1. INTRODUCTION

Alginates are unbranched polysaccharides consisting of 1 →4 linked b-D-mannuronic acid (M) and its C-5 epimer a-L-guluronic acid (G). The natural copolymer is an important component of algae such as kelp, and is also an exopolysaccharide of bacteria including Pseudomonas aeruginosa. It is comprised of sequences of M (M blocks) and G (G-blocks) residues interspersed with MG sequences (MG-blocks)[1]. While it is possible to obtain alginates from both algal and bacterial sources, commercially available alginates currently come only from algae. The copolymer composition, sequence and molecular weights vary with the source and species that produce the copolymer. Due to the abundance of algae in water bodies, there is a large amount of alginate material present in nature.

Industrial alginate production is approximately 30,000 metric tons annually, and is estimated to comprise less than 10% of the biosynthesized alginate material[2] and 30% of this is utilized by the food industry, the rest being used in industrial and pharmaceutical application.

The interest in formulated dosage forms, where the drug release can be controlled, has increased steadily during the last 60 years. In most cases the purpose is to make a product that maintains a prolonged therapeutic effect at a reduced dosing frequency[3].

The naturally occurring alginate polymers have a wide potential in drug formulation due to their extensive application as food additives and their recognized lack of toxicity[4]. Alginates can be tailor-made to suit the demands of applicants in both the pharmaceutical and biomedical areas[5].

This group of polymers possesses a number of characteristics that makes it useful as a formulation aid, both as a conventional excipient and more specifically as a tool in polymeric-controlled drug delivery. Alginate is a naturally occurring anionic polymer typically obtained from brown seaweed, and has been extensively investigated and used for many biomedical applications, due to its biocompatibility, low toxicity, relatively low cost, and mild gelation by addition of divalent cations such as Ca2+. Alginate hydrogels can be prepared by various cross-linking methods, and their structural similarity to extracellular matrices of living tissues allows wide applications in wound healing, delivery of bioactive agents such as small chemical drugs and proteins, and cell transplantation[6].

Alginate wound dressings maintain a physiologically moist microenvironment, minimize bacterial infection at the wound site, and facilitate wound healing. Drug molecules, from small chemical drugs to macromolecular proteins, can be released from alginate gels in a controlled manner, depending on the cross-linker types and cross-linking methods[7].

In addition, alginate gels can be orally administrated or injected into the body in a minimally invasive manner, which allows extensive applications in the pharmaceutical arena.

This review article represents brief discussion on source, chemistry and properties with extensive focus on gelling technique.

2. BACKGROUND OF ALGINATE:

The British chemist E. C. C. Stanford first described alginate (the preparation of “algic acid” from brown algae) with a patent dated 12 January 1881[8]. After the patent, his discovery was further discussed in papers from 1883[9]. Stanford believed that alginic acid contained nitrogen and contributed much to the elucidation of its chemical structure. In 1926, some groups working independently[10, 11] discovered that uronic acid was a constituent of alginic acid. The nature of the uronic acids present was investigated by three different groups shortly afterwards [12-15] which all found d-mannuronic acid in the hydrolysate of alginate. The nature of the bonds between the uronic acid residues in the alginate molecule was determined to be b1,4, as in cellulose.

In a paper chromatographic study of uronic acids and polyuronides, they discovered the presence of an uronic acid different from mannuronic acid in the hydrolysates of alginic acid [15]. This new uronic acid was identified as l-guluronic acid. The quantity of l-guluronic acid was considerable, and a method for quantitative determination of mannuronic and guluronic acid was developed.

Alginate then had to be regarded as a binary copolymer composed of a-l-guluronic and b-d-mannuronic residues. As long as alginic acid was regarded as a polymer containing only d-mannuronic acid linked together with b-1, 4 links, it was reasonable to assume that alginates from different raw materials were chemically identical and that any given sample of alginic acid was chemically ho- mogeneous. From a practical and a scientific point of view, the uronic acid composition of alginate from different sources had to be examined, and methods for chemical fractionation of alginates had to be developed. These tasks were undertaken mainly by Haug and coworkers[16]. The discovery of alginate as a block-copolymer, the correlation between physical properties and block structure, and the discovery of a set of epimerases converting mannuronic to guluronic acid in a sequence-dependent manner also are discussed further in later sections.

3. MANUFACTURING:

Alginate was first described by the British chemist E. C. C. Stanford in 1881and exists as the most abundant polysaccharide in the brown algae comprising up to 40% of the dry matter [8]. It is located in the intercellular matrix as a gel containing sodium, calcium, magnesium, strontium and barium ions[17].It is because of its ability to retain water, and its gelling, viscosifying and stabilising properties, that alginate is widely used industrially. Several bacteria also produce alginate exocellularly,[17-19]and Azotobacter vinelandii has been evaluated as a source for industrial production. But at present, all commercial alginates are extracted from algal sources.

The extraction of alginate from algal material is schematically illustrated in Fig. 1. Because alginate is insoluble within the algae with a counter ion composition determined by the ion exchange equilibrium with seawater, the first step in alginate production is an ion-exchange with protons by extracting the milled algal tissue with 0.1–0.2 M mineral acid. In the second step, the alginic acid is brought into solution by neutralisation with alkali such as sodium carbonate or sodium hydroxide to form the water soluble sodium alginate. After extensive separation procedures such as sifting, floatation, centrifugation and filtration to remove algal particles, the soluble sodium alginate is precipitated directly by alcohol, by calcium chloride or by mineral acid, converted to the sodium form if needed and finally dried and milled. Besides Na-alginate, other soluble alginates are produced such as the potassium and ammonium salts.

The only derivative of alginates today having a commercial value is the propylene glycol alginate (PGA). This product is processed by an esterification of alginate with propylene oxide. PGA is used in beers and salad dressings due to its higher solubility at low pH.

Fig. 1 Principal scheme for the isolation of alginate from seaweeds

Following increased popularity of alginate as an immobilisation matrix, Pronova Biomedical A/S now commercially manufactures ultrapure alginates highly compatible with mammalian biological systems. These qualities are low in pyrogens, and facilitate sterilization of the alginate solution by filtration due to low content of aggregates.

4. CHEMISTRY:

Until Fischer and Dorfel identified the L-guluronate residue[20], D-mannuronate was regarded as the major component of alginate. Fractional precipitation with manganese and calcium salts demonstrated later that alginates are actually block copolymers, and that the ratio of guluronate to mannuronate varies depending on the natural source[21] . Alginate is now known to be a whole family of linear copolymers containing blocks of (1, 4)-linked β-D-mannuronate (M) and α-L-guluronate (G) residues. The blocks are composed of consecutive G residues (GGGGGG), consecutive M residues (MMMMMM), and alternating M and G residues (GMGMGM) (Fig. 2). Alginates extracted from different sources differ in M and G contents as well as the length of each block, and more than 200 different alginates are currently being manufactured[22]. The G-block content of L. hyperborean stems is 60%, and for other commercially available alginates is in the range of 14.0–31.0% [23]. Only the G-blocks of alginate are believed to participate in intermolecular cross-linking with divalent cations (e.g., Ca2+) to form hydrogels. The composition (i.e., M/G ratio), sequence, G-block length, and molecular weight are thus critical factors affecting the physical properties of alginate and its resultant hydrogels[24]. The mechanical properties of alginate gels typically are enhanced by increasing the length of G-block and molecular weight. It is important to note that different alginate sources provide polymers with a range of chemical structure (e.g., bacterial alginate produced from Azotobacter has a high concentration of G-blocks and its gels have a relatively high stiffness[25]. The physical properties significantly control the stability of the gels, the rate of drug release from gels, and the phenotype and function of cells encapsulated in alginate gels.

Fig. 2 Chemical structures of G-block, M-block, and alternating block in alginate.

5. GELLING TECHNIQUES:

Alginate is typically used in the form of a hydrogel in biomedicine, including wound healing, drug delivery and tissue engineering applications. Hydrogels are three-dimensionally cross-linked networks composed of hydrophilic polymers with high water content. Hydrogels are often biocompatible, as they are structurally similar to the macromolecular-based components in the body, and can often be delivered into the body via minimally invasive administration [26]. Chemical and/or physical cross-linking of hydrophilic polymers are typical approaches to form hydrogels, and their physicochemical properties are highly dependent on the cross-linking type and cross-linking density, in addition to the molecular weight and chemical composition of the polymers[27, 28]. Here, we summarize various approaches to cross-link alginate chains to prepare gels, and how these approaches alter the hydrogel properties relevant to biomedical and pharmaceutical applications.

5.1 Ionic Gels:

Ionic gels are formed by adding multivalent ions (typically calcium) to form ionic bonds between adjacent alginate chains. The divalent cations are believed to bind solely to guluronate blocks of the alginate chains, as the structure of the guluronate blocks allows a high degree of coordination of the divalent ions. The guluronate blocks of one polymer then form junctions with the guluronate blocks of adjacent polymer chains in what is termed the egg-box model of cross-linking, resulting in a gel structure (Fig. 3). Due to the very rapid and irreversible binding reaction between multivalent cations and alginates, a direct mixing of these two components rarely produces homogeneous gels. For most gelling purposes, ability to control the introduction of the crosslinking ions is essential. This control is possible by the two fundamentally different methods of preparing an alginate gel: the diffusion method and the internal setting method. The diffusion method is characterised by letting a crosslinking ion (e.g. Ca²⁺) diffuse from an outer reservoir into an alginate solution (Fig. 4(a). Internal setting (sometimes also referred to as in situ gelation) differs from the former in that the Ca²⁺ions are released in a controlled fashion from an inert calcium source within the alginate solution (Fig. 4(b). Controlled release is usually obtained by a change of pH and/or by a limited solubility of the calcium salt source.

Fig. 3 Alginate hydrogels prepared by ionic cross-linking (egg-box model). Only guluronate blocks participate in the formation of a corrugated egg-box-like structure with interstices in which calcium ions are placed.

Fig.4 The two principal methods for the manufacture of alginate gels: (a) diffusion setting, (b) internal gelling

5.1.1 Diffusion setting:

This method has gained great popularity as an immobilization technique, and is used in several methods for the restructuring of foods such as artificial berries, pimiento strips and onion rings[29].Diffusion setting is characterized by rapid gelling kinetics, and this high-speed setting is indeed utilized for immobilization purposes where each droplet of alginate solution makes one single gel bead entrapping the active agent. Rapid gelling is also beneficial in the restructuring of foods when a given size or shape is to be achieved. It has been shown that as long as alginates with an average molecular weight above 100kDa are used, the molecular weight dependence in this system is negligible[30].The resulting gel strength is, however, strongly dependent on chemical composition and sequence. As already mentioned, a good correlation is found between gel strength and the average G-block length larger than 1 (N_{G > 1}) as shown in Fig. 5. Marked effect of gel strength are observed when N_G>1 changes from 5 to 15, which coincides with typical G-block lengths found in commercially available alginates[2].

Fig.5 Mechanical properties of alginate gels as function of average G-block length

An important feature in the diffusion setting method is that the final gel can exhibit an inhomogeneous distribution of alginate, with the highest concentration at the surface and gradually decreasing towards the centre of the gel. Extreme alginate distributions have been reported [31] with a fivefold increase at the surface (from the concentration in the alginate solution prior to gelation) and virtually zero concentration in the centre. This result has been explained by the fact that diffusion setting will create a sharp gelling zone moving from the surface towards the centre of the gel. The activity of alginate (and of the gelling ion) will equal zero in this zone, and alginate molecules will diffuse from the internal, non-gelled part of the gelling body towards the zero activity region [32, 33].It is important to know that homogeneity can be controlled and the parameters governing the final alginate distribution. Maximum inhomogeneity is reached by letting a low molecular weight alginate gel at low concentration of the gelling ion and in the absence of non-gelling ions. Maximum homogeneity is reached by gelling a high molecular weight alginate at high concentrations of both gelling and non-gelling ions[32].The importance of the non-gelling ions in alginate gelling systems is also observed in the stability of the gels. It has been shown that alginate gels start to swell markedly when the ratio between non-gelling and gelling ions becomes too high, and that the observed destabilization increases with decreasing F_G_[32].

5.1.2 Internal setting:

As mentioned earlier, this system is based on an addition of an inactive form of the cross linking ion into an alginate solution. In the case of calcium the insoluble CaCO_{3 or} the slightly soluble CaSO₄may be used, or the Ca^{2+ ions} may be complexed in a chelating agent (EDTA, citrate, etc.). The activation of the cross linking ions is usually linked to a change in pH caused by the addition of organic acids or lactones. Lowering of the pH readily releases Ca²⁺from CaCO₃and complexing compounds. Chelating agents do, however, have discrete pH ranges where the complexed ions are released; in the case of EDTA, pH has to be lowered to around 4.0 to obtain a release of calcium ions. By using salts like CaCO₃and CaSO₄, gels can be prepared over a much wider pH range[34]. GDL can also be used in the production of the ionic gels. In this case TCP (Tri Calcium Phosphate) is initially dispersed in the alginate solution prior to D-glucono- δ- lactone (GDL). Less GDL is added to form the ionic gels compared to the amount used for the acid gels because the pH only needs to decrease sufficiently to allow the calcium ions to dissociate from the TCP (Tri Calcium Phosphate) and form divalent ionic bonds between alginate chains. The time dependent changes in pH observed during the formation of the 1% (w/w) acid and ionic alginate gels are shown in Fig. 6. This figure highlights how the pH of the alginate solution remained above the pKa value (pH >3.5) for the formation of the ionic gels, and below it (pH <3.5) for the formation of the acid gels. Significant changes in the pH are observed in the initial 500 s; thereafter the pH values begin to reach a steady plateau.

The main difference between internal and diffusion setting is the gelling kinetics. With internal setting, the tailor-making of an alginate gelling system towards a given manufacturing process is possible. For example, in the alginate/CaCO₃/D-glucono- δ- lactone (GDL)-system, reducing the average particle size of the carbonate, and thereby increasing the total surface available to the acid, reduces the transition time[34].The modulus of the final gel, however, approaches the same value independent of gelling kinetics. Other mediators can be necessary for the control of gelling kinetics. In the case of CaSO₄, the solubility is so high that gelation would occur spontaneously if complexing agents such as polyphosphates were not present. Internal setting almost always gives homogeneous gels. An exception is when large particle size calcium salt is used in combination with a low molecular weight alginate. In a combination like this, in homogeneities can be observed due to salt sedimentation in a low-viscosity solution[34].

Fig.6 Time dependent changes in pH during the formation of 1% (w/w) acid and ionic alginate gels. The acid gel was formed by adding 14% (w/w) of GDL to the alginate solution. The ionic gel was formed by adding 0.236% (w/w) of GDL to the alginate solution which also contained 1.5 g/l of TCP.

The gel strength of internally set alginate gels depends more on molecular weight than diffusion set gels [35].Whereas gels made by the latter method describe almost a step function where gel strength gets independent of molecular weight at around 100kDa[30] (weight average degree of polymerization, DP_{w ~} 500), the internally set gels still depend on molecular weight even at 300kDa (DP_w ~ 1500). This is, at least partly, due to the fact that the internally set gels are more calcium limited compared to the gels made by diffusion, which means that the non-elastic fraction (sol and loose ends) will be higher in the internally set gels at a given molecular weight.

Observations have shown that the internally set gels are more exposed to syneretic effects than gels set by diffusion. As a rule of the thumb, [Ca²⁺] = 0.5[G] represents the limit at which syneresis becomes prominent in internally set gels[36].There is no detailed understanding of this difference at the moment, but part of the explanation is certainly due to the different modes of gelation. As outlined earlier, diffusion setting gives a gelling zone which moves towards the centre of the gelling body. Here, the alginate molecules become saturated with Ca²⁺and their activity drops towards zero. Internal setting implies a process where gelation starts simultaneously at a large number of locations. This puts some topological strains on the alginate molecules, but their activity and translational mobility do not equal zero. One can therefore imagine that after the primary gel network has been formed, there will still exist elastic segments with free G- blocks that could create new junction zones given the proximity of another free G-block and the presence of calcium ions. If the concentration of Ca²⁺increases, a second class of junction zones may therefore be formed which will contract the gel network resulting in volume reduction.

6.1 Alginic acid gels:

It has been known and utilized for several decades that alginates precipitate at pH below the pK_avalue[37].In fact, the discovery that alginates were block co-polymers originated from the discovery that the different types of blocks had different solubilities at low pH[38]. It is also well known that, under controlled conditions, alginates may form acid gels at these low pH. Acid gels are created by reducing the pH below the pKa of the uronic groups to allow hydrogen bonds to form between adjacent alginate chains. When the concentration of protons is large enough to decrease the pH of the solution below the pKa value of uronic groups on the alginate chains (pKa guluronic acid = 3.65, pKa mannuronic groups = 3.38), they become protonated and hydrogen bonds form between the polymer chains producing weak acid gels. In the case of the acid gels this can be done by dispersing GDL powder in the alginate solution. As the GDL dissolves, it dissociates and releases protons uniformly throughout the alginate solution. When sufficient GDL is added to lower the pH below the pKa value of the uronic groups on the alginate chains acid gels are produced. GDL can also be used in the production of the ionic gels. In this case TCP (Tri Calcium Phosphate) is initially dispersed in the alginate solution prior to GDL. Less GDL is added to form the ionic gels compared to the amount used for the acid gels because the pH only needs to decrease sufficiently to allow the calcium ions to dissociate from the TCP (Tri Calcium Phosphate) and form divalent ionic bonds between alginate chains. It has been shown[39] that gel strength of acid gels prepared by this method becomes independent of pH below 2.5. GDL is added as dry powder, and a sol/gel transition is observed within 30–60 minutes, depending on the chemical composition and molecular weight of the alginate. The time dependent changes in pH observed during the formation of the 1% (w/w) acid and ionic alginate gels are shown in Fig. 6. This figure highlights how the pH of the alginate solution remained above the pKa value (pH >3.5) for the formation of the ionic gels, and below it (pH <3.5) for the formation of the acid gels. Significant changes in the pH are observed in the initial 500 s; thereafter the pH values begin to reach a steady plateau.

6.2 Similarities and Dissimilarities between Ionic and acid gels:

One of the first observed features of the alginic acid gels was that they were considerable more turbid and had a brittle texture compared to the ionic gels. Compression analysis showed that acid gels failed at 1/10 of the load necessary to break ionic gels, and that the relative deformation at breaking point was approximately 50% of that observed for the ionic gels (data not included). The material properties of the final gels produced were investigated by large deformation experiments using the Instron universal testing machine. The maximum stress values for the 1.5% and 0.75% (w/w) ionic gels measured using this technique is significantly larger than the corresponding values for the acid gels. These results showed that the gel strength of the ionic gels was significantly greater than for corresponding acid gels as expected [35, 40]. The compression data (stress as a function of applied strain) are shown in Fig.7. The mean gel hardness values are displayed in table 1. From the studies of ionic gels, It is known that they are extremely dependent on how they are made; i.e. they exhibit non-equilibrium properties. To explore the extent of non- equilibrium properties of the alginic acid gels, they were prepared by two different methods. The first method was, as already described, the addition of GDL. The second method was to dialyse a pre-formed ionic gel with acid to replace the cross-linking ions with protons. The ionic gel shows a considerably higher modulus before it is converted to the acid form (Table 2). Some syneresis of the gels was observed concomitant with the Ca²⁺ exchange with protons. A correlation factor was defined by studying alginic acid gels at different concentrations in order to be able to compare E_app at a give polymer concentration. By adapting this factor, no difference in modulus between the two methods was found (Table 2). Thus, acid gels are more of an equilibrium nature compared to ionic gels.

Table 1 Maximum stress values calculated from the applied breaking force for 1.5% and 0.75% (w/w) ionic and acid alginate gels, and the corresponding NMR R2 values (taken from the linear regression fits to the data shown in Fig. 5). All measurements were carried out at 37 ◦C on gels stored for 24 h[55].

Gel	Gel hardness (Pa)	NMR R2 (s−1)
1.5% Ionic	(4.9±0.4)×104	3.8 ± 0.4
1.5% Acid	(0.8±0.1)×104	3.4 ± 0.3
0.75% Ionic	(1.5±0.13)×104	1.9 ± 0.6
00.75% Acid	(0.3±0.06)×104	2.3 ± 0.2

Table 2 Eapp (kPa) for gels made from three different high-G alginates at 2% (w/v) concentration

Molecular weight, Mw (kDa)	Ca-alginate gel	Ca-gel to alginic acid gel	Syneresis correction	Direct addition of GDL
160	105±4.6	52±4.3	15.5±0.3	15.0±1.1
210	116±11	64±8.1	17.1±1.8	17.8±1.4
320	127±6.4	79±5.8	19.8±1.3	20.4±0.7

Fig. 7 True stress calculated from the compressive force applied to 12mmdiameter cylinders of (a) 0.75% (w/w) acid, (b) 1.5% (w/w) acid, (c) 0.75% (w/w) ionic and (d) 1.5% (w/w) ionic gels as a function of applied true strain

Fig.8 E_appof alginic acid gels as function of guluronic acid content (data points). Alginate concentration =10 mg/ml. Dotted line indicates expected moduli for Ca-alginate gel.

Fig. 8 shows the apparent Young’s modulus observed for acid gels made from various alginate samples (data points). The dotted line shows, approximately, what would be observed if ionic gels were made from alginates with different content of guluronate. It is obvious that chemical composition and sequence to a large extent determine the elastic modulus of the resulting alginic acid gel, and as in the case of ionically crosslinked gels, alginates with a high content of guluronate give the strongest gels. The acid gel differs from the ionic gel in that the modulus actually increases when going down from approximately 35% guluronate. This result suggests that also polymannuronic sequences are able to take part in intermolecular junction zones but to a lower extent compared to polyguluronate. Fig. 9 shows the effect of molecular weight on alginic acid gel formation. This figure shows the elastic response, measured as G and apparent E, of alginic acid gels made from alginates with two different levels of G and at several molecular weights. It can be seen that there is a marked Mw dependence within the high-G acid gels, which actually does not seem to level off, but perhaps becomes somewhat lower at molecular weights above 200 kDa. If the content of G is lowered from 70 to 50%, virtually no molecular weight dependence is observed. The only difference is that the average G-block length has been reduced from 14 to 8 and that there has been an increase in alternating sequences (FGM/MG) from 0.10 to 0.20. This result can be interpreted as showing the importance of the length of G-blocks in the initial quasi-ordered junction zone formation (co-operative binding), or the importance of the alternating sequences in limiting the formation of random aggregates. For the ionic gels[41] it has been reported that the cooperative binding of Ca-ions increases with increasing G-block length up to a degree of polymerisation (Dp) of around 20. These gels also show a sharp levelling off with respect to Mw above 100 kDa[42].

Fig. 9 E_app (A) and G’ (B) for alginic acid gels made from high G -alginates (closed symbols) and medium G alginates (Open symbols). Alginate concentration= 10 mg/mL.

With the exception of some pharmaceutical uses, the number of application of acid gels is rather limited to date. One critical drawback of ionically cross-linked alginate gels is the limited long-term stability in physiological conditions, because these gels can be dissolved due to release of divalent ions into the surrounding media due to exchange reactions with monovalent cations. In addition, the calcium ions released from the gel may promote hemostasis, while the gel serves as a matrix for aggregation of platelets and erythrocytes[43]. These features may be beneficial or negative, depending on the situation, but a desire to avoid these biological reactions, along with more general limitations of ionically cross-linked gels has led to interest in covalently cross-linked alginate hydrogels.

6.3 Covalent cross-linking:

Covalent cross-linking has been widely investigated in an effort to improve the physical properties of gels for many applications, including tissue engineering. The stress applied to an ionically cross-linked alginate gel relaxes as the cross-links dissociate and reform elsewhere, and water is lost from the gel, leading to plastic deformation. While water migration also occurs in covalently cross-linked gels, leading to stress relaxation, the inability to dissociate and reform bonds leads to significant elastic deformation[44]. However, covalent cross-linking reagents may be toxic, and the unreacted chemicals may need to be removed thoroughly from gels. Covalent cross-linking of alginate with poly (ethylene glycol)-diamines of various molecular weights was first investigated in order to prepare gels with a wide range of the mechanical properties. While the elastic modulus initially increased gradually with an increase in the crosslinking density or weight fraction of poly(ethylene glycol) (PEG) in the gel, it subsequently decreased when the molecular weight between cross-links (Mc) became less than the molecular weight of the softer PEG[45]. It was subsequently demonstrated that the mechanical properties and swelling of alginate hydrogels can be tightly regulated by using different kinds of cross-linking molecules, and by controlling the cross-linking densities. The chemistry of the cross-linking molecules also significantly influences hydrogel swelling, as would be expected. The introduction of hydrophilic cross-linking molecules as a second macromolecule (e.g. PEG) can compensate for the loss of hydrophilic character of the hydrogel resulting from the cross-linking reaction[46].

The use of multi-functional cross-linking molecules to form hydrogels provides a wider range and tighter control over degradation rates and mechanical stiffness than bi-functional cross-linking molecules. For example, the physical properties and degradation behavior of poly (aldehyde guluronate) (PAG) gels prepared with either poly (acrylamide-co-hydrazide) (PAH) as a multi-functional cross-linker or adipic acid dihydrazide (AAD) as a bifunctional cross-linker were monitored in vitro. PAG/PAH gels showed higher mechanical stiffness before degradation and degraded more slowly than PAG/AAD gels. The enhanced mechanical stiffness and prolonged degradation behavior could be attributed to the multiple attachment points of PAH in the gel even at the same concentration of overall functional groups[47]. Photo cross-linking is an exciting approach to in situ gelation that exploits covalent cross-linking. Photo crosslinking can be carried out in mild reaction conditions, even in direct contact with drugs and cells, with the appropriate chemical initiators. Alginate, modified with methacrylate and cross-linked by exposure to a laser (argon ion laser, 514 nm) for 30 s in the presence of eosin and triethanol amine, forms clear and flexible hydrogels. The gels were useful for sealing corneal perforation in vivo, indicating a potential clinical use in sutureless surgery[48]. Photo cross-linking reactions typically involve the use of a light sensitizer or the release of acid, which may be harmful to the body. An alternative photo cross-linking approach uses polyallylamine partially modified with phenoxycinnamyldiene acetylchloride, which dimerizes on light exposure at about 330 nm, and releases no toxic byproducts during the cross-linking reaction[49]. The mechanical properties of the gels formed from this photosensitive polyallylamine and alginate were significantly improved by light irradiation, and the gels were freely permeable to cytochrome c and myoglobin[50].

6.4 Thermal gelation:

Thermo-sensitive hydrogels have been widely investigated to date in many drug delivery applications, due to their adjustable swelling properties in response to temperature changes, leading to on-demand modulation of drug release from the gels[51]. Poly (N isopropyla crylamide) (PNIPAAm) hydrogels are the most extensively exploited thermo-sensitive gels, and these undergo a reversible phase transition near body temperature in aqueous media (lower critical solution temperature near 32 ◦C). The transition temperature can be altered by copolymerization with hydrophilic monomers such as acrylic acid and acrylamide[52]. Despite the potential importance of thermo-sensitive hydrogels in biomedical applications, few systems using alginate have been reported yet, as alginate is not inherently thermo-sensitive. However, semi-interpenetrating polymer network (semi-IPN) structures were prepared via in situ copolymerization of N-isopropyla crylamide (NIPAAm) with poly (ethylene glycol)-co-poly (_-caprolactone) (PEG-co-PCL) macromer in the presence of sodium alginate by UV irradiation (Fig. 10). The swelling ratio of the gels increased with the concentration of sodium alginate at a constant temperature, and decreased with an increase in temperature. The use of sodium alginate in semi-IPN structures also improved the mechanical strength and the cumulative release of BSA from the gels, indicating potential in drug delivery applications[53]. Graft copolymerization of NIPAA m onto the alginate backbone after reaction with ceric ions also provided a useful means to prepare temperature-responsive alginate gels, with sensitivity near body temperature (Lee and Mooney, unpublished data) (Fig. 11).

Fig. 10 Schematic description of thermo-sensitive semi-IPN alginate hydrogels

Fig.11 Thermal gelation of an aqueous alginate-g-NIPAAm solution at 37 ◦C.

6.5 Cell cross-linking:

While a number of chemical and physical methods have been reported to form alginate gels, the ability of cells to contribute to gel formation has been largely ignored. When alginate is modified with cell adhesion ligands, the ability of cells to bind multiple polymer chains can lead to long-distance, reversible network formation even in the absence of chemical cross-linking agents. Cells added to an RGD-modified alginate solution form a uniform dispersion within the solution, and this system subsequently generates the cross-linked network structure via specific receptor-ligand interactions without using any additional cross-linking molecules[54]. In contrast, cells added to non-modified alginate solutions aggregate and form a non-uniform structure, due to the dominance of cell-cell interactions in that system. This gelation behavior is shear reversible and can be repeated multiple times. Once the gel structure is broken down by applying shear forces, cross linked structures are recovered within a few minute. This behavior is governed by the weak and reversible ligand receptor interactions in the system. This system might be ideal for cell delivery in tissue engineering because a gel can flow like a liquid during injection into the body, but solidify once it is placed in the body. Further, it was reported that cells can provide additional mechanical integrity to RGD alginate gels that are ionically cross-linked with calcium ions, again via binding interactions between cells and the adhesion ligands coupled to the alginate chains.

7. CONCLUSION:

Alginate is a wonder molecule of this era. Plenty of information has been obtained over the last few decades on its properties and application. Over the time alginate got wide spread applications owing to favorable properties, including thickening, gel forming, stabilizing and biocompatibility properties. The molecule can be tailor-made for a number of biomedical applications.

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