Morphological and Rheological Characterization of Chitosan Liquid-Crystalline Solutions and Gels Hugo M. Lisboa 1,2 , João P. Borges 2 , Marcus V. L. Fook 1 , Ana M. Ramos 3 , Maria T. Cidade 2 1 CertBio, Unidade Académica de Engenharia dos Materiais, Universidade Federal Campina Grande, Campina Grande, PB, Brasil 2 Departamento de Ciência dos Materiais and Cenimat/I3N, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, UNL, 2829-516, Caparica, Portugal 3 Departamento de Química and REQUIMTE, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, UNL, 2829-516, Caparica, Portugal Abstract Chitosan is a biopolymer used in biomedical applications, which has the ability to form lyotropic mesophases (chiral nematic) in several solvents. However, chitosan chiral nematic phase is only observed at high polymer concentrations where the system is in a non-homogeneous gel-like state which has limited or no practical interest. In this work the formation of chitosan liquid-crystalline solutions and gels was investigated. For the first time, a chiral nematic mesophase was observed in low viscosity solutions of chitosan, the novelty of this study. Malic and hydrochloric aqueous solutions with different concentrations of chitosan were prepared and the gel formation was followed by rheological measurements (crossover between the values of G’ and G’’ and evolution of tan vs c for different ). The concentration, C Gel , at which the gel is formed, was found to be dependent on the acid used, the malic aqueous solutions presenting higher C Gel . For the chitosan/malic acid system the chiral nematic mesophase appeared in solution and was preserved in the gel (for C>C Gel ) while for the chitosan/hydrochloric acid system the mesophase was only observed in the gel. This behavior was correlated with the type of interaction polymer/solvent. Swirl-like fingerprint textures typical of cholesteric mesophases were observed by Polarized Optical Microscopy (POM) and the helical pitch was determined from POM images.
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Morphological and Rheological Characterization of Chitosan Liquid-Crystalline Solutions and Gels
Hugo M. Lisboa1,2, João P. Borges2, Marcus V. L. Fook1, Ana M. Ramos3, Maria
T. Cidade2
1 CertBio, Unidade Académica de Engenharia dos Materiais, Universidade Federal Campina Grande, Campina Grande,
PB, Brasil2 Departamento de Ciência dos Materiais and Cenimat/I3N, Faculdade de Ciências e Tecnologia, FCT, Universidade
Nova de Lisboa, UNL, 2829-516, Caparica, Portugal3Departamento de Química and REQUIMTE, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa,
UNL, 2829-516, Caparica, Portugal
AbstractChitosan is a biopolymer used in biomedical applications, which has the ability to form lyotropic
mesophases (chiral nematic) in several solvents. However, chitosan chiral nematic phase is
only observed at high polymer concentrations where the system is in a non-homogeneous gel-
like state which has limited or no practical interest. In this work the formation of chitosan liquid-
crystalline solutions and gels was investigated. For the first time, a chiral nematic mesophase
was observed in low viscosity solutions of chitosan, the novelty of this study. Malic and
hydrochloric aqueous solutions with different concentrations of chitosan were prepared and the
gel formation was followed by rheological measurements (crossover between the values of G’
and G’’ and evolution of tan vs c for different ). The concentration, CGel, at which the gel is
formed, was found to be dependent on the acid used, the malic aqueous solutions presenting
higher CGel. For the chitosan/malic acid system the chiral nematic mesophase appeared in
solution and was preserved in the gel (for C>CGel) while for the chitosan/hydrochloric acid
system the mesophase was only observed in the gel. This behavior was correlated with the type
of interaction polymer/solvent. Swirl-like fingerprint textures typical of cholesteric mesophases
were observed by Polarized Optical Microscopy (POM) and the helical pitch was determined
from POM images. The existence of mesophases in the gel, in both chitosan aqueous solutions,
was also confirmed by rheometry. The results here obtained can be of key importance in the
field of Tissue Engineering. The biomedical interest in liquid crystalline systems of chitosan lies
in the possibility of mimicking the structure of the main component of the extra-cellular matrix in
connective tissues, collagen type I, which has analogous liquid crystalline ordering in acidic
At the gel point, G’ and G’’ curves become more or less parallel to each other and the power law
behavior (G’ G’’ ~ ωm) in frequency is observed. Above this point G’ becomes higher than G’’.
This sol-gel transition is observed for a polymer concentration of about 6.0 wt% for the malic
acid solutions and for concentrations of about 2.6 wt% for the hydrochloric acid solutions, as
can be seen in Figures 5 and 6. To determine more accurately this value, the evolution of the
loss tangent as a function of polymer concentration (at several frequencies) is presented in
Figure 8. These curves converge to a sole one for concentrations equal or higher than the
gelation concentration, CGel, when the G’(ω) and G’’(ω) become parallel and the loss tangent
values are independent of the frequency. The found gelation concentrations, CGel, are equal to
2.6 wt% for the hydrochloric acid solutions, and very close to 6.0 wt% for the malic acid
solutions.
(a)
(b)
Figure 8 – Influence of chitosan concentration on the variation of tan δ at different frequencies,
for both (a) malic and (b) hydrochloric acid solutions.
The viscosity curves, along with the curves obtained in oscillatory measurements, Figures 3 and
6, give, also, an indication of the concentration for which a transition from an isotropic to an
anisotropic solution or gel occurs. In fact, a careful observation of the curves in Figures 3 and 5,
(b), shows that, for the malic acid, a biphase appears for concentrations between 3.6 and 4.0 wt
%, with a critical concentration (appearance of the anisotropic phase, C*) between 4.0 and 4.4
wt%, followed by a decrease of the viscosity for 4.8 wt% solutions and, again, an increase of the
viscosity for higher concentrations. This same result may be seen in Figure 9 (a) where the
dependence of the viscosity with the concentration is presented; this transition is perfectly clear,
even though the curve is not the usual one. In fact this tiny decrease could be thought as an
experimental error, however, it is perfectly reproducible. This observation is in accordance with
the well known concentration dependence of the viscosity in the isotropic-nematic transition
found in lyotropic liquid crystalline polymers (Doi, 1981, Larson, 1999). This situation is
explained by the decrease of the viscosity at the transition due to the fact that the molecules can
slide past each other more readily in the nematic state than in the isotropic one, due to their
orientation. It is important to note that our lyotropic system presents, at rest, cholesteric
mesophase, however it is known that the helix unwound under shear forces, giving rise to a
nematic structure, at shear rates below the lowest values considered in our experiments
(Cidade et al., 1995). The same phenomena occurs for the hydrochloric acid solutions, Figures
4 and 6 (b), with C* between 2.6 and 2.8 wt%. The viscosity as function of the concentration
curve is not so clear in this case (see figure 9 (b)), probably due to the coexistence of the
nematic-isotropic transition and the sol-gel transition. In fact, even present, the nematic phase
behavior is masked by the physical cross-linking occurring in the gel phase. However, in the
absence of the nematic phase a continuous increase of the viscosity could be anticipated, which
is not the case, until concentrations equal or higher than about 4%, meaning that, at these
higher concentrations the nematic arrangement is no longer possible. The relaxation times
presented in Table 1, in the case of malic acid solutions are in accordance, as expected, with
these values of C*; in fact the relaxation time increases with concentration until a value of 4.0 wt
%, then decreases until around 4.8 wt% to increase again for higher concentrations. In the case
of hydrochloric acid since the anisotropic phase appears already in the gel phase, an indication
of C* cannot be given by this method.
(a)
(b)
Figure 9 – Influence of the chitosan concentration on the viscosity of malic (a) and hydrochloric
(b) acid solutions.
These critical concentrations, confirmed by the ones found using optical microscopy, clearly
show that a transition from an isotropic to an anisotropic phase occurs in these systems. A point
of interest is the fact that while in the malic acid system the isotropic-nematic transition appears
before the sol-gel transition (in the solution state), in the case of hydrochloric acid C* is higher
than CGel, which means that the anisotropic phase appears after the gel is formed. Montembault
and coworkers (Montembault et al., 2005a, Montembault et al., 2005b) have shown that
gelation’s mechanism of chitosan in acidic solutions is attributed to the formation of physical
junctions due both to hydrophobic interactions involving N-acetyl groups and hydrogen bonding.
The balance between these hydrophobic interactions and H-bonds depend on the DA. For low
DA (< 25 %), as in the case of the present work, the initial charge density of the polymer is high,
the content of hydrophobic functions is low and hydrogen bonding plays a more important role in
the gelation mechanism (Montembault et al., 2005b). Malic acid is an organic carboxylic diacid
with three functionalities (two carboxyl and one hydroxyl groups) capable of establishing H-
bonds with either amine or hydroxyl groups of chitosan chain which is not the case of the
mineral hydrochloric acid. Therefore, as it is a weaker acid, and its molecule has a higher
volume, it acts like a spacer, reducing the possibility of proximity of the chains and then a lower
number of intermolecular H-bonds between acetylated groups of chitosan chains are
established when this biopolymer is dissolved in malic acid, resulting in a higher CGel. The low
concentration of hydrochloric acid may also be the reason for lower Cgel. As it can observed at
table 2, the solution’s pH increase sharply between concentrations of 2.0 and 2.8 wt%, and that
this is the limit concentration for chitosan solubilization in hydrochloric acid, resulting the
formation of the gel.
Table 2 – Concentration, pH and ionic strength for both chitosan systems
If the protonation ability of chitosan’s amino groups by the two acids is considered, the values of
the acid dissociation constant, pKa is - 6.2 for hydrochloric acid (Robinson et al., 1971) and 3.4
and 5.2 for the first and the second carboxylic group of malic acid to be dissociated (Dawson et
al., 1959). These values mean that hydrochloric acid has a higher capacity to protonate
chitosan, which introduce a large amount of positive charges in a smaller number of polymer
chains, originating the consequent repulsion phenomena between them. Probably, these might
be the explanation for the observed higher C* in the chitosan/malic acid system. In this system,
for this frame of time, the possibility of malic acid to behave as a crosslinker for chitosan, as
reported in literature (Chang et al., 2007) does not seem to be very significant, according to the
results obtained, which must be due to the low concentrations of the acids used. Currently there
is still no agreement on the conformation of chitosan chains. While some authors [Cölfen et al.,
2001, Fee et al., 2003, Kasaai et al., 2006, Terbojevich et al., 1991) point to a rigid-
rod conformation others [Berth et al., 1998, Brugnerotto et al., 2001, Lamarque et al., 2005,
Mazeau et al., 2004, Rinaudo et al., 1993, Shatz et al., 2003, Velásquez et al., 2008, Vold,
2004, Morris et al., 2009) adopt a semi-flexible coil model. Through Flory’s model (Flory,
1956) it is possible to estimate the theoretical value of the critical concentration, with this value
being 17.2 wt%, in both acids. The difference between our experimental values (C* ~ 2.6-2.8 wt
% for hydrochloric acid; C* ~ 4.0 – 4.4 wt% for malic acid) and this may be related to the fact
that this model assumes that the chitosan chains are in a rigid rod conformation, so this result
indicates that the chitosan in the present system is in semi-flexible coil conformation. For the
range of pH and ionic strength used in the present work (see table 2), and accordingly to the
work of Chen and Tsaih [Chen et al., 1998], chitosan chains with a molecular weight higher than
223 000 assume a less stretched conformation, indicating the adoption of a semi-flexible coil
conformation instead of a rigid-rod.
CONCLUSIONS
In this work, hydrochloric and malic acid solutions of chitosan were microscopically and
rheologically characterized, which allowed for the determination of the concentration at which a
gel is formed (CGel) and also the concentration at which a chiral nematic phase appears (C*) in
the solution or gel. CGel was determined from the crossover between the G’ and G’’ as a function
of the angular frequency, as well as by the evolution of the tan as a function of the
concentration for different angular ferquencies, while C* was determined by the evolution of the
viscosity curve and relaxation times, obtained, once again, from the crossover mentioned
before. CGel obtained are equal to 2.6 wt% for the hydrochloric acid solutions, and very close to
6.0 wt% for the malic acidsolutions. Concerning C* the values obtained were between 4.0 and
4.4 wt% for the malic acid solutions and between 2.6 and 2.8 for the hydrochloric acid solutions.
A point of interest is the fact that while in the malic acid system the isotropic-nematic transition
appears before the sol-gel transition (in the solution state), in the case of hydrochloric acid C* is
higher than CGel, which means that the anisotropic phase appears after the gel is formed. The
explanation for this fact lies in the different acid strength of the two acids and their capability in
forming hydrogen bonds with chitosan. Typical swirl-like fingerprint patterns, observed under
polarized optical microscopy, showed that the mesophases presented by the chitosan aqueous
solutions were of the chiral nematic type. The pitch of the mesophase has shown to decrease
with increasing polymer concentration with a power law dependence of 3. The possibility of
obtaining liquid crystalline phases of chitosan from low viscosity acidic solutions opens new
perspectives in materials science, and especially in the Tissue Engineering field. These
mesophases can be “freezed” in a gel, for example, by a pH-triggered sol-gel transition. The
obtained gels can mimic the organization of collagen I and are good analogues of the
extracellular matrix, with a structure close to that of biological tissues. These materials can be
used either in tissue repair or as models for the culture of cells in 3D, to study their migration
and signaling activities, in a manner close to physiological conditions.
Acknowledgements Hugo Lisboa acknowledges Fundação para a Ciência e Tecnologia for grant SFRH/ BDE/
15557/2005
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