You are on page 1of 9

International Journal of Biological Macromolecules 21 (1997) 47 – 55

Alginate based new materials
Kurt Ingar Draget *, Gudmund Skjak-Bræk, Olav Smidsrød ˚
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 specifications 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 [1–4] 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 affinity 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

1. i. However.1. Fig. resembling a foreign body/inflammatory reaction.g. 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. Here. A biological effect of alginate was indeed suggested in the first animal transplantation trials of encapsulated Langerhans islets for diabetes control (see later). at least partly. Alginates highly compatible with biological systems. Fig. Alginate based new materials 2.e. Alginate in solution 2. like D-glucuronic acid (C6-oxidized cellose). Biological effects Future alginate based materials are not restricted to the solid phase. are now being commercially manufactured through an advanced Fig. with its high variety of possible chemical compositions and molecular weights. not taking part in the gel network. In addition to ionic gels. leached out of the capsules and directly triggered an immune response [14]. seems to be more of an equilibrium type.11] have shown that. 2. This result also directly explained the observed capsule overgrowth. 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. overgrowth of alginate capsules by phagocytes and fibroblasts. 3). in bioassays showed that the inducibility depended upon the content of mannuronate in the alginate sample [13] (Fig. alginates can form acid gels at pH below the pKa value of the uronic acid residues.48 K. the most important element in stabilising such gels are the G-blocks. can be expected to have different effects on e. The presence of immunologic response can. The alginate molecule itself. Draget et al. Elastic modulus of diffusionally set calcium alginate gels as function of the average length of homopolymeric G-blocks. contrary to the ionic gel. as in the case of the ionically crosslinked gels. . / International Journal of Biological Macromolecules 21 (1997) 47–55 ing and hence also enhanced mechanical rigidity. M-blocks also support acid gel formation and the acid gel. both cytokines having pro-inflammatory activities. 1.I. ultrapure qualities low in pyrogens and low in aggregates facilitating sterilisation by filtration of the alginate solution. is therefore found with alginates rich in G residues [9]. 2. be linked to i(1“4) glycosidic linkages since also other homopolymeric di-equatorial polyuronates. Alginate monomer (a) and chain (b) conformations and a schematic alginate chain sequence (c). and for increasing non-specific immunity [15].1. biological systems and give different technological properties in the liquid phase. Recent publications [10. 2 displays the effect of G-blocks larger than one unit (NG \ 1) on gel strength. mannuronate rich fragments. Induction of tumour necrosis factor (TNF) and interleukin 1 (IL-1). was reported [12].

2. having a commercial value.I.1. Production of tumour necrosis factor (TNF) from human monocytes as response to alginates with different content of mannuronic acid residues. these enzymes govern the sequential structure and the gel-forming capacities of the polymers [16]. Furthermore. The first 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. Two new families of derivatives have emerged recently. there is an apparent endpoint where the M blocks become too short to support further enzyme activity. In the search for new alginate based materials. An epimerase gene family in A. Draget et al. the recent breakthrough in genetics of alginate producing bacteria also opens up the prospect of polysaccharide engineering either by modifying the producing organism. one must take into account that microbially produced alginates also have a large potential as industrial polymers. Of particular interest are esters with pharmacologically active alcohols where the modified polymer functions as a carrier for drug release [19]. one might therefore obtain materials not only with an increased average content of guluronic acid. giving rise to polymers with enhanced gelling properties [16]. One obvious advantage is their structural diversity. PGA is used in beers and salad dressings due its higher solubility at low pH. both with respect to the chemical composition which ranges from pure mannuronan to polymers containing more than 80% guluronate residues. 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. When the epimerases attack long homopolymeric sequences of mannuronic acid. Commercially produced alginates are therefore mixtures of subpopulations of molecules. Since guluronic acid residues in alginate are introduced on a polymer level by mannuronan C-5 epimerases. purification process developed by Pronova Biopolymer A/S.18]. and to sequence which ranges from typical block structure alginates in A. By treating an alginate sample with C-5 epimerase. is the propylene glycol alginate (PGA). . Chemically modified alginates The only derivative of alginates today. 2. 6inelandii to poly-alternating polymers in Pseudomonas species. implying that spheres and fibres easily can be moulded and enforced by photo-polymerisation [20]. In the second category. We have already proven that the enzymes can be used to modify alginate in vitro. but also with a more uniform distribution of composition and block-structure [17. Such materials might be suitable for encapsulation of cells as discussed later. This product is processed by an esterification of alginate with propyleneoxide. the alginate is still capable of forming gels with calcium ions. or by using recombinant enzymes in vitro. 6inelandii has recently been identified. 3. Provided the degree of substitution is kept low. / International Journal of Biological Macromolecules 21 (1997) 47–55 49 Fig. sequenced and cloned and presents many possibilities for both basic and applied research. 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.K.

It is important to realize that there is a direct dependence between the mechanical strength of 2.g. Distribution of alginate material in gels One of the main features of the diffusional setting method is that the distribution of alginate within the final product is generally not uniform. “. 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. . This method can be applied in the manufacturing of beads. swelling/shrinking. transparency. 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.1. with a high concentration at the surface and gradually decreasing towards the centre of the gel [25] (Fig.50 K. Maximum homogeneity is reached by a high molecular weight alginate gelled with high concentrations of both gelling and non-gelling ions. porosity/diffusion. In the following chapters.2. the technological properties of the final product are largely governed by alginate variables such as chemical composition and sequence and molecular weight and molecular weight distribution. leaching of alginate from the gels and immunology of the leached material. an obvious drawback is an increased potential swelling due to an increased osmotic pressure and less elastic strength inside the liquid capsule. in contrast to internal setting. 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.3. 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. beads. The activity of alginate (and of the gelling ion) will equal zero in this zone. 4). / International Journal of Biological Macromolecules 21 (1997) 47–55 2.g.2. 0.3. 4. Here. 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. Ca2 + . leading to an entrapment of the biocatalyst. making it difficult to obtain the required prolonged mechanical stability. [21] gives a good summary on this subject.3. Ca-alginate concentration profiles in diffusionally set alginate gel cylinders at different NaCl concentrations: . no NaCl.2 M. alginate distribution. e. fibres and films) Diffusional setting. is basically the alginate gelling method where the crosslinking ions. 2. 2. . The liquid core capsule may be exchanged with a solid core capsule to increase stability [29. are allowed to diffuse into a solution of soluble alginate [22]. and alginate molecules will diffuse from the internal. Diffusional setting is always used when alginate acts as an immobilization matrix [23]. Diffusional setting (e.I.27]. This is due to a rapid and gentle gel formation when droplets of soluble alginate (carrying a biologically active substance) hit the gelling liquid.05 M. only some selected examples on the possible optimization of different systems for certain applications will be presented. Diffusional setting is also utilized in several applications involving the restructuring of foods [24]. Inhomogeneity might or might not be beneficial in the final product. Alginate gels In the solid phase. It is therefore important to know that homogeneity can be controlled and which parameters that govern the final alginate distribution. Draget et al. fibres and films. 5). 0. Thu et al.30] (Fig. Important technological properties are gel strength. Fig.

is the reduction in osmotic pressure due to the elastic response of the polymer network. 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].g. / International Journal of Biological Macromolecules 21 (1997) 47–55 51 therefore was to modify the porosity of the highG alginate capsules. Another challenge Fig. Dyion is dependent upon the ionic strength of the solute.K. poly-L-lysine. may give improved binding of PLL and increased mechanical stability. 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. Dyel. To avoid attachment of cells to transplanted capsules due to the positive charges of unreacted PLL. higher moduli are obtained compared to gels made from alginates less enriched in G residues. and also higher diffusion rates [31]. 2. balances the ‘swelling pressure’ so that the total of these three terms equals zero at equilibrium. with a high surface charge density. increasing ionic strength reduces the difference in chemical potential of water due to a more even distribution of the . in such a way that small molecules. When gels are made from an alginate rich in guluronic acid residues. However. Draget et al.3. an outer layer of alginate may be added. were allowed to diffuse almost unrestricted within the capsule and at the same time to keep the large antibodies out. The third term. 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. The proposed explanation for this behaviour is that high-G gels. which gave the desired mechanical properties. Fraction of intact alginate/PLL capsules measured as release of encapsulated blue dextran after 1 year in saline.30]. A possible application for covalently crosslinked alginate gels is therefore as a water absorbent in hygiene and pharmaceutical applications. The forces affecting the swelling of a polymer network can be split into three terms. Proposed model for network structure in gels made from alginates with guluronic acid blocks of different lengths. 5. PLL) at the surface of the alginate bead [29.3. and they exhibit unique swelling properties when rehydrated. 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]. Of these two terms. like glucose and insulin. Porosity may be modified by making a complex between polyanionic alginate and a polycation (e. Fig.I. 6). These Na-alginate gels can be dried. an alginate gel and the porosity of the gel network. 6. the Donnan equilibrium). An inhomogeneous alginate bead.

caused by contamination of reducing agents like polyphenols . it is impossible to obtain a homogeneous gel by simply adding Ca2 + to an alginate solution. the so-called internal gelation [22]. it is important to realize the limitations of alginate based systems. 2. Salt tolerance of covalently crosslinked Na-alginate and polyacrylate gel beads measured as swelling at different ionic strengths. and partly to reduce costs by exchanging a relative expensive polymer like purified agar and agaroses with the cheaper alginate. These alginate gels will therefore exhibit reduced swelling at physiological ionic conditions compared to deionized water. To achieve this it is essential that the nutrient containing gel can be controlled in terms of microbial contamination. mobile ions between the inside and the outside of the gel. 7).4. and for immobilization purposes we have previously recommended sterile filtering rather than autoclaving in order to reduce polymer breakdown and maintain the mechanical properties of the final gel [35]. (i) pH of the final gel is controlled by the relative molar amounts of CaCO3/GDL. Draget et al. Due to the very rapid and irreversible binding of Ca2 + to the guluronic acid blocks. non-syneretic nutrient gels are easily made as long as some precautions are taken. As a function of pH. The motivation behind this is partly to be able to take advantage of the cold setting properties of the alginate gel. due to the inherent stiffness of the alginate molecule itself [32] (Fig. 8). 7. gel strength. homogeneous. 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. applying a high molecular weight alginate giving the nutrient solution a high relative viscosity. 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. 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. a sterile cultivation media for plant cell and tissue culture has been established [34]. homogeneity and ageing phenomena (syneresis).I. When sterilizing alginate solutions and gels. alternatively. or. but this reduction will be less pronounced than for other water absorbing materials. By applying CaCO3 and D-glucono-l-lactone (GDL). In this system. degradation of alginate is at its minimum around neutrality and increases in both directions [36] (Fig.52 K. The increased instability at pH values less than five is explained by a proton catalysed hydrolysis.38]. / International Journal of Biological Macromolecules 21 (1997) 47–55 Fig. Degradation by free radicals is mainly due to the oxidative-reductive depolymerization reactions (ORD) [39–41]. such as crosslinked acrylates. Alginate is soluble in water at room temperature. Solid media for biological cultivation have traditionally been sterilized by autoclaving which also solubilizes the agar or agarose gelling agent. (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. whereas the reaction responsible for the degradation at pH 10 and above is the i-alkoxy-elimination [37. Sedimentation is most easily controlled by using low particle size CaCO3. No significant 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].

By comparing the chemical composition and molecular weight of the alginate mate- Fig. Dotted line refers to expected results with Ca2 + crosslinking.I. has supported the results from the equilibrium studies of these gels [11]. and that G-blocks are most important in acid gel formation as they are in the ionically crosslinked gels (Fig.e. 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. From earlier studies. 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. Moduli of alginic acid gels as function of guluronic acid content (data points).5 h in a traditional 60Co source giving less free radicals produced from O2 in the electron accelerator. Lately.5. have until recently been rather low. i. It is generally believed that under these conditions. A study of the swelling and partial solubilization of alginic acid gels at pH 4. O2 is rapidly depleted with a formation of the very reactive OH’ free radical. Draget et al. 9. the effect of k-irradiation is often disastrous. 8. These results showed that although a substantial polymer breakdown occurred. Alginic acid gels The alginic acid gels represent a new possibility for alginate based materials. Since all these depolymerization reactions increases with temperature.11]. This difference can be attributed to the much shorter period Fig.K. Nor is the sterilization of dry alginate powders simple and straight forward. sterilization doses by 60Co irradiation reduces the molecular weight to the extent that the gelling capacity is almost completely lost [42]. Degradation of alginate isolated from Laminaria digitata measured as the change (D) in intrinsic viscosity ([p]) after 5 h at different pH and at 68°C. The fundamental understanding. especially within drug delivery systems (DDS). / International Journal of Biological Macromolecules 21 (1997) 47–55 53 from the brown algae. autoclaving is generally not recommended for the sterilization of alginate solutions. of k exposure needed for sterilization in the electron accelerator (approximately 1 min) compared to 1. 2. Equilibrium results show that the acid gel requires homopolymeric regions in the alginate. the gelling capacity was preserved. 9). just above the pKa-value of the uronic acid residues. and hence also the number of applications. .

[21] Thu B. Zheng T. Strand W. (Eds. Christensen BE. Zheng T. [23] Smidsrød O. Sandford P. Carbohydr Res 1977. Rian A. Merideth N. Soon-Shiong P. Sanderson GR. Skjak-Bræk. ˚ Valla S. Nelson R. Trends Biotechnol 1990. Sun AM. Ystad. Int J Biol Macro˚ mol 1986. new possibilities for system optimization open up. [12] Soon-Shiong P. both rate of swelling.25:273.118:255.8:71. Espevik T. Macromolecules 1969. For applications of alginic acid gels such as a matrix in DDS systems.2:42. Chem Soc Farad Trans 1974. In: Harris P. 1996:19. [17] Ertesvag H. ˚ ˚ J Bacteriol 1994.176:2846.23:758. The Royal Society of Chemistry. Mendez R. as well as for other glycans. Acta Chem Scand 1972. Romeo A. Zanetti F. Espevik T.36:1741.54 K.21:39. Concluding remarks From the examples presented in this paper. / International Journal of Biological Macromolecules 21 (1997) 47–55 [2] Grasdalen H. [10] Draget KI. Alginate gelation technologies. ˚ [24] Sime WJ. Draget KI. Skjak-Bræk G. Desai N. Harris M.68:23.14:19. [4] Grasdalen H.26:2455.29:209. Høidal HK. pp. [7] Smidsrød OJ. Smidsrød O.). Haug A. G. Espevik T. the kinetics of swelling and dissolution of the gel is perhaps more important than their equilibrium properties. Whittington SG. Lipids and Polysaccharides. Skjak˚ Bræk G. [26] Mikkelsen A. Mendez R. Gaserød O. Elgsæter A. Carbohydr Res 1983. Bucke C. Acta Chem Scand 1970. ˚ Buitelaar RM. Carbohydr Res 1972. Proc Nat Acad Sci USA 1993. Immobilized Cells.). Acknowledgements The authors acknowledge the financial support given by Pronova Biopolymer A/S and the Norwegian Research Council. Smidsrød O. [19] Della Valle F. [20] Soon-Shoing P. [27] Soon-Shiong P. Kinetic studies have also revealed that for acid gels rich in G residues. Smidsrød O. [9] Smidsrød O. Sandford P. [13] Soon-Shiong P. Smidsrød O. In: Wijffels RH. Generally. Smidsrød O.24:726.-R. Basics and Applications Amsterdam: Elsevier Science. Carbohydr ˚ Polym 1989. Moloney MK. Yao Z. Lancet 1994. Acta Chem Scand 1972. In: Food Colloids — Proteins. . Transplant Proc 1991. Tramper J. [28] Lim F. Lanza RP. Doseth B. Smidsrød O. Sweden. Carbohydr ˚ Polym 1994. Mol Microbiol 1995. ˚ Heintz R. Valla S. Yao Q. swelling potential and leakage of dissolved alginate material increases with increasing content of mannuronate residues. ˚ ˚ Smidsrød O.25:31. Carbohydr Res 1979. [6] Smidsrød O. 1990:53. Hals IK. Carbohydr Polym 1993. Heintz R. ˚ rial leaching out from the acid gels with the same data for the whole alginate. [14] Stokke BT. [25] Skjak-Bræk G. by polysaccharide engineering. 279 – 293. Biopolymers 1995. Larsen B. By being able to obtain and systematize detailed information all the way from the genetics and biosynthesis to physical characteristics and technological properties. Heintz R. [16] Skjak-Bræk G. Yao QX. These dependencies become less pronounced as the content of M residues increases. ˚ Smidsrød O.17:1031. Feldman E. The importance of long guluronic acid blocks in the stabilization of acid gels is therefore also shown by such experiments. Grasdalen H.343:950. both molecular weight and bead particle size seem to be of major importance [11].8:330.90:5843. Painter T.10:31. [8] Kohn R. References [1] Grasdalen H. Skjak-Bræk G. Smidsrød O.56:C11. Carbohydr Polym 1996.0. Murphy M. Larsen B. Espevik T. Smidsrød O. Haug A. Cambridge. 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. Science [29] Thu B. Food Gels. 3. Sandford P. Biomaterials 1996. it is obvious that there is a substantial demand for tailor-made alginates. England. [11] Draget KI.16:719. Carbohydrates Europe ˚ 1996. Skjak-Bræk G. [3] Penman A. Larsen B. Skjak-Bræk G. it should be possible to tailor make DDS systems with distinct medical release profiles. Elsevier: Essex. (Eds.57:263. Otterlei M. Smidsrød O. [22] Smidsrød O. Hirsch M. Larsen B. Draget et al. Smidsrød O. Larsen B. European Patent Applied 87401464. Doseth B. Skjak-Bræk G.I. PCT US 927/09364. Yao Z. Bruheim P. [18] Ertesvag H. Merideth N. Based on these results. [15] Skjak-Bræk G. Heinz RE. and the application potential increases. Schmehl M.26:79. Skjak-Bræk G. Skjak-Bræk G. [5] Larsen B.

Draget et al.17:2628. [42] Leo WJ. Myhre S.31:79. Larsen B.I. Acta Chem Scand 1963.17:1466. Smidsrød O.17:1653. Smidsrød O. [41] Smidsrød O. Larsen B. [35] Draget KI. Elgsæter A. Acta Chem Scand 1963. ˚ [31] Martinsen A. Smidsrød O.132:552. [36] Haug A. Carbohydr Res 1967. Larsen B. Storrø I.21:2859. Østgaard K. Haug A.39:186. US Pat. . Macro˚ molecules 1993. Larsen B.K. Larsen B.26:3589. Biotechnol Prog 1990. 1992. Smidsrød O. [40] Smidsrød O. Haug A. Smidsrød O. Acta Chem Scand 1963. 55 [37] Haug A. McLoughlin AJ. Bruheim P. Skjak-Bræk G. Skjak-Bræk G. Haug A. Moe ST. Acta Chem Scand 1963. Malone DM. Espevik T. [32] Skjak-Bræk G. J Plant ˚ Physiol 1988.17:1473.5:482. [39] Smidsrød O. 5144016. / International Journal of Biological Macromolecules 21 (1997) 47–55 [30] Thu B.6:51. Soon-Shiong P. Skjak-Bræk G. Biomaterials 1996. ˚ [33] Moe ST. Østgaard K.17:1069. [38] Haug A. Skjak-Bræk G. Larsen B. [34] Draget KI. Acta Chem Scand 1967. Appl Microbiol Biotechnol 1989. . Biotechnol Bioeng ˚ 1992.