Protein Preferential Solvation in (Sucralose + Water) Mixtures.

Addition of sugars such as sucrose to aqueous protein solutions generally stabilizes proteins against thermal denaturation by preferential exclusion of sugars from proteins (preferential hydration of proteins). In this study, we investigated the effect of sucralose, a chlorinated sucrose derivative, on protein stability and preferential solvation. Circular dichroism and small-angle X-ray scattering measurements showed that sucrose increased the denaturation temperature of myoglobin and was preferentially excluded from the protein, whereas sucralose decreased the denaturation temperature of myoglobin and was preferentially adsorbed to the protein. No clear evidence was obtained for the indirect effects of sucralose on protein destabilization via the structure and properties of solvent water from the physicochemical properties (mass density, sound velocity, viscosity, and osmolality) of aqueous sucralose solutions; therefore, we concluded that a direct protein-sucralose interaction induced protein destabilization.

Gelation and Orientation Dynamics Induced by Contact of Protein Solution with Transglutaminase Solution.

Gel growth induced by contact of polymer solutions with crosslinker solutions yields an emerging class of anisotropic materials with many potential applications. Here, we report the case of a study on the dynamics in forming anisotropic gels using this approach with an enzyme as a trigger of gelation and gelatin as the polymer. Unlike the previously studied cases of gelation, the isotropic gelation was followed by gel polymer orientation after a lag time. The isotropic gelation dynamics did not depend on concentrations of the polymer turning into gel and of the enzyme inducing gelation, whereas, for the anisotropic gelation, the square of the gel thickness was a linear function of the elapsed time, and the slope increased with polymer concentration. The gelation dynamics of the present system was explained by a combination of diffusion-limited gelation followed by free-energy-limited orientation of polymer molecules.

Preparation of Various Nanomaterials via Controlled Gelation of a Hydrophilic Polymer Bearing Metal-Coordination Units with Metal Ions.

We investigated the gelation of a hydrophilic polymer with metal-coordination units (HPMC) and metal ions (PdII or AuIII). Gelation proceeded by addition of an HPMC solution in N-methyl-2-pyrrolidone (NMP) to a metal ion aqueous solution. An increase in the composition ratio of the metal-coordination units from 10 mol% to 34 mol% (HPMC-34) increased the cross-linking rate with AuIII. Cross-linking immediately occurred after dropwise addition of an HPMC-34 solution to the AuIII solution, generating the separation between the phases of HPMC-34 and AuIII. The cross-linking of AuIII proceeded from the surface to the inside of the HPMC-34 droplets, affording spherical gels. In contrast, a decrease in the ratio of metal-coordination units from 10 mol% to 4 mol% (HPMC-4) decreased the PdII cross-linking rate. The cross-linking occurred gradually and the gels extended to the bottom of the vessel, forming fibrous gels. On the basis of the mechanism for the formation of gels with different morphologies, the gelation of HPMC-34 and AuIII provided nanosheets via gelation at the interface between the AuIII solution and the HPMC-34 solution. The gelation of HPMC-4 and PdII afforded nanofibers by a facile method, i.e., dropwise addition of the HPMC-4 solution to the PdII solution. These results demonstrated that changing the composition ratio of the metal-coordination units in HPMC can control the gelation behavior, resulting in different types of nanomaterials.

Formation of Multi-Channel Collagen Gels Investigated Using Particle Tracking Microrheology.

Collagen is one of the most common materials used to form scaffolds for tissue engineering applications. The multi-channel collagen gel (MCCG) obtained by the dialysis of an acidic collagen solution in a neutral buffer solution has a unique structure, with many capillaries of diameters several tens to a few hundred micrometers, and could be a potential candidate as a biomimetic scaffold for three-dimensional tissue engineering. In the present study, the formation of MCCG was investigated by in situ rheological measurements based on a particle tracking method (particle tracking microrheology, PTM). PTM enabled us to measure changes in the rheological properties of collagen solutions under the continuous exchange of substances during dialysis. When an observation plane was set perpendicular to the direction of gel growth, we first observed convectional flow of the collagen solution, followed by phase separation and gelation. We showed that the structure of the MCCG originated from the transient structure formed during the initial stage of viscoelastic phase separation and was fixed by the subsequent gelation.

Gel Volume Near the Critical Point of Binary Mixture Isobutyric Acid-Water.

The volume of a cylindrical polyacrylamide gel was measured when immersed in a binary mixture of isobutyric acid-water at different temperatures and weight fractions of isobutyric acid. Near the upper critical solution temperature of the binary mixture, the curve for gel volume vs. isobutyric acid weight fraction has a shoulder or a peak near the critical weight fraction. On the other hand, in a region away from the critical temperature, the gel volume decreased monotonically with increasing isobutyric acid weight fraction. The cloud point temperature of the binary mixture inside the gel was lower than that outside the gel. Thermodynamic description for the gel in the critical mixture is derived on the basis of the Ising model. By the description, the experimental results are explained consistently. The theoretical analysis shows that the shoulder and the peak appearing in the swelling behavior of the gel are respectively induced by the criticalities of the binary mixture outside and inside the gel. It also shows that the cloud point temperature lowering of the binary mixture inside the gel is attributed to the effective enhancement of the temperature of the binary mixture inside the gel induced by the presence of the gel polymer.

Adsorption dynamics of tannin on deacetylated electrospun Konjac glucomannan fabric.

A nonwoven fabric of Konjac glucomannan (KGM) for the adsorption of tannin was fabricated by using electrospinning and then followed by deacetylation with alkaline solution. To analyze the adsorption dynamics of tannin, the time course of the adsorption of tannin on the nonwoven KGM fabric was measured by immersing the fabric in tannin solution at different concentrations of tannin and amounts of the fabric. The initial and late stages of the adsorption behavior could be expressed, respectively, by using a diffusion-limited equation and a stoichiometric equation. A discussion on the dependence of the control parameters on the adsorption behavior is presented. The results represent the first step to provide an effective adsorption procedure for tannin in the use of modified KGM fabric.

Temperature Dependence of Electrophoretic Mobility and Hydrodynamic Radius of Microgels of Poly(N-isopropylacrylamide).

Electrostatic interactions in charged microgels, which are dominated by the microgel net charge, play a crucial role in colloidal stabilization and loading of small, charged molecules. In this study, the temperature dependences of electrophoretic mobility µ and hydrodynamic radius Rh were measured for a slightly ionized poly(N-isopropylacrylamide) (PNIPA) microgel in a dilute suspension. A decrease in Rh was observed in the temperature range between 30 °C and 35 °C, corresponding to the lower critical solution temperature of PNIPA, and an increase in |µ| was observed in a higher temperature range between 34 °C and 37 °C. The analysis based on electrophoresis theory for spherical polyelectrolytes indicated that the net charge of the microgel decreased as the microgel was deswollen.

Small-angle X-ray and light scattering analysis of multi-layered Curdlan gels prepared by a diffusion method.

Curdlan, a microbial polysaccharide, forms a multi-layered gel consisting of four layers with different turbidity when its alkaline solution is dialyzed against aqueous solutions containing Ca2+ (diffusion-set gel). The present study clarified the microstructure of each layer of the diffusion-set Curdlan gel by small-angle X-ray scattering (SAXS) and small-angle light scattering (SALS). The SAXS data showed that Curdlan chains assume a helical ordered conformation in the gel and that the gel consists of the fibrils formed by the association of Curdlan chains and the aggregates of fibrils. The SAXS results also indicated that the gelation is induced by the formation of a network of Ca2+-cross-linked fibrils in the outer region of the gel, whereas by the network formation of the aggregation of fibrils in the neutralization process in the inner region of the gel. A structural anisotropy of the gel was investigated by analysis of two-dimensional SAXS images, showing that the fibril is oriented circumferentially in the outer region of the cylindrical gel, whereas it is oriented randomly in the inner region of the gel. The SALS data showed that a characteristic length of an inhomogeneous structure in the turbid layers is of the order of micrometers. The observed spatial variation of the microscopic structure is caused by the difference in the paths of pH and [Ca2+] traced in the gelation process.

Study of plasma coagulation induced by contact with calcium chloride solution.

Blood coagulation capability is one of the most important factors for the diagnosis of patients with thrombosis. Regarding the blood coagulation as an example of gelation of soft matter, we can apply an analytical method to this phenomenon and pick up some relevant parameters. In various systems, gelation dynamics induced by contact between a polymer solution and a crosslinker solution are well explained by the "moving boundary picture" (Yamamoto et al., J. Phys. Chem. B, 2010, 114, 10002-10009). The aim of this paper is to clarify whether this picture can be applied to a clinically important biological system used for blood coagulation tests. We have measured the time course of the thickness of a plasma gel layer formed when plasma comes in contact with calcium chloride solution in a rectangular cell and analyzed theoretically on the basis of the moving boundary picture. The entire process was well expressed using a scaled equation involving three parameters characterizing the plasma, k, Kin, and ß, where k is the time required to reach the incipient stage of three-dimensional network formation, the parameter Kin is proportional to calcium chloride concentration and ß is a constant. These results indicate the direct applicability of the general theory of gelation dynamics induced by contact between two solutions to the in vitro coagulation (gelation) of plasma, and the fitting parameters may be used for diagnosis.

Conformation of membrane-bound proteins revealed by vacuum-ultraviolet circular-dichroism and linear-dichroism spectroscopy.

Knowledge of the conformations of a water-soluble protein bound to a membrane is important for understanding the membrane-interaction mechanisms and the membrane-mediated functions of the protein. In this study we applied vacuum-ultraviolet circular-dichroism (VUVCD) and linear-dichroism (LD) spectroscopy to analyze the conformations of α-lactalbumin (LA), thioredoxin (Trx), and ß-lactoglobulin (LG) bound to phosphatidylglycerol liposomes. The VUVCD analysis coupled with a neural-network analysis showed that these three proteins have characteristic helix-rich conformations involving several helical segments, of which two amphiphilic or hydrophobic segments take part in interactions with the liposome. The LD analysis predicted the average orientations of these helix segments on the liposome: two amphiphilic helices parallel to the liposome surface for LA, two hydrophobic helices perpendicular to the liposome surface for Trx, and a hydrophobic helix perpendicular to and an amphiphilic helix parallel to the liposome surface for LG. This sequence-level information about the secondary structures and orientations was used to formulate interaction models of the three proteins at the membrane surface. This study demonstrates the validity of a combination of VUVCD and LD spectroscopy in conformational analyses of membrane-binding proteins, which are difficult targets for X-ray crystallography and nuclear magnetic resonance spectroscopy.

Glassy Behavior of a Tin Dioxide Nanoparticle Suspension.

Dilute suspensions of charged colloidal particles with a short-range attraction and long-range repulsion can exhibit a variety of arrested states. In many applications using suspensions of charged nanoparticles, the optimization of the process requires the understanding of the mechanism underlying the stability and the rheological properties of the suspensions. In an attempt to clarify the solidification mechanism for dilute suspensions of tin dioxide (SnO2) nanoparticles, we present dynamic viscoelasticity, dynamic and static light scattering, and small-angle X-ray scattering experiments on a SnO2 nanoparticle suspension with a nanoparticle concentration of 25.0 wt % (volume fraction φ = 0.045). The behaviors of the observed dynamic and static structure factors reveal that the aging of SnO2 nanoparticles is Wigner glassy rather than gel-like.

Universality and specificity in molecular orientation in anisotropic gels prepared by diffusion method.

Molecular orientation in anisotropic gels of chitosan, Curdlan and DNA obtained by dialysis of those aqueous solutions in gelation-inducing solutions was investigated. In this diffusion method (or dialysis method), the gel formation was induced by letting small molecules diffuse in or out of the polymer solutions through the surface. For the gels of DNA and chitosan, the polymer chains aligned perpendicular to the diffusion direction. The same direction of molecular orientation was observed for the Curdlan gel prepared in the dialysis cell. On the other hand, a peculiar nature was observed for the Curdlan gel prepared in the dialysis tube: the molecular orientation was perpendicular to the diffusion direction in the outermost layer of the gel, while the orientation was parallel to the diffusion direction in the inner translucent layer. The orientation parallel to the diffusion direction is attributed to a small deformation of the inner translucent layer caused by a slight shrinkage of the central region after the gel formation. At least near the surface of the gel, the molecular orientation perpendicular to the diffusion direction is a universal characteristic for the gels prepared by the diffusion method.

Slow knot formation by suppressed self-reptation in a collapsed polymer chain.

Chain-expansion processes from knotted globules have been measured for poly(methyl methacrylate) (PMMA) in the mixed solvent tert-butyl alcohol (TBA) + water (2.5 vol %) by static light scattering. The solution was quenched from the Θ temperature of 41.5 °C to 37.0 °C, aged there for a time period t(p,) and then returned rapidly to the Θ temperature. The chain-expansion process was determined as a time evolution of the expansion factor α(2) after the temperature increase. The measurement was carried out by changing the aging time t(p) from 240 to 7200 min, and the molecular weight from M(w) = 4.0 × 10(6) to 1.5 × 10(7), by taking advantage of the extremely slow chain aggregation in the solution. The chain-expansion process obtained for M(w) = 1.22 × 10(7) became slow with increasing t(p), which revealed the knot formation in single globules. The characteristic time of the chain expansion from globules aged for t(p) = 7200 min was found to depend on the molecular weight as M(w)(2.7). This exponent, which is close to 3, demonstrated a disentanglement process due to self-reptation. The present data were compared with the previous data of the chain expansion from compact globules aged at 25.0 °C. The comparison made at M(w) = 1.22 × 10(7) and at the same values of t(p) revealed that the chain expansion from the globules aged at 25.0 °C was much faster than that from the globules at 37.0 °C, indicating a lower knot density in the more compact globules. It was conjectured that the knot formation due to self-reptation would be suppressed in a compact globule because an entire conformational change required by knot formation would become difficult to occur in the confined space of high segment concentration, particularly for a long polymer chain. The chain collapse of PMMA in the mixed solvent has been observed to occur extremely slowly at the later stage. This slow process was explained by the suppressed self-reptation.

Studies on the formation mechanism and the structure of the anisotropic collagen gel prepared by dialysis-induced anisotropic gelation.

We have found that dialysis of 5 mg/mL collagen solution into the phosphate solution with a pH of 7.1 and an ionic strength of 151 mM [corrected] at 25 °C results in a collagen gel with a birefringence and tubular pores aligned parallel to the growth direction of the gel. The time course of averaged diameter of tubular pores during the anisotropic gelation was expressed by a power law with an exponent of 1/3, suggesting that the formation of tubular pores is attributed to a spinodal decomposition-like phase separation. Small angle light scattering patterns and high resolution confocal laser scanning microscope images of the anisotropic collagen gel suggested that the collagen fibrils are aligned perpendicular to the growth direction of the gel. The positional dependence of the order parameter of the collagen fibrils showed that the anisotropic collagen gel has an orientation gradient.

A galactosamine-mediated drug delivery carrier for targeted liver cancer therapy.

In order to minimize the side effect of cancer chemotherapy, a novel galactosamine-mediated drug delivery carrier, galactosamine-conjugated albumin nanoparticles (GAL-AN), was developed for targeted liver cancer therapy. The albumin nanoparticles (AN) and doxorubicin-loaded AN (DOX-AN) were prepared by the desolvation of albumin in the presence of glutaraldehyde crosslinker. Morphological study indicated the spherical structure of these synthesized particles with an average diameter of around 200 nm. The functional ligand of galactosamine (GAL) was introduced onto the surfaces of AN and DOX-AN via carbodiimide chemistry to obtain GAL-AN and GAL-DOX-AN. Cellular uptake and kinetic studies showed that GAL-AN is able to be selectively incorporated into the HepG2 cells rather than AoSMC cells due to the existence of asialoglycoprotein receptors on HepG2 cell surface. The cytotoxicity, measured by MTT test, indicated that AN and GAL-AN are non-toxic and GAL-DOX-AN is more effective in HepG2 cell killing than that of DOX-AN. As such, our results implied that GAL-AN and GAL-DOX-AN have specific interaction with HepG2 cells via the recognition of GAL and asialoglycoprotein receptor, which renders GAL-AN a promising anticancer drug delivery carrier for liver cancer therapy.

Reversible chain association/dissociation via a CO2 responsive crosslinking/decrosslinking system.

Reversible chain association/dissociation phenomenon via CO(2) responsive crosslinking/decrosslinking was detected in aqueous solutions of polyallylamine (PAA). The chain association/dissociation behavior was reversible and useful in the synthesis of porous crosslinked polystyrene, which suggested potential utility in the area of CO(2)-responsive separable adhesives, switches and sensors.

Anisotropic structure of calcium-induced alginate gels by optical and small-angle X-ray scattering measurements.

It was more than 50 years ago that an appearance of birefringence in alginate gels prepared under cation flow was reported for the first time, however, the anisotropic structure of the alginate gel has not been studied in detail. In the present study, anisotropic Ca-alginate gels were prepared within dialysis tubing in a high Ca(2+)-concentration external bath, and optical and small-angle X-ray scattering (SAXS) measurements were performed to characterize the structure of the gel. The observations of the gel with crossed polarizers and with circular polarizers revealed the molecular orientation perpendicular to the direction of Ca(2+) flow. Analyses of the SAXS intensity profiles indicated the formation of rod-like fibrils consisting of a few tens of alginate molecules and that the anisotropy of the gel was caused by the circumferential orientation of the large fibrils. From the observed asymmetric SAXS pattern, it was found that the axis of rotational symmetry of the anisotropic structure was parallel to the direction of Ca(2+) flow. The alignment factor (A(f)) calculated from the SAXS intensity data confirmed that the orientation of the fibrils was perpendicular to the direction of Ca(2+) flow.

Dynamics in the process of formation of anisotropic chitosan hydrogel.

To control the dynamics of dialysis-induced anisotropic gel formation, we have derived a theoretical expression for the development of the gel layer for a simple case where no cross-link sites for cross-linking agents exists and the inflow and the outflow of low molecular weight components through the dialysis membrane modify the state of polymer molecules to meet the gelation condition. A series of experiments using chitosan solution were done as a model case. The experimental results were compared with asymptotic expressions of the time development equation predicted by theory, and the compatibility of the theoretical picture was examined.

Culture scale-up studies as seen from the viewpoint of oxygen supply and dissolved carbon dioxide stripping.

Oxygen supply and dissolved carbon dioxide (dCO(2)) stripping are two of the most important control parameters in cell culture. In this study, we investigated the effect of scale-up on the volumetric gas transfer coefficient with bioreactors of different sizes (working volume: 80 L, 500 L, 2000 L, and 10,000 L; aspect ratio: 1.0-1.6). Sparging air into water increased the volumetric oxygen transfer coefficient (k(L)a), an index of oxygen supply efficiency, by scale-up roughly in proportion to the depth of the water. A corresponding increase in k(L)a was found in a real cell culture of Chinese hamster ovary cells. dCO(2) stripping efficiency was evaluated in water tests using changes in k(L)a(co2), an index defined in relation to k(L)a. k(L)a(co2) increased following surface aeration, but the rate of increase was reduced by scale-up, which was attributed to a decrease in the liquid surface-to-volume ratio. A similar decrease in efficiency was observed in a 2000 L bioreactor by increasing the liquid volume at constant liquid surface area. The observed scale-up effects are discussed based on a simple theoretical consideration.

Estimation of dissolved carbon dioxide stripping in a large bioreactor using model medium.

Dissolved carbon dioxide (dCO(2)) accumulation is one of the most serious problems in the scale-up of industrial cell culture. To predict the effects of dCO(2) stripping in different culture conditions and at different scales, we examined a method of estimation of dCO(2) stripping using a model medium. The operational parameters (e.g., sparging and agitation rate) and the size of the bioreactor (working volume: 80 L, 500 L, 2000 L; aspect ratio: 1.0 approximately 1.6) were varied, and the model medium was prepared by adjusting pH, density, viscosity, surface tension, and buffer conditions. dCO(2) stripping efficiency was evaluated using the index k(L)a(CO)((2)), which was defined in accordance with the volumetric oxygen transfer coefficient k(L)a. The model medium exhibited dCO(2) stripping behavior similar to real culture medium in all experimental conditions tested. It is expected that the use of the model medium to estimate dCO(2) stripping in real cultures will be valuable for determining the culture conditions in bioreactors in scale-up.