PRODUCTION OF EXTREMELY-SMALL HYDROGEL MICROSPHERES BY UTILIZING WATER-DROPLET DISSOLUTION IN A POLAR SOLVENT

Sari Sugaya, Masumi Yamada, and Minoru Seki* Chiba University, JAPAN

ABSTRACT

In this study, we demonstrated a new method to produce extremely small hydrogel beads by utilizing water-droplet disso-lution in a weak polar solvent. Water droplets containing hydrogel polymer were generated in the continuous phase of the solvent, and water was dissolved from the droplets to the continuous phase while the hydrogel polymer was concentrated. The concentrated droplets were then gelled, forming hydrogel beads significantly smaller than the initial droplet size. We employed methyl acetate as the solvent, having a high solubility of water (8%), and successfully produced Ca-alginate and chitosan microbeads with sizes smaller than 10 µm. KEYWORDS: Hydrogel microbeads, Polar solvent, Alginate, Chitosan

INTRODUCTION

Hydrogel beads are highly useful in biomedical fields and utilized for DDS vehicles, tissue engineering scaffolds, column packing materials, etc. To engineer hydrogels with a microscale resolution, a number of techniques have been developed [1, 2]. Among various types and shapes of hydrogel materials, small-size hydrogel beads are advantageous for biomedical ap-plications due to the efficient supply of oxygen and nutrients. Alginate is one of the most widely used hydrogel materials, and numerous studies to fabricate alginate hydrogel beads have been reported. Researchers have developed microfluidic sys-tems to synthesize small size alginate hydrogel beads [3-5]; however, production of hydrogel beads smaller than 20 !m in di-ameter has not been reported yet, since it is difficult to produce small-size droplets of highly viscous aqueous solutions using a relatively narrow microchannel or micronozzle. In this report, we propose a new method to produce hydrogel beads with sizes less than 10 !m in diameter utilizing the water dissolution into a polar solvent (methyl acetate), enabling the production of hydrogel beads significantly smaller than the initially generated droplets.

EXPERIMENTAL

The principle of producing Ca-alginate hydrogel beads is shown in Figure 1. Methyl acetate was employed as the weak polar solvent, which dissolves water at 8% in it. Alginate is a biocompatible polysaccharide and gelled in the presence of multivalent cations like Ca2+. By introducing methyl acetate and a sodium alginate (NaA) solution into the microchannel, w/o droplets are generated at the first confluence. The generated droplet volume is decreased while droplets are flowing in the continuous phase of methyl acetate. On the other hand, hydrophilic hydrogel polymer is concentrated in the shrunk drop-let, and then gelled by introducing a gelation solution containing Ca2+, producing hydrogel beads smaller than the initial droplet size (Fig. 1).

PDMS microchannel was fabricated by soft lithography and replica molding. The channel depth was uniform, 55 !m. The width of the droplet-generation channel (the first confluence) was 50 !m. The lengths of the water extraction channel and the gelation channel were 70 and 75 mm, respectively. Methyl acetate (flow rate of Q1), a NaA solution (Q 2), and a gela-

Figure 1: Schematic image showing the production process of extremely small Ca-alginate hydrogel microbeads utilizing the dissolution of water droplets into methyl acetate. Na-alginate (NaA) droplets are formed at the first confluence, and their volumes are gradually decreased during flowing through methyl acetate. By adding CaCl2 aq. at the second confluence, NaA droplets are gelled by the diffused Ca2+ and hydrogels beads are formed.

978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001 18 15th International Conference onMiniaturized Systems for Chemistry and Life Sciences

October 2-6, 2011, Seattle, Washington, USA

tion solution containing Ca2+ (Q 3) were introduced by using syringe pumps (Fig. 2 (a)). W/O droplets of a 0.025~0.15% NaA solution were generated by the flow focusing. At the second confluent point, 1 M CaCl2 aq. was introduced. Methyl acetate and the CaCl2 solution formed a laminar flow, and Ca2+ was diffused and droplets were gelled. The diameter and the volume of the generated droplets and hydrogel beads were measured by image processing.

We also demonstrated the production of chitosan hydrogel beads. Chitosan is also a polysaccharide, which is soluble in acetic solutions and forms hydrogels in neutral or alkaline conditions. A chitosan sol solution dissolved in 0.6% acetic acid (Q 2) and 1 mM NaOH solution (Q 3) were used.

RESULTS AND DISCUSSION

Schematic diagrams of the microchannel design and the micrographs of the droplet behaviors are shown in Fig. 2. W/O droplets were generated, and droplet sizes were gradually decreased as flowing in methyl acetate. The initial average diame-ters of the droplets were 70.2 !m and 63.8 !m when Q 2 were 0.9 and 0.3 !L/min, respectively, while the average diameters of the beads were 10.5 and 10.0 !m, respectively; the volumes of the beads were decreased to ~0.5% of those of the initial droplets. We therefore confirmed that the dissolution of water droplets in methyl acetate was possible, which was utilized to produce small hydrogel beads.

Figure 3 shows the obtained hydrogel beads and their drying and re-swelling processes. The obtained beads were mono-disperse with a CV value of ~2% (Fig. 3 (e)). To examine if the obtained microbeads contain water, the microbeads were dried and re-swelled repeatedly. As a result, the beads were shrunk and deformed by drying, and recovered their shapes and

Figure 2: Formation and shrinkage of the NaA droplets in methyl acetate. (a) Schematic diagram of the microchannel de-sign, (b) Time-course decrease in the droplet volumes in the channel, and micrographs showing (c) the droplet formation, (d) flowing droplets, and (e) CaCl2 aq. introduction at the second confluence. NaA droplets were formed in methyl acetate and their volumes were decreased. Q1 = 30, Q2 = 0.9 or 0.3, Q3 = 15 !L/min.

Figure 3: Micrographs showing the hydrogel beads: (a, b) obtained beads after washing in DI water, (c) dried beads by heat-ing, and (d) re-swelled beads by re-suspended in water. (e) Size distribution of the obtained beads (CV of 2.13 %). Q1 = 30, Q2 = 0.9, Q3 = 15 !L/min. Scale bars, 20 !m.

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volumes when re-swelled by adding water (Fig. 3 (c, d)). These drying and re-swelling processes of the beads were repeata-ble and this result indicated that the obtained beads had a hydrogel characteristic.

To control the hydrogel bead sizes, NaA solutions with different concentrations were used. As shown in Fig. 4, the diam-eters of the obtained microbeads were changed from 6 to 10 µm by adjusting the conc. of NaA, while the initially generated droplet sizes were not changed dramatically. The NaA conc. in the droplet is an important factor dominating the hydrogel bead sizes.

As another application, chitosan hydrogel beads were prepared in the same manner. As shown in Fig. 5, we successfully formed monodisperse chitosan hydrogel beads, with the aver-age diameter of 8.6 !m (CV: 5.8%) when 0.05% chitosan solution was used. The presented process of producing extremely-small hydrogel beads utilizing the weak polar solvent would be applied to prepare various types of hydrogel beads.

CONCLUSION

A new method of producing extremely-small hydrogel beads (smaller than 10 µm) are pre-sented, which are difficult to obtain by using previously-reported microfluidic approaches. Further process optimization will enable the productions of hydrogel beads with various sizes, which would be highly useful as the models of cells, DDS carriers, matrices for biological im-mobilization, and so on. ACKNOWLEDGEMENTS

This study was supported in part by Grants-in-aid for Scientific Research A (20241031) and for Young Scientists (B) (23700554) from Japan Society for Promotion of Science (JSPS), and for Improvement of Research Environment for Young Researchers from Japan Science and Technology Agency (JST). REFERENCES [1] A. Khademhosseini and R. Langer, “Microengineered hydrogels for tissue engineering,” Biomaterials, vol. 28, pp. 5087-

8092 (2007). [2] B. V. Slaughter, S. S. Khurshid, O. Z. Fisher, A. Khademhosseini, and N. A. Peppas, “Hydrogels in regenerative medi-

cine,” Adv. Mater., vol. 21, pp. 3307-3329 (2009). [3] S. Sugiura, T. Oda, Y. Izumida, Y. Aoyagi, M. Satake, A. Ochiai, N. Ohkohchi, and M. Nakajima, “Size control of cal-

cium alginate beads containing living cells using micro-nozzle array,” Biomaterials, vol. 26, pp. 3327-3331 (2005). [4] W. -H. Tan and S. Takeuchi, “Monodisperse alginate hydrogel microbeads for cell encapsulation,” Adv. Mater., vol. 19,

pp. 2696-2701 (2007). [5] K. Huang, M. Liu, C. Wu, Y. Yen, and Y. Lin, “Calcium alginate microcapsule generation on a microfluidic system fab-

ricated using the optical disk process,” J. Micromech. Microeng., vol. 17, pp. 1428-1434 (2007). CONTACT *Minoru Seki, Tel: +81-43-290-3436; mseki@faculty.chiba-u.jp

Figure 5: Micrographs showing the chitosan hy-drogel beads. Q1 = 30, Q2 = 0.3, Q3 = 15 !L/min.

Figure 4: NaA droplets in the microchannel and the obtained hydrogel microbeads at different conc. of NaA. These micro-graphs show the NaA droplets at the first confluence, in the water extraction channel, and at the second confluence, and the beads. NaA conc. were (a) 0.025% and (b) 0.15%, respectively. Average diameter of the droplets and the beads are shown as indicated. Q1 = 30, Q2 = 0.9, Q3 = 15 !L/min.

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