Carbohydrate Polymers 154 (2016) 8695

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Polyelectrolyte complexes via desalting mixtures of hyaluronic acid

and chitosanPhysicochemical study and structural analysis
G. Laleve a,b , G. Sudre a , A. Montembault a , J. Meadows b , S. Malaise b , A. Crpet a ,
L. David a , T. Delair a,
a
Univ. Lyon, Universit Claude Bernard Lyon 1, CNRS, IMP, UMR 5223, 15 Boulevard Latarjet, F-69622, Villeurbanne, France
b
Laboratoire ObvieLine, Sinclair IS Pharma, 8 Chemin du Jubin, F-69570, Dardilly, France

a r t i c l e i n f o a b s t r a c t

Article history: Polyelectrolyte complexes (PECs) were prepared from Chitosan (CS) and Hyaluronic Acid (HYA) homoge-
Received 24 March 2016 neous mixtures of aqueous solutions. The method consisted of preparing a homogeneous mixture of the
Received in revised form 20 July 2016 two polysaccharides via charge screening at high salt concentrations. Then, the mixture was dialyzed,
Accepted 3 August 2016
leading to the controlled self-assembly of the two polyelectrolytes. Critical parameters like the chitosan
Available online 3 August 2016
degree of acetylation (DA) and molar mass (Mw), the residual salt concentration and the molar charge
ratio r = nNH3 + (CS)/nCOO (HYA) accounted for the transition from homogeneous aqueous solutions to
Keywords:
colloidal suspensions (r = 0.1) or gel coacervates (r = 0.5). The inuence of the DA and Mw of CS was eval-
Chitosan
Hyaluronic acid
uated by visual observations, light scattering and rheological measurements. For low values of r, Small
Polyelectrolyte complexes Angle X-ray Scattering (SAXS) experiments revealed that the HYA nanostructure was weakly affected
Coacervate by the presence of PECs. On the contrary, the structure was impacted when increasing r, revealing a
Hydrogel heterogeneous aggregate morphology with ladder-like chain interactions.
Nanostructure 2016 Elsevier Ltd. All rights reserved.

1. Introduction of chitin, its parent polymer extracted from the exoskeleton of

Polysaccharide biomaterials have a wide potential of applica-
tions in health sciences, for drug delivery (Hamman, 2010; Luo &
Wang, 2014) or tissue engineering (Li, Ramay, Hauch, Xiao, & Zhang,
2005; Tan, Chu, Payne, & Marra, 2009; Yamane et al., 2005) because
polysaccharides are generally regarded as safe, biocompatible
and exhibit a variety of suitable properties for biomedical appli-
cations. Polyelectrolytes are polymers bearing ionizable groups.
These groups dissociate in aqueous solutions leading to charged
polymer chains and counterions, dispersed or nanostructured in
the solution. This specicity of polysaccharide polyelectolytes has
been widely used since they can form complexes with salts (Hori,
Winans, & Irvine, 2009), proteins (Water et al., 2014) or other poly-
electrolytes (Coimbra et al., 2011; Delair, 2011; Li et al., 2005; Wu
et al., 2007).
Among them, chitosan (CS) receives a growing attention, as
the only cationic and naturally occurring polysaccharide at acidic
pH (pKa = 6.36.7). It is obtained from the partial deacetylation
Corresponding author.
E-mail address: Thierry.Delair@univ-lyon1.fr (T. Delair).
crustaceans, endoskeleton of cephalopods or cell walls of fungi.
Chitosan is a copolysaccharide of N-acetyl-d-glucosamine and
d-glucosamine. It is dened by its degree of acetylation (DA) cor-
responding to the molar fraction of acetylated residues and is
known for its bioactivity, biodegradability and mucoadhesion as
well as a low toxicity. It has been associated to negatively charged
polysaccharides like alginates (Li et al., 2005), carboxymethyl cel-
lulose (Chen & Fan, 2007), hyaluronic acid (Al-Qadi, Alatorre-Meda,
Zaghloul, Taboada, & Remunn-Lpez, 2013; Tan et al., 2009),
dextran sulfate (Drogoz, David, Rochas, Domard, & Delair, 2007),
heparin (Costalat, Alcouffe, David, & Delair, 2015; Costalat, David,
& Delair, 2014), to form a wide variety of biomaterials such as
nanoparticles (Costalat, David et al., 2014; Delair, 2011), scaffolds
(Coimbra et al., 2011), bers (Desorme et al., 2013; Ma et al., 2012),
hydrogels (Martnez-Ruvalcaba, Chornet, & Rodrigue, 2007), etc.
Hyaluronic acid (HYA) is an anionic polysaccharide at pH val-
ues above its pKA = 2.9. Its repeat disaccharide unit is composed
of d-glucuronic acid and N-acetyl-d-glucosamine, linked by -1,4
and -1,3 glycosidic bonds. HYA is widely present in the extracel-
lular matrix of various tissues of the human body, such as in skin,
cartilage, synovia and vitreous humor. HYA exhibits some inter-
esting features such as anti-inammatory properties (Foschi et al.,

http://dx.doi.org/10.1016/j.carbpol.2016.08.007
0144-8617/ 2016 Elsevier Ltd. All rights reserved.
 G. Laleve et al. / Carbohydrate Polymers 154 (2016) 8695 87

1990) and plays an important role in tissue regeneration (Chen Table 1

Macromolecular characterization of homologous series of chitosans. The degree of
& Abatangelo, 1999). Due to the outstanding properties of each
acetylation (DA) was obtained by 1 H NMR; The molar mass (Mw) and dispersity ()
polysaccharide, biomaterials containing hyaluronic acid and chi- by SEC-MALLS.
tosan are actually widely studied. Recently, Kaderli et al. published
DA (%) Mw (kg mol1 )
a study about the efcacy of HYA-CS hydrogel for osteoarthritis
therapy on rabbit model which provided promising results (Kaderli 1 56 3 1.5 0.2
et al., 2015). 85 5 1.7 0.2
150 8 1.9 0.2
Polyelectrolyte complexes are generally formed by mixing
5 55 3 1.5 0.2
aqueous solutions of two oppositely charged polymers and the 75 4 1.6 0.2
entropic gain generated by the liberation of the counterions drives 160 8 1.6 0.2
the association reaction. The main limitation when associating 14 28 1 1.4 0.1
100 5 1.7 0.2
HYA and CS is the spontaneous association leading to precipitates.
160 8 1.9 0.2
Hence, there is a need to control the complexation to develop mate- 28 19 1 1.3 0.1
rials with high potential for biomedical applications. 107 5 1.6 0.2
For this purpose, we investigated a controlled complexation 170 9 1.9 0.2
between HYA and CS by slowing down the process of associa- 49 17 1 1.3 0.1
120 6 1.2 0.1
tion. In particular, we used the dialysis method developed in our
185 9 1.6 0.2
team to induce and control the self-assembly of the two polyelec-
trolytes (Costalat, Alcouffe, David, & Delair, 2014; Costalat et al.,
2015; Costalat, David, et al., 2014). In the case of polysaccharides,
additional interchain interactions like hydrophobic interactions solution. Repeated washings with deionized water were necessary
and/or H-bonding may play a role. Complexation depends on var- to remove the excess of ammonia and recover neutral pH. Then,
ious parameters either intrinsic, like the polymer charge density chitosan was freeze-dried.
(Dautzenberg, 1997; Webster, Huglin, & Robb, 1997) and chain stiff- The puried chitosan was N-reacetylated in controlled con-
ness (Narambuena, Leiva, Chvez-Pez, & Prez, 2010), or extrinsic ditions in order to obtain a homogeneous series of samples of
such as the molar charge ratio (Mrquez-Beltrn et al., 2012), different DAs. Chitosan was dissolved in a water/1,2-propanediol
pH (Kayitmazer, Koksal, & Iyilik, 2015), temperature (Chollakup, mixture (1/1 (v/v)). Then, acetic anhydride was dissolved in
Smitthipong, Eisenbach, & Tirrell, 2010), ionic strength (Webster propanediol and added dropwise. The propanediol minimized the
et al., 1997) and the polymer concentration (Schatz, Domard, hydrolysis of acetic anhydride. These homogenous conditions allow
Viton, Pichot, & Delair, 2004). Macroscopically, polyelectrolyte a statistical distribution of N-acetyl residues within the chains with
complexes can be divided into three categories: soluble complexes no O-acetylation (Vachoud, Zydowicz, & Domard, 1997). The N-
(Dautzenberg, 1997; Kabanov & Zezin, 1984), colloidal suspen- reacetylated chitosan was isolated by precipitation with aqueous
sions (Delair, 2011; Schatz et al., 2004) and biphasic systems ammonia. Washings were achieved by centrifugation and/or dial-
ranging from precipitates (Chollakup et al., 2010) to coacervates ysis with deionized water.
(de Kruif, Weinbreck, & de Vries, 2004; Water et al., 2014) com- The N-reacetylated chitosans were nally depolymerized in
posed of a complex-rich phase and a polymer-poor phase. Recently, controlled conditions by nitrous deamination to produce chitosans
Kayitmazer et al. (2015) published an in-depth study of the com- with lower molar masses. Chitosan was dissolved at 0.5% (w/v) in
plex coacervation between hyaluronic acid and chitosan, in the case a 0.2 M acetic acid/0.15 M sodium acetate buffer. A 1 g L1 sodium
of rather diluted systems and HYA chain lengths with Mw between nitrite aqueous solution was added to chitosan solutions to obtain
50 kg mol1 and 750 kg mol1 . a nitrite/glucosamine units molar ratio of 0.1 (for DAs from 1% to
Here, we show that the intrinsic parameters of chitosan like 15%), and 0.2 (for DAs from 30% to 50%) to optimize the nitrous
the DA and the Mw, as well as the residual salt concentration led deamination reaction kinetics. The reaction was performed under
to the formation of materials of different nanostructures. These high mechanical stirring for different times in order to reach the
nanostructures were investigated by Static Light Scattering and desired molar mass. Low molar mass chitosans were recovered by
Small Angle X-ray Scattering (SLS and SAXS) and they were com- precipitation with aqueous ammonia, then washed repeatedly with
pared with results obtained in our team for chitosan-heparin and deionized water until neutrality and nally freeze-dried.
chitosan-dextran sulfate systems.

2. Experimental
2.1. Materials
Sodium hyaluronate produced by fermentation of Streptococ-
cus Equi was purchased from HTL-biotechnology (France) with a
molar mass Mw 1000 kg mol1 measured by SEC-MALLS. Chi-
tosan obtained from shrimp shell chitin with a medium molar
mass and low Degree of Acetylation was purchased from Mahtani
chitosan Pvt. Ltd. India (batch type 243, DA 1% measured by 1 H
NMR, Mw = 150 kg mol1 and dispersity = Mw/Mn = 1.9 measured
by SEC-MALLS). The chitosan raw material was further puried
by dissolution at 0.5% (w/v) in diluted acetic acid aqueous solu-
tion and by ltering the resulting solution on successive cellulose
Millipore membranes with decreasing porosity ranging from 3 m
to 0.22 m, allowing the elimination of all insolubles. The puri-
ed chitosan was precipitated with a 37% ammonium hydroxide
2.2. Methods
2.2.1. Chitosan characterization
The degree of acetylation was calculated by the Hirai et al.
method by 1 H NMR spectroscopy on a Brucker Avance III 400 MHz
5 mm at 300 K (Hirai, Odani, & Nakajima, 1991).
The weight average molar mass Mw and the dispersity () were
measured by size exclusion chromatography (2500 and 6000 PW
TSK gel columns from Tosohaas) coupled online with a differential
refractometer (Wyatt Optilab T-rEx) and a multi angle laser light
scattering detector (Wyatt Dawn EOS) operating at  = 633 nm. A
degassed 0.2 M acetic acid/0.15 M ammonium acetate buffer with
a pH 4.5 was used as the eluent. The ow rate was maintained
at 0.5 mL/min. The refractive index increments (dn/dc) were indi-
cated independently for each degree of acetylation according to a
previous study (Schatz, Viton, Delair, Pichot, & Domard, 2003). The
physicochemical parameters of the chitosan samples are reported
in Table 1.
88 G. Laleve et al. / Carbohydrate Polymers 154 (2016) 8695
The residual water contents of HYA and CS were determined by
thermogravimetric analysis (TA Instrument TGA Q500).
2.2.2. Preparation of polyelectrolyte solutions and homogeneous
mixtures by charge screening method
The hyaluronic acid and chitosan solutions were prepared sep-
arately before mixing. Sodium hyaluronate was directly dissolved
in a 0.65 M sodium chloride solution to obtain a 4.5% (w/v) HYA
solution. Mechanical stirring at room temperature was performed
overnight to obtain a homogeneous and transparent viscous solu-
tion. Chitosans were solubilized in a 0.65 M sodium chloride
solution, acetic acid was added to achieve the stoichiometric proto-
nation of the NH2 sites in glucosamine residues. CS concentration
depended on the expected charge ratio r = nNH3 + /nCOO (molar mix-
ing ratios between CS positive charges and HYA negative charges)
while HYA concentration in the nal mixture was maintained at
3% (w/v). Mechanical stirring at room temperature was performed
overnight.
CS solutions were then added to HYA solution respecting a
1:2 vol mixing ratio and mechanical stirring was maintained for
at least 2 h. The selected sodium chloride concentration induced
a charge screening for all the DAs, sufcient to prevent the
spontaneous association of the two polyelectrolytes. The nal
homogeneous mixture, containing HYA, CS and NaCl had a pH
around 5.
The resulting mixtures were nally placed into Spectra/Por
1 membranes with a molar mass cut-off MWCO = 3500 g mol1
purchased from Spectrumlabs. They were then dialyzed against
deionized water (V 100 times the volumes of the polymer solu-
tion), containing or not 0.15 M sodium chloride, for a minimum of
6 h under gentle mechanical stirring (the bath was changed every
2 h). This dialysis procedure was dened according to Costalat et al.
and allowed the system to reach the desired salt concentration
(Costalat, David et al., 2014). The progressive elimination of sodium
chloride induced the self-assembly of the two polysaccharides.
2.2.3. Measurements of colloid diameters
PECs colloidal suspensions were diluted about 500 folds in
Versol sterile water and characterized by dynamic light scatter-
ing using a Malvern Nanosizer SZ equipped with a 5 mW He/Ne
laser beam ( = 633 nm) operating at 25 C with a scattering angle
of 173 . The self-correlation function was expanded in a power
series (Cumulants method). 3 samples of the same formulation
were tested and measurements were repeated at least 10 times.
2.2.4. Rheological measurements
The viscosity of the systems before and after dialysis was mea-
sured using an AR2000 rheometer from TA instruments. We used
a 25 mm cone-plate geometry with an angle of 4 and a trunca-
tion of 116 m. Alternatively, a Couette device was used for low
viscosity materials. The temperature was xed at 298 K thanks
to a Peltier plate and the shear rate varied between 103 s1 and
100 s1 . The viscosity 0 was then determined from the Newto-
nian plateau present at low shear rate. Viscoelastic characterization
of coacervates was performed by frequency sweep measurements
using a 25 mm steel plate geometry equipped with a solvent trap
to avoid the drying of the samples. The strain was xed at 1% and
the angular frequency varied from 100 to 0.1 rad/s.
2.2.5. Small angle X-ray scattering
SAXS experiments were performed at the European Synchrotron
Radiation Facility (Grenoble, France) on the BM2-D2AM beamline.
First, SAXS analysis was performed on HYA and CS solutions.
The roles of the salt and polymer concentrations were investigated.
The HYA and CS samples were placed and analyzed in silica tubes
(external diameter 3 mm, wall thick-ness 0.2 mm, 76 mm long,
from Deutero GmbH) with rubber caps to avoid water evaporation.
For the PECs, according to their physical forms, either solid-
like gels or colloids, they were placed either in a home-made
sample holder of 2 mm thickness trapped in Kapton tape or sil-
ica tubes respectively. The incident photon energy was set to
17 keV. We used a 2D CCD X-ray detector from Ropper scientic.
The sample-to-detector distance was about 1.15 m and the beam
stop had a diameter of 3 mm. The q-calibration (q = 4 sin()/;
2 = scattering angle) was realized thanks to a silver behenate pow-
der standard. The scattering contribution of the empty cell and of
the solvent (glass tube lled with deionized water) was subtracted
from the scattering intensity of the samples. The image data treat-
ments took into account the distortion of the camera tapper, the
at eld response and the dark image. The intensity was further
normalized by the incident ux and the sample thickness. The scat-
tering proles of the intensity I as a function of the scattering vector
q are obtained by azimuthally averaging the corrected images.
3. Results and discussion
3.1. Study of HYA CS complexation
Since polyelectrolyte complexation is entropically driven, the
direct mixing of HYA and CS solutions in water led to a fast asso-
ciation and appearance of white precipitates. This observation
was true for every chitosan precursor solution, irrespective of the
degree of acetylation and molar mass. However, the whitening
effect was less pronounced when the DA was high and the Mw
was low.
A method for the control of the complexation of two polyelec-
trolytes was recently developed in our team (Costalat, David et al.,
2014). This was achieved by adding sodium chloride to the poly-
electrolyte solutions in order to screen the electrostatic charges of
both polyelectrolytes. This method was also investigated by Kaderli
et al. for the development of HYA-CS hydrogels for osteoarthrosis
therapy where a minimum salt concentration was added to avoid
precipitation (Kaderli et al., 2015).
We prepared a series of samples combining HYA with each chi-
tosan sample by mixing the two solutions in the presence of salts.
The investigated parameters were the chitosan DA, the molar mass
and the charge ratio r. DA = 1% and DA = 5% were eliminated from
the study, because they could not be dissolved in NaCl solutions
irrespective of the Mw. Thus, we focused on chitosans with DAs
higher than 10%. A visual assessment was performed on the differ-
ent solutions and mixtures before and after the dialysis process in
order to screen a variety of experimental conditions. So we could
select the appropriate polymer and salt concentrations to avoid the
CS precipitation and the spontaneous precipitation due to uncon-
trolled complexation between the two polysaccharides prior the
dialysis step. After dialysis, we could observe the inuence of the
charge ratio, the salt concentration in the dialysis bath as well as
the degree of acetylation and the molar mass of chitosan on the
formed polyelectrolyte complexes.
3.1.1. Polyelectrolyte complexes obtained at low charge ratio
(r = 0.1)
3.1.1.1. In the absence of salt in the dialysis bath, i.e. dialysis against
water, the polyelectrolyte assembly led to insoluble PECs dispersed
in the aqueous phase. For each DA, the complexation led to turbid
and viscous dispersions i.e. dispersions with a high light scattering
(Fig. 1a [1][6]) whatever the molar mass of chitosan. However,
we could notice a slight decrease of the light scattering with higher
DAs and lower molar masses.
 G. Laleve et al. / Carbohydrate Polymers 154 (2016) 8695
Fig. 1. Visual observations ([a] and [b]) and average diameter by DLS ([c] and [d]) of colloidal suspensions containing HYA at 3% (w/v) and CS at ratio r = 0.1 and [NaCl] in the
dialysis bath = 0 mol L1 (left) or [NaCl] in the dialysis bath = 0.15 mol L1 (right). CS characteristics are Mw = 160 kg mol1 and DA = 14% [1], Mw = 28 kg mol1 and DA = 14%
[2], Mw = 170 kg mol1 and DA = 28% [3], Mw = 19 kg mol1 and DA = 28% [4], Mw = 185 kg mol1 and DA = 48% [5], Mw = 17 kg mol1 and DA = 49% [6].
The average diameters of the colloids were highly impacted by
the DA of chitosan and ranging between 300 nm and 900 nm. For
DA = 14%, large objects were obtained with a low polydispersity
whatever the molar mass. For DA = 28% and DA = 49%, the colloids
were smaller and the polydispersity higher (Fig. 1c). These dif-
ferences may be attributed to the charge density of CS. At low
DA, a high charge density on the chitosan macromolecule allowed
interactions with many HYA molecules, leading to larger and sta-
ble objects. Conversely, at high DAs, i.e. low charge densities,
the chitosan macromolecules interacted with few HYA molecules,
resulting in small objects of varying sizes. Interestingly, the molar
mass of chitosan had no major effect on the nal particle size, which
is consistent with preceding works that showed the molar mass
of the polymer in default had a limited inuence on particle size
(Schatz et al., 2004).
3.1.1.2. In the presence of salt, i.e. 0.15 M sodium chloride during
dialysis. For DA = 14%, the results were similar to those obtained
without salt. The PECs dispersions were turbid and viscous, what-
ever the Mw of chitosan (Mw = 28 kg mol1 and Mw = 170 kg mol1
in Fig. 1b [1] and [2] respectively).
For DA = 28% and Mw = 170 kg mol1 , the complexation led also
to a turbid and viscous system (Fig. 1b [3]). When Mw was
decreased to 18 kg mol1 , the system was not turbid anymore but
opalescent (Fig. 1b [4]), i.e. with a lower light scattering intensity.
For DA = 49% and Mw = 185 kg mol1 the system was opales-
cent (Fig. 1b [5]) while for Mw = 17 kg mol1 , soluble PECs were
obtained and the solution was totally transparent (Fig. 1b [6]).
The presence of 0.15 M NaCl as residual salt concentration after
dialysis led to larger diameters and a bi-populated particle dis-
persion (Fig. 1d). The highest polydispersity was observed for the
opalescent and transparent systems. The bi-populated dispersions
might originate from an alteration of the colloidal stability of the
smaller particles that agglomerated into larger ones, as a result of
the decrease in intensity of the stabilizing electrostatic forces in the
presence of salt. Alternatively, we could also consider that, in the
presence of the screening salt, a lower polycation/polyanion associ-
89
ation would occur, leading to loose and hydrated chain aggregates
responsible for the observed high colloidal sizes.
In conclusion, in the absence of salt in the dialysis bath, insoluble
complexes were formed whatever the molar mass and DA of chi-
tosan (Fig. 2a). In the presence of salts in the dialysis baths, the
partial desalting of the homogenous mixture of polysaccharides
yielded various polyelectrolyte complexes either precipitated or
water soluble PECs depending on the DA and molar mass of chi-
tosan (Fig. 2b). As a general trend, lower molar masses associated to
higher DAs led to water soluble complexes as described by Kabanov
et al. for synthetic polymers (Kabanov & Zezin, 1984). The formation
of these soluble complexes required conformational adaptation of
the macromolecules and/or rearrangements within the complexes
that are only possible in the presence of salt.
3.1.2. Polyelectrolyte complexes obtained at higher charge ratios
(r = 0.5)
For charge ratios r = 0.5, we could not clearly point out the
inuence of the salt concentration since at the end of the dialysis
process, either coacervates or turbid solutions were already formed
in both the absence and presence of salt in the dialysis bath.
The association of HYA and CS at r = 0.5 led to two types of
materials. For the higher molar masses, we observed the forma-
tion of a biphasic system with a dense white gel phase, also called
coacervate and a transparent supernatant, irrespective of DA (see
Fig. 3a). Coacervation resulted from a syneresis phenomenon. Dur-
ing the dialysis process, associations were progressively formed via
electrostatic attractive interactions, as a result of the de-salting pro-
cess. As the complexes formed, additional short-range interactions
could develop such as hydrogen bonding or hydrophobic interac-
tions. Water molecules were expulsed out of the complex structure
leading to the contraction of the system. The supernatant is a liq-
uid phase of low viscosity containing mainly water and a small
quantity of polyelectrolyte (the concentration of polymer in the
supernatant was measured by weight residue after freeze-drying
and found lower than 1% (w/v)). For lower molar masses, turbid sus-
90 G. Laleve et al. / Carbohydrate Polymers 154 (2016) 8695
[a]
Fig. 2. Behavior of HA-CS associations at r = 0.1 as a function of the DA and the molar mass of CS in absence of salts [a] or in presence of 0.15 M NaCl [b] residual concentration
in the dialysis bath.
Fig. 3. Mixture containing HYA and CS (Mw = 170 kg mol1 ; DA = 14%) before and after the dialysis process against deionized water at charge ratio r = 0.5 (a). Turbid suspensions
obtained after dialysis for chitosans of low molar masses at r = 0.5 (b).
pensions were obtained, except for DA = 48%, for which a solution
containing opalescent gel-like aggregates was obtained (Fig. 3b).
3.2. Rheological measurements
The objective of this section is to correlate the visual obser-
vations with the rheological properties of the solutions and gels.
For this purpose, we investigated the impact of the charge ratio,
the DA and the Mw of chitosan on the Newtonian viscosity or the
viscoelastic shear moduli (G and G ).
3.2.1. At low charge ratio (r = 0.1)
As seen in Fig. 4a and b, the viscosities of the blends containing
HYA and CS before the dialysis process were mainly determined by
the HYA component, and were independent of the DA of chitosan
and slightly impacted by CS molar mass. Indeed, the Newtonian
viscosities 0 of the blends ranged between 800 Pa s and 900 Pa s for
chitosans of Mw about 170185 kg mol1 , and between 650 Pa s and
700 Pa s for chitosans of Mw around 1728 kg mol1 , irrespective of
DA.
As a general trend, the formation of the PECs led to a decrease
in the viscosities of the solutions. As expected, this decrease was
stronger when the dialysis was carried out against water than
against 0.15 mol L1 sodium chloride. Indeed, as seen in Section
3.1, in the absence of salt, the complexes always precipitated out,
whereas in the presence of salt, more material remained in solu-
tion. Finally, the DA of chitosan also impacted the variations in
viscosities: the lower the DA, the higher the loss of viscosity due to
increased complexation. This is consistent with the strong solubil-
ity of reacetylated chitosans at a DA > 40% in acidic sodium chloride
aqueous solutions (Costalat, David et al., 2014). The molar mass of
chitosan also impacted the extent of the viscosity loss upon com-
plexation: the higher molar mass induced the larger viscosity loss
[b]
(compare Fig. 4a and b). This result is also consistent with our visual
observations showing that the turbidity of the complex dispersion
was always higher for the higher molar masses of the polycation.
3.2.2. At higher charge ratio (r = 0.5)
Again, the viscosities of the blends before the dialysis process
were not impacted by the DA of chitosan but by its Mw, similarly to
the low charge ratio systems. Newtonian viscosities were around
1000 Pa s and around 750 Pa s for respectively the highest and low-
est molar masses of CS (Fig. 4c), when the viscosity of a HYA solution
in the same concentration (i.e 3% w/v) without chitosan was around
250 Pa s. After dialysis, for the low molar masses, as the viscosities
were too low to be measured with a cone-plate system, we mea-
sured the viscosity of the turbid suspensions using a Couette device.
For DAs 14% and 28%, the decrease in viscosity was drastic and the
Newtonian viscosity 0 was found about 1.8 Pa s and 4 Pa s respec-
tively (Fig. 4d). This was due to the presence of a large quantity of
polyelectrolyte complexes, colloids or aggregates, with no soluble
residual polyelectrolytes, reducing the viscosity of the system. For
DA = 49%, there was no Newtonian plateau and 0 was assimilated
to the viscosity at 0.1 s1 . Similarly to the charge ratio r = 0.1, we
pointed out that viscosity loss was reduced with an increase in the
DA of chitosan.
The coacervates, obtained with the high molar masses were
retrieved by elimination of the supernatant. The storage and loss
moduli of these coacervates were then evaluated from a constant-
strain frequency sweep within frequency ranges of 1000.1 rad/s.
A piece of coacervate was cut and the residual supernatant was
removed with absorbing paper before analysis. G and G values
are in that case only qualitative because they highly depended on
the percentage of syneresis which we were not able to control from
an experiment to another. G and G were found similar for DA = 15%
and DA = 30% and the trend for DA = 49% was an increase of the mod-
 G. Laleve et al. / Carbohydrate Polymers 154 (2016) 8695
[a]
[c]
Fig. 4. Comparison of the viscosity loss for high Mw ([a]) and low Mw ([b]) depending on the DA and the sodium chloride concentration for PECs with r = 0.1. Diagrams
representing the Newtonian viscosity of the blends before the dialysis process ([c]) and after dialysis ([d]) as a function of the CS molar mass and DA at charge ratio r = 0.5.
High molar masses yielded coacervates hydrogels.
Fig. 5. Curves representing the storage modulus (G ) and the loss modulus (G )
moduli as a function of the angular frequency . Symbols , and for G , G and
tan for DA = 14% and Mw = 170 kg mol1 respectively; Symbols ,  and for G ,
G and tan for DA = 28% and Mw = 175 kg mol1 respectively; Symbols ,  and
for G , G and tan for DA = 49% and Mw = 185 kg mol1 .
uli. The most important result was that every coacervates exhibited
a gel viscoelastic behavior dominated by elasticity with values of
tan = G /G comprised between 0.2 and 0.4 (Fig. 5).
91
[b]
[d]
3.3. SAXS analysis of HYA and CS solutions
3.3.1. Structure of HA in solution
In a rst step, we studied the conformation of the chains of HYA
in solution. Two important parameters were the polymer concen-
tration and the residual salt concentration in the solution. Since
HYA is the excess polymer, knowing the structure prior the dial-
ysis process was important for the analysis of the SAXS data we
obtained for PECs.
3.3.1.1. Inuence of the concentration. We prepared HYA solutions
at concentrations varying between 0.3% and 4%. The corresponding
SAXS proles I(q) are presented in Fig. 6a for a q-range extending
from 0.01 to 0.32 1 .
For each concentration, we observed the so-called polyelec-
trolyte peak, characteristic of a periodic organization of the
polyelectrolyte chains in solution, resulting from the repulsive elec-
trostatic interactions between the polymer chains (Essa, Spiteri,
Williams, & Boue, 2009). The evolution of the location of this
peak with the polymer concentration is related to the hydropho-
bicity/hydrophilicity of the polyelectrolyte in aqueous solutions
(Popa-Nita, Rochas, David, & Domard, 2009). On increasing the HYA
concentration, the SAXS diagrams revealed that the polyelectrolyte
peak was shifted to higher q values, corresponding to smaller inter-
chains distances (or correlation distances). At high q values, we
used a law I(q) = C/q giving coefcients (Table 2): slightly
increased with the concentration until 3% with values close to
92 G. Laleve et al. / Carbohydrate Polymers 154 (2016) 8695

Fig. 6. SAXS analysis of HYA in solution at various concentrations in% (w/v) [a]. Evolution of the position of the polyelectrolyte peak qmax as a function of HYA concentration
Cp [b]. Drawings are adapted from Ref. Dobrynin and Rubinstein (1999). (The molar mass of the averaged residue of Sodium Hyaluronate is taken to be 194 g mol1 ).

Table 2
Values of coefcients determined from the slope at high q values depending on
the HYA concentration.

HA% (w/v) 0.3% 0.5% 1% 2% 3% 4%

1.20 1.31 1.32 1.35 1.37 1.84

1.21.4 reecting a rod-like conformation probably coming from

the helical structure of HYA in solution (Heatley & Scott, 1988).
underwent an abrupt change at 4% corresponding to a conforma-
tional change and the apparition of a more aggregated structure.
We performed a tting on the different curves using a lorentzian
function, which led to the position qmax of the maximum intensity
of the peak. Then, the position qmax was plotted as a function of the
HYA concentration (Fig. 6b) and compared with previous results of
Popa-Nita et al. (2009).
For polyelectrolytes in solution, the scaling model proposed by
Dobrynin and Rubinstein (1999) consisted in considering the poly-
electrolyte chains conformation as beaded necklaces. Depending Fig. 7. Inuence of the salt concentration on the chains conformation of HYA in
on the polymer concentration, it predicts a transition from a string- solution at 3% (w/v).
controlled conformation (where the correlation length is larger
than the string length; qmax Cp 1/2 ) to a beaded necklace model Table 3
where the chain conformation is governed by the bead size (where Values of Gyration radius determined with a Guinier law at low q values >13Rg
is of the order of the distance between the beads; qmax Cp 1/3 ). depending on the salt concentration.
In our system, i.e. for HYA salt-free solutions, the concentration HA% (w/v) Concentration on NaCl (mol L1 ) Rg ()
corresponding to this transition was found about 0.06 mol L1 , in
3 0.100 20
agreement with previous results, and showing that hyaluronate is
3 0.150 26
more hydrophilic than the CS of same acetylation degree (i.e. 50%). 3 0.650 38
In the following complexation study, the HYA concentration was
3% (w/v) (i.e. a residue molar concentration of Cp = 0.15 mol L1 )
so the conformation of HYA chains was in the beads-controlled
revealed the presence of a pseudo-isosbestic point at q 0.083 1 ,
regime. The corresponding correlation length , i.e. the distance
reecting the existence of mainly two possible structural states.
between the beads can be determined with equation  = 2/q and
The rst state corresponds to the classical polyelectrolyte con-
was found close to 76 in absence of added salts.
formation of a salt-free polyelectrolyte solution, characterized by
the presence of the polyelectrolyte peak. The other state was
3.3.1.2. Inuence of the salt concentration. As we prepare the HYA observed in the presence of high concentration of salts. The scatter-
and CS mixture in presence of salts, we studied the evolution of the ing intensity increased in the low q region, showing the presence
HYA chain conformation in the presence of salts. of bigger objects which was attributed to the presence of aggre-
We prepared HYA solutions at 3% (w/v) with various amounts gates at the nanoscale due to a more hydrophobic behavior. The
of sodium chloride from 0 to 0.65 mol L1 . The synchrotron SAXS gyration radii (Rg ) were determined using a Guinier law I(q) = I0
diagrams (Fig. 7) revealed an outstanding reproducibility. In the exp (q2 Rg 2 /3) and results are reported in Table 3. The results
salt-free solution, we found again the well-dened polyelectrolyte showed an evolution to higher values of Rg when the salt concen-
peak. As the salt concentration was increased, the scattered inten- tration was increased. We also noted that for a salt concentration
sity increased and the polyelectrolyte peak disappeared for sodium of 0.65 mol L1 , a Rg value of 38 corresponding to the half of
chloride concentration higher than 0.1 mol L1 . These diagrams also the correlation length at [HYA] = 3% was found. Starting from a
 G. Laleve et al. / Carbohydrate Polymers 154 (2016) 8695 93

[a] [b]

[c]

Fig. 8. Inuence of the DA for PECs containing HYA at 3% (w/v) and CS at a constant charge ratio r = 0.1 ([a] and [b]) and r = 0.5 ([c]) and comparison to a salt-free HYA
solution. In [a] and [b], the CS concentration (w/v) was 0.16%, 0.21% and 0.30% for DAs 14%, 28% and 49% respectively. In [c], two curves showing the differences observed for
coacervates at DA = 14% from two different batches. The CS concentration (w/v) was 0.82%.

beaded-controlled regime, nanoscale aggregates are growing with
increasing the salt concentration until entering in contact. Since
[HYA] and [NaCl] were maintained at 3% (w/v) and 0.65 mol L1
respectively in the mixtures before dialysis, this conformational
state corresponded to the starting point of the complexation for
HYA in our study.
3.3.2. Study of the structure of PECs
The HYA concentration was maintained at 3% (w/v) for the
whole study. The nanostructure of a series of PECs was then studied
varying the charge ratio r. The CS concentration was varied depend-
ing on the expected ratio and the DA. This part of the work focuses
on the comparison of the PECs nanostructure at two charge ratios
(r = 0.1 and r = 0.5).
3.3.2.1. Charge ratio r = 0.1. Polyelectrolyte complexes were
formed by desalting a homogeneous mixture of HYA and CS
with a charge ratio r = 0.1. The starting salt concentration in the
HYA and CS solutions was 0.65 mol L1 and desalting was then
achieved by dialysis against deionized water. The resulting PECs
(opalescent/turbid systems) were analyzed by SAXS. Here, we
compared the structure of PECs with different DAs. The diagram of
a 3% (w/v) salt-free HYA solution was added for comparison.
Whatever the molar mass of the chitosan, the SAXS diagrams
revealed the existence of the polyelectrolyte peak (Fig. 8a and b).
The position of this peak was almost independent on the varia-
tion of the DA of CS. As the CS concentration was low compared
to the HYA concentration, we can assume that every CS macro-
molecule was complexed to HYA. Thus, the polyelectrolyte peak can
be attributed to uncomplexed or partially-complexed HYA chains.
Compared to the 3% HYA solution, qmax was slightly shifted to lower
q values, i.e. to higher values of the correlation length . Such evo-
lution of the correlation length could be due to an increase in the
persistence length by polyelectrolyte association and/or by dilution
effect of HYA. The position qmax of the PECs was found at 0.071 1
for every DA and corresponds to that obtained with a 2% (w/v)
salt-free HYA solution.
At high q values, a law I(q) = C/q gave = 1.32 whatever the
DA. This behavior was similar to HYA solutions and corresponds to
the HYA nanostructure. At low q values, an increase of the upturn
of scattered intensity was observed and can be interpreted as a
signature of aggregation with decreasing DA. These observations
at the nanoscale were consistent with the macroscopic observa-
94 G. Laleve et al. / Carbohydrate Polymers 154 (2016) 8695
tions, which showed a decrease in the light scattering contrast with
increasing DA.
3.3.2.2. Charge ratio r = 0.5. Polyelectrolyte complexes were also
formed by completely desalting a homogeneous mixture of HYA
and CS at a charge ratio r = 0.5. The starting salt concentration was
0.65 mol L1 and desalting was achieved by dialysis against deion-
ized water. The resulting PECs (coacervates) obtained for the higher
molar masses were also analyzed by SAXS. On Fig. 8c, different
scattering diagrams are obtained for samples prepared in similar
conditions (pH, ionic strength). The discrepancy of these results
illustrates the strong sensibility of the structure of the PECs towards
the experimental preparation parameters.
However, the SAXS diagrams did not exhibit the existence of
the classical polyelectrolyte peak. HYA repulsive interactions were
highly reduced due to the polyelectrolyte associations obtained at
high charge ratio. As a consequence, the neutralized hyaluronan
chains no longer exhibited a patterned polyelectrolyte organization
anymore. The pattern could be divided in three scattering domains.
At high q values, the slope was found close to q1 , which is similar
to the behavior already described by our team for chitosan-heparin
and chitosan-dextran sulfate complexes and can be attributed to
a stable morphology constituted of large rod-like objects corre-
sponding to ladder-like chain associations (Costalat et al., 2015).
At intermediate q values, the slope was comprised between q0.5
and q0.8 ; the absence of plateau probably originates from the con-
tribution of larger objects which contribution is characterized by
an increase of the slope in the low q region.
4. Conclusions
Polyelectrolyte complexation with a slow kinetic process using
the desalting method was performed to control the hyaluro-
nan/chitosan assembly. Sodium chloride was used to screen the
electrostatic charges of the polyions, at a suitable concentration
to avoid CS precipitation. The assembly via attractive electrostatic
interactions was restored by dialysis to reduce the salt concen-
tration. We investigated the parameters impacting the assembly
process. We showed a direct correlation between the visual obser-
vations, to be better quantied with light scattering and the
rheological properties of the PECs. The nanostructure was also
investigated by Small Angle X-Ray Scattering.
Mainly colloidal suspensions resulted from HYA/CS assembly
at low charge ratio r, whereas coacervates were obtained when
increasing r. The properties of the nal materials were highly
dependent on the chitosan molar mass and degree of acetylation,
as well as on the residual salt concentration.
Prior to the dialysis, aggregated systems were already formed
due to the high HYA concentration which impacted the nano-
structure of HYA in solution. On adding a low quantity of CS,
the SAXS diagrams revealed that the conformation of HYA chains
remained mainly unchanged. The PECs nanostructure was close to
the nanostructure of an HYA solution. This was also demonstrated
by rheological measurement since the PECs have a typical viscous
behavior.
When the charge ratio was increased, the PECs were either col-
loidal suspensions or coacervates, depending on CS molar mass. In
both cases, the PEC formation led to very aggregated systems, also
revealed by SAXS analysis. Because both hyaluronic acid and chi-
tosan exhibit interesting biological properties, the study and the
understanding of such associations is one of the main goals in the
polyelectrolyte domain. The controlled production of either col-
loidal suspensions or coacervates could break open developments
in the eld of biomaterials.
Acknowledgments
The authors thank the Laboratoire ObvieLine a Sinclair Pharma
company and the ANRT Association nationale de la recherche et de
la technologie through the Cifre program for the nancial support.
They also want to thank the CRG group at ESRF (France), in particu-
lar Cyrille Rochas (CERMAV, CNRS France) for his assistance during
SAXS experiments. The authors would like to acknowledge the Cen-
tre for the Characterization of Polymers by Liquid Chromatography
of the Institut de Chimie de Lyon for their expertise and assistance
in molar mass determination by SEC measurements.
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