Alginates for Gel Immobilization, Encapsulation and Scaffolds

One of the universal carbohydrate polymers used for cell immobilization is calcium alginate. This polyelectrolyte and hydrocolloid is extracted from seaweed. It has become one of the most important materials in commercial use.  Here, we describe and discuss the properties of alginates for gel immobilization, which are exploited in a wide range of industries including the food industry, biotechnology, consumer healthcare and pharmaceutical industries.

The unique properties of alginate makes it now the most widely used polymer for immobilization and microencapsulation (Funduenanu et al., 1999).

One of the principle reasons it is chosen is because it has low toxicity and is relatively robust although it will breakdown eventually. It is also natural and suitable for vegan applications. 

Introduction

Alginic acid is a heteropolysaccharide of L-guluronic acid (G) and D-mannuronic acid (M). It is mainly extracted from algae and brown algae in particular although some bacteria produce a version such as Pseudomonas aeruginosa. The main genus of seaweed algae is Phaeophycea which grows in shallow waters of temperate zones. The source dictates the composition and sequence of these two sugars in alginic acid and it varies widely. Different species of brown seaweeds, such as Laminaria hyperborea, Ascophyllum nodosum and Macrocystis pyrifera, are responsible for producing these alginates with widely different chemical composition. The source of alginate also determines both chemistry and physical performance of any gel created. It is one of the issues with alginate that there is such variability in composition but managed properly offers a range of gelling opportunities (Onsøyen, 1997).

Structure

The uronic acid monomers are arranged in blocks of various patterns along the chain. Homopolymeric regions are interspersed with regions of alternating structure which are often described as heteropolymeric (Smidsrod et al., 1990). Each block of monomers can be up to 20 sugar units long. There are different sequences and compositions of these monomers in the various types of alginate available. The regions are referred to as M blocks for poly(mannuronic acid), G blocks for poly(guluronic acid), and MG blocks for poly(mannuronic acid) alternating with guluronic acid (Clare, 1993). 

The differences in the nature of the linkage between M blocks and G blocks is reflected in the conformation of these sections in the polymer chain. The M block section is flat while the G block section is buckled (Clare, 1993). This sectional nature of the alginate polymer confers different backbone chain flexibility to the polymers in solution. The differences in flexibility are not due to differences in H-bonding which is present to the same extent for each monomer. The difference in flexibility arises from a greater restriction about the carbon to oxygen bonds joining the monomers. The α-(1–4) linkage of the guluronic acid residues introduces greater steric hindrance from the carboxyl groups and so high M content alginate chains are more flexible in solution than high G content alginate chains.

Alginates work well with other polymers. Being a highly anionic polyelectrolyte, it combines well with strongly cationic polymers such as chitosan. They also work well in combination with pectin.

The structure of alginate and its applications other than immobilization of enzymes and cells are largely discussed in the article on alginates.

Alginates for Gel Immobilization

Alginates have long been used for immobilizing cells and enzymes because they form a polymeric gel mesh ideally suited for this purpose. The properties of immobilized systems needs to be investigated as part of the scale-up and applicability of these immobilized systems.

Calcium alginate gels are prepared usually by casting a solution of sodium alginate (1% w/w) into a bath of calcium chloride (2% w/v) at room temperature and collecting the gel formed. It can also be produced by casting calcium chloride solution into an alginate bath so a number of options exist for preparing these gel structures.

Calcium binds the alginates polymers into a structure which has similarities to that formed by low-methoxyl pectin. There are differences however and the gel structure is slowly being teased out.

The diffusivity of proteins is always of interest to those wishing to select the right alginate for the purpose. One study looked at different M and G levels to understand how easy it was for proteins like bovine serum albumin (BSA) to diffuse into and through alginate gels. Protein diffusion was greatest in gels of a low guluronic acid content (low G content). The explanation for this was based on the rigidity of the alginate polymer which is greater when bound to calcium because guluronic acid has a stronger preference for these cations and they are known to  produce much stiffer gels. So, the greater the flexibility of the polymer the higher the diffusivity of the protein or indeed any other material most likely entrained in the gel. Its useful to know this when developing alginate gel spheres (Amsden & Turner, 2000) for encapsulating materials.

Modified forms of alginate are also useful in encapsulation. Dodecenyl succinic anhydride (SAC12) encapsulation of lipase showed a 3-fold increase in specific activity towards water-soluble substrates (Falkeborg et al., 2015).

Cells are often immobilised because it prevents their free movement, stabilises them and they operate as miniature reactors. Cells are not uniformly dispersed but non-uniformly with a larger number of cells on the surface of the beads than within the core (Zohar-Perez et al., 2004). 

Gelled Foams

Dried alginate-based gelled foams have been studied in applications such as scaffolding in tissue engineering, regenerative medicine and wound healing (Hegge et al., 2010; 2011; Andersen et al., 2012). PGA is a potential candidate for this type of behaviour its surface active properties are used to stabilize the discontinuous phase such as air, lipids or solids whilst the conserved G-blocks encourage gelation in the continuous phase. It could mean oil droplets are entrained within the foam as a consequence of this biphasic behaviour.

Applications

Food preservation – microspheres with antibacterial properties are used to prolong fruit shelf-life. Polylysine is an effective antimicrobial agent. An antibacterial agent is prepared of sodium alginate and ε-polylysine into microspheres (DSA-Pl MPs). The dialdehyde form of sodium alginate is produced by periodate oxidation of the polymer. The polylysine is conjugated onto the DSA backbone using a Schiff’s base reaction to produce the DSA-PL conjugates. Then emulsification followed by addition of Ca²⁺ cations in a calcium chloride bath. Spherical microspheres are produced with a relatively narrow size distribution and good dispersity suited to coating fruit. The microspheres are prone to acid degradation. They also have exceptional antimicrobial activity when sprayed onto fruit (Ge et al., 2022).

Immobilized Enzymes

Numerous examples of enzymes encapsulated in alginate gels are known. the most common laboratory approach is drop-wise addition of an aqueous solution of sodium alginate with the enzyme into a bath of what is termed a ‘hardening solution’ of calcium ions. Calcium almost instantly cross-links the alginate to form small spheres which precipitate and are collected with the entrapped enzyme.

Lipase is often immobilised in gels and alginate is no exception (Won et al., 2005). The Candida rugosa lipase (EC 3.1.1.3) was trapped using drop-wise addition into an aqueous solution of sodium alginate.

A number of parameters were checked concerning immobilization including alginate concentration, CaCl₂ concentration, ratio by weight of enzyme to alginate (E/A) and bead size on loading efficiency (percent of total enzyme entrapped) and immobilization yield (specific activity ratio of entrapped lipase to free lipase). In this study, an increase in alginate concentration raised loading efficiency, but decreased immobilization yield. The researchers also thought that the concentration of  CaCl₂  would have a similar effect but this was relatively small in the concentration range of 50–300 mM. The ratio by weight of enzyme to alginate did not have much affect on loading efficiency and immobilization yield.

One issue with Ca-alginate beads is leakage of the entrapped material out of the gel. To avoid this, the beads of entrapped enzyme are coated with chitosan or silicate.

Dye removal from waste streams is a good example of this type of application. Laccase, an enzyme from white-rot fungi was encapsulated into alginate-chitosan beads using an emulsification-internal gelation method (Lu et al., 2007). The optimal immobilization condition was 2% sodium alginate, 2% CaCl₂, 0.3% chitosan and 1:8 ratio by volume of enzyme to alginate. Laccase stability improved with immobilization. The loading efficiency and immobilised yield was 88.12% and 46.93%, respectively. In terms of enzyme performance both immobilised and free enzyme had low decolourization efficiency using the dye Alizarin Red. The addition of the chemical 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) improved reaction rates. Immobilization also meant retention of 35.7% activity after three uses in the model reactor.

Amylases have also been immobilized (Ertan et al., 2006) for starch hydrolysis. The α‐amylase from Aspergillus sclerotiorum was immobilised in a similar manner to lipase. The optimum CaCl₂ concentration was 3% w/v. The highest level of enzyme activity was observed with a  loading enzyme concentration of 140 U/mL, and bead (diameter 3 mm) amount of 0.5 g. Only 35% of the initial activity was lost after 7 passes.

A number of enzymes can be immobilised together too (Blandino et al., 2003). The example cited is of glucose oxidase and catalase.

Other Applications of Alginates include:

Drug Delivery Systems

Alginate gels can be used as a carrier matrix for controlled release of drugs or bioactive compounds. The gel structure can be engineered to control the diffusion and release rate of the encapsulated substances, providing sustained and targeted drug delivery.

Biocompatibility

Alginate is generally biocompatible and does not induce significant immune responses. This property makes alginate solutions suitable for various biomedical applications, including tissue engineering, wound healing, and regenerative medicine.

Viscosity and Rheology

Alginate solutions exhibit high viscosity, which can be advantageous in several biotechnological processes.

Bioinks

Bioink in 3D Bioprinting: Alginate hydrogels can be used as bioinks in 3D bioprinting, allowing the precise deposition of cells and biomaterials in a controlled manner to fabricate complex 3D structures. The viscosity of alginate solutions can be adjusted to ensure proper flow and printing characteristics.

Printability: Alginates possess shear-thinning behavior, meaning their viscosity decreases under shear stress, facilitating the extrusion process during bioprinting. This characteristic enables the bioink to flow through the printing nozzle more easily, while maintaining its shape after deposition due to gelation. The shear-thinning behavior of alginates allows for the deposition of intricate structures with high fidelity and resolution.

Tunable Mechanical Properties: The mechanical properties of alginate-based bioinks can be adjusted by varying the concentration of alginate and the degree of crosslinking. By controlling these factors, it is possible to create bioinks with a wide range of stiffnesses and viscoelastic properties that mimic different tissues and support the specific needs of the printed constructs.

Cell Encapsulation: Alginates offer the ability to encapsulate living cells within the hydrogel matrix during the printing process. This encapsulation protects the cells, maintains their spatial organization, and provides a suitable microenvironment for their growth and differentiation. The porous structure of alginate hydrogels allows for the exchange of nutrients and waste materials, promoting cell viability and functionality.

Biofunctionalization: Alginates can be modified and functionalized to incorporate bioactive molecules, growth factors, and signaling cues within the bioink. These modifications enable the incorporation of specific signals that guide cell behavior, such as promoting cell adhesion, differentiation, and tissue regeneration.

Scaffold Fabrication

Alginate solutions can be used to create porous scaffolds with a defined structure. By adjusting the viscosity and crosslinking conditions, alginate scaffolds can be tailored to support cell attachment, proliferation, and tissue regeneration.

Scaffolds provide a three-dimensional structure that supports cell attachment, proliferation, and tissue regeneration. Alginate scaffolds offer several advantages for cell culture and tissue engineering applications. 

1. Biocompatibility: Alginates are generally biocompatible and do not induce significant immune responses. This property is essential for creating scaffolds that can support cell growth and tissue regeneration without adverse effects. Alginate scaffolds provide a favorable environment for cells, allowing them to adhere, spread, and proliferate.
2. Porous Structure: Alginate scaffolds can be designed to have a porous structure, which allows for efficient nutrient and oxygen diffusion throughout the scaffold. The porosity of the scaffold can be controlled by adjusting the concentration and viscosity of the alginate solution, as well as the crosslinking method.
3. Gelation and Crosslinking: Alginate solutions undergo gelation when exposed to divalent cations, such as calcium ions. This gelation process can be utilized to create stable and mechanically robust scaffolds. Alginate crosslinking can be achieved through different methods:

• Ionic Crosslinking: Calcium ions are commonly used to induce gelation of alginate solutions. By immersing the alginate scaffold in a calcium chloride solution, crosslinks are formed between alginate chains, resulting in a solid gel structure. The concentration of calcium ions and the immersion time can be adjusted to control the mechanical properties of the scaffold.
• Covalent Crosslinking: Alginate can be chemically modified to introduce functional groups that can react and form covalent bonds with other molecules or polymers. This method provides additional control over the mechanical and degradation properties of the scaffold.

4. Tunable Properties: Alginate scaffolds offer the advantage of tunable properties to meet specific requirements for different tissue engineering applications. Key properties that can be controlled include:

• Mechanical Strength: The mechanical properties of the scaffold can be adjusted by varying the concentration of the alginate solution, the degree of crosslinking, or incorporating reinforcing materials. This allows the scaffold to match the mechanical properties of the native tissue it is intended to replace or support.
• Degradation Rate: Alginate scaffolds can be designed to degrade at specific rates. The degradation rate affects the scaffold’s longevity, as well as the release of encapsulated bioactive molecules or growth factors. Degradation can be controlled by adjusting the crosslinking density or incorporating degradable components within the scaffold.

5. Cell Adhesion and Signaling: Alginate scaffolds can be modified to enhance cell adhesion and provide specific signaling cues to promote cell growth and tissue regeneration. The surface of the scaffold can be functionalized with cell adhesion peptides, such as RGD (Arg-Gly-Asp), to facilitate cell attachment. Additionally, growth factors or other bioactive molecules can be incorporated into the alginate matrix to provide localized signaling cues for cell proliferation, differentiation, and tissue regeneration.

Overall, alginate scaffolds offer a versatile platform for cell growth and tissue engineering applications. Their biocompatibility, porous structure, gelation properties, tunable characteristics, and the ability to incorporate bioactive molecules make them valuable tools in creating scaffolds that support cell attachment, proliferation, and the development of functional tissues.

Chelation and Metal Ion Sequestration

Alginate has a strong affinity for divalent cations such as calcium, which enables the formation of gel structures. However, alginate can also chelate or sequester other metal ions, which is useful in various biotechnological applications:

Heavy Metal Removal

Alginate can be used as an adsorbent for heavy metal ions in wastewater treatment and environmental remediation processes. The chelation properties of alginate enable the binding and removal of toxic metal contaminants.

Alginate solutions as has been discussed already, can be used to encapsulate enzymes or other sensitive biomolecules, protecting them from degradation or denaturation including the toxic nature of heavy metal ions. The chelation of metal ions by alginate can thus provide stabilization and support the activity of the encapsulated biomolecules.

Overall, the behavior of alginate solutions in the biotechnology industry, particularly their gel-forming ability, biocompatibility, viscosity, and metal ion chelation properties, makes them valuable for various applications, ranging from tissue engineering and drug delivery to bioprinting and environmental remediation.

References

Amsden, B., & Turner, N. (1999). Diffusion characteristics of calcium alginate gels. Biotechnology and Bioengineering, 65(5), pp. 605-610

Blandino, A., Macias, M., & Cantero, D. (2003). Calcium alginate gel as encapsulation matrix for coimmobilized enzyme systems. Applied Biochemistry and Biotechnology, 110, pp. 53–60.

Ertan, F., Yagar, H., & Balkan, B. (2007). Optimization of α‐amylase immobilization in calcium alginate beads. Preparative Biochemistry & Biotechnology, 37(3), pp. 195-204 (Article).

Falkeborg, M., Paitaid, P., Shu, A. N., Pérez, B., & Guo, Z. (2015). Dodecenyl succinylated alginate as a novel material for encapsulation and hyperactivation of lipases. Carbohydrate Polymers, 133, pp. 194-202.

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Lu, L., Zhao, M. & Wang, Y. (2007) Immobilization of Laccase by Alginate–Chitosan Microcapsules and its Use in Dye Decolorization. World J. Microbiol. Biotechnol. 23, pp. 159–166 (Article). 

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Won, K., Kim, S., Kim, K.J., Park, H.W., Moon SJ (2005) Optimization of lipase entrapment in Ca–alginate gel beads. Process Biochem. 40 pp. 2149–2154 (Article).

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