Behaviour of Amphiphilic Polysaccarides in Aqueous Medium Cqb081d

35Rinaudo, M.: Behaviour of amphiphilic polysaccharides in aqueous mediumjunio, 2008

D.R. TIP Revista Especializada en Ciencias Qumico-Biolgicas, 11(1):35-40, 2008

BBBBBEHAEHAEHAEHAEHAVIOURVIOURVIOURVIOURVIOUR OFOFOFOFOF AMPHIPHILICAMPHIPHILICAMPHIPHILICAMPHIPHILICAMPHIPHILIC POLPOLPOLPOLPOLYSACCHARIDESYSACCHARIDESYSACCHARIDESYSACCHARIDESYSACCHARIDESINININININ AQUEOUSAQUEOUSAQUEOUSAQUEOUSAQUEOUS MEDIUMMEDIUMMEDIUMMEDIUMMEDIUM

Marguerite RinaudoMarguerite RinaudoMarguerite RinaudoMarguerite RinaudoMarguerite RinaudoCentre de Recherches sur les Macromolcules Vgtales

(CERMAV-CNRS) affiliated with Joseph Fourier University, BP53, 38041Grenoble cedex 9 (France). E-mail: [email protected]

Nota: Artculo recibido el 02 de mayo de 2008 y aceptado el 27 de mayode 2008.

ARTCULO ORIGINAL

AAAAABSTRACTBSTRACTBSTRACTBSTRACTBSTRACTThis paper describes the rheological and thermal behaviour of alkylated chitosan and hyaluronan derivativesand methylcellulose, Their behaviour is characterized by the formation of hydrophobic domains which may beconsidered as crosslinked points. Then, depending on the thermodynamic conditions (temperature, saltconcentration), an aqueous solution transforms to a 3D-physical network formed on the basis of hydrophobiccooperative interactions.Key Words: Alkylated chitosan, alkylated hyaluronan, amphiphilic polysaccharides, methylcellulose, rheologicalbehaviour.

RRRRRESUMENESUMENESUMENESUMENESUMENEste trabajo describe el comportamiento reolgico y trmico de quitosano y hialuronano alquilados y demetilcelulosa. Dicho comportamiento est caracterizado por la formacin de dominios hidrofbicos que puedenconsiderarse como puntos de entrecruzamiento. Por tanto, en dependencia de las condiciones termodinmicas(temperatura, salinidad), las disoluciones acuosas pueden transformarse en una red fsica tridimensional basadaen interacciones cooperativas de carcter hidrofbico.Palabras Clave: Quitosano alquilado, hialuronano alquilado, polisacridos anfiflicos, metilcelulosa, comportamientoreolgico.

n this paper, different amphiphilic polysaccharidesbased on chitin, cellulose and hyaluronan are describedand few of their properties are given. First, chitin is apoly ((1-4)-N-acetyl-D-glucosamine); it is a natural

polysaccharide of major importance, first identified in 1884(Figure 1a). This biopolymer is synthesized by an enormousnumber of living organisms (schrimps, crabs).1 As manypolysaccharides, chitin has some hydrophobicity and in aqueousNaOH it can form a lower critical solution temperature (LCST)solution with a critical temperature around 30C.2 Thischaracteristic temperature is very important to characterize theinfluence of temperature on the behaviour of a polymer insolution: at a temperature lower than the LCST, the polymer issoluble and one gets one phase; over the LCST, two phases

IIIIIIIIIINTRODUCTIONNTRODUCTIONNTRODUCTIONNTRODUCTIONNTRODUCTION

separate corresponding to a concentrated and a diluted solutionsat equilibrium. After deacetylation in alkaline conditions, onegets chitosan which becomes soluble in aqueous acid solutiondue to the protonation of the NH2 group at C-2 position of theD-glucosamine. The different chitosans are characterized bytheir average number of acetyl groups per monomeric unit(degree of acetylation,DA)(Figure 1a). Using the reductiveamination, a series of amphiphilic chitosan derivatives wereproduced from aldhehydes with different chain lengths (Cn from3 to 14) at controlled DS (usually lower than 10% to maintainwater solubility in acidic conditions)3,4. Alkylated chitosans withgood solubility in acidic conditions (pH < 6) have a number ofvery interesting properties.

Hyaluronic acid (HA), a linear polysaccharide composed ofrepeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronic acid (Figure 1b), is the only non sulfatedglycosaminoglycan found in the extracellular matrix. HA is an

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attractive polymer for the development of new biocompatiblematerials with many applications in viscosupplementation, tissueengineering, and drug delivery5,6. This biopolymer has been thesubject of various chemical modifications leading for example tochemically cross-linked materials with successful application inthe treatment of osteoarthritis and HA-drug conjugates havingcontrolled-release and cell-targeted properties7, 8. We haverecently prepared new water-soluble alkylated derivatives ofhyaluronan (HA-C10).9,10

The third polymer examined is the methylcellulose; this derivativeof cellulose is a LCST polymer (the LCST is located at 292C)which gives a strong physical gel on heating over 50C 11. It isoften used in food applications.

In this paper, it is intended to summarize the behaviour of thesethree systems we have previously studied and which are basedon polysaccharide derivatives. We intend to show the originalproperties that such amphiphilic copolymer like systems whichcan be considered as smart materials being thermosensitive, saltand /or pH-sensitive.

EEEEEXPERIMENTALXPERIMENTALXPERIMENTALXPERIMENTALXPERIMENTALMethylcellulose used is a commercial sample (Methocel A4MPremium from Dow Chemical; the average degree of substitutionwas 1.8) (Figure 2). All the solutions tested were prepared bydirect dissolution at ambient temperature in 0.1M NaCl solutionand fixed at 6g/L.

Alkyl-chitosans were prepared by Rinaudo et al. 4; they are

dissolved in acidic conditions (acetic acid 0.3M) in the presenceor the absence of an external salt (sodium acetate).

Alkyl-hyaluronan under its sodium salt form is dissolved inwater; NaCl is added when necessary to get a 0.15 M NaCl saltconcentration, reflecting the biological conditions. HA isproduced from a bacterial strain by ARD Cy (Pomacle, France).The dynamic rheological measurements were performed on a AR1000 from TA Instruments using a plate-cone geometry; the conehas 4 cm as diameter and 3 59angle. The temperature wascontrolled by the Pelletier plane in the range of 20 or 37C up to65 or 70C with a controlled rate of temperature variation (3/minfor methylcellulose). The strain percent was selected to be in thelinear domain. A film of silicone oil was carefully disposes on theexamined solution such as to prevent its evaporation.

DSC experiments were performed on methylcellulose with amicrocalorimeter DSCIII (SETARAM, France) in the same rangeof temperature as adopted for rheological measurements andwith the same solutions but with a rate of temperature change of0.5 /min for heating and cooling cycles.

RRRRRESULTSESULTSESULTSESULTSESULTS ANDANDANDANDAND DISCUSSIONDISCUSSIONDISCUSSIONDISCUSSIONDISCUSSIONA- Alkylated chitosansA- Alkylated chitosansA- Alkylated chitosansA- Alkylated chitosansA- Alkylated chitosansAlkyl-chitosans were prepared by reductive amination on thefree NH2 in C-2 position of the D-glucosamine units with adegree of substitution around 0.05 to preserve the water solubilityin acidic conditions. They were described previously in ourlaboratory3,4. There are two important effects of the grafting ofalkyl-chains on chitosan as soon as the alkyl-chain length islarge enough (carbon chain n = 10 or better 12):

- first, these derivatives exhibit surface activity and they werecompared with corresponding low molecular weightsurfactants12,13; for the same amount of alkyl chains with thesame length, they have a relatively low effect on the decreaseof the surface tension but they improve much the stability ofthe interfacial film; it was clearly demonstrated that a simplesurfactant and modified chitosan have completely differentbehaviour12-14. It is interesting to mention that alkyl-chitosansare compatible with neutral and cationic surfactants; it wasdemonstrated that cationic surfactant adsorbed on the alkylchain grafted on chitosan, promotes its solubilisation.

Figure 1.Chemical structure of the repeat unit of chitin (whenFigure 1.Chemical structure of the repeat unit of chitin (whenFigure 1.Chemical structure of the repeat unit of chitin (whenFigure 1.Chemical structure of the repeat unit of chitin (whenFigure 1.Chemical structure of the repeat unit of chitin (when

DA>0.9) and chitosan (when DA0.9) and chitosan (when DA0.9) and chitosan (when DA0.9) and chitosan (when DA0.9) and chitosan (when DA


- secondly, they increase considerably the viscosity of aqueoussolution due to hydrophobic inter-chain interactions; especiallyfor C-12 chain length and a DS~0.05, a physical gel is obtainedin dependence on the pH15 and on salt concentration4. Figure3 indicates that in absence of external salt, the behaviour is thatof a solution with the elastic modulus G lower than the viscousmodulus G in all the range of frequencies covered; in thepresence of salt able to screen the electrostatic repulsionsbetween chains, hydrophobic interactions dominate and a gel-like behaviour is observed with G>G.

These gels are characterized by rheology and correspond to astorage modulus G larger than the loss modulus G; thephysical gel formation results from a balance betweenelectrostatic repulsions between the positively charged chitosanchains and hydrophobic attraction due to alkyl chains mainly inrelation with their length3,4. The hydrophobic domains formed inthe systems are important to adsorb hydrophobic moleculessuch as pyrene (a fluorescent probe used to evidence thesedomains in dilute solution);it has been previously shown 4 thatthese associations can be destroyed by addition of cyclodextrinswhich are known to complex the alkyl chains of surfactants intheir hydrophobic cavity16 .

Alkylated hyaluronanThe chemical structure of hyaluronan (also named hyaluronic

acid or HA) is prepared under the sodium salt form. It is a verysoluble polymer in water8. Based on our previous knowledge onamphiphilic polymers, it was decided to modify HA and to graftalkyl chains of different lengths and different amounts on the HAbackbone. These derivatives were synthesized by Creuzet et al.9and Rinaudo et al.10 under mild aqueous conditions. The firststep was to prepare a HA derivative selectively modified withadipic dihydrazide (HA-ADH) with a substitution degree of 0.08;then, alkyl chains with different chain lengths were introducedwith a substitution degree around 0.05 9, 10. These polymers beingstill water-soluble exhibit associating properties in the presenceof external salt. Formation of hydrophobic domains isdemonstrated from fluorescence measurements in the presenceof pyrene as a probe. Then, it is shown that the viscosity ofpolymer solutions in 0.15 M NaCl becomes non-Newtonian forHA grafted with C-10 and C-12 chains and increases rapidlywhen the polymer concentration increases over 1g/L. When thepolymer concentration increases, a gel-like behaviour inducedby external salt addition was observed from rheologicalmeasurements (Figure 4). In absence of salt, the alkylatedhyaluronan behaves as a viscoelastic solution but when externalsalt is added, a gel formation occurs due to the screening ofelectrostatic repulsions between polyanionic chains.

B- Gelation of methylcelluloseB- Gelation of methylcelluloseB- Gelation of methylcelluloseB- Gelation of methylcelluloseB- Gelation of methylcelluloseCommercial water soluble methylcelluloses (with a degree of

Figure 3. Rheological behaviour of alkylated chitosan (chit-C12) at 7g/L in 0.3M acetic acid (G Figure 3. Rheological behaviour of alkylated chitosan (chit-C12) at 7g/L in 0.3M acetic acid (G Figure 3. Rheological behaviour of alkylated chitosan (chit-C12) at 7g/L in 0.3M acetic acid (G Figure 3. Rheological behaviour of alkylated chitosan (chit-C12) at 7g/L in 0.3M acetic acid (G Figure 3. Rheological behaviour of alkylated chitosan (chit-C12) at 7g/L in 0.3M acetic acid (G ! , G, G, G, G, G" , , , , , * ! ))))) and in 0.3M aceticand in 0.3M aceticand in 0.3M aceticand in 0.3M aceticand in 0.3M aceticacid/0.2M sodium acetate (G acid/0.2M sodium acetate (G acid/0.2M sodium acetate (G acid/0.2M sodium acetate (G acid/0.2M sodium acetate (G ! , G, G, G, G, G" , , , , , * " ).).).).).

0.01000 0.1000 1.000 10.00frequency (Hz)

1.000E-4

1.000E-3

0.01000

0.1000

1.000

10.00

100.0

G'

(Pa

)

1.000E-4

1.000E-3

0.01000

0.1000

1.000

10.00

100.0G

'' (Pa

)

0.01000

0.1000

1.000

10.00

100.0

|n*|

(P

a.s

)

Chit C12 -presence of saltChit C12-absence of salt


Figure 4. Rheological behaviour of alkylated hyaluronan (HA-C10) at 8g/L in water (GFigure 4. Rheological behaviour of alkylated hyaluronan (HA-C10) at 8g/L in water (GFigure 4. Rheological behaviour of alkylated hyaluronan (HA-C10) at 8g/L in water (GFigure 4. Rheological behaviour of alkylated hyaluronan (HA-C10) at 8g/L in water (GFigure 4. Rheological behaviour of alkylated hyaluronan (HA-C10) at 8g/L in water (G! , G, G, G, G, G" , , , , , * " ) and in 0.15M NaCl (G) and in 0.15M NaCl (G) and in 0.15M NaCl (G) and in 0.15M NaCl (G) and in 0.15M NaCl (G! ,,,,,GGGGG" , , , , , * ! ).).).).).

0.01000 0.1000 1.000 10.00frequency (Hz)

1.000E-5

1.000E-4

1.000E-3

0.01000

0.1000

1.000

10.00

100.0G

' (P

a)

1.000E-5

1.000E-4

1.000E-3

0.01000

0.1000

1.000

10.00

100.0

G'' (P

a)

0.1000

1.000

10.00

100.0

|n*|

(P

a.s

)

HA 8g/L in H2OHA 8g/L in 0.15M NaCl

Figure 5. Influence of temperature on the rheological behaviour of a 6g/L solution of methylcellulose in 0.1M NaCl.Figure 5. Influence of temperature on the rheological behaviour of a 6g/L solution of methylcellulose in 0.1M NaCl.Figure 5. Influence of temperature on the rheological behaviour of a 6g/L solution of methylcellulose in 0.1M NaCl.Figure 5. Influence of temperature on the rheological behaviour of a 6g/L solution of methylcellulose in 0.1M NaCl.Figure 5. Influence of temperature on the rheological behaviour of a 6g/L solution of methylcellulose in 0.1M NaCl.

(a)

(b)

90.00

80.00

70.00

60.00

50.00

40.00

30.00

20.00

10.00

0

G' (

Pa)

35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75temperature (C)

H2O


Figure 6. DSC curves for methylcellulose at 6g/L in 0.1M NaCl on heating and cooling at 0.5C/min.Figure 6. DSC curves for methylcellulose at 6g/L in 0.1M NaCl on heating and cooling at 0.5C/min.Figure 6. DSC curves for methylcellulose at 6g/L in 0.1M NaCl on heating and cooling at 0.5C/min.Figure 6. DSC curves for methylcellulose at 6g/L in 0.1M NaCl on heating and cooling at 0.5C/min.Figure 6. DSC curves for methylcellulose at 6g/L in 0.1M NaCl on heating and cooling at 0.5C/min.

substitution in the range of 1.7) are amphiphilic polymers andthey associate when temperature increases due to hydrophobicinteractions. The characteristic temperatures for phase transitiondepend on the rate of temperature change; in addition, a largehysteresis is observed between gel formation on heating andmelting of the gel on cooling curves as obtained in rheometry orDSC. An example is given in Figures 5 and 6.

The mechanism was previously analyzed and a two stepsgelation was clearly demonstrated11, 17,18,19: a clear gel forms over30 C in relation with the presence of highly substituted zoneson the cellulosic backbone and a phase separation with formationof a turbid stronger gel is obtained over 50 C. In fact, thisbehaviour is directly related with the chemical modificationprocess. Commercial methylcelluloses are produced fromcellulose in a heterogeneous process in which amorphous zonesare more substituted than crystalline ones; the resultingmicrostucture is a blockwise distribution of the methylsubstituents along the cellulose backbone. This was provedwhen methylcelluloses were prepared under homogeneousconditions and have a completely different phase diagram19. Itis concluded that the interchain interactions are based onhydrophobic interactions depending on the degree of substitutionof cellulose but also on the distribution of methyl substituentsalong the cellulosic backbone.

Figure 5 demonstrates the strong increase of the storage modulusG when temperature increases; a strong gel is formed withmethylcellulose at 70 C (with G independent on the frequencyin the range covered in this experiment and G>G)19. Aftercooling down to 37C, the rheological parameters remain higherthan after direct dissolution at low temperature in relation withthe temperature range for clear gel stability. The change in thebehaviour of methylcellulose is also clearly shown by DSC aspresented in figure 6; on heating, the phase transition has a peakat 59.5C (formation of the turbid gel corresponding to a largeincrease in G) and on cooling the peak occurs at 31.7C (gel-soltransition). The temperature of 37C is still in the range of the cleargel.

CCCCCONCLUSIONONCLUSIONONCLUSIONONCLUSIONONCLUSIONThis paper summarizes the behaviour of three derivatives ofpolysaccharides which have the chemical structure of amphiphilicblock and randomly grafted copolymers. These amphiphilicpolymers have surface active properties (and behave as protectivecolloids) but especially they increase the viscosity of aqueoussolution in dependence of the temperature, pH and saltconcentration.

In given thermodynamic conditions, a reversible 3D-network isformed. Figure 7 presents a schematic description of these

Heat flow(mw)

Onset point: 37,03 CPeak 1 top: 31,71C

Enthalpy/ J/g: -0,1185 (Exothermic effect)

Onset point: 56,58 CPeak 1 top: 59,52C

Enthalpy/ J/g: 0,0948 (Endothermic effect)

25 30 35 40 45 50 55 60 65 70 75 Temperature/ C

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

-0.08

-0.10

-0.12

-0.14

-0.16

-0.18

-0.20


physical gel: (a) represents the alkyl chain grafted chitosan orhyaluronan; (b) corresponds to gelation of methylcellulose.

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5. Rinaudo, M. Characterization and Properties of Some Polysaccharides

Figure 7. Schematic representation of physical gel formed byFigure 7. Schematic representation of physical gel formed byFigure 7. Schematic representation of physical gel formed byFigure 7. Schematic representation of physical gel formed byFigure 7. Schematic representation of physical gel formed byhydrophobic interactions on alkylated chain grafted copolymershydrophobic interactions on alkylated chain grafted copolymershydrophobic interactions on alkylated chain grafted copolymershydrophobic interactions on alkylated chain grafted copolymershydrophobic interactions on alkylated chain grafted copolymers(a) and on block wise copolymers (b).(a) and on block wise copolymers (b).(a) and on block wise copolymers (b).(a) and on block wise copolymers (b).(a) and on block wise copolymers (b).

(a)

(b)

Used as Biomaterials. In Macromolecular Symposia (WorldPolymer Congress-MACRO 2006) vol. 245/246, 549-557 (2006).

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7. Milas, M., et al. Comparative rheological behavior of hyaluronanfrom bacterial and animal sources with cross-linked hyaluronan(hylan) in aqueous solution. Biopolymers 59(4), 191-204(2001).

8. Milas, M. & Rinaudo, M. Characterization and properties ofhyaluronic acid (hyaluronan) . In Polysaccharides: structuraldiversity and functional versatility. 2nd ed. (ed. Severian, D.)Chap. 22, pp. 535-549 (Marcel Dekker, New York, 2005).

9. Creuzet, C., Kadi, S., Rinaudo, M. & Auzely-Velty, R. Newassociative systems based on alkylated hyaluronic acid.Synthesis and aqueous solution properties. Polymer 47(8),2706-2713 (2006).

10. Rinaudo, M., Auzely, R., Kadi, S., Bresin, A. & Kubik, E. Newderivatives of hyaluronic acid, their preparation process andtheir uses. International patent n WO2007/059890 (2007).

11. Hirrien, M., Chevillard, C., Desbrires, J., Axelos, M.A.V. &Rinaudo, M. Thermogelation of methylcelluloses: new evidencefor understanding the gelation mechanism. Polymer 39(25),6251-6259 (1998).

12. Babak, V., Lukina, I., Vikhoreva, G., Desbrires, J. & Rinaudo, M.Interfacial properties of dynamic association between chitinderivatives and surfactants. Colloids and Surfaces, A:Physicochemical and Engineering Aspects 147 (1-2), 139-148(1999).

13. Babak, V.G., Rinaudo, M., Desbrires, J., Vikhoreva, G.A. &Michalski, M.-C. The effect of alkyl chain length of a polysoapon the surface activity of its complexes with cationic surfactants.Mendeleev Communications (4), 149-151 (1997).

14. Nonaka K.I., Kazama, S., Goto, A., Fukuda, H. & Yoshioka, H. SpinProbe Study on the Interaction of Chitosan-Derived PolymerSurfactants with Lipid Membrane. Journal of Colloid andInterface Science 246(2), 288-295 (2002).

15. Desbrires, J., Rinaudo, M. & Chtcheglova, L. Reversiblethermothickening of aqueous solutions of polycations fromnatural origin. Macromolecular Symposia 113, 135-149 (1997).

16. Eli, W., Chen, W. & Xue, Q. The association of anionic surfactantswith -cyclodextrin. An isothermal titration calorimeter study.Journal of Chemical Thermodynamics 31(10), 1283-1296(1999).

17. Desbrires, J., Hirrien, M. & Rinaudo, M. Relation between theConditions of Modification and the Properties of CelluloseDerivatives: Thermogelation of Methylcellulose. ACSSymposium Series 688, 332-348 (1998).

18. Vigouret, M., Rinaudo, M. & Desbrires, J. Thermogelation ofmethylcellulosein aqueous-solutions. Journal de ChimiePhysique et de Physico-Chimie Biologique 9(5), 858-869(1996).

19. Hirrien, M., Desbrires, J. & Rinaudo, M. Physical propertiesof methylcelluloses in relation with the conditions forcellulose modification. Carbohydrate Polymers 31(4), 243-252 (1996).

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Behaviour of Amphiphilic Polysaccarides in Aqueous Medium Cqb081d