Tsutomu Ono

Synthesis and transport characterization of alginate/aminopropyl-silicate/alginate microcapsule: application to bioartificial pancreas.

To develop a novel type of immunoisolation membrane for a microcapsule-shaped bioartificial pancreas, we attempted to use a sol-gel synthesized silicate. An aminopropyl-silicate membrane derived from 3-aminopropyltrimethoxysilane and tetramethoxysilane was formed on Ca-alginate gel beads via electrostatic interaction. The positively charged amino groups remaining on the surface of the resultant gel beads were neutralized by immersion in an aqueous Na-alginate solution. From measurements of the partition coefficients and effective diffusivities of different substances to the gel beads, it was found that the aminopropyl-silicate membrane prepared under optimized composition of silicon alkoxide precursors successfully rejected gamma-globulin, giving good permeability to substances having a low molecular weight. Islets could be encapsulated within the newly developed microcapsules while retaining their ability to secrete insulin.

In vitro and in vivo evaluation of alginate/sol-gel synthesized aminopropyl-silicate/alginate membrane for bioartificial pancreas

Alginate/aminopropyl-silicate/alginate (Alg/AS/Alg) membrane was prepared on Ca-alginate gel beads by a sol-gel process. The membrane has identical to Si-O-Si identical to bonds as well as electrostatic bonds between amino groups of AS and carboxyl groups of alginate. Permeability and stability were investigated for the membrane. Furthermore, rat islets encapsulated in the membrane (499 +/- 32 microns in diameter, 1000 islets/recipient) were transplanted to the peritoneal cavities of the mice with streptozotocin-induced diabetes. Our data show that the membrane had the molecular weight cut-off point of between 70 and 150 kDa, and hardly inhibited the permeation of glucose and insulin. The Alg/AS/Alg microcapsule was more stable than the well-known Alg/poly-L-lysine (PLL)/Alg microcapsule. After 30 days of soaking in stimulated body fluid, the percentages of intact microcapsule were 98.4 +/- 0.5 (mean +/- SEM)% and 88.0 +/- 1.5% (p < 0.001) for the Alg/AS/Alg and Alg/PLL/Alg microcapsules, respectively. The maximum maintenance period of normoglycemia was 105 days without administration of immunosuppressive drugs.

Extraction Behavior of Hemoglobin Using Reversed Micelles by Dioleyl Phosphoric Acid

A new surfactant, dioleyl phosphoric acid (DOLPA), has been applied to the extraction of hemoglobin using reversed micelles. The reversed micelles formed by DOLPA can easily extract hemoglobin from aqueous to reversed micellar solutions. DOLPA is the first surfactant to extract hemoglobin completely without using any cosurfactants. On the basis of the difference between DOLPA and AOT reversed micelles in the forward extraction behavior of hemoglobin, the nature of the interfacial complex that would be formed between surfactants and hemoglobin at the oil−water interface was found to be the dominant factor in determining the extraction efficiency of hemoglobin by reversed micelles. In addition, back‐transfer studies of hemoglobin from the DOLPA reversed micelles were also carried out by the phase transfer method. It was found that hemoglobin, once dissolved into the DOLPA reversed micelles, is not transferred to a fresh aqueous solution even when the conditions are adjusted to not allow the forward transfer of hemoglobin. However, the addition of several kinds of alcohol drastically improved the yield in the back‐transfer of hemoglobin. The efficiency in the back‐transfer of hemoglobin strongly depends on the aqueous conditions that are in contact with the reversed micelles, such as pH, ionic strength, and alcohol concentration. A pH higher than the pI of hemoglobin, a salt concentration lower than that of the water pool, and the proper concentration of alcohol are required for the recovery aqueous phase to ensure the back‐transfer of hemoglobin from the DOLPA reversed micelles.

Control of molecular weight cut-off for immunoisolation by multilayering glycol chitosan-alginate polyion complex on alginate-based microcapsules

Glycol chitosan is a positively charged polysaccharide which is water-soluble at pH 7.4, and is able to form a polyion complex (PIC) with anionic polymers, such as alginate. The authors attempt to develop a novel type of alginate-based microcapsule using this glycol chitosan for a islets-encapsulated bioartificial pancreas. The number of layers composed of glycol chitosan-alginate (GC-Alg) PIC were optimized, in order to cut off immunoglobulin transport and to protect encapsulated islets from the host immune reaction, and the transport characteristics were evaluated of glucose, bovine serum albumin (BSA) and gammaglobulin. To add mechanical stability to the microcapsule, calcium ions, which crosslinked the alginate polymers close to the interface between core Caalginate and multilayered membrane, were partially substituted with barium ions after the formation of multilayered Ca-alginate gel beads. The partition coefficients of BSA and gamma-globulin were decreased with the increasing number of layers. The immunoisolation was achieved againstgamma-globulin with four layers of the GC-Alg PIC membrane, while BSA could permate the membrane. The four-layered Ba-alginate gel bead had a good permeability for glucose, giving a diffusion coefficient corresponding to 80% of that in pure water. Insulin secretion from the islets in the four-layered Ba-alginate microcapsule was satisfactorily observed with the fractional stimulation ratio of 2.17. This result indicates that the encapsulated islets maintained their viability even after encapsulation. It was, thus, shown that the Ba-alginate microcapsule with four layers of the GC-Alg PIC membrane is promising as the microencapsulation material for a bioartificial pancreas.Glycol chitosan is a positively charged polysaccharide which is water-soluble at pH 7.4, and is able to form a polyion complex (PIC) with anionic polymers, such as alginate. The authors attempt to develop a novel type of alginate-based microcapsule using this glycol chitosan for a islets-encapsulated bioartificial pancreas. The number of layers composed of glycol chitosan-alginate (GC-Alg) PIC were optimized, in order to cut off immunoglobulin transport and to protect encapsulated islets from the host immune reaction, and the transport characteristics were evaluated of glucose, bovine serum albumin (BSA) and gamma-globulin. To add mechanical stability to the microcapsule, calcium ions, which crosslinked the alginate polymers close to the interface between core Ca-alginate and multilayered membrane, were partially substituted with barium ions after the formation of multilayered Ca-alginate gel beads. The partition coefficients of BSA and gamma-globulin were decreased with the increasing number of layers. The immunoisolation was achieved against gamma-globulin with four layers of the GC-Alg PIC membrane, while BSA could permeate the membrane. The four-layered Ba-alginate gel bead had a good permeability for glucose, giving a diffusion coefficient corresponding to 80% of that in pure water. Insulin secretion from the islets in the four-layered Ba-alginate microcapsule was satisfactorily observed with the fractional stimulation ratio of 2.17. This result indicates that the encapsulated islets maintained their viability even after encapsulation. It was, thus, shown that the Ba-alginate microcapsule with four layers of the GC-Alg PIC membrane is promising as the microencapsulation material for a bioartificial pancreas.

Protein Refolding by Reversed Micelles Utilizing Solid-Liquid Extraction Technique

This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid-liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid-liquid extraction. The solid-liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid-liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates.

Extraction and Activity of Chymotrypsin Using AOT−DOLPA Mixed Reversed Micellar Systems

Novel reversed micellar solutions formulated with a mixture of AOT (dioctyl sulfosuccinate) and DOLPA (dioleyl phosphoric acid) show good potential for use in reversed micellar protein extraction operations. Chymotrypsin is easily extracted from an aqueous phase into organic isooctane containing 10 mM AOT and DOLPA in a 4:1 ratio. The extraction ability of the mixed reversed micelles of 10 mM was higher than that of 200 mM AOT alone. The results of extraction indicated that the AOT−DOLPA mixed reversed micelles are very useful for separating and enriching chymotrypsin. Back‐extraction of chymotrypsin from the organic phase to a fresh aqueous phase is also accomplished by adding an alcohol to the organic phase. Although the back‐transfer of chymotrypsin from the reversed micelles formed by AOT alone is very slow and difficult, in the AOT−DOLPA mixed reversed micelles, the back‐extraction can be achieved completely by addition of 10% (v/v) isobutyl alcohol to the reversed micellar phase. The time to attain to the equilibrium of back‐extraction was reduced from more than 24 to 2 h by adding the alcohol. On the basis of the activity data, the best composition of AOT and DOLPA was a 4:1 ratio and the total surfactant concentration was 10 mM. The activity of chymotrypsin recovered from the mixed reversed micelles was higher than that of the initial protein before the forward‐transfer. This result means that the novel mixed reversed micellar solutions are useful not only in separation but also in purification of proteins.

Proliferation and Insulin Secretion Function of Mouse Insulinoma Cells Encapsulated in Alginate/Sol-Gel Synthesized Aminopropyl-Silicate/Alginate Microcapsule

Alginate/aminopropyl-silicate/alginate microcapsules, ca. 15 μm in membrane thickness and ca. 500 μm in diameter, were prepared via sol-gel process. The aminopropyl-silicate membrane was derived from two silicone alkoxide precursors, tetramethoxysilane and 3-aminopropyl-trimethoxysilane on Ca-alginate micro gel beads. Pancreatic β-cell line (MIN6) cells were encapsulated in the microcapsule. The encapsulated MIN6 cells proliferated and formed spheroidal tissues in vitro. The diameter of the MIN6 spheroids increased to approximately 250 μm with an increase in the incubation period until the day 35. Storeptozotocin-induced diabetic mice became normoglycemia after implantation of the MIN6-enclosing microcapsules. The normoglycemic state remained until the retrieval of the implanted microcapsules for 1 month. These results indicate that the potential use of the alginate/aminopropyl-silicate/alginate microcapsule as a vehicle for a genetically engineered cell-enclosing therapeutic material delivery system.

Application of reversed micelles in bioengineering

Reverse micelles have found wider biological applications recently in terms of providing an enabling hydrophilic environment for solubilized functional biomaterials in organic media. Further, separation and purification of target proteins from aqueous solution using micellar aggregates created in hydrophobic media as well as efficient refolding of proteins in micellar solutions have become possible.

Important parameters affecting efficiency of protein refolding by reversed micelles.

Refolding of denatured RNase A as a model of inclusion bodies was performed by reversed micelles formulated with sodium di‐2‐ethylhexyl sulfosuccinate (AOT) in isooctane. In the novel refolding process, a solid‐liquid extraction was utilized as an alternative to the ordinary protein extraction by reversed micelles based on a liquid‐liquid extraction. First, the effects of operational parameters such as concentration of AOT, Wo (= [H2O]/[AOT]), and pH were examined on the solubilization of solid denatured proteins into a reversed micellar solution. The solubilization was facilitated by a high AOT concentration, a high Wo value, and a high pH in water pools. These conditions are favorable for the dispersion of the solid protein aggregates in an organic solvent. Second, the renaturation of the denatured RNase A solubilized into the reversed micellar solution was conducted by addition of glutathione as a redox reagent. A complete renaturation of RNase A was accomplished by adjusting the composition of the redox reagent even at a high protein concentration in which protein aggregation would usually occur in aqueous media. In addition, the renaturation rates were improved by optimizing water content (Wo) and the pH of water pools in reversed micelles. Finally, the recovery of renatured RNase A from the reversed micellar solution was performed by adding a polar organic solvent such as acetone into the reversed micellar solution. This precipitation method was effective for recovering proteins from reversed micellar media without any significant reduction in enzymatic activity.

Permeability of alginate/sol–gel synthesized aminopropyl-silicate/alginate membrane templated by calcium-alginate gel

An alginate/aminopropyl-silicate/alginate membrane was prepared on calcium-alginate gel beads via electrostatic interaction and sol–gel processes. The effects of the chemical composition and the molecular weight (MW) of the core alginate on the permeability of the alginate/aminopropyl-silicate/alginate membrane were investigated for five different types of alginates. Our data shows that an immunoisolatable MW cut-off point was achieved for the membranes formed on the alginates, MW=7.0×104 with molar ratio of M/G (mannuronic acid to guluronic acid), M/G=1.30, MW=1.1×105 with M/G=1.30, and MW=7.0×104 with M/G=0.65. For these three membranes, the membrane templated by the alginate of MW=1.1×105 had a higher MW cut-off point than that templated by the alginate of MW=7.0×104. In addition, the MW cut-off point increased with an increase in the content of guluronic acid. The membranes formed on the alginates of MW=2.0×104 with M/G=1.30, and MW=7.0×104 with M/G=2.25 did not have an immunoisolatable MW cut-off point.