Preparation of Sodium Alginate Hydrogel Microparticles by Electrospinning Using Various Types of Salts

Preparation of Sodium Alginate Hydrogel Microparticles by Electrospinning Using Various Types of Salts Sun Gil Kim1, Young Jae Lee1, Eun Joo Shin2, Yeong Soon Gal3, Yong Rok Lee4, Tae Hwan Oh1, Han Gon Choi5, Jung-Ae Kim5, Chul Soon Yong5, Sung Soo Han1, Seok Kyun Noh4, and Won Seok Lyoo1* Division of Advanced Organic Materials, School of Textiles, Yeungnam University, Gyeongsan 712-749, Korea Division of Clothing and Textiles. Dong-A University, Pusan 604-714, Korea 3 Polymer Chemistry Laboratory, College of Engineering, Kyungil University, Gyeongsan 712-701, Korea 4 School of Display and Chemical Engineering, Yeungnam University, Gyeongsan 712-749, Korea 5 College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Korea 1 2

Received: 21 September 2009, Accepted: 12 May 2010 SUMMARY Sodium alginate (SA) hydrogel microparticles were simply obtained by using electrospinning method at diverse processing parameters such as polymer concentration, applied voltage, tip to collector distance (TCD), and type and amount of divalent cations (crosslinking and gelation agents). In the case of TCD, there is no significant on the morphological shape of SA hydrogel microparticle. On the contrary, the particle size was decreased with an increase in the applied voltage. But at a high applied voltage, the polydispersity of SA hydrogel microparticle was increased and broad range of particle sizes was observed. Nearly monodisperse microparticles were prepared at applied voltage of 10kV. We also obtained SA hydrogel microparticles by the exchange of sodium ions with divalent cations such as Sr2+ and Ba2+, which presented uniform spherical shape, in comparison with that prepared using Ca2+.

INTRODUCTION Alginates are a family of linear polysaccharides synthesized by brown algae and some bacteria. They contain varying amounts of (l-4)linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. The residues may vary widely in composition and sequence and are arranged in a pattern of blocks along the chain. The homopolymeric regions of M and G blocks are interspersed with regions of alternating structure (M, G blocks). The composition and extent of the sequences and the molecular weight determine the physical properties of the alginates. The molecular variability is dependent on the organism and tissue from which the alginates are isolated1.

One of the most important and useful properties of alginates is the ability to form gels in the presence of some multivalent metal ions such as calcium. The controlled addition of these ions technically leads to insoluble alginate gel formation. The affinity of alginates for calcium ions and their gel-forming properties are mainly related to the overall fraction of G residues, the molecular weight of the polymer, and the calcium ion concentration at the time of gelation. When two G residues are adjacent in the polymer, they form a binding site for calcium. Therefore, the content of G blocks is a key structural feature contributing to gel strength2. However, it is now realized that M and G also contribute to the overall binding of calcium by sodium alginates (SA)3-4.

*Corresponding author: Prof. Won Seok Lyoo (e-mail: [email protected])

Smithers Rapra Technology, 2010

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Alginate hydrogels have the potential to be used as either controlled release matrices or membrane systems for therapeutic drugs. In the membrane system, alginates could be applied as a coating material on the solid units. The preparation of coated matrices can involve chemical procedures, such as crosslinking reactions of the matrix surface or of a polymer, which coats the matrix surface5-6. Thus, in this study, electrospinning of SA solution was conducted to obtain SA hydrogel microparticles having various dimensions at diverse process parameters such as polymer concentration, applied voltage, tip to collector distance (TCD), and type and amounts of gelation agent such as divalent cations. We have also attempted to optimize the electrospinning conditions for SA hydrogel microparticles and to study the effect of gelation agent.

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Sun Gil Kim, Young Jae Lee, Eun Joo Shin, Yeong Soon Gal, Yong Rok Lee, Tae Hwan Oh, Han Gon Choi, Jung-Ae Kim, Chul Soon Yong, Sung Soo Han, Seok Kyun Noh, and Won Seok Lyoo

EXPERIMENTAL Materials Sodium alginate (SA) (number-average molecular weight 120,000 ~ 190,000 g/mol, ratio of mannuronic acid to guluronic acid residues (M/G) = 1.56, low viscosity 20 ~ 40 cps for 1% aqueous solution at 25 °C) was purchased from Aldrich Chemical Co. (St. Louis, MO). Calcium chloride, strontium chloride, and barium chloride as gelation agents were purchased from Junsei Chemical Co., Tokyo, Japan. Other extra-pure grade reagents were used without further purification.

Solution Preparation SA was dissolved in water 70 °C for 1  h and maintained for 30 min to ensure homogenization. Concentration of SA solution was varied from 1 to 3 wt.%. Gelating agent was dissolved in water at room temperature for 1 hr. Concentration of salt solution was varied from 0.1 to 1.0 M.

Electrospinning The experimental setup device used for the electrospinning process is shown in Scheme 1. The electrospinning

setup consists of a syringe and needle, an aluminium collecting drum, highvoltage supply (Dongyang, Co., Daegu, Korea). SA solution in a capillary tube formed a droplet by the syringe pump. By applied voltage, the droplet was dripped into salt solution, and allowed to solidify for 1 h at room temperature. Applied voltage was varied from 10 to 20kV. TCD was varied from 10 to 20 cm. All electrospinning procedures were carried out at room temperature.

Characterization The morphology and diameter of the SA hydrogel microparticles were observed and determined with the use of optical microscope (OM; Eclipse E200, Nikon, Tokyo, Japan) and scanning electron microscope (SEM) (S-4100, Hitachi Co., Tokyo, Japan). The particle diameters of some samples were also measured directly with a video microscope (VMS; VS-32, Sometech, Seoul, Korea). Differential scanning calorimetry (DSC) measurements were performed under a nitrogen flow with a Q100 DSC apparatus from TA Instruments (New Castle, Delaware). All samples were heated from 0 to 240 °C at a heating rate of 10 °C /min.

Scheme 1. Experimental setup for the electrospinning process

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RESULTS AND DISCUSSION To prepare SA hydrogel microparticles by electrospinning, we performed a series of experiments with varying electrospinning parameter, including solution concentration of SA, type and amount of gelation agent, applied voltage, and TCD. Electrospinning conditions used in this study are listed in Table 1. Figure 1 showed the OM and SEM photographs of SA hydrogel microparticles prepared from the electrospinning at various SA concentrations. Concentration of CaCl2 as gelation agent, applied voltage, and TCD were fixed on 0.1 M, 15kV, and 15 cm, respectively. The difference between the OM and SEM photographs obtained from the same samples might be explained by the fact that the OM photographs were obtained using the uniformly swollen SA hydrogel microparticles in aqueous medium (crosslinking solution) but SEM photographs were obtained when the swollen SA hydrogel microparticles were freeze-dried with liquid nitrogen and then sputter-coated with gold (E1030, Hitachi, Tokyo, Japan) during the preparation of specimens. Accordingly, SA hydrogel microparticles were collapsed during the drying process because of the force generated by shrinkage during the sputtering. The size of SA hydrogel microparticle was increased as the SA concentration was increased. However, in SEM image, morphological structure of 1 wt.% SA was destroyed owing to lower SA concentration. We found that the more viscous the solution became, the more uniform the particles were formed. In addition, the diameters of the particles became bigger as the concentration of the SA solution was increased. Too low SA concentration resulted in very soft and unstable particles while increased SA concentration to above 2.0 wt.% hardened the particles. At even higher concentration of 2.0 wt.% and above, there were evidently sufficient molecular chain entanglements in the SA solution to prevent the breakup of the electrically driven jet and to allow the electrostatic stress to

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Preparation of Sodium Alginate Hydrogel Microparticles by Electrospinning Using Various Types of Salts

further elongate the jet. When only 1.0 wt.% SA concentration was used, the particles produced were easily broken because of their low mechanical strength. This is presumably due to the fact that the increasing the number of biopolymer molecules per unit solution increases, the binding sites for Ca2+ ions also increases. That is, a more densely crosslinked gel structure is probably formed8-9.

Table 1 Electrospinning conditions Materials

Type of solvent Conc. of SA aqueous solution Gelling agent TCD (cm) Applied voltage (kV)

Sodium alginate (SA) (chemical grade, viscosity: 20 ~ 40 cps Mn: 120,000 ~ 190,000 g/mol, M/G ratio = 1.56) Distilled water 1, 2, 3 wt.% CaCl2 : 0.1, 0.2, 0.4, 0.8, 1.0 M SrCl2, BaCl2 : 0.1 M 10, 15, 20 10, 15, 20

Figure 1. Photographs of SA hydrogel microparticle dripped into 0.1 M CaCl2 solution at different SA concentrations: (a) 1 wt.%; (b) 2 wt.%; (c) 3 wt.% (upper photographs were by OM and under photographs were by SEM)

Figure 2. Photographs of SA hydrogel microparticle dripped into 0.1 M CaCl2 solution at different TCDs: (a) 10 cm; (b) 15 cm; (c) 20 cm (upper photographs were by OM and under photographs were by SEM)

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Sun Gil Kim, Young Jae Lee, Eun Joo Shin, Yeong Soon Gal, Yong Rok Lee, Tae Hwan Oh, Han Gon Choi, Jung-Ae Kim, Chul Soon Yong, Sung Soo Han, Seok Kyun Noh, and Won Seok Lyoo

The morphology of the SA hydrogel microparticles prepared at TCD of 10, 15, and 20 cm, respectively. In the case of TCD, there is no significant effect on the SA hydrogel microparticle morphology. This indicates that under the present conditions, the TCD is not a principal factor for controlling the particle size. Such a small change in the TCD would cause little change in the potential differences at the interface between the SA solutions. Figure 3 showed the OM and SEM photographs of SA hydrogel

microparticles prepared from the electrospinning at various applied voltages. Concentration of SA, CaCl2 as gelation agent, and TCD were fixed to 2 wt.%, 0.1 M, and 10 cm, respectively. In a higher applied voltage, the size of SA hydrogel microparticle slightly decreased. The behaviour observed in this experiment is considered to be a general phenomenon, which is found in voltage effect of electrospinning10. The spinning voltage played a significant role on the fibre structure and diameter. It is known that the electrostatic force was gradually increased with

increasing the applied voltage. The split ability of droplet was reinforced due to the increasing the electrostatic force. When the applied voltage was increased, the jet velocity was increased and the solution was removed from the tip more quickly. At higher voltage, the particle diameter decreased due to the increases in the pulling and stretching forces. The effects of concentration of CaCl2 solution as crosslinking and gelation agents on the morphology of the resultant particles are show in

Figure 3. Photographs of SA hydrogel microparticle dripped into 0.2 M CaCl2 solution at different applied voltages: (a) 10 kV; (b) 15 kV; (c) 20 kV (upper photographs were by OM and under photographs were by SEM)

Figure 4. Photographs of SA hydrogel microparticle dripped into CaCl2 solutions with different concentrations of divalent cations: (a) 0.2 M; (b) 0.4 M; (c) 0.8 M; (d) 1 M (upper photographs were by VMS and under photographs were by SEM)

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Figure 4. Structural and mechanical properties of SA hydrogels can also be tuned by adjusting the ionic strength of the jellification medium or the multi-valent metal ion source. Concentration of SA, applied voltage, and TCD were fixed to 1 wt.%, 15kV, and 15 cm, respectively. In this case of concentration of salt solution, there is no significant effect on the SA hydrogel microparticle morphology. It was known that low solubility calcium salts or inert electrolytes reduce the gelation rate and increase both the structural uniformity and the mechanical strength of gels11-13. Figure 5 presents the OM and SEM photographs of SA hydrogel microparticles prepared from the electrospinning using various types of salts. Concentration of SA, applied voltage, and TCD were fixed to 1 wt.%, 15kV, and 15 cm, respectively. SA hydrogel microparticle obtain by the exchange of sodium ions with divalent cations such as Sr2+, and Ba2+ presented uniform spherical shape, in comparison with SA hydrogel microparticle prepared by Ca 2+ . Monovalent cations and Mg2+ ions do not induce gelation14.

Effects of gelation salts and applied voltage on the average diameter of SA hydrogel microparticles are shown in Figure 6. Concentration of SA and TCD were fixed to 2 wt.% and 15 cm, respectively. In all cases, the particle size decreased with an increase in applied voltage but at a high applied voltage of electrospinning, the polydispersity was increased and broad range of particle sizes were observed. Monodisperse particle was prepared at an applied voltage of 10kV. This was probably because of the high instability of the jet at the tip, and was similar to modes of electrostatic atomization described elsewhere15. The electrospinning process could be sustained in a variety of modes characterized by the shape of surface from which the liquid jet originated. It has been verified experimentally that the shape of the initiating drop changes with spinning condition (voltage, viscosity, feed rate)16. It was known that the electrostatic force was gradually increased with increasing the applied voltage. The split ability of the droplet was reinforced due to the increasing the electrostatic force. Increasing the voltage causes the rate at which the solution was removed

from the capillary tip to exceed the rate of delivery of the solution to the tip needed to maintain the conical shape of the surface of droplet. The position of ejection of the jet on the surface of droplet was changed very rapidly and led to a somewhat unsteady flow of the spinning solution. The large dispersity in particle diameter generated by the process was probably a result of this instability. The DSC curves of crosslinked SA hydrogel microparticles are presented in Figure 7. All the SA hydrogel microparticles showed endothermic peaks around 100 °C ascribed to the dehydration in the particles, and displayed exothermic peaks in the range of 200 ~ 280 °C. Two exothermic peaks were attributed to the disintegration of molecular side chains and part of main chains, respectively 17-18. Crosslinked SA hydrogel by divalent ion of Ca2+ showed a strongest characteristic exothermic peak at around 270 °C that is attributed to the part of main chains disintegration. From these results, it was supposed that the crosslinking force of Ca2+ ion is stronger than those of Ba2+ and Sr2+.

Figure 5. Photographs of SA hydrogel microparticle dripped into diverse crosslinking solutions: (a) 0.1 M CaCl2; (b) 0.1 M BaCl2; (c) 0.1 M SrCl2 (upper photographs were by OM and under photographs were by SEM)

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Sun Gil Kim, Young Jae Lee, Eun Joo Shin, Yeong Soon Gal, Yong Rok Lee, Tae Hwan Oh, Han Gon Choi, Jung-Ae Kim, Chul Soon Yong, Sung Soo Han, Seok Kyun Noh, and Won Seok Lyoo

Figure 6. Average diameter of SA hydrogel microparticle with applied voltage

stable morphology was revealed. The gelation of SA is mainly achieved by the exchange of sodium ions with divalent cations such as Ca2+, Ba2+, and Sr2+. It was found the crosslinking force values follow the order Ca2+ > Ba2+ > Sr2+.

Acknowledgements This work was supported by grant No. RTI04-01-04 from the Regional Technology Innovation Program of the Ministry of Knowledge Economy (MKE).

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Figure 7. The DSC thermograms of SA hydrogel microparticle dripped into diverse crosslinking solutions: 0.2 M (a) CaCl2; (b) BaCl2; (c) SrCl2

CONCLUSIONS In order to produce SA hydrogel microparticles, we used electrospinning technique. The particle size and its distribution could be controlled by adapting various electrospinning conditions. The particle size was decreased with increasing applied voltage. However, in the case of higher

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