good resolution and has the additional advantage that a washing step is not required to remove fluorescent probe, that is, not bound to the (His)(6)-protein. (C) 2010 Elsevier Ltd. All rights reserved.

An increasing interest in bio-hybrid systems and cell-material interactions is evident in the last years. This leads towards the development of new nano-structured devices and the assessment of their biocompatibility. In the present study, the development of free-standing single layer poly(lactic acid) (PLA) ultra-thin films is described, together with the analysis of topography and roughness properties. The biocompatibility of the PLA films has been tested in vitro, by seeding C2C12 skeletal muscle cells, and thus assessing cells shape, density and viability after 24, 48 and 72 h. The results show that free-standing flexible PLA nanofilms represent a good matrix for C2C12 cells adhesion, spreading and proliferation. Early differentiation into myotubes is also allowed. The biocompatibility of the novel ultra-thin films as substrates for cell growth promotes their application in the fields of regenerative medicine, muscle tissue engineering, drug delivery, and-in general-in the field of bio-hybrid devices.

An efficient procedure through deprotection of 2,4,6-trimethoxypyrimidine derivative afforded porphyrinato zinc bearing multi-barbiturates acting as multiple hydrogen bonding sites at the meso positions. Addition of excess amphiphilic triaminopyrimidine derivative, as a complementary motif to the barbiturate, in an aqueous solution of porphyrin conjugated with multiple barbiturates at the meso positions resulted in precipitation. The cast film from the chloroform solution of the precipitate indicated the formation of a well-defined porphyrin assembly. (c) 2010 Elsevier Ltd. All rights reserved.

An ultra-thin polymer film (nanosheet) composed of polysaccharides (i.e., polysaccharide nanosheet) provides sufficient adhesiveness, flexibility and robustness to act as an effective wound dressing. We have recently demonstrated the sealing effect of a nanosheet on a murine cecal puncture. Nevertheless, a small percentage of bacteria penetrated the nanosheet because of its ultra-thin structure. Here, we have developed an antibiotic-loaded nanosheet to inhibit bacterial penetration and investigated its therapeutic efficacy using a model of a murine cecal puncture. Tetracycline (TC) was sandwiched between a poly(vinylacetate) (PVAc) layer and the polysaccharide nanosheet (named PVAc-TC-nanosheet). Under physiological conditions, TC was released from the nanosheet for 6 h. Microscopic observation between the interface of the PVAc-TC-nanosheet and bacteria demonstrated how its potent anti-microbial effect was achieved. In vivo studies show that overlapping therapy with the PVAc-TC-nanosheet (thickness: 177 nm) significantly increases mouse survival rate after cecal puncture as well as suppressing an increase in the intraperitoneal bacterial count and leukocyte count. (C) 2010 Elsevier Ltd. All rights reserved.

Background. Recent developments in nanobiotechnology have led us to develop a method of producing a free-standing polymer nanosheet composed of polysaccharides (ie, polysaccharide nanosheet) with a thickness of tens of nanometers. Owing to its enormous aspect ratio, the polysaccharide nanosheet is semi-absorbent and has a physical adhesive strength 7 5-fold greater than that of conventional films of > 1 mu m thickness. Herein, we have investigated the therapeutic sealing effect of this polysaccharide nanosheet on murine cecal puncture as a wound dressing material.
Methods. Murine cecum was punctured and then overlapped with the polysaccharide nanosheet Thereafter, we evaluated its sealing effect on bacterial peritonitis as well as the protection offered by the polysaccharide nanosheet against bacterial permeability using an in vitro transmembrane assay
Results. The 39-nm-thick polysaccharide nanosheet overlapped tightly the perforated recant. No adhering agents were required because of the ability of the polysaccharide nanosheet to adhere to the tissue surface by physical adsorption (eg, van der Waals interaction) Sealing the perforated cecum with the polysaccharide nanosheet increased survival rate without postoperative adhesion by comparison with untreated mice (90 vs 30%, P < .01) These data were supported by the improvement in peritonitis related to bacterial counts, white blood cell counts, and the serum tumor necrosis factor level. Moreover, using an. in vitro transmembrane assay, we showed that the polysaccharide nanosheet inhibited effectively bacterial penetration.
Conclusion. We have demonstrated the potential clinical benefits of the nanosheet-type biomaterial that can be used for repairing a cecal colotomy without chemical bonding agents (Surgery 2010;148 48-58)

We have constructed liposomes with hemostatic activity as a platelet substitute using moderately thrombocytopenic rats. The liposomes were conjugated with the dodecapeptide (HHLGGAKQAGDV: H12), which is a fibrinogen gamma-chain C-terminal sequence (gamma 400-411). To visualize liposome accumulation at the site of vascular injury by in vivo computed tomography, a water-soluble contrast dye, N,N'-bis[2-hydroxy-1-(hydroxylmethyl)ethyl]-5-[(2S)-2-hydroxylpropanoylamino]-2,4,6-triiodoisophthalamide (iopamidol), was encapsulated into the H12-conjugated liposomes. We achieved direct visualization of specific accumulation of the H12(iopamidol)liposomes at the jugular vein injured by ferric chloride and succeeded in semiquantitative analyses of the accumulated amount of H12-liposomes in the injured site. We therefore propose that H12-liposomes that are specifically recruited to, and exert their hemostatic activity at the site of vascular injury, have a significant potential as a carrier and/or as an ideal platelet substitute. Furthermore, the H12-(iopamidol)liposomes would also be clinically useful as diagnostic agents for pathological thrombus detection and as contrast dyes for hepatosplenography.
From the Clinical Editor: The authors have constructed liposomes with hemostatic activity as a platelet substitute using moderately thrombocytopenic rats. They propose that H12-liposomes that are specifically recruited to, and exert their hemostatic activity at the site of vascular injury, have a significant potential as a carrier and/or as an ideal platelet substitute. Furthermore, the H12-(iopamidol)liposomes would also be clinically useful as diagnostic agents for thrombus detection and as contrast dyes for hepatosplenography. (C) 2010 Elsevier Inc. All rights reserved.

Sakai H, Okuda N, Sato A, Yamaue T, Takeoka S, Tsuchida E. Hemoglobin encapsulation in vesicles retards NO and CO binding and O-2 release when perfused through narrow gas-permeable tubes. Am J Physiol Heart Circ Physiol 298: H956-H965, 2010. First published December 31, 2009; doi:10.1152/ajpheart.00741.2009.-Intravenous administration of cell-free Hb induces vasoconstriction and circulatory disorders, presumably because of the intrinsic affinities to endogenous nitric oxide (NO) and carbon monoxide (CO) as vasorelaxation factors and because of the facilitated O-2 release that might induce autoregulatory vasoconstriction. We examined these gas reactions when Hb-containing solutions of four kinds were perfused through artificial narrow tubes at a practical Hb concentration (10 g/dl). Purified Hb solution, polymerized bovine Hb (Poly(B)Hb), encapsulated Hb [Hb-vesicles (HbV), 279 nm], and red blood cells (RBCs) were perfused through a gas-permeable narrow tube (25 mu m inner diameter) at 1 mm/s centerline velocity. The level of reactions was determined microscopically based on the visible-light absorption spectrum of Hb. When the tube was immersed in NO and CO atmospheres, both NO binding and CO binding of deoxygenated Hb (deoxy-Hb) and Poly(B)Hb in the tube was faster than those of HbV and RBCs, and HbV and RBCs showed almost identical binding rates. When the tube was immersed in a N-2 atmosphere, oxygenated Hb and Poly(B)Hb showed much faster O-2 release than did HbV and RBCs. Poly(B)Hb showed a faster reaction than Hb because of the lower O-2 affinity of Poly(B)Hb than Hb. The diffusion process of the particles was simulated using Navier-Stokes and Maxwell-Stefan equations. Results clarified that small Hb (6 nm) diffuses laterally and mixes rapidly. However, the large-dimension HbV shows no such rapid diffusion. The purely physicochemical differences in diffusivity of the particles and the resulting reactivity with gas molecules are one factor inducing biological vasoconstriction of Hb-based oxygen carriers.

We developed pH-responsive liposomes containing synthetic glutamic acid-based zwitterionic lipids and evaluated their properties both in vitro and in vivo with the aim of constructing an efficient liposome-based systemic drug delivery system. The glutamic acid-based lipids; 1,5-dihexadecyl N-glutamyl-L-glutamate (L1) and 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (L2) were synthesized as a pH-responsive component of liposomes that respond to endosomal pH. The zeta potential of liposomes containing L1 or L2 was positive when the solution pH was below 4.6 or 5.6, respectively, but negative at higher pH values. The pH-responsive liposomes showed improved fusogenic potential to an endosome-mimicking anionic membrane at acidic pH, where the zeta potential of the liposomes was positive. We then prepared doxorubicin (DOX)-encapsulating liposomes containing L1 or 12, and clarified by confocal microscopic studies that the contents were rapidly transferred into both the cytoplasm and nucleus. Release of DOX from the endosomes mediated by the pH-responsive liposomes dramatically inhibited cancer cell growth. The L2-liposomes were slightly more effective than L1-liposomes as a drug delivery system. Intravenously injected L2-liposomes displayed blood persistence comparable to that of conventional phospholipid (PC)-based liposomes. Indeed, the antitumor efficacy of L2-liposomes was higher than that of PC-based liposomes against a xenograft breast cancer tumor in vivo. Thus, the high performance of L2-liposomes results from both efficient intracellular drug delivery and comparable blood persistence in comparison with the conventional PC-based liposomes in vitro and in vivo. (C) 2009 Elsevier B.V. All rights reserved.

We investigated the ability of cationic liposomes composed of 1,5-dihexadecyl N-arginyl-L-glutamate (Arg-Glu2C(16)) to carry nucleic acids into neuronal cells. Such liposomes have been shown to have a remarkable capacity for transfecting immortalized cell lines. Lipoplexes between the Arg-Glu2C(16) liposomes and plasmid DNA encoding green fluorescent protein (GFP) were analyzed in terms of lipoplex formation, intracellular DNA trafficking, transfection efficiency, and cytotoxicity in neuronal SH-SY5Y cells. A maximum number of cells expressing GFP was obtained with lipoplexes at a lipid-to-DNA ratio of 15. With these lipoplexes, 16% of the cells were GFP- positive, which was approximately fourfold higher than the level obtained with a commercially available transfection reagent, Lipofectamine 2000. Furthermore, as a result of the low cytotoxicity of the Arg-Glu2C(16) lipoplexes, the proportion of GFP- positive cells could be increased to 25% by increasing the concentration of lipoplexes that was applied to the cells. We have demonstrated that Arg-Glu2C(16), as a model cationic amino acid-based lipid, has a high capability as a gene carrier, even for neuronal transfection.
From the Clinical Editor: In this study, specific cationic liposomes were characterized as nucleic acid transfection agents for neuronal cells. A fourfold higher transfection rate with low cytotoxicity was reported compared to Lipofectamine 2000, a commercial reagent. The authors conclude that the studied cationic liposomes have a high capability as a gene carrier for neuronal transfection. This may become clinically significant in future gene therapy efforts of neuronal diseases. (C) 2010 Elsevier Inc. All rights reserved.

We report an artificially fabricated extracellular matrix (ECM)-like nanostructure, which utilizes self-assembly of collagen and hyaluronic acid in a quasi two-dimensional space of tens-of-nm thickness, displaying different cellular adhesive characteristics depending on the structural properties between the fibril and non-fibril elements. This nano-fibrous assembly will facilitate a mechanobiological approach for the development of tissue-engineering scaffolds.

A free-standing biodegradable nanosheet composed of poly(L-lactic acid) (PLLA) was shown to have excellent sealing efficacy for a gastric incision as a novel wound dressing material that did not require adhesive agents, and the PLLA nanosheet-induced wound repair showed neither scars nor tissue adhesion. This material may, therefore, be an ideal alternative to conventional tissue repairing procedures using suture/ligation in surgery.

A free-standing biodegradable nanosheet composed of poly(L-lactic acid) (PLLA) was shown to have excellent sealing efficacy for a gastric incision as a novel wound dressing material that did not require adhesive agents, and the PLLA nanosheet-induced wound repair showed neither scars nor tissue adhesion. This material may, therefore, be an ideal alternative to conventional tissue repairing procedures using suture/ligation in surgery.

We have studied biocompatible spherical carriers carrying a dodecapeptide, HHLGGAKQAGDV (H12), on their surface as platelet substitutes. This peptide is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411) and specifically recognizes the active form of glycoprotein IIb/IIIa on activated platelets. Our purpose is to assess the possibility of making a novel platelet substitute consisting of disk-shaped nanosheets having a large contact area for the targeting site, rather than conventional small contact area spherical carriers. ne H12 peptide was conjugated to the surface of the free-standing nanosheets made of biodegradable poly(D,L-lactide-co-glycolide) (PLGA). These H12-PLGA nanosheets were fabricated onto 3 mu m disk-shaped patterned hydrophobic octadecyl regions on a SiO(2) substrate. By way of comparison, spherical H12-PLGA microparticles with the same surface area and conjugation number of H12 were also prepared. The resulting H12-PLGA nanosheets specifically interacted with the activated platelets adhered on the collagen surface at twice the rate of the H12-PLGA microparticles under flow conditions, and showed platelet thrombus formation in a two-dimensional spreading manner. Thus, H12-PLGA nanosheets might be a suitable candidate novel platelet alternative substitute for infused human platelet concentrates for the treatment of bleeding in patients with severe thrombocytopenia.

We have studied biocompatible spherical carriers carrying a dodecapeptide, HHLGGAKQAGDV (H12), on their surface as platelet substitutes. This peptide is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411) and specifically recognizes the active form of glycoprotein IIb/IIIa on activated platelets. Our purpose is to assess the possibility of making a novel platelet substitute consisting of disk-shaped nanosheets having a large contact area for the targeting site, rather than conventional small contact area spherical carriers. ne H12 peptide was conjugated to the surface of the free-standing nanosheets made of biodegradable poly(D,L-lactide-co-glycolide) (PLGA). These H12-PLGA nanosheets were fabricated onto 3 mu m disk-shaped patterned hydrophobic octadecyl regions on a SiO(2) substrate. By way of comparison, spherical H12-PLGA microparticles with the same surface area and conjugation number of H12 were also prepared. The resulting H12-PLGA nanosheets specifically interacted with the activated platelets adhered on the collagen surface at twice the rate of the H12-PLGA microparticles under flow conditions, and showed platelet thrombus formation in a two-dimensional spreading manner. Thus, H12-PLGA nanosheets might be a suitable candidate novel platelet alternative substitute for infused human platelet concentrates for the treatment of bleeding in patients with severe thrombocytopenia.

Recent developments in nanotechnology have led to a method for producing free-standing polymer nanosheets as a macromolecular organization. Compared with bulk films, the large aspect ratio of such nanosheets leads to unique physical properties, such as transparency, noncovalent adhesion, and high flexibility. Here, a biomedical application of polymer nanosheets consisting of biocompatible and biodegradable polysaccharides is reported. Micro-scratch and bulge tests indicate that the nanosheets with a thickness of tens of nanometers have sufficient physical adhesiveness and mechanical strength for clinical use. A nanosheet of 75 nm thickness, a critical load of 9.1 x 10(4) N m(-1), and an elastic modulus of 9.6 GPa is used for the minimally invasive repair of a visceral pleural defect in beagle dogs without any pleural adhesion caused by wound repair. For the first time, clinical benefits of sheet-type nano-biomaterials based on molecular organization are demonstrated, suggesting that novel therapeutic tools for overlapping tissue wounds will be possible without the need for conventional surgical interventions.

Polyethylene glycol-modified vesicles (liposomes) encapsulating hemoglobin (HbV) are artificial oxygen carriers that have been developed as a transfusion alternative. The HbV suspension in an albumin solution is nearly Newtonian; other biopolymers, hydroxyethyl starch (HES), dextran (DEX), and modified fluid gelatin, induce flocculation of HbVs through depletion interaction and render the suspensions as non-Newtonian. The flocculation level increased with hydrodynamic radius (R(h)) or radius of gyration (R(g)) of series of HES or DEX with different molecular weights at a constant polymer concentration (4 wt %). However, the flocculation level differed markedly among the polymers. A crowding index (C(i)) representing the crowding level of a polymer solution is defined as (excluded volume of one polymer) x (molar concentration) x Avogadro's number, using R(h) or R(g). All polymers' flocculation level increases when C(i) approaches 1: when the theoretical total excluded Volumes approach the entire solution Volume, the excluded HbV particles are forced to flocculate.

Freestanding quasi-two-dimensional ultrathin films (e,g., 41 nm thick polymer nanosheets) were produced. on which stimuli-responsive 47 nm chick polymer brushes were constructed by atom transfer radical polymerization (ATRP) of poly(N-isopropylacrylamide). The resulting surfaces of the multilayered polysaccharide ultrathin films were evaluated by ellipsometry, IR imaging, In situ variable-temperature atomic force microscopy (AFM), and contact angle measurements. The morphological transformation of the freestanding polymer nanosheet bearing thermoresponsive polymer brushes was observed macroscopically through reversible structural color changes at the air-water interface. The dynamic shape change of the nanosheetwas also monitored with the addition of a surfactant such as sodium n-dodecylsulfate to reduce the hydrophobicity of the surface, It was then demonstrated chat the highly flexible freestanding polymer nanosheet is capable of acting as a unique platform for inducing stimuli-responsive I behavior in nanomaterials.

We have synthesized a series of cationic amino acid-based lipids having a spacer between the cationic head group and hydrophobic moieties and examined the influence of the spacer on a liposome gene delivery system. As a comparable spacer, a hydrophobic spacer with a hydrocarbon chain composed of 0, 3, 5, 7, or 11 carbons, and a hydrophilic spacer with an oxyethylene chain (10 carbon and 3 oxygen molecules) were investigated. Plasmid DNA (pDNA)-encapsulating liposomes were prepared by mixing an ethanol solution of the lipids with an aqueous solution of pDNA. The zeta potentials and cellular uptake efficiency of the cationic liposomes containing each synthetic lipid were almost equivalent. However, the cationic lipids with the hydrophobic spacer were subject to fuse with biomembrane-mimicking liposomes. 1,5-Dihexadecyl-N-lysyl-N-heptyl-L-glutamate, having a seven carbon atom spacer, exhibited the highest fusogenic potential among the synthetic lipids. Increased fusion potential correlated with enhanced gene expression efficiency. By contrast, an oxyethylene chain spacer showed low gene expression efficiency. We conclude that a hydrophobic spacer between the cationic head group and hydrophobic moieties is a key component for improving pDNA delivery. (C) 2009 Elsevier B.V. All rights reserved.

Hemoglobin vesicles (HbVs), liposomal oxygen carriers containing human hemoglobin, are candidates for development of a clinically useful transfusion alternative. Our previous in vivo animal studies of massive HbV dosages demonstrated the lack of any suppressive effect on hematopoiesis. Before starting clinical trials, we aimed to examine the details of the biocompatibility of HbVs with human hematopoietic stem/progenitor cells. We investigated the effect of HbVs at a concentration of up to 3 vol/vol (%) on expansion of human umbilical cord (CB) hernatopoietic stem/progenitor cells, using a coculture system of human TERT-transfected bone marrow stromal cells and CD34(+) cells in vitro. The exposure of CB CD34(+) cells to HbVs up to 14 days suppressed the expansion of total cells and the CD34(+) cells themselves, whereas the empty liposomes, that did not contain Hb, had modestly inhibitory effects on the expansion of these cells. As a result, the number of colonies obtained from the expanded CD34(+) cells was inhibited by the exposure to HbVs. In contrast, exposure to HbVs for 3 days had no effect on the expansion of CD34(+) cells and only slightly decreased the number of total cells. Our in vitro experimental condition does not fully recreate the physiological condition, and the effect of the direct contact of HbV would be magnified because of the absence of shielding by the vasculature and the lack of the reticuloendothelial system and blood stream. However, the present data raise some concern regarding hematopoiesis, and one has to pay attention to this in future human clinical trials. ASAIO Journal 2009; 55:200-205.

Sheet-shaped carriers, having both obverse and reverse surfaces and thus a large contact area for targeting a site, have several advantages over spherical-shaped carriers, which have an extremely small contact area for targeting sites. Here, we proposed a novel method to prepare a free-standing ultrathin and biocompatible nanosheet having heterosurfaces, by a combination of four processes: (1) specific adsorption of recombinant human serum albumin (rHSA) molecules onto a patterned octadecyltrimethoxysilane self-assembled monolayer region (ODS-SAM), (2) preparation of nanosheets of rHSA molecules bearing thiol groups (SH-rHSA) via two-dimensionally disulfide crosslinking, (3) surface modification of the resulting nanosheet, and. (4) preparation of the free-standing nanosheet by detachment from the ODS-SAM. The SH-rHSA molecules at pH 5.0 and a concentration of 1 mu g/ml, were specifically adsorbed on the patterned ODS-SAM regions by hydrophobic interaction, and were two-dimensionally crosslinked in the presence of copper ion as an oxidant. The rHSA-nanosheets were then simply detached from the ODS-SAM by treatment with surfactant. We succeeded in the preparation of rectangular (10 mu m X 30 mu m) and ultrathin (4.5 +/- 1.0 nm) rHSA-nanosheets on a patterned ODS-SAM, and could also obtain free-standing rHSA-nanosheets having heterosurfaces by Surface modification with fluorescent latex beads. Thus, the rHSA-nanosheets having heterosurfaces could be regarded as a new biomaterial for drug carriers, hemostatic reagents, wound dressing for burn injury, and so forth. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 89A: 233-241, 2009

Background: The dodecapeptide HHLGGAKQAGDV (H12), corresponding to the fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411), is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated H12-coated nanoparticles (polymerized albumin or liposome) as platelet function-supporting synthetic products. Objectives: To strengthen the hemostatic ability of H12-coated particles as a platelet substitute, we exploited installation of a drug delivery function by encapsulating adenosine diphosphate (ADP) into liposomes [H12-(ADP)-liposomes]. Methods and results: Via selective interaction with activated platelets through GPIIb/IIIa, H12-(ADP)-liposomes were capable of augmenting agonist-induced platelet aggregation by releasing ADP in an aggregation-dependent manner. When intravenously injected into rats, liposomes were readily targeted to sites of vascular injury as analyzed on computed tomography. In fact, comparable to fresh platelets, liposomes exhibited considerable hemostatic ability for correcting prolonged bleeding time in a busulphan-induced thrombocytopenic rabbit model. In addition, the liposomes showed no activating or aggregating effects on circulating platelets in normal rabbits. Conclusion: H12-(ADP)-liposome may thus offer a promising platelet substitute, being made with only synthetic materials and exerting hemostatic functions in vivo via reinforcement of primary thrombus formation by residual platelets in thrombocytopenia at sites of vascular injury, but not in circulation.

Whereas several biomedical applications of carbon nanotubes have been proposed, the use of boron nitride nanotubes (BNNTs) in this field has been largely unexplored despite their unique and potentially useful properties. Our group has recently initiated an experimental program aimed at the exploration of the interactions between BNNTs and living cells. In the present paper, we report on the magnetic properties of BNNTs containing Fe catalysts which confirm the feasibility for their use as nanovectors for targeted drug delivery. The magnetisation curves of BNNTs characterised by the present study are typical of superparamagnetic materials with important parameters, including magnetic permeability and magnetic momentum, derived by employing Langevin theory. In-vitro tests have demonstrated the feasibility for influencing the uptake of BNNTs by living cells by exposure to an external magnetic source. A finite element method analysis devised to predict this effect produced predictive data with close agreement with the experimental observations.

Mass-produced carbon nanotubes (CNTs) are strongly aggregated and highly hydrophobic, and processes to make them water soluble are required for biological applications. Both covalent and non-covalent strategies are pursued for obtaining stable, highly concentrated CNT aqueous dispersions. Covalent functionalization has the great disadvantage of producing an irreversible chemical modification of nanotubes, thus alterating their mechanical, chemical and electric properties. On the other hand, non-covalent functionalization is often obtained by employing surfactants that sensibly affect cell viability. Moreover, derivatization with biological moieties is often impossible through non-covalent CNT dispersion. This paper proposes a non-covalent dispersion of multi-wall CNT based on a lipidic mixture that can guarantee high concentration and high stability as well as high cytocompatibility. Moreover, CNTs wrapped with a lipid membrane are realized to demonstrate that the proposed CNTs can be functionalised with a dodecapeptide that specifically recognizes activated platelets without chemical modification of the nanotube itself.

The chemical and physical modification of a material surface is important for practical applications. In this study, we utilized the bilateral nature of a free-standing polysaccharide nanosheet as a platform for surface modification, selectively modifying the upper and lower surfaces with different sizes of latex beads. By means of a water-soluble sacrificial layer, we adsorbed latex beads with the diameter of 200 rim onto the obverse surface of the polysaccharide nanosheet as well as latex beads with that of 2 mu m onto the reverse side. The optical characteristics of the nanosheet were analyzed on the basis of 'thin film interference theory' by making stepwise increments in thickness through sequentially overlaying free-standing 30nm polysaccharide nanosheets onto a SiO(2) substrate. The surface of the polysaccharide nanosheet was quite flat and smooth. Having confirmed by optical and scanning electron microscopy that we had achieved selective surface modification of the polysaccharide nanosheet with micro/nano-latex beads, we noted a unique optical property when the nanosheet was adsorbed on the SiO(2) substrate. The localization of the beads on the nanosheet surface produced structural colors in the films due to an optical interference. This selective surface modification technique will be utilized to create 'smart' nanocomposites possessing different surface functions. (C) 2008 Elsevier B.V. All rights reserved.

Polyethylene glycol (PEG)-modified hemoglobin (Hb) vesicles (HbVs) are artificial oxygen carriers encapsulating a concentrated Hb solution in phospholipid vesicles. In our previous studies, HbV showed a sufficient resuscitative effect comparable to that of red blood cells in hemorrhagic shock animal models during several hours' observation. However, the profiles of the recovery, including hematopoiesis and elimination of HbV, remain unknown. This study conducted 14-day observations of Wistar rats after hemorrhagic shock and fluid resuscitation with HbV suspended in recombinant human serum albumin. Shock was induced by 50% blood withdrawal from a femoral artery. The rats showed hypotension, metabolic acidosis, and hyperventilation. After 15 min, they received HbV or shed autologous blood through a femoral vein. Both groups showed rapid recovery of hemodynamic and blood gas parameters. No meaningful difference was found between groups. After decannulation and awakening, the rats were housed in cages. The reduced hematocrit of the HbV group returned to the original level in 7 days. Plasma enzyme levels were slightly higher in both groups at 1 day because of systemic reperfusion injury. Splenomegaly was considerable in the HbV group because of the HbV accumulation and extramedullar hematopoiesis, but it subsided within 14 days. Along with the HbV elimination in the spleen and liver, immunohistochemistry with anti-PEG antibody revealed that PEG-conjugated lipid had disappeared within 14 days. In conclusion, HbV showed a sufficient resuscitative effect comparable to that of red blood cell transfusion. Phagocytized HbV disappeared within 14 days. Elevated hematopoiesis contributed to complete hematocrit recovery within 7 days.

Polymerized albumin particles (poly Alb) with recombinant glycoprotein Ibα (rGPIbα-poly Alb) are a promising candidate for a platelet substitute. Thus, we focused on the lateral motion of poly Alb in the presence of red blood cells, because the lateral motion plays an important role in aggregate formation. We visualized the microscopic motion of poly Alb toward the immobilized ligand (von Willebrand factor, VWF) surface in a model arteriole with red blood cells with a high-speed camera. At a higher shear rate of 1,500 s-1, the concentration profile of poly Alb appeared to peak near the wall. This profile enhances the interaction between the particles and wall. Particularly the migration angle, being the angle of the poly Alb velocity vector, was enlarged near the wall and contributed to transfer of poly Alb toward the immobilized VWF surface. This tendency is desirable to achieve the adhesion of particles on the wall. © Japanese Society of Biorheology 2009.

Polymeric ultra-thin films, so called nanosheets, show peculiar properties making them potentially useful for several applications in biomedicine. Moreover, the possibility to functionalize these films by using different agents opens new and partially unexplored scenarios, including micro/nano sensing and actuation. This paper compares two different methods for the preparation of free-standing nanosheets, loaded with magnetic particles for no-contact manipulation in liquid environment. Morphology and functionalities of the two types of nanosheets have been characterized and compared. These magnetic nanosheets could find applications as free-standing carriers to be released and controlled in endoluminal surgery or as plasters with nanometric thickness to be delivered in situ on surgical incisions.

Platelet glycoprotein GPIaIIa is an adhesive protein that recognizes collagen. We have investigated polymerized albumin particles conjugated with recombinant GPIaIIa (rGPIaIIa-poly Alb) for their platelet-like function. To evaluate the feasibility of these particles to achieve the hemostatic process, we measured the deformability (Young's modulus and spring constant) and the adhesive force of the particles using atomic force microscopy, which can measure the mechanical properties of individual cells. Our results showed that the Young's modulus of these particles was 2.3-fold larger than that of natural platelets and 12-fold larger than that of human red blood cells. The Young's modulus of the particles may have been determined by the properties of the polymerized albumin particle, although the glycoprotein of the platelet surface also contributed to the higher modulus. Our results also showed that the adhesive force of the rGPIaIIa-poly Alb with the collagen ligand was 52% of that of natural platelets. These two mechanical properties (deformability and adhesive force) of cells or particles, such as rGPIaIIa-poly Alb, are important specifications for the optimum design of platelet substitutes. © Japanese Society of Biorheology 2009.

Sheet-shaped carriers having both obverse and reverse surfaces (thus, a large contact area for targeting a site and adhesive properties without any chemical cross-linker onto tissue surface) have several advantages as surgical dressings. These advantages include active targeting over spherically shaped carriers, which thus have an extremely small contact area for targeting sites. Here, we propose a novel methodology for preparation of a freestanding, ultra-thin, and biocompatible polymer nanosheet having heterosurfaces, fabricated through macromolecular assembly. In the context of biomedical applications, the targeted properties include injectable sheet-shaped drug carriers having precisely controlled size by exploiting micropatterned substrate, and giant polymer nanosheets composed of biocompatible polysaccharides. A huge aspect ratio, in excess of 10(6), is particularly applicable for novel surgical dressings. These biocompatible polymer nanosheets having heterosurfaces can thus be regarded as new biomaterials for minimally invasive treatment.

Hemoglobin vesicles (HbVs) are artificial oxygen carriers encapsulating purified and concentrated Hb solution in phospholipid vesicles (liposomes). We examined in-vitro reaction profiles of a formulation of HbV with NO and CO in anaerobic and aerobic conditions using stopped-flow spectrophotometry and a NO electrode. Reaction rate constants of NO to deoxygenated and oxygenated HbV were considerably smaller than those of cell-free Hb because of the intracellular NO-diffusion barrier. The reaction of CO with deoxygenated HbV was slightly slower than that of cell-free Hb solely because of the co-encapsulated allosteric effector, pyridoxal 5'-phosphate. The NO depletion in an aerobic condition in the presence of empty vesicles was monitored using a NO electrode, showing that the hydrophobic bilayer membrane of HbV, which might have higher gas solubility, does not markedly facilitate the O-2 and NO reaction, and that the intracellular Hb is the major component of NO depletion. In conclusion, HbV shows retarded gas reactions, providing some useful information to explain the absence of vasoconstriction and hypertension when they are intravenously injected. (c) 2008 Elsevier B.V. All rights reserved.

A hydrogen-bonded helical columnar liquid crystal was synthesized, in which the helical structure is induced by a centered triphenylene derivative bearing chiral side-chains. The triphenylene derivative, 2,6,10-tris(carboxymethoxy)-3,7,11-tris((S)-(-)-2-methyl-1-butanoxy)triphenylene (TPC4(S)), and a dendric amphiphile, 3,5-bis-(3,4-bis-dodecyloxy-benzyloxy)-N-pyridine-4-yl-benzamide (DenC12), were mixed in a 1:3 ratio to obtain a complex, TPC4(S)-DenC12. Analyses by H-1-NMR spectroscopy, diffusion ordered spectroscopy (DOSY), CD spectroscopy, infrared (IR) spectroscopy, polarized optical microscopy (POM), differential scanning calorimetry (DSC), and X-ray diffractometry revealed that TPC4(S)-DenC12 self-assembles to form helical columnar stacks in solution and a helical columnar liquid crystal in bulk. The hydrogen bonding between TPC4(S) and DenC12 is essential for the helical columnar organization, and the preference for a one-handed helical conformation is likely derived from the steric interaction between the chiral side-chains and the dendric amphiphiles in the packing of the hydrogen-bonded columnar assemblies. Copyright C(-) 2008 John Wiley & Sons, Ltd.

Our purpose was to produce a platelet substitute that could enhance haemostatic ability using rabbits with severe thrombocytopenia. We have developed polymerized albumin particles (polyAlb) for treatment of bleeding and focused on a dodecapeptide, HHLGGAKQAGDV (H12), as a useful ligand for activated platelet. This sequence occurs only at the carboxy-terminus of the fibrinogen gamma-chain (gamma 400-411). H12 was conjugated to the surface of polyAlb modified with poly(ethylene glycol) (PEG) chains to produce blood-compatible particles (H12-PEG-polyAlb) that had prolonged blood residence time and enhanced stability in vitro and in vivo. The H12-PEG-polyAlb was administered intravenously to rabbits with severe thrombocytopenia, and the ear bleeding time was measured in order to evaluate the haemostatic effect. The H12-PEG-polyAlb significantly shortened the ear bleeding time of severely thrombocytopenic rabbits and showed no effect on the inhibition or promotion of endogenous and exogenous coagulation activities. Furthermore, we could assess the haemostatic capacity of the H12-PEG-polyAlb, based on the relationship between transfused platelet count and the bleeding time. The H12-PEG-polyAlb may be a suitable candidate for an alternative to human platelet concentrates infused to treat bleeding in patients with severe thrombocytopenia.

We synthesized cationic lipids bearing lysine, histidine, or arginine as a cationic headgroup for use in gene transfer studies. The cationic assemblies formed from lysine- or arginine-type lipids gave unilamellar vesicles (similar to 100 nm diameter), whereas the morphology of the histidine-type lipids was tube-like. The competences of the cationic assemblies were sufficient to form lipoplexes, and the resulting lipoplexes were evaluated in terms of gene expression efficiencies with COS-7 cells. The lysine- or arginine-type lipids exhibited higher gene expression efficiencies than that of Lipofectamine2000, a conventional transgenic reagent, indicating that stable lipoplexes could be prepared between spherical cationic assemblies and plasmid DNA. The gene expression efficiency in relation to the cationic headgroup of the lipids was as follows: lysine >= arginine > histidine. In addition, gene expression efficiency was enhanced by decreasing the length of the alkyl chain of the hydrophobic moiety. Unlike Lipofectamine2000, no reduction in transfection efficiency in the presence of fetal bovine serum was observed for the lipoplexes formed using synthetic cationic lipids. Moreover, the synthetic cationic lipids revealed remarkably low cytotoxicity compared with Lipofectamine2000. In conclusion, cationic assemblies formed from 1,5ditetradecyl-N-lysyl-L-glutamate or 1,5-ditetradecyl-N-arginyl-L-glutamate can be used as an effective plasmid DNA delivery system.

The aim of this paper is to develop highly magnetized, biodegradable and biocompatible, polymeric nanoparticles for drug delivery in cell therapy. Alginate magnetic nanoparticles are realized by an emulsion/reticulation technique, after the dispersion of magnetite in an alginate solution. Such nanoparticles are characterized in terms of external morphology (FIB imaging), microstructure (TEM imaging), size distribution, zeta potential, magnetic properties (SQUID analysis) and drug release behaviour. Magnetization curves show the typical trend of superparamagnetic materials. Important parameters, such as magnetic permeability and magnetic momentum, are derived by employing Langevin theory. Experimental results reveal that a bi-exponential model fully describes the drug release. Finally, in vitro experiments on NIH/3T3 cells are carried out and demonstrate that our magnetic alginate nanoparticles can effectively drive the drug delivery towards an external magnetic field source.

Sheet-shaped carriers, having obverse and reverse surfaces and thus a large contact area as a targeting site, have several advantages over spherical-shaped carriers, which have an extremely small contact area. Herein is proposed a novel method for the preparation of free-standing nanoparticle-fused nanosheets having uniform micrometer shape, nanometer thickness and heterogenous surfaces, using a water-soluble sacrificial film. This was achieved by combination of four processes: (1) specific adsorption of latex beads at pH 5.0 and a concentration of 1.0 x 10(11) mL(-1) onto a patterned dodecyltrimethoxysilane self-assembled monolayer (DTS-SAM) region by a conventional dry patterning process, (2) fabrication of the latex bead-sheet via thermal-fusion at 110 degrees C for 60s, (3) preparation of the free-standing nanosheet by detachment from the DTS-SAM, and (4) hetero-modification of the resulting nanosheet using a water-soluble poly(acrylic acid) as a sacrificial supporting film. Thus, this sheet-shaped carrier having hetero-surfaces can be regarded as a new material for delivery of drugs, hemostatic reagents and as wound dressings for bum injury, etc. (C) 2007 Elsevier B.V. All rights reserved.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, Cm) is a natural compound which possesses antioxidant, anti-inflammatory and anti-tumor ability. Here, phospholipid vesicles or lipid-nanospheres embedding Cm (CmVe or CmLn) were formulated to deliver Cm into tissue macrophages through intravenous injection. Cm could be solubilized in hydrophobic regions of these particles to form nanoparticle dispersions, and these formulations showed ability to scavenge reactive oxygen species as antioxidants in dispersions. At 6 h after intravenous injection in rats via the tail vein (2 mg Cm/kg bw), confocal microscopic observations of tissue sections showed that Cm was massively distributed in cells assumed as macrophages into the bone marrow and spleen. Taken together, these results indicate that the lipid-based nanoparticulates provide improved intravenous delivery of Cm to tissues macrophages, specifically bone marrow and splenic macrophages in present formulation, which has therapeutic potential as an antioxidant and anti-inflammatory. (C) 2007 Elsevier B.V. All rights reserved.

One physiological significance of the red blood cell (RBC) structure is that NO binding of Hb is retarded by encapsulation with the cell membrane. To clarify the mechanism, we analyzed Hb-vesicles (HbVs) with different intracellular Hb concentrations, [Hb] in, and different particle sizes using stopped-flow spectrophotometry. The apparent NO binding rate constant, k(on)'((NO)), of HbV at [Hb](in) = 1 g/dl was 2.6 x 10(7) M-1 s(-1), which was almost equal to k(on)((NO)) of molecular Hb, indicating that the lipid membrane presents no obstacle for NO binding. With increasing [Hb] in to 35 g/dl, k(on)'((NO)) decreased to 0.9 x 10(7) M-1 s(-1), which was further decreased to 0.5 x 10(7) M-1 s(-1) with enlarging particle diameter from 265 to 452 nm. For CO binding, which is intrinsically much slower than NO binding, k(on)'((CO)) did not change greatly with [Hb] in and the particle diameter. Results obtained using diffusion simulations coupled with elementary binding reactions concur with these tendencies and clarify that NO is trapped rapidly by Hb from the interior surface region to the core of HbV at a high [Hb] in, retarding NO diffusion toward the core of HbV. In contrast, slow CO binding allows time for further CO- diffusion to the core. Simulations extrapolated to larger particles (8 mu m) showing retardation even for CO binding. The obtained k(on)'((NO)) and k(on)'((CO)) yield values similar to those reported for RBCs. In summary, the intracellular, not extracellular, diffusion barrier is predominant due to the rapid NO binding that induces a rapid sink of NO from the interior surface to the core, retarding further NO diffusion and binding.

A free-standing polysaccharide nanosheet prepared by using a spin-coating-assisted layer-by-layer method is transferred from silicone rubber onto human skin (see figure) by fabricating a three-layered 'nano-adhesive plaster' involving a water-soluble sacrificial layer. By using this novel technique, conventional ultrathin films can be made without the need for a solid substrate.

Hemoglobin vesicles (HbV) or liposome-encapsulated Hbs are artificial oxygen carriers that have been developed for use as transfusion alternatives. The extremely high concentration of the HbV suspension (solutes, ca. 16 g/dL; volume fraction, ca. 40 vol %) gives it an oxygen-carrying capacity that is comparable to that of blood. The HbV suspension does not possess a colloid osmotic pressure. Therefore, HbV must be suspended in or co-injected with an aqueous solution of a plasma substitute (water-soluble polymer), which might interact with HbV. This article describes our study of the rheological properties of HbV suspended in a series of plasma substitute solutions of various molecular weights: recombinant human serum albumin (rHSA), dextran (DEX), modified fluid gelatin (MFG), and hydroxylethyl starch (HES). The HbV suspended in rHSA was nearly Newtonian. Other polymersHES, DEX, and MFGinduced HbV flocculation, possibly by depletion interaction, and rendered the suspensions as non-Newtonian with a shear-thinning profile (10(-4)-10(3) s(-1)). These HbV suspensions showed a high storage modulus (G') because of the presence of flocculated HbV. However, HbV suspended in rHSA exhibited a very low G'. The viscosities of HbV suspended in DEX, MFG, and high-molecular-weight HES solutions responded quickly to rapid step changes in shear rates of 0.1-100 s(-1) and a return to 0.1 s(-1), indicating that flocculation is both rapid and reversible. Microscopically, the flow pattern of the flocculated HbV that perfused through microchannels (4.5 mu m deep, 7 mu m wide, 20 cmH(2)O applied pressure) showed no plugging. Furthermore, the time required for passage was simply proportional to the viscosity. Collectively, the HbV suspension viscosity was influenced by the presence of plasma substitutes. The HbV suspension provides a unique opportunity to manipulate rheological properties for various clinical applications in addition to its use as a transfusion alternative.

BACKGROUND: Second-gene ration platelet (PLT) substitutes for treatment of bleeding were studied and the focus was on a dodecapeptide, HHLGGAKQAGDV (H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411) and exists only in a fibrinogen domain.
STUDY DESIGN AND METHODS: H12 was conjugated to the surface of polymerized albumin particles (polyAlb) modified with polyethylene glycol (PEG) chains to produce biocompatible particles (H12-PEG-polyAlb) that had prolonged blood circulation t(1/2) and were more stable in vitro and in vivo compared with H12-polyAlb (not modified with PEG). H12-PEG-polyAlb was administered intravenously into thrombocytopenic rats and the t(1/2) of the particles and the tail bleeding time were measured to evaluate the prolongation in the hemostatic effect.
RESULTS: H12-PEG-polyAlb particles modified with PEG prolonged the t1/2 and maintained specific binding ability to activated PLTs. The particles dose dependently shortened the tail bleeding time of thrombocytopenic rats 6 hours after injection.
CONCLUSION: H12-PEG-polyAlb may be a suitable candidate for treatment of bleeding into thrombocytopenic patients as an alternative to PLT concentrate transfusion.

Self-assembled porphyrins via noncovalent bonding have attracted wide-ranging researchers in material science. We reported herein the synthesis of the tetraphenyl porphyrin derivatives bearing uracyl groups as acceptor-donor-acceptor (ADA) type hydrogen bonding units, through the condensation of 5,10- or 5,15-bis (3-amino-4-ethylhexylphenyl) porphyrin derivatives with 6-carboxyuracyl derivatives. When two porphyrins having uracyl groups at the different substituted positions were respectively mixed with a melamine derivative in benzene, H-1 NMR spectra showed that the 5,15 substituted uracyl porphyrin formed a hydrogen-bonded suprastructure with the melamine derivative as a complementary molecule to the uracyl moiety, although the other 5,10-substituted uracylporphyrin could not form such a structure. The SEM observation indicated that the mixture with the 5,15-substituted uracyl porphyrin and the melamine with long alkyl chains formed a sheet-like structure. Copyright (C) 2007 John Wiley & Sons, Ltd.

An advantage of using vesicles (liposomes) as drug delivery carriers is that their pharmacokinetics can be controlled by surface characteristics, which can permit specific delivery of the encapsulated agents to organs or cells it? vivo. Here we report a vesicle formulation which targets the bone marrow after intravenous injection in rabbits. Surface modification of the vesicle with an anionic amphiphile; L-glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5- dihexadecyl ester (SA) results in significant targeting of vesicles to bone marrow. Further incorporation of as little as 0.6 mol% of poly(ethylene glycol)-lipid (PEG-DSPE) passively enhanced the distribution of SA-vesicles into bone marrow and inhibited hepatic uptake. In this model, more than 60% of the intravenously injected vesicles were distributed to bone marrow within 6 h after administration of a small dose of lipid (15 mg/kg b.w.). Histological evidence indicates that the targeting was achieved due to uptake by bone marrow macrophages (BMM phi). The efficient delivery of encapsulated scintigraphic and fluorescent imaging agents to BMM phi suggests that vesicles are promising carriers for the specific targeting of BMM phi and may be useful for delivering a wide range of therapeutic agents to bone marrow. (c) 2007 Elsevier Ltd. All rights reserved.

Tricarboxylic triphenylene (TPC5) and monopyridyl dendron (DenC12) were mixed in 1:2, 1:3, and 1:4 molar ratios, and investigation by IR, DSC, and XRD studies proved that TPC5 and DenC12 self-assembled to form a hexagonal columnar liquid crystal with 1:3 molar stoichiometry via complementary hydrogen-bonds.

We studied the peroxidase activity of ferrylhemoglobin radical (Hb(Fe4+ = 0*)) generated by the reaction of metHb (Hb(Fe3+)) with hydrogen peroxide (H2O2). To clarify the behaviors of ferrylHb radical, it was isolated from the reaction mixture of metHb and H2O2 by GPC at 4 degrees C. The radical species underwent rapid autoreduction to metHb at 37 degrees C accompanied with denaturation; however, it was stable for several minutes at VC. In ESR measurements, the signal of the ferrylHb radical immediately disappeared in the presence of L-Tyrosine (L-Tyr), and simultaneously, the signal of the ferric heme increased. This suggested that the ferrylHb radical immediately converted to metHb by L-Tyr even at 4 degrees C. Furthermore, dimerized L-Tyr was detected in the reaction mixture. This showed that the ferrylHb radical was reduced to metHb by electron donation from L-Tyr. The enzymatic reaction using L-Tyr as the substrate resulted in the elimination of H2O2 in this system.

Hemoglobin vesicle (HbV), a liposomal oxygen carrier containing human hemoglobin, was intravenously infused into rats. After the infusion of saline, the HbV or empty vesicle (EV), numbers of red cells, leukocytes and platelets in peripheral blood were unchanged during the observation period of one week in addition to each time point among three groups. However, the lymphocyte ratio transiently decreased and the granulocyte ratio increased in the HbV and EV groups at 6 h after the infusion. Those changes returned to the initial value one day after the infusion and those were maintained for the subsequent observation period. No dramatic change was seen in the ratio of CD4(+)/CD8(+) T cells. A transient decrease of the complement titer was observed three days after the infusion of HbV and EV, although the consumption of complement titer was not detected in rat serum by mixing HbV or EV in vitro, indicating that the transient decrease of complement titer in vivo was not due to the consumption of complement due to the interaction with HbV or EV. Multiple infusions of HbV caused the decrease of complement titer only after the first infusion and no allergic reaction was observed. No anaphylactic shock was observed in rats administered with EV several times, while ovalbumin (OVA) sensitized rats died with symptoms of respiratory distress after the second OVA administration. These results indicate that HbV could be administered without serious clinical symptoms or adverse reactions.

We have synthesized a 5,15 meso-substituted methyluracyl porphyrin derivative bearing 6-methyluracyl units directly at the meso positions. The atropisomerization was regulated by steric replusion between the methyl substituents. When the atropisomers were mixed with alkylated melamine as a complementary hydrogen-bonding unit, the hydrogen-bonded assemblies were analyzed by diffusion-ordered spectroscopy (DOSY) in solution, which clarified that the alpha alpha isomer formed a face-to-face dimer, whereas the alpha beta isomer took a zigzag structure.

Hemoglobin (Hb) vesicles have been developed as cellular-type Hb-based O-2 carriers in which a purified and concentrated Hb solution is encapsulated with a phospholipid bilayer membrane. Ferrous Hb molecules within an Hb vesicle were converted to ferric metHb by reacting with reactive oxygen species such as hydrogen peroxide (H2O2) generated in the living body or during the autoxidation of oxyHb in the Hb vesicle, and this leads to the loss of O-2 binding ability. The prevention of metHb formation by H2O2 in the Hb vesicle is required to prolong the in vivo O-2 carrying ability. We found that a mixed solution of metHb and L-tyrosine (L-Tyr) showed an effective H2O2 elimination ability by utilizing the reverse peroxidase activity of metHb with L-Tyr as an electron donor. The time taken for the conversion of half of oxyHb to metHb (T-50) was 420 min for the Hb vesicles containing 4 g/dL (620 mu M) metHb and 8.5 mM L-Tyr ((metHb/L-Tyr) Hb vesicles), whereas the time of conversion for the conventional Hb vesicles was 25 min by stepwise injection of H2O2 (310 mu M) in 10 min intervals. Furthermore, in the (metHb/L-Tyr) Hb vesicles, the metHb percentage did not reach 50% even after 48 h under a pO(2) of 40 Torr at 37 degrees C, whereas T-50 of the conventional Hb vesicles was 13 h under the same conditions. Moreover, the T-50 values of the conventional Hb vesicles and the (metHb/L-Tyr) Hb vesicles were 14 and 44 h, respectively, after injection into rats (20mL/kg), confirming the remarkable inhibitory effect of metHb formation in vivo in the (metHb/L-Tyr) Hb vesicles.

BACKGROUND: Hemoglobin-vesicles (HbVs; diameter, 251 +/- 81 nm) are artificial O-2 carriers. Their efficacy for acute exchange transfusion has been characterized in animal models. However subsequent profiles of recovery involving the degradation of HbV in the reticuloendothelial system (RES) and hematopoiesis remain unknown.
STUDY DESIGN AND METHODS: Isovolemic 40 percent exchange transfusion was performed in 60 male Wistar rats with HbV suspended in 5 g per dL recombinant human serum albumin (rHSA; HbV/rHSA, [Hb] = 8.6 g/dL), stored rat RBCs suspended in rHSA (sRBC/rHSA), or rHSA alone. Hematological and plasma biochemical analyses and histopathological examination focusing on the spleen were conducted for the subsequent 14 days.
RESULTS: The reduced hematocrit (Hct) level (26%) for the HbV/rHSA and rHSA groups returned to its original level (43%) in 7 days. Plasma erythropoietin was elevated in all groups: the rHSA group showed the highest value on Day 1 (321 +/- 123 mIU/mL) relating to the anemic conditions (HbV/rHSA, 153 +/- 22; sRBC/rHSA, 63 +/- 7; baseline, 21 +/- 3). Simultaneously, splenomegaly occurred in all the groups as HbV/rHSA > rHSA > sRBC/rHSA. Histopathologically, the accumulated HbV in the spleen was undetectable by Day 14, but hemosiderin was deposited in slight quantities for both the HbV/rHSA and sRBC/rHSA groups. Considerable amounts of erythroblasts were apparent in the spleens of both the rHSA and the HbV/rHSA groups.
CONCLUSION: HbVs were phagocytized and degraded in RES, a physiological compartment for the degradation of RBCs, and the elevated erythropoietic activity resulted in the complete recovery of Hct within 7 days in the rat model.

Hemoglobin vesicles (HbVs), cellular-type artificial oxygen carriers containing human hemoglobin, were assessed for their biocompatibility by mixing with human plasma in vitro . Among three kinds of HbVs (PEG-DPEA-HbV, PEG-DPPG-HbV and DPPG-HbV), PEG-DPEA-HbV did not affect the extrinsic or intrinsic coagulation activities of the plasma, while PEG-DPPG-HbV and DPPG-HbV tended to shorten the intrinsic coagulation time. The kallikrein-kinin cascade of the plasma was slightly activated by PEG-DPPG-HbV and DPPG-HbV, but not by PEG-DPEA-HbV. The complement consumption of the plasma was observed by incubation with DPPG-HbV, but not with PEG-DPEA-HbV or PEG-DPPG-HbV. These results indicate that PEG-DPEA-HbV has a higher biocompatibility with human plasma.

A calix[4]arene-porphyrin duplex was prepared by mixing equimolar amounts of hydroxy or carboxy calix[4]arene derivatives and pyridyl or o-aminophenylporphyrin derivatives. Electrospray ionization mass spectrometry (ESI-MS) was applied to screen a suitable pair of a calix[4]arene and a porphyrin. meso-Tetra(2-pyridyl)porphyrin formed a duplex with tetrahydroxy or tetrahydroxymethylcalix[4]arene via triple or quadruple hydrogen bonds. H-1 NMR spectra showed that the tetrahydroxycalix[4]arene was symmetrically located upon the porphyrin ring, whereas trihydroxycalix[4]arene was slanted on the porphyrin ring. The duplex of meso-tetra(2-pyridyl)porphyrinatocobalt(II) and tetrahydroxymethylcalix[4]arene formed a complex with benzylimidazole, which was capable of reversible dioxygen-binding. The capping structure upon porphyrin provided by calix[4]arene raised the life-time of dioxygen-adduct compared with the porphyrin without calix[4]arene.

We studied prototypes of platelet substitutes that bear on their surface a dodecapeptide, HHLG-GAKQAGDV (1112). The peptide is a fibrinogen gamma chain carboxy-terminal sequence (gamma 400-411) and recognizes specifically the active form of glycoprotein (GP) IIb/IIIa on the surface of activated platelets. We conjugated H12 to the end of poly(ethylene glycol) chains on the surface of a phospholipid vesicle with an average diameter of 220 nm to prepare H12-PEG-vesicles. The half-life of the H12-PEG-vesicles was significantly prolonged by PEG modification, and the ability of H12 on the surface of the vesicle to recognize GPIIb/IIIa was maintained even though the surface was modified with PEG chains. The H12-PEG-veiscles enhanced the in vitro thrombus formation of platelets that were adhering to a collagen-immobilized plate, when thrombocytopenia-imitation blood was passed over the plate. Based on the flow cytometric analyses of PAC-1 binding and P-selectin expression, the H12-PEG-vesicles were shown not to cause platelet activation. Furthermore, the H12-PEG-vesicles dose-dependently shortened the tail bleeding time of thrombocytopenic rats. It was confirmed that the H12-PEG-vesicles had a hemostatic effect and may be a suitable candidate for an alternative to human platelet concentrates transfused into thrombocytopenic patients.

BACKGROUND: Prototypes of platelet (PLT) substitutes have been studied and the focus was on a dodecapeptide, HHLGGAKQAGDV (H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411) and exists only in the fibrinogen domain.
STUDY DESIGN AND METHODS: H12 was conjugated to the surface of polymerized albumin particles (polyAlb) as biocompatible and biodegradable particles with a mean diameter of 260 +/- 60 nm, and the hemostatic ability of H12-conjugated polyAlb (H12-polyAlb) under flow conditions and thrombocytopenic rats have been studied
RESULTS: H12-polyAlb enhanced the in vitro thrombus formation of activated PLTs on a collagen-immobilized plate when exposed to the flowing thrombocytopenic imitation blood. Furthermore, the analysis of the tail bleeding time of rats that were made thrombocytopenic by busulfan injection showed that H12-polyAlb had a hemostatic effect. Based on the bleeding time and the amount injected, the hemostatic capacity of 20 H12-polyAlb was estimated to correspond to that of one PLT.
CONCLUSION: These results were important first steps toward the development of PLT substitutes and indicated that H12-polyAlb may be a suitable candidate for an alternative to human PIT concentrates transfused into thrombocytopenic patients in the future.

Objectives: The aim of this study was to investigate and compare the effects of a traditionally formulated, low-viscosity, right-shifted polymerized bovine hemoglobin solution and a highly viscous, left-shifted hemoglobin vesicle solution (HbV-HES) on the oxygenation of critically ischemic peripheral tissue.
Design. Randomized, prospective study.
Setting. University laboratory.
Subject. A total of 40 male golden Syrian hamsters.
Interventions: Island flaps were dissected from the back skin of anesthetized hamsters. The flap included a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. One hour after completion of the preparation, the animals received a 33% blood exchange with 6% hydroxyethyl starch 200/0.5 (HES, n = 9), HbV suspended in HES (HbV-HES, n = 8), or polymerized bovine hemoglobin solution (n = 9).
Measurements and Main Results., Three hours after the blood exchange, microcirculatory blood flow (laser-Doppler flowmetry) was increased to 262% of baseline for HbV-HES (p < .01) and 197% for polymerized bovine hemoglobin solution (p < .05 vs. baseline and HbV-HES). Partial tissue oxygen tension (bare fiber probes) was only improved after HbV-HES (9.4 torr to 14.2 torr, p < .01 vs. baseline and other groups). The tissue lactate/pyruvate ratio (microdialysis) was elevated to 51 in the untreated control animals, and to 34 +/- 8 after HbV-HES (p < .05 vs. control) and 38 +/- 11 after polymerized bovine hemoglobin solution (not significant).
Conclusions. Our study suggests that in critically ischemic and hypoxic collateralized peripheral tissue, oxygenation may be improved by normovolemic hemodilution with HbV-HES. We attributed this improvement to a better restoration of the microcirculation and oxygen delivery due to the formulation of the solution.

Hemoglobin vesicles (HbV) are artificial oxygen carriers that encapsulate a concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane. The oxygen transporting ability of HbV in vivo has been demonstrated by the transfusion of HbV into hemorrhagic shock rodent models. However, the compatibility of HbV with human blood cells must be evaluated. Preincubation of platelets with concentrations of 20% or 40% HbV had no effect on the binding of PAC-1, a monoclonal antibody that detects activation-dependent conformational changes in alpha(IIb)beta(3) on platelets, or the surface expression of CD62P in whole blood. ADP-induced increases in PAC-1 binding were significantly enhanced by exposing the platelets to concentrations of either 20% or 40% HbV, whereas the ADP-induced increases in CD62P expression were not affected by HbV treatment at either concentration. Preincubation of platelet-rich plasma (PRP) with HbV minimally reduced the spontaneous release of TXB2 and RANTES, but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with ADP. Similarly, preincubation of PRP with HbV minimally reduced the spontaneous release of RANTES but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with collagen, although collagen-induced serotonin release tended to decrease with HbV pretreatment. These data suggest that the exposure of human platelets to high concentrations of HbV (up to 40%) in vitro did not cause platelet activation and did not adversely affect the formation and secretion of prothrombotic substances or proinflammatory substances triggered by platelet agonists, although one of the earliest events in ADP-induced platelet activation was slightly potentiated by HbV pretreatment at the doses tested. Taken together, these results imply that HbV, at concentrations of up to 40%, do not have any aberrant interactions with either unstimulated or agonist-induced platelets.

A hemoglobin vesicle (HbV; diameter 252 +/- 53 nm) or liposome-encapsulated Hb is an artificial oxygen carrier developed for use as a transfusion alternative, and its oxygen-transporting capacity has been well characterized, although critical physiological compartments for the Hb degradation after a massive infusion of HbV and the safety outcome remain unknown. In this study, we aimed to examine the compartments for its degradation by daily repeated infusions (DRI) of HbV, focusing on its influence on the reticuloendothelial system ( RES). Male Wistar rats intravenously received the HbV suspension at 10 ml/kg/day for 14 consecutive days. The cumulative infusion volume ( 140 ml/kg) was equal to 2.5 times the whole blood volume ( 56 ml/kg). The animals tolerated the DRI well and survived, and body weights continuously increased. One day after DRI, hepatosplenomegaly occurred significantly through the accumulation of large amounts of HbV. Plasma clinical chemistry was overall normal, except for a transient elevation of lipid components derived from HbV. These symptoms subsided 14 days after DRI. Hemosiderin deposition and up-regulation of heme oxygenase-1 coincided in the liver and spleen but were not evident in the parenchyma of these organs. Furthermore, the plasma iron and bilirubin levels remained unchanged, suggesting that the heme-degrading capacity of the RES did not surpass the ability to eliminate bilirubin. In conclusion, phospholipid vesicles for the encapsulation of Hb would be beneficial for heme detoxification through their preferential delivery to the RES, a physiological compartment for degradation of senescent RBCs, even at doses greater than putative clinical doses.

The recombinant human serum albumin (rHSA) dimer, which was cross-linked by a thiol group of Cys-34 with 1,6-bis(maleimido)hexane, has been physicochemically characterized. Reduction of the inert mixed-disulfide of Cys-34 beforehand improved the efficiency of the cross-linking reaction. The purified dimer showed a double mass and absorption coefficient, but unaltered molar ellipticity, isoelectric point (pl: 4.8) and denaturing temperature (65degreesC). The concentration dependence of the colloid osmotic pressure (COP) demonstrated that the 8.5 g dL(-1) dimer solution has the same COP with the physiological 5 g dL(-1) rHSA. The antigenic epitopes of the albumin units are preserved after bridging the Cys-34, and the circulation lifetime of the I-125-labeled variant in rat was 18 h. A total of 16 molecules of the tetrakis{(1-methylcyclohexanamido)phenyl}porphinatoiron(II) derivative (FecycP) is incorporated into the hydrophobic cavities of the HSA dimer, giving an albumin-heme hybrid in dimeric form. It can reversibly bind and release O-2 under physiological conditions (37degreesC, pH 7.3) like hemoglobin or myoglobin. Magnetic circular dichroism (CD) revealed the formation of an O-2-adduct complex and laser flash photolysis experiments showed the three-component kinetics of the O-2-recombination reaction. The O-2-binding affinity and the O-2-association and -dissociation rate constants of this synthetic hemoprotein have also been evaluated. (C) 2004 Elsevier B.V. All rights reserved.

We synthesized 3-dithiolthione-substituted polythiophene, poly [3-(1',2-dithiol-3'-thione-4'-yl)thiophene], by cyclization reaction of poly [3-bis(methoxycaronyl)methyl thiophene]. Its redox behavior and electronic states showed that the oxidation of the polythiophene followed the oxidation of the dithiolthione ring to the radical cation of the dithiolthione ring.

Ferric methemoglobin is reduced to its ferrous form by photoirradiation either by direct photoexcitation of the heme portion to induce electron transfer from the surrounding media (Sakai at al. (2000) Biochemistry 39, 14595-14602) or by an indirect electron transfer from a photochemically reduced electron mediator such as flavin. In this research, we studied the mechanism and optimal condition that facilitates photoreduction of flavin mononucleotide (FMN) to FMNH2 by irradiation of visible light, and the succeeding reduction of concentrated metHb in phospholipid vesicles to restore its 02 binding ability. Visible light irradiation (435 nm) of a metHb solution containing FMN and an electron donor such as EDTA showed a significantly fast reduction to ferrous Hb with a quantum yield (Phi) of 0.17, that is higher than the method of direct photoexcitation of heme ( Phi = 0.006). Electron transfer from a donor molecule to metHb via FMN was completed within 30 ns. Native-PAGE and IEF electrophoresis indicated no chemical modification of the surface of the reduced Hb. Coencapsulation of concentrated Hb solution (35 g/dL) and the FMN/EDTA system in vesicles covered with a phospholipid bilayer membrane (Hb-vesicles, HbV, diameter: 250 nm) facilitated the metHb photoreduction even under aerobic conditions, and the reduced HbV restored the reversible O-2 binding property. A concentrated HbV suspension ([Hb] = 8 g/dL) was sandwiched with two glass plates to form a liquid layer with the thickness of about 10 mum (close to capillary diameter in tissue, 5 mum), and visible light irradiation (221 mW/cm(2)) completed 100% metHb photoreduction within 20 s. The photoreduced FMNH2 reacted with O-2 to produce H2O2, which was detected by the fluorescence measurement of the reaction of H2O2 and p-nitrophenylacetic acid. However, the amount of H2O2 generated during the photoreduction of HbV was significantly reduced in comparison with the homogeneous Hb solution, indicating that the photoreduced FMNH2 was effectively consumed during the metHb reduction in a highly concentrated condition inside the HbV nanoparticles.

It is known that damage to the intestinal mucosa followed by systemic inflammatory response is one of the leading causes of shock related morbidity and mortality. In this study, we examined the ability of an artificial oxygen carrier hemoglobin vesicle (HbV) to sustain systemic and intestinal perfusion during hemorrhagic shock. In rabbits, hemorrhagic shock (40% of the estimated blood volume) was resuscitated with 5% albumin (alb group), HbV suspended in 5% albumin (HbValb group), or washed red blood cells suspended in 5% albumin (RBCalb group). Plasma tumor necrosis factor (TNF)-alpha level was measured in rats under the same experimental protocol. No significant intergroup differences were seen in systemic hemodynamics. In contrast, parameters of intestinal perfusion significantly deteriorated in the alb group but were equally well sustained in the HbValb and RBCalb groups. Also, a significant increase in plasma TNF-alpha level was seen in the alb group but not in the RBCalb or HbValb groups. These results indicate the proficient oxygen transporting capability of HbV and its potential efficacy in shock resuscitation.

Phospholipid vesicles encapsulating Hb (Hb-vesicies: HbV) have been developed for use as artificial O-2 carriers (250 nm phi). As one of the safety evaluations, we analyzed the influence of HbV on the organ functions by laboratory tests of plasma on a total of 29 analytes. The HbV Suspension ([Hb] = 10 g/dl) was intravenously infused into male Wistar rats (20 ml/kg; whole blood = 56 ml/kg). The blood was withdrawn at 8 h, and 1, 2, 3, and 7 days after infusion, and the plasma was ultracentrifuged to remove HbV in order to avoid its interference effect oil the analytes. Enzyme concentrations, AST, ALT, ALP, and LAP showed significant, but minor changes. and did not show a sign of a deteriorative damage to the liver that was one of the main organs for the HbV entrapment and the succeeding metabolism. The amylase and lipase activities showed reversible changes, however, there was no morphological changes in pancreas. Plasma bilirubin and iron did not increase in spite of the fact that a large amount of Hb was metabolized in the macrophages. Cholesterols, phospholipids, and beta-lipoprotein transiently increased showing the maximum at 1 or 2 days, and returned to the control level at 7 days. They should be derived from the membrane components of HbV that are liberated from macrophages entrapping HbV. Together with the previous report of the prompt metabolism of HbV in the reticuloendothelial system by histopathological examination, it can be concluded that HbV infusion transiently modified the values of the analytes without any; irreversible damage to the corresponding organs at the bolus infusion rate of 20 ml/kg. (C) 2003 Elsevier Ltd. All rights reserved.

A method to quantitatively measure the bacterial endotoxin content (lipopolysaccharide, LPS) in phospholipid vesicles or liposomes is necessary because the conventional Limulus amebocyte lysate (LA-L) test does not provide an accurate measurement due to the hydrophobic interaction of LPS and vesicles that shields the activity of LPS to clot the LA-L coagulant. This interference was evident from isothermal titration calorimetry results in our study that clearly demonstrated the insertion of the LPS molecule into the phospholipid bilayer membrane. Hemoglobin-vesicles (HbVs; particle diameter = 251+/-80 nm; [Hb] = 10 g/dL) are artificial oxygen carriers encapsulating a conc. Hb solution in phospholipid vesicles, and their oxygen transporting ability has been extensively studied. To accurately measure the LPS content in the HbV suspension, we tested the solubilization of HbV with deca(oxyethylene) dodecyl ether (C12E10), used to release the LPS entrapped in the vesicles, as a pretreatment for the succeeding LAL assay of the kinetic-turbidimetric gel clotting (detecting wavelength, 660 nm). The C12E10 surfactant interferes with the gel clotting in a concentration-dependent manner, and the optimal condition was determined in terms of minimizing the dilution factor and C12E10 concentration. We clarified the condition that allowed the measurement of LPS at > 0.1 endotoxin units (EU)/mL in the HbV suspension. Moreover, the utilization of histidine-immobilized agarose gel effectively concentrated the trace amount of LPS from the C12E10-solubilized HbV solution and washed out C12E10 as an inhibitory element. The LA-L assay with the LPS-adsorbed gel resulted in the detection limit of 0.0025 EU/mL. Pretreatment with C12E10 would be applicable not only to HbVs but also to other drug delivery systems using phospholipid vesicles encapsulating or incorporating functional molecules. (C) 2004 Wiley-Liss, Inc.

Objective: Hemoglobin-vesicle (HbV) has been developed to provide oxygen-carrying ability to plasma expanders. Its ability to restore the systemic condition after hemorrhagic shock was evaluated in anesthetized Wistar rats for 6 hrs after resuscitation. The HbV was suspended in 5 g/dL recombinant human serum albumin (HbV/rHSA) at an Hb concentration of 8.6 g/dL.
Design: Prospective, randomized, controlled trial.
Setting: Department of Surgery, School of Medicine, Keio University.
Subjects: Forty male Wistar rats.
Interventions: The rats were anesthetized with 1.5% sevoflurane inhalation throughout the experiment. Polyethylene catheters were introduced through the right jugular vein into the right atrium for infusion and into the right common carotid artery for blood withdrawal and mean arterial pressure monitoring.
Measurements and Main Results: Shock was induced by 50% blood withdrawal. The rats showed hypotension (mean arterial pressure = 32 +/- 10 mm Hg) and significant metabolic acidosis and hyperventilation. After 15 mins, they received HbV/rHSA, shed autologous blood (SAB), washed homologous red blood cells (wRBC) suspended in rHSA (wRBC/rHSA, [Hb] = 8.6 g/dL), or rHSA alone. The HbV/rHSA group restored mean arterial pressure to 93 +/- 8 mm Hg at 1 hr, similar to the SAB group (92 +/- 9 mm Hg), which was significantly higher compared with the rHSA (74 9 mm Hg) and wRBC/rHSA (79 8 mm Hg) groups. There was no remarkable difference in the blood gas variables between the resuscitated groups; however, two of eight rats in the rHSA group died before 6 hrs. After 6 hrs, the rHSA group showed significant ischemic changes in the right cerebral hemisphere relating to the ligation of the right carotid artery followed by cannulation, whereas the HbV/rHSA, SAB, and wRBC/rHSA groups showed less changes.
Conclusions: HbV suspended in recombinant human serum albumin provides restoration from hemorrhagic shock that is comparable with that using shed autologous blood.

The stability of some porphyrin-calix[4]arene sodium-ion complexes were determined by a collision-activated decomposition (CAD) method utilizing electrospray ionization mass spectrometry (ESI-MS). Comparing the values of E-1/2, the collision energy at which the relative intensity of the complex ion is 0.5, we found that the porphyrin-calix[4]arene complex with the higher value of E-1/2 corresponded to that with the larger association constant (K-ass), as measured by H-1-NMR in CDCl3. Both our ESI-MS and NMR studies proved that the number of hydrogen bonds and the rigidity of the calix[4]arene stabilized the complex. The ESI-MS technique could be successful in screening the binding affinity in host-guest systems with a small amount of sample. Copyright (C) 2004 John Wiley Sons, Ltd.

In order to perform a fundamental study of platelet substitutes, novel particles that bound to activated platelets were prepared using two oligopeptides conjugated to latex beads. The oligopeptides were CHHLGGAKQAGDV (H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411), and CGGRGDF (RGD), which contains a fibrinogen alpha-chain sequence (alpha 95-98 RGDF). Both peptides contained an additional amino-terminal cysteine to enable conjugation. Human serum albumin was adsorbed onto the surface of latex beads (average diameter 1 mum) and pyridyldisulfide groups were chemically introduced into the adsorbed protein. H 12 or RGD peptides were then chemically linked to the modified surface protein via disulfide linkages. H12- or RGD-conjugated latex beads prepared in this way enhanced the in vitro thrombus formation of activated platelets on collagen-immobilized plates under flowing thrombocytopenic-imitation blood. Based on the result of flow cytometric analyses of agglutination, PAG-1 binding, antiP-selectin antibody binding, and annexin V binding, the H12-conjugated latex beads showed minimal interaction with non-activated platelets. These results indicate the excellent potential of H12-conjugated particles as a candidate for a platelet substitute. (C) 2003 Elsevier Inc. All rights reserved.

Hemoglobin (Hb) vesicles (particle diameter, ca. 250 nm) have been developed as Hb-based oxygen carriers in which a purified Hb solution is encapsulated with a phospholipid bilayer membrane. The oxidation of Hb to nonfunctional ferric Hb (metHb) was caused by reactive oxygen species, especially hydrogen peroxide (H2O2), in vivo in addition to autoxidation. We focused on the enzymatic elimination of H2O2 to suppress the metHb formation in the Hb vesicles. In this study, we coencapsulated catalase with Hb within vesicles and studied the rate of metHb formation in vivo. The Hb vesicles containing 5.6 x 10(4) unit mL(-1) catalase decreased the rate of metHb formation by half in comparison with Hb vesicles without catalase. We succeeded in prolonging the oxygen-carrying ability of the Hb vesicle in vivo by the coencapsulation of catalase.

We are aiming to improve the encapsulation efficiency of proteins in a size-regulated phospholipid vesicle using an extrusion method. Mixed lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-dipalmitoyl-L-glutamate-N-succinic acid (DPEA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5,000)] (PEG-DSPE) at a molar ratio of 5, 5, 1, and 0.033 were hydrated with a NaOH solution (7.6 mM) to obtain a polydispersed multilamellar vesicle dispersion (50 nm to 30 mum diameter). The polydispersed vesicles were converted to smaller vesicles having an average diameter of ca. 500 nm with a relatively narrow size distribution by freeze-thawing at a lipid concentration of 2 g dL(-1) and cooling rate of -140 degreesC min(-1). The lyophilized powder of the freeze-thawed vesicles was rehydrated into a concentrated protein solution (carbonyl hemoglobin solution, 40 g dL(-1)) and retained the size and size distribution of the original vesicles. The resulting vesicle dispersion smoothly permeated through the membrane filters during extrusion. The average permeation rate of the freeze-thawed vesicles was ca. 30 times faster than that of simple hydrated vesicles. During the extrusion process, proteins were encapsulated into the reconstructed vesicles with a diameter of 250 20 nm.

We are aiming to improve the encapsulation efficiency of proteins in a size-regulated phospholipid vesicle using an extrusion method. Mixed lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-dipalmitoyl-L-glutamate-N-succinic acid (DPEA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5,000)] (PEG-DSPE) at a molar ratio of 5, 5, 1, and 0.033 were hydrated with a NaOH solution (7.6 mM) to obtain a polydispersed multilamellar vesicle dispersion (50 nm to 30 mum diameter). The polydispersed vesicles were converted to smaller vesicles having an average diameter of ca. 500 nm with a relatively narrow size distribution by freeze-thawing at a lipid concentration of 2 g dL(-1) and cooling rate of -140 degreesC min(-1). The lyophilized powder of the freeze-thawed vesicles was rehydrated into a concentrated protein solution (carbonyl hemoglobin solution, 40 g dL(-1)) and retained the size and size distribution of the original vesicles. The resulting vesicle dispersion smoothly permeated through the membrane filters during extrusion. The average permeation rate of the freeze-thawed vesicles was ca. 30 times faster than that of simple hydrated vesicles. During the extrusion process, proteins were encapsulated into the reconstructed vesicles with a diameter of 250 20 nm.

The recombinant fragment of the platelet membrane glycoprotein Ia/IIa (rGPIa/IIa) was conjugated to the polymerized albumin particles (polyAlb) with the average diameter of 180 nm. The intravenous administration of rGPIa/IIa polyAlb to thrombocytopenic mice ([platelet] = 2.1 +/- 0.3 x 10(5) particles/muL) with three doses of ca. 2.4 x 10(10), 7.2 x 10(10), and 2.4 x 10(11) particles/kg, respectively, significantly reduced their bleeding time to 426 +/- 71, 378 +/- 101, and 337 +/- 46 s, respectively, whereas that of the control groups (PBS) was 730 +/- 198 s. The injection of rGPIa/IIa-polyAlb (2.4 x 10(11) particles/kg) was approximately equal to the effect of the injection of the mouse platelets at a dose of 2.0 x 10(10) particles/kg. It was confirmed that rGPIa/IIa-polyAlb had a recognition ability against collagen and could contribute to the hemostasis in the thrombocytopenic mice as a platelet, substitute. (C) 2003 Elsevier Science (USA). All rights reserved.

ヘモグロビン(Hb)を濃度高くリン脂質小胞体の内水相に内包したヘモグロビン小胞体(HbV)が人工酸素運搬体として開発され,赤血球と同等の酸素運搬機能と安全性が動物投与試験から明らかにされた.界面活性剤deca(oxyethylene)dodecyl ether(C12E10))でHbVを溶解してリポポリサッカライド(LPS)を遊離させた後,リムルステスト試薬と混合し,ゲル化反応を比濁時間分析法によって解析する方法を検討した.0.1EU/mLまでの検出限界を得ることができ,添加回収法によりその妥当性を検証することができた

We encapsulated a purified and concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane to form Hb vesicles (particle diameter, ca. 250 nm) for the development of artificial oxygen carriers. Reaction of Hb inside the vesicle with hydrogen peroxide (H2O2) is one of the important safety issues to be clarified and compared with a free Hb solution. During the reaction of the Hb solution with H2O2, metHb (Fe-III) and ferrylHb (Fe-IV=O) are produced, and H2O2 is decomposed by the catalase-like reaction of Hb. The aggregation of discolored Hb products due to heme degradation is accompanied by the release of iron (ferric ion). On the other hand, the concentrated Hb within the Hb vesicle reacts with H2O2 that permeated through the bilayer membrane, and the same products as the Hb solution are formed inside the vesicle. However, there is no turbidity change, no particle diameter change of the Hb vesicles, and no peroxidation of lipids comprising the vesicles after the reaction with H2O2. Furthermore, no free iron is detected outside the vesicle, though ferric ion is released from the denatured Hb inside the vesicle, indicating the barrier effect of the bilayer membrane against the permeation of ferric ion. When vesicles composed of egg york lecithin (EYL) as unsaturated lipids are added to the mixture of Hb and H2O2, the lipid peroxidation is caused by ferrylHb and hydroxyl radical generated from reaction of the ferric iron with H2O2, whereas no lipid peroxidation is observed in the case of the Hb vesicle dispersion because the saturated lipid membrane of the Hb vesicle should prevent the interaction of the ferrylHb or ferric iron with the EYL.

The recombinant fragment of the platelet membrane glycoprotein, rGPIbalpha, was conjugated to phospholipid vesicles with the average diameter of ca. 1 mum using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). We used five kinds of rGPIbalpha-vesicles with different fluorescent anisotropies of 1,6-diphenyl-1,3,5-hexatriene (DPH) to study the rolling properties of the vesicles on the von Willebrand factor (vWf)-immobilized surface. Under flow conditions, the rolling velocity of the rGPIbalpha-vesicles decreased with the increasing membrane flexibility. It is considered that the vesicles with a high membrane flexibility have a high deformability and can be flattened to a high degree during rolling on the vWf surface, thus resulting in the large contact area. We obtained a recipe to control the rolling velocity of artificial platelets by membrane flexibility. (C) 2002 Elsevier Science (USA). All rights reserved.

Hemoglobin-vesicles (HbV) have been developed for use as artificial O-2 carriers in which a purified Hb solution is encapsulated within a phospholipid bilayer membrane. In this study, bovine Hb (BHb) was tested as a source of HbV instead of human Hb (HHb). We compared the preparation process and characteristics of BHbV with those of HHbV. The purification of BHb was effectively performed simply with an ultrafiltration system including a process for removing virus and scrapie agent. The removal ratio of the phospholipid components of bovine red blood cells was over 99.99%, and the protein purity was over 99.9%. The deoxygenated and carbonylated BHb showed denaturation transition temperatures at 83 and 87degreesC, respectively, which are higher than those of HHB (80 and 78degreesC, respectively), and resistant to pasteurization (60degreesC, 10 h). The purified BHb was concentrated to over 40 g/dl, and encapsulated in a phospholipid bilayer membrane to form BHbV with a diameter of about 280 nm. The O-2 affinity (P-50) of the BHbV was regulated by coencapsulation of an appropriate amount of Cl- (as NaCl), which binds to BHb as an allosteric effector, in the range 16-28 Torr, comparable to human blood (P-50 = 28 Torr). This is quite simple in comparison with HHb which requires phosphate derivatives such as pyridoxal 5-phosphate as a replacement for 2,3-diphoshoglyceric acid. The viscosity and colloid osmotic pressure of the BHbV when suspended in 5% human serum albumin are 3.5 cP and 20 Torr, respectively, comparable to those of human blood. In conclusion, BHb can be used as a source for the production of HbV, not only because of its abundance in the cattle industry, but also because of the physicochemical advantages of the purification process, thermal stability, and regulation of O-2 affinity in comparison with HHb.

Hemoglobin (Hb) has been widely studied as a raw material for various types of oxygen carriers. In the purification of Hb from red blood cells including virus inactivation and denaturation of other proteins and the long-term storage of Hb vesicles (HbV), a deoxygenation process is one of the important processes because of the high stability of deoxygenated Hb to heating and metHb formation. Though an oxygenated Hb solution can be deoxygenated with an artificial lung, it is difficult to reduce the oxygen partial pressure of the Hb solution to less than 10 Torr. We developed an electrochemical system for complete deoxygenation of the Hb solution at the cathode compartment using hydrogen containing nitrogen gas at the anode compartment. Oxygen in the Hb solution was reduced to OH- at the cathode compartment within several minutes at a potential value of - 1.67 V and was finally converted to water by neutralization with H+ from the anode in the whole system. The resulting completely deoxygenated Hb could tolerate heat treatment at 62 degreesC for 10 h with no denaturation of deoxygenated Hb. The metHb formation rate of reoxygenated Hb at 37 degreesC was not changed after heat treatment. Furthermore, vesicular stomatitis virus (VSV) could be inactivated at an inactivation degree of more than 5.96 log by heat treatment.

In the purification process of hemoglobin (Hb) from red blood cells, we stabilized Hb as carbonylhemoglobin (HbCO) against pasteurization at 60°C. In this study, the process of carbonylation (HBO2 → HbCO) was tested with a membrane oxygenator (CX-II08
membrane area, 0.8m2
maximum circulation rate, 1.21/min) under the conditions of a solution flow rate of 100-1000 ml/min and a CO gas flow rate of 30-100 ml/min. Comparing the overall O2 transfer coefficient of carbonylation with that of deoxygenation (N2 flow) revealed that the resistance to O2 transfer of carbonylation was about 35 times smaller, indicating that carbonylation hindered O2 rebinding (deoxyHb → HbO2). On the other hand, the O2 released in the course of carbonylation hindered carbonylation at the beginning, because rebinding of O2 is competitive with carbonylation. The time required for carbonylation was significantly shortened from 1000 to 150s when the solution flow rate was increased from 50 to 400 ml/min
however, the CO gas flow rate did not affect it very much. Increasing the Hb concentration from 7.5 to 15 g/dl accelerated carbonylation by 1.3 times. Even though further study is necessary to select a suitable polymer membrane to avoid protein adsorption, a membrane oxygenator will be effective for the large-scale carbonylation of Hb as a starting material of HbV in the production process.

A novel disulfide-containing aniline, 1,4-dihydrobenzo[d] [1,2] dithiin-5-ylamin (1), was synthesized by the alkylbromination of 1,2-dimethyl-3-nitrobenzene, followed by the formation of a disulfide bond via thioesterification and then the reduction of the nitro group. The monomer showed a reductive and oxidative potential of the disulfide bond at -0.63 and 1.25 V (vs Ag/Ag+), respectively. Although the monomer showed the oxidative potential of aniline at 0.85 V (vs SCE), the electro- or chemical-oxidative polymerization of the monomer resulted in the formation of only the oligomer, suggesting that the disulfide-containing condensed ring would prevent further propagation of the polymerization due to steric hindrance. However, the nearly ideal copolymerization of 1 with aniline (An) was carried out by chemical oxidative polymerization. The resulting copolymers (1/An = 1/1 and 2/1, by mole) had weight-averaged molecular weights ((M) over bar (w)) of 10 000 and 6600, respectively and good redox activities coupled with those of the polyaniline and disulfide group. The copolymer showed conductivities of 5.1 x 10(-2) S/cm for 1/1 and 1.3 x 10-2 S/cm for 2/1 after being redoped with camphorsulfonic acid. It is suggested that these copolymers might be good candidates for the cathode materials of secondary polymer batteries.

A novel disulfide-containing aniline, 1,4-dihydrobenzo[d] [1,2] dithiin-5-ylamin (1), was synthesized by the alkylbromination of 1,2-dimethyl-3-nitrobenzene, followed by the formation of a disulfide bond via thioesterification and then the reduction of the nitro group. The monomer showed a reductive and oxidative potential of the disulfide bond at -0.63 and 1.25 V (vs Ag/Ag+), respectively. Although the monomer showed the oxidative potential of aniline at 0.85 V (vs SCE), the electro- or chemical-oxidative polymerization of the monomer resulted in the formation of only the oligomer, suggesting that the disulfide-containing condensed ring would prevent further propagation of the polymerization due to steric hindrance. However, the nearly ideal copolymerization of 1 with aniline (An) was carried out by chemical oxidative polymerization. The resulting copolymers (1/An = 1/1 and 2/1, by mole) had weight-averaged molecular weights ((M) over bar (w)) of 10 000 and 6600, respectively and good redox activities coupled with those of the polyaniline and disulfide group. The copolymer showed conductivities of 5.1 x 10(-2) S/cm for 1/1 and 1.3 x 10-2 S/cm for 2/1 after being redoped with camphorsulfonic acid. It is suggested that these copolymers might be good candidates for the cathode materials of secondary polymer batteries.

Background &amp
Aims: Liver is a major organ for heme detoxification under disease conditions, but its self-protective mechanisms against the toxicity are unknown. This study aimed to examine roles of carbon monoxide (CO), the gaseous product of heme oxygenase (HO), in ameliorating hepatobiliary dysfunction during catabolism of heme molecules in endotoxemic livers. Methods: Vascular resistance and biliary flux of bilirubin-IXα, an index of HO-derived CO generation, were monitored in perfused livers of endotoxemic rats. Livers were perfused with HbO2, which captures nitric oxide (NO) and CO, or metHb, a reagent trapping NO but not CO. Results: In endotoxin-pretreated livers where inducible NO synthase and HO-1 overproduced NO and CO, HbO2 caused marked vasoconstriction and cholestasis. These changes were not reproduced by the NO synthase inhibitor aminoguanidine alone, but by coadministration of zinc protoporphyrin-IX, an HO inhibitor. CO supplementation attenuated the events caused by aminoguanidine plus zinc protoporphyrin-IX, suggesting that simultaneous elimination of these vasorelaxing gases accounts for a mechanism for HbO2-induced changes. This concept was supported by observation that metHb did not cause any cholestasis
the reagent captures NO but triggers CO overproduction through rapid degradation of the heme by HO-1. Conclusions: These results suggest protective roles of CO against hepatobiliary dysfunction caused by heme overloading under stress conditions.

[Poly(ethylene-lycol)]-modified hemoglobin vesicles (PEG-HbV), a type of encapsulated hemoglobin, have been developed as artificial oxygen carriers and it is important to evaluate their blood compatibility. We studied the effects of PEG-HbV on human polymorphonuclear neutrophils (PMNs) in vitro, focusing on the functional responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP) as an agonist. The pretreatment of the PMNs with PEG-HbV up to a concentration of 60 mg/dl Hb did not affect the fMLP-triggered chemotactic activity. In parallel to these results, the fMLP-induced upregulation of CD11b (Mac-1) levels on the PEG-MV-pretreated PMNs was comparable to that of untreated cells. Furthermore, the pretreatment of the PMNs with the PEG-HbV even at 600 mg/dl Hb did not affect the gelatinase B (Matrix methalloproteinase-9 (MMP-9)) release, suggesting that the fMLP-induced release of secondary and tertiary granules was normal. In addition, the fMLP-triggered superoxide production of the PMNs was unchanged by the pretreatment with the PEG-HbV at 600 mg/dl Hb. Thus. these results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on the fMLP-triggered functions of human PMNs.

To increase the safety of stroma-free hemoglobin solution (SFH) as an artificial oxygen carrier source, we investigated the effect of heat treatment on virus inactivation in hemoglobin solution. The hemoglobin solution spiked with vesicular stomatitis virus (VSV) was treated at 60 degreesC for 1 hr under either an air or CO atmosphere. VSV was inactivated at >5.8 log(10) and >6.0 log(10) under the air and CO atmosphere, respectively. Although the methemoglobin rate increased after the heat treatment under the air atmosphere, no methemoglobin formation was observed by the treatment under the CO atmosphere. Isoelectric focusing analysis revealed the denaturation of hemoglobin after the heat treatment under the air, while hemoglobin banding was not altered in the carbonylated condition. Some protein bands other than hemoglobin were weakened or disappeared on SDS-PAGE after the heat treatment under both conditions. In addition, the hemoglobin concentration in the SFH was higher after the heat treatment than before the treatment. These findings indicate that the heat treatment under the CO atmosphere inactivates viruses without hemoglobin denaturation, and hence, this method is a promising approach to prepare a safer SFH as artificial oxygen carriers.

Albumin polymers, having an average diameter of 1020 ± 250 nm, were prepared by the disulfide polymerization of recombinant human serum albumin (rHSA) by controlling of the pH and temperature. Fibrinogen could be conjugated on the surface of an albumin polymer using N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Under flow conditions, the fibrinogen-conjugated albumin polymers (fibrinogen-albumin polymers) were irreversibly attached to the platelet-immobilized surface in the reconstituted blood at a low platelet concentration ([platelet] = 5.0 × 104/μL, a 5-fold diluted platelet concentration), and the attachment was suppressed by the addition of anti-GPIIb/IIIa monoclonal antibodies. It was confirmed that fibrinogen-albumin polymers specifically interacted with GPIIb/IIIa expressed on the surface of the activated platelets. Although platelets with a low platelet concentration were hardly attached to the platelet-immobilized surface under the flow conditions, the addition of fibrinogen-albumin polymers enhanced the attachment of the remaining platelets to the surface, indicating that the fibrinogen-albumin polymers would help the hemostatic ability of platelets at the site of vascular injury of patients in thrombocytopenia.

We studied the effects of hemoglobin-vesicles modified with PEG (PEC-HbV), a type of liposome-encapsulated hemoglobin (LEH), on human platelet functions in vitro. The effect of a low concentration of PEG-HbV (Hb; 5.8 mg/dl) was assessed by examining an agonist-induced aggregation response, and that of relatively high concentrations of PEG-HbV (Hb; 0.29, 1 and 2 g/dl) by measuring the release of RANTES (Regulated upon activation, normal T-cell expressed and presumably secreted) from platelets, which is regarded as a marker of platelet activation. The preincubation of platelets with PEG-HbV at 5.8 mg/dl of Hb did not affect platelet aggregation induced by collagen, thrombin and ristocetin. The pretreatment of platelet-rich plasma (PRP) with PEG-HbV at concentrations up to 2 g/dl of Hb had no aberrant effects on the collagen-induced RANTES release. Furthermore, the collagen-induced release of RANTES from PRP was not affected by longer incubation with PEG-HbV at 2 g/dl of Hb. The basal levels of RANTES from PRP were unchanged in the presence of PEG-HbV. These results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on platelet functions in the presence of plasma.

Ferric metHb can be photoreduced to the ferrous state by direct photoexcitation in the near-ultraviolet region. In this research, we studied the mechanism and facilitating conditions for the photoreduction and the resulting restoration of O-2 binding. MetHb in phosphate-buffered saline or pure water in a CO atmosphere was photoreduced to form HbCO by illuminating the N band (365 nm), one of the porphyrin pi --> pi* transitions, whereas the photoreduction did not occur in Ar, N-2, or O-2. The transient absorption spectrum exhibited the generation of deoxyHb within 30 ns in both the CO and Ar atmospheres; however, only in CO did the subsequent CO binding inhibit the back reaction. The photoreduction rate was dependent on the pH and ligand anions, showing that aquametHb in the high-spin state was predominant for the photoreduction. Axial ligand-to-metal charge-transfer (LMCT) bands overlap with the Soret and Q bands in metHb; however, the excitation of these bands showed little photoreduction, indicating that the contribution of these LMCT bands is minimal. Excitation of the N band significantly contributes to the photoreduction, and this is facilitated by the external addition of mannitol, hyaluronic acid, Trp, Tyr, etc. Especially, Trp allowed the photoreduction even in an Ar atmosphere, and the reduced Hb can be converted to HbO(2) by O-2 bubbling. One mechanism of the metHb photoreduction that is proposed on the basis of these results consists of a charge transfer from the porphyrin ring to the central, ferric iron to form the porphyrin pi cation radical and ferrous iron by the N band excitation, and the contribution of the amino acid residues in the globin chain as an electron donor or an electron pathway.

To conjugate water-soluble macromolecules on the surface of phospholipid vesicles, we synthesized a poly(ethylene glycol) (PEG)-lipid having four acyl chains using a lysine (Lys)-type monodendron structure. One end of the diamino-PEG was amidified with Lys, and then two amino groups of the Lys moiety were amidified with two Lys derivatives which had been acylated with two stearoyl groups. The other end of the PEG was activated with a triazine group or a pyridyldithio group. The hydrate of the lipid mixture of dipalmitoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol, and the PEG-lipid at a molar ratio of 5/5/1/0.3 was extruded in order to prepare the phospholipid vesicles with the average diameter of 270 +/- 20 nm. The coupling ratio of cytochrome c with the PEG-lipid was monitored by HPLC, detecting the pyridyl 2-thione liberated from the pyridyldithio group and determining it to be 26% on the basis of the incorporated PEG-lipid.

We synthesized a series of amphiphiles with poly(ethylene glycol) [MW 2000 (PEG20), 5000 (PEG50), 12 500 (PEG125)] as a headgroup and one, two, or four palmitoyl chains (1C16, 2C16, or 4C16), using a lysine monodendron as a connector. The relationship between the hydrophilic-hydrophobic balance of the multiacyl PEG-lipids and the physicochemical characteristics in self- or co-assembly with phospholipids were studied. The PEG-lipids were easily synthesized by combination of a general liquid-phase peptide synthesis and the acylation of an amino acid. The PEG part of the PEG-lipid films was crystallized to show a typical spherulite pattern. The thermal properties and microscopic observation revealed the phase separation of PEG and acyl chain parts. The critical micelle concentrations (cmcs) mainly depend on the number of acyl chains rather than the molecular weight of the PEG chain, although the area per molecule is dependent on the molecular weight of the PEG chain rather than the number of the acyl chains. The gel-to-liquid crystalline phase transition temperature was increased with the increasing number of acyl chains and the decreasing molecular weight of the PEG chain. The PEG-lipids in the aqueous dispersions assemble to take fibrous structures with bimolecular thickness because of the intermolecular hydrogen bonding. The PEG-lipids were immobilized onto the surface of the phospholipid vesicles by simply adding their aqueous dispersions to the vesicle dispersion; however, they dissociated from the vesicles on dilution of the mixed dispersion because they were incorporated into the vesicles in an equilibrium state. To prevent the dissociation of the PEG-lipids, at least two and four acyl chains were required for PEG with M-W 5000 and 12 500, respectively. The aggregation of the vesicles by the addition of water-soluble polymers was significantly inhibited with the increasing molecular weight of the PEG chain. For the tight immobilization of the PEG-lipids with the long PEG chain onto the vesicular surface, an increased number of acyl chains is necessary, and the surface modification with the long PEG chains significantly increases the dispersion stability of the vesicles.

Highly sulfonated poly(thiopenylenesulfonic acid) (PTPSA) was prepared from a soluble polysulfonium salt precursor and mixed with poly(alkylenecarbonate) (PAC, alkylene ethylene, PEG; propylene, PPC; butylene, PBC). The PTPSA and PAC were Employed as a proton source for conduction and a flexible base matrix :for proton dissociation and conduction pathway, respectively. For the composites of PTPSA/PAC (3/1, 2/1, 2/1 and 1/2, by weight), the effect of alkyl chain lengths of PAC, temperature dependence of proton-conductivity, and water content on the composites was studied. The proton-conductivity of the PTPSA/PAC composites Increased with the increasing PTPSA portion. The PTPSA/PBC (3/1) composite shows the highest proton-conductivity of 1.5 x 10(-6) Scm(-1) at 30 degreesC. However, the increased incorporation of PTPSA does not contribute to producing the higher proton conduction the composites. Moreover, the PTPSA/PBC composite including 15 wt% of water retained the proton-conductivity of 7.2 x 10(-2) Scm(-1) at 240 degreesC, which could be the conductivity comparative to the Nafion membrane and thermal stability. Copyright (C) 2000 John Wiley & Sons, Ltd.

The critical micelle concentrations of 1,2-dipalmitoyl-sn-glycero-3-phosphoethaanolamine-N-[monomethoxy poly(ethyleneglycol) (5000)] (PEG-DPPE) and its distearoyl analogue (PEG-DSPE) were 70 and 9 mu M, respectively, in buffer solutions ([Tris] = 20 mM, [NaCl] = 140 mM, pH 7.4) at 37 degrees C. When these PEG-Lipid micelle dispersions were mixed with the dispersions of phospholipid vesicles comprised of a C16 membrane, of which the carbon number is 16, or a C18 membrane, the PEG-lipid micelles were dissociated into monomers and then spontaneously incorporated into the surface of the preformed vesicles. The incorporation rates and the enthalpy changes during incorporation were measured with an isothermal titration microcalorimeter. The incorporation rate of PEG-DPPE was faster than that of PEG-DSPE, because the dissociation rate of the PEG-DPPE micelles was faster than that of PEG-DSPE micelles. The incorporation equilibrium constant of PEG-DSPE was larger than that of PEG;DPPE due to its slow dissociation rate from the membrane, caused by the stronger hydrophobic interaction. The combination of PEG-DSPE and the C18 membrane was the most thermodynamically stabilized pair. Furthermore, the dispersion stability of the surface-modified vesicles prepared by this spontaneous incorporation was analyzed by using the critical molecular weight of the polymer for the aggregation of vesicles. The aggregation of the vesicles was successfully supressed with an increase in the molecular weight of the PEG in the PEG-lipid and its incorporation ratio.

The stability of hemoglobin vesicles (HbV) as an oxygen infusion was tested during the storage for 1 year at 4, 23, and 40 degrees C. The surface of the HbV was modified with poly(ethylene glycol) (PEG), and the suspension was deoxygenated with nitrogen bubbling. The samples stored at 4 and 23 degrees C showed a stable dispersion state for 1 year, though the sample stored at 40 degrees C showed the precipitation and decomposition of vesicular components, a decrease in pH, and 4% leakage of total Hb after 1 year. The PEG chains on the vesicular surface stabilize the dispersion state and prevent the aggregation and fusion due to their steric hindrance. The original metHb content (ca. 3%) before the preservation gradually decreased to less than 1% in all the samples after 1 month due to the presence of homocysteine inside the vesicles which consumed the residual oxygen and gradually reduced the trace amount of metHb. The rate of metHb formation was strongly dependent on the partial pressure of oxygen, and no increase in metHb formation was observed due to the intrinsic stability of the deoxygenated Hb. Preservation at 4 and 23 degrees C slightly reduced P-50 (increased the oxygen affinity) from 38 Torr to 32 and 31 Torr, respectively. These results indicate the possibility that HbV suspension can be stared at room temperature for at least I year.

A series of hemoglobin (Hb)-based O(2) carriers, acellular and cellular types, were synthesized and their physicochemical characteristics were compared. The acellular type includes intramolecularly cross-linked Hb (XLHb), polyoxyethylene (POE)-conjugated pyridoxalated Hb (POE-PLP-Hb), hydroxyethylstarch-conjugated Hb (HES-XLHb), and glutaraldehyde-polymerized XLHb (Poly-XLHb). The cellular type is Hb-vesicles (HbV) of which the surface is modified with POE (POE-HbV). Their particle diameters are 7 +/- 2, 22 +/- 2, 47 +/- 17, 68 +/- 24, and 224 +/- 76 nm, respectively, thus all the materials penetrate across membrane filters with 0.4 mu m pore size, though only the POE-HbV cannot penetrate across the filter with 0.2 mu m pore size. These characteristics of permeability are important to consider-an optimal particle size in microcirculation in vivo. POE-PLP-Hb ([Hb] = 5 g/dL) showed viscosity of 6.1 cP at 332 s(-1) and colloid osmotic pressure (COP) of 70.2 Torr, which are beyond the physiological conditions (human blood, viscosity = 3-4 cP, COP = ca. 25 Torr). XLHb and Poly-XLHb showed viscosities of 1.0 and 1.5 cp, respectively, which are significantly lower than that of blood. COP of POE-HbV is regulated to 20 Torr in 5% human serum albumin (HSA). HES-XLHb and POE-HbV/HSA showed comparable viscosity with human blood. Microscopic observation of human red blood cells (RBC) after mixing blood with POE-PLP-Hb or HES-XLHb disclosed aggregates of RBC, a kind of sludge, indicating a strong interaction with RBC, which is anticipated to modify peripheral blood flow in vivo. On the other hand, XLHb and POE-HbV showed no rouleaux or aggregates of RBC. The acellular Hbs (P(50) = 14-32 Torr) have their specific O(2) affinities determined by their structures, while that; of the cellular POE-HbV is regulated by coencapsulating an appropriate amount of an allosteric effector (e.g., P(50) = 18, 32 Torr). These differences in physicochemical characteristics between the acellular and cellular types indicate the advantages of the cellular type from the physiological points of view.

Albumin microspheres (AMS), of which the average diameter is 240 ± 10 nm, were prepared by pH control and heat treatment. Cytochrome c and rGPIbα
a water-soluble fragment of the α chain of a recombinant platelet glycoprotein (GP)Ib containing a von Willebrand factor (vWf)-binding site were selected as a receptor protein. Cytochrome c was used as a probe protein for monitoring. Onto the surface of the AMS and those proteins, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was reacted through the amide linkage to obtain PD-AMS, PD-cytochrome c, and PD-rGPIbα, respectively. The latter two were further reduced to SH-cytochrome c and SH-rGPIbα by dithiothreitol and conjugated with PD-AMS by a thiol - disulfide exchange reaction. The resulting AMS contain cytochrome c or rGPIbα of about 25000 or 2500 molecules, respectively. The addition of ristocetin to the rGPIbα-AMS in the presence of vWf caused specific aggregation. Furthermore, the rGPIbα-AMS enhanced the ristocetin induced platelet aggregation in a low platelet concentration (4.0 × 107/mL).

A novel polyaromatic acid, poly(thiophenylenesulfonic acid) (PTPSA), prepared from a soluble precursor of poly(arylenesulfonium salt) was employed as a dopant for polyaniline (PAn). The electro-oxidative polymerization of aniline in PTPSA aqueous solution gives an electroactive PAn/PTPSA composite with three redox couples at 0.16, 0.53, and 0.71V (vs. SCE). Composites of different molar ratio ([SO3H]/[An] = 0.10, 0.21, 0.42, 0.50, 0.83, 1.67) was prepared in order to confirm the transformation from the emeraldine base to its PTPSA salt, where a quantitative doping reaction was observed by means of UV-VIS and IR measurements. The thennogravimetric analysis revealed that the composites do not decompose up to 200 degrees C in nitrogen atmosphere. The composite ([SO3H]/[An] = 0.50) shows an electrical conductivity of 1.5 S . cm(-1) at 30 degrees C and 3.0 x 10(-1) S . cm(-1) at 170 degrees C. The composite retains its conductivity after heating at 150 degrees C for 24 h.

The photophysical and photochemical properties of 5,10, 15,20-tetrakis{alpha,alpha,alpha,alpha-o-[2',2'-dimethyl-20'-((2"-(trimethylammonio)ethyl) phosphonatoxy)alkanamido]phenyl}porphinatozinc(II) (zinc lipidporphyrins, ZnLPs (C-10, C-18)) have been studied in homogeneous DMSO solution and compared with those of 5, 10,15,20-tetrakis{alpha,alpha,alpha,alpha-o-pivalamidophenyl}porphinatozinc(II) (ZnTpivPP) and tetrakis-phenylporphinatozinc(II) (ZnTPP). The fluorescence quantum yields of the ZnLPs were lower than that of ZnTPP, but their fluorescence lifetimes were relatively long. The electron transfer reactions from the photoexcited states of these Zn porphyrin complexes to several quinone derivatives were evaluated by fluorescence spectroscopy and laser hash photolysis. The efficiencies of oxidative quenching of the excited porphyrins via a dynamic process were significantly decreased by the presence of the bulky substituents on one side of the porphyrin macrocycle. This steric effect of the porphyrin side-chains was quantitatively examined by the Marcus classical treatment. (C) 1999 John Wiley & Sons, Ltd.

Amidic glycolipids, 1,5-bis-O-aIkyl-N-maltooligonoyl-L-glutamate (1), having various lengths of two hydrocarbon chains (carbon number, m: 14, 16, 18) and maltooligotose with (glucose unit, n: 3, 5, 7) and a N-glycosidic lipid, 1,5-bis-O-octadecyl-N-maltopentaonosyl-L-glutamate (2) have been synthesized. The assembling structures were analyzed by microscopic observation, such as negatively stained TEM, cryo-TEM, and AFM. The glycolipid 1a (rn,n. 14,5) showed a fiber-like structure in all the observed temperatures, while 1b (16,5) showed a fiber-like structure when the hydrating temperature was above the gel-to-liquid crystalline phase transition temperature (T-c; 45 degrees C) and a large disk-like structure when incubated below the T-c. The glycolipid 1c (18,5) took a large disk-like structure after hydration of the powder above the T-c. The glycolipids 1d (18,3) and 1e (18,7) showed a mixture of large disks and large vesicles and a mixture of small disks and micelles, respectively. The N-glycosidic lipid, 2, with no amide linkage made a vesicular structure only. The preparation procedure using high shear stress, such as extrusion and sonication, converted the large disk of 1c to smaller assemblies, such as small disk-, cone-, and granule-like assemblies, depending on the preparation conditions. The glycolipid molecules in the planer part of the disk were packed so tightly that molecular mobility was very low even above the T-c(58 degrees C), and the reactivity of the saccharide chain against Concanavalin A was also very low, indicating that the high reactivity probably comes from the loose packing of saccharide chains around the edge part of the assemblies.

Phospholipid vesicles encapsulating purified hemoglobin [Hb vesicles (HbV); diameter 259 +/- 82 mm; oxygen affinity 31 mm Hg; [Hb] 5 and 10 g/dL] were developed to provide oxygen-carrying capacity to plasma expanders. Their function as a blood replacement was tested in the subcutaneous microvasculature of awake hamsters during severe hemodilution in which 80% of the red blood cell mass was substituted with suspensions of the vesicles in 5% human serum albumin (HSA) solution. Vesicles were tested with membranes that were unmodified (HbV/HSA) or conjugated with polyethyleneglycol (PEG) on the vesicular surface (PEG-HbV/HSA). The viscosity of 10 g/dL HbV/HSA was 8 cP at 358 s(-1) owing to the intervesicular aggregation, while that of 10 g/dL PEG-HbV/HSA was 3.5 cP, since PEG chains inhibit aggregation. Both materials yielded normal mean arterial pressure, heart rate, and blood gas parameters at all levels of exchange, which could not be achieved with HSA alone. Subcutaneous microvascular studies showed that PEG-HbV/HSA significantly improved microhemodynamic conditions (flow rate, functional capillary density, vessel diameter, and oxygen tension) relative to unmodified HbV/HSA. Even though the enhancement of PEG modification did not achieve the functional characteristics of the blood-perfused microcirculation, PEG reduced vesicular aggregation and viscosity, improving microvascular perfusion relative to the unmodified type. These results highlight the significance of microvascular analysis in the design of red cell substitutes and the necessity of surface modification of HbV to prevent aggregation. (C) 1998 John Wiley & Sons, Inc.

A synthetic glycolipid, 1,5-bis-O-octadecyl-N-maltopentaonoyl-L-glutamate (1), was dispersed in aqueous media above its phase transition temperature to form sheet-like assemblies. They were changed to disk- and ellipsoid-like assemblies by extrusion and sonication, respectively. The structures, stabilities, and thermal properties were studied with microscopic, optical, and calorimetric methods. The disk-like assemblies were homogeneous and the most stable. They formed a unique complex with Concanavalin A (ConA) to become a rouleaux-like structure. (C) 1998 Elsevier Science Limited.

The hemoglobin vesicle (HbV) is a red cell substitute encapsulating purified concentrated Hb in a phospholipid vesicle. In order to improve the oxygen carrying capability of HbV, the pH value of the Hb solution should be adjusted to 7.0 in the HbV preparation, and then the pH value should be adjusted to 7.4 where HbV functions as an oxygen carrier, because the maximum value of [Hb]/[Lipid] was obtained in which the pH of the Hb solution was 7.0, and the metHb formation rate was suppressed in the pH 7.4. Generally, the pH control of the inner aqueous phase of HbV is difficult by changing the pH in the outer phase. We could control the pH of the Hb solution from 7.4 to 7.0 by dissolving CO2 into the Hb solution, and after the preparation of HbV, the pH of HbV is changed to 7.4 by reducing the pressure. The resulting pH-controlled HbV by CO2 gas showed a high [Hb]/[Lipid] value of 1.7 with a low rate of metHb formation.

The oxygen-releasing behavior of hemoglobin vesicles (HbV) was measured in order to study the difference in oxygen dynamics inside and outside the cellular Hb using a conventional stopped now method and a newly developed stopped flow flash photolysis method. The partial pressure of oxygen in the solution outside the HbV was monitored with the lifetime of the triplet state of meso-tetraphenylporphinatozinc(II) bound to human serum albumin excited by the laser flash. The change in the partial pressure of oxygen outside the HbV showed a biphasic profile and was slower than that inside the HbV. The first phase shows the oxygen-releasing process from Hb near the phospholipid bilayer membrane, and the second phase is considered the process in which oxygen diffuses to the bulk aqueous region and reaches the equilibrium value.

Human serum albumin (HSA) incorporating synthetic tetraphenylporphinatoiron(II) derivatives (FeP1 or FeP2) can bind and release oxygen reversibly under physiological conditions (in aqueous media, pH 7.4, 37 degrees C). The maximal binding ratio of FeP1/HSA was estimated to be eight, and the stepwise equilibrium constants for FeP1 binding to HSA (K-1-K-8) ranged from 1.2x10(6) to 1.3x10(4) M-1. The major binding sites of FeP1 are presumably identical to those of hemin, bilirubin and long-chain fatty acids. The O-2-binding ability of the HSA-FeP can be regulated by changing the molecular structure of the incorporated hemes. The half-lifetime of the O-2-coordinated FeP2 in HSA was significantly longer than that of HSA-FeP1.

A hydrophobic tetraphenylporphyrinatoiron(II) derivative bearing a covalently bound axial imidazole [Fe(II)P] was efficiently and noncovalently bound into human serum albumin (HSA) up to an average of eight Fe(II)P molecules per HSA molecule. The aqueous solutions of the HSA-Fe(II)P complex provided a reversible and relatively stable oxygen adduct under physiological conditions (pH 7.4 and 37 °C). The half-life of the oxygen adduct (τ( 1/4 )) was 1 h at 37 °C in an air atmosphere. With Fe(II)-TpivPP (the so-called 'picket-fence heme') having no axial base, an oxygenated HSA-Fe(II)TpivPP complex was obtained using a 20-fold molar excess of 1,2-dimethylimidazole, but the τ( 1/4 ) was very short (ca. 10 min at 37 °C). The oxygen affinity [P( 1/4 )(O2)] and oxygen transporting efficiency (OTE) of HSA-Fe(II)P at 37 °C were 30 Torr and 22%, respectively. Furthermore, the oxygen-binding and dissociation rate constants (k(on) and ko(off)) are extremely high in comparison with those of hemoglobin. The HSA molecule binding eight Fe(II)P molecules can transport about 3.4 mL/dL of oxygen under physiological conditions, corresponding to about 60 % of the oxygen transporting amount of human blood.

The hemoglobin vesicle (HbV) is a red cell substitute encapsulating purified concentrated Hb in a phospholipid vesicle. In order to suppress metHb formation or autoxidation, for the long-term maintenance of the oxygen transporting capability, a series of thiols (cysteine, Cys
glutathione, GSH
homocysteine, Hcy
and acetylcysteine, Acy) were studied as reductants of metHb. Hcy and GSH showed a good suppressive effect on metHb formation, while Cys adversely accelerates the metHb formation at a rate twice that of the Hb solution without any reductants and Acy showed no change. The significant suppression by the coaddition of superoxide dismutase (SOD) and catalase to Cys indicated that Cys was easily oxidized by oxygen and simultaneously generates a large amount of active oxygens. The effective suppression of metHb formation by SOD and catalase was not observed for HbV containing no reductants, indicating that the generation of active oxygens from Hb itself is not significant. The coencapsulation of Hcy with Hb resulted in a low rate of metHb formation in HbV (initial rate, 1%/h) in vitro at oxygen partial pressure (P(O2)) of 142 Torr. The rate increased with decreasing P(O2), showed a maximum (2.2%/h) around P(O2) = 23 Torr, and then decreased to 0%/h at 0 Torr. From these results, it is suggested that the fast metHb formation rate in the blood circulation of Wistar rats injected with 20 vol % of the HbV solution would be mainly caused by the exposure of HbV to the low P(O2).

Encapsulation of hemoglobin within a liposome is one of the strategies in the development of artificial oxygen carriers. It maintains the oxygen transporting properties of hemoglobin and, at the same time, eliminates the side effects of cell free hemoglobin. Hemoglobin vesicles (HbV) are a type of liposome encapsulated hemoglobin. They have a particle size of approximately 250 nm, a hemoglobin concentration of 10 g/dl, and the oxygen affinity, P50, is regulated to 32 Torr. In this study the authors examined the oxygen transporting capability of HBV in vivo, by performing exchange transfusions in rats. Exchange transfusion (90% of the estimated circulatory volume) with HbV suspended in 5% albumin (containing 160 mEq/L, sodium and 107 mEq/L, chloride) was carried out in mate Wistar rats. Mean arterial pressure and heart rate were monitored through the arterial catheter. Arterial blood samples for gas analyses were also obtained from the arterial catheter. Abdominal aortic blood flow was measured by an ultrasonic pulsed Doppler flowmeter as an indicator of cardiac output. The oxygen tension of blood withdrawn from the right atrium was measured as an indicator of mixed venous oxygen tension. These values were employed to calculate oxygen delivery and consumption. Renal cortical and skeletal muscle tissue oxygen tensions were monitored as indicators of tissue perfusion. Five percent albumin and washed rat red blood cells suspended in 5% albumin containing 10 g/dl of hemoglobin, were employed as controls. At the completion of a 90% exchange transfusion, renal cortical and skeletal muscle tissue oxygen tensions, along with oxygen delivery and consumption, were sustained almost equally well with the HbV suspension compared to the washed rat red blood cell suspension, but declined significantly with the albumin suspension. The results indicate that the oxygen transporting capability of HbV was almost equivalent to that of rat red blood cells.

Poly(ethylene glycol) (PEG(5000))-conjugated phosphatidylethanolamine was introduced onto the surface of hemoglobin vesicles (HbV); phospholipid vesicles encapsulating concentrated Hb (d = 0.257 +/- 0.087 mu m; P-50 = 32 Torr). The obtained PEG-modified HbV (HbV-PEG) was studied for use as a red cell substitute from the viewpoint of rheology, surface properties, and hemodynamics. The viscosity of the unmodified HbV suspended in saline ([Hb] = 10 g/dL) was 2.6 cP (shear rate = 358 s(-1), 37 degrees C), less than that of human blood (4 cP). However, when suspended in a 5 g/dL albumin solution (HbV/albumin), it increased to 8 cP due to the molecular interaction between albumin and vesicles, and the viscosity increased with decreasing shear rate, e.g., 37 cP at 0.58 s(-1). As for the HbV-PEG/albumin, on the other hand, the viscosity was 3.5 cP at 358 s(-1) and was comparable with that of human blood. Optical microscopy showed formless flocculated aggregates of the unmodified HbV, while no aggregates were confirmed for the HbV-PEG. The steric hindrance of PEG chains seemed to be effective in preventing intervesicular access and the resulting aggregation To estimate the flow profiles in the capillaries, the suspensions were allowed to penetrate through isopore membrane filters (pore size = 0.4-8 mu m, cf. capillary diameter = 4-10 mu m). The penetration rate of the HbV-PEG/albumin was higher than that of the unmodified HbV/albumin due to the suppression of aggregation, whereas both of them were significantly higher than that of human blood due to the smaller size of vesicles than RBC. Ninety percent exchange transfusion was performed with the HbV-PEG/albumin or HbV/albumin in anesthetized Wistar rats (n = 6). The blood flow in the abdominal aorta increased 1.5 times, and the total peripheral resistance decreased in the HbV-PEG/albumin-administered group in comparison with the HbV/albumin group. As for the blood gas parameters, the base excess and pH remained at higher levels in the HbV-PEG/albumin group, and the O-2 tension in mixed venous blood for the HbV-PEG/albumin group tended to be maintained at a higher level than that for the HbV/albumin group. Thus, the PEG modification of HbV reduced the viscosity by the suppression of aggregation and resulted in prompt blood circulation in vivo.

The hemoglobin vesicle (HbV) is a red cell substitute encapsulating purified concentrated Hb in a phospholipid vesicle. In order to suppress metHb formation for the long term maintenance of oxygen transporting capability in vivo, thiols (cysteine, Cys; homocysteine, Hey) were studied as reductants of metHb. Hey showed a suppressive effect on metHb formation, while Cys adversely accelerates metHb formation at the rate of twice the Hb solution without any reductants. The suppression of Cys-induced metHb formation by the addition of superoxide dismutase (SOD) and catalase indicated that Cys was easily oxidized by oxygen and simultaneously generated a large amount of active oxygens. The rate of metHb formation was influenced by PO2 and pH. Furthermore, the reducing systems (methylene blue (MB), NADH or ascorbic acid) were added to the outer aqueous phase of HbV, and the artificial reduction systems constructed through the bilayer membrane were evaluated.

To evaluate the oxygen transporting capability of Hemoglobin vesicles (HbV) the physiological responses to 40% and 90% exchange transfusions with HbV in anesthetized rat were observed. Hb concentration of HbV dispersions is 10g/dL. HbV dispersed in phosphate buffered saline and HbV dispersed in 5% albumin solution were used as samples for 40% and 90% exchange transfusions, respectively. HbV surface-modified with polyoxyethylene (HbV-Poe) was also used in the 90% exchange transfusion. As controls, phosphate buffered saline, 5% albumin solution, and HbV containing methemoglobin and therefore deprived of oxygen transporting capabilities (metHbV) were administered as non-oxygen carrying fluids and washed rat red blood cells (ratRBC) as an oxygen carrying fluid. Measurements included mean arterial pressure, arterial blood gas analyses, aortic blood flow and renal cortical tissue oxygen tension. At the completion of the exchange transfusion renal cortical tissue oxygen tensions along with oxygen delivery and consumption were sustained almost equally well with the HbV dispersion compared to the washed rat red blood cell dispersion, but declined significantly in the phosphate buffered saline and albumin solutions. These results indicated that the oxygen transporting capability of HbV was almost equivalent to that of rat red blood cells. in the HbV-Poe group, aortic blood flow was sustained higher in comparison to the HbV group. As for the blood gas parameters, pH and venous oxygen tensions in the HbV-Poe group tended to be higher than those in the HbV group.

The intervesicular aggregation of phospholipid vesicles is induced by the addition of water-soluble polymers such as poly(ethylene glycol), dextran, etc. due to the interaction between the vesicular surface and the water-soluble polymers. The interaction can be expressed by the critical molecular weight (M(c)) of the water-soluble polymers for the aggregation of vesicles. The surface modification of vesicles with glycolipids (O-1,O-5-bis(octadecyl) N-maltooligonoyl-L-glutamate) accelerates the aggregation of vesicles induced by dextran; therefore, M(c) significantly decreased due to the surface modification. No dependence of phospholipid concentration and dextran concentration in an aqueous phase on the M(c) indicates that dextran does not act as a cross-linking agent among the vesicles. A clear dependence of the density of the saccharide chains on the vesicular surface on the M(c) suggests that dextran should adsorb on the surface of the vesicles by the interaction with the oligosaccharide chains on the surface and cause vesicular aggregation. A lower critical solution temperature was observed for this kind of interaction, and the critical temperature was controlled by changing the molecular weight of dextran.

The two kinds of veri cell substitutes, hemoglobin-vesicles (HbV) and lipidheme-vesicles (LihV, totally synthetic oxygen carrier), were evaluated in terms of physicochemical properties such as binding and dissociating reactions of ligands (CO, O-2 and NO), rheological and structural properties. Carbonylation of Hb during the purification of Hb and the preparation of HbV is effective to prevent Hb denaturation. The rates of oxygenation of both HbV and LihV are faster than that of I ed blood cells (RBC). Their oxygen affinities (P-50, HbV, 32 mmHg; LihV, 43 mmHg, cf. RBC, 28 mmHg) can be controlled to transport a sufficient amount of oxygen comparable with that of RBC. The smaller sizes of vesicles are advantageous for prompt ligand reaction and low viscosity. Both HbV and RBC show about 100 times less vasoconstrictive effects than stripped Hb. HbV shows only one sixth of the slow binding rate of NO (=endothelial derived relaxation factor) in comparison with stripped Hb. Inhibition of vasoconstriction by those vesicles is discussed from the kinetic data.

Hemoglobin vesicles, which encapsulated concentrated hemoglobin (Hb) with a bilayer of dipalmitoylphosphatidylcholine/cholesterol/palmitic acid, were prepared under various preparation conditions in order to decrease the number of bilayers (n) constructing the vesicle and increase the Ho concentration in the interior of the vesicle ([Hb](in)). n decreased when the surface potential of the bilayer became more negative because of the electrostatic repulsion between the bilayers, while with a changing xi-potential of Hb from positive to negative, [Hb](im) showed a precipitous fall because of the electrostatic repulsion between Hb and the surface of the bilayer. A temperature decrease leads to a quality increase in the Hb vesicles ([Hb]/[lipid]) in spite of the [Hb](in) decrease by the viscosity increase of the Hb solution. This is explained by the effective reduction of n due to the reduction in membrane fluidity and the protonation of Hb.

Hemoglobin vesicles (HbV) as red cell substitutes were prepared from a purified carbonylhemoglobin (HbCO) solution and a lipid mixture composed of phospholipids, cholesterol, and cr-tocopherol. The diameter was controlled to 251 +/- 87 nm using an extrusion method; the vesicles penetrated through the membrane filters with regulated pore sizes. After the ligand exchanging reaction (HbCO --> HbO(2)), the oxygen affinity (P-50) Of HbV was 32 Torr, which was controlled with the coencapsulation of pyridoxal 5'-phosphate. The rate of metHb formation in HbV was nonenzymatically reduced with the coencapsulation of DL-homocysteine. The Hb concentration of the HbV suspension, which was dispersed in a phosphate buffered saline solution (pH 7.4), was controlled at 10 g/dL. At this concentration, the total lipid concentration was 6.2 g/dL and the viscosity, 2.6 cP (230 s(-1)), was lower than that of the blood (4.4 cP). The HbV suspension showed a typical non-Newtonian flow for a particle dispersion and agreed well with the Casson model. The viscosity at shear rates lower than 23 s(-1) showed a maximum with increasing the mixing ratio of human blood, plasma, or albumin, while no maximum was observed for the mixture with washed red blood cells. The aggregates of HbV are formed by interaction with plasma proteins, including albumin, while the aggregates reversibly dissociate at higher shear rate.

Hemoglobin vesicles (HbV) as red cell substitutes were prepared from a purified carbonylhemoglobin (HbCO) solution and a lipid mixture composed of phospholipids, cholesterol, and cr-tocopherol. The diameter was controlled to 251 +/- 87 nm using an extrusion method; the vesicles penetrated through the membrane filters with regulated pore sizes. After the ligand exchanging reaction (HbCO --> HbO(2)), the oxygen affinity (P-50) Of HbV was 32 Torr, which was controlled with the coencapsulation of pyridoxal 5'-phosphate. The rate of metHb formation in HbV was nonenzymatically reduced with the coencapsulation of DL-homocysteine. The Hb concentration of the HbV suspension, which was dispersed in a phosphate buffered saline solution (pH 7.4), was controlled at 10 g/dL. At this concentration, the total lipid concentration was 6.2 g/dL and the viscosity, 2.6 cP (230 s(-1)), was lower than that of the blood (4.4 cP). The HbV suspension showed a typical non-Newtonian flow for a particle dispersion and agreed well with the Casson model. The viscosity at shear rates lower than 23 s(-1) showed a maximum with increasing the mixing ratio of human blood, plasma, or albumin, while no maximum was observed for the mixture with washed red blood cells. The aggregates of HbV are formed by interaction with plasma proteins, including albumin, while the aggregates reversibly dissociate at higher shear rate.

1,2-Bis(2,4-octadecadienoyl)-sn-glycero-3-phosphocholine (DODPC), constructing the bilayer membrane of a vesicle, was polymerized by UV irradiation. The freeze-dried vesicles were completely dissolved in chloroform to obtain cross-linked phospholipid polymer (polyDODPC). The polyDODPC consisted of high molecular weight components and oligomers. The polyDODPC was dispersed by sonication, and two size distributions of the reconstituted vesicles were confirmed, larger vesicles from high molecular weight polyDODPC and smaller vesicles from oligomers. When the polyDODPC vesicles of various sizes and unimodal size distribution were reconstituted by an extrusion method, the vesicles of the same size as the vesicles at polymerization showed excellent stability compared with the other sizes. Interestingly, the original size of the polyDODPC vesicles could be completely restored by a cholate dialysis method. It was also clarified that vesicles larger than the original size were reconstituted from the higher molecular weight component, and the smaller vesicles were from the oligomers. Furthermore, a linear phospholipid polymer obtained from 1-palmitoyl-2-(2,4-octadecadienoyl)-sn-glycero-3-phosphocholine could not restore the original size. Those results indicate that the original size of the vesicles can be restored completely by all polyDODPCs having the original composition of large and small molecular weights and cross-linked structure.

Three single chain lithocholic acid derivatives with a steroidal moiety in the center of the hydrocarbon chain and two head groups at both ends of the chain were synthesized and characterized. Due to their high compatibility with phospholipids, they can be incorporated homogeneously into the phospholipid bilayer membrane. The resulting mixed vesicles showed high stability against aggregation by monitoring the turbidity change in the vesicle suspensions. The entire membrane-spanning packing thus locates the steroidal moiety at the center of the bilayer. The interaction of the steroidal moiety with Lipid molecules at the center of the bilayer resulted in higher mobility of lipid molecules below the T-c of the host phospholipid membrane as clarified by temperature-dependent H-1 NMR, fluorescence depolarization anisotropy, and differential scanning calorimetry (DSC). The results were compared with those of cholesterol, whose steroidal moiety is located near the surface of the membrane.

In order to change carbonylhemoglobin (HbCO) to oxyhemoglobin(HbO(2)), the photodissociation of HbCO and O-2-binding to hemoglobin were studied using Hb liquid membrane system in O-2 atmosphere. The reduction rate of HbCO increased with illuminance and saturated nearly at 2.0 . 10(5) 1x. under a constant O-2 pressure of 760 Torr. The reduction rate of HbCO was a 5-fold low when the partial O-2 pressure was decreased from 760 to 0 Torr under the illuminance. In the static liquid membrane the exchanging rate of CO ligand in HbCO was decreased with the increment of the thickness of the liquid membrane, while the stirred liquid membrane showed a reverse result. The ligand exchange apparatus was made based on the obtained results, and the high flow rate of Hb solution enhanced the reduced amount of HbCO. Moreover, when the ligand exchange of Hb-vesicle dispersion was compared with an Hb solution, it proceeded more efficiently due to the encapsulation of concentrated Hb into a small compartment dispersed in a buffer solution.

Solid hemoglobin (Hb) films were prepared by casting from a mixed solution of human hemoglobin and maltose (wt ratio of Hb/maltose is 60/40). The behavior of O-2 and CO in coordinating to Hb in the films was then observed. The coordination equilibrium constants of both ligands were smaller by about an order of magnitude, and the rate constants (K-on, and k(off)) were smaller by about 13 orders of magnitude, than those of aqueous solutions of Hb. Interestingly, O-2 and CO saturation was limited to 52% and 86%, respectively, under a partial pressure of 760 Torr (1 Torr=133.322 Pa) of each gas, in spite of the one-to-one ligand binding ratio in aqueous solutions. This is due to the restraint of the structural change from a tense to a relaxed state in the solid phase. The limited saturation of the coordination in the solid phase was found to increase to 100% when a small amount (5.9%) of water was added; a significant structural change from the tense to the relaxed states followed.

A lithocholic acid derivative having high compatibility with phospholipid was synthesized and incorporated to the phospholipid bilayer membrane. A significant influence of it on the molecular packing and the stability enhancement of the vesicle suspension were clarified by a fluorescence depolarization method and a turbidity measurement, respectively.

The suppression of methemoglobin (metHb) formation by glutathione (GSH) is difficult because GSH is oxidized not only by the reduction of metHb, but also by oxygen to generate active oxygen species, such as superoxide (O2-.) and hydrogen peroxide (H2O2), which contribute to metHb formation. An effective nonenzymatic reduction of metHb was achieved at a high concentration of Hb (40 wt%, 2.48 x 10(-2) M subunits (1 M = 1 mol dm-3)) because the reduction of metHb was accelerated, and at a low partial pressure of oxygen (pO2 = 40 Torr (1 Torr = 133.322 Pa)) because the oxidation of GSH was effectively suppressed. Hb-vesicles, which encapsulate concentrated Hb (40 wt%, 2.48 x 10(-2) M subunits), transport oxygen in the blood stream at relatively low pO2 (110 Torr in artery and 40 Torr in venous, 58 Torr in average). The rate of metHb formation in Hb-vesicles was effectively decreased from 3.8 x 10(-7) to 1.6 x 10(-7) M s-1 by coencapsulating GSH at 58 Torr.

In order to prepare Hb-vesicles having a high oxygen-transporting ability, it is important to encapsulate concentrated Hb with a mixed lipid membrane having low lamellarity (n). The concentration of Hb in the Hb-vesicles ([Hb](in)) increased with the Hb concentration used for the preparation of Hb-vesicles. Hb-vesicles with smaller lamellarity could be obtained y lowering the preparation temperature. The encapsulation of Hb was significantly influenced by the ionic strength and the solution pH because lipids and Hb are electrolytes. The lamellarity decreases with increasing solution pH because of electrostatic repulsion between the surfaces of lipid membranes. On the other hand, [Hb](in) increases when the solution pH is lowered below the pI of Hb (7.0 at 25 degrees C) due to electrostatic interaction between Hb and the surface of the membrane. This interaction decreases with the ionic strength of the Hb solution, leading to a lower [Hb](in).

The performance of Hb-vesicles depends on the weight ratio of Hb to lipid ([Hb]/[Lipid]). This value is improved by lowering the number of bilayer membrane of the vesicle and raising the concentration of Hb in the interior of the vesicle. Maximum [Hb]/[Lipid] ratio was obtained at ca. pH 7, that would relate to the isoelectric point (pl) of Hb at 25 degrees C. On the other hand, the [Hb]/[Lipid] ratio decreased with ionic strength and increased with lowering temperature. The Hb-vesicles encapsulating 40 g/dl Hb with only one bilayer membrane were isolated by using the difference in the density of the vesicles.

Hb小胞体中の高純度Hbのメト化抑制のために、α-tocopherol(α-Toc)とGlutathione(GSH)の使用を検討した。卵黄レシチンを膜成分として用いた系では、α-Toc添加により脂質の過酸化が抑制され、met-Hb生成速度は低下する。しかしDPPCなどの飽和型脂質を用いた系の方がmetHb生成速度は低くなる。<br>GSHはmetHbを還元する一方、酸素酸化過程で生じる活性酸素種が逆にmetHb生成を促進する。Hb濃度を高くして還元速度を高め、酸素分圧を低くしてGSHの酸素酸化を抑制することにより、GSHによるmetHb生成を効果的に抑制した。Hb溶液系で最適なGSH添加条件を明らかにし、これをHb小胞体に適用することによりmetHb生成速度をGSH未添加系の半分以下に低下させることができた。

ヘモグロビン小胞体の二分子膜成分に合成glycolipidを導入し、オリゴ糖鎖で小胞体表面を修飾した。小胞体表面から水相に伸びたオリゴ糖鎖の排除体積効果のため、Ca²⁺(5mM)やデキストラン(平均分子量40,000)添加により惹起される小胞体の凝集を抑制することができた。更に凍結融解後も粒径分布は変化はなく、ヘモグロビンの漏出、酸素親和度の変化は殆ど認められなかった。従って合成glycolipidの導入により、凍結保存可能なヘモグロビン小胞体が調製できた。

Polypyrrole (PPy) formation in the interior of a porous electrode by electropolymerization was considered to be impossible. When a concentrated pyrrole solution was soaked into a sintered-Ta electrode and electrolyzed in a pyrrole-free solution, PPy was formed in the interior. PPy occupied 70% of the inner space, and no defect was observed by SEM.

Both PPy film and Ta2O5 layer are simultaneously formed on Ta-electrode by one process of electrical oxidation in an aqueous solution. Prepara tion of the structure (PPy/Ta2O5/Ta) can also takes place by the electrolysis using Ta-electrode on which PPy is formed previously by electropolymerization. Since PPy film obtained by the former method has higher electric conductivity (110 S/cm) and better adhesion than the later, (PPy/Ta2O5/Ta)structure formed simul taneously is confirmed better electrical properties as a capacitor.

A tantalum electrode on which polypyrrole (PPy) had been previously formed by electropolymerization was galvanostatically electrolyzed in an aqueous solution of 0.01 wt% phosphoric acid. This process contains the irreversible oxidation of a PPy film, the decomposition of solvent, and the formation of Ta2O5 by the reaction of OH− coming through the PPy film, with Ta electrodes. A three layer‐structure (PPy/Ta2O5/Ta) was confirmed by electron spectroscopy for chemical analysis (ESCA). A PPy film containing CIO4− as dopant [PPy(CIO4−)] was significantly deteriorated in comparison with PPy(TsO−) at the electrolysis. Therefore, the (PPy(TsO−)/Ta2O5/Ta) system showed better electrical characteristics as a capacitor than the (PPy(CIO4−)/Ta2O5/Ta) system showed better electrical characteristics as a capacitor than the (PPy(ClO4−)/Ta2O5/Ta) system. Copyright © 1993 John Wiley &amp
Sons, Ltd.

脂質分子膜で包まれたHb小胞体の酸素輸送効率を向上させるため、少ない層数(n)の分子膜で高濃度Hb溶液を被覆した小胞体調製が課題となる。層数(小胞体粒径200nmφ)と小胞体内のHb濃度([Hb]_in)を測定する方法を確立し、調製条件との関係を解析した。その結果、Hb濃度増大に伴って[Hb]_inは向上し、低温調製(4℃)により層数は低下しHb内包効率([Hb]/[Lipid]比)は向上する。また、この系は電解質系であるため、系のpHやイオン強度(塩濃度)の影響を受ける。[Hb]/[Lipid]比は等電点付近(pI=7.0)で最も高くなり、イオン強度が高くなると低下する。

1,2-Di-O-octadecyl-3-O-alpha(beta)-maltopentaosyl-rac-glycerol (DOMPG) was incorporated into lipid vesicles to modify the surface of the vesicles. The aggregation of small unilamellar vesicles below the gel-to-liquid crystalline phase transition temperature and the aggregation of negatively-charged vesicles by the addition of calcium ion were effectively inhibited.

Electropolymerization of pyrrole on tantalum (Ta) electrodes was carried out in Britton-Robinson buffer solutions containing 0.1 M (1 M=1 mol dm-3) potassium chloride (KCl) under galvanostatic conditions. A polypyrrole (PPy) film and a tantalum oxide (Ta2O5) layer are formed on a Ta electrode by one electrochemical oxidation process. The conditions of this simultaneous formation were investigated in respect with the current density, pyrrole concentration, pH, and total charge. Under certain conditions ([py]=0.5 M, pH=1.8, current density=3.5 mA cm-2, charge 1 C), a 5.6-mu-m thick PPy film was efficiently formed on a homogeneous 45 nm thick Ta2O5 layer.

The ion-ion interaction of the ion pairs between radical anions of pi-conjugative molecules (polynuclear aromatic hydrocarbons, AR) such as anthracene, phenanthrene or naphthalene, and sodium ion in CH3(OCH2CH2)xOCH3 was evaluated from ionic conductivity measurement. The rate of ion-dissociation increased as either the number of (oxyethylene) unit(x) or the electron affinity of AR increased. This indicates that the continuous (oxyethylene) units are more favorable for ion-dissociation and a molecule having higher ability to become a radical anion shows higher rate of dissociation.

Polymerized phospholipid vesicle encapsulating Hb (polylipid/Hb vesicle) was prepared from a mixture of unsaturated phospholipid, cholesterol and unsaturated fatty acid and polymerization by gamma-ray irradiation. The average radius of resulting vesicles was 203 +/- 39 nm and concentrated Hb (30 wt%) was efficiently encapsulated. gamma-Ray polymerization proceeds theoretically at low temperature (4-degrees-C). P50 and oxygen transporting efficiency were adjusted to 40 mmHg and 40%, respectively. Oncotic pressure and solution viscosity can be controlled to the same values as blood.

The carbonyl hemoglobin (CO‐Hb), which was used to prevent denaturation (metHb) during the preparation of samples, was encapsulated into lipid vesicles constituted from unsaturated phospholipid, cholesterol and unsaturated fatty acid. Unsaturated components were polymerized by γ‐irradiation to enhance the stability of bilayer membrane. An aqueous dispersion of resulting Hb vesicles was freeze‐dried in the presence of saccharides (50–200 mM) to obtain a dehydrated powder of Hb vesicles. Change in the vesicle size, the leakage of encapsulated Hb and the oxidation of Hb to metHb were not observed. Therefore, the long‐term storage of Hb vesicles can be realized as a dry powder. Copyright © 1992 John Wiley &amp
Sons, Ltd.

Ion‐conductive polymer which shows an ionic conductivity (σi) of 1.4 × 10−4S/cm at 25°C when mixed with LiClO4 (molar ratio in Li/OE = 0.05) was used as a separator of electrodes in a lithium secondary battery. The effect of high ionic conductivity on the performance of the battery was studied. The polymer structure was $$ \\left[ {\\rm H}\\rlap{-} ({\\rm CH}_2 {\\rm CHR}\\rlap{-} )_{\\rm n} {\\rm H},{\\rm R} = - {\\rm CH}_2 ({\\rm OCH}_{\\rm 2} {\\rm CH}_{\\rm 2} )_6 {\\rm OCH}_3 \\right],$$ and the cathode was comprised of poly(1,3,4‐thiadiazole disulfide), graphite powder and the polymer electrolyte. The cell [(−)Li/polymer electrolyte/graphite–poly(disulfide) (+)] had an open circuit voltage (Voc) of 3.25 V, a plateau voltage of 2.75 V, a discharge density (id) of 0.05 mA/cm2 with the cathode utilization of 63%, and achieved over several tens of cycles at 25°C. Copyright © 1992 John Wiley &amp
Sons, Ltd.

Electropolymerization of pyrrole was carried out on tantalum as a base metal electrode. The Ta electrode retards polymerization at lower pyrrole concentrations because of the formation of insulating Ta2O5. However, at high concentrations of pyrrole, the polymerization proceeds efficiently to yield a polypyrrole (PPy) film, similar to that on a Pt electrode. The PPy film on a Ta electrode shows the same electroactivity and electric conductivity as that on a Pt electrode. Electropolymerization on Ta is accompanied by a decrease in electrode area due to the formation of the Ta2O5, the behavior of which is the only difference from that on Pt.

A composite film of Na+-form Nafion and diendoacetylated poly(oxyethylene) (MBAR(w) = 400) (POE400E) was cast from a dimethylformamide solution at 120-degrees-C. The ionic conductivity of this thin film was measured. The resulting film showed good flexibility, transparency, and no leakage of POE400E up to the POE incorporation of 55 wt%. At this composition, sodium ionic conductivity was 1.0 x 10(-6) S cm-1, which is relatively high as a single-ion conductor because of the high dissociation of the sodium perfluorosulfonate groups. The ionic conductivity increases with POE incorporation. This can be explained from a decrease in the glass transition temperature (T(g)) of POE regions and the connection of ion clusters swollen by POE.

Poly(oxyethylene) (POE) was incorporated into the ionic clusters of ionomer, ethylene and methacrylic acid (7.2% neutralized with KOH) copolymer membrane. The changes of properties were studied from SAXS, DSC, IR and ionic conductivity. The IR study suggested that the coordinated structures in ionic clusters of the membrane were destroyed by POE incorporation, and also SAXS suggested that ionic clusters were swollen by POE incorporation. The ionic conductivity, a carrier being K+ in this system, increases from 10(-16) S/cm to 10(-9) S/cm at 30 degrees C by the incorporation of POE (20.5 wt%). On the other hand, a large amount of POE (63 wt%) could be incorporated into ionomer membrane by the esterification of methacrylic acid groups (93%) with POE. When LiClO4 was added, ionic conduction occurred in the phase-separated POE domain, which had a low glass transition temperature (-55.2 degrees C), showing an ionic conductivity 2.6 x 10(-6) S/cm at 25 degrees C.

The mixed liposomes, composed of a polymerizable lipid, 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phosphorylcholine (DODPC), and nonpolymerizable membrane constituents, DPPC, cholesterol, and sodium didodecyl phosphate, were prepared by an extrusion method. After polymerization, nonpolymerizable constituents were removed to obtain the polymerized framework of the liposome (the skeletonized liposome). The release of small molecules from the skeletonized liposomes through resulting holes was analyzed. A 5(6)-carboxyfluorescein (CF) and saccharides with various molecular weights were applied as release molecules. The molecular weight of dextran, whose retention ratio in the skeletonized liposomes is 50%, relates to the apparent size of the holes, i.e., the apparent domain size of the phase separation of a polymerized mixed liposome. The size of the holes increases with an increase in the mole fraction of nonpolymerizable lipids. This also depends on the polymerization temperature and the structure of nonpolymerizable lipids.

In order to improve the ionic conductivity of solid polymer electrolyte by controlling ion(alkali metal ion)–dipole(ether oxygen) interaction, two kinds of modification were adopted on oligo(oxyethylene) (OOE). One is the capping of terminal hydroxyl groups of OOE with methyl or acetyl groups. The other is the replacement of the center ethylene group of OOE with methylene or propylene group. Ion–dipole interaction was analyzed by measuring the ion dissociation, ion conduction and Tg of Nafion/modified OOE composite films. The modification of the end groups was more effective than that of the center group in increasing ionic conductivity. The methyl group is superior to the acetyl group as the end group of OOE for lithium ion conduction. Copyright © 1991 John Wiley &amp
Sons, Ltd.