International Journal of Biological Macromolecules 21 (1997) 47 55

Alginate based new materials

Kurt Ingar Draget *, Gudmund Skjak-Brk, Olav Smidsrd
Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian Uni6ersity of Science and Technology, N-7034 Trondheim, Norway Received 19 July 1996; received in revised form 18 December 1996; accepted 19 February 1997

Abstract Present and future applications of alginates are mainly linked to the most striking feature of the alginate molecule; i.e. a sol/gel transition in the presence of multivalent cations, e.g. Ca2 + , almost independent on temperature. These very mild conditions, combined with the fact that alginates are highly characterised and understood both in the liquid and in the gel phase, makes this biopolymer unique compared to other gelling polysaccharides. Only pectins resemble alginate in the sol/gel transition behaviour, but this system can hardly be said to be as well characterised and understood as the alginates. The properties of alginate solutions and gels suggest biomedical and pharmaceutical uses. In this paper, the question of the specications required by a polymer for applications in some biomedical areas will be discussed. 1997 Elsevier Science B.V. Keywords: Alginates; Gels; Polysaccharides

1. Introduction Alginates must be regarded as a family of copolymers since the fraction and sequence of the two monomers, h-L-guluronic acid (G) and i-Dmannuronic acid (M) (Fig. 1a), varies over a wide range. The fact that G and M are C5 epimers results in a switch-over of the monomer chair conformation, giving rise to all four possible glycosidic linkages and at the molecular level (Fig.
* Corresponding author. Tel.: + 47 73598260; fax: + 47 73591283; e-mail: KDRAGET@KJEMI.UNIT.NO

1b), large effects like cavity formations between G residues are observed. By chemical fractionation and nuclear magnetic resonance (NMR) characterisation [14] coupled with statistical modelling [5,6], it has been shown that the occurrence of G and M within the alginate molecule is block-wise (Fig. 1c) and not random. Gelation of alginates is based on the afnity of alginates towards certain ions and the ability to bind these ions selectively and cooperatively. Selective ion binding is strictly linked to the content of guluronate residues (G), or more precisely, the length of the G-blocks [7,8]. Increased ionic bind-

0141-8130/97/$17.00 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 4 1 - 8 1 3 0 ( 9 7 ) 0 0 0 4 0 - 8

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ing and hence also enhanced mechanical rigidity, is therefore found with alginates rich in G residues [9]. Fig. 2 displays the effect of G-blocks larger than one unit (NG \ 1) on gel strength. The increased gel strength with increasing G content (and increasing length of G blocks) stems from association of long G-blocks and a shortening of the elastic segments. In addition to ionic gels, alginates can form acid gels at pH below the pKa value of the uronic acid residues. Recent publications [10,11] have shown that, as in the case of the ionically crosslinked gels, the most important element in stabilising such gels are the G-blocks. However, M-blocks also support acid gel formation and the acid gel, contrary to the ionic gel, seems to be more of an equilibrium type.

Fig. 1. Alginate monomer (a) and chain (b) conformations and a schematic alginate chain sequence (c).

2. Alginate based new materials

2.1. Alginate in solution 2.1.1. Biological effects Future alginate based materials are not restricted to the solid phase. The alginate molecule itself, with its high variety of possible chemical compositions and molecular weights, can be expected to have different effects on e.g. biological systems and give different technological properties in the liquid phase. A biological effect of alginate was indeed suggested in the rst animal transplantation trials of encapsulated Langerhans islets for diabetes control (see later). Here, overgrowth of alginate capsules by phagocytes and broblasts, resembling a foreign body/inammatory reaction, was reported [12]. Induction of tumour necrosis factor (TNF) and interleukin 1 (IL-1), both cytokines having pro-inammatory activities, in bioassays showed that the inducibility depended upon the content of mannuronate in the alginate sample [13] (Fig. 3). This result also directly explained the observed capsule overgrowth; mannuronate rich fragments, not taking part in the gel network, leached out of the capsules and directly triggered an immune response [14]. The presence of immunologic response can, at least partly, be linked to i(14) glycosidic linkages since also

other homopolymeric di-equatorial polyuronates, like D-glucuronic acid (C6-oxidized cellose), also exhibit this feature [15]. In vivo animal models have now revealed the immunologic potential of polymannuronate in such diverse areas as for protection against lethal bacterial infections and irradiation, and for increasing non-specic immunity [15]. Alginates highly compatible with biological systems, i.e. ultrapure qualities low in pyrogens and low in aggregates facilitating sterilisation by ltration of the alginate solution, are now being commercially manufactured through an advanced

Fig. 2. Elastic modulus of diffusionally set calcium alginate gels as function of the average length of homopolymeric G-blocks.

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Fig. 3. Production of tumour necrosis factor (TNF) from human monocytes as response to alginates with different content of mannuronic acid residues.

giving rise to polymers with enhanced gelling properties [16]. Another interesting prospect is the use of C-5-epimerization to reduce the compositional heterogeneity in an alginate sample since the average composition of alginate may vary from plant to plant and from one tissue to another. Commercially produced alginates are therefore mixtures of subpopulations of molecules. When the epimerases attack long homopolymeric sequences of mannuronic acid, there is an apparent endpoint where the M blocks become too short to support further enzyme activity. By treating an alginate sample with C-5 epimerase, one might therefore obtain materials not only with an increased average content of guluronic acid, but also with a more uniform distribution of composition and block-structure [17,18]. Such materials might be suitable for encapsulation of cells as discussed later.

purication process developed by Pronova Biopolymer A/S. In the search for new alginate based materials, one must take into account that microbially produced alginates also have a large potential as industrial polymers. One obvious advantage is their structural diversity, both with respect to the chemical composition which ranges from pure mannuronan to polymers containing more than 80% guluronate residues, and to sequence which ranges from typical block structure alginates in A. 6inelandii to poly-alternating polymers in Pseudomonas species. Furthermore, the recent breakthrough in genetics of alginate producing bacteria also opens up the prospect of polysaccharide engineering either by modifying the producing organism, or by using recombinant enzymes in vitro. Since guluronic acid residues in alginate are introduced on a polymer level by mannuronan C-5 epimerases, these enzymes govern the sequential structure and the gel-forming capacities of the polymers [16]. An epimerase gene family in A. 6inelandii has recently been identied, sequenced and cloned and presents many possibilities for both basic and applied research. We have already proven that the enzymes can be used to modify alginate in vitro,

2.1.2. Chemically modied alginates The only derivative of alginates today, having a commercial value, is the propylene glycol alginate (PGA). This product is processed by an esterication of alginate with propyleneoxide. PGA is used in beers and salad dressings due its higher solubility at low pH. Two new families of derivatives have emerged recently. The rst one is based on a patent where a wide range of alginate esters can be prepared by treating the quaternary ammonium salt of alginic acid with alkylating agents in organic aprotic media such as DMSO. Of particular interest are esters with pharmacologically active alcohols where the modied polymer functions as a carrier for drug release [19]. In the second category, a photo-crosslinkable alginate is prepared by grafting the alginate with acrylate or allyl groups with a subsequent covalent crosslinking by UV photopolymerisation yielding strong and highly deformable alginate gels. Provided the degree of substitution is kept low, the alginate is still capable of forming gels with calcium ions, implying that spheres and bres easily can be moulded and enforced by photo-polymerisation [20].

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2.2. Alginate gels

In the solid phase, the technological properties of the nal product are largely governed by alginate variables such as chemical composition and sequence and molecular weight and molecular weight distribution. Important technological properties are gel strength, porosity/diffusion, alginate distribution, swelling/shrinking, transparency, leaching of alginate from the gels and immunology of the leached material. Thu et al. [21] gives a good summary on this subject. In the following chapters, only some selected examples on the possible optimization of different systems for certain applications will be presented.

Inhomogeneity might or might not be benecial in the nal product. It is therefore important to know that homogeneity can be controlled and which parameters that govern the nal alginate distribution. Maximum inhomogeneity is obtained with a low molecular weight alginate in a solution containing a low concentration of the gelling ion and in the absence of non-gelling ions. Maximum homogeneity is reached by a high molecular weight alginate gelled with high concentrations of both gelling and non-gelling ions.

2.3. Diffusional setting (e.g. beads, bres and lms)

Diffusional setting, in contrast to internal setting, is basically the alginate gelling method where the crosslinking ions, e.g. Ca2 + , are allowed to diffuse into a solution of soluble alginate [22]. This method can be applied in the manufacturing of beads, bres and lms. Diffusional setting is always used when alginate acts as an immobilization matrix [23]. This is due to a rapid and gentle gel formation when droplets of soluble alginate (carrying a biologically active substance) hit the gelling liquid, leading to an entrapment of the biocatalyst. Diffusional setting is also utilized in several applications involving the restructuring of foods [24].

2.3.2. Capsules for immobilization and implantation Perhaps the most renowned application of alginate as an immobilization matrix is the use of encapsulated pancreatic islets for the treatment of type I diabetes [13,27]. The original method [28] was based on a liquid core capsule where the crosslinking ions (Ca2 + ) were exchanged with non-gelling Na + ions by sequestering agents like citrate. Here, an obvious drawback is an increased potential swelling due to an increased osmotic pressure and less elastic strength inside the liquid capsule, making it difcult to obtain the required prolonged mechanical stability. The liquid core capsule may be exchanged with a solid core capsule to increase stability [29,30] (Fig. 5). It is important to realize that there is a direct dependence between the mechanical strength of

2.3.1. Distribution of alginate material in gels One of the main features of the diffusional setting method is that the distribution of alginate within the nal product is generally not uniform, with a high concentration at the surface and gradually decreasing towards the centre of the gel [25] (Fig. 4). This has been explained by the fact that diffusion setting creates a sharp gelling zone which moves 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 [26] resulting in a depletion of alginate in the centre part of the gel.

Fig. 4. Ca-alginate concentration proles in diffusionally set alginate gel cylinders at different NaCl concentrations:
, 0.2 M; , 0.05 M; , no NaCl.

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therefore was to modify the porosity of the highG alginate capsules, which gave the desired mechanical properties, in such a way that small molecules, like glucose and insulin, were allowed to diffuse almost unrestricted within the capsule and at the same time to keep the large antibodies out. Porosity may be modied by making a complex between polyanionic alginate and a polycation (e.g. poly-L-lysine; PLL) at the surface of the alginate bead [29,30]. An inhomogeneous alginate bead, with a high surface charge density, may give improved binding of PLL and increased mechanical stability. To avoid attachment of cells to transplanted capsules due to the positive charges of unreacted PLL, an outer layer of alginate may be added.
Fig. 5. Fraction of intact alginate/PLL capsules measured as release of encapsulated blue dextran after 1 year in saline.

an alginate gel and the porosity of the gel network. When gels are made from an alginate rich in guluronic acid residues, higher moduli are obtained compared to gels made from alginates less enriched in G residues, and also higher diffusion rates [31]. The proposed explanation for this behaviour is that high-G gels, with their long Gblocks and their short elastic segments become more of a stiff open and static network compared to the more dynamic and entangled network structure of the low-G gels with their relative long elastic segments [21] (Fig. 6). Another challenge

Fig. 6. Proposed model for network structure in gels made from alginates with guluronic acid blocks of different lengths.

2.3.3. Superswelling alginate materials Pre-formed Ca-alginate gels can also be covalently crosslinked with epichlorohydrin with a subsequent removal of Ca2 + ions by EDTA [32]. These Na-alginate gels can be dried, and they exhibit unique swelling properties when rehydrated. The forces affecting the swelling of a polymer network can be split into three terms. Two of these terms favour swelling and can be said to constitute what might be called swelling pressure: (a) the mixing term (Dymix is the osmotic pressure generated by polymer/solvent mixing) and (b) the ionic term (Dyion is the osmotic effect of an unequal distribution of the polymer counterions between the inside and the outside of the gel; the Donnan equilibrium). Of these two terms, the ionic part has been shown to contribute approximately 90% of the swelling pressure even at one molar ionic strength for highly ionic gels like Na-alginate [33]. The third term, Dyel, is the reduction in osmotic pressure due to the elastic response of the polymer network, balances the swelling pressure so that the total of these three terms equals zero at equilibrium. A possible application for covalently crosslinked alginate gels is therefore as a water absorbent in hygiene and pharmaceutical applications. However, Dyion is dependent upon the ionic strength of the solute; increasing ionic strength reduces the difference in chemical potential of water due to a more even distribution of the

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Fig. 7. Salt tolerance of covalently crosslinked Na-alginate and polyacrylate gel beads measured as swelling at different ionic strengths.

mobile ions between the inside and the outside of the gel. These alginate gels will therefore exhibit reduced swelling at physiological ionic conditions compared to deionized water, but this reduction will be less pronounced than for other water absorbing materials, such as crosslinked acrylates, due to the inherent stiffness of the alginate molecule itself [32] (Fig. 7).

2.4. Sterile alginate gels for biological culti6ation

Some attempts have been made to apply alginate as an alternative to agar as a solidifying agent in biological cultivation media. The motivation behind this is partly to be able to take advantage of the cold setting properties of the alginate gel, and partly to reduce costs by exchanging a relative expensive polymer like puried agar and agaroses with the cheaper alginate. To achieve this it is essential that the nutrient containing gel can be controlled in terms of microbial contamination, gel strength, homogeneity and ageing phenomena (syneresis). Due to the very rapid and irreversible binding of Ca2 + to the guluronic acid blocks, it is impossible to obtain a homogeneous gel by simply adding Ca2 + to an alginate solution. The most common way to overcome this problem is to introduce Ca2 + into the

alginate solution in an inactive form with a subsequent controlled release and gel formation; the so-called internal gelation [22]. By applying CaCO3 and D-glucono-l-lactone (GDL), a sterile cultivation media for plant cell and tissue culture has been established [34]. In this system, homogeneous, non-syneretic nutrient gels are easily made as long as some precautions are taken. (i) pH of the nal gel is controlled by the relative molar amounts of CaCO3/GDL, (ii) syneresis is controlled by adjusting the amount of CaCO3 to the amount of guluronate residues and (iii) homogeneous gels are obtained as long as sedimentation of CaCO3 particles is prevented prior to gel setting. Sedimentation is most easily controlled by using low particle size CaCO3, or, alternatively, applying a high molecular weight alginate giving the nutrient solution a high relative viscosity. No signicant inferiority was found between the cultivation results obtained on these alginate based solid media compared to the results obtained on the agar based control media [34]. Solid media for biological cultivation have traditionally been sterilized by autoclaving which also solubilizes the agar or agarose gelling agent. Alginate is soluble in water at room temperature, and for immobilization purposes we have previously recommended sterile ltering rather than autoclaving in order to reduce polymer breakdown and maintain the mechanical properties of the nal gel [35]. When sterilizing alginate solutions and gels, it is important to realize the limitations of alginate based systems. Alginate is a single stranded polymer and susceptible to a variety of depolymerization processes: The glycosidic linkages are cleaved by both acid and alkaline degradation mechanisms and by oxidation with free radicals. As a function of pH, degradation of alginate is at its minimum around neutrality and increases in both directions [36] (Fig. 8). The increased instability at pH values less than ve is explained by a proton catalysed hydrolysis, whereas the reaction responsible for the degradation at pH 10 and above is the i-alkoxy-elimination [37,38]. Degradation by free radicals is mainly due to the oxidative-reductive depolymerization reactions (ORD) [3941], caused by contamination of reducing agents like polyphenols

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from the brown algae. Since all these depolymerization reactions increases with temperature, autoclaving is generally not recommended for the sterilization of alginate solutions. Nor is the sterilization of dry alginate powders simple and straight forward; the effect of k-irradiation is often disastrous. It is generally believed that under these conditions, O2 is rapidly depleted with a formation of the very reactive OH free radical. We have recently been testing short term k exposure in an electron accelerator as an alternative to long term exposure from a traditional 60 Co-source. These results showed that although a substantial polymer breakdown occurred, the gelling capacity was preserved. From earlier studies, sterilization doses by 60Co irradiation reduces the molecular weight to the extent that the gelling capacity is almost completely lost [42]. This difference can be attributed to the much shorter period

Fig. 9. Moduli of alginic acid gels as function of guluronic acid content (data points). Dotted line refers to expected results with Ca2 + crosslinking.

of k exposure needed for sterilization in the electron accelerator (approximately 1 min) compared to 1.5 h in a traditional 60Co source giving less free radicals produced from O2 in the electron accelerator.

2.5. Alginic acid gels

The alginic acid gels represent a new possibility for alginate based materials, especially within drug delivery systems (DDS). The fundamental understanding, and hence also the number of applications, have until recently been rather low. Lately, we have published some papers which describe some of the most important features of the alginic acid gel at equilibrium and during re-solvatation [10,11]. Equilibrium results show that the acid gel requires homopolymeric regions in the alginate, and that G-blocks are most important in acid gel formation as they are in the ionically crosslinked gels (Fig. 9). A study of the swelling and partial solubilization of alginic acid gels at pH 4, i.e. just above the pKa-value of the uronic acid residues, has supported the results from the equilibrium studies of these gels [11]. By comparing the chemical composition and molecular weight of the alginate mate-

Fig. 8. Degradation of alginate isolated from Laminaria digitata measured as the change (D) in intrinsic viscosity ([p]) after 5 h at different pH and at 68C.

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K.I. Draget et al. / International Journal of Biological Macromolecules 21 (1997) 4755 [2] Grasdalen H, Larsen B, Smidsrd O. Carbohydr Res 1979;68:23. [3] Penman A, Sanderson GR. Carbohydr Res 1972;25:273. [4] Grasdalen H. Carbohydr Res 1983;118:255. [5] Larsen B, Smidsrd O, Painter T, Haug A. Acta Chem Scand 1970;24:726. [6] Smidsrd O, Whittington SG. Macromolecules 1969;2:42. [7] Smidsrd OJ. Chem Soc Farad Trans 1974;57:263. [8] Kohn R, Larsen B. Acta Chem Scand 1972;26:2455. [9] Smidsrd O, Haug A. Acta Chem Scand 1972;26:79. [10] Draget KI, Skjak-Brk G, Smidsrd O. Carbohydr Polym 1994;25:31. [11] Draget KI, Skjak-Brk G, Christensen BE, Gaserd O, Smidsrd O. Carbohydr Polym 1996;29:209. [12] Soon-Shiong P, Otterlei M, Skjak-Brk G, Smidsrd O, Heintz R, Lanza RP, Espevik T. Transplant Proc 1991;23:758. [13] Soon-Shiong P, Feldman E, Nelson R, Heintz R, Yao Z, Yao Q, Zheng T, Merideth N, Skjak-Brk G, Espevik T, Smidsrd O, Sandford P. Proc Nat Acad Sci USA 1993;90:5843. [14] Stokke BT, Smidsrd O, Zanetti F, Strand W, Skjak Brk G. Carbohydr Polym 1993;21:39. [15] Skjak-Brk G, Espevik T. Carbohydrates Europe 1996;14:19. [16] Skjak-Brk G, Smidsrd O, Larsen B. Int J Biol Macro mol 1986;8:330. [17] Ertesvag H, Doseth B, Larsen B, Skjak-Brk G, Valla S. J Bacteriol 1994;176:2846. [18] Ertesvag H, Hidal HK, Hals IK, Rian A, Doseth B, Valla S. Mol Microbiol 1995;16:719. [19] Della Valle F, Romeo A, Hirsch M.-R. European Patent Applied 87401464.0. [20] Soon-Shoing P, Desai N, Sandford P, Heintz R. PCT US 927/09364. [21] Thu B, Smidsrd O, Skjak-Brk, G. In: Wijffels RH, Buitelaar RM, Bucke C, Tramper J. (Eds.), Immobilized Cells; Basics and Applications Amsterdam: Elsevier Science, 1996:19. [22] Smidsrd O, Draget KI. Alginate gelation technologies. In: Food Colloids Proteins, Lipids and Polysaccharides, Ystad, Sweden, The Royal Society of Chemistry, Cambridge, pp. 279 293. [23] Smidsrd O, Skjak-Brk G. Trends Biotechnol 1990;8:71. [24] Sime WJ. In: Harris P. (Eds.), Food Gels. Elsevier: Essex, England, 1990:53. [25] Skjak-Brk G, Grasdalen H, Smidsrd O. Carbohydr Polym 1989;10:31. [26] Mikkelsen A, Elgster A. Biopolymers 1995;36:1741. [27] Soon-Shiong P, Heinz RE, Merideth N, Yao QX, Yao Z, Zheng T, Murphy M, Moloney MK, Schmehl M, Harris M, Mendez R, Mendez R, Sandford P. Lancet 1994;343:950. [28] Lim F, Sun AM. Science [29] Thu B, Bruheim P, Espevik T, Smidsrd O, Soon-Shiong P, Skjak-Brk G. Biomaterials 1996;17:1031.

rial leaching out from the acid gels with the same data for the whole alginate, an enrichment in mannuronic acid residues was found together with a reduction in the average length of G-blocks and a lowering of the molecular weight. The importance of long guluronic acid blocks in the stabilization of acid gels is therefore also shown by such experiments. For applications of alginic acid gels such as a matrix in DDS systems, the kinetics of swelling and dissolution of the gel is perhaps more important than their equilibrium properties. Generally, both rate of swelling, swelling potential and leakage of dissolved alginate material increases with increasing content of mannuronate residues. Kinetic studies have also revealed that for acid gels rich in G residues, both molecular weight and bead particle size seem to be of major importance [11]. These dependencies become less pronounced as the content of M residues increases. Based on these results, it should be possible to tailor make DDS systems with distinct medical release proles.

3. Concluding remarks From the examples presented in this paper, it is obvious that there is a substantial demand for tailor-made alginates, as well as for other glycans, by polysaccharide engineering. By being able to obtain and systematize detailed information all the way from the genetics and biosynthesis to physical characteristics and technological properties, new possibilities for system optimization open up, and the application potential increases.

Acknowledgements The authors acknowledge the nancial support given by Pronova Biopolymer A/S and the Norwegian Research Council.

References
[1] Grasdalen H, Larsen B, Smidsrd O. Carbohydr Res 1977;56:C11.

K.I. Draget et al. / International Journal of Biological Macromolecules 21 (1997) 4755 [30] Thu B, Bruheim P, Espevik T, Smidsrd O, Soon-Shiong P, Skjak-Brk G. Biomaterials 1996;17:1069. [31] Martinsen A, Storr I, Skjak-Brk G. Biotechnol Bioeng 1992;39:186. [32] Skjak-Brk G, Moe ST. US Pat. 5144016, 1992. [33] Moe ST, Skjak-Brk G, Elgster A, Smidsrd O. Macro molecules 1993;26:3589. [34] Draget KI, stgaard K, Smidsrd O. Appl Microbiol Biotechnol 1989;31:79. [35] Draget KI, Myhre S, Skjak-Brk G, stgaard K. J Plant Physiol 1988;132:552. [36] Haug A, Larsen B. Acta Chem Scand 1963;17:1653.

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[37] Haug A, Larsen B, Smidsrd O. Acta Chem Scand 1963;17:1466. [38] Haug A, Larsen B, Smidsrd O. Acta Chem Scand 1967;21:2859. [39] Smidsrd O, Haug A, Larsen B. Carbohydr Res 1967;5:482. [40] Smidsrd O, Haug A, Larsen B. Acta Chem Scand 1963;17:1473. [41] Smidsrd O, Haug A, Larsen B. Acta Chem Scand 1963;17:2628. [42] Leo WJ, McLoughlin AJ, Malone DM. Biotechnol Prog 1990;6:51.