Smart systems based on polysaccharides

5 Smart systems based on polysaccharides M. N. G U P T A and S. R A G H AVA, Indian Institute of Technology Delhi, India

5.1

What are smart materials?

5.1.1

Smart water soluble polymers and smart hydrogels

Responding to changes in their external environment is a hallmark of living systems. On a larger time scale, evolution is believed to be adapting to the (changing) environment. On a shorter time scale, the design of living systems ensure that they not only cope up but adequately deal with what is happening around them (Vincent, 2000; Jeong and Gutowska, 2002). This is true at all levels of organization: species → organism → organ → tissues → cells → molecules. Biological molecules like proteins are considered very smart machines. This is reflected in their regulatory features such as allosteric regulation (Urry, 1993; Alberts et al., 1994). In recent years, two kinds of smart materials (Table 5.1) have attracted great attention: smart water soluble polymers and smart hydrogels. The former can change their solubility in a medium in response to one or more of a stimulus/stimuli (Table 5.2, Figure 5.1) (Roy and Gupta, 2003). The nature of these stimuli can vary: changes in pH, temperature, presence of a chemical species are the most common stimuli. Some other stimuli that have been used in this context are: electric field, solvents, light, and pressure (Kim and Table 5.1 Kinds of smart materials Smart polymeric materials

Nature of stimulus

Reference

Chitosan Alginates Carrageenans Methylcellulose Gellan Xyloglucan

pH Ion, pH Ion Temperature Ion Temperature

BeMiller (1965) Smidsrød and Draget (1997) Van de Velde and Ruiter (2002) Haque and Morris (1993) Masteiková et al. (2003) Kumar et al. (2002)

129

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Natural-based polymers for biomedical applications Table 5.2 Types of stimuli Smart polymer

Insoluble

Soluble

Chitosan

> pH 6.5

< pH 5.5

Alginate

Ca2+ < pH 2.0

EDTA > pH 2.0

Carrageenan

K+

Water

+ Stimulus Water molecules – Stimulus

Swollen polymer

Collapsed polymer

5.1 Smart polymeric material.

Park, 2002). These polymers are also called reversibly soluble-insoluble polymers. The second kind, smart hydrogels, change their shape/volume in response to similar stimuli (as used in the case of smart polymers) (Peppas, 1995; Hoffman, 2002). These changes are accompanied by uptake/release of large amounts of solvent. Such smart polymeric materials can be fashioned out of naturally occurring sources or can be synthesized using normal chemistry which is used for synthesizing polymers (Roy et al., 2004).

5.1.2

Polysaccharides as smart materials

Among the naturally occurring polymers which can be used as such as smart polymers or can be turned into smart hydrogels, polysaccharides constitute the most common and important molecules (Cascone et al., 2001). This chapter discusses some of the important polysaccharides. In each case, the ways to obtain these carbohydrates and the structural basis for smartness (with the nature of stimulus/stimuli identified) are briefly discussed. This is followed by a discussion on their various applications in the context of their smart behaviour. It is also brought out that the smartness is a seamless feature that runs through the various formats in which such materials are used. Such formats include tablets, films, microspheres and nanoparticles.

Smart systems based on polysaccharides

5.2

Chitin and chitosan

5.2.1

Natural occurrence and purification

131

Chitin is a naturally occurring polyaminosaccharide. It occurs in the shells of crustaceans, exoskeletons of insects and cell walls of fungi. It is synthesized (and degraded) in the biosphere at the rate of >10 gt/yr which makes it an important renewable biomass. Commercially, wastes from the seafood processing industry constitute the source for chitin. Chitinases occur fairly widely and account for the biodegradable nature of chitin (Cosio et al., 1982). Alkaline N-deacetylation of chitin produces chitosan, which consists of ≥ 80–85% free amino groups. Chitin degradation in nature is quite slow. An estimate in 1999 showed that shell fish processing discards constitute 50– 90% of the total solid waste landing in USA. At the global scale, the estimate of this type of waste was 5.118 × 106 Mt/y. Shrimp and crab shell waste constitute the most widely used source for isolation and purification of chitin (Shahidi et al., 1999). It is also isolated from fungal mycelia. The purification protocol of chitin from seafood waste follows the sequence of steps shown in Figure 5.2. Chitin subjected to 40–45% NaOH deacylates and produces chitosan. The deacylation degree can vary but in order to produce soluble chitosan (at low pH), about 80-85% deacetylation is necessary. It may be noted that some deacetylation happens during extraction of chitin itself so any chitin would have some limited degree of deacetylated amino groups. Chitosan can be purified by solubilizing in acids followed by filtration. Spray drying of the filtrate produces the chitosan powder. Ku
era (2004) has described a method for obtaining crosslinked chitosan directly from fungal mycelium. Of all the commercially produced polysaccharides (e.g. cellulose, dextran, pectin, alginate, agar, agarose, starch, carrageenans and heparin), chitosan is the only basic polysaccharide. Both chitin and chitosan are nontoxic with LD50 of chitosan being 16g/kg body weight (similar to salt or sugar!). Chitosan can be sterilized by any of the sterilization methods without affecting even its physical properties (Singh and Ray, 2000). The reactivity of free –NH2 group, nontoxic nature, biodegradability and sterilizability has resulted in numerous applications of chitosan in a variety of areas. Of the two, only Waste

dil NaOH

Deproteinization

dil HCl

Chitin

5.2 Protocol for purification of chitin.

Demineralization

Decolorization

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chitosan shows smartness as a pH-sensitive polymer and hence this chapter will focus more on applications of chitosan based materials.

5.2.2

Structure

Chitin is constructed from units of N-acetyl-D-glucose-2-amine. These are linked together in β(1→4) fashion (in a similar manner to the glucose units which form cellulose) (see Figure 5.3a). In effect chitin may be described as cellulose with one hydroxyl group on each monomer replaced by an acetylamine group. This allows for increased hydrogen bonding between adjacent polymers, giving the polymer increased strength. Chitin does not dissolve in water. Chitosan is obtained by means of alkaline N-deacetylation of chitin (see Figure 5.3b). This is done by removing acetyl groups from some of the Nacetyl glucosamine residues, leaving exposed amine groups capable of attaining positive charges in aqueous solutions at low pH; hence, chitosan can be dissolved at low pH. This active amine group provides many unique chemical and physical properties to the chitosan polymer.

5.2.3

Smart behavior of chitosan

Chitosan contains free amino groups with pKa ≈ 6.5. Hence at pH < 6.5, chitosan chains carry enough positive charge. This positive charge makes O

O

CCH3 NH O

H

CCH3 CH2OH

H OH H

O

O

H O

CH2OH

O

OH

H

H

NH

NH

H

H

OH

O

H

O

n

CH2OH

CCH3 O (a) NH2 O

H

CH2OH

H

O

OH H

H O

O CH2OH

O

OH

H

H

NH2 (b)

5.3 (a) Structure of chitin; (b) chitosan.

NH2

H

H

OH

O

H CH2OH

O

n

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chitosan a cationic polyelectrolyte, which is soluble in water (and in dilute solutions of many organic acids such as formic acid, acetic acid, tartaric acid, citric acid). If the pH of an aqueous solution of chitosan is raised to above 7.5, the polymer precipitates as all the amino groups have ionized (–NH 3+ → –NH 2 ) and the polymer carries no charge. This process, being a simple ionization is reversible and makes chitosan a reversibly solubleinsoluble polymer or a pH responsive smart polymer (Terbojevich and Muzzarelli, 2000). Many methods of preparing chitosan hydrogels have been described (Draget et al., 1992). Such gels include chitosan-oxalate gels, chitosan-naphthalene sulphuric acid gels and gels prepared by crosslinking chitosan with Mo (+6). In most of these cases, unfortunately the chemistry of preparation is less than clear. The chitosan-Mo gels were found to swell nine times when placed in distilled water, the swelling capacity decreasing to 0 in 100 mM sodium chloride solution.

5.2.4

Applications

Chitin and chitosan constitute one of the most widely studied polymers from an application point of view. The application areas include waste water treatment, food industry, agriculture, paper and pulp industry, cosmetics, medicine, tissue engineering, bioseparation and biocatalysis (Dutta, 2005). It is not possible to cover all these applications here. The following overview of application focuses mostly on those applications which exploit the smart behavior of chitosan. Applications in enzymology One of the early applications of the smart behavior of chitosan is in the area of bioseparation of a lectin from wheat germ (Senstad and Mattiasson, 1989). Lectins are proteins of nonimmune origin that recognize free carbohydrate, or as part of glycoconjugates, in a specific fashion. This property makes these molecules as excellent tools in biology (Liener et al., 1986; Van Damme et al., 1997). The lectin from wheat germ is specific for N-acetylglucosamine. When chitosan solution was added to a crude homogenate of wheat germ, the polymer selectively complexed with the lectin. The ‘affinity complex’ could be precipitated by raising the pH and the lectin recovered after dissociation from the complex. This approach, called affinity precipitation, is a powerful tool in downstream processing of proteins/ enzymes (Gupta and Mattiasson, 1994). More details of this and other bioseparation techniques mentioned here can be found in Chapter 2. Subsequently, the similar approach was followed for purification of lectin from tomato and potato as these lectins also have similar specificity (Tyagi et al., 1996). The same principle was

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extended to developing an interesting version of a bioseparation technique called aqueous two-phase affinity extraction (Walter and Johansson, 1994; Hatti-Kaul, 2000). The technique exploits partition of a protein in a twophase polymer/polymer or polymer/salt system. PEG/salt is a frequently used two-phase system. One of the key constraints has been that it is difficult to separate partitioned protein from PEG. Incorporation of chitosan in the PEG phase, not only enhances the partition of protein (having binding affinity towards chitosan), affinity precipitation of the affinity complex from the PEG phase leaves the latter free for reuse (Teotia and Gupta, 2001a, b). In both approaches, the application of chitosan as a smart polymer can be extended beyond proteins which recognize chitosan (Mondal and Gupta, 2006). Apart from free –NH2 group, chitosan also has numerous hydroxyl groups. These two functionalities are valuable for linking any affinity ligand to chitosan. As the density of these affinity ligands on the polymer can be controlled and generally is not very high, such conjugation does not abolish the smart behavior of chitosan. It is possible that the pH of phase transition may change somewhat. The macro-(affinity ligand) so synthesized can be used either in affinity precipitation or aqueous two-phase extraction. As larger numbers of affinity ligands are available (Gupta, 2002), this creates a vast opportunity for chitosan in the area of bioseparation. Today, powerful technologies exist by which peptide libraries can be created for obtaining an affinity ligand for practically any enzyme/protein (Mondal and Gupta, 2006). This creates unlimited scope for chitosan (and similar materials) to be used in bioseparation. The smart behavior of chitosan can also be used to design smart biocatalysts. Enzymes as biocatalysts are superior to chemical catalysts as these proteins can act at normal temperature and pressure and show high specificity. It is now also known that apart from aqueous milieu, enzymes can also function in neat solvents, reverse micelles and gaseous phase (Gupta, 1992; Gupta, 2000). One factor, which has limited their application, has been cost. Immobilization is a well-established technique for converting enzymes into reusable catalysts and consists of adsorbing, entrapping, encapsulating or covalently linking enzymes to polymeric matrices (Cao, 2005; Guissan, 2006). Conventionally, these polymeric matrices are insoluble materials like agarose or polyacrylamide. The concept works well except that as most of the enzyme molecules are within the polymeric network, the ‘mass transfer limitation’ is especially severe for macromolecular substrates. Considering that most of the biomass is macromolecular in nature, immobilized enzymes have not shown good performance in the area of biomass conversion. When the biomass is insoluble like lignocellulosic material, this conventional heterogeneous biocatalyst design is not much use. Smart polymers like chitosan as watersoluble matrices for enzyme immobilization provide an interesting option in the biocatalyst design. An enzyme linked to chitosan can operate at pH

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below 6.3 as a soluble (homogeneous) biocatalyst. After the reaction is over, the biocatalyst can be recovered by raising the pH and reuse can be evaluated (Roy et al., 2004). It is interesting to note that while both chitosan and chitin based insoluble matrices have been extensively used for enzyme immobilization (Krajewska, 2004), there is only one application of using chitosan for designing a smart biocatalyst. Laccase from Coriolopsis gallica was linked with chitosan via carbodiimide coupling. The conjugate showed reversible soluble-insoluble behavior. The immobilized enzyme had enhanced stability at both pH 1 and pH 13. This successful design should encourage use of chitosan as a smart matrix for obtaining smart biocatalysts for hydrolysis of macromolecular substrates. Pharmaceutical, biomedical and miscellaneous applications of chitosan Chitosan forms gels at low pH range and is reported to have antacid and antiulcer activities in the stomach. Both physical gels and chemically crosslinked gels are degraded by lysozyme and this allows the design of enzyme degradable hydrogels for drug delivery purpose. Chitosan malate granules as carriers have been reported to work well for sustained release effects for drugs. As these granules do not dissolve at the acidic pH of the stomach this is a cost-effective way of prolonging residence time of drugs in the stomach since drugs are shielded from deactivating enzymes and the acidic pH (Henriksen et al., 1993). Numerous studies related to this application have been reported (Singh and Ray, 2000). In case of injured tissues, chitosan and its derivatives help blood coagulation and accelerate wound healing. Chitosan implants in the cornea are reported to encourage neovascularization (Singh and Ray, 2000). Chitooligosaccharides and chitosan lactate have been shown to be useful in replacing other chemical preservatives for processed food materials. Chitosan films have been used as food wraps (Shahidi et al., 1999). Extended shelf life has been reported by the use of chitosan films in the case of fruits, vegetables and fish. The cationic nature of chitosan at pH 4.5 results in its complexing (and precipitating) milk fat globule fragments. This constitutes an industrially viable process for removing fat from whey (Shahidi et al., 1999). Chitosan as a food additive is reported to possess antioxidative and hypocholesterolemic effects (Shahidi et al., 1999). The interaction of this positively charged polymer with negatively charged skin and hair forms the basis of its usefulness in skin care and health care products. For the former application, it is also used as a matrix for minerals, liposomes, fragrances and pigments. Its films or gels on its own and with cross-links with anions have moisture retaining capacity which is valuable in skin care applications.

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Later discussion on composite materials will discuss how the stimuliresponsiveness of chitosan as a part of composite materials leads to some further very interesting applications.

5.3

Alginates

5.3.1

Natural occurrence and purification

Alginic acid occurs as the main cell wall constituent of brown macro algae in the form of mixed salts of Na+, Mg2+, and Ca2+ ions. Apart from these sea weeds, alginates are produced by the microorganism Azotobacter vinelandii and some Pseudomonas strains. Commercially available alginates are mostly isolated from Laminaria hyperborea, Macrocystis pyrifera and Ascophyllum nodosum. Some other minor sources are Laminaria digitate, Laminaria japonica, Eclonia maxima, Lesonia negrescens and Sargassum sp. (Smidsrød and Skjåk-Bræk, 1990). The soluble alginic acid is extracted from algae with 0.1–0.2 N mineral acid. Mechanical treatment of the suspension is necessary to facilitate diffusion of alginic acid out of the algal mass. This step removes other salts and polymers. Sodium alginate is obtained by neutralization with sodium hydroxide. The alginate is precipitated by the addition of CaCl2 or ethanol (Smidsrød and Draget, 1997). Polyphenols are present in most of the alginate preparations. While these contaminants cannot be removed completely, some of the applications for alginate require that their level is brought down to less than a few percent. Bleaching with H2O2 and NaClO2, repeated precipitation with ethanol or acetone and treatment with activated carbon or polyvinylpyrrolidone help in removal of polyphenols. Their presence can be evaluated by fluorescence spectroscopy (Skjåk-Bræk et al., 1989). Samples with different chain length of the polymer can be prepared by ultrasonication (Martinsen et al., 1989). The total worldwide production of alginates has been estimated to be around 30 000 Mtons per year. The algae are mostly harvested from cold and temperate waters of North Europe, South American west coast, Southern Australia, Japan, and China (Smidsrød and Draget, 1997). Alginates show polydispersity with respect to average molecular weights which are generally in the range of 50–500 kDa.

5.3.2

Structure

Alginates are linear unbranched polymers containing β (1→4) linked Dmannuronic acid (M) and α (1→4) linked L-guluronic acid (G) residues (see Figure 5.4). These monomers occur in the alginate molecule as regions made up exclusively of one unit or the other, referred to as M blocks or G blocks, or as regions in which the monomers approximate an alternating sequence.

Smart systems based on polysaccharides COOH C

O

O

H

H

C

C

OH C

OH

OH

C

C

H

H

H

COOH C H

H C

H

C H

C H

O H

C O

O

H

OH

COOH

137

C O

C OH

OH

C

C

H

H

H

5.4 Structure of alginate.

The NMR demonstrated that ring conformations were 4C1 for mannuronic acid and 1C4 for guluronic acid. The D-mannuronic acid exists in the 1C conformation and in the alginate polymer is connected in the β-configuration through the 1- and 4- positions. The L-guluronic acid has the 1C conformation and is α (1→ 4) linked in the polymer. Because of the particular shapes of the monomers and their modes of linkage in the polymer, the geometries of the G block regions, M block regions, and alternating regions are substantially different. Much of the early work (in the 1960s) on alginates and their applications should be credited to the Norwegian Institute of Seaweed Research. Schematically, a typical alginate would look like: M-M-M-M-M……..M-G-M-G-M-G……..G-G-G-G-G…….. M block MG block G block It was found that the ratio of total M/total G is different in different species. Ascophyllum nodosum alginate has M/G = 2.7 whereas alginate from Laminaria hyperborea shows the extreme of M/G = 0.6. Interestingly, young tissues are rich in M blocks and the percentage of G blocks increases as tissue grows older. The bacterial alginates show the presence of O-acetyl groups. Interestingly, A. vinelandii initially produces poly M and extracellular enzyme mannuronan C-5 epimerase converts some M into epimer C-5 guluronic acid; O-acetyl groups wherever present inhibit epimerization. It has been shown that algal alginate’s composition can also be modified by this epimerase (Smidsrød and Draget, 1997).

5.3.3

Smart behavior of alginates

The pKa values for –COOH groups in M and G are 3.38 and 3.65, respectively. This results in precipitation of the soluble polymer below pH 2. However, most of the applications of alginate arise from the fact that it forms insoluble gels/precipitates with divalent metal ions, especially Ca2+. The affinity order for alginates is (Smidsrød and Skjåk-Bræk, 1990):

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Natural-based polymers for biomedical applications

Pb2+ > Cu2+ > Cd2+ > Ba2+ > Sr2+ > Ca2+ > Co2+ = Ni2+ = Zn2+ > Mn2+ Some multivalent ions like Ti3+ and Al3+ stabilize Ca2+-alginate gels. Running a sodium alginate solution as drops into a CaCl2 solution gives rise to fairly spherical beads of Ca-alginate. If another species like a drug, protein or cell is added to the sodium alginate solution, the species is entrapped in Caalginate beads. This has been exploited in a large number of applications related to drug release systems and whole cell immobilization (Smidsrød and Skjåk-Bræk, 1990). As chelators like EDTA can remove Ca2+ easily, alginates can be considered as Ca2+-responsive polymers. Common buffers like phosphate or citrate also chelate Ca2+. As Na and Mg alginates are soluble, these ions are called antigelling ions. It is necessary to keep Na+:Ca2+ ratio below 25:1 for G-rich and below 3:1 for M-rich alginates. High G-alginates result in Ca-alginate beads which are more porous, have higher mechanical stability and greater tolerance to salts and chelating compounds. These beads also show minimum volume change on swelling-deswelling (drying and resuspension in aqueous solutions) as compared to beads made from low G-alginates (Smidsrød and Skjåk-Bræk, 1990; Smidsrød and Draget, 1997). A good discussion on the fine structure of alginate gels is given in an excellent overview by Smidsrød and Draget (1997).

5.3.4

Applications

Alginate is a nontoxic biocompatible polymer and food grade alginate preparations are easily available. Thus, it is not surprising that this polymer is also used widely for numerous applications. In food materials, most of the uses of alginate originates in enhancing the viscosity. As a natural cold soluble hydrocolloid, it is used as a stabilizer/thickener in low fat margarines/ low fat spreads, salad mayonnaise/dressing, beverages, bakery products, ice creams, pet foods and restructured food (e.g. pimento fillings for olives!). Alginate (and its blends) are also used in jams, marmalades, textile printing, paper coating and as lubricants and binding agents in welding rod coatings (http://www.fmcbiopolymer.com/PopularProducts/FMCAlginates/Introduction/ tabid/795/Default.aspx). In biotechnology, the major applications of alginate involve its use as a material for entrapment. For whole cell immobilization, the simplicity of entrapment protocol has made Ca2+-alginate a favorite choice (Smidsrød and Skjåk-Bræk, 1990). For entrapment of other molecules of smaller size, e.g. drugs and proteins, composite materials containing alginate have been used more often (see Section 5.6 on composite materials). Let us look at the applications of alginate which directly exploit the smart nature of alginate. Alginate shows inherent selectivity in binding to quite a

Smart systems based on polysaccharides

139

few enzymes. This possibility has turned alginate into a very valuable polysaccharide in the area of bioprocessing in general and bioseparation in particular. The applications of alginate in bioseparation of proteins was reviewed recently (Jain et al., 2006). Hence, only a summary of the results will be provided. An affinity complex of the target enzyme with alginate can be precipitated by Ca2+ from the crude protein extracts. As already discussed (in Section 5.2 on chitin and chitosan), this strategy is known as affinity precipitation. The enzymes which have been purified by this simple elegant method include pectinase, lipase, α-amylase, β-amylase, pullulanase and phospholipase D (Jain et al., 2006). A recent interesting observation is that microwave pretreatment of alginate resulted in higher selectivity of the polymer towards pectinase and 20-fold purification (as compared to 10-fold purification observed by using untreated alginate). Similarly, just as described for chitosan, alginate can also be incorporated into PEG–salt two-phase systems and used for purification of enzymes by affinity partitioning. Again, the affinity complex of alginate-target enzyme can be separated by exploiting the Ca2+-responsive property of alginate. As the conjugation chemistry with alginate is already available (Draget et al., 1988), it is easy to link any affinity ligand to alginate and extend its use as a soluble affinity material for other large numbers of enzymes and proteins (for a discussion on the use of affinity based separations, see for example, Gupta, 2002). The concept of smart biocatalyst design has already been discussed in the context of chitosan. The first such design was in fact reported with alginic acid (Charles et al., 1974). The polymer was linked to lysozyme. Later on Dominguez et al. (1988) covalently coupled β-galactosidase with alginate but no details of its catalytic performance for lactose hydrolysis were unfortunately provided. One reason why alginate has not been used more for smart biocatalyst design is that charged matrices (like alginate) bind a lot of proteins and other molecules (substrates/products) nonspecifically by electrostatic interactions. Even then, considering that synthetic polymers like methacrylates have been used quite extensively in smart biocatalyst design (Roy et al., 2004), alginate in that respect may be an underexploited polymer. Pharmaceutical applications Fathy et al. (1998) have used tiaramide, a nonsteroidal antinflammatory drug (with a short half life), in alginate beads as a sustained release formulation. Pharmacokinetic parameters measured during in vivo experiments showed that high G alginate gave the best results. Earlier, Downs et al. (1992) described a slow release system for growth factors and concluded that entrapment in alginate beads constitutes an effective localized and slow release delivery system for biologically active molecules. Bodmeier et al. (1989) exploited

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Natural-based polymers for biomedical applications

the fact that Ca2+-alginate beads remained intact in 0.1 N HCl but dissolved in intestinal fluids to develop an oral formulation for delivery of micro- and nanoparticles as drugs. Alginate has been used fairly extensively in tissue engineering. Wang et al. (2003) showed that Ca-alginate is a good substrate for rat marrow cell proliferation. Yang et al. (2002) evaluated galactosylated alginate as a scaffold for hepatocyte attachment. It was shown that tissue engineered cardiac graft consisting of cardiomyocytes in alginate scaffold prevented damage after myocardial infarction in rats. The process for cardiac cell seeding and distribution in 3D alginate scaffolds has also been optimized (Dar et al., 2002). Alginate as a pseudochaperonin Alginate has been found to be a good additive for facilitating protein refolding (Mondal et al., 2006). Normally it is believed that polymers bind to hydrophobic patches in unfolded protein to prevent aggregation. The success of alginate as ‘pseudochaperonin’ indicates that polyelectrolytes may also serve the purpose by interacting with some charged residues in the unfolded protein.

5.4

Carrageenans

5.4.1

Natural occurrence and purification

Unlike land plants, marine algae produce large amounts of sulphated polysaccharides. The family of sulphated polysaccharides called carrageenans is one such class. More than 600 years ago, in the village of Carraghen situated on the south Irish Coast, flans were made by cooking the Irish moss (red seaweed species, Chondrus crispus) in milk. The use of Irish moss polysaccharides as a thickner, textile sizing and beer clarification has been mentioned (Velde and Ruiter, 2002). Commercial production began in the 1930s in USA when purified carrageenans were produced (Van de Velde et al., 2002). This family of polysaccharides is today produced from genus Chondrus, Eucheuma, Gigartina, and Iridaca of red seaweed Rhodophyceae. Purification A typical flowsheet for purification of carrageenan is shown in Figure 5.5 (http://www.fmcbiopolymer.com/PopularProducts/FMCCarrageenan/ Introduction/tabid/804/Default.aspx). The concentrated carrageenan solution is either converted into gel by running into KCl solution or precipitated by adding isopropyl alcohol.

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141

Harvest the seaweed, quickly dry it and bale it

Mechanical grinding and sieving to remove impurities (e.g. sand and salt)

Extensive washing

Hot extraction to solubilize carrageenan

Centrifugation to remove dense particles and filtration to remove small particles

Evaporation of water

5.5 Flowsheet for purification of carrageenan.

5.4.2

Structure

Carrageenan is a mixture of linear polymers of sulfated galactans which constitute cell wall material of marine red algae. Mostly, the chain consists of alternating units of 3-linked-β-D-galactopyranose (G-unit) and 4-linkedα-D-galactopyranose (D-unit) or 4-linked 3,6-anhydrogalactose (DA-unit). Other carbohydrate residues like xylose, glucose, their uronic acids and some other groups like pyruvic acid and methyl ethers (as substituents) are also present. The sulphate content is in the range of 22%–38% (w/w) (Van de Velde et al., 2002; Michel et al., 2006). Any algal extract contains a mixture of structural variants of carrageenan, the chemical structure depending upon the algal source and even the life stage of the algae and extraction procedures. The earliest investigations classified the carrageenans based on their solubility in KC1 solution as κ-carrageenan (insoluble) and λ-carrageenan (soluble). Later, through a vast amount of studies using various chemical and instrumental techniques such as alkali-treatment, methylation, partial acid hydrolysis, enzymic degradation and 13C-NMR and IR spectroscopy, this has been replaced, for the most part, by classification based on chemical structure. As a result, the carrageenans are divided into three families according to the position of sulfate groups in the 1,3- and 1,4-linked galactose residues. This classification is in terms of the nature of the repeating disaccharide made from D/DA and G units. The carrageenan preparations are called κ, ι, and λ corresponding to one, two and three sulphate groups per disaccharide (see Figure 5.6) (Michel et al., 2006). The presence of substituent groups, replacing hydroxyl groups, or other modifications of this disaccharide unit, such as anhydride ring formation, gives rise to the structural variants present in carrageenans. Therefore, carrageenans are a mixture of structurally related

142

Natural-based polymers for biomedical applications (a) Kappa (κ) CH2OH –O

CH2 O

3SO

O

H

O

H

O H

H H

H

H

O H

OH

OH

H

(b) Iota (ι) CH2OH

CH2 O

–O SO 3

O

H

O

H

O H

H

H

H

H

O OH

H

H

OSO3–

(c) Lambda (λ) CH2OSO3–

CH2OH O

O H

O

H

H

H H

H H

H

O H

OSO3–

H

OSO3–

5.6 Structure of carrageenans.

polysaccharides differing primarily in the proportions of galactose, ester sulfate (also in the position and content) and 3,6-anhydro galactose depending upon the species of carrageenophytes. κ-carrageenans are soluble in hot water, sodium ι-carrageenan is soluble in cold and hot waters and λ-carrageenan is partially soluble in cold water and completely soluble in hot water. The IUPAC names for κ, ι, and λ-carrageenan are carrageenose 4′ sulphate, carrageenose 2,4′ sulphate, and carrageenan 2,6,2′ trisulphate, respectively (Van de Velde and Ruiter, 2002). Commercially available food grade carrageenans have average molecular weight in the range of 400–600 kDa.

Smart systems based on polysaccharides

5.4.3

143

Smart behavior of carrageenan

κ-Carrageenan forms gels with helical structures in the presence of K+ ions. Other monovalent ions which induce gel formation of κ-carrageenan solutions are Rb+, Cs+ and NH +4 (van de Velde et al., 2002). Ca2+ produces more compact and brittle gels. ι-Carrageenan forms dry and elastic gels with Ca2+ whereas λ-carrageenan forms free flowing, non-gelling, viscous (pseudoplastic) solutions in water. Gels prepared with κ- and ι-carrageenan are thermoreversible. These can be melted upon heating and reset upon cooling. Increase in concentration of the respective cations increases gelling temperature. It has been shown that 0.3% κ-carrageenan at about 38°C is about >75% precipitated with 0.2% KCl. The precipitate could be dissolved in distilled water. Hence, in a way, κ-carrageenan can be considered a K+-responsive smart polymer (Roy and Gupta, 2003). Mitsumata et al. (2003) have described the pH response of complex hydrogels made up of κ-carrageenan, chitosan, and CM-cellulose. The maximum degree of swelling was observed in the range of pH 11-12.

5.4.4

Applications

It was estimated that the worldwide sale of carrageenan in 2000 was around US$310 million (Van de Velde and Ruiter, 2002). The commercial applications of carrageenan revolve around their use as gelling, thickening and stabilizing agents. Processed food products such as ice creams, whipped cream, yogurt, jellies and sauces are some illustrative examples (Van de Velde et al., 2002). There are a number of reasons which make carrageenan an ideal component in food. Traditional use for > 600 years obviously initiated these applications in the industrial society. It is regarded as a GRAS item and has FDA (USA) approval for use as a food additive. In fact, the WHO expert committee recommended that it is not necessary to specify a daily limit for carrageenans (Van de Velde et al., 2002). However, a minimum average molecular weight of 100 kDa is prescribed since cecal and colonic ulceration was reported with fragments of carrageenan (Van de Velde and Ruiter, 2002). The carrageenan binds water nicely and this helps in formulations which have to contain aqueous fluids. Although not a surfactant, it does stabilize emulsions and suspensions. At high temperature, it melts and has lower viscosity. This allows processing and good heat transfer while dealing with food systems. Below 49°C, it solidifies and forms gels. The gels are stable at room temperature. Carrageenans have a better textural, mouthfeel, flavor, and processing properties as compared to starch. Thus, they can replace starch as thickener in many food preparations. In fact, κ-carrageenan increases the viscosity of starch systems manifold. Similarly, it shows synergism with locus bean gum and konjac flour and stronger elastic gels are obtained.

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An important property of carrageenan which makes this a better hydrocolloid to be used (in food and other systems) is the way it interacts with other proteins, especially caseins, the milk proteins. The positive charges on casein micelles interact electrostatically with the negatively charged sulfate of carrageenan and leads to stable and strong gels. Chocolate milk and flans are two examples of products which are based upon this interaction. Apart from food systems it is also being used in toothpastes and air fresheners. Hand lotions, shampoos and contraceptive gels represent growing/ potential market segments for carrageenans (http://www.micchem.com/products/ Carrageenan.htm). Carrageenans have been used for immobilization of whole cells and enzymes (Van de Velde et al., 2002). As the enzymes, in general, can leak out through porous carrageenan gels, gel hardening by use of K+ (high concentration), Ca2+, Al3+, Fe2+, galactomannans or glucomannans is required for this application. Van de Velde et al. (2002) have listed the applications of enzymes immobilized in carrageenan gels for various biotransformations. In addition, some bioanalytical applications, in H2O2 determination (immobilized catalase), pesticide analysis (co-immobilized choline oxidase and choline esterase), lecithin analysis in food and drugs (co-immobilized choline oxidase and choline esterase) and monitoring the rancidity of olive oils (immobilized tyrosine), have been mentioned (Van de Velde et al., 2002). Among the applications of whole cell immobilization in κ-carrageenan are waste water treatment, asymmetric synthesis and production of vinegar, milk prefermentation and production of beer and ethanol (Van de Velde et al., 2002). Applications of carrageenan as an excipient in drug formulations and other medical applications have been covered by Van de Velde and Ruiter (2002). Thommes et al. (2007) have recently examined the effect of drying on extruded pellets in which κ-carrageenan was used as a pelletization aid. It was found that heating above 80°C decreased the disintegration time. This has implications in the context of the drug release properties of κ-carrageenan pellets. More importantly, these authors suspect fragmentation of κ-carrageenan. In view of carrageenan fragments being not acceptable by WHO (as already mentioned), this is of serious concern and needs to be investigated carefully. The smart nature of κ-carrageenan as a polymer, has been exploited for bioseparation of pullulanase (Roy and Gupta, 2003) and yeast alcohol dehydrogenase (Mondal et al., 2003). In both cases, affinity precipitation (Roy and Gupta, 2002) was used as the bioseparation technique. The precipitation of κ-carrageenan was carried out by K+ addition. While the polysaccharide as such showed the selective affinity for pullulanase, in the other case, κ-carrageenan was used as a smart carrier for the dye cibacron blue which functioned as an affinity ligand. As other dyes in particular and affinity ligands in general can be linked to κ-carrageenan in a similar way, the strategy can be extended for purifying other enzymes as well.

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145

Other miscellaneous smart polysaccharides and their applications

While the three polysaccharides discussed previously have been more extensively used, some other polysaccharides (though used less frequently) have also been used for some interesting applications. Colon-specific drug release systems exploit the change in the pH along the gastrointestinal tract between 2 (stomach) and 10 (colon). Aguilar et al. (2007) mention the use of several polysaccharides (amylose, guar gum, pectin, inulin, chondroitin sulphate, dextran and locust bean gum) in designing colon-specific drug release systems. Zhang et al. (2007) have recently described a dextran based antigen/ antibody hydrogel. The presence of free antigen affected the antigen/antibody internal interactions and resulted in changes in the permeability of solutes through the membrane. However, it may be noted that it is not dextran from which the smart behavior originated, it was the smartness of the well known biological affinity pair of antigen/antibody. Themoreversibility of xyloglucan gels have been exploited in quite a few cases for obtaining drug release systems. This polysaccharide is obtained from tamarind seeds. It consists of a [1→4]-β-D-glucan backbone with [1→6]-α-D-xylose branches partially substituted by [1→2]-β-D-galactoxylose. Treatment of naturally occurring xyloglucan by β-galactosidase gives a thermally reversible xyloglucan gel whose sol-gel temperature can be varied by varying degree of hydrolysis. It is believed that xyloglycan gels may be useful for rectal and intraperitoneal drug delivery. Their usefulness in oral drug delivery has also been explored (Kumar et al., 2002). Gellan gum (produced by Pseudomonas elodea) is a linear anionic polymer of a repeating tetrasaccharide unit of glucose (two units), glucuronic acid and rhamnose. In the native state, some of the glucoses are acylated with acetyl and L-glyceryl groups. The viscosity of gellan gum dispersions is dependent upon pH, temperature, and the presence of cations. The gum is used in the food industry, as a growth media for bacteria and in plant tissue culture. Again, it has been used for designing sustained release systems for drugs (Kumar et al., 2002). Vigo (1998) has viewed the variety of structures which could be created by interacting cellulose with other polymers. A recent work shows that methylcellulose is an effective thermosensitive flocculant (Franks, 2005). Zohuriaan-Mehr et al. (2006) have described a hybrid hydrogel of gum arabic-acrylate which showed swelling-deswelling response to pH, salinity, Ca2+ and organic solvents. Garner et al. (1999) have described a polypyrrole-heparin composite in which exposed heparin could be varied by either application of negative potentials or by exposure to an aqueous reductant. While there is no ambiguity about what constitutes a smart material, it is

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sometime possible to confer smartness on a seemingly nonsmart material. For example, a cyclodextrin microgel was found to show a pH-dependent host-guest inclusion effect for a dye (Liu et al., 2004). Considering that cyclodextrins are already exploited extensively, this creates another dimension which will further their usefulness in many areas.

5.6

Polysaccharide-based composite materials

The previous discussion has focused on polysaccharide materials. In material science, it is not uncommon to blend, complex, copolymerize different materials to improve upon the desirable property of a polymeric material. In the area of smart materials also, many composite materials have been obtained from different polymeric materials. This section focuses on such materials wherein at least one component is a polysaccharide.

5.6.1

Examples

Many stimuli-responsive hydrogels have been prepared by combining polysaccharides (chitosan, alginate, cellulose and dextran) with thermoresponsive materials. The areas of potential application for such composite materials include drug delivery, tissue engineering and wound healing. In some cases where both components are smart, the composites show dual stimuli-responsive behavior. The preparative strategies used for obtaining such composite materials include graft copolymerization, blending, formation of polyelectrolyte complexes and core-shell type polymerization. A recent review (Prabaharan and Mano, 2006) deals with these approaches quite well and describes some of the composite materials. A non-toxic and biocompatible material was obtained by grafting poly(Nisopropyl acrylamide (NIPAAm)) monomer onto chitosan using ceric ammonium nitrate as the initiator. The copolymer had a lower critical solution temperature (LCST) of 32°C with a swelling ratio higher at pH 4 than at pH 7. The pH dependent swelling behavior was more noticeable at 25°C than at 32°C (Chung et al., 2005). Also, chitosan-g-pNIPAAm particles prepared by emulsion copolymerization have been reported. Again, the particles displayed dual stimuli-response as far as swelling behavior was concerned. A semiinterpenetrated network was obtained by the free radical polymerization of NIPAAm in the presence of chitosan by using tetraethyleneglycoldiacrylate as the crosslinking agent. The resulting material showed a dramatic response (in terms of degree of swelling) to pH. Response behaviors of such a semiinterpenetrated network and corresponding full-interpenetrated network have been found to be very different (Verestiue et al., 2004). A limiting factor for the use of pNIPAAm hydrogels in their applications in the areas of sensors, actuators and chemical valves has been the slow

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deswelling rate of pNIPAAm hydrogels. Semi-IPN hydrogels prepared from linear alginate and cross-linked pNIPAAm have shown better response rates. The cellulose-reinforced hydrogels showed the interesting property of pore size control with temperature. The pNIPAAm grafted to dextran formed micelles (spheres with mean diameter of < 30 nm) in aqueous media which, in principle, can be used for drug delivery of lipophilic drugs. Graft copolymers combining mostly pNIPAAm with other polysaccharides have also been prepared by radiation (e.g. UV, γ-irradiation) based methods and condensation reactions. Worth mentioning are the comb-type graft hydrogels obtained from alginate and pNIPAAm. These macroporous hydrogels showed rapid swelling/deswelling response to changes in both pH and temperature. Physically or chemically cross-linked polymeric blend based hydrogels have also been described. An interesting example is that of porous hydrogels with cell attachment and growth sites. The IPN hydrogels prepared from Ca2+-alginate and pNIPAAm showed different pore morphologies depending upon the temperature. The porous hydrogels became nonporous beyond their LCST temperature. The mechanical strength of these hydrogels also increased dramatically in their more compact form beyond their LCST. While glutaraldehyde has been more frequently used for obtaining chemically crosslinked blends (such as chitosan/pNIPAAm blends), genipin has also been used as a crosslinker for obtaining a blend of chitosan and poly(vinyl pyrrolidine) (PVP). Low pH and high temperature led to greater swelling which was also enhanced as PVP content was increased (Khurma et al., 2005). Dual sensitive polyelectrolyte complexes (PEC) have been prepared by combining cationic chitosan and anionic alginate with polymers carrying opposite charges. PECs are reported to have applications as membranes, antistatic coatings, sensors and medical prosthetic materials (Etienne et al., 2005; Casalbore-Miceli et al., 2006; Vasiliu et al., 2005). Core-shell type copolymers constitute a highly complex design in composite materials with the attractive property that the responsiveness is tunable (Prabaharan and Mano, 2006). Smart microgels with a thermoresponsive core with pH sensitive shells made up of pNIPAAm and chitosan have been described. Similarly, composites with a cross-linked copolymer of NIPAAm and chitosan as core and acrylate copolymers as shells have also been described. The main focus of studies on such microgels has been, of course, on studying their swelling/deswelling behavior at various pH values. The main application of these composite materials has been to design drug delivery systems which release drugs in a controlled fashion at a specific site. The biocompatible nature of chitosan and alginate has resulted in their being components of many composites synthesized for this application. A PEG-g-chitosan preparation which was an injectable liquid at low temperature but turned into a semisolid gel at body temperature showed linear release of BSA up to 70 hours (Bhattarai et al., 2005). A thermosensitive hydrogel

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made up of chitosan and β-glycerophosphate was found to work well as a site directed, injectable and controlled-release (over a 1 month period) system in a preclinical trial for paclitaxal delivery to localized solid tumors (Prabaharan and Mano, 2006). A number of studies have been reported with composites of alginate and pNIPAAm. In addition to response to the presence of Ca2+, the effect of pH on ionization of carboxyl groups of alginate was also exploited in such designs. The temperature determined the drug release rate and this dependence on the temperature itself could be varied by changing the pH. Some of the other thermoresponsive composites which showed promising results as drug delivery materials are ethylcellulose/pNIPAAm microcapsules and NIPAAm grafted on dextran methacrylate (Ichikawa and Fukumori, 2000; Huang and Lowe, 2005). One of the challenges in tissue engineering is to find a material which could serve as a cell culture carrier and allow harvesting in the case of highly adhesive mesenchymal stem cells. Chitosan-g-pNIPAAm has turned out to be a useful material; the cells could be harvested simply by lowering the temperature. The injectable composite material served as a good scaffold for chondrogenic differentiation of human stem cells (Prabaharan and Mano, 2006). Non-woven fabrics made of thermosensitive composites based upon chitosan also show promise as wound healing dressing materials. Such materials, interestingly, showed higher bacteriostatic property as compared to chitosan (Prabaharan and Mano, 2006). Various starch-based composites have been described in the literature (Marques et al., 2002). An extensive list of work on starch-based composites by the group of Prof. Reis can be accessed at http://www.3bs.uminho.pt. The target applications are in drug delivery (Malafaya et al., 2001) and tissue engineering (Gomes et al., 2001, Salgado et al., 2002). Such composite materials also include starch-chitosan hydrogels (Baran et al., 2004). The starch-based thermoplastic hydrogels used as bone cements and drug delivery carriers may also be mentioned here (Pereira et al., 1998). Finkenstadt (2005) has reviewed the applications of polysaccharides in designing biosensors, environmentally sensitive membranes and components in high-energy batteries. These applications are based upon their electroactive nature which is exploited by using them for doping, blending or grafting into other materials. While direct exploitation of smart behavior is not yet seen, it may turn out to be an asset. For example, electroactive polypyrroles required a negatively charged counterion hyaluronic acid to exhibit full conductivity. The composite laminate showed sharper responses in terms of cell compatibility, nontoxicity and increased vascularization as compared to the material without hyaluronic acid. Considering the intended application for tissue engineering, it may be possible to build-in specific cell responses toward stimuli.

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Cascone and Maltinti (1999) have evaluated blends of poly(vinyl alcohol) (PVA) with chitosan or dextran as drug delivery systems for growth hormone. The hormone helps in wound healing and tissue repair. The blended hydrogels were superior to pure PVA hydrogels with respect to release of PVA as such over a period of time. They are also less expensive than similar blends of PVA with collagen and hyaluronic acid which have been described earlier (Giusti et al., 1993; Guerra et al., 1994). It was found that either chitosan or dextran content controlled the hormone release. The dextran containing hydrogels reached the swelling equilibrium faster than chitosan containing blends. This has implication for drug release kinetics (Cascone and Maltinti, 1999). The initial step of water uptake is followed by the drug release step. Hence, the GH release clearly shows two-step kinetics in the case of chitosanPVA hydrogel whereas GH release appears as a single phase process for dextran-PVA hydrogel (Cascone and Maltinti, 1999). The same group, more recently, evaluated hydrogel blends of PVA with hyaluronic acid, dextran and gelatin as potential tissue engineering scaffolds (Cascone et al., 2004). PVA has been a material of choice as PVA hydrogels have water contents similar to those of natural tissues. Besides, PVA hydrogels are biocompatible, sterilizable and easy to mould into a desired shape. The blending was aimed at improving mechanical stability and biocompatibility. It was found that hydrogels containing dextran/ PVA in the ratio of 40:60 showed the highest porosity among all the blends tested. Overall, the blends showed the desired porosity for fibroblast growth. Whether these porosities will actually translate into support for cell adhesion and proliferation needs to be tested. Zhang et al. (2004) have synthesized dextran-maleic anhydride/pNIPAAm smart hybrid gels by photocrosslinking. FT-IR, DSC, swelling kinetics showed that the composite hydrogels were responsive to both temperature and pH. The LCST could be adjusted by changing the ratio of the two components during synthesis. Blending with dextran made these composite gels partially biodegradable. Finally, the work of Kaffashi et al. (2005), while preliminary in nature, illustrates the possibility of blending naturally occurring gums with more well defined polymer. These workers blended gelatin with tragacanth gum. This gum, isolated from the Astragalus plant consists of polygalacturonic acids. The smartness of the hydrogel was not investigated but at a conceptual level, this raises several interesting possibilities as a variety of plant gums have been described in the literature (Aspinall, 1969; Verbeken et al., 2003). Some more examples of composite materials based upon polysaccharides are shown in Table 5.3.

5.7

Future trends

Currently, materials based upon smart polysaccharides are extensively used in the food industry and to a lesser extent in some other industries. In

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Table 5.3 Some more examples of composite materials based upon polysaccharides Composite

Application

Reference

Chitin and chitosan based materials Poly-l-lysine coated covalently on chitosan beads

Adsorption of bilirubin

Chandy and Sharma (1992)

DNA-chitosan complexes

Removal/ concentration of carcinogenic heterocyclic amines

Hayatsu et al. (1997)

Chitosan conjugated magnetite

Recovery of recombinant E. coli

Honda et al. (1999)

Chitosan-sialic acid branched polysaccharides

Soluble hybrids Bound lectins

Sashiwa et al. (2000)

Chitosan-magnetite aggregates containing Nitrosomonas europaea cells

Ammonia removal from waste water

Liu et al. (2000)

Chitosan-hydroxyapatite composites

Bone substitute as bioceramics

Finisie et al. (2001)

Chitosan attached to sugar, dendrimers, cyclodextrins, crown ethers

Miscellaneous applications Sashiwa and Aiba including drug delivery (2004) systems and other medical applications

Alginate-chitosan-poly (lactic co-glycolic acid) composite microspheres

Protein delivery systems

Zheng et al. (2004)

Nanostructured poly (lactic-co-glycolic acid)/chitin matrix

Tissue engineering

Min et al. (2004)

Self-assembled Immobilized chitosan/poly organophosphorus (thiophene-3-acetic acid) hydrolase for detection layers of paraoxon

Alginates Dried calcium alginate/ magnetite spheres

Constantine et al. (2003)

Support for chromatographic Burns et al. (1985) separations and enzyme immobilization

A mixed gel of colloidal silica and alginate

Ethanol production by yeast immobilized in the mixed gel

Fukushima et al. (1988)

Chitosan-alginate coacervate capsules

Encapsulation of cells/ tissues/pharmaceuticals

Daly and Knorr (1988)

Alginate-starch copolymers

Affinity adsorption of α-amylase

Somers et al. (1993)

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Table 5.3 (Continued) Composite

Application

Reference

Xanthan-alginate spheres

Encapsulation of urease

Elcin (1995)

Polyethyleneiminemodified barium alginate

Immobilization of cephalosporium acremonium for production of cephalosporin C

Park and Khang (1995)

Alginate beads coated with chitosan or DEAE-dextran

Protein release system

Huguet et al. (1996)

Alginate-polythylene glycol gels

Cultivation of mammalian cells

Seifert and Phillips (1997)

Alginate-polylysine capsules

Immunoprotection of endocrine cells

De Vos et al. (1997)

Poly(methylene co-guanidine) coated alginate

Encapsulation of urease

Hearn and Neufeld (2000)

Alginate-chitosan beads

Immobilization of antibodies

Albarghouthi et al. (2000)

Alginate-Konjac glucomannanchitosan beads

Controlled release system for proteins

Wang and He (2002)

Multilayer alginate/ protamine microsized capsules

Encapsulation of α-chymotrypsin

Tiourina and Sukhorukov (2002)

Magnetized alginate

Magnetic resonance imaging

Shen et al. (2003)

Magnetic alginate particles Purification of α-amylase

Safarikova et al. (2003)

Alginate-chitosan coreshell microcapsules

Enzyme immobilization

Taqieddin and Amiji (2004)

Additive for low fat beef frankfurters

Candogen and Kolasarici (2003)

κ-Carrageenan-g-poly acrylamide

Adsorption of fluids and adhesion

Meena et al. (2006)

Carrageenan-g-poly (Sodium acrylate)/ kaolin hydrogels

Superabsorbent composites

Pourjavadi et al. (2007)

Carrageenans Carrageenan-pectin gels

other areas like biosensors, molecular gates and valves, the synthetic thermostable polymer pNIPAAm has dominated. Increasingly, the composites of synthetic polymers and polysaccharides are being investigated for their applications as well as in designing drug release systems. In tissue engineering

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and other usages wherein biocompatibility is a key factor, polysaccharides scores over synthetic smart polymers. Again, composites may be the ideal materials. The information given in this chapter hopefully will motivate research workers to more vigorously exploit polysaccharides in designing smart materials. There are two more reasons to use polysaccharides more often. The current realization that marine biodiversity offers a rich source of materials should lead to a search for a near ideal polysaccharide for a particular purpose. Nature already had made those ‘combinatorial libraries’ of diverse structures. Second, in the drive towards a sustainable society, biodegradable materials from renewable resources constitute an important class. Polysaccharides are from renewable sources and are biodegradable to a varying extent. A survey of the recent patented literature shows that a trend of using polysaccharides for niche applications is emerging. A recent US patent uses cellulose derivatives for forming an ink receptive top layer on materials used for recording inkjet images (Baker, 2003). Another US patent (Ni and Yates, 2004) uses sodium alginate to improve gelation properties of pectic substances for delivery of basic fibroblast growth factor. Some more examples can be found in a review by Al-Tahami and Singh (2007). Given rich structural biodiversity, easy possibility of conjugation/complexation of other substances, biodegradability and biocompatibility (to a varying degree depending upon the particular polysaccharide), polysaccharides and composites based upon polysaccharides are bound to find increasing numbers of applications in diverse areas. Their smartness in many cases is an additional attractive feature.

5.8

Acknowledgement

The preparation of this chapter and the research work from the authors’ laboratory mentioned in this chapter were supported by the Department of Science and Technology (Government of India) core group grant on ‘applied biocatalysis’ and Department of Biotechnology (Government of India) project grants. The support by the Indian Council of Medical Research in the form of Senior Research Fellowship to SR is also acknowledged.

5.9

Sources of further information

A search on Google Scholar™ beta with the phrase ‘Stimuli-sensitive polysaccharides’ yielded about 6530 hits. The sources varied from biotechnology journals to microbiology journals or medical journals. This reflects the wide range of relevance of this broad class of materials. It also conveys that this area has become truly an area which can immensely benefit from multidisciplinary efforts. Some of the sources which we would like to recommend are:

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General references on smart materials Roy I and Gupta M N (2003), ‘Smart polymeric materials: Emerging biochemical applications’, Chem Biol, 10, 1161–1171. Hoffman A S (2002), ‘Hydrogels for biomedical applications’, Adv Drug Deliv Rev, 43, 1–12. Peppas N A (1985), Hydrogels in medicine and pharmacy, Boca Raton, FL, CRC Press.

General references on bioseparation by using smart polysaccharides Gupta M N (ed.) (2002), Methods in Affinity-based Separation of Proteins/ enzymes, Switzerland, Birkhauser Verlag. Mondal K, Roy I and Gupta M N (2006), ‘Affinity based strategies for protein purification’, Anal Chem, 78, 3499–3504. Roy I, Mondal K and Gupta M N (2007), ‘Leveraging protein purification strategies in proteomics’, J Chromatogr B, 849, 32–42. Mondal K and Gupta M N (2006), ‘The affinity concept in bioseparation: Evolving paradigms and expanding range of applications’, Biomol Eng, 23, 59–76.

Chitosan and chitin Kumar M N V R (1999), ‘Chitin and chitosan fibres: A review’, Bull Mater Sci, 22, 905–915. Shahidi F, Kamil J, Arachchi V and Jeon Y J (1999), ‘Food applications of chitin and chitosans’, Trends Food Sci Technol, 10, 37–51. Muzzarelli R A A (1977), Chitin, Oxford, Pergamon Press. Skjåk-Bræk G, Anthonsen T and Sandford P (eds) (1989), Chitin and Chitosan, London, Elsevier. http://wwwcsi.unian.it/chimicam/chimicam.html

Alginates Gerbsch N and Buchholz R (1995), ‘New processes and actual trends in biotechnology’, FEMS Microbiol Rev, 16, 259–269. (A good and informative review of immobilization techniques with special emphasis on alginate.) Martinsen A, Skjåk-Bræk G and Smidsrød O (1989), ‘Alginate as immobilization material: I. Correlation between chemical and physical properties of alginate gel beads’, Biotechnol Bioeng, 33, 79–89.

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Skjåk-Bræk G, Murano E and Paoletti S (1989), ‘Alginate as immobilization material. II: Determination of polyphenol contaminants by fluorescence spectroscopy, and evaluation of methods for their removal’, Biotechnol Bioeng, 33, 90–94. Smidsrød O and Skjåk-Bræk G (1990), ‘Alginate as immobilization material for cells’, TIBTECH, 8, 71–78.

κ-carrageenans Van de Velde F and De Ruiter G A (2002), ‘Polysaccharides from eukaryotes’, in Biopolymers, Vol 6, Polysaccharides II, Weinheim, Wiley-VCH, 245– 274. Van de Velde F, Lourenço N D, Pinheiro H M and Bakker M (2002), ‘Carrageenan: A food-grade and biocompatible support for immobilisation techniques’, Adv Synth Catal, 344, 815–835. http://www.fmcbiopolymer.com/PopularProducts/FMCCarrageenan/ Introduction/tabid/804/Default.aspx.

Composites Kumar M N V R, Kumar N, Domb A J and Arora M (2002), ‘Pharmaceutical polymeric controlled drug delivery systems’, in Advances in Polymer Science, Vol 160, Heidelberg, Springer Verlag. Aguilar M R, Elvira C, Gallardo A, Vázquez B and Román J S (2007), ‘Smart polymers and their applications as biomaterials’, Topics in Tissue Engineering, 3, 1–27. Al-Tahami K and Singh J (2007), ‘Smart polymer based delivery systems for peptides and proteins’, Recent Pat Drug Del Formul, 1, 65–71.

5.10

References

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