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Carbohydrate Polymers
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a r t i c l e
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Article history:
Received 28 September 2009
Received in revised form 28 April 2010
Accepted 29 April 2010
Available online 7 May 2010
Keywords:
Chitin/chitosan production
Chito-oligomers
Physical hydrogels
Multi-membrane systems
Biological properties
a b s t r a c t
Chitosan is a complex linear amino co-polymer which, depending on a great variety of parameters,
exhibits a more or less amphiphilic structure. This makes this polysaccharide one of the most versatile
macromolecules for which it is sometimes difcult to control all the factors responsible of its properties.
The purpose of this paper is to present a review of about 30 years of research in our team in the context of a precise scientic strategy. Thus, these years were devoted to improve the production of chitin
and chitosan, produce series of co-polymers and co-oligomers, improve their characterizations, reveal a
general law of behavior, generate nano-particles, physical gels and derived forms, show a continuum of
structure from solutions to other physical states, propose the concept of materials decoys of biological
media, etc.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Starting in 1982, our works focused on the study of the
physicochemical properties of chitosan in solution through the
investigations on various interaction mechanisms and the elaboration of new materials with applications in numerous elds. Due to a
non-accessible glass transition temperature before thermal decomposition, solutions were at the core of our activities related to the
elaboration and the study of the properties of other physical forms
like gels or solids (bers, lms, particles, capsules) processed from
this physical state.
An accurate study of chitosan properties requires the use of wellcharacterized high quality products. For numerous applications it
is also essential to produce a large spectrum of structures with perfect control of the chemical architectures, molecular weights and
molecular weight distributions. In the rst case, it is necessary to
manage both the degree of acetylation (DA) and the distribution
of the two kinds of residues along the polymer backbone. This is
quite difcult for long co-polymer chains, and, up to now, the production of only statistic distributions is under control. The case of
co-oligomers, more accessible in theory, remains complex. Series of
fully acetylated/de-acetylated oligomers, or statistic co-oligomers
of chosen DA were produced as well as pure co-oligomers of controlled architecture and DA.
All these considerations required that we could also control
the production of both chitin and chitosan. They were necessarily
founded on the fact that the control of both the chemical structures and the derived materials should allow us to nd interesting
applications, especially to illustrate new concepts in the eld of life
sciences.
2. Results and discussions
2.1. Production of chemical structures
The rst challenge was to produce a fully de-acetylated chitosan
with the highest possible molecular weight to access almost all
the structures made from glucosamine and N-acetyl glucosamine
residues. This required extracting chitin with a maximum preservation of its structure (molecular weight, DA). Two industrial sources
were considered related to the two crystalline allomorphs, and ,
present in biomass, in shrimp shells and squid pens, respectively.
We rst studied the extraction of chitin from shrimp shells
(Percot, Viton, & Domard, 2003a; Percot, Viton, & Domard, 2003b).
We noticed that after peeling of fresh shrimps, the best preservation of the cuticles is achieved by adding solid sodium chloride
(10 kg NaCl/500 g shells). Then, the demineralization was performed under soft conditions: at room temperature, in the presence
of a stoichiometric amount of 0.25 M HCl with regards to the
calcium carbonate content, for only 15 min; all acid excess only contributing to the polymer degradation. The de-proteinization was
later classically processed at 70 C for 24 h in 1 M NaOH. These conditions allowed a good preservation of the chitin structure, with a
high molecular weight and a DA remaining above 95%. A kinetic
study of de-proteinization (Percot et al., 2003a,b) was then performed. Three different rate constants were observed and their
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of other physical forms we may study like gels and solids based
on chitosan. These nano-aggregates are also identied by electron
microscopy when we achieve a critical pH just before the polymer
precipitation. In the latter case, they have a size within 250350 nm
(Schatz, Pichot, 2003; Schatz, Viton, 2003). Their formation is also
favored by an increase of concentration, which contributes to the
hydrophobisation, especially over C*. Thus, for a DA over 70%, at
Cp > C*, they are observed at any pH by QELS or electron microscopy
(Popa-Nita, Alcouffe, Rochas, David, & Domard, 2010), by means of
the interesting technique of wet STEM. Moreover, they look like
coreshell structures. Over C*, recent studies by means of SAXS
(synchrotron beam), showed the presence of a scattering peak (the
so-called polyelectrolyte peak). The value of the scattering vector
qmax at the maximum intensity was studied as a function of DA, Cp
and Mw . Its variation with Cp , for different DAs, illustrated a power
law which exponent (a) showed a transition between 1/2 and 1/3,
in relation with the model of the necklace, proposed to illustrate the conformation of polyelectrolytes (Dobrynin & Rubinstein,
1999). When a is 1/2, the polymer is highly hydrophilic and the
conformation is controlled by the strings. On the contrary, for 1/3,
it becomes highly hydrophobic and is controlled by the pearls.
The critical concentration Cb between both values revealed a second critical phenomenon to be related to C**, the second critical
concentration, we already identied in previous studies (Boucard,
Viton, & Domard, 2005; Montembault, Viton, & Domard, 2005a;
Montembault, Viton, & Domard, 2005b; Montembault, Viton, &
Domard, 2005c) where a nano-phase separation occurs with one
phase constituted of more or less isolated and hydrophilic chains
and a second of hydrophobic nano-aggregates. The variation of Cb
with DA followed again the general law of behavior (Popa-Nita,
Rochas, 2009).
2.3. From solutions to other physical forms
The knowledge of the properties in solution including changes
of chain conformation and molecular organization is quite useful to
understand the formation of derived forms. It is thus relatively easy
to form a physical hydrogel of chitosan with no use of an external
cross-linking agent provided we consider three essential conditions. The rst is to be initially at a concentration over C* to favor
polymer chain junctions. The second is to displace the H/H balance to overcome C**, and the third is to generate a bi-dimensional
solgel transition. We identied two different ways.
In the rst case, the second criterion was fullled using a
hydro-alcoholic solvent inducing a lower dielectric constant of the
medium, thus becoming more hydrophobic than pure water. In this
medium the acid used to dissolve chitosan was less dissociated,
even in the case of HCl. Then, the third criterion could be achieved
by means of a simple evaporation of water and the non-dissociated
acid (acetic acid, hydrochloric acid), both allowing to also overcome
C**, provided the alcohol used had a higher boiling temperature
than water. A kinetics study conrmed the hydrophobic role of an
increase of temperature favoring a faster gelation with two different energies of activation and a transition at near 40 C. We also
noticed a decrease of the neutralization degree at which gelation
occurred, as a function of DA, illustrating again the general law of
behavior. This law was also observed when studying the variation
of the storage modulus of the gels increasing with DA, thus showing the important role played by acetyl groups in the formation of
hydrophobic junctions (Montembault et al., 2005a,b,c). The alcohol
used was shown to participate in the gelation and only a few alcohols allowed gelation to undergo (propandiol, glycerol . . .). SAXS
showed that during the evaporation of water and even over the gel
point, the polyelectrolyte peak was always present, conrming the
formation of a polyelectrolyte alcohol gel (Boucard et al., 2005).
Therefore these gels could be neutralized in an alkaline medium
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material. After the beginning of the gel dehydration at the end of the
dermis reconstruction, the gel becomes multi-lamellar, the access
is then possible to cells nding nutriments as well as growth factors brought initially by blood. The cells beginning to penetrate the
material can also produce nitrites favoring the local degradation
of the gel with the production of oligomers, and so on. Then, the
material becomes a sort of articial skin model.
3. Conclusion
Progress over these 30 years can be considered in various steps.
One rst concerns the increase of the molecular weight of the
extracted chitin from either shrimp shells or squid pens. The objective would be to avoid any degradation before treatment. Thus, it
would be important to treat the raw material immediately after
its collection from living animals. We could thus approach a Mw of
3 106 g/mol close to that supposed in the native chitin.
The second is related to the control of the hydration of the reaction medium during the de-acetylation of chitin, to preclude the
level-off due to the complete consumption of the reactive. Then,
thanks to the recent studies and starting with a chitin of Mw near
3 Mg/mol we could overcome the million g/mol for a fully deacetylated chitosan, thus accessing to new physical properties for
all the physical forms.
The production of co-oligomers of perfectly controlled architecture and size remains a challenge that must be regarded as
particularly promising for their specic biological properties, but
also their physicochemical properties: for example, the possibility to form self-assembling materials (nano-particles, organo-gels,
etc.). They are also interesting to produce a new series of copolymers, especially that concerning the block co-polymers useful
for their typical amphiphilic properties.
Solution properties of chitosan reveal the important role played
by all parameters inuencing the balance between hydrophilic and
hydrophobic interactions, at the origin of their nano-structuration
associated to the existence of a general law of behavior. This opens
the understanding of all the interactions involving chitosan and
allows us to show a continuum of structure from the solutions to
any physical states. These advances are at the origin of the understanding of some biological properties also allowing us to propose
new concepts; especially that of materials decoys of biological
media. All these results should open a large and interesting access
to a wide eld of new properties and interpretations.
Acknowledgements
We are particularly grateful to all those having contributed to
these works, with a special attention to Dr. C. Rochas (Universit J. Fourier, Grenoble, France, Dr. M.T. Corvol, Universit Paris
V, France) and all the cited PhD students.
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