**3. Composition of alginate**

#### **3.1 Types of alginate polymers**

Alginate is an unbranched copolymer of β-d-mannuronic acid (M group) and α-l-guluronic acid (G group) commercially derived from algae [8]. Hydroxyl and carboxyl groups are abundantly distributed across the polymer, giving rise to a plethora of chemically modifiable sites [21]. Commercially, alginate is blocked in either consecutive M, consecutive G, or alternating M & G [22]. Given that it is a polymer with versatile properties, alginate can be easily changed to exhibit a variety of properties tailored to the individual needs of an implant. Pre-existing properties of alginate are able to be chemically enhanced through manipulating the percentage of M and G in the material, adding immunoprotective layers that impede diffusion, as well as divalent cation crosslinking. Under dicationic conditions, viscous alginate transforms into a gel, which can then be used as an encapsulation medium for islet cells [23–25]. M groups lie with the main ring in an equatorial conformation, with each mer connected covalently through an ether (**Figure 2**). With dications situating on top and on bottom of each layer, a "swelling" effect is noted as there is no discrete pocket between mers resulting in each linear polymer stacking on top of the crosslinking dication. In contrast, G groups lie with the main ring axial (**Figure 3**). These mers are also attached through an ether. However, these ethers create pockets large enough for dications to situate without creating a swollen stacking effect. Manipulation of molecular weight and composition (%G or %M) leads to varying strength and porosity of the gel [26, 27]. Generally, alginates with higher %G have higher comparative levels of elasticity and stability [22].

Along with *Macrocystis porifera* and *Ascophyllum nodosum*, another type of brown seaweed that is a main source of alginate is called *Sargassum polycystum* C. Agardh. One study extracted alginate from *S. polycystum* to examine its chemical makeup and viscosity in the leaf, stem and thallus regions [28]. Thallus of seaweed refers to undifferentiated tissues where nutrient uptake and gas exchange occurs [29]. Of the structures studied for *S. polycystum*, the leaf region was found to have the highest alginate yield followed by the stem region and the thallus [28]. In contrast, the viscosity of alginate was highest in the stem region, followed by the leaf region and thallus [28]. Of the M/G ratios observed, the stem region had the lowest M/G ratio and the highest level of G-block content, optimal properties of forming a rigid and stable gel [28]. Similarly, other commercial brown seaweeds such as the stems of *Laminaria hyperborea* have a high G-block content (70%) and a low M/G ratio (0.43) [30].

#### **3.2 UPLVM**

Purified alginate containing almost entirely M blocks with little to no G blocks will typically not yield the ideal type of hydrogel desired through dication crosslinking. The lack of pockets available for crosslinking will result in a form of layers of alternating alginate polymers and dications. In terms of structural integrity, UPLVM alginate will be less porous and strong, allowing higher malleability. In addition, although there has been no clear consensus, studies have suggested that a greater percentage of M blocks in alginate cause an increased immune response compared to UPLVG alginate, as indicated by increased production of TNF-⍺, IL-1 and IL-6 [31].

#### **3.3 UPLVG**

Alginate with a higher %G blocks (>60% G) will result in a material a higher polymer strength and available crosslinking sites for barium atoms [27]. Since there are sites for divalent cation network crosslinking, the cations will situate themselves in the "egg carton" that the G blocks form. The cations in this position

**41**

*Current Perspective and Advancements of Alginate-Based Transplantation Technologies*

create a resistance to shear and frictional forces that the layers undergo as alternating molecular weight increases [32]. Therefore, this orientation allows for higher density, strength and porosity. The carboxylate anions that lie equatorial in G blocks are usually protonated under acidic conditions, which are deprotonated as the basicity of the solution increases [33]. The alcohol groups also present in the polymer provide a hydrogen that is capable of being deprotonated in basic conditions. Although this pH change resistance results in an increased stability of G-rich polymers against depolymerization, pH changes in solution will result in swelling of

Alginate is commercially sold as a mixture of M & G blocks with varying purification. NovaMatrix in Norway is a widely used company that manufactures and distributes purified alginates [34]. Their UP LVG (ultrapure low viscosity, high G) alginate has at least a 1.5:1 ratio of G:M blocks, with UP LVM (ultrapure low viscosity, high M) alginate having a ratio of G:M of less than 1. NovaMatrix recommends a shelf life of 5 years for their UP LVG alginate while only 3 years for UP LVM alginate. Both types of alginate should ideally be stored in a fridge and maintained at a relatively neutral pH (between 5 and 8) so as to decrease the rate of depolymerization. Higher temperatures will result in an increased rate of polymerization, which is expanded even further in the presence of an acid or a base. A neutral pH will prevent acid or base catalysts from recruiting oxygen to initiate oxidative-reductive depolymerization. Both M-rich and G-rich alginates react with no significant difference when base catalyzed, but M-rich alginates are hypothesized to be more vulnerable to acid catalyzed depolymerization, as supported by a catalytic constant that is twice as large for M-rich alginates than that of G-rich alginates [35]. In addition, in the presence of oxygen and an acidic or alkaline environment, the phenol and carboxylate groups will react in undesired

Alginate derivatives have also been created to encompass certain properties. One example of this is amphiphilic alginate derivatives that utilize the alginate backbone and synthesize it with hydrophobic moieties [8, 36]. An example of this is sodium alginate being conjugated with long alkyl chains via ester bonds [36]. The microparticles for the derivative are prepared using dispersion in a sodium chloride solution; this allows for encapsulation of proteins and their subsequent release by adding surfactants to disrupt intermolecular hydrophobic junctions or esterases that hydrolyze the ester bond between the alginate backbone and alkyl chains [36]. Other types of derivatives include dodecylamine conjugated to an alginate backbone via amide linkage which leads to long-term stability in aqueous media compared to other alginate derivatives composed of dodecyl esters which have

*DOI: http://dx.doi.org/10.5772/intechopen.87120*

alginate capsules [34].

*Chemical structure of G-block monomer.*

ways, resulting in free radical depolymerization.

**3.5 Alginate derivatives**

**3.4 Mixtures**

**Figure 3.**

**Figure 2.** *Chemical structure of M-block monomer.*

*Current Perspective and Advancements of Alginate-Based Transplantation Technologies DOI: http://dx.doi.org/10.5772/intechopen.87120*

**Figure 3.** *Chemical structure of G-block monomer.*

create a resistance to shear and frictional forces that the layers undergo as alternating molecular weight increases [32]. Therefore, this orientation allows for higher density, strength and porosity. The carboxylate anions that lie equatorial in G blocks are usually protonated under acidic conditions, which are deprotonated as the basicity of the solution increases [33]. The alcohol groups also present in the polymer provide a hydrogen that is capable of being deprotonated in basic conditions. Although this pH change resistance results in an increased stability of G-rich polymers against depolymerization, pH changes in solution will result in swelling of alginate capsules [34].

#### **3.4 Mixtures**

*Alginates - Recent Uses of This Natural Polymer*

elasticity and stability [22].

ratio (0.43) [30].

**3.2 UPLVM**

and IL-6 [31].

**3.3 UPLVG**

into a gel, which can then be used as an encapsulation medium for islet cells [23–25]. M groups lie with the main ring in an equatorial conformation, with each mer connected covalently through an ether (**Figure 2**). With dications situating on top and on bottom of each layer, a "swelling" effect is noted as there is no discrete pocket between mers resulting in each linear polymer stacking on top of the crosslinking dication. In contrast, G groups lie with the main ring axial (**Figure 3**). These mers are also attached through an ether. However, these ethers create pockets large enough for dications to situate without creating a swollen stacking effect. Manipulation of molecular weight and composition (%G or %M) leads to varying strength and porosity of the gel [26, 27]. Generally, alginates with higher %G have higher comparative levels of

Along with *Macrocystis porifera* and *Ascophyllum nodosum*, another type of brown seaweed that is a main source of alginate is called *Sargassum polycystum* C. Agardh. One study extracted alginate from *S. polycystum* to examine its chemical makeup and viscosity in the leaf, stem and thallus regions [28]. Thallus of seaweed refers to undifferentiated tissues where nutrient uptake and gas exchange occurs [29]. Of the structures studied for *S. polycystum*, the leaf region was found to have the highest alginate yield followed by the stem region and the thallus [28]. In contrast, the viscosity of alginate was highest in the stem region, followed by the leaf region and thallus [28]. Of the M/G ratios observed, the stem region had the lowest M/G ratio and the highest level of G-block content, optimal properties of forming a rigid and stable gel [28]. Similarly, other commercial brown seaweeds such as the stems of *Laminaria hyperborea* have a high G-block content (70%) and a low M/G

Purified alginate containing almost entirely M blocks with little to no G blocks will typically not yield the ideal type of hydrogel desired through dication crosslinking. The lack of pockets available for crosslinking will result in a form of layers of alternating alginate polymers and dications. In terms of structural integrity, UPLVM alginate will be less porous and strong, allowing higher malleability. In addition, although there has been no clear consensus, studies have suggested that a greater percentage of M blocks in alginate cause an increased immune response compared to UPLVG alginate, as indicated by increased production of TNF-⍺, IL-1

Alginate with a higher %G blocks (>60% G) will result in a material a higher polymer strength and available crosslinking sites for barium atoms [27]. Since there are sites for divalent cation network crosslinking, the cations will situate themselves in the "egg carton" that the G blocks form. The cations in this position

**40**

**Figure 2.**

*Chemical structure of M-block monomer.*

Alginate is commercially sold as a mixture of M & G blocks with varying purification. NovaMatrix in Norway is a widely used company that manufactures and distributes purified alginates [34]. Their UP LVG (ultrapure low viscosity, high G) alginate has at least a 1.5:1 ratio of G:M blocks, with UP LVM (ultrapure low viscosity, high M) alginate having a ratio of G:M of less than 1. NovaMatrix recommends a shelf life of 5 years for their UP LVG alginate while only 3 years for UP LVM alginate. Both types of alginate should ideally be stored in a fridge and maintained at a relatively neutral pH (between 5 and 8) so as to decrease the rate of depolymerization. Higher temperatures will result in an increased rate of polymerization, which is expanded even further in the presence of an acid or a base. A neutral pH will prevent acid or base catalysts from recruiting oxygen to initiate oxidative-reductive depolymerization. Both M-rich and G-rich alginates react with no significant difference when base catalyzed, but M-rich alginates are hypothesized to be more vulnerable to acid catalyzed depolymerization, as supported by a catalytic constant that is twice as large for M-rich alginates than that of G-rich alginates [35]. In addition, in the presence of oxygen and an acidic or alkaline environment, the phenol and carboxylate groups will react in undesired ways, resulting in free radical depolymerization.

#### **3.5 Alginate derivatives**

Alginate derivatives have also been created to encompass certain properties. One example of this is amphiphilic alginate derivatives that utilize the alginate backbone and synthesize it with hydrophobic moieties [8, 36]. An example of this is sodium alginate being conjugated with long alkyl chains via ester bonds [36]. The microparticles for the derivative are prepared using dispersion in a sodium chloride solution; this allows for encapsulation of proteins and their subsequent release by adding surfactants to disrupt intermolecular hydrophobic junctions or esterases that hydrolyze the ester bond between the alginate backbone and alkyl chains [36]. Other types of derivatives include dodecylamine conjugated to an alginate backbone via amide linkage which leads to long-term stability in aqueous media compared to other alginate derivatives composed of dodecyl esters which have

a risk of hydrolysis [36]. Amphiphilic alginate derivatives that are water soluble have also been developed using cholesteryl groups which are synthesized using N,N′-dicyclohexylcarbodiimide as a coupling agent and 4-(N,N′-dimethylamino) pyridine as a catalyst at room temperature [37]. Other derivatives include cellinteractive alginates which are composed of cell-adhesive peptides. To prepare these, carbodiimide chemistry is used to combine peptide side-chains with carboxylic groups on sugar molecules [36]. This is important because alginate must acquire the proper ligands to participate in cell-to-cell interactions since it does not innately have cell-adhesivity to mammalian cells [36]. The peptide sequence arginineglycine-aspartic acid (RGD) has been studied widely due to the copious integrin receptors for this ligand on a variety of cell types [38]. This, among other types of cell-interactive alginates, has been used for scaffolds for tissue engineering and 2-D and 3-D cell culture matrices.
