Cationic curdlan: Synthesis, characterization and application of quaternary ammonium salts of curdlan

Article abstract of DOI:10.1016/j.carbpol.2015.01.050

Water-soluble curdlan derivatives containing quaternary ammonium groups with a degree of substitution up to 0.15 were synthesized using different cationic agents in alkaline medium. The chemical structure of curdlan derivatives was confirmed by FTIR, ¹³C and ¹H NMR spectroscopy. The influence of some reaction conditions (temperature, time, and molar ratio) on the degree of substitution and the viscosimetic behaviour were studied. The degree of substitution increased with the amount of the cationization agent per anhydroglucose unit and was higher when the glycidyl reagents were used, compared with the case when the reagents contained chloro-hydroxypropyl groups. The viscosity behaviour of these new derivatives of curdlan in aqueous solutions and the values of intrinsic viscosities calculated using different semi-empirical equations denote a high hydrodynamic dimension of the macromolecular coils. The interaction of these cationic curdlan derivatives with an anionic curdlan (monobasic curdlan phosphate) was studied in situ by turbidimetric measurements and after 24 h by optical density and dynamic light scattering. The formation of polyelectrolyte complexes was influenced by the degree of substitution, the nature of the quaternary substituent, and by the ionic strength of the aqueous solution. The morphology of the polyelectrolyte complexes particles in dry state was examined by atomic force microscopy.

Full text of DOI:10.1016/j.carbpol.2015.01.050

Carbohydrate Polymers 123 (2015) 396–405
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Cationic curdlan: Synthesis, characterization and application of
quaternary ammonium salts of curdlan
∗
Dana M. Suflet , Irina Popescu, Irina M. Pelin, Alina Nicolescu, Gabriela Hitruc
Petru Poni Institute of Macromolecular Chemistry, Aleea Grigore Ghica Voda 41A, 700487 Iasi, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 August 2014
Received in revised form 14 January 2015
Accepted 16 January 2015
Available online 7 February 2015
Water-soluble curdlan derivatives containing quaternary ammonium groups with a degree of substi-
tution up to 0.15 were synthesized using different cationic agents in alkaline medium. The chemical
13
1
structure of curdlan derivatives was confirmed by FTIR, C and H NMR spectroscopy. The influence
of some reaction conditions (temperature, time, and molar ratio) on the degree of substitution and the
viscosimetic behaviour were studied. The degree of substitution increased with the amount of the cation-
ization agent per anhydroglucose unit and was higher when the glycidyl reagents were used, compared
with the case when the reagents contained chloro-hydroxypropyl groups. The viscosity behaviour of
these new derivatives of curdlan in aqueous solutions and the values of intrinsic viscosities calculated
using different semi-empirical equations denote a high hydrodynamic dimension of the macromolecular
coils. The interaction of these cationic curdlan derivatives with an anionic curdlan (monobasic curdlan
phosphate) was studied in situ by turbidimetric measurements and after 24 h by optical density and
dynamic light scattering. The formation of polyelectrolyte complexes was influenced by the degree of
substitution, the nature of the quaternary substituent, and by the ionic strength of the aqueous solution.
The morphology of the polyelectrolyte complexes particles in dry state was examined by atomic force
microscopy.
Keywords:
Cationic curdlan derivative
Quaternary ammonium salt of curdlan
Polyelectrolyte complexes
©
2015 Elsevier Ltd. All rights reserved.
1
. Introduction
gum (Banerjee et al., 2013), konjac glucomannan (Yu, Huang,
Ying, & Xiao, 2007), hemicellulose (Ren, Sun, Liu, Chao, & Luo,
2006), pullulan (Souguir, Roudesli, Picton, Le Cerf, & About-Jaudet,
2007), or starch (Auzely-Velty & Rinaudo, 2003; Heinze, Haack, &
Rensing, 2004; Kavaliauskaite, Klimaviciute, & Zemaitaitis, 2008).
These derivatives are widely used as flocculants or as thickener
in different fields including waters treatment, papermaking, food,
pharmaceutical, and cosmetic industries (Prado & Matulewicz,
2014).
Curdlan, a bacterial polysaccharide formed by pure culture
fermentation of Agrobacterium biobar 1 (identified as Alcaligenes
faecalis var. myxogenes) is a polysaccharide with a linear structure
composed entirely of d-glucose linked by ␤-glucosidic bonds in
(1 → 3) positions. Native curdlan was found to have immunomod-
ulatory effects and anti-tumour activity (Chen & Seviour, 2007;
Mantovani et al., 2008; Vetvicka & Vetvickova, 2012), but its water
insolubility generally attributed to the triple-helical structure and
the extensive number of intra/intermolecular hydrogen bonds
limits its applications. The introduction of ionic groups on the glu-
cosidic chain led to the obtaining of water-soluble derivatives. The
water-soluble derivatives of curdlan with sulphate (Osawa et al.,
1993; Yoshida, Hatanaka, & Uryu, 1990), carboxymethyl (Jin, Zhang,
Yin, & Nishinari, 2006; Zhang & Edgar, 2014a), phosphate (Suflet,
Polysaccharides with quaternary ammonium groups (deriva-
tives of cellulose, guar gum, starch, chitosan, hyaluronan,
dextran, pullulan) have some important properties as for
example: hydrophilicity, biodegradability, biocompatibility, and
bacteriostatic properties, very useful for bio-applications (Klein,
2
010; Rinaudo, 2008). These cationic polysaccharides can
be obtained by the reaction of the native polymers with
various reagents with ammonium groups. The commercial
reagents, glycidyltrimethylammonium chloride or 3-chloro-2-
hydroxypropyltrimethylammonium chloride, are the most used to
prepare quaternary ammonium salts of polysaccharides such as
agarose (Prado, Matulewicz, Bonelli, & Cukierman, 2011), cellu-
lose (Song, Sun, Zhang, Zhou, & Zhang, 2008; Yan, Tao, & Bangal,
2
&
009), micro/nanocrystalin cellulose (Zaman, Huining, Chibante,
Ni, 2012), cellulose nanofibers (Khatri, Mayakrishnana, Hiratac,
Wei, & Kima, 2013), chitin (Dinga et al., 2012), chitosan (Xu
et al., 2011), dextran (Nichifor, Stanciu, & Simionescu, 2010), guar
∗ Corresponding author. Tel.: +40 232 217454; fax: +40 232 211299.
E-mail address: dsuflet@icmpp.ro (D.M. Suflet).
http://dx.doi.org/10.1016/j.carbpol.2015.01.050
0
144-8617/© 2015 Elsevier Ltd. All rights reserved.

D.M. Suflet et al. / Carbohydrate Polymers 123 (2015) 396–405
397
Cl
CH3
O
HCl
+Cl
O
+Cl
Cl
+
N
HO
N
N
CH3
CH3
CH3
CH3
CH3 CH3
TEA
CH3 CH3
ECH
GTEAC
CHTEAC
Scheme 1. The synthesis of glycidylpropyl triethylammonium chloride and N-(3-chloro-2-hydroxypropyl)-triethylammonium chloride.
OR
1
1
. aq. NaOH
O
R O
1
2
. GTMAC or CHPTMAC
Cl
CH
3
CH
.
....
O ....
R
R
= H or .
1
2
+
3
N
OR
OH
1
OH
OH
CH3
O
NCurd I
HO
....
.
O ...
OH
OR2
1
. aq. NaOH
Curd
O
Cl
CH
2
. GTEAC or CHPTEAC R O
3
2
.
= H or
+
.
....
O ....
N
CH
3
OR
3
2
CH
NCurd II
Scheme 2. Reaction of curdlan with different quaternary ammonium agents.
Nicolescu, Popescu, & Chitanu, 2011), and amino/ammonium
groups (Kurita, Matsumura, Takahara, Hatta, & Shimojoh, 2011;
Numata, Sugikawa, Kaneko, & Shinkai, 2008; Wang et al., 2012;
Zhang & Edgar, 2014b, Chen, Wang, & Zhang, 2013) have already
been studied, but from our knowledge the cationic curdlan deriva-
tives with ammonium quaternary groups have not been reported
yet.
anionic partner. The interaction between them was investigated
in situ both in the absence and in the presence of added salts, by
turbidimetric measurements and the PECs were characterized after
24 h by optical density and dynamic light scattering. The morphol-
ogy of the PECs particles in dry state was examined by atomic force
microscopy.
In this paper, new curdlan derivatives with quaternary ammo-
nium groups were synthesized using different etherifying agents
such as 2,3-epoxypropyl trialkylammonium chloride or (3-chloro-
2. Experimental
2.1. Materials
2
-hydroxypropyl) trialkylammonium chloride. The quaternary
ammonium groups generally bring antimicrobial effects to the
polymers (Nichifor et al., 2010). Thus, the introducing of quaternary
ammonium groups on curdlan chain could extend its use in other
areas of medicine. The obtained derivatives were characterized by
Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance
Curdlan (Curd) was purchased from Wako Pure Chemical
Ind., Japan, and was used as received. Epichlorohydrin (ECH),
triethylamine (TEA), glycidyltrimethylammonium chloride ≥90%
solution (GTMAC), N-(3-chloro-2-hydroxypropyl) trimethylam-
monium chloride 60% solution (CHPTMAC), sodium hydroxide
and sodium chloride were bought from Sigma–Aldrich, Germany.
Methanol, hydrochloric acid (0.1 N), and acetone were purchased
from Chimopar, Romania. The monobasic curdlan phosphate
sodium salt (PCurd) with Mw = 178 kDa (determined by size
exclusion chromatography in aqueous salt solution) having a
substitution degree of about 1 (determined by potentiometric titra-
tion) was synthesized in our laboratory using a method that was
described elsewhere (Suflet et al., 2011). All reagents were used
without further purification and all experiments were performed
using twice-distilled water.
(
NMR) spectroscopy. The influence of reaction conditions such as
temperature, time, or molar ratio on the degree of substitution (DS)
was investigated. The viscosity behaviour and the complexation
capacity of these new polysaccharides were also studied.
As it is known, polyelectrolyte complexes (PECs) or thin films
formed using the layer-by-layer technique can be used in differ-
ent pharmaceutical and biomedical applications (Boudou, Crouzier,
Ren, Blin, & Picart, 2009; Tang, Wang, Podsiadlo, & Kotov, 2006),
where the biodegradability, biocompatibility and non-toxicity of
these systems is a priority. In this respect, the polysaccharides
are good candidates to be used in these fields. Moreover, curdlan
and its derivatives, with antitumoral and antibacterial properties,
are suitable to prepare new materials with tailored properties for
biomedical applications (Laroche & Michaud, 2007; Luo & Wang,
2.2. Methods
2.2.1. Synthesis of quaternization reagents with ethyl groups
2
014; Zhan, Lin, & Zhang, 2012; Zhang & Edgar, 2014a). There-
fore, the obtaining of PECs based on the same polysaccharidic chain
curdlan) was also investigated in this paper: the new cationic curd-
Synthesis of glycidylpropyl triethylammonium chloride (GTEAC)
was performed in our laboratory by the reaction between
epichlorohydrin and triethylamine (El-Dougdoug, 1999). Briefly, an
equimolar mixture of ECH and TEA in water was stirred at room
temperature for about 6 h. Two separated phases were obtained
(
lan derivatives with quaternary ammonium groups were used as
cationic partners, and monobasic curdlan phosphate was used as

3
98
D.M. Suflet et al. / Carbohydrate Polymers 123 (2015) 396–405
Fig. 1. Influence of the temperature and reaction time on the DS of 2-hydroxypropyl-3-trimethylamoniumm curdlan (NCurd I) (a) and 2-hydroxypropyl-3-triethylamoniumm
curdlan (NCurd II) (b) for QR: AGU = 1:3. Influence of the molar ratio between QR:AGU unit on the DS of curdlan derivatives (c).
and the lower phase (mainly GTEAG) was used directly in the next
step without further purification.
polyelectrolyte solution of PCurd, was added using an all-purpose
titrator 716 DMS Titrino (Metrohm, Switzerland). PCurd was added
to the complementary polyelectrolyte solution both in the absence
Synthesis of N-(3-chloro-2-hydroxypropyl)-triethylammonium
chloride (CHPTEAC) was also performed in our laboratory follow-
ing the reactions from Scheme 1. The GTEAC obtained previously
was reacted with concentrated HCl solution till the pH was stabi-
lized at 3. The reaction mixture was used in the next step (Nichifor
et al., 2010).
−
3
−2
and in the presence of added salt (10 and 10 M NaCl) with an
addition rate of 0.5 ml/min, under magnetic stirring. The turbid-
ity values were measured during the titration, after the addition of
each 0.02 ml titrant and 60 s equilibrium time between the addition
steps. For the study of PECs dispersions by dynamic light scattering
and optical density methods, other series of samples were prepared
by addition of different volumes of titrant solution (PCurd) on the
complementary polyelectrolyte solutions (NCurd), until a certain
ratio between them was achieved. The resulting dispersions were
stirred for the next 60 min and the measurements were performed
after 24 h of storage.
2.2.2. Synthesis of quaternary ammonium curdlan derivatives
The synthesis of 2-hydroxypropyl-3-trialkylamonium curdlan
(
NCurd) was performed as follows: 1 g of dried curdlan was dis-
solved in 40 ml 0.5 N NaOH (24 h at room temperature) and then
the quaternization agent was added dropwise under constant stir-
ring (Scheme 2). The reaction was allowed to proceed at different
◦
temperatures (50, 60, or 70 C) and periods of time (4, 5, or 6 h).
2.2.4. Analysis and characterization of quaternary ammonium
The reaction mixture was precipitated in methanol/acetone (1:1,
v/v), re-dissolved in water, neutralized with 0.1 N HCl, and then
purified by diafiltration in a Millipore cell with PM10 membrane
till the diafiltrate conductivity was lower than 5 ␮S/cm. The qua-
ternary ammonium curdlan was recovered from aqueous solution
by freeze-drying, when white powders were obtained.
curdlan derivatives
FTIR spectra were recorded on KBr pellet using a Vertex 70
Bruker spectrometer. For the NMR analysis, all the compounds were
dissolved in D O, and the spectra were recorded on a Bruker Avance
2
1
DRX 400 instrument operating at 400.1 and 100.6 MHz for H and
13
C nuclei. 1D NMR signal assignments were made based on 2D
NMR homo- and heteronuclear correlations. H,H-COSY and H,C-
HSQC experiments were recorded using standard pulse sequences
in the version with z-gradients, as delivered by Bruker with TopSpin
1.3 PL10 spectrometer control and processing software. All NMR
measurements were done at room temperature. C NMR spectra
were recorded using between 20,000 and 40,000 scans.
2
.2.3. Preparation of polyelectrolyte complexes
The aqueous solution of polyanion (PCurd) and polycations
−
3
(
5
NCurd I and NCurd II) with concentrations of 5 × 10
and
−
4
13
× 10 equiv./L, respectively, were prepared at room temperature
+
and the concentration of the charged groups in solutions (n and
n ) was determined using Particle Charge Detector PCD 03 (Mütek
GmbH, Germany). For the in situ study of PEC formation, the titrant,
−
Conductometric titration was performed using a 712 Conduc-
tometer (Metrohm, Switzerland), equipped with 6.0908.110 cell. A

D.M. Suflet et al. / Carbohydrate Polymers 123 (2015) 396–405
399
Fig. 2. Deconvolution of FTIR spectra of the native curdlan (Curd) and the quater-
nized curdlan samples (NCurd I and NCurd II).
solution of 0.1 N KOH was used in order to estimate DS by con-
ductometric titration, according to the method already verified
(Suflet, Chitanu, & Popa, 2006). Viscometric measurements were
performed with an Ubbelohde viscometer with type 0a capillary, at
Fig. 3. NMR spectra of the native curdlan and the quaternized curdlan samples
◦
2
5 ± 0.01 C using an AVS 350 Schott (Germany) automatic viscosity
◦
1
13
(
NCurd I and NCurd II) in D2O at 25 C: H NMR (a) and C NMR (b).
measuring system.
2
.2.5. Characterization of PECs
The turbidimetric titrations involve measurements of the
solution” followed by intensive rinsing with distilled water and
◦
then immersed in NH OH/H O /H O mixture solution, at 70 C, in
4
2
2
2
intensity variation of a light flow passing through a solution
with/without colloidal particles. Turbidity measurements were
made with a Brinkmann PC900 colorimeter (USA) equipped with
a 40% neutral density filter and a probe tip of 1 cm light path.
The device indicates the value of transmitted light (transmittance,
T%) and the system’s turbidity, t%, is obtained as a difference:
t% = 100 − T%. Optical density at 500 nm (OD500) was also used as
a measure of the turbidity for the PEC dispersions obtained at
an ultrasonic bath, followed by an intensive rinse with water. The
clean silicon wafers were immersed in PECs dispersions for 30 min,
then washed three times in twice distilled water, and finally dried at
room temperature. The shapes of PECs particles were examined by
means of a Scanning Probe Microscope Solver PRO-M platform (NT
◦
MTD, Russia), in tapping mode in air, at room temperature (23 C).
−
+
different ratios between the polyelectrolytes charges (n /n ). A
Thermo Fisher Scientific Evolution 201 (USA) spectrophotometer
was used for the UV–vis measurements. Dynamic light scattering
3. Results and discussion
3.1. Characterization of curdlan derivatives
(
DLS) was used to determine the hydrodynamic diameter, D , of
h
the PEC particles. DLS measurements were carried out with a Delsa
Nano Submicron Particle Size (USA). For atomic force microscopy
The DS was influenced by several factors including the molar
ratio between the quaternized reagent (QR) and the anhydroglu-
cose unit (AGU), the reaction time, and the temperature, as
presented in Fig. 1. The DS values of quaternized samples were
evaluated by conductometric titration.
(
AFM) investigations, silicon wafer supports were used after care-
ful cleaning in two steps, as described elsewhere (Popescu, Suflet,
Mihai, 2012). Briefly, supports were firstly immersed in “piranha
&

4
00
D.M. Suflet et al. / Carbohydrate Polymers 123 (2015) 396–405
Fig. 4. Plot of viscosity data for NCurd in free-salt aqueous solution in terms of Huggins (a), Rao (b) and Heller (c) equations. Plot of viscosity data in the presence of 0.1 M
NaCl in terms of Huggins equation (d).
As it was expected, the DS values of the quaternized curd-
lan samples slightly increased with the increase of temperature
and reaction time. Furthermore, when quaternary agents with gly-
cidyl groups (GTMAC, GTEAC) were used in the reaction, higher
DS were obtained, compared with the reagents containing chloro-
hydroxypropyl groups (CHPTMAC, CHPTEAC). Due to the structure
of curdlan (triple-helical crystalline structure and a large number
of hydrogen bonds) its reactivity is lower compared with other
polysaccharides, therefore low DS (maximum 0.15) were obtained.
The increase of QR:AGU ratio until 3:1 led to the increase of the DS,
but over this ratio the DS was not improved. Furthermore, if the
QR:AGU ratio was increased over 5:1, the cross-linking of curdlan
solution occurred.
The modified curdlan derivatives were analyzed through NMR
1
13
spectroscopy (Fig. 3). As compared with the H and C NMR spectra
of curdlan, the spectra of the two derivatives retain the char-
acteristic signals of the parent polysaccharide, but also present
additional signals of the R1 and R2 substituents (Scheme 2). Thus,
in the 1H NMR spectra of both derivatives, the curdlan back-
bone protons have the signals in the interval 3–4.2 ppm, while
the signal corresponding to the H-1 proton is overlapped by the
water signal. The methyl protons have characteristic signals at
3.26 ppm (for R1 substituent) (Pretsch, Buhlmann, & Badertscher,
2010) and 1.36 ppm (for R2 substituent) (Nichifor et al., 2010;
Souguir et al., 2007). The protons, namely CH2 from the ethyl
group, CH -7 and CH -9, have signals in the same interval as
2
2
In order to confirm the substitution reaction, the FTIR spec-
tra were recorded (Supplementary Data S1). The native curdlan
the curdlan backbone protons and because of their overlapping
it was not possible to detect the exact chemical shifts. The H-
8 proton has a characteristic signal around 4.40 ppm in both
derivatives.
−1
showed a broad band at 3433 cm , assigned to stretching vibration
−
1
modes of O H groups. The bands from 2889, 1377, and 1078 cm
were assigned to stretching vibration of CH2 groups. In Fig. 2
In the 13C NMR spectra, the methyl carbons have characteristic
signals at 54.3 ppm (for R1 derivative) and 6.9 ppm (for R2 deriva-
tive). The methylene carbon from the ethyl group has the signal
at 53.7 ppm, while the signals for CH -7 and CH -9 are assigned
−
1
the deconvolution of the peaks from 1300 to 1500 cm region is
presented and it can be observed that the most notable difference
between native curdlan and NCurd spectra is in the interval from
2
2
−1
1
476 to 1490 cm , which corresponds to the methyl and ethyl
at 73.2 ppm and 68.2 ppm, overlapped with the CH-2 and CH-4
carbons from curdlan. The signal corresponding to CH-8 carbon
appears around 64.5 ppm, in both derivatives (Pelosi, Bulone, &
Heux, 2006; Saito, Yokoi, & Yoshioka, 1989).
groups bound to the quaternary nitrogen (Nichifor et al., 2010; Song
et al., 2008). Other bands characteristic to quaternary compounds
of curdlan overlap with the bands of the parent polysaccharide.

D.M. Suflet et al. / Carbohydrate Polymers 123 (2015) 396–405
401
Table 1
Experimental parameters from the viscometric data.
Sample
0 M salt
Rao
0.1 M salt
Huggins
[ꢀ] (L/g)
Heller
2
2
[ꢀ] (L/g)
a
c* × 10
[ꢀ] (L/g)
KHe
c* × 10
kH
NCurd I (DS = 0.15)
NCurd II (DS = 0.12)
17.70
11.88
0.13
0.12
5.65
8.42
18.23
13.33
0.37
0.42
5.20
7.50
0.34
0.20
0.27
3.33
Fig. 5. The variation of the turbidity when the polyanion solution (5 × 10−3 equiv./L) was added on the polycation solution (5 × 10−4 equiv./L): NCurd I (a) and NCurd II (b).
3
.2. Viscometric behaviour
In these equations ꢀr and ꢀsp are the relative and specific viscos-
ity of the polymer solution (L/g), [ꢀ] is the intrinsic viscosity (L/g), c
is the polymer concentration (g/L), a and KHe are constants describ-
ing the interactions in the solution. Fig. 4b and c presents the linear
plots for both polycations.
Viscosity studies provide an evaluation of the macroscopic
behaviour of polymer solutions and allow the estimation of the
intrinsic viscosity of quaternary ammonium curdlan salts. The
viscosity of diluted polymer solutions is described by Huggins equa-
tion (Eq. (1)).
The properties of polyelectrolytes solutions are strongly
dependent on the ionic strength of the aqueous medium. Thus, with
the increase of the amount of added salt into the polyelectrolyte
solution, the electrostatic repulsions are gradually screened, so that
at high salt concentration the macroion behaves as neutral polymer
(Cohen & Priel, 1989; Cohen, Priel, & Rabin, 1988; Mandel, 1987). In
Fig. 4d it is shown the linear dependence ꢀsp/c = f(c) of NCurd sam-
ples in aqueous solution with added salt (0.1 M NaCl). The values
of parameters from the viscometric data were calculated from the
Rao and Heller equations for the salt-free solutions and from the
Huggins equation in the presence of added salt and are presented
in Table 1. The lower intrinsic viscosity value of NCurd II compared
to NCurd I is due to the increase of the number of carbon atoms of
alkyl radical of quaternary groups that decrease the hydrophobic-
ity of the polymer. This behaviour was confirmed in literature for
other cationic polysaccharides (Nichifor et al., 2011).
Intrinsic viscosity is a measure of hydrodynamic volume of
macromolecules in diluted solution at concentration lower than the
overlap concentration (c*; c* = 1/[ꢀ]) where the polymer chains are
separated and the intrinsic viscosity depends only on the dimen-
sions of the individual polymer chains. The c* values for quaternary
curdlan derivatives are presented in Table 1. The high values of
[ꢀ] will anticipate the decrease of the c*, which denotes the high
hydrodynamic dimension of the macromolecular coils of these
new derivatives of curdlan. Also, based on experimental obser-
vations, in aqueous solution with added salt (0.1 M NaCl), the
physical meaning of the dimensionless Huggins coefficient (kH)
can be summarized as follows: in the case of NCurd I sample, the
ꢀ
sp
ꢀred
=
= [ꢀ](1 + ckH[ꢀ] + · · ·)
(1)
c
where ꢀred and ꢀsp are the reduced and specific viscosity of the
polymer solution (L/g), [ꢀ] – the intrinsic viscosity (L/g), c – the
polymer concentration (g/L), kH – Huggins coefficient, an indication
of polymer–solvent affinity. In Fig. 4a the Huggins plots (ꢀsp/c vs.
c) for NCurd solutions in pure water are shown. The quaternary
ammonium salt samples exhibit typical polyelectrolyte behaviour.
The exponential increase of the reduced viscosity with polymer
dilution was ascribed to the expansion of the polyelectrolyte chains
due to the increase of counterion dissociation and as a result of
the intramolecular repulsive forces. This polyelectrolyte behaviour
confirms that the substitution reaction of curdlan occurred.
Due to the non-linear shape of the ꢀsp/c vs. c curves (Fig. 4a),
the extrapolation to zero concentration is not possible, and conse-
quently cannot be used to determine the intrinsic viscosity. This
problem can be solved by means of semi-empirical equations like
Rao (Eq. 2) or Heller (Eq. 3) equations (Durand, 2007) used for neu-
tral polymer and successfully extended to some polyelectrolytes
in aqueous solutions (Ghimici & Nichifor, 2006; Nichifor, Stanciu,
Ghimici, & Simionescu, 2011).
1
1
[ꢀ]c
a − 1
2.5
=
−
(2)
(3)
2
(ꢀ 1r /2 − 1)
ꢀ
ꢁ
1
2
1
ꢀsp
1
1
0
.27 value of kH implying that this system is a powerful solvent
+
=
− KHe
ln(1 + ꢀsp)
[ꢀ]c
for this derivative, while the higher value of kH (3.33) observed

4
02
D.M. Suflet et al. / Carbohydrate Polymers 123 (2015) 396–405
Fig. 6. OD500 (a, b) and hydrodynamic diameter (c, d) of the PEC dispersions as a function of the ratio between charges.
for NCurd II sample was specific for polymers in “poor solvent”,
when the polymer–polymer interactions become favourable over
polymer–solvent interactions. In the last case the system is very
sensitive to the formation of aggregates (Ma & Pawlik, 2007; Nandi,
Bhattarai, & Das, 2007).
and in the presence of added salt, as shown in Fig. 5. The deviation
from a 1:1 stoichiometric charge ratio could be due to the differ-
ence in the distribution of charges on the polysaccharidic backbone
between the opposite charges of polyelectrolytes (Le Cerf et al.,
2014; Mende, Buchhammer, Schwarz, Petzold, & Jaeger, 2004),
especially because, in our case, the substitution degree of the poly-
cation was much lower than that for the polyanion. In the absence of
salt, the NCurd I sample with DS = 0.15 presents an abrupt increase
of the turbidity which corresponds to the ratio between charges
3.3. Study of PEC formation
Oppositely charged polysaccharides in aqueous medium inter-
−
+
act spontaneously to form polyelectrolyte complexes, a process
known to be very important in biological systems. Also, it is known
that the formation of PECs is governed by the characteristics of
individual components (e.g., properties and position of ionic sites,
charge density, macromolecular chain rigidity) and also by the
chemical environment (e.g., solvent, ionic strength, pH and tem-
perature) (Thünemann, Müller, Dautzenberg, Joanny, & Löwen,
(n /n ) of around 1. With the decrease of the DS, the increase of
−
+
the turbidity was located at lower n /n ratios, meaning that the
neutral part of the polymer chains contributed to the insolubility
of formed PECs. When NCurd II was used as polycation in com-
−
+
plexation, the end-point was observed at n /n ratios lower than 1,
meaning that a part of the cationic charges is not accessible for the
polyanion due to the steric hindrance of the longer ethyl groups. As
in the case of NCurd I, with the increase of the DS of NCurd II the
2
004; Tsucuchida & Abe, 1982). Hereinafter, two samples from each
−
+
derivative of NCurd I and NCurd II with DS between 0.15 and 0.07
were used in order to study the PECs formation with a polyanionic
curdlan derivative with DS = 1.
end-point was closer to n /n = 1 and the turbidity before and after
the end point increased.
The addition of a low molecular electrolyte in the system
influences the PECs formation: thus the low concentration of
added salt determines the shielding of charges and increases the
chains flexibility, leading to an easier rearrangement of the chains
in the PECs aggregates, instead of the high concentration of salt
which shields the electrostatic attraction between polyanion and
3.3.1. In situ study of PEC formation
The formation of PECs was followed first by turbidimetric titra-
tion during the addition of the anionic partner (PCurd) solution to
the cationic partner (NCurd I or NCurd II) solution, in the absence

D.M. Suflet et al. / Carbohydrate Polymers 123 (2015) 396–405
403
Fig. 7. AFM images of complex particles obtained for a ratio between charges for NCurd I (a–f) and NCurd II (g–l) in the absence and presence of added salt.
polycation, inhibiting the formation of PECs. For this reason, at
the equivalent point the turbidity is increased by the addition of
dispersion turbidity) and of Dh values as a function of the ratio
between charges is presented.
For the dispersions prepared in the absence of salt, but also for
those prepared in the presence of salt, the end point defined as the
−
3
−2
1
0
M NaCl, but it is decreased when 10 M NaCl was added.
abrupt increase of the OD
(Fig. 6a and b) was located at almost
500
−
+
3.3.2. The study of PEC particles after 24 h of storage
the same values of n /n ratios as in the titration in situ (Fig. 5).
The shape of the turbidimetric curves after 24 h of storage was also
the same with those obtained in situ. These findings suggest the
stability of the formed PECs.
Previous studies showed that a stirring time is necessary to
reach the equilibrium in the process of PECs formation as stable
dispersions (Popescu et al., 2012). Thus, the samples were stored
for 24 h in order to determine the turbidity of the PEC dispersions
The DLS measurements of the PEC particles (Supplementary
Data S2) obtained in the absence of salt show that the abrupt
increase of the Dh is located at higher n /n ratios compared to
and the hydrodynamic diameter (D ) of the particles at equilib-
h
−
+
rium. In Fig. 6, the variation of OD500 values (as a measure of the

4
04
D.M. Suflet et al. / Carbohydrate Polymers 123 (2015) 396–405
the variation of OD500. This indicates that the primary complexes
initially formed, which are visualized by the abrupt increase of the
turbidity, have low dimensions (around 200 nm for the quaternary
curdlan samples with higher DS and around 600 nm for the samples
with lower DS). With the further addition of the titrant (PCurd),
the anionic polymer chains linked the primary complexes with
the formation of much larger aggregates (Buchhammer, Mende, &
Oelmann, 2003). The formation of the aggregates was observed at
was also studied in the absence and presence of added salt, and
the values of intrinsic viscosities in salt-free solutions were calcu-
lated using different semi-empirical equations. The high values of
[ꢀ] (lower c* values) denote a high hydrodynamic dimension of the
macromolecular coils of the new derivatives of curdlan.
The obtaining of polyelectrolyte complexes based on the curdlan
derivatives was also investigated. The new cationic curdlan deriva-
tives with quaternary ammonium groups were used as cationic
partners, and monobasic curdlan phosphate was used as anionic
partner in PECs. The interaction between them was investigated
in situ by turbidimetric measurements and the formed PECs were
characterized after 24 h by optical density and dynamic light scat-
tering.
−
+
n /n = 1 for the derivatives with methyl groups (Fig. 6c), and 0.9
for the derivatives with ethyl groups (Fig. 6d). The aggregates are
larger with the increase of the DS of the NCurd samples (6000 nm
for NCurd I with DS = 0.07 compared with 9000 for NCurd I with
DS = 0.15). In the case of the derivative with methyl groups (NCurd
I) the size of the aggregates slowly increase with further addition of
polyanion, but the OD500 values decrease, showing that the primary
complexes from the solution are embedded into the aggregates. In
the case of the derivative with ethyl groups (NCurd II), both the
The end-point of interpolyelectrolyte interaction followed in
situ by turbidimetry was influenced both by the structure and by
the degree of substitution of quaternary derivatives. Thereby, the
−
+
end-point was closer to n /n = 1 with the increase of the DS of
−
+
−
+
Dh and the OD500 slowly decrease after n /n = 0.9, probably due
to the rearrangement of the aggregates. The results obtained by
DLS measurements are in agreement with the high hydrodynamic
dimension of the macromolecular coils of quaternary derivatives
calculated from viscosimetric data.
NCurd and this was observed at lower n /n ratios, in the case of
NCurd II compared to NCurd I, meaning that a part of the cationic
charges are not accessible due to the steric hindrance of the longer
ethyl groups.
A good accordance was observed between in situ turbidimet-
ric measurements and after 24 h storage, when the end-point was
In order to reduce the dimensions of the PECs, their formation
was also performed in the presence of salt. Thus, in the systems
−
+
located at almost the same values of n /n ratios, suggesting the
stability of the formed PECs. The variation of the hydrodynamic
diameter showed that the PECs initially formed were the primary
complexes with Dh around 200–500 nm both in the absence and
the presence of added salt. With further addition of the titrant,
the anionic polymer chains linked these primary complexes with
the formation of much larger aggregates with Dh over 6000 nm in
free-salt system. When in the system was added salt, the excess of
anionic charges were shielded, the linking of primary complexes
does not occurs and the size particles of PECs remain at the initial
sizes (500 nm). The presence of added salt had a lower effect in the
case of NCurd II when the PECs sizes were also reduced, but not less
than 3000 nm. These new cationic curdlan derivatives could be used
to obtain micro/nanoparticles by chemical cross linking or by elec-
trostatic interactions (formation of PECs) able to load various active
principles for pharmaceutical, cosmetic or medical applications.
−
3
−2
with NCurd I, the addition of 10 M and 10 M NaCl led to the
formation of small particles (300–500 nm) even with the excess
of polyanion. When the derivative with ethyl groups (NCurd II)
with high DS was used as cationic partner, the addition of salt also
reduced the Dh of the PECs with excess of PCurd, but not under
3
000 nm.
AFM was used as a direct method to obtain information about
the morphology of the PECs particles, knowing that the complexes
exist in variable shapes and forms, such as round spherical particles,
linear chains, segments, and aggregating flocks (Sæther, Holme,
Maurstad, Smidsrød, & Stokke, 2008; Sun, Mao, Mei, & Kissel, 2008;
Volod’ko et al., 2014; Zhao et al., 2009). Hence, before the end-point
−
+
(
n /n < 0.9), the small sizes of PECs nanoparticles (primary com-
plexes) were confirmed by AFM both in the absence and in the
presence of added salt (Fig. 7a–c). In the case of PECs formation
with NCurd I, in the absence of salt, when polyanion was added
−
+
in excess (n /n > 0.9), the AFM micrographs indicated large com-
plexes with various shapes, flocks, and aggregates. The presence
of added salt in the system led to the formation of small particles
even with excess of polyanion (Fig. 7d–f). In the case of NCurd II,
the addition of salt did not have the same effect on the PECs dimen-
sions when the relative large particle sizes were obtained (Fig. 7k
and l). The AFM images of PECs particles are in concordance and
confirm the results obtained by DLS measurements.
Acknowledgements
Paper dedicated to the 65th anniversary of “Petru Poni” Institute
of Macromolecular Chemistry of Romanian Academy, Iasi, Roma-
nia. I.M. Pelin acknowledges the financial support of the Romanian
Ministry of Education, CNCS-UEFISCDI, project number PN-II-RU-
PD-2012-3-0073.
Appendix A. Supplementary data
4
. Conclusions
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.
Cationic curdlan derivatives with a degree of substitution of
2
015.01.050.
up to 0.15 were synthesized using different quaternary ammo-
nium reagents in alkaline medium. The DS values were influenced
by several factors including the molar ratio between quaternized
reagent and anhydroglucose unit (AGU), reaction time, or temper-
ature. DS values increase with the amount of cationization agent
per anhydroglucose unit. Furthermore, when quaternary agents
with glycidyl groups (GTMAC, GTEAC) were used in the reaction,
higher DS were obtained, compared with the reagents containing
chloro-hydroxypropyl groups (CHPTMAC, CHPTEAC). The increase
of QR:AGU ratio until 3:1 led to the increase of the DS, but over
this ratio the DS was not improved, even more, if the QR:AGU
ratio was increased over 5:1, the cross-linking of curdlan solution
occurred. The viscosity behaviour of these new polysaccharides
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