Biomacromolecules 2005, 6, 61-67
61
Role of the pH on Hyaluronan Behavior in Aqueous Solution
Iuliana Gatej,†,‡ Marcel Popa,‡ and Marguerite Rinaudo*,†
Centre de Recherches sur les Macromolécules Végétales, CNRS, affiliated to Joseph Fourier University,
BP 53, 38041 Grenoble Cedex 9, France, and Université Technique “Gh.Asachi”,
67 Boulevard D.Mangeron, 6600 Iasi, Romania
Received May 19, 2004; Revised Manuscript Received July 20, 2004
In this paper, we have examined the behavior of hyaluronan solutions at different pH values. A slight
degradation is observed in acidic conditions (pH ) 1.6) and basic medium (pH ) 12.6) from molecular
weight distribution analysis, but the rheological behavior is relatively not influenced much by the pH at the
exclusion of two domains: around pH ) 2.5, a gel-like behavior is shown and is attributed to cooperative
interchain interactions due to the reduction of the polymer net charge and may be the protonation of the
acetamido groups; for pH > 12, the decrease of viscosity is mainly attributed to a reduction of the stiffness
of the polymeric backbone in alkaline conditions due to the partial breakage of the H-bond network.
Introduction
Hyaluronan (also called hyaluronate, hyaluronic acid, or
HA) was previously extracted from bovine vitreous humor,
rooster combs, or umbilical cords; then it was very expensive
and certainly associated with some proteins. Now the same
polysaccharide was recognized to be produced by bacteria
such as Streptococcus zooepidemicus on a large scale with
a good yield and a large degree of purity. Then the price
decreased, allowing the development of its applications, but
its contribution depends on the conditions of use.
The chemical structure of HA is represented as a linear
polyelectrolyte based on β1-4-D-glucuronic acid and β1-3N-acetyl-D-glucosamine alternated in the repeat unit. The
main uses of HA are ophthalmic surgery,1,2 arthritic treatment, and, more recently, cosmetics.3,4 The work developed
for a few years in our laboratory concerned mainly the
bacterial HA under the native form in neutral pH. Loosely
cross-linked HA (named hylan) allowing better rheological
performances and especially a gel-like behavior in a large
range of frequencies is also produced for viscosupplementation in arthrosis treatment.5
This paper concerns the role of pH on the physicochemical
properties of HA in aqueous solutions.
Experimental Section
HA is a bacterial sample produced by ARD Cy (Pomacle,
France). It is prepared under the sodium salt form6 and
characterized by steric exclusion chromatography (SEC)
using a Waters Alliance GPCV2000 (U.S.A.) equipped with
three detectors on-line: refractometric and viscometric
detectors associated with a multiple-angle laser light scattering detector from Wyatt (U.S.A.).7 The concentration
* Corresponding author. Tel.: 33476037627; 33476547203. E-mail:
[email protected].
† CNRS.
‡ Université Technique “Gh.Asachi”.
injected is in the range of 0.5 g/L, and the volume injected
is 108 µL on two columns in series (Shodex OH-pack 805
and 806). The eluent is 0.1 M NaNO3, and the temperature
for elution is 30 °C; the weight-average molecular weight
Mw and the polydispersity index I (I ) Mw/Mn) are given as
characteristics of the polymers. The initial values are Mw )
1.334 × 106 and I ) 1.49.
For rheology, the HA solutions are prepared at a concentration of 10 g/L in 0.15 M NaCl; the pH was controlled by
successive additions of HCl for the acidic medium and NaOH
for basic conditions.
The rheological behavior was studied using an AR 1000
rheometer from TA Instruments at 20 °C, when not precise.
Plane-cone geometry is used with a 3.59° angle and 4-cm
diameter. Dynamic experiments were performed in the linear
domain at 5% deformation. The complex viscosity |η*| (Pa)
is given at a low frequency corresponding to the Newtonian
domain when it exists or at a fixed frequency (0.1 rad/s).8
The control of the structure of the polysaccharides can
also be performed by 1H NMR (nuclear magnetic resonance)
in the presence of a standard to calibrate the signal corresponding to the -CH3 of the N-acetylglucosamine unit. The
standard generally used is 5 mM sodium succinate or
dimethyl sulfoxide (DMSO) in D2O when the polymer
concentration for NMR is around 5 mg/mL. But the NMR
signals of specific groups, especially in 1H NMR, are
quantitative only when they are mobile, that is, not involved
in an ordered secondary structure such as a helical structure
which can be stabilized by H bonds in stereoregular polymers
or by specific interactions.9-11 1H NMR spectra were
acquired on a Bruker AC300 spectrometer. Chemical shifts
are given relative to external tetramethylsilane (TMS ) 0
ppm); the methyl signal from N-acetamido is at 1.93 ppm,
and the -CH3 from DMSO is at 2.61 ppm.
Results and Discussion
A. Acidic Medium. The rheological behavior of the initial
solution of HA is given in Figure 1. The initial pH of this
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Biomacromolecules, Vol. 6, No. 1, 2005
Gatej et al.
Figure 1. Rheological behavior of initial HA at 10 g/L in 0.15 M NaCl at 20 °C and pH ) 6.05.
Figure 2. Complex viscosity as a function of the pH in acidic
conditions. T ) 20 °C, polymer concentration Cp ) 10 g/L, and
frequency is 1 rad/s.
solution is 6.05. Figure 1 is characteristic of a viscoelastic
solution with a transition located at ω0 over which G′
becomes larger than G′′, meaning that entanglements exist
giving an elastic character in this range of frequencies. The
complex viscosity presents a plateau in the domain of low
frequencies.
In Figure 2, the complex viscosity is represented at a fixed
frequency as a function of pH; at pH ) 2.42, the complex
viscosity passes through a large maximum which is interesting to investigate. At this pH, the dynamic experiments
demonstrate that G′ > G′′ in a large range of frequencies
corresponding to a gel-like behavior. This process was
previously described without any analysis (Figure 3).12-14
For pH values in the range of 2.86 up to 6.05 and at 1.6,
the behavior in dynamic experiments remains similar just
modified by a slight change in the ionic concentration and a
decrease of the net charge. Addition of NaOH in the pH )
1.6 solution to increase the pH to 3.34 allows the initial
behavior to nearly be recovered but at higher salt content.
The complex viscosities for some solutions at different pH
values are compared in Figure 4. This indicates that the
mechanism of association assumed to be the cause of the
large maximum in the range of pH ) 2.5 is reversible
(comparison of pH ) 3.34 and 1.6) and that it corresponds
to a critical balance of charges in the polymer; the reduction
of the carboxylic group dissociation favors the H-bond
formation (in relation with the intrinsic pK of this polyelectrolyte determined equal to 2.9 ( 0.1),15 but also the
protonation of the -NH- group giving a positive net charge
able to complex with the negative charge of few -COOH
was suggested to interpret this mechanism even if the
protonation constant for the acetamido group is not known
at the time. Then the effect observed can be interpreted as
due to an isoelectric point located around pH ) 2.5. From
SEC experiments, it can be shown that after a short time at
pH ) 1.6, the molar mass of the polymer decreases (one
finds Mw ) 974 000 and I ) 1.59).
In Table 1, the main rheological characteristics of the
solutions are given for the different pH values tested.
B. Characterization of the Gel-like Behavior. Dynamic
experiments were performed as a function of temperature
up to 50 °C at 1 Hz and pH ) 2.42. The results are given in
Figure 5 where a characteristic temperature of ∼40 °C is
shown to correspond to the transition from G′ > G′′ to G′′
> G′. From this temperature dependency, it comes that a
gel-like structure stabilized by H bonds is formed when the
-COO- dissociation decreases as mentioned in the previous
paragraph. The cooperative interactions cause large thermosensitive interactions, as it was shown that the pH was
Role of the pH on HA Behavior in Solution
Biomacromolecules, Vol. 6, No. 1, 2005 63
Figure 3. Rheological behavior of HA at 10 g/L in 0.15 M NaCl at pH ) 2.42 and 20 °C showing a gel-like behavior in a large range of
frequencies.
Figure 4. Complex viscosity of HA solution for different pH values: (O) 1.6; (b) 2.86; (0) 3.34; (9) 5.21; and (]) 6.05. Cp ) 10 g/L in 0.15 M
NaCl at 20 °C.
not modified by temperature increase. From NMR of the
acetamido CdO resonance, a large sharpening of the signal
at a temperature larger than 40 °C due to the dissociation of
H bonds between acetamido NH and carboxylate groups was
previously demonstrated.11
In a second step, one investigates the role of polymer
concentration on the gel-like behavior at pH ) 2.57. Addition
of solvent (0.15 M NaCl) at different contents allows the
polymer concentration at a given pH to be varied; for these
experiments, the systems were heated moderately after
dilution and cooled for equilibration. A few data are given
in Figure 6, where it is shown that with dilution a viscoelastic
solution behavior is obtained down to 6.66 g/L in the
frequency domain covered; specifically, a Newtonian plateau
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Biomacromolecules, Vol. 6, No. 1, 2005
Gatej et al.
Figure 5. Evolution of the elastic modulus (G′) and viscous modulus (G′′) as a function of temperature for HA at pH ) 2.42. Conditions: HA
solution Cp ) 10 g/L in 0.15 M NaCl at 1 rad/s.
Figure 6. Influence of the polymer concentration on the rheological behavior of solution. The G′ and G′′ are represented and show a rapid
evolution of the viscoelastic behavior with polymer concentration. (O, b) 10 g/L; (], [) 6.66 g/L; (0, 9) 5 g/L.
exists only for the lower polymer concentration (e5 g/L).
For concentrations of 5 g/L and lower, the viscosity drops
suddently as indicated in Table 3.
Then one can conclude that when the pH of the solution
is decreased by progressive addition of HCl, a thermosensitive gel is formed around pH ) 2.5 as a result of the
Biomacromolecules, Vol. 6, No. 1, 2005 65
Role of the pH on HA Behavior in Solution
Table 1. Rheological Characteristics for HA in the Acidic Medium
pH
G′a (Pa)
G′′a (Pa)
ω0 (rad/s)
|η*|b (Pa‚s)
6.05
5.61
5.21
4.38
3.34c
2.86
2.57
2.42
2.1
1.6
1.39
1.02
0.93
1.17
0.39
0.77
31.81
38.89
25.32
0.47
4.51
3.47
3.20
4.03
1.95
2.92
19.37
21.93
16.26
1.84
18.64
21.19
22.70
22.14
43.04
30.40
0.13
0.10
0.18
30.58
5.45
4.21
3.86
4.72
2.15
3.38
133
175
108
2.20
a Values taken at 1 rad/s. b Values taken at 0.1 rad/s. c Solution at pH
) 1.6 added with NaOH.
Figure 7. Complex viscosity as a function of the pH in basic
conditions. T ) 20 °C, polymer concentration Cp ) 10 g/L, and
frequency is 1 rad/s.
decrease of the carboxylate dissociation, favoring intermolecular interactions. A further decrease of the pH (see
pH ) 1.6) causes the gel-sol transition; this transition may
be related to the protonation of the acetamido groups causing
an electrostatic repulsion between cationic polymers. As
mentioned before, a balance of opposite charges is suggested
as contributing to gelation also favored by the local stiffness
of the HA chain.7,16,17
C. Basic Conditions. Progressive addition of NaOH on
the HA initial solution shows that viscosity decreases slightly
down to 11.58 followed by a transition in the range of pH
) 12 which corresponds to the pK of the -OH groups (Table
2; Figure 7). This transition was previously described by
Reed et al. who demonstrated a decrease of the radius of
gyration of HA molecules without any change of the molar
mass for progressive addition of NaOH.18 From Table 2, it
is observed that the transition is nearly reversible; the values
obtained for the different parameters for solutions at pH )
11.5 and pH ) 11.58 are nearly identical, as well as the
results for pH ) 3.82 (Table 2) and pH ) 3.34 (Table 1;
Figure 8). Two hypotheses are able to justify a decrease of
the viscosity at a pH larger than 12: first a decrease of the
dimensions of the molecules based on a decrease of the
stiffness and, second, a degradation of the backbone of the
polymer with a decrease of the molecular weight. To
investigate the mechanism involved in the quasi-reversible
change in the viscosity at high pH, the SEC characterization
of the different samples was performed. As shown in Figure
9, the molecular weight distribution is only slightly modified
by addition of NaOH up to pH ) 12.6 followed by rapid
reneutralization; Mw becomes Mw ) 1.183 × 106 and I )
1.58 and are unable to justify the decrease of the viscosity
Figure 8. Complex viscosity of the HA solution for different pH values: ([) 12.33; (]) 12.6; (9) 11.5; (0) 10.95; (O) 6.15; and (b) 3.82. Cp )
10 g/L in 0.15 M NaCl at 20 °C.
Biomacromolecules, Vol. 6, No. 1, 2005
66
Gatej et al.
Figure 9. Molar mass distributions in neutral conditions for initial pH (1; Mw ) 1.334 × 106; I ) 1.49), for HA having to be adjusted to pH ) 1.6
(2; Mw ) 9.74 × 105; I ) 1.59); and pH ) 12.6 (3; Mw ) 1.183 × 106; I ) 1.58) before reneutralization.
Table 2. Rheological Characteristics for HA in Alkaline Conditions
pH
G′ (Pa)
G′′ (Pa)
ω0 (rad/s)
|η*|a (Pa‚s)
6.15b
1.68
1.14
1.05
0.77
0.83
0.77
0.69
0.47
0.10
0.61
0.50
4.94
3.74
3.51
2.83
3.19
3.07
2.85
2.22
0.87
2.58
2.18
16.27
21.03
22.17
26.33
29.90
29.74
31.17
73.03
6.42
4.54
4.26
3.31
3.72
3.52
3.23
2.40
0.89
2.90
2.48
6.96
7.52
8.70
10.95
11.23
11.58
12.33
12.6
11.5c
3.82a
33.64
39.25
a Addition of HCl on the pH ) 12.33 solution. b Initial solution. c Addition
of HCl on the pH ) 12.6.
Table 3. Influence of Polymer Concentration on the Gel-like
Behavior
pH
polymer concentration
(g/L)
G′ (Pa)
at 100 rad/s
ω0
(rad/s)
|η*| (Pa‚s)
at 0.1 rad/s
2.57
2.7
2.34
2.6
10
6.66
5
2
102.5
21.35
4.02
0.13
3.74
133.5
6.25
0.73
0.033
observed; it is recalled that the rheology is nearly reversible.
From these data, we are able to conclude that a reversible
change in the stiffness of the HA molecule must occur when
it passes through basic conditions at pH > 12.5.
NMR experiments were developed to confirm this hypothesis; the mobility of the chain was tested from the
relative height of NMR signals corresponding to the -CH3
group in the N-glucosamine unit in the presence of a wellcontrolled amount of DMSO used as a standard to calibrate
the proton signals. In Figure 10, the 1H NMR spectrum taken
at 80 °C is given showing the increases of chain mobility in
the presence of sodium hydroxide (a) and when compared
with the spectrum after reneutralization (b). In fact, the local
mobility of the chain is related to the ratio of the integrals
of the signal for the -CH3 protons at 1.9 ppm for HA and
that of -CH3 at 2.6 ppm (for DMSO taken as the internal
reference); this ratio passes from 0.91 in alkaline conditions
(a) to 0.74 after reneutralization (b), respectively, indicating
a larger mobility of HA molecules in the alkaline medium.
In fact, for HA, some authors propose the existence of an
Figure 10. 1H NMR spectra for HA in D2O in the presence of DMSO
as the reference (a) in the presence of NaOH pH > 12.5 and (b)
after reneutralization.
ordered conformation in solution which is stabilized by H
bonds.11,19,20 These bonds can be released in the presence of
NaOH18 or urea.21 However, Sicinska et al.22 concluded from
a detailed 1H and 13C NMR study of the repeating HA
disaccharide that interactions with water predominated, and
they found no evidence for long-lived intramolecular hydrogen bonds in aqueous solution for this disaccharide. On
the opposite, on HA, the apparent intensity of the signal
corresponding to the protons of the acetyl groups as well as
those of the sugar units depends on the NaOH concentration
Biomacromolecules, Vol. 6, No. 1, 2005 67
Role of the pH on HA Behavior in Solution
and on the temperature adopted for 1H NMR measurements.
The exact conformation in aqueous solution is not yet
established, but it may be possible that for the polymeric
chain, dynamic H-bonded regions exist, controlling the
average stiffness of the molecule. This hypothesis was
proposed in the literature;23-25 then the fraction of H-bonded
domains depends on the pH which would transfom the -OH
groups in dissociated alcoholate (pK ∼ 12) at high pH,
increasing the net charge of the HA and destabilizing the
H-bond network.
(9)
(10)
(11)
(12)
Conclusion
(13)
The behavior of HA in aqueous solution depends on the
pH; in this paper, the rheological behavior was examined
and analyzed. It is shown that, in a large range of pH values,
the rheological behavior remains nearly unchanged (2.8 <
pH < 12); in the range of pH ) 2.5, a thermoreversible gellike behavior appears attributed to a cooperative interaction
mechanism. A H-bond network may be formed when the
net charge of the polymer decreases (reduction of the
-COOH dissociation) and, maybe, when some protonation
of the amido group occurs. At lower pH (pH ) 1.6), the
polymer is resolubilized. This sol-gel transition is also pHreversible. A reversible conformational transition to a random
coil is observed for pH > 12.5 due to the -OH groups
dissociation in alkaline conditions; this mechanism reduces
the number of H bonds between -OH and acetamido groups
which control the local stiffness of the HA molecule.
(19)
References and Notes
(20)
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