_• Polymer Gels and Networks 2 (1994) 65-71 © 1994 Elsevier Science Limited

Printed in Great Britain. All rights reserved 0966-7822/94/$07.00

ELSEVIER

Peculiar Rheological Behaviour of Tetrabutylammonium Hyaluronate in Mixture with its

n-Propyl Ester Derivative in Dimethylsulfoxide

Roberto Gilli, Antonel la Flaibani & Luciano Navarini*

POLY-bi6s Research Centre, LBT-Area di Ricerca, Padriciano 99, 1-34012 Trieste, Italy

(Received 26 July 1993; accepted 14 October 1993)

ABSTRACT

Steady shear and dynamic oscillatory tests were performed both on tetrabutylammonium hyaluronate and on its n-propyl ester derivative in dimethylsulphoxide (DMSO) in the high polymer concentration regime. The viscoelastic behaviour of the individual polymers is typical of an entangled network of disordered polysaccharides. The supramolecular structure of the tetrabutylammonium hyaluronate in DMSO reflects the occurrence of intermolecular association. A peculiar rheological res- ponse of the mixtures in DMSO solution has been found.

INTRODUCTION

Hyaluronan (hyaluronic acid, hyaluronate) is a regularly alternating glycosaminoglycan of the extracellular matrix in mammals and also in the exocellular products of some bacteria. The disaccharide repeating unit is composed of fl(1---~4) D-glucuronic acid (GIcA) linked with f l ( l ~ 3 ) N-acetyl-D-glucosamine. The chain stiffness and the dis- ordered elongated conformation adopted by hyaluronan in aqueous systems I account for the peculiar viscoelastic properties that are relevant to its performance in biological fluids. 2 It has been shown that the inter-residue hydrogen bond pattern, which may contribute to the chain stiffness, is affected by the solvent. On the basis of 1-H NMR studies on sodium hyaluronate oligosaccharides, two different models

* To whom correspondence should be addressed.

65

66 R. Gilli, A. Flaibani, L. Navarini

have been suggested for water and DMSO, respectively? '4 It has also been reported that the solution behaviour of the tetrabutylammonium (TBA) hyaluronate in water is different from that in DMSOJ In particular, NMR and viscosity data have provided evidence for associa- tion of the polyanionic chains at high polymer concentration in DMSO but not in water. It has been suggested that the chain-chain association in DMSO may reflect the role of the tetrabutylammonium ions in favouring intermolecular interactions by parallel screening of the electrostatic repulsions. C(6) GIcA ester derivatives of hyaluronic acid (a new class of biocompatible, bioresorbable uncharged polymers) show a different behaviour in DMSO. Such behaviour has turned out to be similar to that of TBA hyaluronate in water. The lack of association of the ester derivatives in DMSO has been related to the higher segmental flexibility as deduced from NMR data. 5 In order to provide further evidence of the different solution behaviour of TBA hyaluronate and its ester derivatives in DMSO, steady shear and dynamic oscillatory experiments have been performed at high polymer concentration. In fact, the polymer chain association might affect the network structure in the entangled regime and, accordingly, its viscoelastic response. Moreover, the possibility of modulating the extent of TBA hyaluronate association in DMSO has been explored by studying the rheological behaviour in the presence of ester derivatives under the same ex- perimental conditions. The mixtures have shown an unexpected, reproducible rheological behaviour.

E X P E R I M E N T A L

Yetrabutylammoniun hyaluronate (TBA-HA) (Mw = 220000) and the n-propyl ester of hyaluronan (HYAFF9) (Mw--115 000), obtained and prepared as described elsewhere, 5 were solubilized in 0-1 M tet- rabutylammonium bromide (TBABr) in DMSO under prolonged stir- ring at room temperature. Mixtures of the two polymers were prepared by solubilizing the powder blends in 0.1M TBABr in DMSO under prolonged stirring at room temperature. No opalescence was observed. The individual (for pure systems) or the overall (for mixtures) polymer concentration was 6% (w/w) in all experiments. A RV20/CV20 Haake rheometer equipped with a concentric cylinder geometry (sensor system ZB15 with radii ratio 1.078) was employed in steady shear viscosity and dynamic oscillatory measurements. The temperature was set at 25 + O.OI°C by means of a Haake circulating thermocryostat. Dynamic oscillatory experiments were performed within the established linear viscoelastic regime.

Peculiar rheological behaviour of tetrabutylammonium hyaluronate 67

100 I I

• o

, o,o,,O o o • - OQ m A . _ Q ~ O O

eo~ ,~ 10 O A

AA A ~z5

,..-2 Z~&Z~ 0 n,,,

A

1 I I

0 1 1 1 0 1 0 0

~o/rad s-'

Fig. 1. Viscoelastic spectrum of n-propy] hya]uronic acid ester derivative (HYAFF9) in DMSO 0.1 M T B A B r (polymer concentration 6% (w/w)).

RESULTS AND DISCUSSION

In Fig. 1 the viscoelastic spectrum of HYAFF9 in DMSO is shown. In the whole range of frequency investigated a liquid-like behaviour (loss modulus, G">storage modulus, G') is observed. By increasing the frequency, only the tendency of the moduli to approach each other is discernible, without any evidence of crossover. The complex viscosity, rl*, is slightly frequency-dependent and tends to approach a constant value at the lowest frequencies. The overall behaviour is the typical response of an entangled polymer network in the terminal region of frequencies just before the crossover towards the plateau region. The spectrum of TBA hyaluronate is reported in Fig. 2. At low frequencies, G" predominates thus indicating that the network rearranges by flow within the period of one oscillation. Upon increasing the frequency, G' approaches G" and the moduli cross at about 5.0rads -1. At high- frequency values, a solid-like behaviour is observed, with G' greater than G" and both moduli showing little change with frequency. The complex viscosity adopts a profile similar to that of HYAFF9. This kind of viscoelastic behaviour is typically exhibited by random coil polysac- charides in concentrated solutions and it closely resembles that of hyaluronic acid in water. 6

68 R. Gilli, A. Flaibani, L. Navarini

1 0 0 0 i i

m. 000000 a. 0 ° ° ° ° ° *

" """6 I 100 * o A o A : 0 " ~ 0 0 ZXA

Lb z~ A

10 I I 0,1 1 10 100

(.0 / r a d . s -~

Fig. 2. Viscoelastic spectrum of t e t rabu ty lammonium hyaluronate in D M S O 0.1 M T B A B r (polymer concentrat ion 6% (w/w)).

The viscoelastic behaviour of the TBA-HA sample differs mainly quantitatively (about one order of magnitude), rather than qualita- tively, from that of HYAFF9. In spite of the different molecular weights of the two polymers, alignment and interactions of stiff chain segments (polymer-polymer association) cannot be excluded a priori , and this may account for the 'stout' entangled network formed by the tet- rabutylammonium hyaluronate in DMSO. Under this hypothesis, it would seem rather plausible that the n-propyl groups in the HYAFF9 sample play a role in preventing polymer-polymer associations. In the attempt to clarify this point, rheological measurements have been performed on mixtures of the two polymers in DMSO under identical experimental conditions. Unexpectedly, the steady shear viscosity of the mixtures deviates remarkably (positive deviation) from additivity by varying the composition. As shown in Fig. 3, the steady shear viscosity at 2-1 s -~ reaches a maximum when the mixture composition ranges from 40% to 50% (w/w) TBA-HA. In this range of composition, the flow curves show a shear-thinning behaviour in the whole range of shear rates investigated (0.3-300s -]) without evidence for an upper Newtonian plateau, at variance with those of the pure systems. Despite the different profile of the flow curves, the gradient of the double logarithmic plot approaches the value of about -0 .7 as generally found

Peculiar rheological behaviour of tetrabutylammonium hyaluronate 69

10 3 , , , I

10'

I0' K , i ,

0 20 40 60 80 I00

TBA-HA/% W/W

Fig. 3. Steady shear viscosity, r~, at 2.1s -~ as a function of mixture composition (% (w/w) TBA-HA) (Polymer concentration 6% (w/w)).

for entangled systems. The result was fully reproducible, even for slightly differing sample preparation conditions. These findings clearly indicate an interaction between the two polymers in DMSO solution. However, in order to ascertain whether the interaction is synergistic, dynamic oscillatory tests were performed. The viscoelastic spectrum of the mixture containing 40% (w/w) TBA-HA is reported in Fig. 4. In the whole range of frequencies, a solid-like behaviour with G' greater than G" is evident. The frequency dependence and the absolute values of the moduli are comparable to those of the TBA-HA at the highest frequencies only. Moreover, at low frequency values, the presence of HYAFF9 in the mixture with TBA-HA strongly enhanced the elastic component of the system. In such a range of frequencies, both G' and G" tend to approach each other, although the crossover frequency cannot be determined due to instrumental limitations. The complex viscosity is strongly frequency-dependent and adopts an approximate power law in the whole range of frequencies. Similar results have been obtained on the 1:1 mixture. The overall response resembles that of an entangled system, although it does not appear to be a mere composite of those of the individual polymers.

This unexpected interaction between the two polymers could be ascribed to the mutual exclusion of incompatible coils or to the association of stiff 'in register' chain segments that forms mixed

70 R. Gilli, A. Flaibani, L. Navarini

1000 I I

~ 0 o 0 o00°° *~ 0 O0

z~Az~ 00000@@ .. 000 @00000

O00~_@l@ o 100 • O O O 0 ~ L ~ A

~ zx Z~

zx

~ zxzx z~

A

10 I I 0.1 I 10 100

o ~ / r ad .s -~

Fig. 4. Viscoelastic spectrum of mixture TBA-HA (40% (w/w)) and HYAFF9 (60% (w/w)) in DMSO 0.1 M TBABr (polymer concentration 6% (w/w)).

aggregates. In view of the chemical and geometrical compatibility between the two polymers and their chain stiffness, the latter hypothesis cannot be fully disregarded, although the intermolecular interactions are probably weak and not specific (no evidence of gel formation). However, it is clear that the presence of HYAFF9 in the mixtures strongly modifies the nature and the extent of intermolecular associa- tions. One plausible explanation may reside in the competition between chain-chain association and chain-solvent association. The TBA-HA is soluble in DMSO thanks to the condensation of the bulky and highly hydrophobic tetrabutylammonium counterions onto the polymer.

Accordingly, in parallel to the screening of the electrostatic repul- sions exerted by the TBA ions and the corresponding stabilization of intramolecular hydrogen bonding (enhanced stiffness), solvent- mediated chain-chain interactions may promote the association of the polymer at high polymer concentrations. The uncharged low-polarity HYAFF9 molecules are characterized by a higher segmental flexibility in DMSO 7 and therefore the loss of conformational entropy involved in the association is not energetically counterbalanced even at high polymer concentrations. The same mechanism for the association of TBA-HA in DMSO may be invoked to interpret the observed peculiar rheological response of the mixtures. In fact, in the mixtures, the

Peculiar rheological behaviour of tetrabutylammonium hyaluronate 71

TBA-HA chain-chain interactions may be enhanced due to the competition of HYAFF9 in chain-solvent interactions, with consequent increase in the effective concentration of both polymers. It remains to be demonstrated whether a similar effect occurs by changing the chemical identity of the ester derivatives. Further studies are in progress.

ACKNOWLEDGEMENTS

The research activities leading to this work were partially developed with relation to a contract with the National Program of Research on Advanced Biotechnologies by the Fidia S.p.A. from the Italian Ministry of University and Scientific Technological Research. We are indebted to Prof. S. Paoletti and to Prof. V. Crescenzi for helpful discussions.

REFERENCES

1. Gamini, A. Paoletti, S. & Zanetti, F., Chain rigidity of polyuronates: Static light scattering of aqueous solutions of hyaluronate and alginate. In Laser Light Scattering in Biochemistry, Vol. 99, ed. S. E. Harding, D. B. Sattelle & V. A. Bloomfield. Royal Soc. Chem., Cambridge, 1992, pp. 294-311.

2. Balazs, E. A. & Gibbs, D. A., The rheological properties and biological function of hyaluronic acid. In Chemistry and Molecular Biology o f the Intercellular Matrix, ed. Balazs, E. A. Academic Press, London, 1970, 3, pp. 1241-1253.

3. Scott, J. E., Heatley, F. & Hull, W., Secondary structure of hyaluronate in solution. Biochem. J., 220 (1984) 197-205.

4. Cowman, M. K., Cozart, D., Nakanishi, K. & Balazs, E. A., 1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solu- tion: The amide proton environment. Arch. Biochem. Biophys., 230 (1984) 203-12.

5. Kvam, B. J., Atzori, M., Toffanin, R., Paoletti, S. & Biviano, F., 1H and 13C-NMR studies of solutions of hyaluronic acid esters and salts in methyl sulfoxide: Comparison of hydrogen-bond patterns and conformational behaviour. Carbohydr. Res., 230 (1992) 1-13.

6. Morris, E. R., Rees, D. A. & Welsh, E. J., Conformation and dynamic interactions in hyaluronate solutions. J. Mol. Biol., 138 (1980) 383-400.