Polyelectrolyte Complexes from Polysaccharides: Formation and

Sep 20, 2007 - ... from Polysaccharides: Formation and Stoichiometry Monitoring ..... J. V. Araujo , N. Davidenko , M. Danner , R. E. Cameron , S. M. ...
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Langmuir 2007, 23, 10950-10958

Polyelectrolyte Complexes from Polysaccharides: Formation and Stoichiometry Monitoring Alexandre Drogoz,†,‡ Laurent David,‡ Cyrille Rochas,§ Alain Domard,‡ and Thierry Delair*,† Unite´ mixte CNRS-BioMe´ rieux, UMR 2714, ENS Lyon, 46, alle´ e d’Italie, 69364 Lyon Cedex 07, France, Laboratoire des Mate´ riaux Polyme` res et des Biomate´ riaux, UMR CNRS 5223 IMP UniVersite´ de Lyon, UniVersite´ Lyon 1, Baˆ t ISTIL, 15 Bd. A. Latarjet, 69622 Villeurbanne Cedex, France, and Laboratoire de Spectrome´ trie Physique, UMR 5588 UniVersite´ Joseph Fourier, Grenoble1- CNRS BP 87 38402, Saint Martin d’He` res, France ReceiVed March 27, 2007. In Final Form: July 9, 2007 Colloids were obtained from non-stoichiometric polyelectrolyte complexes with two polysaccharides of opposite charge: chitosan and dextran sulfate (DS) as the polycation and polyanion, respectively. The complexes were elaborated by a one-shot addition of the polymer in default to the one in excess. The colloids were positively or negatively charged according to the nature of the polymer in excess. Dynamic light scattering (DLS) demonstrated that particles were formed at a very early stage in the complexation process. The consumption of the excess polyelectrolyte was monitored with a dye assay specific for dextran sulfate (toluidine blue) or chitosan (orange II). From these experiments, two different mechanisms of colloidal PEC formation were evidenced, according to the nature of the polymer in excess. On adding chitosan to DS in excess, regular consumption of the polyanion was observed at a constant stoichiometry, in the 1.5 to 1.85 range (sulfate residues for one glucosamine group), according to the molar mass of the polycation. When DS was added to chitosan in excess, the overall stoichiometry varied from ca. 6 (glucosamine residues for one sulfate group) down to 1 as the charge molar mixing ratio R ) n+/n- decreased from 20 to 1. The existence of various mechanisms, according to the nature of the polymer in excess, could be attributed to the differences in chemical reactivity (strong vs low) of the ion in excess and the conformation and flexibility of the macromolecular chains related to their electrostatic potential.

1. Introduction Polyelectrolyte complexes (PECs) result from the complete or partial ionic condensation between oppositely charged polymers.1 The concomitant release of corresponding counterions is the main driving force of the reaction because it corresponds to an increase in entropy of the system. Other interactions may be involved in the formation of PEC structures, such as hydrogen bonding, hydrophobic, or other van der Waals interactions. Watersoluble polyelectrolyte complexes were formed with polyelectrolytes having a weak charge density and large differences in molecular dimensions when they were mixed in non-stoichiometric ratios.2,3 Alternatively, polyions having a high charge density and/or similar high molar masses led to insoluble and highly aggregated complexes, according to the scrambled-egg model, by the incorporation of several polyelectrolyte chains.4 Colloidal dispersions in the submicrometer range could be obtained by polyelectrolyte complexation, provided polymer concentrations remained low in the medium. Various synthetic polyions were used, such as poly(diallyldimethylammonium chloride) associated with various copolymers of maleic anhydride5,6 or poly(sodium 2-acrylamido-2methylpropenesulfonate).7 * Corresponding author. E-mail: [email protected]. † Unite ´ mixte CNRS-BioMe´rieux, UMR 2714. ‡ UMR CNRS 5223 IMP Universite ´ de Lyon. § UMR 5588 Universite ´ Joseph Fourier. (1) Dautzenberg, H. In Physical Chemistry of Polyelectrolytes; Surfactants Science Series; Radeva, T., Ed.; Marcel Dekker: New York, 2001; Vol. 99, p 743. (2) Dautzenberg, H.; Karibyants, N. Macromol. Chem. Phys. 1999, 200, 118. (3) Kabanov, V.; Zezin, A. B. Makromol. Chem. Suppl. 1984, 6, 259. (4) Dautzenberg, H.; Hartmann, J.; Grunewald, S.; Brand, F. Ber. BunsenGes. Phys. Chem. 1996, 100, 1024. (5) Buchhammer, H.-M.; Mende, M.; Oelmann, M. Colloids Surf., A 2003, 218, 151. (6) Muller, M.; Reihs, T.; Ouyang, W. Langmuir 2005, 21, 465.

With these types of polymers, fairly isodisperse colloids could be obtained, especially when the polymer in excess was added to the one in default.5,7 Polysaccharides were also used to obtain colloids in the submicrometer range with chitosan (a β, (1f4)-linked copolymer of 2-amino-2-deoxy-β-D-glucan (GlcN) and 2-acetamido-2deoxy-β-D-glucan (GlcNAc)) as the polycation and various polyanions such as DNA,8 dextran sulfate,9-11 poly-γ-L-glutamic acid,12 carboxymethyl konjac glucomannan,13 and carboxymethyl cellulose.14 Very often, these new materials were designed for applications such as DNA8/drug delivery13,14 and cell interactions12 in the field of bioscience. The charge neutralization can be monitored by viscosimetry,15,16 with the viscosity minimum corresponding to the end of the titration of one polyion by the other. The end point of the titration can also be observed by potentiometry or conductometry. In the latter case, the variations in conductivity depend on the nature of the free ions in solution. Such characterization methods, based on the variations of a physicochemical property of the (7) Dragan, E. S.; Schwarz, S. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 5244. (8) Liu, G. W.; De Yao, K. J. Controlled Release 2002, 83, 1. (9) Schatz, C.; Domard, A.; Viton, C.; Pichot, C.; Delair, T. Biomacromolecules 2004, 5, 1882. (10) Schatz, C.; Lucas, J. M.; Viton, C.; Domard, A.; Pichot, C.; Delair, T. Langmuir 2004, 20, 7766. (11) Sarmento, B.; Ribeiro, A.; Veiga, F.; Ferreira, D. Colloids Surf., B 2006, 53, 193. (12) Lin, Y. H.; Chung, C. K.; Chen, C. T.; Liang, H. F.; Chen, S. C.; Sung, H. W. Biomacromolecules 2005, 6, 1104. (13) Du, J.; Sun, R.; Zhang, L. F.; Xiong, C. D.; Peng, Y. X. Biopolymers 2005, 78, 1. (14) Watanabe, J.; Iwamoto, S.; Ichikawa, S. Biosci. Biotechnol. Biochem. 2005, 69, 1637. (15) Takahashi, T.; Takayama, K.; Machida, Y.; Nagai, T. Int. J. Pharm. 1990, 61, 35. (16) Lee, K. Y.; Park, W. H.; Ha, W. S. J. Appl. Polym. Sci. 1997, 63, 425.

10.1021/la7008545 CCC: $37.00 © 2007 American Chemical Society Published on Web 09/20/2007

Polyelectrolyte Complexes from Polysaccharides

continuous phase (viscosity, pH, and conductivity), give a global evaluation of the complexation reaction but do not give access to the complex composition. This type of information can be obtained by elemental analysis of the complex,17,18 solid-state NMR,18 or the depletion method.15,19,20 The depletion method relied on the measurement of the amount of remaining polyelectrolyte in the supernatant, leading to the amount of polymer effectively involved in the complex. Karibyan et al. reported discrepancies between results obtained by potentiometry and the depletion method.20 Whereas the first method provided 1:1 stoichiometry for the complexation of polystyrene sulfonate and poly(diallyldimethylammonium chloride) (PDADMAC), the second revealed that the stoichiometry decreased from 2.5 to 1 on adding the polycation to the polyanion. In the present work, the formation of chitosan-dextran suflate colloidal PECs was monitored by the depletion technique during the neutralization process. We observed that non-stoichiometric complexes were formed, whose mechanism of formation and composition depended on the nature of the polymer in excess, the molar mass of the polyions, and the charge density of chitosan. Hence, we focused our work on the investigation of the mode of complexation and on the characterization of the colloids in terms of physicochemistry and chemical composition in order to obtain better knowledge of these new bionanomaterials. 2. Experimental Section 2.1. Materials. Two weakly acetylated chitosans were used: one with a low weight-average molecular weight (Mw) (Aber technologies, batch A32E03, DA ∼1%, Mw ∼13 000 g‚mol-1) and another with a relatively high molar mass (Mahtani chitosan PVT, batch 124, DA ∼5%, Mw ∼349 000 g‚mol-1). Prior to use, the polymers were purified as follows: dissolution in a 0.1 M acetic acid solution, filtration through Millipore membranes of decreasing porosity (from 3 to 0.22 µm), precipitation with an ammonia/methanol mixture, rinsing with deionized water until neutrality, and lyophilization. Purified high-Mw chitosan was N-acetylated in homogeneous media at different DAs with acetic anhydride. The reaction was performed in a hydroalcohol mixture according to the procedure previously described by Vachoud et al.21 After re-acetylation, chitosans were neutralized, rinsed with deionized water, and then freeze dried. In addition, hydrolysis9,10 with control of the kinetics was carried out to produce low-molecular-weight polymers. For this purpose, chitosans were dissolved at 0.5% (w/v) in a 0.2 M acetic acid/0.1 M sodium acetate buffer. A 0.15 M sodium nitrite solution was added to chitosan solutions to obtain a nitrite/glucosamine units molar ratio of 0.5. The reaction was performed under moderate magnetic stirring for various reaction times (1-15 h). Low-molarmass chitosans were recovered by precipitation with an ammonia/ methanol mixture, purified by several washings with deionized water until neutrality was achieved, and finally lyophilized. Dextran sulfate was from Sigma and was used without further purification. The manufacturer provided the water content. The molecular characteristics were determined by gel permeation chromatography, according to our previous work.10 The weightaverage molar mass was Mw ) 1.51 ( (0.20 × 106) g/mol with a polydispersity index Ip of 1.52 ( 0.09, and the z-average root mean square of the radius of gyration was Rg,z ) 72.5 ( 3.0 nm. The degree of sulfation (i.e., the number of sulfate functions per glucosidic unit) was 2.2 ( 0.2, as determined by colloidal titration using toluidine blue.10 (17) Kikuchi, Y.; Fukuda, H. Makromol. Chem. 1974, 175, 3593. (18) Chen, W. B.; Wang, L. F.; Chen, J. S.; Fan, S. Y. J. Biomed. Mater. Res. A 2005, 75, 128. (19) Hugerth, A.; Caram-Lelham, N.; Sundelof, L. O. Carbohydr. Polym. 1997, 34, 149. (20) Karibyants, N.; Dautzenberg, H.; Colfen, H. Macromolecules 1997, 30, 7803. (21) Vachoud, L.; Zydowicz, N.; Domard, A. Carbohydr. Res. 1997, 302, 169.

Langmuir, Vol. 23, No. 22, 2007 10951 Table 1. Physicochemical Characteristics of Chitosans Determined by SEC and Capillary Viscometry in an Acetic Acid/Ammonium Acetate Buffer (pH 4.5, µ ) 0.15 M) Mw × 105 (g‚mol-1) DA(%)

DPwa

Ipb

Low Molar Masses 83 ( 4 1.25 ( 0.07 196 ( 5 1.68 ( 0.01

Rg,z (nm)

0.13 ( 0.070 0.32 ( 0.080

1 5

1.32 ( 0.03 1.36 ( 0.03

Medium Molar Masses 5 809 ( 19 1.48 ( 0.01 48.0 ( 1.4 11.5 820 ( 18 1.43 ( 0.05 44.5 ( 1.5

3.49 ( 0.08

5

c

93 ( 6

High Molar Mass 2139 ( 49 1.86 ( 0.04 94.5 ( 2.5

a Weight-average degree of polymerization. b Polydispersity index, IP ) Mw/Mn. c The molecular weight was too low to determine the gyration radius by static light scattering.

Orange II and toluidine blue were from Aldrich (Saint Quentin Fallavier, France). 2.2. Methods. Characterization of Chitosan. The degree of acetylation was determined on purified chitosans by 1H NMR spectroscopy (Varian, 500 MHz), according to the method developed by Hirai et al.22 The water content was determined by thermogravimetric analysis (DuPont Instrument 2950). Mw, Rg,z, and Ip were measured by gel permeation chromatography (3000 and 6000 PW TSK gel columns, i.d. ) 7.8 mm, length ) 300 mm) coupled online with a differential refractometer (Waters 410) and a multiangle laser-light-scattering spectrometer (MALLS, Wyatt, Dawn DSP) equipped with a 5 mW He/Ne laser operating at λ ) 632.8 nm. Analyses were performed in microbatch mode using the K5 flow cell. Light intensity measurements were derived following the classical Rayleigh-Debye equation, allowing us to deduce Mw and Rg,z. A degassed 0.2 M acetic acid/0.15 M ammonium acetate buffer (pH 4.5) was used as the eluent. The flow rate was maintained at 0.5 mL/min. Refractive index increments (dn/dc) were determined independently for each sample in the same solvent with an interferometer (NFT Scan Ref) operating at λ ) 632.8 nm. The physicochemical properties of the chitosan samples are reported in Table 1. Preparation of Polyelectrolyte Solutions. Chitosan was dispersed in deionized water at various concentrations, taking into account the initial water content. Dissolution was achieved under moderate stirring by adding a stoichiometric amount of 0.1 M hydrochloric acid with respect to the free amines for each chosen degree of acetylation. Then, the pH of the solutions was adjusted to 4.0 with 0.1 M sodium hydroxide or hydrochloric acid, and the added amount was considered in calculating the final concentration and the total ionic strength (0.050 M). Before use, every solution of chitosan was filtered on 0.22 µm pore size Millipore membranes. Dextran sulfate solutions, at different concentrations, were prepared in deionized water. Sodium chloride was added to obtain the required ionic strength. Polyelectrolyte Complex Formation. Colloidal PECs were formed under non-stoichiometric conditions (R ) n+/n- * 1, where R is the charge molar mixing ratio) by a one-shot addition of the polymer in default to the polymer in excess under magnetic stirring (1250 rpm) at room temperature. Both polyion solutions were at identical pH and ionic strength (0.05 M). The final particle dispersion volume was 30 mL at a solid content of 0.1 wt %. Under these conditions, the volume of excess polymer solution was always higher than the volume of the default polymer. Particles were separated from the polyelectrolyte mixture by centrifugation at 7800g and 20 °C for 30 min. The supernatant was removed, the pellet was resuspended in deionized water, and the centrifugation was repeated. The final resuspension was carried out in a volume of deionized water so as to reach 1% solid content. Physicochemical Characterization of the Complex Dispersions. Quasi-Elastic Light Scattering. Dynamic light scattering measurements of PEC dispersions were carried out at 90° using two instruments: (22) Hirai, A.; Odani, H.; Nakajima, A. Polym. Bull. 1991, 26, 87.

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Figure 1. Chemical structures of (a) orange II dye and (b) toluidine blue. - A Malvern Zetasizer HS3000 equipped with a 10 mW He/Ne laser beam operating at λ ) 633 nm. All measurements were performed at 25.0 ( 0.2 °C. The self-correlation function was expanded in a power series (cumulants methods).23 The polydispersity value provided by the software is a dimensionless value defined by µ2/(Γ)2, where µ2 is the second cumulant of the correlation function and Γ is the average decay rate. Each measurement is the average of 3 series of 10 measurements each. For a monodisperse colloid, the polydispersity index should be inferior to 0.05, but values up to 0.5 can be used for comparison purposes.24 - An ALV-DLS/SLS-5022F instrument equipped with a 22 mW He/Ne laser beam. All measurments were performed at 25 ( 0.1 °C and analyzed using the CONTIN algorithm. Particle Solid Content. The solid content was defined as the ratio of the weight of particles dried at 150 °C for 30 min (after purification) to the initial weight of solution. Electrophoretic Mobilities. Electrophoretic mobilities (µE) of the particles were determined at 25 °C with a Malvern Zetasizer HS3000 equipped with a 10 mW He/Ne laser beam operating at λ ) 633 nm. µE was expressed as the average of 15 measurements with a relative error of ∼5%. Prior to any measurement, the colloid was purified, as described above, by two centrifugation/redispersion steps to remove the remaining excess polymer. Electrophoretic mobilities and particle size measurements were carried out by dilution of the dispersions in 10-3 M sodium chloride in Milli-Q-grade water. FTIR Spectroscopy. Samples for FTIR were dried and ground. The powder was mixed with KBr (1:100 w/w) and pressed into a pellet. Analysis was performed on an FTIR spectometer (Nicolet Nexus) at CNRS Service Central d’Analyses 69390, Vernaison, France. The samples were scanned from 400 to 4000 cm-1, and 256 scans were recorded. XPS Analysis. X-ray photoelectron spectroscopy (XPS) measurements were performed using a Thermo VG ESCALAB 250 instrument (based at ITODYS Lab, University Paris VII) equipped with a monochromatic Al KR X-ray source (1486.6 eV, 650 µm spot size). The samples were mounted onto double-sided adhesive tape. The pass energy was set at 150 and 40 eV for the survey and narrow scans, respectively. Additional high-resolution C 1s and N 1s regions were recorded using a pass energy of 10 eV. Charge compensation was achieved with a combination of electron and argon ion flood guns. The partial pressure for the argon flood gun was 2 × 10-8 mbar. These standard conditions of charge compensation resulted in a negative but perfectly uniform static charge. Data acquisition and processing were achieved with Avantage software, version 2.2. Spectral calibration was determined by setting the main C 1s component due to C-C/C-H bonds to 285 eV. The surface composition was determined using the peak areas normalized to the sensitivity factors (Scofield sensitivity factors corrected for the analyzer transmission function). Chitosan Assay. The stoichiometry of the complex was determined by the depletion method based on the quantification of residual polymers in the supernatants after complexation. Each value is the average of three independent measurements. The residual concentration of chitosan was determined with orange II, an anionic dye (Figure 1 a) that can interact with ammonium groups of chitosan.25 The samples were prepared as follows: 2 mL of particles were centrifuged at 12 000g for 30 min, and the supernatant was carefully recovered before filtration on a 0.22 µm (23) Koppel, D. E. J. Chem. Phys. 1972, 57, 4814. (24) Coombes, A. G. A.; Scholes, P. D.; Davies, M. C.; Illum, L.; Davis, S. S. Biomaterials 1994, 15, 673. (25) Gummow, B. D.; Roberts, G. A. F. Makromol. Chem. 1985, 186, 1239.

Drogoz et al. Millipore membrane to eliminate any remaining particles. Then, the supernatant was diluted 60-fold in the complexation medium (0.05 M sodium chloride, pH 4). For 1 mL of diluted supernatant, 100 µL of orange II (10-3 M) was added and mixed for 15 min. The concentration of orange II was chosen so as to be in excess with regard to the amino groups of chitosan to be assayed. After centrifugation (12 000g for 30 min) to remove the chitosan-orange II complexes, the absorbance was measured at 484 nm, corresponding to the unbound dye. The chitosan concentration was determined from the optical density data via calibration curves established with solutions of chitosan of identical molar mass and DA. In this assay, there is an inverse proportionality relationship between the orange II absorbance and the chitosan concentration. The absorbance measurement was performed with a µQuant UV/vis spectrometer from Bio-tek Instruments. Dextran Sulfate Assay. The residual amount of dextran sulfate was determined by the titration of sulfate functions, performed with the cationic dye toluidine blue10 (Figure 1b). The calibration curves and samples were also prepared in the same way as described for chitosane titration except that the absorbance was measured at 584 nm.

3. Results and Discussion 3.1. Formation of Polyelectrolyte Complex Dispersions. In previous studies,9,10 we showed that colloidal complexes could be obtained from chitosan and dextran sulfate. The optimal experimental conditions required the use of a salt concentration of 0.05 M NaCl; otherwise, no colloid could be obtained, probably because of the chain stiffness resulting from electrostatic repulsive forces, as observed by Dautzenberg for synthetic polymers.26 The effect of ionic strength being established and the molar mass of the polymer in excess strongly impacted the size of the resulting particles.9,10 We also showed that the surface charge of the colloid was generated by excess polymer. Therefore, positively charged particles were produced for R > 1 (i.e., when chitosan was the polymer in excess); conversely, negatively charged particles were produced for R < 1 (i.e., when dextran sulfate was in excess). In Figure 2, we reported the impact of the initial polymer concentration on the diameter of positive particles (R ) 2) determined by QELS (obtained from the second-order results of the cumulant methods). When at least one of the two counterparts remained at or under a limit concentration of 0.1 wt %, increasing the concentration of the other partner up to 0.5 wt % slightly impacted the particle size, with the polydispersity index remaining in the 0.2-0.4 range in accordance with our preceding results.9,10 The values of the polydispersity indexes showed that the particles were rather broadly distributed in size as a result of the complexity of the processes involved in the formation of colloidal PECs, as suggested by Dautzenberg.20 Optimal experimental conditions were obtained at initial concentrations of 0.1 wt % for both polyions because (i) the long-term colloidal stability of the resulting colloid was the best (no settlement of the dispersion was observed over 3 weeks of storage at room temperature under gentle stirring) and (ii) from a practical standpoint the added volumes were high enough to minimize the impact of experimental variations of added volumes. Moreover, under these conditions, the volume of the solution for the polymer in excess was higher than that of the solution of the polymer in default. This optimized polymer concentration of 0.1 wt % will be used for the formation of colloids in the remainder of this study. We used DLS to investigate the positive particle formation process as a function of the molar mixing ratio R (n+/n-). Chitosan (DA ) 5%, Mw ) 132 × 103 g‚mol-1) solutions were analyzed at decreasing polysaccharide concentrations. The analysis of the (26) Dautzenberg, H. Macromolecules 1997, 30, 7810.

Polyelectrolyte Complexes from Polysaccharides

Langmuir, Vol. 23, No. 22, 2007 10953

Figure 2. Diameter of complex particles versus chitosan (CS) (DA ) 5%, Mw ) 132 000 g‚mol-1) and dextran sulfate (DS) concentrations. Table 2. Scattering Light Intensity versus Molar Mixing Ratio (R ) n+/n-) for Positive Particles Based on Chitosan DA ) 5%, Mw ) 132 000 g/mol, and Dextran Sulfate molar mixing ratio R (n+/n-)

scattering light intensity (103 Hz)

starting chitosan solution 100 80 60 40 20 10 5 3 1.5

50 144 513 763 979 1760 3200 7800 8760 11 800

correlation functions revealed two populations, one centered near 10 nm in diameter and the other around 300 nm, probably corresponding to aggregated molecules of chitosan. On filtration of the initial solution of chitosan through a 0.22 µm filter, the relative intensity of the second population decreased in comparison with the first. On adding dextran sulfate at a mixing ratio of R (n+/n-) ) 100 and thus for small amounts of polyanion, a sharp increase in scattering intensity was observed (Table 2) as a result of the particle formation. The scattered light intensity increased from 50 to 11 800 with increasing amounts of added dextran sulfate from R ) 100 to 1.5. It is worth noting that because of the high scattering intensities, samples at R e 5 had to be diluted. The intensity values reported in Table 2 take into account the dilution factor. Over this range of R values, two different populations of objects were observed to be similar to those in the starting chitosan solution. The measured average particle diameter was similar to the larger population of the chitosan solution, and its relative ratio increased with decreasing R, as a result of the formation of new particles and/or an increase in the density (and polarizability) of the scattering objects. In Figure 3a, we reported the variations of the mean diameter of the crude (noncentrifuged) particles from R ) 100 to 1.5. The diameter, around 410 nm, remained constant until R g 10. At R ) 8, a sharp decrease was observed until R ) 5, and then the particle size increased again, indicating a flocculation process that was largely observed at R ) 1. As seen from Figure 3b, the size distribution for a colloidal dispersion obtained for R ) 2 was monomodal. For R < 1,

particles obtained in the presence of excess polyanion were rather homogeneous in size, but flocculation was observed for R g 0.6 (i.e., almost at half-neutralization). After cleaning the dispersion by centrifugation, supernatant removal, and redispersion of the pellet in milli-Q-grade water, the electrophoretic mobilities were measured at pH 5.5 for various charge ratios R. As reported in Figure 4 for the cationic particles, on decreasing R (which corresponds to the addition of dextran sulfate to excess Chitosan), the mobilities barely decreased before R ) 3. Below R ) 3, a drastic reduction in mobility was observed, which explained that the colloid flocculation close to R ) 1 resulted from charge neutralization. For the negative particles, from R ) 0.1 to 0.5, a constant electrophoretic mobility of -3.4 µm‚cm/v‚s was observed. For R > 0.5, no value was available because it was not possible to obtain homogeneous colloidal dispersions but only aggregated material. Considering the constant electrophoretic mobility observed over 0 < R < 0.5, the flocculation observed for R > 0.5 probably occurred by particle bridging with chitosan chains rather than by charge neutralization and the loss of electrostatic stabilization. 3.2. Characterization of the Complexes. Infrared Analysis. The infrared spectra of the complexes and the parent polysaccharides (chitosan DA ) 5%, Mw ) 132 × 103g/mol, and DS) are reported in Figure 5. We observed specific bands for chitosan at 1651 and 1596 cm-1 corresponding to the amide I band27 and the NH3+ deformation,12 respectively. Dextran sulfate was characterized by a band at 1229 cm -1 due to sulfate asymmetric stretching28 and a minor band at 1638 cm-1 (Figure 5). In the complex of both polysaccharides, a band appeared at 1626 cm-1, but it could result from a contribution of chitosan via the 1651 and 1596 cm-1 bands and/or dextran sulfate via the 1638 cm-1 band. A band in the trace of the complex appeared at 1522 cm-1 along with the concomitant evolution of the structure of the band of dextran sulfate at 1230 cm-1 corresponding to sulfate asymmetric stretching.28 On increasing the polyanion content in the complex, this band at 1230 cm-1 broadened and differentiated into two peaks, as observed for PECs obtained with dextran sulfate and polyethylenimine (PEI).28 The absorption at 1522 cm-1 can be attributed to an ammonium moiety associated with (27) Stoilova, O.; Koseva, N.; Manolova, N.; Rashkov, I. Polym. Bull. 1999, 43, 67. (28) Tiyaboonchai, W.; Woiszwillo, J.; Middaugh, C. R. J. Pharm. Sci. 2001, 90, 902.

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Drogoz et al.

Figure 3. DLS experiments. (a) Average diameter of particles based on chitosan (DA ) 5%, Mw ) 132 000 g‚mol-1) and dextran sulfate as a function of the mixing ratio (R). (b) Size distribution for R ) 2.

sulfate, as previously reported for the formation of soluble complexes of dextran sulfate and chitosan, even in the presence of sodium chloride or urea.29

Figure 4. Electrophoretic mobilities of complexes versus the mixing ratio R. Particles based on chitosan DA ) 5%, Mw ) 132 000 g‚mol-1.

a sulfate group, in accordance with observations by Gamzazade et al.29 for soluble complexes of chitosan and dextran sulfate. This band was also observed in PECs of chitosan with polycarboxylates,30,31 but for particles obtained with dextran sulfate and chitosan, Sarmento et al. did not mention it.11 Similar results (i.e., with the same absorption band at 1522 cm-1 and the same evolution of the structure of the band of dextran sulfate at 1230 cm-1) were obtained for anionic PECs (data not shown). These results suggest the formation of ion pairs between the amino groups of chitosan and the sulfate moieties of dextran (29) Gamzazade, A. I.; Nasibov, S. M. Carbohydr. Polym. 2002, 50, 339. (30) Shi, X. W.; Du, Y. M.; Sun, L. P.; Zhang, B. Z.; Dou, A. J. Appl. Polym. Sci. 2006, 100, 4614. (31) Simsek-Ege, F. A.; Bond, G. M.; Stringer, J. J. Appl. Polym. Sci. 2003, 88, 346.

XPS (X-ray Photoelectron Spectroscopy) Analysis of Dry Particles. For DS and chitosan (DA ) 5%, Mw ) 132 × 103 g‚mol-1), data of both negatively and positively charged complexes are reported in Table 3. These data provide evidence for both nitrogen and sulfur, though, as expected, the relative N/S atomic ratio was smaller for the negatively charged particles. These results suggest that the overall stoichiometries (N/S) are close to 1 and 0.6 for the particles obtained at a charge ratio R of 2 (positive) and 0.3 (negative), respectively. Interestingly, sodium was detected only at a charge ratio of 0.3, corresponding to excess dextran sulfate whose counterion was sodium. This fact suggests that the surface of negative particles is mainly composed of dextran sulfate. 3.3. Stoichiometry of the Complexation Process. We used the depletion method to monitor the complex composition during its processing under experimental conditions suitable for the formation of particles in the submicrometer size range. The remaining excess polymer in the supernatant was quantified, after phase separation by centrifugation. This value was substracted from the initial input in the reaction medium, to lead to the amount of polyion within the colloidal PEC. In every case, the polymer in default could not be detected during particle formation.

Polyelectrolyte Complexes from Polysaccharides

Langmuir, Vol. 23, No. 22, 2007 10955

Figure 5. FT-IR (a) spectra of chitosan DA ) 5%, Mw ) 132 000 g/mol, dextran sulfate, and the polyelectrolyte complex (PEC) at a charge ratio (() of 2. Table 3. XPS Analysis of Chitosan-DS Complexes Based on Chitosan DA ) 5%, Mw ) 132 000 g‚mol-1, and Dextran Sulfate

a

atom

%a

%b

C O N S Na

49.58 38.35 3.53 5.74 2.8

52.09 39.46 4.3 4.15 undetected

Negative particle (R ) 0.3). b Positive particle (R ) 2).

To monitor the consumption of excess polymer during the titration process (i.e., with varying R), a molar ratio F was defined to assess the fraction of functional groups of the polymer in excess that was involved in the complex. Hence, F is the ratio of the concentration of complexed functional groups to the initial concentration of functional moieties of the polymer in excess. In addition, the stoichiometry S was defined as the ratio of the number of two oppositely charged functional groups in the PEC. In other words, S is the overall number of complexed functional groups of the polymer in excess with respect to the number of functional groups of the polymer added in default on the experimental basis that 100% of the default polymer was involved in the complexes. Particles Obtained in the Presence of Excess Dextran Sulfate. In these experiments, excess dextran sulfate was assayed using toluidine blue, as described in the Experimental Section. The two molar ratios defined above will be written as F: ratio of the complexed sulfate moieties ([SO4-]complexed) to the overall sulfate concentration available in the starting solution ([ SO4-]initial); F ) [ SO4-]complexed/ [ SO4-]initial and S: stoichiometry ratio (i.e., the number of complexed sulfate groups to the number of ammonium moieties ([NH3+]introduced) added); S ) [ SO4- ]complexed/ [NH3+]introduced. The assay was performed for three chitosan samples: Mw ) ∼132 × 103 g‚mol-1, DA ) 5 or 11.5%, and Mw ) 32 × 103 g‚mol-1 DA ) 5%. As reported in Figure 6, F increased

monotonously with the charge ratio R, hence one can regard the mechanism of interaction between the polyions to be identical over the entire investigated R range. The S values at the plateau (Figure 6), corresponding to an average of 1.5 to 1.8 sulfate moieties linked to one amine group, was close to the values of the slope of the linear variations of F versus R. Considering an average degree of sulfation of dextran sulfate of 2.2, the complexes formed in this work were non-stoichiometric. A strict chargeto-charge neutralization would lead to S ) 1 whereas a residueto-residue complexation would provide S ) 2.2. The observed intermediate values (S ) 1.5-1.8) suggest that both processes took place, with a predominance of the residue-to-residue binding for the chitosan of the lowest molar mass (S ) 1.85, Mw ) 32 × 103 g‚mol-1, Figure 6b) but a favored charge-to-charge neutralization at higher molar mass (S ) 1.5, Mw ) 132 × 103 g‚mol-1, Figure 6a). The residue-to-residue interactions took place when chitosan molecules had an expanded conformation (i.e., at low molar mass or high DA).32 An S value of 1.5 found for chitosan with a molar mass of ca. 132 × 103 g‚mol-1, DA ) 5, and R ) 0.3 was very close to the atomic ratio determined by XPS (sulfur/nitrogen ) 1.67, Table 3). S reached the plateau for R close to or above 0.1. Below this critical value, S was reproducibly either lower (Figure 6a) or higher (Figure 6b) than the plateau value. This could be explained considering that, at low chitosan concentrations, the large excess of dextran sulfate would “freeze” the conformation of chitosan chains. Hence, the low-molar-mass chitosan with an expanded conformation would undergo quasi-perfect residue-to-residue binding (S close to 2.2, Figure 6b). For the higher-molar-mass chitosan samples with a random coil conformation (Rg ) 45 nm, Table 1), the surrounding dextran sulfate molecules would preferentially bind at the periphery of the polycation coils. As a consequence, non-neutralized segments of chitosan would be entrapped within the core of the object, leading to values of (32) Lamarque, G.; Lucas, J. M.; Viton, C.; Domard, A. Biomacromolecules 2005, 6, 131.

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Figure 6. Determination of the complex stoichiometry (S) and the complexation factor (F) of negative particles. (a) DA ) 5%, Mw ) 132 000 g‚mol-1. (b) DA ) 5%, Mw ) 32 000 g‚mol-1.

Figure 7. Determination of the complex stoichiometry (S) and the complexation factor (F) of positive particles. (a) DA ) 5%, Mw ) 132 000 g‚mol-1. (b) DA ) 1%, Mw ) 13 000 g‚mol-1.

S < 1 observed in Figure 6a. A similar situation was described by Chen et al.33 in the framework of the so-called “entrapment segment” model. Above the limit concentration ratio corresponding to R ) 0.1, dextran sulfate was no longer in sufficient excess to prevent the molecular adaptation of chitosan molecules from taking place. Hence, the charge-to-charge and residue-toresidue processes could occur independently of the initial conformation of the polycation. Particles Obtained in the Presence of Excess Chitosan. In these experiments, the excess chitosan was assayed with orange II, a negatively charged dye, as described in the Experimental Section. As in the preceding case, the two ratios F and S were used for data analysis, but their definitions were modified to take into account that chitosan was the polymer in excess: F ) [NH3+]complexed/[NH3+]initial and S ) [NH3+ ]complexed/[SO4-]introduced. F represents the fraction of ammonium moieties involved in the complex, and S shows the number of ammonium groups necessary to bind one sulfate counterpart. It is clear from Figure 7 that the variations of F versus R were not linear and the stoichiometries were not constant, converse (33) Chen, J. H.; Heitmann, J. A.; Hubbe, M. A. Colloids Surf., A 2003, 223, 215.

to the previous case. In fact, two domains of behavior were identified for each DP and DA, with a critical R value of Rc ) 4 or 8 for the higher (Figure 7a) and lower DPs (Figure 7b), respectively. (See also Supporting Information.) When R was above Rc, F remained constant, only 15-25% of the amine groups were involved in a complex, and the S ratio monotonously decreased from 6–4 down to 1. For R e Rc, F increased up to 100% consumption of the available ammonium groups with an overall stoichiometry S ) 1, corresponding to an ideal chargeto-charge neutralization process. Moreover, the XPS results for chitosan with a molar mass of 132 × 103 g‚mol-1, DA ) 5%, and R ) 2 showed a sulfur/nitrogen atomic ratio close to 1, in accordance with the stoichiometry determined by the assays. From the results obtained above for both positive and negative particles, the mechanisms of formation of the complexes differed according to the nature of the polyelectrolyte used in excess. The experimental conditions were designed to ensure a constant ionic strength of (50 × 10-3M) and a full ionization of the two polyelectrolytes during the complexation process with solutions maintained at pH 4. The formation of polyelectrolyte complexes with dextran sulfate in excess took place at constant stoichiometry. One can consider that the two polyions interacted to form primary

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Scheme 1. Proposed Mechanism for the Formation of Colloidal PECs from Chitosan and Dextran Sulfate in Excess

Scheme 2. Proposed Mechanism for the Formation of Colloidal PECs from Dextran Sulfate and Chitosan in Excess

complexes (Scheme 1), which rearranged to colloidal species. These complexes resulted from two modes of interactions between the polyions: a residue-to-residue interaction or a charge-tocharge neutralization. The first process was favored for chitosan with the lowest DP and may be explained by the guest-host model introduced by Kabanov.3,34 In this model, the high-molarmass polymer in excess (host polyelectrolyte, here dextran sulfate) complexed the low-molar-mass polymer (guest, here chitosan), according to the ladder-like residue-to-residue process. This process requires high differences in molar mass between both partners. Moreover, chitosan molecules at low DP are stiff, disfavoring the conformational adaptations required for chargeto-charge interactions as reported by Tuschida.35 When chitosan is in excess, two different modes of complexation should be considered, according to R, either above or below the critical value Rc. For R above Rc, the fully dissociated (34) Kabanov, V. A.; Zezin, A. B. Pure Appl. Chem. 1984, 56, 343. (35) Tsuchida, E.; Abe, K. AdV. Polym. Sci. 1982, 45, 1.

sulfate moieties cross linked various chitosan coils by chargeto-charge neutralization (step 1, Scheme 2). The formation of these primary objects consumed between 15 and 25% of the initial chitosan feed. Another way of describing this first step would be to consider that DS molecules reacted on the aggregated fraction of chitosan macromolecules. The formation of these primary objects led locally to high concentrations of chitosan, which could perform as preferential sites for trapping dextran sulfate (step 2, Scheme 2), explaining the plateau of F versus R and the linear decrease of the stoichiometry (S vs R). For R < Rc (Scheme 2), the consumption of the free chitosan in solution was observed at an overall stoichiometry of 1. Interestingly, the value of Rc depended on the amount of chitosan involved in the first two steps. R ) 4 for an initial consumption of ca. 25% of chitosan (Figure 7a), and R ) 8 (Figure 7b) for an initial consumption of ca. 15% of the chitosan sample of the lowest molar mass. Finally, it is worth noting that, if a single mechanism were involved in the complexation of dextran sulfate with chitosan

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in excess, F would be a linear function of 1/R (as F was a linear function of R for the negatively charged particles obtained with dextran sulfate in excess), which was not verified. Finally, the electrophoretic mobilities of the particles reflected the nature of the polymer used in excess. The variations of these values with R (Figure 4) well illustrate the differences in the mechanisms of formation of the colloids. When dextran sulfate was in excess, the mobilities remained constant in the R range investigated whereas a decrease in mobility was observed (particularly sharp for R ) 3 and lower) when chitosan was in excess.

4. Conclusions Colloidal PECs in the submicrometer range could be obtained from dextran sulfate and chitosan. DLS experiments showed that the colloidal particles formed early in the neutralization process, even at very low concentrations of added polyion (i.e., at high (low) values of R for cationic (anionic) particles). We showed that the nature of the polymer in excess strongly affected the mechanism by which the colloids were formed. When chitosan was in excess, the stoichiometry (S) decreased during the complexation process until the charge ratio R reached a critical value Rc for which S remained constant (S ) 1) up to the exhaustion of the free polycation. Two different mechanisms could be considered: (i) For R > Rc, cross linking of the chitosan macromolecules by the polyanion molecules reaching the polycation solution. As a result, local overconcentrations of cationic charges were formed, which were preferentially neutralized by dextran sulfate. (ii) For R < Rc, complexation occurred

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with an overall stoichiometry of 1. When dextran sulfate was in excess, PEC formation could occur by two mechanisms: random charge neutralization, resulting in the cross linking of chitosan chains, or residue-to-residue interactions. The second mechanism could take place because dextran sulfate is a flexible polyion. Hence, the formation of the first sulfate/ammonium ion pair could induce the macromolecules to zip together. The level of occurrence of this mechanism increased with the increased stiffness of chitosan (i.e., either via a reduction in molar mass or an increase in DA). It is noteworthy that to maintain the colloidal stability the charge ratio R (n+/n-) should remain under 0.6. Our work gives the first insight into the complexation mechanisms and an elaboration of colloidal PECs from oppositely charged polysaccharides. Further work is ongoing to characterize the complexes better in terms of morphology, colloidal stability, and interactions with biomolecules. Acknowledgment. This work was financially supported by a grant from the Fondation Me´rieux. We thank Bonhomme´ Anne for IR spectra, Lucas Jean-Michel for GPC data, Veron Laurent for NMR spectra, and Mohamed Chehimi for XPS analysis and fruitful discussions. Supporting Information Available: Stoichiometry monitoring data for the various chitosan samples provided for cationic and anionic colloids. This material is available free of charge via the Internet at http://pubs.acs.org. LA7008545