Alginate Modification and Lectin-Conjugation Approach to Synthesize the Mucoadhesive Matrix

Abstract

Alginates are natural anionic polyelectrolytes investigated in various biomedical applications, such as drug delivery, tissue engineering, and 3D bioprinting. Functionalization of alginates is one possible way to provide a broad range of requirements for those applications. A range of techniques, including esterification, amidation, acetylation, phosphorylation, sulfation, graft copolymerization, and oxidation and reduction, have been implemented for this purpose. The rationale behind these investigations is often the combination of such modified alginates with different molecules. Particularly promising are lectin conjugate macromolecules for lectin-mediated drug delivery, which enhance the bioavailability of active ingredients on a specific site. Most interesting for such application are alginate derivatives, because these macromolecules are more resistant to acidic and enzymatic degradation. This review will report recent progress in alginate modification and conjugation, focusing on alginate-lectin conjugation, which is proposed as a matrix for mucoadhesive drug delivery and provides a new perspective for future studies with these conjugation methods.

1. Introduction

Alginates are examples of anionic polymers obtained from brown seaweed. To date, alginates have been used in numerous biomedical applications due to their biocompatibility, low toxicity, and abundant sources. Alginates are attractive for wound healing, tissue engineering, and drug delivery applications and have become more favored because of their hydrogel formation ability in mild conditions [1,2]. Because of their well-known advantages, the recent trend of added-value alginates is rising. Functionalization is a potential way to generate alginate derivatives and gives new additional properties to the biopolymer. The functionalization has provided the possibility to form ionic and covalent crosslinking. They result in improved physical and chemical properties and additional biological activity, thus factually accommodating the proposed application demand. Alginate derivatives can be synthesized through chemical modifications, including esterification, amidation, acetylation, phosphorylation, sulfation, graft copolymerization, and oxidation and reduction [3].

On the other hand, lectins have earned drug targeting researchers’ attention as they provide target specificity [4]. They are known as proteins that can recognize and bind to specific sugar complexes attached to proteins and lipids. These bindings are supported by the carbohydrate recognition domain (CRD) within the structure [5]. Additionally, the binding specificity and endocytosis of lectins depended on that structure. One study has suggested that lectins be coupled with macromolecular carriers, allowing cellular uptake and controlled intercellular route [6]. The adhesivity of lectin wheat germ agglutinin (WGA) coupled with a model drug, fluorescent bovine serum albumin (FBSA), is investigated, and glycine-FBSA is used as a control [7]. The WGA is managed to increase the interaction with Caco-2 single cell 2–8 times higher than glycine. The cell adhesion specificity corresponds to the amount of WGA and the molecular size of the formed WGA-FBSA coupled polymer. In the subsequent report, WGA is occupied as the targeting moiety for poly(lactic-co-glycolic acid) (PLGA) microparticle loaded with three fluorescent model drugs; fluorescein sodium, sulforhodamine, and boron-dipyrromethene [8]. The crosslinking between PLGA and WGA is established with carbodiimide chemistry, and the binding rate of WGA-PLGA is recorded as having up to a 3-fold increase compared to the non-grafted PLGA.

The mucoadhesive property in drug delivery is to localize and prolong an active ingredient at a particular site in the body. Extending the contact time of the delivered active ingredients will improve the poor adsorption drug’s bioavailability, increasing the therapy’s effectiveness. Both alginate and lectin show adhesiveness characteristics. These biopolymers could perform interfacial interactions with mucus membranes (e.g., gastrointestinal, buccal, nasal, etc.), especially for lectin; its specific adhesivity is based on its glycan preference. Additionally, the mucoadhesive ability depends on the nature of the mucosal tissue and the physicochemical properties of the polymeric drug delivery formulation [9].

One of the goals to be achieved from alginate functionalization is to enhance chemical interaction with cells through mucoadhesion. Studies show that the low affinity of alginate matrices can be boosted with an attachment of cell targeting or signaling moieties [1]. With their bioadhesive properties, lectins are proposed as a targeting media in drug delivery [10]. Together with lectins, alginates could form molecules with an improved acidic and enzymatic degradation resistance, making them suitable for oral delivery. Hence, a particular set of alginate characteristics is often required to perform well in a specific application. Thus, chemical modification is one helpful way to fulfill the necessity.

Here we report the strategies to functionalize the alginate biopolymer based on available functional groups and the following conjunction of mucoadhesive moieties. This new smart biopolymer is expected to serve as a molecule vehicle and adhesive moiety to deliver molecules to a particular site.

2. Alginate Structure and Properties

2.1. Chemical Structure of Alginates

Alginates are marine biopolymers, originated from brown seaweed (Phaeophyceae), including Laminaria hyperborea, Laminaria digitata, Laminaria japonica, Ascophyllum nodosum, and Macrocystis pyrifera [2]. Alginate is also found in bacteria. The main difference between algae and bacterial alginate is the presence of O-acetyl groups at C₂ and/or C₃ in the bacterial alginates. They are linear water-soluble polysaccharides of 1-4 linked α-L-guluronic acid (G) and β-D-mannuronic acid (M), with free active functional hydroxyl and carbonyl groups (Figure 1). The composition, sequence, and molecular weight of alginates depend on the biopolymer source. Their M/G ratio typically defines them; the distribution of M and G units along the chain with a molecular weights ranges between 32,000 and 400,000 g/mol [11].

The alginate functional properties depend on the composition (M/G ratio), the internal structure, and the molecular weight, while its solubility relies upon the pH and the carbonyl group’s counterions existed on the solution [3]. Sodium alginate can slowly dissolve in water and form a viscous solution, but it is insoluble in alcohol. The aqueous solution of sodium alginate has shear-thickening properties and also presents pH responsive characteristics [12].

2.2. Biocompatibility and Degradability of Alginates

Alginate is favored for cell encapsulation due to its low toxicity and its gelation ability under cell suitable conditions, because the distribution of M and G fragments along its chain varies with the alginate source and processing methods. The various characteristic affects its biocompatibility, purification, molecular weight, and intrinsic viscosity [13,14]. Further purification is suggested to reduce the risk of immune response to developed antibodies against the encapsulation material [15]. Several impurities have been found in sodium alginate, such as heavy metals, endotoxins, proteins, and polyphenols [2].

The four main degradation processes of alginate are physical, thermal, chemical, and biodegradation. Physical degradation is mostly due to the rupture of glycosidic bonds, by ultrasound, for example [16,17]. The radiation of ultraviolet light can promote photochemical degradation to alginate [18]. This degradation is performed under photochemical UV/TiO₂ reaction at pH 7. The degradation leads to the formation of alginate oligomers without changing its chemical structure. The third mechanism is chemical degradation, the most often used method to low molecular weight alginate. This type of degradation can be achieved by oxidation reaction. The oxidation of alginate is discussed in the following chapters. The biodegradability of alginate hydrogel can be affected by the crosslinking agent utilized in the gelation process [19]. Lower molecular weight alginate, obtained by oxidation, is more biodegradable than the original sodium alginate.

2.3. Mucoadhesive Properties of Alginates

Alginate is one of the water-soluble bioadhesive polymers that have been used in drug delivery systems. The surface properties of alginate are studied based on the thermodynamically and mechanical approach to evaluate its mucoadhesive properties [20]. For alginate, the mucus-polymer interaction is mainly affected by the polar force. This study also notes that polysaccharides, such as alginate and scleroglucan, show better biocompatibility than xanthan, carbopol, poly co-(methyl vinyl ether-maleic anhydride (Gantrez), and hydroxypropylmethyl cellulose (HPMC).

Studies have been conducted to improve the adhesion of alginate. The improvement aims to strengthen the mucus-polymer bond by chemical modification [21,22]. The modification introduces the thiol or sulfhydryl groups (-SH) to the alginate side chains. The modification product is known as a thiolated alginate. Cysteine is covalently bound to alginate via the carbodiimide method. Thus, the improvement of thiolated alginate mucoadhesiveness is due to the covalent bond between cysteine and mucus [23]. Such interaction displays high cohesive properties to ensure the active ingredients’ localization at a given target side. However, increasing the polymer and mucin mixture viscosity of this modified alginate is observed by rheological study with mucin [22].

Other studies successfully promote a better polymer-mucus interaction by the physical modification of alginate [9,24]. This modification provided higher non-covalent molecular interaction by adding another bioadhesive polymer. Because this mixture does not change the functional groups of alginates, the gelation ability of alginate is not affected. Nevertheless, the selective hydroxyl group’s chemical modification of alginate will also keep the gelation ability of alginate intact.

3. Alginate Application in Drug Delivery

Alginates are generally used in the food, beverage, cosmetic, paper, textile printing, and pharmaceutical industries. They have been utilized as stabilizers, thickeners, emulsifiers, and hydration and gelling agents. The main use of alginate in the biomedical industry is mainly focused on hydrogels used in wound healing, drug delivery, and tissue regeneration. The broad range of applications is due to its biocompatibility, low toxicity and relatively low-cost consumption, and structural similarity to the extracellular matrices of living tissue [2].

3.1. Alginate Hydrogel

Alginate hydrogels are biocompatible and structurally identical to the macromolecular-based component of the biological body. Hydrogels are defined as three-dimensional networks in which hydrophobic polymers chains are crosslinked together [25]. The crosslinking of alginates, specifically, can be conducted via ionic, covalent, and cell pathways as well as by free radical polymerization [26,27]. However, ionic crosslinking is the typical way to synthesize alginate gels, and calcium has been traditionally used to establish crosslinking. The tendency to use ionic crosslinking is supported by the fact that it offers an adequate method for active substance entrapment without affecting their bioactivity [28].

A critical factor in controlling the gelation process using divalent cations is the gelation rate. Gel uniformity and greater mechanical integrity are expected from a slower gelation system [29]. When crosslinking alginates, calcium carbonate and calcium sulfate have shown different gelation rates [30]. With a rapid gelation rate, the calcium chloride is suitable for ionic crosslinker for a bioencapsulation scheme, whereas others are more favored in tissue engineering scaffolds. The gelation rate also depends on the temperature; low temperature reduces the Ca²⁺ reactivity, leading to low gelation rates, well-ordered network structures, and improved mechanical properties [26].

On the other hand, the alginates’ M/G ratio and molecular weight contribute to the variation of the physicochemical properties of their ionic-crosslinked gel. The geometries of the G-block, M-block, and alternating regions are different due to particular shapes and modes of linkage in the alginate sequence. Thus, their intermolecular crosslinking with divalent ions is dissimilar and gives other gel characteristics. A higher content of G-block results in higher tensile strength and modulus and more extensibility than for alginates richer in M-blocks [31]. In addition, a rheological study of high molecular weight alginate fluid gels showed rapid gelation kinetics and higher viscosities than lower molecular weight [32]. The alginate source and the gelling ion conditions (type and concentration) affect the gel stability and permeability. A study on calcium, barium, and strontium ions has been conducted, forming alginate microbeads [33]. These three different cations reacted differently with the G- and M-blocks of alginate, resulting in diverse stability, permeability, gel strength, and distribution of alginates in the gel beads.

Besides ionic crosslinking, many alternative approaches to produce alginate gels have been investigated. Alginate can be covalently crosslinked using carbodiimide chemistry, and one study found that the crosslinker type and the crosslinking density adequately affect hydrogel properties [34]. The crosslinking density affected the mechanical properties of hydrogels, and the type of crosslinking molecules influenced the swelling properties. Another representative strategy prompting the covalent crosslinking in alginate is free radical polymerization. Alginate-methacrylate hydrogels can be prepared through photopolymerization, obtaining mechanical properties that are dominated by the molecular conformation and electron density of the methacrylate reactive groups [35]. Nevertheless, if the alginate polymer chain’s surface is decorated with cell adhesion ligands, cell cross linking most likely also produces alginate gels [36].

The properties of alginate hydrogels, such as high-water content, nontoxicity, soft consistency, biocompatibility, and biodegradability, make them sufficient candidates as drug carriers. As reported previously, alginate hydrogel could synthesize into nanogel, and it has good stability in biological fluid because of the low deriving for aggregation [37]. An alginate nanogel loaded with gold nanoparticles creates a thermos-responsive platform suitable for chemo-photothermal therapy for breast cancer [38]. Another study also developed a pressure trigger controlled drug released from alginate-cyclodextrin nanogel [39]. This carrier manages to enhance the apoptosis mechanism for colon cancer drug delivery. An improved drug loading efficiency of alginate nanogel for cancer therapy can be achieved using additional keratin to create a composite platform [40].

3.2. Alginate Ester

Alginate ester is synthesized through esterification. Esterification is the earliest functionalization of alginate, and it is generally carried out in non-aqueous systems. This functionalization could perform to both available functional groups, either carbonyl or hydroxyl. An additional catalyst is needed for esterification of the hydroxyl groups for a more selective reaction [41].

A full chemoselective carbonyl alginate ester was introduced with the use of tetrabutylammonium (TBA) salt of alginate [42]. This amphiphilic alginate is suggested for use as an amorphous solid dispersions (ASDs) matrix. The presence of alginate esters, ethyl, butyl, and benzyl are aimed at enhancing drug solubility. The introduction of butyl groups to alginate through esterification forms hydrophilic alginate without losing their gelation ability in the presence of calcium chloride [43]. By using the same functionalization method, a methyl alginate ester was synthesized and used as an excipient for direct compression in immediate drug-release tablet production [44]. Another study created a nanoparticle platform based on oleate alginate ester for curcumin delivery [45]. Alginate is active with formamide so it can actively react with methyl oleate; alginate ester is formed after 48 h of reaction, and the excess formamide can be removed with soxhlet apparatus.

The attachment of the hydrophobic moieties on the alginate backbone creates a micelle carrier system. Such a system is one way to enhance the solubility of poorly water-soluble drugs and drug loading encapsulation, and is a controlled release mechanism [46]. However, further investigation is necessary to improve the solubility enhancement factor and to find a compatible way to facilitate ASD formation. Moreover, the development of a specific esterification method is needed to create a tunable hydrophobicity–hydrophilicity balance characteristic [42].

3.3. Alginate Dialdehyde

Alginate cannot degrade in mammals due to a lack of alginate-degrading enzymes [2]. Ionically crosslinked alginate hydrogels, on the other hand, can be dissolved by ionic exchange reaction, but the high molecular weights (>50 kDa) of the parent alginate restrain the renal clearance. To address the limitation, alginate dialdehyde (ADA), which Malaprade first reported, has been widely investigated [47,48,49]. This alginate derives from partial oxidation of alginate chains, involving C₂-C₃ bond cleavage and transforming into an open ring containing two aldehyde groups. This functionalization is selective modification of the hydroxyl moieties. ADA has a lower molecular weight and is more soluble in aqueous media. Its stiffness and persistence length decreases with an increase in the degree of oxidation [50].

ADA consists of multiple aldehyde groups, a reactive group that can form covalent bonds with free amino groups in gelatin, and chitosan. The development of Schiff’s base crosslinking hydrogels between ADA and polymer containing free amino groups has been investigated [51,52]. A study found that the degree of crosslinking of ADA/PEG(polyethylene glycol)-gelatin hydrogels are higher than ADA/PEG-chitosan hydrogels [53]. This degree of crosslinking is defined as the number of groups that interconnect two materials, generally expressed in mole percent. This can be determined by trinitrobenzene sulfonic acid (TNBS) assay or ninhydrin assay [52,54]. Furthermore, the rheological, degradation, and comprehensive properties of the ADA/PEG-chitosan hydrogel are suggested to be more suitable for the self-crosslinking injectable scaffold.

The synthesis of hydrogels derived from ADA and gelatin has been reported; the crosslinking between ADA and gelatin was increased by increasing the degree of oxidation of ADA [55]. Such in situ forming hydrogel is used as wound dressing material; it is molded in accordance with the wound shape to enable conformability of the dressing for their application [56]. The advantage of alginates as a wound dressing is the ability to absorb excess wound fluid and maintain physiological moisture and an aseptic environment [57]. Another study successfully performed bone regeneration with RGD-alginate and found that the alginate’s system showed an excellent bone formation ability [58]. The application of injectable hydrogel from ADA and gelatin for meniscal injury treatment has also been investigated [59]. Moreover, the same hydrogel could serve as a drug delivery vehicle in the formed nanogel and successfully delivered curcumin as an active ingredient [5].

4. Alginate Modification and Coupling Strategies

Alginate and its derivatives have been widely employed as drug carriers. To improve alginate characteristics and to add specific active sides, structural modification is one strategy that can be applied (Figure 2).

The modification of alginates has been intensively studied to fulfill a range of application requirements. Various chemical and physical modifications have been carried out. These opportunities have arisen because the alginate’s structure displays plenty of free available carboxylic and hydroxyl groups that allow different chemical modification strategies [47].

4.1. Carboxylic Groups

The monomer structure of alginates, α-L-guluronic acid (G), and β-D-mannuronic acid (M) consist of a 1:2 ratio of the carboxylic acid group to the hydroxyl one. The carboxylic acid group in the alginate’s backbone is the one that mainly dictates the solubility. Thus, it is essential to set the pH above a particular critical value in an aqueous environment, so the carboxylic acid groups are deprotonated.

4.1.1. Carbodiimide

Carbodiimide is a widely known method to modify alginates that converts the carboxylates into ester and amide derivatives. The technique utilized a particular ratio of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) with N-hydroxysuccinimide (NHS) or hydrochloride (HCl). Alginate modifications with bioactive peptides for tissue engineering, for example, are usually achieved by carbodiimide chemistry. Using carbodiimide chemistry, the mannuronan part of alginate is modified up to 0.2% degree of substitution by peptide [60].

The synthesis of gelatin-alginate tissue adhesive is synthesized using this methodology [61]. The resulting polymer is suitable as a bioadhesive to bind soft tissue and locally deliver an antibiotic drug with good cytotoxicity results [62]. Others have also reported the application of carbodiimide chemistry to synthesized dodecanol amphiphilic alginate as an emulsifier and hydrogels of tyramine-alginate for bioactive scaffolds, and for creating cell-laden bioinks from thiol-ene crosslinked alginate [63,64,65,66].

The general crosslinking mechanism of carbodiimide chemistry is depicted in Figure 3. The EDC/NHS coupling mechanism is associated with urea derivatives in the first step of the reaction [2]. In some cases, this by-product formation is a disadvantage and has contributed to the destruction of the gelling properties of alginate derivatives [67].

4.1.2. Ugi Multicomponent Reaction

The urge to explore the possibility of creating alginate hydrogel with varied cross linkers is massive. This variety of crosslinkers brought the opportunity to provide controlled mechanical features. The ester linkages, crosslinking with poly(ethylene glycol)-diamines, or crosslinking provided by adipic dihydrazide and lysine, mainly establish crosslinking-density controlled mechanical properties [55,68,69,70]. The crosslinking is formed from a protonated imine and a carboxylate, which reacts with an isocyanide, resulting in a bis-amide (Figure 4) [71]. This method was conceived from the possibility of achieving more regulated hydrogel rheological features [72].

Matsumoto et al. later adopted the Ugi multicomponent reaction to synthesize amphiphilic alginate [73]. This alginate acid ester displayed tunable organic solubility and thermal properties.

4.2. Hydroxyl Groups

Four different modification methods could perform alginate derivatization through the hydroxyl groups. Each way provides a distinct character of alginate derivatives, which open up the possibilities for a range of applications.

4.2.1. Acetylation and Esterification of Hydroxyl Groups

Among the earliest attempts of alginate modifications, investigators have introduced the synthesis of acetylated alginate derivatives. Pioneer researchers conducted a series of acetylation reactions. Chamberlain et al. performed acetylation of alginic acid yarn, where the hydroxyl group was first made available by the swelling water process [74]. The relatively mild method was reported to overcome the degradation that occurred during the previous procedure. Alginic acid has been swollen with acetone instead of water before it was reacted with ketene [75]. Skjåk-Bræk et al. reported a more detailed analysis of the acetyl substitution of alginates [76]. The acetylation is also reported to increase the swelling ability of alginate calcium gels and decrease their viscosity with the increasing acetylation degree [77]. More recently, acetylation has been applied to tetrabutylammonium (TBA)-alginate in an organic solvent, leading to a maximum degree of acetylation [78].

The esterification can be applied to modify both the carboxylic acid (half modified) and the hydroxyl group of alginates. In this part, we discuss the ester alginates formation through the hydroxyl group. Alginate has been hydrophobically modified with the dodecyl chain at 12% mol/mol saccharide units [79]. The alginate acetate was synthesized in dimethyl sulfoxide, using a tetrabutylammonium (TBA)-alginate as the substrate [78]. The study hypothesized that the 1,3 axial interactions between C₂-OH groups with C₄ protons or C₃-OH groups with C₅ protons within the alginate chain structure are responsible for the acetylation [42].

This modification is a carboxyl group chemoselective reaction (Figure 5), making it suitable to synthesize benzyl, butyl, ethyl, and methyl alginates. This alginate carboxylate ester is inorganic solvent-soluble and might be ideal for preparing a solid dispersion drug delivery matrix. All in all, the modification strategy in organic reagents would extend the possibility of new pathways for alginate functionalization.

Additionally, ester formation can also be explored with an epoxide ring-opening mechanism to prepare hydrophobically modified alginate [80]. The condensation of sodium alginate with dodecyl glycidyl ether (DGE), at pH 9 and 80 °C, produces a self-assembled amphiphilic alginate derivative. These polymers have been studied as water-insoluble substance carriers [81]. This could increase clofazimine and Amphotericin B apparent water solubilities and exhibit a sustained-release effect. In a subsequent report, an improved synthesized method has been reported with the addition of sodium dodecyl sulfate (SDS) as a surfactant to increase polymer production yields [82].

4.2.2. Phosphorylation

Other functional groups, in addition to the hydroxyl functionalities of alginate, have been achieved with phosphorylation. These reactions generated alginate derivatives that possessed lower molecular weight than their origin and a decreased gelation ability.

Coleman et al. utilized the heterogeneous urea/phosphate reaction to successfully creating the phosphorylation of alginate hydroxyl groups [83]. The functionalization of the alginate M unit preferentially occurred at the C3 equatorial carbon of the polysaccharide ring, while the reaction on the G unit is still unclear. Due to alginate chain degradation upon the modification, the hydrogel was synthesized by blending the phosphorylated product with native alginate. This study discusses the enhancement of the hydrogel due to the participation of phosphate ions in interchain crosslinking and their effect on the induction of HAP surface mineralization.

Another study developed an injectable hydrogel from sodium alginate O-phosphorylation, which was achieved using a mixture of H₃PO₄/P₂O₅/Et₃PO₄/hexanol [84]. An internal gelation process produced the hydrogel of the resulting phosphorylated alginate calcium complex and sodium alginate. The suitability of the resulting injectable hydrogel for artificial soft tissue replacement was studied on MC3T3 cells.

4.2.3. Sulfation

Sulfation is a modification method that involves the formation of a C-O-S bond; it is mainly utilized to synthesize heparin-like molecules (Figure 6). Because alginate is the only polysaccharide containing carboxylic acid functionalities in each of its uronic acid units, it is suitable for generating heparin-like molecules by sulfation reaction. Several studies have reported the development of heparin-like polysaccharides by sulfation, mainly using chlorosulfonic acid as the sulfation agent. Recently, one study discussed the microspheres of the sulfated alginate derivative to promote a drug-controlled release behavior in human induced pluripotent stem cell (hiPSC)-derived endothelial cells [85]. The most common method used to synthesize the microspheres of sulfated alginate utilizes the lower density of sulfate and heparin groups. This modification is aimed at the bioactivity enhancement of immobilized vascular endothelial growth factor (VEGF). Mhanna et al. used a different approach using a tetrabutylammonium (TBA) alginate salt to increase their solubility in pyridine. However, this method required more studies related to the reproducibility of sulfation degree [86].

Another strategy employed a carbodiimide-H₂SO₄ intermediate or TBA salt in DMF [87,88]. Both are described methods in strong acid conditions that may not always favor the specific application. A deteriorating effect on the gelation ability is also found in sulfated alginate, resulting in a lower stiffness gel and increased swelling rate compared to unmodified alginate. However, heparin-like alginate hydrogels for encapsulation are still of great interest in their applications [89].

4.2.4. Oxidation and Reduction

The oxidation of alginate has been significantly studied in the field of soft tissue engineering. Several oxidation agents have been used to carry out the reaction, but sodium meta periodate (NaIO₄) is the reagent most frequently used to serve this purpose. The oxidation can be performed in pure water or ethanol/water dispersion in a subduing environment. The ring-opening leads to the formation of di-aldehyde, the so-called alginate dialdehyde (ADA). A decrease of molecular weight and gelation ability is expected, depending on the degree of oxidation [67,90]. ATR-FTIR spectroscopy is used to characterize the newly formed aldehyde, although the band is weak, in some cases, due to the formation of hemiacetals of the free aldehyde group [48]. The proton at 90 °C or carbon NMR spectroscopy is another option for confirming the new group formation [11].

The newly formed aldehyde groups are characterized mainly by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. A new characteristic band of aldehyde is observed in the range of 1725–1751 cm⁻¹ [48,90]. This band is weak and, in some cases, not even detected. The phenomenon is related to the hemiacetal formation of a high concentration of aldehyde groups in oxidized urinates [47,48]. The presence of aldehyde can also be investigated with ¹³C NMR, and the aldehyde signal clearly appears at 90–95 ppm when the degree of oxidation is 19 mol% or above [47,91].

The degree of oxidation is defined as a percentage of oxidized uronic acid residues in the ADA. Such a degree can be determined by UV-Vis spectrophotometry or potentiometric titration [92,93]. The degree of oxidation controls the physicochemical properties of the ADA and resultant hydrogels. Because the oxidation reaction significantly affects the guluronic acid residues, only ADA with a maximum 10% degree of oxidation can form hydrogels with calcium ions [47].

Without further modification, alginate dialdehyde has been used to synthesize hydrogels with gelatin in the presence of borax [55], while Emami et al. studied the physical properties of these hydrogels by controlling the concentration of alginate solution and oxidation condition [90]. With the addition of 2-hydroxyethyl methacrylate (HEMA) and the presence of hydroxyapatite (HAp), a porous alginate hydrogel is synthesized [94]. The hydrogel is synthesized with the cyrogelation method, where the additional compounds enhance the mechanical properties and biocompatibility.

ADA-GEL (gelatin) hydrogel is also reported as bioink, where the gelatinization is obtained by crosslinking with divalent ion [95]. The ADA with a 30% degree of oxidation is utilized to produce stable ADA-GEL scaffolds, where barium is present in the cross-linker solution. Nevertheless, ADA-GEL stiffness can be tuned by controlling the concentration of ADA, type of crosslinker, and the ratio of divalent ions in the crosslinker. The general procedure of periodate oxidation followed by reductive amination on the MGM fragment of alginate is depicted in Figure 7.

The advantage of oxidation alginate derivatives is their highly reactive alginate dialdehyde moieties compared to the original sodium alginate. This now available active site of alginate derivative brings an opportunity for further modification to achieve the required characteristics for specific applications. The reductive amination is most explored as the following procedure to modify the alginate dialdehyde. Sodium cyanoborohydride (NaBH₃CN), among other hydrides, is the reagent most employed for reductive amination to avoid reducing the unreacted aldehyde. Other reagents, such as picoline borane complex (pic-BH₃) or α-picoline borane, are also reported as mild and efficient reductive agents [96,97].

4.2.5. Graft Copolymerization

Grafting can be performed through the living-free radical method. A single electron transfer living radical polymerization (SET-LRP) is performed on TBA alginate salt that is to be grafted with methylmethacrylate (MMA) [98]. The TBA salt is soluble in an organic solvent, which allows anhydrous esterification. Thus, it can be reacted with the initiator for the SET-LRP procedure. The SET-LRP resulted in a self-assembled amphiphilic alginate derivative. Free radical grafting polymerization has been performed through atom transfer radical polymerization (ATRP) [99]. The method is utilized to synthesize a macrophage-targeted carrier from sodium alginate to be grafted with allylamine with the presence of ammonium persulfate (APS). The resulting polymer is then grafted with mannose as the targeted ligand.

5. Lectin-ADA Conjugation

5.1. Lectin

Lectins are found in various natural sources such as plants, animals, and microbes, where they have a substantial biological role in relation to cells and proteins. They are protein or glycoprotein complexes of non-immune origin that have the capacity to selectively noncovalent bind to sugars. They can agglutinate cells, which distinguishes them from other carbohydrate-binding proteins. Based on the binding site, the lectins can be classified into morolectin (monovalent), hololectin (bivalent), chimerolectin (consist of one or more substrate binding sites together with another catalytic domain), and superlectin (multivalent) [5]. The lectins can also be classified based on their sequence and structural homology [4]. C-type lectins require calcium for recognition, while some S-type (galalectins) require free thiols for stability. It is also a P-type, one that recognizes Man-6-P, I-type groups from immunoglobulins that could distinguish carbohydrate and be marked as lectins that bind to sialic acid.

As a class of proteins with structural diversity, lectins can attach precisely to carbohydrate molecules of glycoproteins on the cell surface. These proteins represent a distinct binding process in the same way as carbohydrate-binding modules (CBM) but only interact with complex glycan structures [5]. These reactions are reversible equilibrium means, represented by:

Ka = [LS]/[L] × [S],

(1)

The association constants, the number of combining sites/mole, and other thermodynamic and kinetic parameters of the interaction can also be investigated by physicochemical approaches, such as spectrophotometry, spectrofluorimetry, and nuclear magnetic resonance (NMR) [100].

5.2. Lectin Biomedical Applications

Lectins play many roles in biomedicine and diagnostics, such as being a mediated therapeutic against cancer, an antiviral drug, antibacterial agent, insecticide, and antifungal agent [5]. Mushroom lectins (MLs) have been studied intensively, and the glycan-binding protein extracted from various medical/edible mushrooms has shown antioxidant, antidiabetic, antimicrobial, and antiviral activities [101]. Because they are known for differentiating a sugar residue and cell agglutination, they have become a tool to investigate carbohydrates on the cell surface and isolate and characterize glycoproteins [102].

Previous literature has studied the application of lectins for molecule recognition. WGA has been used to study the protein interactions with peptidoglycan, and it has been studied intensively using nuclear magnetic resonance spectrometry. These studies have identified aromatic residues that act as the key players for sugar binding at positions 21, 23, and 30 in hevein domains [103,104]. Furthermore, the signal assignment from ¹H NMR, when comparing spectra between the absence and presence of the lectin, showed a broadening of the GMDP ligand signals in the presence of the lectin [105].

Lectins are used as diagnostic tools, such as Lens culinaris agglutinin (LCA), and bind specifically to α1-6 fucose and have been used to detect hepatocellular carcinoma (HCC) [106]. Lectins have also been investigated for ovarian cancer diagnosis, such as WGA, LCA, Ulex europaeus agglutinin (UEA), Vicia villosa lectin (VVL), and Glycine max agglutinin (GMA) [107]. Six different lectins: Peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), Solanum tuberosum lectin (STL), LCA, UEA I, and WGA have been investigated for their binding affinity towards the 5637, HT-1376, and SV-HUC cells (urothelial cell lines). This study finds that WGA has shown the highest bioadhesive potential, while PNA can be a potent tissue discriminator for cancer diagnosis. Furthermore, Pinellia ternata lectin (PTL), Aleuria aurantia lectin (AAL), and Sambucus nigra agglutinin (SNA) could potentially be used in pancreatic cancer [108].

5.3. Lectin Mediated Drug Delivery

A lectin-modified polymer-based drug carrier has been intensively studied to achieve a high local drug concentration [10]. In this case, the lectin direct targeting method is a valuable strategy for oral or other routes, controlled release, and diagnostic tools. These lectin-mediated drug systems for local delivery into the colon via a gastrointestinal tract (GIT) have been found to be more effective than time-pH-based systems, enzyme-triggered systems, or pressure-based systems [109]. The side specificity of drug targeting improves drug pharmacokinetics, prevents side effects on healthy tissue, and increases drug uptake by targeted cells [110]. Other research has also pointed out that the lectin grafted liposomes are a potential drug delivery system for a sustained release [111].

The binding and uptake ability of bovine serum albumin-wheat germ agglutinin (BSA-WGA) conjugates have been studied using the Caco-2 cell model and were found to overcome the mucosal barrier [7]. Later, a poly(lactic-co-glycolic acid) microcarrier grafted with wheat germ agglutinin was successfully synthesized [8]. The resulting polymer can be used to deliver a fluorescent model drug. It has been found that the system is able to be utilized for fluorescence detection and microscopic imaging for cellular interaction. The binding affinity of Ricinus communis agglutinin (RCA 120)-conjugated gold nanoparticles have been analyzed using HeLa, 293, and 293T cell lines [112]. The study suggested that the lectin-conjugate nanoparticles detect glycosyl complex differential expression on the cellular surface, especially when strongly accumulated onto HeLa (cervical cells).

Studies have been conducted to investigate the bioadhesion of Lycopersicon esculentum agglutinin (LEA). The lectin was extracted from tomato and specifically bound tetra-(N-acetylglucosamine), (GluNAc)₄. Their bioadhesion towards Caco2 cells and fixed pig enterocytes after conjugation to coated polystyrene microspheres have been demonstrated [113]. An overview of different carrier-lectin conjugates used in targeting delivery, along with the method for the conjugation, is provided in

Table 1. An overview of the research on lectin-mediated drug delivery system.

Carrier	Lectin	Glycan Preference	Carrier-Lectin Conjugation Method	Targeted Cell/Organ	Ref.
PLGA nanoparticles	Wheat germ agglutinin (WGA)	Sialic acid and N-acetyl glucosamine (GlcNac)	Carbodiimide	Caco-2 cells Caco-2 and HT-29 cells (colon cancer) A549 cells (Type II alveolar epithelial cells) Wistar rats	[8,113,114,115]
PLGA nanoparticles containing isopropyl myristate (IPM)	Wheat germ agglutinin (WGA)	Sialic acid and N-acetyl glucosamine (GlcNac)	Carbodiimide	A549 cells, H1299 cells (non-small cell lung carcinoma), CCL-186 cells (lung fibroblast IMR-90)	[116]
Carboxymethylated kappa-carrageenan microparticles	Wheat germ agglutinin (WGA)	Sialic acid and N-acetyl glucosamine (GlcNac)	Glutaraldehyde linker	Caco-2 cells	[117]
Poly-l-glutamic acid (PGA) and α-poly-(l)-glutamic acid (PGA)	Wheat germ agglutinin (WGA)	Sialic acid and N-acetyl glucosamine (GlcNac)	Carbodiimide	5637 cells (urothelial carcinoma)	[118,119]
Methoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) nanoparticles	Wheat germ agglutinin (WGA)	Sialic acid and N-acetyl glucosamine (GlcNac)	Carbodiimide	U87MG cells (glioblastoma)	[120]
Thiolated alginate nanoparticles	Wheat germ agglutinin (WGA)	Sialic acid and N-acetyl glucosamine (GlcNac)	Carbodiimide	HT-29	[121]
Chitosan-Ca-alginate (CTS-Ca-ALG) microparticles	Wheat germ agglutinin (WGA)	Sialic acid and N-acetyl glucosamine (GlcNac)	Carbodiimide	Caco-2 cells	[122]
Liposomes	Wheat germ agglutinin (WGA)	Sialic acid and N-acetyl glucosamine (GlcNac)	Carbodiimide	A549 cells	[123]
Liposomes	Concanavalin A (Con A), Wheat germ agglutinin (WGA), and Soybean agglutinin (SBA)	α-D-mannose, α-D-glucose, and N-acetyl glucosamine (GlcNac)	Neutravidin-Biotin-complexes linker	A549 cells	[124]
Liposomes	Wheat germ agglutinin (WGA), Tomato lectin (TL), and Ulex europaeus agglutinin 1 (UEA1)	Sialic acid, N-acetyl glucosamine (GlcNac), and α-L-fucose	Carbodiimide	Mice	[125]
Liposomes nanocarriers	Wheat germ agglutinin-carbopol (WGA-CP) conjugate	N-acetyl glucosamine (GlcNac)	Carbodiimide	Caco-2 cells and intestinal membrane of rats	[126]
Gliadin nanoparticle (GNP)	Ulex europaeus agglutinin 1 (UEA1)	α-L-fucose	Carbodiimide	Bovine submaxillary gland mucin (BSM)	[127]
Poly(lactide) (PLA) microspheres	Lycopersicon esculentum agglutinin (LEA)	N-acetyl-d-glucosamine and α-L-fucose	PVA linker	Rat intestinal mucosa	[128]
Ethylcellulose (EC) and chitosan floating-mucoadhesive microp[articles	Concanavalin A (Con A)	α-D-mannose and α-D-glucose	Carbodiimide	Pig gastric mucosa	[129]

.

5.4. Lectin-Alginate Dialdehyde Conjugate

Carbodiimide chemistry is a popular method of crosslinking a macromolecular carrier with lectins. Previous studies agree that the process is non-destructive toward the bioadhesion of lectins [8]. This method is favored because it can maintain the bioactivity of substrates. However, carbodiimide is associated with urea derivatives formation, which leads to the destruction of the hydrogel properties of alginates [38].

Crosslinking strategies involving polysaccharides, alginate in particular, usually start with two or more steps. The first step is to provide the biopolymer with sufficiently reactive groups. In these cases, periodate oxidation is one way to create these groups. With access to controlling the degree of oxidation, this method creates highly reactive aldehyde functionalities. By using a proper amount of periodate, a sufficient number of aldehydes will be obtained without losing the desired characteristic of alginates. The resulting biopolymer is known as alginate dialdehyde (ADA) and can be used in coupling reactions with amine-containing molecules, in this case, lectins. Covalent linkages are expected to form after amines react with formyl groups under reductive amination conditions using a suitable reducing agent.

The two-step modification reactions, periodate oxidation and reductive amination, are used to synthesize alginate-derived surfactants [130]. The polymeric surfactants consist of alginate dialdehyde coupled with a linear alkyl amine in the presence of sodium cyanoborohydride. By using a mild reduction agent, α-picoline borane, two studies generate a functional alginate dialdehyde. The first study coupled one model molecule, l-Tyrosine methyl ester, to determine the optimum coupling condition, and reached between 2–6% degree of substitutions [67]. This optimum condition was then applied to the ADA by coupling three different bioactive peptides (GRGDSP, GRGDYP, and KHIFSDDSSE) with an 8% degree of oxidation and substitution range from 3–6%. The dental stem cells (RP89) and myoblasts (C2C12) were used for cell adhesion studies, and they demonstrated the bioactivity of GRGDSP. Secondly, alginate derivate hydrogel was obtained from an amide bond formation between ADA and 4-(2-aminoethyl)benzoic acid [131]. The resulting polymer degradation behavior was dictated by the degree of oxidation of the ADA, and no correlation was found with the alginate M/G ratio. The hydrogel of ADA coupled 4-(2-aminoethyl)benzoic acid showed resistance to the simulated acid condition of the gastric fluid.

Periodate oxidation and reductive amination can successfully synthesize new biomaterial from alginate derivatives without affecting the bioactivity of peptides [67]. The proposed modification technique can manage the gelling properties by controlling the degree of oxidation. The lectins are grafted to the active aldehyde sides of the alginate derivative through reductive amination, a conjugation method that is able to maintain the lectin-specific bioadhesion properties.

6. Conclusions and Future Outlook

The availability of hydroxyl and carboxyl groups in the alginates chain for chemical reactions opens up the possibility for advanced chemical modification. Chemical modification is the preferred method for generating the alginate derivatives, having enhanced and altogether new characteristics. The range of this modification establishes a compelling potential for tailoring the next generation of alginate-based biomaterial for particular applications [1,3].

The advent of carbonyl activating reagents, such as 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) with N-hydroxysuccinimide (NHS) in carbodiimide chemistry, has permitted selective modification of alginates. This allows modification of bulk properties and the attachment of functional substituent. Further, ¹H-nuclear magnetic resonance investigation revealed the formation of urea derivative by-products from the coupling reaction between alginates and arginine-glycine-aspartate (RGD) [60]. Filtration could only partly remove this urea derivative and leave the N-acyl urea bound to alginates to stay on the resulting polymer.

On the other hand, the oxidation of alginates is a functionalization by the reaction on the hydroxyl groups. This selective modification on the hydroxyl moieties is able to maintain the capacity of alginate derivatives to spontaneously form hydrogels in the presence of divalent ions [3]. Together with reductive amination, these methods are suggested as a more efficient way to functionalize the alginate’s biopolymer [67]. The oxidation gives access to controlled biopolymer degradability, forming a cross-linking reaction and bioactive conjugation via reductive amination on the now-available aldehyde functional groups.

The alginate functionalization with peptide through oxidation and reductive amination is one of the most attractive developments in the alginate bioconjugation field. This finding opens up new possibilities to conjugate the alginate with bioactive substances through the hydroxyl groups. These are forming more degradable polymers without losing their attractive features.

Despite the alginate derivative’s potential in numerous biomedical applications, the regiocontrol and chemoselectivity issues in alginate derivative synthesis raise challenges, as do the number of clinical translations. However, as further remarkable features of alginate-based materials in the biomedical field emerge, more studies are warranted to improve the synthesis strategies, analytics, characterization, and technology of alginates and their derivatives-based material.