Carbohydrate Polymers 123 (2015) 396–405

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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

Article history: Received 21 August 2014 Received in revised form 14 January 2015 Accepted 16 January 2015 Available online 7 February 2015 Keywords: Cationic curdlan derivative Quaternary ammonium salt of curdlan Polyelectrolyte complexes

a b s t r a c t 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, 13 C and 1 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 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. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Polysaccharides with quaternary ammonium groups (derivatives 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, 2010; 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-2hydroxypropyltrimethylammonium chloride, are the most used to prepare quaternary ammonium salts of polysaccharides such as agarose (Prado, Matulewicz, Bonelli, & Cukierman, 2011), cellulose (Song, Sun, Zhang, Zhou, & Zhang, 2008; Yan, Tao, & Bangal, 2009), 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: dsufl[email protected] (D.M. Suflet). http://dx.doi.org/10.1016/j.carbpol.2015.01.050 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

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 immunomodulatory 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 glucosidic 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,

D.M. Suflet et al. / Carbohydrate Polymers 123 (2015) 396–405

Cl

CH3 O

Cl

+

O

N

397

Cl

HCl

Cl

HO

+

N CH3

CH3

CH CH3 3

CH3

+

N CH3

CH CH3 3

ECH TEA

GTEAC

CHTEAC

Scheme 1. The synthesis of glycidylpropyl triethylammonium chloride and N-(3-chloro-2-hydroxypropyl)-triethylammonium chloride.

OR1 1. aq. NaOH 2. GTMAC or CHPTMAC

O

R1 O .....

OR1

OH O

HO

Cl O ....

R1 = H or .

CH3 +

N OH

CH3 CH3

NCurd I O ...

..... OH

OR2

1. aq. NaOH

Curd

2. GTEAC or CHPTEAC

R2 O

O

.....

O .... OR2 NCurd II

CH3

Cl

R2 = H or .

+

N OH

CH3 CH3

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 derivatives with ammonium quaternary groups have not been reported yet. In this paper, new curdlan derivatives with quaternary ammonium groups were synthesized using different etherifying agents such as 2,3-epoxypropyl trialkylammonium chloride or (3-chloro2-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 (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 different 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, 2014; Zhan, Lin, & Zhang, 2012; Zhang & Edgar, 2014a). Therefore, the obtaining of PECs based on the same polysaccharidic chain (curdlan) was also investigated in this paper: the new cationic curdlan derivatives with quaternary ammonium groups were used as cationic partners, and monobasic curdlan phosphate was used as

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 morphology of the PECs particles in dry state was examined by atomic force microscopy. 2. Experimental 2.1. Materials 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) trimethylammonium 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 titration) 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. 2.2. Methods 2.2.1. Synthesis of quaternization reagents with ethyl groups 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

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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. Synthesis of N-(3-chloro-2-hydroxypropyl)-triethylammonium chloride (CHPTEAC) was also performed in our laboratory following the reactions from Scheme 1. The GTEAC obtained previously was reacted with concentrated HCl solution till the pH was stabilized at 3. The reaction mixture was used in the next step (Nichifor et al., 2010). 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 dissolved in 40 ml 0.5 N NaOH (24 h at room temperature) and then the quaternization agent was added dropwise under constant stirring (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). 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 quaternary ammonium curdlan was recovered from aqueous solution by freeze-drying, when white powders were obtained. 2.2.3. Preparation of polyelectrolyte complexes The aqueous solution of polyanion (PCurd) and polycations (NCurd I and NCurd II) with concentrations of 5 × 10−3 and 5 × 10−4 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,

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 and in the presence of added salt (10−3 and 10−2 M NaCl) with an addition rate of 0.5 ml/min, under magnetic stirring. The turbidity 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.

2.2.4. Analysis and characterization of quaternary ammonium 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 D2 O, and the spectra were recorded on a Bruker Avance DRX 400 instrument operating at 400.1 and 100.6 MHz for 1 H and 13 C nuclei. 1D NMR signal assignments were made based on 2D NMR homo- and heteronuclear correlations. H,H-COSY and H,CHSQC 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. 13 C NMR spectra were recorded using between 20,000 and 40,000 scans. Conductometric titration was performed using a 712 Conductometer (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 quaternized curdlan samples (NCurd I and NCurd II).

solution of 0.1 N KOH was used in order to estimate DS by conductometric titration, according to the method already verified (Suflet, Chitanu, & Popa, 2006). Viscometric measurements were performed with an Ubbelohde viscometer with type 0a capillary, at 25 ± 0.01 ◦ C using an AVS 350 Schott (Germany) automatic viscosity measuring system. 2.2.5. Characterization of PECs The turbidimetric titrations involve measurements of the 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 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 (DLS) was used to determine the hydrodynamic diameter, Dh , of the PEC particles. DLS measurements were carried out with a Delsa Nano Submicron Particle Size (USA). For atomic force microscopy (AFM) investigations, silicon wafer supports were used after careful cleaning in two steps, as described elsewhere (Popescu, Suflet, & Mihai, 2012). Briefly, supports were firstly immersed in “piranha

Fig. 3. NMR spectra of the native curdlan and the quaternized curdlan samples (NCurd I and NCurd II) in D2 O at 25 ◦ C: 1 H NMR (a) and 13 C NMR (b).

solution” followed by intensive rinsing with distilled water and then immersed in NH4 OH/H2 O2 /H2 O mixture solution, at 70 ◦ C, in 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).

3. Results and discussion 3.1. Characterization of curdlan derivatives The DS was influenced by several factors including the molar ratio between the quaternized reagent (QR) and the anhydroglucose unit (AGU), the reaction time, and the temperature, as presented in Fig. 1. The DS values of quaternized samples were evaluated by conductometric titration.

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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 curdlan samples slightly increased with the increase of temperature and reaction time. Furthermore, when quaternary agents with glycidyl groups (GTMAC, GTEAC) were used in the reaction, higher DS were obtained, compared with the reagents containing chlorohydroxypropyl 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. In order to confirm the substitution reaction, the FTIR spectra were recorded (Supplementary Data S1). The native curdlan showed a broad band at 3433 cm−1 , assigned to stretching vibration modes of O H groups. The bands from 2889, 1377, and 1078 cm−1 were assigned to stretching vibration of CH2 groups. In Fig. 2 the deconvolution of the peaks from 1300 to 1500 cm−1 region is presented and it can be observed that the most notable difference between native curdlan and NCurd spectra is in the interval from 1476 to 1490 cm−1 , which corresponds to the methyl and ethyl 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.

The modified curdlan derivatives were analyzed through NMR spectroscopy (Fig. 3). As compared with the 1 H and 13 C NMR spectra of curdlan, the spectra of the two derivatives retain the characteristic signals of the parent polysaccharide, but also present additional signals of the R1 and R2 substituents (Scheme 2). Thus, in the 1 H NMR spectra of both derivatives, the curdlan backbone 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, CH2 -7 and CH2 -9, have signals in the same interval as the curdlan backbone protons and because of their overlapping it was not possible to detect the exact chemical shifts. The H8 proton has a characteristic signal around 4.40 ppm in both derivatives. In the 13 C NMR spectra, the methyl carbons have characteristic signals at 54.3 ppm (for R1 derivative) and 6.9 ppm (for R2 derivative). The methylene carbon from the ethyl group has the signal at 53.7 ppm, while the signals for CH2 -7 and CH2 -9 are assigned 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).

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401

Table 1 Experimental parameters from the viscometric data. Sample

0 M salt

0.1 M salt

Rao

NCurd I (DS = 0.15) NCurd II (DS = 0.12)

Heller

Huggins

[] (L/g)

a

c* × 10

[] (L/g)

KHe

c* × 10

[] (L/g)

kH

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

2

2

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 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 equation (Eq. (1)). red =

sp = [](1 + ckH [] + · · ·) c

(1)

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 consequently 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 neutral polymer and successfully extended to some polyelectrolytes in aqueous solutions (Ghimici & Nichifor, 2006; Nichifor, Stanciu, Ghimici, & Simionescu, 2011). 1 1/2 2(r

1 2



− 1)

=

1 a−1 − 2.5 []c

1 1 + sp ln(1 + sp )

 =

1 − KHe []c

(2)

(3)

In these equations r and sp are the relative and specific viscosity of the polymer solution (L/g), [] is the intrinsic viscosity (L/g), c is the polymer concentration (g/L), a and KHe are constants describing the interactions in the solution. Fig. 4b and c presents the linear plots for both polycations. 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 samples 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 hydrophobicity 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 dimensions 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 observations, 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 0.27 value of kH implying that this system is a powerful solvent for this derivative, while the higher value of kH (3.33) observed

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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). 3.3. Study of PEC formation Oppositely charged polysaccharides in aqueous medium interact 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 temperature) (Thünemann, Müller, Dautzenberg, Joanny, & Löwen, 2004; 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. 3.3.1. In situ study of PEC formation The formation of PECs was followed first by turbidimetric titration during the addition of the anionic partner (PCurd) solution to the cationic partner (NCurd I or NCurd II) solution, in the absence

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 difference 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 polycation 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 (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 complexation, 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 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

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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 10−3 M NaCl, but it is decreased when 10−2 M NaCl was added.

3.3.2. The study of PEC particles after 24 h of storage 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 and the hydrodynamic diameter (Dh ) of the particles at equilibrium. In Fig. 6, the variation of OD500 values (as a measure of the

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 abrupt increase of the OD500 (Fig. 6a and b) was located at almost 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. 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

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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 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 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. In order to reduce the dimensions of the PECs, their formation was also performed in the presence of salt. Thus, in the systems with NCurd I, the addition of 10−3 M and 10−2 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 3000 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 complexes) 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 complexes 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 dimensions 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.

was also studied in the absence and presence of added salt, and the values of intrinsic viscosities in salt-free solutions were calculated 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 derivatives 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 scattering. 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 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 turbidimetric measurements and after 24 h storage, when the end-point was 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 electrostatic interactions (formation of PECs) able to load various active principles for pharmaceutical, cosmetic or medical applications. Acknowledgements Paper dedicated to the 65th anniversary of “Petru Poni” Institute of Macromolecular Chemistry of Romanian Academy, Iasi, Romania. I.M. Pelin acknowledges the financial support of the Romanian Ministry of Education, CNCS-UEFISCDI, project number PN-II-RUPD-2012-3-0073. Appendix A. Supplementary data

4. Conclusions Cationic curdlan derivatives with a degree of substitution of up to 0.15 were synthesized using different quaternary ammonium 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 temperature. 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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