Advances in Materials Science and Engineering
Volume 2017 (2017), Article ID 9304592, 7 pages
https://doi.org/10.1155/2017/9304592

Research Article

The Size Stability of Alginate Beads by Different Ionic Crosslinkers

Shu-Ling Huang and Yung-Sheng Lin

Department of Chemical Engineering, National United University, Miaoli, Taiwan

Correspondence should be addressed to Yung-Sheng Lin; linys@nuu.edu.tw

Received 7 June 2017; Revised 12 July 2017; Accepted 18 July 2017; Published 7 September 2017

Academic Editor: Luca De Stefano

Copyright © 2017 Shu-Ling Huang and Yung-Sheng Lin. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Few studies have discussed the stability of gelled alginate bead size. Therefore, the present study investigated the dynamic shrinkage of gelled alginate beads affected by two common ionic crosslinkers at different concentrations and temperatures. The results indicate that the gelled alginate beads gradually shrank with longer gelling times. The beads incubated in a Ca²⁺ solution shrank more dramatically than those incubated in a Ba²⁺ solution. Those incubated at room temperature exhibited greater shrinkage than those incubated at a low temperature. A 25% size reduction occurred in the 1% Ca²⁺ solution at room temperature after 300 minutes of gelling time. The alginate beads gelled took at least 120 minutes to become stable after the ionic gelation process.

1. Introduction

Because of easy availability, cost effectiveness, biodegradability, and biocompatibility [1], natural polymers have been used extensively over the past few decades [2–4]. Among them, alginate, a type of polysaccharides composed of 1,4′-β-d-mannuronic acid (M-block) and α-l-guluronic acid (G-block), is plentiful in our environment [5, 6]. Alginate was discovered as a structural component of brown marine algae [7]. Until now, alginate has been processed extensively as capsules, beads, and fibers for various application fields, especially in drug delivery [8]. It is suitable for encapsulating targeted drugs because of favorable properties such as water solubility [9], nontoxicity [10], and biodegradability [11].

Alginate particle size is a crucial factor for many applications [12–15]. The bead size is determined by the manufacturing process and alginate properties [16, 17]. Among these properties, alginate’s affinity with various divalent ions has a crucial influence on the particle size [18]. Divalent cations such as Ca²⁺ and Ba²⁺ are common crosslinkers for producing alginate particles [19]. A previous study indicated that alginate beads gelled in Ba²⁺ solutions were larger than those gelled in Ca²⁺ solutions [20]. Another study found that alginate beads incubated in a 10 or 20 mM Ba²⁺ solution were larger than those incubated in a 50 mM Ca²⁺ solution [18]. These results show that alginate bead size is easily influenced by these two crosslinkers. However, few studies have compared the dynamic size changes of alginate beads gelled by these two crosslinkers in a single experiment. One study demonstrated that alginate bead sizes incubated in Ca²⁺ solution decreased continuously over a 30-minute gelling period [21]. Another study reported that a prolonged gelling time did not affect bead size in Ba²⁺ solution after 2 or 20 minutes [22].

In addition to detecting size changes due to crosslinkers, one study have indicated that the size of the beads diminishes by 8% after being gelled at a high temperature (90°C) [23]. However, another paper claimed that incubation at temperatures between 5°C and 40°C had no significant effect on the bead size [13]. Accordingly, this paper discusses whether bead size is affected by temperature changes.

According to the aforementioned studies, alginate bead size is changed by crosslinkers or temperature. However, until now, the effects of Ca²⁺ and Ba²⁺ and temperature on dynamic alginate size have seldom been reported. The present study observed the dynamic shrinkage of alginate beads due to gelling by Ca²⁺ and Ba²⁺ at various temperatures.

2. Materials and Methods

2.1. Materials

Sodium alginate powder (A0682, M/G = 69/31, MW = 12,000~80,000) was purchased from Sigma Aldrich Chemical Co., Ltd. (St. Louis, USA), and Coomassie Brilliant Blue G-250 was obtained from One Star Biotechnology Co., Ltd. (Taipei, Taiwan). Barium chloride dihydrate and anhydrous calcium chloride were supplied by Eco Chemical Co., Ltd. (Taichung, Taiwan).

2.2. Preparation of Alginate Beads

Sodium alginate solution was prepared in distilled water at a concentration of 2% (w/v), and Coomassie Brilliant Blue G-250 was added at a concentration of 0.05% (w/v) for observation. For simple and straight comparison, CaCl₂ and BaCl₂ were also prepared by 10% w/v and diluted to 1% w/v, respectively. A disposable Terumo® syringe (3 mL) was filled with homogenized alginate solution, which was subsequently extruded using a KDS230 syringe pump (KD Scientific Inc., Holliston, USA). Alginate dispersion was then added dropwise into the cuvettes filled with 3 mL of Ca²⁺ solution and Ba²⁺ solution at a constant injection rate, enabling calcium and barium ion-crosslinked alginate and the gelled beads to be created uniformly [24] (Figure 1). We examined the beads at various predetermined times (5, 20, 30, 60, 120, 180, 240, and 300 minutes) after initializing the gelling process until reaching the equilibrium size state. This experiment was controlled around the set room temperature (25°C) and low temperature (8°C) and repeated at least three times. Student -test was used for the statistical analysis.

Figure 1: Schematic drawing of alginate bead formation.

2.3. Characterization

A digital camera (DP70, Olympus, Taiwan) was employed for imaging to estimate the bead morphology. At a predetermined time, the alginate beads were removed from the crosslinking solution for imaging. To ensure statistical representativeness, three alginate beads were analyzed for each condition. The bead sizes were obtained from recorded photographs and are expressed as the mean ± standard deviation.

3. Results and Discussion

Table 1 shows a series of photographs for four alginate beads gelled by two concentrations (1% w/v and 10% w/v) of two crosslinkers (Ca²⁺ and Ba²⁺) at different gelling times (5, 20, 30, 60, 120, 180, 240, and 300 minutes) at room temperature (25°C). The results indicate that all the alginate beads shrank with longer gelling times. Furthermore, the bead sizes in the Ca²⁺ solution were initially larger than those in the Ba²⁺ solution because the beads incubated in the Ca²⁺ solution did not form sufficiently tightly and had a lower affinity than the beads incubated in the Ba²⁺ solution [25, 26]. At 300 minutes, the beads in high concentrations of crosslinkers were larger than those in low concentrations of crosslinkers. By analyzing the statistical results in Table 1, we plotted Figures 2 and 3 for the dynamic size shrinkage of alginate at room temperature. Figures 2 and 3 show the original bead size and normalized size by the initial bead size, respectively.

Table 1: Photographs of alginate beads incubated in different concentrations of Ca²⁺ and Ba²⁺ at room temperature (25°C) for various gelling times.

Time (minutes)
1% Ca²⁺
10% Ca²⁺
1% Ba²⁺
10% Ba²⁺

5

20

30

60

120

180

240

300

Figure 2: Diameters of alginate beads incubated in different concentrations of Ca²⁺ and Ba²⁺ at room temperature (25°C) for various gelling times.

Figure 3: Normalized diameters of alginate beads incubated in different concentrations of Ca²⁺ and Ba²⁺ at room temperature (25°C) for various gelling times.

According to Figure 2, all alginate beads shrank with longer gelling times, and the beads in the Ca²⁺ solution were larger than those in the Ba²⁺ solution before 30 minutes. To conform to a standard to easily observe the shrinking rates, Figure 3 shows the normalized sizes of the beads obtained at room temperature, thereby demonstrating that the beads incubated in the Ba²⁺ solution did not shrink more notably than those in the Ca²⁺ solution. After 60 minutes, the shrinking curves had become smooth, but 1% of the Ca²⁺ curve required 120 minutes to become smooth. Compared with different concentrations of crosslinkers, the beads in high concentrations of crosslinkers barely shrank. The final gelled bead sizes in high concentrations were between 0.9 (10% Ca²⁺) and 0.93 (10% Ba²⁺), and those in low concentrations were between 0.75 (1% Ca²⁺) and 0.85 (1% Ba²⁺). Therefore, the effect of the Ca²⁺ concentration on the gelation-shrinkage process was more pronounced than that of the Ba²⁺ concentration. The gel-forming ability of alginates depends on the G-blocks binding divalent cations [27]. Sufficient divalent cations are present in high concentrations of crosslinkers, leading to the tight formation of gel and low structural rearrangement to reduce the size. A previous study [18] demonstrated that alginate’s affinity toward various divalent ions can decrease in the order of Ba²⁺ > Ca²⁺. Hence, the beads incubated in the Ba²⁺ solution were able to form tightly, resulting in minimal shrinkage. The outcome is in accordance with Figure 2.

With the same gelling conditions as those in Table 1, Table 2 shows alginate beads at a low temperature (8°C). The outcomes were similar to those when the experiment was conducted at room temperature; the bead sizes in the Ca²⁺ solution were initially larger than those in the Ba²⁺ solution. The images in Table 2 were also subjected to a statistical analysis. Figures 4 and 5 show the original and normalized bead sizes at a low temperature, respectively.

Table 2: Photographs of alginate beads incubated in different concentrations of Ca²⁺ and Ba²⁺ at a low temperature (8°C) for various gelling times.

Time (minutes)
1% Ca²⁺
10% Ca²⁺
1% Ba²⁺
10% Ba²⁺

5

20

30

60

120

180

240

300

Figure 4: Diameters of alginate beads incubated in different concentrations of Ca²⁺ and Ba²⁺ at a low temperature (8°C) for various gelling times.

Figure 5: Normalized diameters of alginate beads incubated in different concentrations of Ca²⁺ and Ba²⁺ at a low temperature (8°C) for various gelling times.

Figure 4 demonstrates the same trend as Figure 2. All the curves for the low temperature were much smoother than those for room temperature, indicating that the beads incubated at a low temperature did not shrink substantially, even with a low concentration of Ca²⁺. This is because the gelled alginate structure has low rearrangement mobility at low temperatures, resulting in a minor size change.

Figure 5 shows the normalized size shrinkage in 8°C. The results indicate that the final gelled bead sizes were between 0.85 (1% Ca²⁺) and 0.95 (10% Ba²⁺). By contrast, Figure 3 shows that the gelled bead sizes were between 0.75 (1% Ca²⁺) and 0.93 (10% Ba²⁺). It reveals that four groups were significantly different from each other with value less than 0.05 in Figure 5. Except groups between 10% Ca²⁺ and 1% Ba²⁺, the other pairs in Figure 3 also showed significant difference with value less than 0.05. The beads incubated in 1% Ca²⁺ solution exhibited considerably more evident size changes than those in the other three conditions, because a temperature increase activates motion in water molecules and a higher temperature raises the shrinkage rate of alginate beads. Shrinking is governed by cooperative diffusion of the gel network. The final gelled alginate beads in low crosslinker concentrations had minor mobility restrictions, resulting in major shrinkage compared with those in high crosslinker concentrations.

The shrinkage, tightness, release behaviors, and swelling properties of alginate beads are all relating to each other. Kaygusuz et al. reported metal ion and surfactant effects on the mechanical strength of alginate beads, and they concluded similar trends in bead sizes of pure Ca²⁺ or Ba²⁺ alginate by changing alginate concentration [28]. Harper et al. reported effects of various cations on the physical properties of alginate films and found Ba²⁺ ions produced strong alginate films [29]. Kaygusuz et al. reported cation effects on the slow release from alginate beads [30]. Encapsulation efficiency of model dye in Ba-alginate beads was much higher than that of Ca-alginate and dye release from Ca-alginate beads was much faster. Darrabie et al. reported the effect of gelling cation on microbead swelling and concluded that Ca-alginate microbeads were more prone to swelling than the corresponding Ba-alginate beads [31].

4. Conclusion

All alginate beads in this study shrank with longer gelling times. However, the beads incubated in the Ca²⁺ solution shrank more substantially than those in the Ba²⁺ solution. The beads incubated at a low temperature exhibited minor size changes compared with those incubated at room temperature, which exhibited dramatic shrinkage such as the 25% size reduction in 1% Ca²⁺ solution. The results show that the alginate beads gelled stably at least 120 minutes later, thereby providing a guideline for utilizing alginate properties in different fields.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

This work was supported by the Ministry of Science and Technology, Taiwan (MOST 105-2221-E-239-031).

References

S. Hua, H. Ma, X. Li, H. Yang, and A. Wang, “pH-sensitive sodium alginate/poly(vinyl alcohol) hydrogel beads prepared by combined Ca²⁺ crosslinking and freeze-thawing cycles for controlled release of diclofenac sodium,” International Journal of Biological Macromolecules, vol. 46, no. 5, pp. 517–523, 2010.

C. Wischke, C. Schneider, A. T. Neffe, and A. Lendlein, “Polyalkylcyanoacrylates as in situ formed diffusion barriers in multimaterial drug carriers,” Journal of Controlled Release, vol. 169, no. 3, pp. 321–328, 2013.

C.-C. Chen, C.-L. Fang, S. A. Al-Suwayeh, Y.-L. Leu, and J.-Y. Fang, “Transdermal delivery of selegiline from alginate-pluronic composite thermogels,” International Journal of Pharmaceutics, vol. 415, no. 1-2, pp. 119–128, 2011.

P. C. Balaure, E. Andronescu, A. M. Grumezescu et al., “Fabrication, characterization and in vitro profile based interaction with eukaryotic and prokaryotic cells of alginate-chitosan-silica biocomposite,” International Journal of Pharmaceutics, vol. 441, no. 1-2, pp. 555–561, 2013.

A. K. Nayak, M. S. Hasnain, S. Beg, and M. I. Alam, “Mucoadhesive beads of gliclazide: design, development, and evaluation,” ScienceAsia, vol. 36, no. 4, pp. 319–325, 2010.

M. D. De'Nobili, L. M. Curto, J. M. Delfino, M. Soria, E. N. Fissore, and A. M. Rojas, “Performance of alginate films for retention of L-(+)-ascorbic acid,” International Journal of Pharmaceutics, vol. 450, no. 1-2, pp. 95–103, 2013.

A. K. Nayak and D. Pal, “Development of pH-sensitive tamarind seed polysaccharide-alginate composite beads for controlled diclofenac sodium delivery using response surface methodology,” International Journal of Biological Macromolecules, vol. 49, no. 4, pp. 784–793, 2011.

P. Degen, S. Leick, F. Siedenbiedel, and H. Rehage, “Magnetic switchable alginate beads,” Colloid and Polymer Science, vol. 290, no. 2, pp. 97–106, 2012.

N. Mennini, S. Furlanetto, M. Cirri, and P. Mura, “Quality by design approach for developing chitosan-Ca-alginate microspheres for colon delivery of celecoxib-hydroxypropyl-β-cyclodextrin-PVP complex,” European Journal of Pharmaceutics and Biopharmaceutics, vol. 80, no. 1, pp. 67–75, 2012.

N. Işiklan, M. Inal, F. Kurşun, and G. Ercan, “PH responsive itaconic acid grafted alginate microspheres for the controlled release of nifedipine,” Carbohydrate Polymers, vol. 84, no. 3, pp. 933–943, 2011.

L. Wang, J. Shansky, C. Borselli, D. Mooney, and H. Vandenburgh, “Design and fabrication of a biodegradable, covalently crosslinked shape-memory alginate scaffold for cell and growth factor delivery,” Tissue Engineering - Part A, vol. 18, no. 19-20, pp. 2000–2007, 2012.

V. J. Mohanraj and Y. Chen, “Nanoparticles - a review,” Tropical Journal of Pharmaceutical Research, vol. 5, no. 1, pp. 561–573, 2006.

P. Smrdel, M. Bogataj, and A. Mrhar, “The influence of selected parameters on the size and shape of alginate beads prepared by ionotropic gelation,” Scientia Pharmaceutica, vol. 76, no. 1, pp. 77–89, 2008.

W. R. Gombotz and S. F. Wee, “Protein release from alginate matrices,” Advanced Drug Delivery Reviews, vol. 31, no. 3, pp. 267–285, 1998.

K. J. Klemmer, D. R. Korber, N. H. Low, and M. T. Nickerson, “Pea protein-based capsules for probiotic and prebiotic delivery,” International Journal of Food Science and Technology, vol. 46, no. 11, pp. 2248–2256, 2011.

B. Strasdat and H. Bunjes, “Incorporation of lipid nanoparticles into calcium alginate beads and characterization of the encapsulated particles by differential scanning calorimetry,” Food Hydrocolloids, vol. 30, no. 2, pp. 567–575, 2013.

B.-B. Lee, P. Ravindra, and E.-S. Chan, “Size and shape of calcium alginate beads produced by extrusion dripping,” Chemical Engineering and Technology, vol. 36, no. 10, pp. 1627–1642, 2013.

Ý. A. Mørch, I. Donati, B. L. Strand, and G. Skjåk-Bræk, “Effect of Ca²⁺, Ba²⁺, and Sr²⁺ on alginate microbeads,” Biomacromolecules, vol. 7, no. 5, pp. 1471–1480, 2006.

K. L. Chen, S. E. Mylon, and M. Elimelech, “Enhanced aggregation of alginate-coated iron oxide (Hematite) nanoparticles in the presence of calcium, strontium, and barium cations,” Langmuir, vol. 23, no. 11, pp. 5920–5928, 2007.

Y. Liu, Y. Tong, S. Wang, Q. Deng, and A. Chen, “Influence of different divalent metal ions on the properties of alginate microcapsules and microencapsulated cells,” Journal of Sol-Gel Science and Technology, vol. 67, no. 1, pp. 66–76, 2013.

P. Smrdel, M. Bogataj, F. Podlogar et al., “Characterization of calcium alginate beads containing structurally similar drugs,” Drug Development and Industrial Pharmacy, vol. 32, no. 5, pp. 623–633, 2006.

V. Vaithilingam, G. Kollarikova, M. Qi et al., “Effect of prolonged gelling time on the intrinsic properties of barium alginate microcapsules and its biocompatibility,” Journal of Microencapsulation, vol. 28, no. 6, pp. 499–507, 2011.

J. W. Woo, H. J. Roh, H. D. Park et al., “Sphericity optimization of calcium alginate gel beads and the effects of processing conditions on their physical properties,” Food Science and Biotechnology, vol. 16, no. 5, pp. 715–721, 2007.

W.-L. Chou, P.-Y. Lee, C.-L. Yang, W.-Y. Huang, and Y.-S. Lin, “Recent advances in applications of droplet microfluidics,” Micromachines, vol. 6, no. 9, pp. 1249–1271, 2015.

Q. L. Loh, Y. Y. Wong, and C. Choong, “Combinatorial effect of different alginate compositions, polycations, and gelling ions on microcapsule properties,” Colloid and Polymer Science, vol. 290, no. 7, pp. 619–629, 2012.

J. J. Chuang, Y. Y. Huang, S. H. Lo et al., “Effect of pH on the shape of alginate particles and its release behavior,” International Journal of Polymer Science, vol. 2017, pp. 1–9, 2017.

G. Orive, R. M. Hernández, A. R. Gascón, and J. L. Pedraz, “Encapsulation of cells in alginate gels,” in Methods in Biotechnology, vol. 22, pp. 345–355, 2006.

H. Kaygusuz, G. A. Evingür, Ö. Pekcan, R. von Klitzing, and F. B. Erim, “Surfactant and metal ion effects on the mechanical properties of alginate hydrogels,” International Journal of Biological Macromolecules, vol. 92, pp. 220–224, 2016.

B. A. Harper, S. Barbut, L.-T. Lim, and M. F. Marcone, “Effect of various gelling cations on the physical properties of “wet” alginate films,” Journal of Food Science, vol. 79, no. 4, pp. E562–E567, 2014.

H. Kaygusuz, F. B. Erim, Ö. Pekcan, and G. Akin Evingür, “Cation effect on slow release from alginate beads: A fluorescence study,” Journal of Fluorescence, vol. 24, no. 1, pp. 161–167, 2014.

M. D. Darrabie, W. F. Kendall, and E. C. Opara, “Effect of alginate composition and gelling cation on microbead swelling,” Journal of Microencapsulation, vol. 23, no. 6, pp. 613–621, 2006.