Gelation kinetics of scleraldehyde–chitosan co-gels

Polymer Gels and Networks 6 (1998) 113—135

Gelation kinetics of scleraldehyde—chitosan co-gels Bo Guo, Arnljot Elgsaeter, Bj+rn T. Stokke* Norwegian Biopolymer Laboratory, Department of Physics, The Norwegian University of Science and Technology, NTNU, N-7034 Trondheim, Norway Received 8 July 1997; received in revised form 9 January 1998; accepted 14 January 1998

Abstract The gelation kinetics of modified scleroglucan carrying various fractions of reactive aldehyde groups in the side-chains, scleraldehyde, and chitosan was determined using oscillatory shear rheometry to study the dynamic viscoelastic properties as a function of reaction time. The experimentally determined initial increase in the storage modulus per unit time, *G@/*t, for various scleraldehyde concentrations, C , degree of aldehyde substitution of scleraldehyde, S# D , and chitosan concentrations, C , revealed the existence of a lower critical concentration A-$ C)* for gelation for both the two polysaccharides, C and C . The observed power law S#,0 C)*,0 coefficients m and n, *G@/*t&(D C )m Cn , were both significantly larger than one in the A-$ S# C)* employed relative concentrations ranges C /C 3(1—20) and C /C 3(1—10), which was S# S#,0 C)* C)*,0 accounted for by the total wastage reactions and network statistics. The rate of gel formation showed a maximum near pH 7 due to a delicate balance among several competing effects: (1) The free-amine concentration of the chitosan increases in the pH range 4.5—6.5 (pK of the ! amino group), (2) the Schiff-base junction formation is acid catalyzed, and (3) the solubility of chitosan decreases when the pH exceeds 7. The increased aggregation resulting from increasing the pH from 7 to 7.6 yielded an increasing fraction of reactive groups involved in wastage reactions, yielding an increase both in C and the power law coefficient n from 1.7 to 2.6. The C)*,0 temperature dependence of *G@/*t could be accounted for by incorporating an apparent activation energy E "43 kJ mol~1. The temperature dependence of G@ of the fully cured ! scleraldehyde—chitosan co-gels was found to give rise to a shift from *G@/*T'0 Pa/min to *G@/*¹(0 Pa/min, when the crosslink density increased. This suggests a change from entropy dominated elasticity at low crosslink density, to an enthalpy dominated elasticity when this

* Corresponding author. 0966-7822/98/$ — see front matter ( 1998 Elsevier Science Ltd. All rights reserved. PII S 0 9 6 6 - 7 8 2 2 ( 9 8 ) 0 0 0 0 5 - 7

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density increases, possibly due to decreasing length of the elastically active chains. ( 1998 Elsevier Science Ltd. All rights reserved

1. Introduction In recent years, great attention has been devoted to biopolymer gels because of their biocompatibility and biological functions, and consequently, potential applications in the biomedical and pharmaceutical fields. Scleroglucan is a fungal polysaccharide secreted by certain fungi of the genus Sclerotium. It is a neutral, water soluble and non-toxic polysaccharide composed of a linear (1,3)-linked bD-Glcp backbone substituted with a (1,6)-linked b-D-Glcp residue attached as a side chain to about every third backbone unit [1,2]. This chemical structure has been shown to have the ability to enhance the immune system sensitivity resulting in improved antitumor, antibacterial, antiviral, anticoagulatory and wound healing response [3]. The general immunostimulating properties of scleroglucan, and other structurally similar polysaccharides, b(1,3)-glucans, are of considerable interest [3—7]. The immunological properties of these materials have in most cases been investigated when the polysaccharide is presented to the biological test system in dissolved form. Our long term goal is to understand the immunological properties of the b(1,3)glucans crosslinked into a gel. To reach this goal, we utilize the recently reported approach involving crosslinking aldehyde functionalized scleroglucan by the amino group of chitosan [8,9]. Unmodified scleroglucan in solution does not gel when blended with chitosan. Periodate selectively oxidises the scleroglucan side-chains because the main chain residues do not possess any vicinal-hydroxyls. The periodate oxidation leads to quantitative introduction of aldehyde groups on the side chains. Crosslinks can be formed between these aldehyde groups on the scleroglucan chains and the free amino groups on the chitosan chains via the Schiff-base reaction mechanism. The details of the crosslinking mechanism described in the litterature predict that several parameters can be used to control the gelation process of the scleraldehyde—chitosan system and, eventually, the mechanical and swelling properties of this gel system. The objective of this work is to study the properties of this new gel system, with emphasis on gelation kinetics. The rheological measurements were carried out in order to investigate systematically the effects on the gelation rate of both scleroglucan and chitosan concentrations, the degree of scleroglucan oxidation, pH value in pregel solution and temperature. The temperature dependence of the storage modulus as a function of crosslink density following 24 h of gelation was determined to address the thermodynamic nature of the elasticity. 2. Background Methods for direct, reliable determination of the aldehyde content in scleraldehyde are lacking at present. Here, indirect determination of the aldehyde content is carried

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out by measurement of the consumed periodate and formed formic acid, or alternatively, by determination of the carboxylic acid content of fully oxidized samples. The method used here is based on the assumption that both the C(2)—C(3) and C(3)—C(4) of the anhydroglucose units in the scleroglucan sidechain can be accessed by periodate oxidation [10] (Scheme 1). It has been found that some systems containing adjacent functional groups such as diketones also yield carboxylic acids as a result of periodate oxidation [11]. It was observed that the solution became acidic during periodate oxidation of scleroglucan. This is an indication of double oxidation in the scleroglucan side-chain. Either the C(3)—C(4) (rate constant k ) or the C(2)—C(3) (rate 1 constant k@ ) bond is cleaved in the first oxidation step yielding dialdehyde groups at 1 the respective carbons. A second oxidation of the complementary bond (k and k@ , 2 2 Scheme 1) yields aldehyde functionalities at C(2) and C(4) with concomitant release of formic acid from C(3). Complete oxidation of the scleroglucan side-chain thus consumes two moles periodate and forms one mole formic acid. Hence, both consumed periodate and produced formic acid can be used to determine the fraction of sidechains bearing aldehydes. Following Aalmo and Painter [12], the kinetics of intact sidechain residue reduction can be described: d(1!P #F ) t t "!(k #k@ )(1!P #F )(P!GlP ), 1 1 t t t dt

(1)

where P and F are the molar proportions of consumed periodate and produced t t formic acid, P equals the initial concentration of periodate, and Gl is the initial concentration of side-chain residues. Integration and rearranging of Eq. (1) yield:

A

B

1 P!GlP t (2) ln "(k #k@ )t 1 1 (P!2Gl) P(1!P #F ) t t which can be used to calculate the fraction of aldehyde-functionalized side-chains. The total rate for periodate consumption (on integrated form), for rate constant k is 1, 1 2(P!GlP ) t "k t. ln (3) p (P!2Gl) P(2!P ) t

A

B

Scheme 1. Selective, stoichiometric periodate oxidation of sclerogucan side chain and release of formic acid.

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Additionally, the aldehyde content of the functionalized scleroglucan was determined independently by first carrying out further oxidation of the aldehyde groups to carboxylic acid using sodium chlorite under acidic conditions [13,14]. This was followed by determination according to established methods [15] of the carboxylic groups by their ability to bind Mg2`. The crosslinking reaction in the scleraldehyde—chitosan co-gels is based on Schiffbase formation between the aldehyde groups in the side-chains of the scleraldehyde and the amino group of the deacetylated residues of chitosan (Scheme 2) [16]. Using the shorthand notation of A and D to denote 2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-glucopyranose of chitosan, respectively, and referring to the aldehyde-functionalized side-chains of scleroglucan as B, the system can be described as an f -functional polymer reacting with an f -functional polymer. The inherent D B distribution of the functionalities among the individual polymers is not explicitely incorporated in this semi-quantitative account of the system. Following the strategy of Clark [17,18] who used the cascade formalism [19,20], it is assumed that the crosslinking sites can be treated as independent reacting groups. The rate of formation of effective crosslinks, C , assuming second order kinetics is then given as [21]: ~BD~ dC ~BD~"k C C , (4) ~BD~ B D dt where k is the rate constant of the reaction. The actual concentrations of the ~BD~ reacting species are given in terms of initial concentrations, C , and C , and extents B0 D0 of reaction as C "C (1!p !p ), (5a) B B0 B BW C "C (1!p !p ), (5b) D D0 D DW C "C p "C p , (5c) ~BD~ B0 B D0 D where p and p are the extents of reaction of B- and D-groups involved in effective B D crosslink formation, and p and p are the fractions of the initial concentrations BW DW wasted either by ineffective crosslinks (cycles) or other side-reactions. The degree of conversion p of chitosan leading to effective crosslinks is given by rp where r" D B [NH ]/[CHO] is the stoichiometric ratio between the free amino groups and the 2 aldehyde groups of scleraldehyde. The wastage reactions can be written: dp DW"k (1!p !p )(1!p !p )#k (1!p !p ) DW1 D DW B BW DW2 D DW dt

Scheme 2. Schiff-base formation [16].

(6)

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and similarly for p . In Eq. (6), k denotes the rate constant for formation of BW DW1 ineffictive crosslinks via the Schiff-base mechanism, and k equals the rate constant DW2 for any side-reaction that may occur depending only on the concentration of the remaining nonreacted amino groups. This differs from the wastage term given by Clark [17,18] because of the incorporation of the second order term in Eq. (6). The bases for the latter term is that the Schiff-base mechanism only can give rise to cycle-formation between groups located on different types of polymers. The kinetic scheme for the effective crosslinks was then combined with a statistical description of an f -functional polymer crosslinked with an f -functional polymer to express D B the time dependence of the storage modulus after the gel-point had been reached. This occurs at a critical, effective degree of conversion p given by the expression B,C [22,23]: p "(r( f !1)( f !1))~1@2. (7) B,C B D The wastage terms are incorporated in the kinetics of crosslink formation, and therefore not included explicitely in the statistics of network formation as done in other models [22,23]. The storage modulus of the reacting system can then be written G@"/ (p )aR¹, (8) E B where / (p ) is the concentration of effective crosslinks at effective degree of converE B sion p for the scleraldehyde reactive groups, R the molar gass, constant and ¹ the B absolute temperature. In Eq. (8), it is assumed that the front-factor a associated with nonideal entropic elasticity of the elastically active network chains is independent of the stoichiometric ratio and degree of crosslinking. For polysaccharides a typically equals 3—40 [24,25]. This may not be valid for the interconnected scleraldehyde— chitosan networks because the stiffness of the two types of elastic active chains differs greatly (Fig. 1). Various stoichiometric ratios r and actual values of p could therefore B indicate that the front-factor a depends on p , and initial concentrations. The conB centration of effective network strands was calculated using the probability for a randomly selected junction point to have three or four paths to the infinite network [26,27]: / (p )"C p [0.75((1!g0!g1)g1 #(1!g0 !g1 )g1) D B D B D B E B B0 B (9) #(1!g0!g1)(1!g0 !g1 )], D D B B where the probabilities for zero and one path to the infinite network seen through a chain of either type is given, respectively, by g0"l(fB~1), (10a) B B (10b) g1"f (1!l )l (fB~2). B B B B The extinction probability, m , in the cascade formalism, i.e., the probability that B a randomly chosen crosslink does not connect to the infinite network when seen through chains of type B, is given as l "1!p #p (1!p #p l(fB~1) )(fD~1). B B D D D B

(11)

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Fig. 1. Schematic illustration of crosslinking of an f —functional scleraldehyde with an f —functional B D chitosan. The illustration is intended to depict the selective oxidation of the sidechain of scleroglucan yielding aldehyd-groups only in the side-chains, examples of random distribution of the deacetylated (D) and acetylated (A) glucosamine residues in the chitosan, and the large difference in chain stiffness between the two constituents of the network.

The mathematical expressions for the probabilities of one or zero paths to the infinite network, g1 and g0 , and the extinction probability when seen out through the network D D chains of type D are analogous to Eqs. (10) and (11). This model (Eqs. (4)—(11)) allows in principle calculation of the gel storage modulus rate of change close to the gel point, and the influence of wastage reaction on the gelation point. Such calculations would require that sample parameters f , f , the kinetic parameters and the front factor a are B D all known. In the case of gelation of a one-component system, Clark provided the analytical solution of the differential equations in question, and established for this case the relationship between the gelation time and the concentration [18]. In the present case, the numerical solution of the coupled differential equations is most readily obtained using the numerical integration scheme following the same ideas as pursued by Clark [17,18]. Typical examples illustrating the rate of change of G@ near a critical concentration were obtained by first choosing the kinetic parameters k , ~BD~ k ,k ,k , and k , so that the observed properties of the critical gelation DW1 DW2 BW1 BW2 experiment was qualitatively accounted for. These values of the kinetic parameters were subsequently used to illustrate effects of varying the two polysaccharide concentrations on the predicted critical concentrations for gelation and *G@/*t. The initial concentrations of the two types of the reactive groups depend on the polysaccharide concentrations, the degree of substitution, and in the case of chitosan also pH. The concentration of reactive groups on scleraldehyde, C is CHO given as C "C "D C /M , (12) B0 CHO A-$ S# Scl where D is the degree of aldehyde substitution of scleraldehyde, C is the concenA-$ S# tration of scleraldehyde (mg/ml) and M the molar mass of the repeating unit of S#-

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scleraldehyde. The concentration of the primary amines, C "C depends on NH2 D0 the fraction of deacetylated residues of the chitosan sample, F , the difference betD ween the pH in solution and the pK of the amine group, and the chitosan concentra! tion C as C)* 101Ka~1H F C D C)* , C "C " (13) D0 NH2 (1#10pKa~1H) M C)* where M is the average molar mass per hexopyranose residue of the chitosan. Note C)* however, that the ordinary Schiff-base type reaction, i.e. R-CHO # R@-NH P R2 CH"N-R@ # H O, is normally an acid-catalysed process [16] (Scheme 2) and 2 therefore that k (Eq. (4)), as well as the kinetic coefficients in the wastage ~BD~ reactions involving Schiff-base formation may depend on the pH. The temperature dependence on the crosslinking reaction can be described in terms of an activation energy E for the Schiff-base reaction according to the Arrhenius law: ! k "k e~E! @RT, (14) ~BD~ 1 where k is a constant. 1 3. Materials and methods 3.1. Oxidation of scleroglucan side-chains A purified scleroglucan sample (Actigum CS-11, kindly provided by Sanofi Bioindustries, France) was used in this study. The intrinsic viscosity [g] was found to equal 3000 ml/g in double distilled water and 3600 ml/g in 0.01 N NaOH at 20°C and shear rate c5"5 s~1, using a Cartesian Diver viscometer. The weight average molecular weight estimate, M , of the sample obtained using the experimentally determined [g] 8 and reported relation between the [g] and M for schizophyllan, a b(1,3)-glucan with 8 identical chemical composition as scleroglucan [28], equals 2—3]106 g/mol. This sample was used for all experiments except for determination of the gel point, where an ultrasonically depolymerized scleroglucan sample of CS-11, with M " 8 0.56]106 g/mol was employed. Scleroglucan stock solutions were prepared at concentration C "10 mg/ml, by adding 1 g scleroglucan to 100 ml of double distilled S# water under rapid stirring. The samples were left stirring for at least two days to ensure full hydration. The slightly turbid stock solution was diluted further to the required concentrations using double distilled water. Scleraldehyde, CS-CHO-X, where X denotes the nominal % of the sidechains modified to contain aldehyde groups, was obtained by quantitative introduction of aldehyde groups in the side chains of scleroglucan by adding the required volume of a freshly prepared 0.3 M solution of NaIO (p.a., Merck Inc.) and leaving 4 the sample in the dark for 24 h at room temperature [15]. Samples with nominal aldehyde group content ranging from 5 to 60%, i.e., CS-CHO-5 to CS-CHO-60, were prepared.

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3.2. Determination of aldehyde group content in scleraldehyde The content of aldehyde groups in the scleraldehyde samples was determined using two independent indirect methods. The first one determines the consumed amount of periodate by titration of the remaining periodate with sodium thiosulphate, and the produced amount of formic acid by titration with NaOH following the oxidation reaction. For these measurements, 250 ml of 5 mM (polysaccharide repeating units) scleroglucan solution was blended with 250 ml 25.3 mM periodate solution. Excess amount of periodate was used to ensure the complete oxidation. The oxidation reaction was carried out at 6°C. Ten ml reacted mixture was sampled and titrated with sodium thiosulphate and NaOH at different reaction times. The second method used to determine the content of introduced aldehyde groups involves further oxidation of the scleraldehyde samples to sclerox using an excess of sodium chlorite, which subsequently transforms the aldehyde groups into carboxylic groups. The second oxidation step was performed under acidic condition by adding acetic acid to a final concentration of 0.25 M. Solid NaClO was added to a final concentration of 0.5 M to 2 ensure complete conversion of aldehyde groups into carboxylic groups. After the second oxidation, the pH value of the mixture was adjusted to pH"5.6—6.0 using 0.1 M NaOH (Radiometer PHM92 lab pH meter equipped with a pHC2411 combining electrode). The samples were then dialysed against double distilled water for three days, and subsequently freeze-dried. The degree of carboxylation of the sclerox samples was determined by measuring the amount of Mg2` ions bound as counter ions to carboxylic groups as described previously [15]. The Mg2` content was determined by atomic absorption employing a Perkin Elmer 560 Atom absorption spectrometer equipped with a Ca—Mg Intesitron lamp. 3.3. Gelation kinetics of scleraldehyde—chitosan gels The chitosan—scleraldehyde gels were prepared by blending aqueous solutions of CS-CHO-X, and chitosan solution using the two polysaccharide concentrations and percentage of aldehyde groups in the scleraldehyde to vary the cross-link density of the gels. The molar ratios of free amino groups to aldehyde groups, r" [NH ]/[CHO], were used in addition to the polysaccharide concentrations as an 2 index of cross-link densities. A chitosan stock solution at concentration C " C)* 5 mg/ml was prepared by adding 0.20 g of a chitosan Chit-FD-51 with fraction of deacetylated glucosamine residues F "0.51 and intrinsic viscosity [g]"1270 ml/g D (kindly provided by Dr. K.M. Vas rum, Dept. of Biotechnology, NTNU) to 40 ml double distilled water. The pH of the CS-CHO-X and chitosan solutions was adjusted adding NaOH solution. The light transmittance, ¹, at wavelength j"660 nm of the chitosan solutions was measured in quartz cells with 1 cm pathlengths employing a Zeiss PMQ3 spectrophotometer. Two ml of the CS-CHO-X solutions was thoroughly mixed with a specified volume of chitosan solution. One ml of this mixture was rapidly transferred to the sample stage of a Bohlin VOR rheometer. A 30 mm diameter serrated parallel plate geometry (SP30) at a gap distance of 0.9—0.96 cm, and a 4 g cm torsion bar were employed for

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determination of the gelation kinetics. The sample was sealed using a low density, low viscosity silicon oil to minimise potential problems associated with evaporation of the solvent during the gelation experiments. The gelation was determined by measuring the storage, G@, and loss, GA, moduli at intervals of 2—5 min using a selected frequency and temperature. When the gelation kinetics had been followed for 24 h and G@ had reached an apparent plateau, the temperature was increased at 0.5°C min~1 from 20 to 70°C to determine the temperature dependence of G@.

4. Results and discussion 4.1. Degree of aldehyde substitution of scleraldehyde Fig. 2A shows consumed NaIO , and produced formic acid versus reaction time for 4 periodate oxidation of scleroglucan at 6°C. The initial data are well described by Eq. (2) (Fig. 2B), and the slope of a linear least squares fit for the data up to a reaction time of 10 h yielded k #k@ "0.008 mM~1 h~1. Further analysis showed that k (Eq. 1 1 1 (3)) was close to k #k@ , indicating that formation of a cyclic hemiacetal form from 1 1

Fig. 2. (A) Experimentally determined consumption of NaIO (s) in the oxidation reaction (mol/mol), and 4 the formation of formic acid (h) vs reaction time for periodate oxidation of scleroglucan. (B) The initial decay of intact glucose side-chain residues (s) plotted according to Eq. (2) versus reaction time.

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the singly oxidized intermediate (not shown in Scheme 1), is neglible in the present case [12]. The reaction is characterized by an increasing amount of consumed periodate and produced formic acid levelling off after about 24 h. Initially, the ratio between periodate consumption and production of formic acid is larger than two, indicating that single-oxidation also takes place in the sidechain. After 40 h, every mole of scleroglucan repeating unit consumes two moles of periodate and produces one mole of formic acid. Similar data were reported by Yanaki and coworkers for schizophyllan oxidation by periodate [29]. In that case, each mole of glucose units consumed 0.5 moles of periodate and produced 0.25 moles of formic acid. These experimental results are consistent with the double oxidation process for oxidation of scleroglucan by periodate being the dominant process. The results from the Mg2` binding experiments of the fully oxidized scleraldehyde samples confirmed these findings. Table 1 shows the experimentally determined degrees of carboxylation of the sclerox samples and the theoretical predictions. These predictions rest on calculations based on the consumed amount of NaIO in the first 4 oxidation step assuming single oxidation and assuming full conversion in the second oxidation. Comparison of the experimental data for five separate samples with the predicted oxidation of 10% or more, shows that the actual degree of aldehyde substitution is only about half the predicted values. This discrepancy arises because of the double oxidation with concomitant release of formic acid in the first oxidation step. The sample with low degree of conversion, i.e., 5% carboxylic groups introduced, deviated from this result, possibly due to an increased fraction of single oxidation at this lower degree of conversion. This exception might imply that the double oxidation process depends on the total concentration of periodate in the first oxidation step for low molar ratios of NaIO and oxidisable groups. The calculated average functional4 ity f , using the experimentally determined aldehyde content range from 170 to 1300 B for these high molecular weight samples (Table 1). Table 1 Aldehyde contents and average functionalities, f , of the scleraldehyde samples B Sample

CS-CHO-5 CS-CHO-10 CS-CHO-20 CS-CHO-30 CS-CHO-40 CS-CHO-60

f # B

Degree of carboxylation Theoretical! (%)

Experimental" (%)

5 10 20 30 40 60

4.4 5.4 12.4, 18.3, 24.0, 32.8,

11.8 17.4 23.1 34.6

170 210 470 690 910 1300

! Calculated from consumed NaIO and assuming single oxidation (see text). 4 " Determined from binding of Mg2`. More than one listed result represents independent experiments. # Calculated using the average of the experimentally determined aldehyde fractions, and molecular weight of the scleroglucan repeating unit equal to 648 g/mol. The molecular weight M "2.5.]106 g/mol as 8 determined for the unmodified scleroglucan was used.

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4.2. ¹ime and frequency dependence of scleraldehyde—chitosan mixtures Fig. 3A shows a representative time dependence of the storage (G@) and loss (GA) modulus for scleraldehyde—chitosan mixture at the selected oscillatory frequency of 1 Hz. The data show that G@ first increases rapidly and subsequently levels off, whereas GA is always much lower than G@. The frequency dependences of G@ and GA were recorded from 0.006 to 10 Hz after 24 h of gelation (Fig. 3B). The value of G@ is typically at least one order of magnitude larger than GA, and both moduli show a slight increase at the higher frequencies (Fig. 2B). The loss modulus exhibits a minimum in the experimental frequency range. This behaviour is similar to that observed for numerous biopolymer gels. The initial rate of gelation *G@/*t was determined employing a linear least squares fit between G@ and t and using the data

Fig. 3. (A) Storage G@, (s), loss, GA (h) moduli, and phase angle d (*) at u"6.28 s~1 of scleraldehyde—chitosan gels, 8.8 mg/ml CS-CHO-60, 0.45 mg/ml Chito-FD-51 at pH 7.6, vs incubation time at 30°C. (B) Storage, G@, (s) and loss, GA (h) modulus vs u for the same gel as in Fig. 3A 24 h after blending the solutions.

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determined during the first hour (Fig. 3A, Table 2). The gels appeared homogeneous when inspected in a light microscope employing phase contrast, indicating that phase separation is not prominent in the current system. 4.3. Gelation kinetics From the above analyses follows that the main factors affecting the gelation kinetics of the scleraldehyde—chitosan co-gels are the scleraldehyde concentration, the fraction of aldehyde in the scleraldehyde sample, the chitosan concentration, the fraction of deacetylated units of the chitosan sample, and the pH and temperature in the solution. Note also that existence of lower critical concentrations of both scleraldehyde and chitosan are expected, and that concentration effects on the gelation rates should be considered in view of the existence of such critical concentrations [17,18,30,31]. The molecular weight of the two types of polysaccharides are also expected to affect the gelation kinetics, but a systematic study of the effects of varying these parameters was not included in this study. Fig. 4 shows storage modulus, G@(u"6.28 s~1) versus time at 30°C for several different values of the scleraldehyde concentration (Fig. 4A), the chitosan concentration (Fig. 4B), the different degrees of aldehyde substitution of scleraldehyde (Fig. 4C) and the pH in the solutions (Fig. 4D). The data show that G@ increases monotonically with time for all the combinations of the parameters listed above. There is a monotonic increase in the initial rate of change of G@ with increasing C (Fig. 4A), C (Fig. 4B), and fraction of aldehyde groups in S# C)* the scleraldehyde (Fig. 4C). The effect of altered pH is characterized by an increase in the rate of gelation up to about pH 7, followed by a decreasing rate of gelation upon further increase in the pH (Fig. 4D). This pH effect is discussed below. Similar trends were found for the values of G@ after 10 h of gelation. The initial rate of gelation, *G@/*t, was determined by a linear least squares fit of G@ as shown in Fig. 3A, for all the conditions (Table 2). The data show that the set of parameters employed yields values of *G@/*t spanning nearly three orders of magnitude. The data indicated that there were lower critical values for gelation both for the chitosan, C , and scleraldehyde, C , concentrations. The experimentally detemined C)* S# *G@/*t was found to be nearly linearily dependent on the chitosan concentration when C was varying less than 20% (Fig. 5A). The linear least square fit yields the S# intercepts C "0.10 mg/ml for CS-CHO-20 at pH 7, C "0.34 and 0.35 mg/ml C)*,0 C)*,0 for CS-CHO-20 and CS-CHO-60 at pH 7.6, respectively (Fig. 5A). Analogously, linear extrapolations of the kinetic data for constant C , for varying C , to *G@/*t"0 C)* S# Pa/h, yielded intercepts at C "0.93 mg/ml and C "1.67 mg/ml for CS-CHOS#,0 S#,0 60 and CS-CHO-20 samples, respectively (Fig. 5B). However, extrapolation of these experimental data for the CS-CHO-60 sample employing a fitted power law exponent, yields a critial lower concentration of C "0.60 mg/ml, which is even lower than S#,0 the C observed for the CS-CHO-20 sample. These results suggest a lower critical S#,0 concentration both for the scleraldehyde and chitosan concentration. This is in agreement with what is reported for other gels, however, the present co-gels show that the lower critical concentration exist for both of the polymers. Gelation experiments using polymer concentrations close to these observed critical concentrations

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Fig. 4. Effect of varying C (A), C (B), degree of oxidation in the scleroglucan side-chain (C) and pH of S# C)* the pregel solutions (D) on storage, G@, moduli at u"6.28 s~1 vs incubation time t at 30°C. (A) G@ vs t of scleraldehyde—chitosan gels for 1 (s), 2 (h), 5(*), 7.5 (+) and 10 (e) mg/ml CS-CHO-20, and constant chitosan concentration 0.83 mg/ml Chit-FD-51 and at pH 7.6. (B) Series with increasing chitosan concentration with concentrations (C , C )"(8.8, 0.45) (s); (8.1, 0.83), (h); (7.5, 1.15), (*) and (6.9, 1.42), S# C)* (+) mg/ml, respectively, using sample CS-CHO-60 and pH 7.6. (C) Effect of degree of oxidation at C "8.8mg/ml, C "0.83 mg/ml using samples CS-CHO-10 (s), CS-CHO-20 (h), CS-CHO-40 (*) and S# C)* CS-CHO-60 (+), all at pH 7.6. (D) Constant scleraldehyde CS-CHO-60 concentration C "4.9 mg/mL S# and chitosan concentration C "0.83 mg/ml at pH"5.0 (s), 6.6 (h), 7.05 (*) and 7.6 (+). C)*

were attempted employing 1 mg/ml CS-CHO-60. Adding a chitosan solution to aqueous CS-CHO-60 at this low C resulted in precipitation. This suggests that S# Schiff-base formation occurs giving rise to insoluble clusters rather than macroscopic network formation. At a scleraldehyde concentration of C "1 mg/ml, the reduced S# concentration C [g] (in scleraldehyde) equals three indicated that the scleraldehyde S# chains are barely overlapping, which may promote formation of independent clusters rather than overall network formation. The observations described above were compared with the semi-quantitative predictions obtained employing the above kinetic model (Eqs. (4)—(11)). The critical gelation behaviour was studied (see below) and the results used to obtain a set of kinetic constants k ,k ,k ,k and k in the kinetic model for polymer ~BD~ DW1 DW2 BW1 BW2 concentrations just above the gel point (Eq. (7)). The obtained kinetic parameters were subsequently used to calculate examples of the concentration dependence of *G@/*t for degrees of conversion just above the gelation threshold. One example for the

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Table 2 Gelation kinetics parameters for scleraldehyde—chitosan! co-gels at 30°C Varying parameter

Scleraldehyde sample

C S# (mg/ml)

D A-$

D C A-$ S# (mg/ml)

C C)* (mg/ml)

pH

*G@/*t (Pa h~1)

C S#

CS-CHO-60

2.0 5.0 7.5 10.0

0.34 0.34 0.34 0.34

0.67 1.9 2.5 3.4

0.83 0.83 0.83 0.83

7.6 7.6 7.6 7.6

5.7$0.1 22.0$3.0 76.0$9.0 160$18

C S#

CS-CHO-20

1.0 2.0 5.0 7.5 10.0

0.12 0.12 0.12 0.12 0.12

0.12 0.24 0.61 0.91 1.2

0.83 0.83 0.83 0.83 0.83

7.6 7.6 7.6 7.6 7.6

2.0$0.4 4.2$0.6 8.4$0.9 15.0$2.0 28.0$6.0

C S#

CS-CHO-20

3.0 5.0 6.8 7.5 9.0

0.12 0.12 0.12 0.12 0.12

0.36 0.61 0.82 0.91 1.1

0.42 0.42 0.42 0.42 0.42

7.0 7.0 7.0 7.0 7.0

2.2$0.4 7.0$1.1 12.0$1.8 16.0$2.5 28.0$6.0

C C)*

CS-CHO-60

8.8 8.1 7.5 6.9

0.34 0.34 0.34 0.34

3.0 2.7 2.5 2.3

0.45 0.83 1.2 1.4

7.6 7.6 7.6 7.6

C C)*

CS-CHO-20

9.0 8.6 8.3 7.8 7.6 6.9

0.12 0.12 0.12 0.12 0.12 0.12

1.1 1.0 1.0 0.94 0.92 0.84

0.45 0.65 0.83 1.0 1.2 1.4

7.6 7.6 7.6 7.6 7.6 7.6

4.7$1.1 9.6$0.8 22$1 26$1 32$2 44$1

C C)*

CS-CHO-20

8.3 8.3 8.3 8.3 7.6 6.9

0.12 0.12 0.12 0.12 0.12 0.12

1.0 1.0 1.0 1.0 0.92 0.84

0.10 0.21 0.31 0.42 0.83 1.15

7.0 7.0 7.0 7.0 7.0 7.0

2.1$0.5 9.5$2.2 16$4 28$6 66$12 90$17

D A-$

CS-CHO-10 CS-CHO-20 CS-CHO-40 CS-CHO-60

4.2 4.2 4.2 4.2

0.05 0.12 0.24 0.34

0.23 0.51 0.98 1.4

0.83 0.83 0.83 0.83

7.6 7.6 7.6 7.6

3.7$0.9 8.7$0.9 15$1 24$5

49$13 161$17 322$36 404$43

! A chitosan sample with degree of deacetylation F "0.51, and [g]"1270 ml g~1 was used in all D experiments.

f "470 (corresponding to the CS-CHO-20 sample, Table 1), clearly indicates that B there exists lower critical concentrations of both type of polymers needed for gelation, and their interrelation (Fig. 6). Such calculations also show that *G@/*t depends on the ratio between the actual and critical polymer concentrations. For example, the power law of *G@/*t&C2.1 for the CS-CHO-60 sample was most likely observed S#

B. Guo et al. / Polymer Gels and Networks 6 (1998) 113—135

127

Fig. 5. Initial increase in the storage modulus per unit time, *G@/*t at ¹"30°C vs (A) chitosan and (B) scleraldehyde concentration at 30°C. The data for the different values of C were obtained using the C)* scleraldehyde samples CS-CHO-60 (s), and CS-CHO-20 (h) at pH 7.6 and CS-CHO-20 (*) at pH 7 and C equal to about 8 mg/ml (Table 2). The data for C "0.83 mg/ml (B) were obtained using the S# C)* scleraldehyde samples CS-CHO-60 (+), and CS-CHO-20 (e) at pH 7.6. The continuous lines depict linear least squares fits of *G@/*t vs the respective concentrations.

because of the employed polymer concentration range is relatively close to the critical value, C /C 3(1—15) rather than because of the order of the reaction kinetics S# S#,0 [assumed to be second order, Eq. (4)]. Using an experimental approach analogous to the one employed here, large power law coefficients have been attributed to the use of a relatively small concentration range close to the critical polymer concentration for gelation [17,30]. Most of the data for *G@/*t of the scleraldehyde—chitosan gels could be fitted to ‘‘master curves’’ *G@/*t&(C D )2.4 C1.7 at pH 7, and *G@/*t& S# A-$ C) (C D )2.1 C2.7 at pH 7.6. These appearant fits to one master curve for each solution S# A-$ C) pH value are partly because similar ratios between actual and critical polymer concentrations have been used. The finding that the product C D can be used to fit S# A-$ the data is in agreement with the assumption that aldehyde concentration C equals CHO C D (Eq. (12)). The increase in the power law coefficient for the chitosan concentraS# A-$ tion when the pH is changed from 7 to 7.6 could be caused by increasing aggregation of the chitosan, which in turn could lead to an increase in the wastage fraction. The resulting increased C would yield a smaller range of C /C for the employed C)*,0 C)* C)*,0 concentration range at pH 7.6 than pH 7.0. The estimated C "0.1 and 0.34 mg/ml C)*,0 at pH 7 and pH 7.6 (Fig. 5A) supports this interpretation. 4.4. Effect of pregel solution pH on gelation kinetics Attempts to include pH as a parameter alongside C , D , and C in the above C4 A-$ C)* analyses, as suggested by Eq. (13), were not successful, probably because changing the

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B. Guo et al. / Polymer Gels and Networks 6 (1998) 113—135

Fig. 6. A contour plot where each line represent C and C yielding the same increase in the storage C)* S# modulus per unit time *G@/*t. The values of *G@/*t were calculated for the high molecular weight sample using kinetic constants adjusted to account for the critical behaviour of the low molecular weight scleraldehyde sample. The data were normalised with respect to the calculated value at *G@/*t(C " C)* 1 mg/ml, C "10 mg/ml). The labelled lines correspond to the normalised values shown, and the minor S# lines (- - -) are evenly spaced in the intervals. The contour line labelled with zero correspond to a *G@/*t of 0.1% of that at C "1 mg/ml, C "10 mg/ml. C)* S#

pH may give rise to various values of C /C . The effect of pH on the gelation C)* C)*,0 kinetics was therefore considered separately. Another reason for this lack of success may be that pH not only controls the actual charge on the amine groups, as was the basis for including pH in Eq. (13), but also affects the solubility of chitosans [32], and that H O` is an active catalyst in the Schiff-base formation (Scheme 2). Fig. 7 shows 3 the gelation kinetic parameter *G@/*t as function of pH in pregel solutions for 5 mg/ml CS-CHO-60 (s), and 10 mg/ml (h) and 5 mg/ml CS-CHO-20 (e). The data reveal that *G@/*t increases rapidly when the pH is increased from 4.5 and reaches a maximal value at pH 7.0. The experimental data in this pH range fit the power law *G@/*t&101.5 1H for the 5 mg/ml CS-CHO-60 sample, which represents one of the strongest dependences of pH. Further increase of pH yielded an exponential decrease *G@/*t"K 10~1.8 1H. The gelation kinetics data show that there is an optimum pH value in the pregel solution where the maximum gelation rate can be obtained. The maximum gelation rate is found at 0.3 pH units above the reported pK value for the ! amino groups of chitosan, i.e. at 6.6 [33]. When the pH is increased from 4.5 to 7, more amino groups become neutral and reactive. This increases the effective free amine concentration and the rate of

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129

Fig. 7. Initial increase in storage modulus per unit time *G@/*t versus pH in the pregel solution for 5 mg/ml CS-CHO-60, 0.83 mg/ml Chit-FD-51 (s), for 10 mg/ml CS-CHO-20, 0.79 mg/ml Chit-FD-51 (h), and for 5 mg/ml CS-CHO-20, 0.79 mg/ml Chit-FD-51 (e) at 30°C. The solid line depicts a least squares fit of a power law for 5 mg/ml CS-CHO-60. The upper panel shows light transmittancy (*) at j"660 nm of 5 mg/ml Chit-FD-51 versus solution pH.

Schiff-base formation. However, the discrepancy between the power-law coefficient estimated using the data obtained from the pH-series (1.4) and the value obtained by using different values of C at pH 7.0 (1.7) suggests that additional mechanisms C)* also are active in this pH range. The fact that H O` is involved in formation of 3 some of the intermediates in the Schiff-base reaction is one possible explanation (Scheme 2), but this possibility is not pursued quantitatively here. Increasing the pH above 7.0 yields alternative mechanisms that may influence the gelation kinetics: (1) reduced concentration of H O`, and (2) decreased solubility of the chitosan [32]. 3 The solubility of chitosans is reported [32] to depend strongly on the degree of acetylation, and this was explored for the F sample used here by determining the D light transmittancy as a function of pH (Fig. 7, upper panel). The data show that the transmittance remains constant from pH 5.2 to pH 7.0, but decreases rapidly when pH is increased beyond pH 7.0. This indicates that the increase of the chitosan solution turbidity when the pH is increased corresponds to onset and subsequent increase in chitosan aggregation. The influence of this increased aggregation can be described as an increase in the wastage reactions compared to that at pH 7, yielding an increase in C . C)*,0 4.5. Effect of temperature on gelation kinetics The kinetic parameters *G@/*t and the time needed to yield the initial 10 Pa increase in G@, t as a function of gelation temperature, ¹, are shown in Fig. 8. *G{/10 P!

130

B. Guo et al. / Polymer Gels and Networks 6 (1998) 113—135

Fig. 8. (A) Initial increase in the storage modulus per unit time, *G@/*t (s), and the time needed to obtain initial 10 Pa increase in the storage modulus G@, t (h), as a function of gelation temperature. The *G{/10 P! chitosan concentration equals 0.83 mg/ml, CS-CHO-60 sample concentration equals 5 mg/ml at pH 7.6. (B) The same data as in Fig. 8A presented versus 1/¹ according to the Arrhenius law for activation energy. The lines depict least square fit to Arrhenius law.

Increasing ¹ yields an increase in *G@/*t, and decrease in t in the temper*G{/10 P! ature range from 15 to 40°C. This suggests that the crosslinking is favoured when temperature is increased. The influence of T on the gelation kinetics can be used to yield an apparent activation energy for the crosslink reaction process by employing Arrhenius law. Fit of Arrhenius law to either ln(*G@/*t) or ln(1/t ) versus 1/¹ *G{/10 P! (Fig. 8B) yields activation energies E of 43.3 or 43.4 kJ mol~1, respectively. Note that ! the two kinetic parameters *G@/*t and 1/t are measured here because it may *G{/10 P! be important to distinguish between the two approaches for determination of E [34]. ! The finding that the E obtained using the two different approaches are equal suggests ! that both (*G@/*t) and 1/t reflect molecular processes. *G{/10 P! 4.6. Determination of the gel-point In the above experiments, *G@/*t was the experimental parameter used to correlate the various experimental variables. Frequency scans throughout the gelation in order to determine the gel point according to the Winter-Chambon criterion, i.e., the rheological gel-point is where G@(u) and GA(u) are parallel, GA"tan(kn/2)G@&uk, where 0(k(1, did not yield the expected cross-over plots of tan d versus time using u as a parameter [35—38]. One reason for this is probably a large contribution from entanglements at the concentration used for the high M samples. However, shifting 8 the experimental frequency window to lower values may have allowed determination of the critical gel-point, but this is not possible in the present system because, for the lower frequencies, G@ and GA change significantly during the needed data collection time. Determination of the gel-point was therefore explored using an ultrasonically

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131

Fig. 9. (A) Experimentally determined G@(u) (open symbols) and GA(u) (filled symbols) for 4.7 mg/ml sonicated scleroglucan and 0.25 mg/ml (r"[NH ]/[CHO]"0.70) (s, d), 0.225 mg/ml (r"0.63) (h, j), 2 and 0.2 mg/ml (r"0.56) (*, m) Chit-FD-51 after 68 h incubation at 20°C. (B) Power law region of G@(u) and GA(u) of the r"0.63 sample of Fig. 9A.

depolymerized scleroglucan sample. Fig. 9A shows G@(u) and GA(u) for 4.7 mg/ml sonicated scleroglucan mixed at pH 7.0 with Chit-FD-51 concentration of 0.25 mg/ml (r"[NH ]/[CHO]"0.70), 0.225 mg/ml (r"0.63) and 0.2 mg/ml (r"0.56) after 2 incubation for 68 h. The corresponding frequency sweeps from 8]10~3 to 8 Hz reveal an obvious transition from pregel solution (r"0.56) to postgel network (r"0.70) rheological behaviour. The sample measured for chitosan concentration of 0.25 mg/ml (r"0.70) yields that G@ is larger than GA and that there is an apparent plateau at the lower range of the experimentally accessible frequencies. This implies that extrapolation to uP0, should yield that G@ is larger than 0, showing a typical elastomer mechanical properties. At the intermediate crosslink concentration (r"0.63), a clear power law region is seen in Fig. 9B, where both G@(u) and GA(u)

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B. Guo et al. / Polymer Gels and Networks 6 (1998) 113—135

plotted as double logarithmic scale are well fitted to straight lines. The overall slopes (i.e., exponents k) are estimated to equal 0.674$0.004 for GA(u) and 0.648$0.012 for G@(u), with a mean value of 0.661$0.013. Similar values of the exponent k have been observed for several other systems with imbalanced stoichiometry [35—38]. 4.7. Some elastic properties of the scleraldehyde—chitosan gels The emphasis in this paper is on the kinetics of scleraldehyde-chitosan gel formation. However, a few additional data on the gel elasticity determined for the 24 h cured gels will be discussed. The frequency dependence of the storage modulus (Fig. 10A) shows that a nearly frequency independent plateau is obtained in the experimental window; the slopes of G@ vs u being rather small for the 24 h cured gels of 9.7 mg/ml CS-CHO-60. The actual value of G@ at a selected frequency within this series shows

Fig. 10. Experimentally determined G@ versus u (A) and ¹(B) of scleraldehyde—chitosan gels, the concentration for CS-CHO-60 sample is 9.7 mg/ml, pH 7.6 and the chitosan concentration varied from 0.83 mg/ml (s), 1.15 mg/ml (*), to 1.43 mg/ml (h). The gel has been cured for 24 h before the temperature scan, and the corresponding frequency scan of storage modulus G@ before changing the temperature were shown in Fig. 10A.

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133

that increasing the crosslink density yields an increase in G@. The number of observations, however, are too few to allow a quantitative analysis of the correlation between the experimental variables and the (pseudo-)equilibrium gel properties. The temperature dependence of the equilibrium storage modulus G@ for 9.7 mg/ml CS-CHO-60 samples mixed with different amounts of 5.0 mg/ml chitosan solution is shown in Fig. 10B. The temperature coefficient of the storage modulus (*G@/*¹) was calculated using linear regression. The temperature coefficient for scleroglucan— chitosan gels changes from positive for the lowest chitosan concentration (lowest crosslinked gels), to negative in the highest chitosan concentration (highest crosslinked gels). This suggest a change from entropy dominated elasticity at low degree of crosslinking changing to enthalphic elasticity at increasing degree of crosslinking. This change in origin of elasticity may occur because the length of the elastically active chains relative the chain stiffness is expected to decrease, an eventually entering a region where non-entropic energy of deformation modes starts to become significant for the increased crosslink densities.

Acknowledgements The authors would like to thank Mr Clemens J. Belle, Germany, and Mrs Solrun Aarflot, for determining the aldehyde and carboxyl contents on scleroglucan side chains after two steps oxidation modification. One of the authors (B. Guo) is indebted to the Loan Foundation of Educational Ministry of Norway for financial support. We gratefully acknowledge the helpful discussion with Dr Bj+rn E. Christensen, regarding the chemistry of the system. The chitosan sample was kindly provided by Dr Kjell M. Vas rum, Trondheim. The scleroglucan sample was kindly provided by Sanofi Bio industry, France.

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