Modification of Alginates to Modulate Their Physic-Chemical Properties and Obtain Biomaterials with Different Functional Properties

Alginate was discovered in 1881 by Stanford [ 1 ]. The term is usually used to refer to salts of alginic acid, although it can refer to alginic acid and all its derivatives [ 2 ]. Alginates are unbranched polysaccharides linked by 1→4 glycosidic bonds composed of β- d -mannuronic acid (M) and its C-5 epimer α- L -guluronic acid (G) [ 3 ]. These polysaccharides are an important building block in algae and exopolysaccharide bacteria, including Pseudomonas aeruginosa . Only alginates isolated from algae are commercially available. The natural origin of alginates causes variation in their biopolymer compositions, sequences, and molecular weights, depending on the source and species used by the manufacturer. Industrial production of alginates amounts to about 30,000 tons per year, which is less than 10% of the available bio-synthesized polymer [ 4 ]. Modifying the properties and expanding the spectrum of possible applications could lead to wider use of this relatively widely available raw material. Combining the methods and tools of chemistry and biochemistry gives access to modified alginic acid derivatives with controlled properties resulting from the location, nature, and quantity of the introduced substituents. This allows for modulation of their solubility, hydrophobicity, affinity for specific proteins, and many other properties. The modification process is, in many cases, complicated by the variable properties of the starting material (alginic acid), such as its solubility, pH sensitivity, and structural complexity. Therefore, it is necessary to control the course of the chemical reactions and to analyze the final compounds. Despite the difficulties involved, there is much interest in the modification of alginates leading to their controlled derivatization. The importance of alginates as natural polysaccharides in medicine cannot be overstated. One of their most important applications is the use of cross-linked alginates to produce hydrogels for cell encapsulation [ 5 , 6 , 7 ]. A flagship example is the use of alginate gels to encapsulate islets of Langerhans for diabetes treatment [ 8 ]. Alginates are widely used as dressing materials to treat various types of wounds [ 9 ]. However, they intensify the effects of cystic fibrosis, because alginate gels secreted by Pseudomonas aeruginosa form bacterial biofilms [ 10 ]. This literature review discusses methods used to modify alginates. Alginate modifications may have one of two objectives: (1) to introduce completely new properties not found in unmodified alginates or (2) to improve their existing properties. The Structure of Alginates The structure of alginates was established based on partial hydrolysis and subsequent fractionation [ 11 ]. Fractionation leads to a soluble (hydrolysable) fraction and an insoluble fraction (resistant to hydrolysis). The insoluble fractions consist of molecules composed mainly of d -mannuronic acid (M) residues or L -guluronic acid (G) residues. The soluble fractions are rich in compounds containing alternating MG residues [ 12 ]. There are considerable differences between alginates in terms of the sequence of G, M, GG, MM, and GM/MG blocks ( Figure 1 ) and of their chemical compositions, which may depend on the species of algae and even the time of year they were harvested. The calculated dry weight content of alginates is in the range of 17–44% [ 13 , 14 ]. The structure of M, G, and MG blocks is determined based on an examination of 1 H-NMR and 13 C-NMR [ 15 , 16 ]. The results of fractionation of alginate hydrolysis products have been confirmed by computer modeling, providing better understand the alginate microstructure [ 17 , 18 , 19 ].

Polysaccharides, including alginates, undergo hydrolytic cleavage in an acidic environment [ 35 ]. The reaction is performed in three steps: (1) protonating the oxygen atom at a glycosidic bond to form the conjugated acid; (2) hydrolysis of the conjugate to form the non-reducing terminus and the carboxonium ion; and (3) rapid addition of water to the carboxonium ion, leading to the formation of a reducing end ( Figure 4 ). In the form of dry powder, sodium alginate can be stored for several months at room temperature without degradation. The half-life can be extended to years by storage at low temperature. Alginic acid is degraded significantly faster than sodium alginate. This is due to internal acidic catalysis with the participation of the C-5 carboxyl group [ 36 ]. The enzymatic degradation of alginates in the presence of lyases occurs according to the β-elimination mechanism ( Figure 5 ), ultimately leading to the formation of unsaturated compounds [ 37 , 38 ]. The alginate degradation reaction has a similar course in an alkaline environment. The reaction rate increases rapidly above pH 10.0 and below pH 5.0. In the case of decomposition in an environment with a pH above 10.0, the reaction takes place mainly by β-elimination, whereas in the case of an acidic reaction (pH below 5.0) acid-catalyzed degradation occurs as described above [ 39 ]. The β-elimination reaction consists of the cleavage of the proton at the C-5 position, which is supported by the presence of an electron-acceptor substituent (carbonyl group) at the C-6 position [ 40 ]. Alginates are prone to degradation not only in the presence of acids or bases, but also in the presence of reducing compounds at neutral pH. Many reducing compounds, such as hydroquinone, sodium sulfite, sodium bisulfide, cysteine, ascorbic acid, hydrazine sulphate, and leuco-methylene blue, also cause alginate degradation [ 41 ]. Brown algae alginates contain different amounts of phenolic compounds, depending on the species [ 42 ]. The rate of degradation increases with the amounts of phenolic compounds in the alginates. Sterilization techniques, such as high temperature treatment, ethylene oxide treatment, or γ-radiation, cause degradation of alginates [ 43 ]. The susceptibility of alginates to degrade must be taken into account when planning chemical modifications of this polysaccharide. Modifications of alginates may be aimed at improving their existing properties or introducing completely new properties [ 44 ]. Derivatization and strategies for alginate modification depend on three main factors: solubility, reactivity, and methods of characterizing new materials. As already mentioned, alginates can be dissolved in water, organic solvents, or mixed systems. The choice of solvent system may dictate the types of reagents that can be used for the modification. Moreover, the degree of solubility of the alginate in the solvent system may affect the selectivity of the modification. Alginates can be modified on two secondary -OH groups (C-2 and C-3) or on the carboxyl group (C-6). The difference in reactivity between the two types of functional groups can be used to selectively modify one of the two types of functional groups. Regioselective modification of the hydroxyl groups at the C-2 or C-3 position is difficult, due to the slight differences in their reactivity. The selectivity of the modification can be additionally controlled by taking advantage of differences in the reactivity of the M or G residues. This can be achieved, for example, by selectively chelating the G residues in Ca-alginate gels or by taking advantage of the partial dissolution properties of alginates in different solvent systems. To more thoroughly understand and predict the effects of modifying new alginate derivatives, it is often necessary to have multiple alginate samples with a varied and fixed M/G ratio. Typically, it is also necessary to derivatize alginates enriched in M, G, or MG blocks to obtain the data necessary to understand the course of the reactions. The lack of commercial availability of sequence-controlled alginates impedes the complete structural characterization of their derivatives. Due to the complex nature of the alginate backbone, advanced analytical techniques are often needed. 3.1. Reaction of Hydroxyl Groups 3.1.1. Acylation of Hydroxyl Groups Chamberlain et al. [ 45 ] were the first to describe the acetylation of alginic acid hydroxyl groups. It was observed that the hydroxyl groups in dry alginic acid yarn do not react with acetic anhydride, due to the strong hydrogen bonds between the functional groups. However, upon swelling with water the hydroxyl groups become available for the acetylating agent. After the swelling process, the solvent was exchanged by replacing the water with glacial acetic acid and then reacted with acetic anhydride in the presence of H 2 SO 4 as a catalyst. The alginate di-acetate was obtained with 97.3% yield (based on total acetyl content).

The process of introducing sulphate residues into polysaccharides can be carried out by both chemical and enzymatic modification. Polysaccharide sulphates are biocompatible with blood and show anticoagulant activity [ 54 , 56 ]. The first method of incorporating sulphate groups into alginates was based on using chlorosulphonic acid and formamide as a solvent ( Figure 8 ) [ 56 ]. The degree of substitution was 1.41. Cohen et al. proposed a method of obtaining alginate sulphates using carbodiimide coupling reagents [ 57 ]. The hydroxyl groups were esterified with the DCC–sulfuric acid derivative generated in situ. No activation of alginate carboxyl groups was observed ( Figure 9 ). The DCC-H 2 SO 4 adduct reacts with the nucleophilic hydroxyl groups of the alginate. The reaction products were characterized based on 13 C-NMR. No change in the chemical shift was observed in the spectra for C-1 and C-6. It was found that sulphate esters are formed with either one or two hydroxyl groups at C-2 and C-3. The use of a strongly acidic reagent causes a significant reduction in the molecular weight of the polysaccharide. The average molecular weight decreases during the reaction from 100 to 10 kDa, but this has no effect on the M/G ratio. Traditional methods of forming alginate esters with sulfuric acid using sulfuric acid, chlorosulfonic acid, sulfamic acid, sulfuryl chloride, or sulfur trioxide cause degradation. A new synthetic method that can avoid degradation of the alginate was proposed by Fan et al. [ 58 ]. The solution uses sodium hydrogen sulphate IV and sodium nitrate III in water ( Figure 10 ). The DS value was 1.87 for a reaction performed at 40 °C, with a ratio of esterifying agent to uronic acid of 2:1. The anticoagulant properties of sulphated alginate derivatives were found to depend on the degree of DS, the molecular weight of the polysaccharide, and the concentration [ 59 , 60 ]. The most important use of sulfated alginate derivatives is due to the fact that they have properties similar to heparin [ 61 , 62 ]. Additionally, they were used to obtain biologically active conjugates with cell-adhesion molecules, growth factors, and chemokines [ 63 , 64 , 65 ]. Sulfonated alginates can be useful for treating viral infections caused by Flaviviridae , Togaviridae , Rhabdoviridae , and Herpesviridae [ 66 , 67 , 68 ]. It is believed that the negative charge resulting from the presence of carboxylic and sulphated residues is responsible for the antiviral activity, as it favors interaction with the positively charged host cell. As a result, contact with the virus and the host cell is difficult [ 69 ]. Another explanation for the observed antiviral activity relates to the inhibition of viral penetration, as alginates form a physical barrier around the cells [ 70 ]. Alginates with a high content of M blocks have immunomodulatory properties, resulting from the activation of macrophages responsible for the excretion of cytokines and cytotoxic factors [ 71 ].

Click chemistry reactions are another method for chemically modifying alginate derivatives. Click chemistry reactions take place under mild conditions, are not sensitive to presence of water, and do not require the use of complicated methods of isolating the final products. Click chemistry reactions lead to the formation of a stable conjugate. Additionally, click chemistry is characterized by high selectivity leading to formation for a single product [ 96 ]. Click chemistry reactions have also found application in the synthesis of modified biopolymers, including alginates. Due to specific requirements for each reaction in this group the appropriate alginate derivatives have to be applied ( Table 4 ). Although, materials obtained via click chemistry reactions were investigated to assess the possibility of the production of biomaterials from them, it was found that the obtained new alginate derivatives have application potential as materials for cell encapsulation, drug delivery, producing antimicrobial materials, tissue engineering, targeted delivery of systemic small molecules, and wound dressing [ 96 ].

Alginate derivatives are an attractive biomaterial for numerous applications, including tissue engineering, cell encapsulation, and wound healing [ 172 , 173 , 174 , 175 ], in situ formation of gels, controlled drug delivery and release systems, as well as targeted drug delivery [ 176 , 177 , 178 , 179 ]. The pool of pharmaceutical products based on alginates includes: Drugs administered orally (Gastrotuss baby syrup [ 180 ], Algicid suspension/tablets [ 181 ], Gaviscon Double Action Liquid [ 182 ] and Tablets [ 183 ]), creating a mechanical barrier between the stomach and esophagus that prevents reflux, choking, dysphagia, heartburn, belching, and irritability, which accelerates the movement of the stomach and regenerates the mucous membranes of the esophagus Materials applied to the skin (Flaminal Forte gel [ 184 ], Purilon Gel gel [ 185 ], Saf-Gel gel [ 186 ], Hyalogran dressing [ 187 ], SeaSorb dressing [ 188 ], Tromboguard dressing [ 189 , 190 ], Fibracol Plus dressing [ 191 ], Algivon dressing [ 192 ], Guardix-SG [ 193 , 194 ]), which affect dissolution of the dry layer and necrotic tissue, ensure a moist environment at the wound surface, have hemostatic and antibacterial activity, and influence tissue granulation, epithelialization, and healing Rectal agents (Natalsid suppositories [ 195 ]) used for chronic hemorrhoids, proctitis, and chronic anal fissures after rectal surgery Periodontal agents (Progenix putty, Progenix plus injection [ 196 ]), used for bone defects, and Emdogain gel [ 197 , 198 ] used for intraosseous defects and defects of mandibular furcation with minimal atrophy of the interdental bone Agents applied arthroscopically (ChondroArt 3D injection [ 199 ]), used in degenerative diseases of the joints and spine. Numerous studies have shown that alginate derivatives have antibacterial, antiviral, and antifungal properties [ 200 ]. However, the mechanism of antimicrobial action is still unknown. It is believed that their antimicrobial activity may be due to the fact that negatively charged alginates interact with bacterial cells, leading to disruption of the cell wall/membrane and leakage of intracellular substances [ 201 ]. Membranes, caused by the formation of a sticky layer of alginate around the bacteria, make the transport of nutrients to the bacterial cells difficult [ 202 ]. Alginates have been found to have bacteriostatic activity against Pseudomonas , Escherichia , Proteus , and Acinetobacter [ 203 , 204 ]. The antimicrobial activity of alginates depends on their molecular weight, M/G block ratio, structural modifications, the pH of the environment, and the type of formulation use [ 205 ]. Calcium alginate activates platelets and affects thrombin, making it an effective hemostatic [ 206 , 207 ] and suitable for use in wound dressings [ 208 , 209 , 210 ]. Low-molecular-weight sodium alginate lowers blood pressure [ 211 ]. The hypotensive mechanism is due to the antagonism of voltage-gated calcium channels [ 212 ]. Potassium alginate is considered a promising agent for preventing cardiovascular complications related to hypertension, including hypertrophy of the heart and kidneys, and the risk of stroke [ 213 ]. Low molecular weight alginates have anti-oxidative and anti-inflammatory effects, which are associated with a reduction in the biosynthesis of nitric oxide, reactive oxygen species (ROS), prostaglandin E2, and COX-2 cyclooxygenase [ 214 , 215 ]. Their anti-oxidative activity may be related to the stimulation of monocyte secretion by anti-inflammatory cytokines, which is associated with alginates that have a high content of M residues [ 216 ]. Due to their ability to chelate, alginates can bind toxins and heavy metals, which helps protect against carcinogenesis [ 217 , 218 ]. Alginic acid has also been shown to be anti-anaphylactic, inhibiting the release of histamine from mast cells and reducing the expression of histidine decarboxylase and pro-inflammatory cytokines [ 219 , 220 ]. Alginates are considered promising candidates for use in preparations for the treatment of obesity and type 2 diabetes. They attenuate the postprandial glycemic response, by modulating gastric emptying [ 221 ] or inhibiting glucose transporters, and also affect the rate of intestinal glucose absorption [ 222 ]. The hypoglycemic effect of alginates may be related to a reduction in the activity of α-amylase, an intestinal enzyme responsible for the hydrolysis of bonds between glucose residues in carbohydrates [ 223 , 224 , 225 ]. Thanks to their fiber-forming properties and ability to form gels, alginates can mimic the structure of the extracellular matrix, and therefore provide a relatively neutral and moist microenvironment. The usefulness of alginates as macroporous scaffolds conditioning favorable conditions for cellular attachment, proliferation, and differentiation is evidenced by the presence on the market of two 3D products: AlgiMatrix (Thermo Fisher Scientific/Life Technologies, Carlsbad,