Aqueous Two-Phase System Formation in Small Droplets by Shirasu Porous Glass Membrane Emulsification Followed by Water Extraction.

By utilizing water transport phenomena between two different water-in-oil (W/O) emulsion droplets through continuous oil phase, we developed a novel method of aqueous two-phase system (ATPS) formation in small droplets prepared by Shirasu porous glass (SPG) membrane emulsification technique. When we mixed W/O emulsion droplets containing poly(ethylene glycol) (PEG) and dextran (DEX) at concentrations below the threshold of the phase separation, with droplets containing other solutes at high concentrations, water extraction from the droplets containing PEG and DEX to those containing the other solutes occurred, owing to the osmotic pressure difference. This effect increased the concentrations of PEG and DEX in the droplets above the phase separation threshold. We demonstrated the feasibility of the preparation method by varying the pore sizes of the SPG membranes, the solutes, and their concentrations. Only when the concentration of the solute was high enough to extract sufficient amounts of water did the homogeneous disperse phase consisting of PEG and DEX in droplets turn into a PEG-rich phase and DEX-rich phase, showing ATPS. This result was irrespective of the solute itself and pore size of the SPG membrane. In particular, we successfully demonstrated monodisperse ATPS droplets with diameters of approximately 10 µm under a certain condition.

Direct Observation of Splitting in Oil-In-Water-In-Oil Emulsion Droplets via a Microchannel Mimicking Membrane Pores.

Direct observation of double emulsion droplet permeation through a microchannel that mimicked 100 µm membrane pores with a porosity of 66.7% provided insights regarding splitting mechanisms in porous membranes. The microchannel was fabricated by standard soft lithography, and the oil-in-water-in-oil double emulsion droplets were prepared with a glass capillary device. By changing the flow rate from 0.5 to 5.0 × 10-2 m s-1, three characteristic behaviors were observed: (a) passage into one channel without splitting; (b) division into two smaller components; and (c) stripping of the middle water phase of the double emulsion droplets into a smaller double emulsion droplet and a smaller water-in-oil single emulsion droplet. The mechanisms are discussed with respect to the balance of viscous forces and interfacial tension, the contact point with the tip of the channel, and the relative position of the innermost droplet within the middle droplet.

Size-dependent interaction of cells and hemoglobin-albumin based oxygen carriers prepared using the SPG membrane emulsification technique.

Hemoglobin-based oxygen carriers (HBOCs) of various sizes have been developed so far, but their optimum size has not been clarified yet. Here, we examined the effect of HBOCs size on their interaction with cells using Shirasu porous glass (SPG) membrane emulsification technique, which enables precise tuning of particle size. Microspheres composed of bovine hemoglobin (bHb) and bovine serum albumin (BSA) was fabricated with the average diameters of 1.2-18.3 µm and the coefficient of variation of below 13%. Cellular uptake of the microspheres by RAW264.7 was observed at a diameter below 5 µm; however, uptake of the microspheres by HepG2 and HUVEC were not observed at any diameter. No enhancement of the generation of reactive oxygen species in the cytoplasm was detected at diameters above 9.8 µm in the three cell lines, due to their low cellular uptake. In addition, cytotoxicity of the microspheres decreased with increasing microsphere diameter in the three cell lines and microspheres of 18.3 µm showed good cellular compatibility regardless of the oxyhemoglobin percentage. Since cytotoxicity is a crucial factor in their applications, our systemic investigation would provide a new insight into the design of HBOCs.

Membrane-Integrated Glass Capillary Device for Preparing Small-Sized Water-in-Oil-in-Water Emulsion Droplets.

In this study, a membrane-integrated glass capillary device for preparing small-sized water-in-oil-in-water (W/O/W) emulsion droplets is demonstrated. The concept of integrating microfluidics to prepare precise structure-controlled double emulsion droplets with the membrane emulsification technique provides a simple method for preparing small-sized and structure-controlled double emulsion droplets. The most important feature of the integrated device is the ability to decrease droplet size when the emulsion droplets generated at the capillary pass through the membrane. At the same time, most of the oil shell layer is stripped away and the resultant double emulsion droplets have thin shells. It is also demonstrated that the sizes of the resultant double emulsion droplets are greatly affected by both the double emulsion droplet flux through membranes and membrane pore size; when the flux is increased and membrane pore size is decreased, the generated W/O/W emulsion droplets are smaller than the original. In situ observation of the permeation behavior of the W/O/W emulsion droplets through membranes using a high-speed camera demonstrates (1) the stripping of the middle oil phase, (2) the division of the double emulsion droplets to generate two or more droplets with smaller size, and (3) the collapse of the double emulsion droplets. The first phenomenon results in a thinner oil shell, and the second division phenomenon produces double emulsion droplets that are smaller than the original.

Preparation of uniform-sized hemoglobin-albumin microspheres as oxygen carriers by Shirasu porous glass membrane emulsification technique.

We have developed a new type of artificial oxygen carrier composed of bovine hemoglobin (bHb) and bovine serum albumin (BSA) prepared by Shirasu porous glass (SPG) membrane emulsification technique. The resultant emulsion droplets containing 10 wt% bHb and 5-20 wt% BSA were subsequently cross-linked by glutaraldehyde to form the microspheres. Due to the uniform pore structure of SPG membranes, the average diameters of bHb10-BSAm microspheres were successfully controlled at around 5 µm with a coefficient of variation of around 10%. In addition, the biocompatibility of the carriers depended on their oxyhemoglobin percentage regardless of their same size. Finally, the P50 values of these microspheres ranged from 8.08 to 11.60 mmHg, which showed a high oxygen affinity and an oxygen delivery function.

Size-controlled and monodisperse enzyme-encapsulated chitosan microspheres developed by the SPG membrane emulsification technique.

Lysozyme-encapsulated chitosan microspheres with micron-size diameters were successfully prepared for the first time by employing the Shirasu porous glass (SPG) membrane emulsification technique followed by cross-linking with glutaraldehyde, and the relationships between the preparation conditions and characteristics of the microspheres were studied in detail. This preparation method provided size-controllability and monodispersity of the microspheres, owing to the sharpness of the pore sizes of the SPG membranes. It was also possible to predict the average diameters of the enzyme-encapsulated microspheres using no fitting parameters, on the basis that each microsphere is prepared in an emulsion containing chitosan and lysozyme, without any collisions or aggregation occurring. X-ray photoelectron spectroscopy measurements indicated that the amount of encapsulated lysozyme was controlled by the concentrations of chitosan and lysozymes in the dispersion phase used for preparing the emulsions from which the enzyme-encapsulated microspheres are formed. Finally, the apparent activity of the encapsulated lysozymes was measured by the viscosimetric method, using ethyleneglycolchitin. Results showed that about half of the activity of the encapsulated lysozymes was maintained during the preparation procedure when employing the SPG membrane emulsification technique.

Drastic difference in porous structure of calcium alginate microspheres prepared with fresh or hydrolyzed sodium alginate.

Fresh or hydrolyzed sodium alginate was used as a material for preparing calcium alginate microspheres, and a drastic difference in porous structure was observed between them, even though the other materials and the preparation method except for the sodium alginate were exactly the same. When fresh sodium alginate was used, nonporous microspheres were obtained. In contrast, when 82-day-hydrolyzed sodium alginate, whose molecular weight became 7% of the molecular weight of the fresh sodium alginate, was used, porous microspheres with 6.5 times larger BET surface area were obtained. XPS studies indicated that the atomic ratio of Ca, the crosslinker of the alginic acid polymer, was almost the same in both cases. Therefore, the difference in porous structure was not attributed to the amount of crosslinking points, but to the low-molecular-weight compounds formed by hydrolysis, and they would work as pore-generating agents.

Preparation of monodisperse chitosan microcapsules with hollow structures using the SPG membrane emulsification technique.

We describe herein successful preparations of monodisperse chitosan microcapsules with hollow structures using the SPG membrane emulsification technique. Two preparation procedures were examined in this study. In the first method, monodisperse calcium alginate microspheres were prepared and then coated with unmodified chitosan. Subsequently, tripolyphosphate treatment was conducted to physically cross-link chitosan and solubilize the alginate core at the same time. In the second method, photo-cross-linkable chitosan was coated onto the monodisperse calcium alginate microspheres, followed by UV irradiation to chemically cross-link the chitosan shell and tripolyphosphate treatment to solubilize the core. For both methods, it was determined that the average diameters of the chitosan microcapsules depended on those of the calcium alginate microparticles and that the microcapsules have hollow structures. In addition, the first physical cross-linking method using tripolyphosphate was found to be preferable to obtain the hollow structure, compared with the second method using chemical cross-linking by UV irradiation. This was because of the difference in the resistance to permeation of the solubilized alginate through the chitosan shell layers.

Development of a novel fouling suppression system in membrane bioreactors using an intermittent electric field.

A novel membrane bioreactor system that uses an intermittent electric field was successfully developed to suppress membrane fouling, caused mainly by activated sludge. We found that the surface of the activated sludge is negatively charged, and propose the utilization of an electric repulsive force to move the sludge away from the membrane by applying an electric field only when the permeate flux has drastically declined because of membrane fouling. The experimental results showed that a field of 6 V cm(-1), switched on and off every 90 s, significantly improved the removal of the activated sludge from the membrane and accordingly improved the average permeate flux.