Versatile and Efficient Formation of Colloids of Biopolymer-Based

Biomacromolecules 2004, 5, 1882-1892

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Versatile and Efficient Formation of Colloids of Biopolymer-Based Polyelectrolyte Complexes Christophe Schatz,†,‡ Alain Domard,‡ Christophe Viton,‡ Christian Pichot,† and Thierry Delair*,† Unite´ mixte CNRS-BioMe´ rieux, UMR 2142, ENS Lyon, 46, Alle´ e d’Italie, 69364 Lyon Cedex 07 France, and Laboratoire des Mate´ riaux Polyme` res et des Biomate´ riaux, UMR CNRS 5627, ISTIL, Domaine Scientifique de la Doua, 15 Bd. Latarjet, 69622 Villeurbanne Cedex France Received April 9, 2004; Revised Manuscript Received May 31, 2004

The formation of colloids based on polyelectrolyte complexes (PECs) of biopolymers was investigated through the complexation between two charged polysaccharides, chitosan as polycation, and dextran sulfate as polyanion. The slow dropwise addition of components, generally used for the formation of PECs, allowed to elaborate both cationic or anionic particles with an excess of chitosan or dextran sulfate, respectively. The PEC particles featured a core/shell structure, the hydrophobic core resulting from the segregation of complexed segments whereas excess component in the outer shell ensured the colloidal stabilization against further coagulation. Considering the host/guest concept for the formation of PECs, the influence of the molecular weight of components on particles sizes could be well explained by the chain length ratios of the two polymers. As an irreversible flocculation occurred with a dropwise approach for both cationic and anionic PEC particles when the mixing ratio was close to unity, a more versatile, and simpler to setup, method was designed: the one-shot addition of one solution to the other. Because process of addition is faster than the flocculation, cationic or anionic particles could be elaborated irrespective of the order of addition of the reactant. Characterization of these particles by quasielastic light scattering, electrophoresis, and scanning electron microscopy revealed very similar properties to those obtained by a slow dropwise approach. Critical coagulation concentrations of 0.12 and 0.09 M (with sodium chloride) for cationic and anionic particles evidenced a mostly electrostatic stabilization. 1. Introduction Polyelectrolyte complexes (PECs) are formed in solution by strong electrostatic interactions between charged microdomains of at least two oppositely charged polyelectrolytes (PEL). At low ionic strength, the complexation is entropy driven by the release of small counterions, initially bound to the polyelectrolytes.1 According to the chemical composition of PEL, the process of complexation may be cooperative in the sense of secondary interactions, such as hydrogen bonds, can take place and favor the ion-pairing between PEL.2 The conditions of formation, the properties, and applications of PECs were extensively studied but, at the colloidal level, less works detailed the elaboration of PECs, especially with biopolymers. Such structures were investigated with synthetic PEL in the framework of water-soluble PECs by the groups of Kabanov3,4 and Tsuchida.5,6 Indeed, complexation between polyelectrolytes having significantly different molecular weights, weak ionic groups and mixing in nonstoichiometric ratios leads to the formation of watersoluble aggregates on a molecular level. These particles consist in a long host molecule sequentially complexed with shorter guest polyions of opposite charge, according to a “zip” mechanism. Another way to produce quasisoluble PEC * To whom correspondence should be addressed. E-mail: Thierry. [email protected]. † CNRS-BioMe ´ rieux. ‡ UMR CNRS.

particles was comprehensively reported by Dautzenberg.1,7-9 Complexation between polyelectrolytes with comparable large molecular weight and/or strong ionic groups leads to nearly spherical colloidal structures in conditions of high dilution ( 1) or by the reverse addition, a default chitosan to an excess dextran sulfate leading to the formation of anionic particles (n+/n- < 1). To ensure a high reproducibility of QELS measurements, a single solution of

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excess component was used in each experiment and successive dropwise additions of default polymer were performed to get the required mixing ratios. The common feature of the two complexation processes mentioned above was the increase of turbidity assessed by the optical density at λ ) 500 nm as the concentration of titrant increased (Figure 2). This evidenced the formation of increasing amounts of insoluble polyelectrolyte complexes since neither chitosan nor DS absorbed light at λ ) 500 nm. Until the flocculation of the system characterized by a drop of the optical density, the colloidal dispersions were stable, indicating that electrostatic or electrosteric stabilization prevented further coagulation. This stabilization might be ensured by an excess of binding of the major component, likely to form a stabilizing shell around the particles (see below). In this work, the degree of acetylation of chitosan was restricted to 15% as we demonstrated that a low acetyl content favored the formation of less swollen particles.10 Charge density of dextran sulfate was also kept at a constant value corresponding to 2.2-2.4 sulfate functions per glucosidic unit. In all experiments, the initial solutions of polyelectrolytes were adjusted at pH ) 4.0 to ensure the full protonation of chitosan,12 DS being also fully dissociated at this pH. In addition, using solutions having the same pH also allowed us to avoid any neutralization reactions. Finally, initial concentrations of chitosan and dextran sulfate were set at 10-3 and 5 × 10-4 g/mL as we demonstrated that these conditions allowed the formation of small PEC particles.10 3.1.1. Formation of Cationic Particles. Cationic particles were formed by the addition of increasing amounts of dextran sulfate to an excess chitosan (decreasing n+/n- ratios from 20 to ∼1). Figure 2 evidenced an antagonist effect of the molecular weight of chitosan and DS on the PEC particle properties. Indeed, high molecular weight chitosan led to the highest particle sizes and optical densities whatever DS molecular weights and mixing ratios. In contrast, the higher the molecular weight of DS, the smaller the particle sizes and the optical densities. Thus, smallest particles are obtained by the complexation of chitosan having Mw ) 1.6 × 104 g/mol with DS having Mw ) 1.5 × 106 g/mol. Another characteristic of the complexation process is the decrease of hydrodynamic diameters during the titration until a secondary aggregation was obtained preceding the irreversible flocculation. It means that the addition of dextran sulfate allowed both the formation of new particles, evidenced by the increase of the optical density and their rearrangements toward more compact structures. The range of the particle size reduction decreased also with increasing dextran sulfate molecular weight, meaning that colloids formed with high molecular weight polyanions were denser than those obtained with the lowest molar mass. The polydispersity index (PI) of the colloidal dispersions displayed about the same behavior as the hydrodynamic diameter toward the effect of the molecular weight of components. However, its variation as n+/n- decreased was more limited in range. Finally, it is worth noting that very high values of PI, obtained with high molecular weight chitosans, cannot be interpreted since above 0.3 PI loses its physical meaning.

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Figure 2. Sizes, polydispersity indexes, and optical densities of PEC particles obtained by slow addition of a DS solution in a chitosan solution (n+/n- > 1) and by slow addition of a chitosan solution in a DS solution (n+/n- < 1) for (a) low molecular weight chitosan (Mw ) 1.6 × 104 g/mol), (b) high molecular weight chitosan (Mw ) 3.65 × 105 g/mol) and three molecular weight dextran sulfates: (0) Mw ) 5 × 103 g mol-1, (2) Mw ) 104 g mol-1, (4) Mw ) 1.5 × 106 g mol-1. Conditions: pH ) 4.0, µ ) 5 × 10-2 M, flow rate ) 20 mL/h, [chitosan]0 ) 10-3 g/mL, [DS]0 ) 5 × 10-4 g/mL.

On the basis of these results, one can consider that the particle forming process relies on the formation of hydrophobic segments on polymer chains by charge neutralization and segregation of these segments into particles stabilized by free unpaired charges. Thus, for a set chitosan molar mass, the size of the titrant component impacts on the dimension of the objects in such a way that ion-pairing is optimum for charge neutralization. Hence, the longer the segment with ion pairs, the stronger the interactions between these segments and the smaller the particles. On this assumption, two different situations may be considered. First, for low molecular weight polyanions (Mw ∼ 5 × 103 and ∼104 g/mol), chitosan chains in excess amount can act as host polycations (see the respective contour lengths in Table 1). Then, the complexes formed bear many unpaired positive charges still available for complexation. Being more hydrophilic, uncomplexed segments of chitosan constitute the corona of the particles (Figure 3a). Thus, the higher the chitosan chain length, the thicker the corona and the higher the hydrodynamic volume of particles. However, as the chitosan chains

exhibit a relative stiff conformation, one cannot rule out the possibility that the initial larger coil volume for the higher molecular weight chitosan chains contributes to the formation of more swollen core and hence to high particle sizes. Addition of more polyanion led to the observed size reduction by consumption of the residual charges with concomitant collapse of the newly formed hydrophobic segments onto the hydrophobic core of the objects. Second, when using high molecular weight DS (Mw ) 1.5 × 106 g/mol), the polyanion can function as a host molecule capable of accommodating several polycation chains. Then, formed hydrophobic segments are longer and can strongly interact with one another to yield more compact objects (Figure 3b). This requires that not all charges of chitosan be neutralized, otherwise flocculation can occur as we will see later for anionic particles. Finally, it is worth noting that the values of mixing ratio at the flocculation point are close to the unity whatever the molecular weight of initial components evidencing that almost all the charges of the polyions were complexed. However, the high mismatching between charge distance of

Colloids of Polyelectrolyte Complexes


Figure 3. Formation of colloidal polyelectrolytes complexes based on chitosan (light gray) and dextran sulfate (black) through the hydrophobic segregation of complexed segments. Four limit cases are considered according to the charge of the colloid and the molecular weight of components (schemes are not to scale). (a), (c) high Mw chitosan versus low Mw dextran sulfate; (b), (d) low Mw chitosan versus high Mw dextran sulfate.

chitosan and dextran sulfate (Table 1) hinders a regular 1:1 arrangement of the polyions required for a strict ladder-like PEC structure. It means that each DS unit, bearing 2.2-2.4 sulfate functions, should be able to complex more than one glucosamine moiety and thus act as an ionic cross-linker. 3.1.2. Formation of Anionic Particles. Anionic particles were obtained by addition of increasing amounts of chitosan to an excess dextran sulfate (increasing n+/n- ratios from 0.1 to ∼1). As for the cationic particles, the formation of complexes mainly depended on the size of the polymer in excess. Here, the most surprising phenomena was obtained with a DS of 5 × 103 g/mol. Indeed, when a solution of chitosan having low or high molecular weight was added into an excess solution of DS 5k to get n+/n- ) 0.1, the mixture was turbid during less than 5 min and then flocculated. With a DS of 104 g/mol, colloids were formed in a rather small range of molar mixing ratios, flocculation occurring at n+/n- ) 0.5 and 0.3 for low and high molecular weight chitosan, respectively (Figure 2). The poor colloidal stability was confirmed by the irreversible aggregation encountered during the centrifugation of these particles. Using a DS of 1.5 × 106 g/mol, the domain of formation of colloids increased at n+/n- ) 0.6-0.7 according to the chitosan molecular weight (Figure 2). These results evidenced that the key parameter controlling the formation and the stability of anionic particles was the chain size ratio between chitosan and DS. Indeed, two different situations can be

identified depending on whether chitosan may act as host molecules or not. First, low colloidal stability occurred as long as dextran sulfate remained smaller than the titrant, in other words while the titrant (chitosan, Mw ) 1.6 × 104 or 3.65 × 105 g/mol) could host several molecules of polyanion (dextran sulfate, Mw ∼ 5 × 103 or 104 g/mol). Reasons for the premature flocculation observed with a DS 5k and the poor colloidal stability with DS 10k may be the followings. Dextran sulfate being highly flexible, more charged than chitosan and used in excess for the formation of anionic colloids, small molecules of DS 5k would favor a complete ion-pairing with chitosan leading to quasi-neutral complexes without enough unpaired sulfate functions to ensure the stabilization of particles (Figure 3c). Hence, formed complexes quickly aggregated. On increasing the molecular weight of dextran sulfate to 104 g/mol, conformational adaptation of DS for a complete-ion pairing was less favorable and let enough unpaired sulfate groups participate in the colloidal stabilization. The very short decrease of the mean diameter at the onset of the dextran sulfate titration by low molecular weight chitosan (Figure 2a), illustrating a limited compaction upon addition of chitosan, confirms the presence of some free segments of DS in PEC particles, probably located at their surface. Such a decrease of size was not observed with high molecular weight chitosan (Figure 2b), complexation being earlier limited by the flocculation. Hence 104 g/mol is a

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threshold molar mass for dextran sulfate to elaborate anionic PEC particles with the two chitosans used in this study. Conversely, in the reverse process, the complexation of an excess of low molecular weight chitosan by a high molecular weight dextran sulfate, highly stabilized PEC particles were obtained. This can be explained by considering that chitosan is weakly charged and relatively stiff. Hence, charge neutralization cannot be totally achieved and some glucosamine residues remained unpaired ensuring electrostatic stabilization (Figure 3b). Second, when DS could act as a host molecule, i.e., for the highest molecular weight (Mw ) 1.5 × 106 g/mol), chitosan counterparts formed “hydrophobic patches” on complexation with the large DS molecules. Theses patches segregated to form particles consisting in a hydrophobic core and a large hydrophilic shell of uncomplexed dextran sulfate segments (Figure 3.d), leading to larger diameters of particles than those observed with DS 10k (Figure 2). Then, a further addition of chitosan led to a reduction of both size and polydispersity index, by complexing with the negatively charged shell, as previously observed for cationic particles obtained from high molar mass chitosan. Figure 2 also displays that anionic colloids of similar sizes were obtained by complexing high molar mass DS with the two chitosans, evidencing that the hydrodynamic diameter of these PEC particles was indeed determined by the thickness of the large dextran sulfate outer shell. Finally, whether chitosan or dextran sulfate were used as starting solutions, flocculation at a n+/n- ratio close to 1 was irreversible and the addition of excess titrant never led to the recovery of colloids with a reversed surface charge. 3.2. Formation of Particles by the One-Shot Addition of Polyelectrolytes. Since irreversible flocculation was observed when getting slowly close to electrical neutrality, the idea was to go beyond this critical value as quickly as possible in order to achieve a fast charge inversion, which would allow maintaining the colloidal stability. Hence, the titrant solution was added as quickly as possible to the starting solution. This new way to proceed was tested to elaborate both anionic and cationic colloids by adding excess dextran sulfate solution to default chitosan or excess chitosan to default dextran sulfate. The influence of the order of addition of reactants on the formation of colloids was also investigated by adding the polyelectrolyte in default to excess of oppositely charged PEL as for the dropwise addition. The mean diameters of particles obtained by these two processes were determined for the complexation of a low molecular weight chitosan with three DS (Figure 4). Whatever the nature of the polyelectrolyte used as starting solution and the order of addition (excess to default or the opposite) colloidal PECs were obtained at various n+/n- ratios, using the same polyelectrolyte concentration range as before. More surprisingly, the shape of the curves of the variations of the hydrodynamic diameter with varying n+/n- ratios for the oneshot addition looked quite similar to the ones obtained by the slow addition process. This suggests high kinetics for the particle formation process for both, one-shot and slow additions. However, the one-shot addition of the titrant seems to be faster than the reaction, otherwise flocculation would

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Figure 4. Sizes of PEC particles obtained by a one-shot addition of a DS solution in chitosan solutions (2) and by a one-shot addition of a chitosan solution in DS solutions (4) for various weight-average molecular weights of DS: (a) Mw ) 5 × 103 g mol-1 (b) Mw ) 104 g mol-1 (c) Mw ) 1.5 × 106 g mol- 1. Conditions: pH ) 4.0, µ ) 5 × 10-2 M, [chitosan]0 ) 10-3 g/mL, [DS]0 ) 5 × 10-4 g/mL.

have impeded the formation of positive colloids when adding excess chitosan to dextran sulfate (or inversely for anionic colloids) since oppositely charged components are in stoichiometric amount for a brief time during the mixing. The effect of dextran sulfate molecular weight on the particle size was very similar for both slow and fast processes. It was impossible to obtain stable anionic colloids with the lowest molecular weight dextran sulfate (DS 5k). Using DS 10k, the size variations of anionic particles with increasing n+/n- ratios, display a regular increase in diameters, suggesting that the aggregation mode was slightly more predominant in this process than in the dropwise one. Moreover, experimentally, these particles have a very poor colloidal stability since we observed their aggregation during the centrifugation as for those obtained by dropwise addition. Again, it was with DS of the highest molar mass that anionic PECs could be obtained over the broadest range of n+/nratios. Similarly to the dropwise method, a “U” shaped curve was obtained for the variations of the particle size with increasing n+/n- ratios from 0.1 to 0.7 (Figures 4c and 2a),



Figure 5. Influence of dextran sulfate (Mw ) 1.5 × 106 g mol-1) concentration and mixing order on the properties of PEC particles obtained by a one-shot addition with chitosan (DA ) 15%, Mw ) 1.6 × 104 g mol-1) for n+/n- ) 2 (a) and n+/n- ) 0.2 (b). Conditions: pH ) 4.0, µ ) 5 × 10-2 M, [chitosan]0 ) 10-3 g/mL, total volume of solutions (after mixing) ) 5 mL. (light gray) [DS]0 ) 5 × 10-4 g/mL, (dark gray) [DS]0 ) 10-3 g/mL, (black) [DS]0 ) 5 × 10-3 g/mL.

confirming that anionic particles were surrounded by a large corona of uncomplexed DS whose thickness decreased when the concentration of chitosan increased. As a general feature, the average particle sizes obtained by the one-shot method were slightly smaller than by the dropwise process (Figure 2) and so were the polydispersity indexes (inferior to 0.2 in identical experimental conditions). Interestingly, the mean diameter was more elevated when the excess was added to the default (which can also be observed in Figures 5 and 6) for both positively and negatively charged particles. Indeed, these particles are formed by passing through the flocculation point, causing a slight increase of their degree of aggregation and thus of their size. 3.2.1. Influence of External Parameters on the Particle Formation Process. To investigate the influence of the polymer concentration and of the mixing order, a dextran sulfate of 1.5 × 106 g/mol was selected as this polymer led to the most stable colloids for n+/n- ratios of 2, for cationic particles, and 0.2, for anionic particles. Figures 5 and 6, as in Figure 4, confirm that the addition of the excess polymer to the default led to colloids with larger diameters and a slight increase in polydispersity indexes for the cationic particles only. Dextran sulfate initial concentration could be increased at least up to 5 × 10-3 g/mL without affecting the stabilization of the colloids. The increase in particle size, polydispersity indexes and optical density was sharp on reaching this critical concentration, whatever the mixing order and the n+/n- ratios (Figure 5), meaning that a

relatively high concentration of polyelectrolyte favored the degree of aggregation of PEC particles as demonstrated for synthetic polyelectrolytes.28,29 The size increase with dextran sulfate concentration had been observed previously for the dropwise approach.10 If an increase of the chitosan concentration led to higher optical densities as for the dextran sulfate (Figure 6), its effect on the particle size was quite limited in range as already noticed for the slow addition process, for which the opposite tendency was observed, namely a reduction in particle size.10 This evidences that a higher content of chitosan favored the formation of more particles without affecting significantly their sizes. The lower reactivity of chitosan in comparison to the highly charged dextran sulfate may be responsible of this tendency but further investigations are needed to obtain a better understanding of this effect. 3.2.2. Colloidal Characterization. Scanning Electron Microscopy. Two types of particles based on low molecular weight chitosan and high molecular weight DS and obtained by the one-shot addition of default component to excess one were analyzed by scanning electron microscopy. Microphotographs show that particles have almost a spherical shape (Figure 7). It is worth noting that these pictures were obtained after elimination of the free excess polymer in the mixture by two successive centrifugations followed by re-dispersion in water. On crude particles no clear data could be obtained as excess polymer was bound onto the colloid surface, leading to an overestimation of the particle size.10 At n+/nratios of 2 or 0.2, parts a and b of Figure 7 respectively, particles look fairly identical and confirm the values of

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Figure 6. Influence of chitosan (DA ) 15%, Mw ) 1.6 × 104 g mol-1) concentration and mixing order on the properties of PEC particles obtained by a one-shot addition with dextran sulfate (Mw ) 1.5 × 106 g mol-1) for n+/n- ) 2 (a) and n+/n- ) 0.2 (b). Conditions: pH ) 4.0, µ ) 5 × 10-2 M, [DS]0 ) 5 × 10-4 g/mL, total volume of solutions (after mixing) ) 5 mL. (white) [CS]0 ) 5 × 10-4 g/mL, (light gray) [CS]0 ) 10-3 g/mL, (dark gray) [CS]0 ) 5 × 10-3 g/mL, (black) [CS]0 ) 10-2 g/mL.

hydrodynamic diameters obtained by QELS, though the negatively charged one appeared slightly smaller than by light scattering. This might be due to the drying step of the sample preparation, inducing a collapse of the large and mobile outer shell of dextran sulfate. Whether the particles have been obtained by the dropwise or the one-shot method, identical morphologies have been observed (data reported in ref 10). Colloidal Stability Versus pH. The variations of the hydrodynamic diameters and electrophoretic mobilities with pH have been reported in Figure 8 for the same PEC particles as those studied by SEM. The experiments for which the increase of pH was obtained by acetic acid neutralization with sodium hydroxide do not have a constant ionic strength all along the investigated pH range. Hence, another set of data was recorded with a controlled ionic strength set at 10-2 M by the addition of sodium chloride. The decrease in size observed for the cationic particles (Figure 8a) can be interpreted as resulting from the neutralization of the protonated glucosamine units leading to a collapse of chitosan chains in the particle corona. The progressive neutralization of chitosan was confirmed by the concomitant decrease of electrophoretic mobilities (Figure 8b). For pH values higher than 5.5, the effective over-charge of the particles was reduced to such an extent that secondary aggregation occurred, impeding further measurements. With anionic particles, a decrease in particle size was observed until pH 5 (Figure 8a) whereas electrophoretic mobilities remained almost constant in all the investigated pH range (Figure 8b), resulting from the presence of sulfate groups at the surface, always protonated in the pH range investigated. Relatively high values of hydrodynamic diameters in comparison to those obtained during the complexation (Figure 4) have to be related to the low ionic strength

of the media (see below). The decrease in particle size between pH 3.5 and 5 could result from a loss of hydration of the negatively charged shell due to a decrease in the H3O+ ion concentration. Beyond pH 8, colloidal characterization could not be performed as a drastic decrease in optical density due to the dissolution of the complex was observed. Indeed, most of the protonated glucosamine units were neutralized at this pH,30 inducing a decomposition of the PECs into the two hydrophilic macromolecules. As particles were highly diluted, precipitation of chitosan was not observed. Colloidal Stability Versus Ionic Strength. The investigation of the effects of ionic strength on particle diameters and electrophoretic mobilities was undertaken with PEC based on low molar mass chitosan and high molar mass DS. In Figure 9a, a sharp decrease in particle size was observed for anionic particles with increasing salt concentrations, conversely to cationic ones. This size reduction was probably due to a collapse of the large DS-rich corona as a result of the charge screening by increasing the electrolyte concentration. Above 0.075 M in salt, the colloidal stability was lost, and irreversible aggregation was observed. Cationic particles displayed a very limited size reduction with increasing salt concentration and aggregation occurred at 0.1 M NaCl. As already observed in Figure 2a, the size of the chitosan-rich outer shell was smaller than for dextran sulfate coronas. Anyway, for both types of particles, the stabilization mechanism seemed mainly to be of electrostatic type. The variations of electrophoretic mobilities with ionic strength were reported in Figure 9b. For cationic particles, the mobilities logically decreased with increasing salt content. For anionic particles, the mobilities remained almost constant as charge



Figure 8. Hydrodynamic diameters (a) and electrophoretic mobilities (b) of chitosan/dextran sulfate PEC particles (see experimental conditions in Figure 7) as a function of pH in various media: (0) n+/ n- ) 2 in neutralized 10-2 M acetic acid solutions. (9) n+/n- ) 2 in neutralized 10-2 M acetic acid solutions adjusted at µ ) 10-2 M with NaCl. (2) n+/n- ) 0.2 in neutralized 10-2 M acetic acid solutions adjusted at µ ) 10-2 M with NaCl. (4) n+/n- ) 0.2 in 10-2 M phosphate buffer.

Figure 7. Scanning electron microphotographs of chitosan (Mw ) 1.6 × 104 g/mol)/dextran sulfate (Mw ) 1.5 × 106 g/mol) PEC particles, obtained by a one-shot addition of components, for two mixing ratios, (a) n+/n- ) 2 (b) n+/n- ) 0.2. Conditions: [chitosan]0 ) 5 × 10-3 g/mL, [DS]0 ) 5 × 10-4 g/mL, pH ) 4.0, µ ) 5 × 10-2 M. Particles were twice washed before analysis.

shielding could be compensated by the increase in charge density due to the reduction in particle size. Colloidal stability of PEC particles was also assessed from the critical coagulation concentration (CCC), i.e., the concentration of salt at which the particles are no longer stable and begin to aggregate. CCC was determined by plotting the stability ratio (W) as a function of ionic strength using a log-log scale (Figure 10). Physically, 1/W, corresponds to the fraction of particle-particle collision resulting in a sticking event.31 Intersection of the negative slope line with the horizontal one, corresponding to W ) 1 (for high ionic strength), provided the values of CCC which were found to be close to 0.12 and 0.09 M for cationic and anionic particles, respectively. These relatively low values, in good agreement

with previous size variations (Figure 9a), are in accordance with a mostly electrostatic stabilization for both types of particles. Finally, on a process standpoint, particles were easily and reproducibly produced on a larger scale, up to 40 mL of solutions for a n+/n- ratio of 2 (no higher volume was actually tested) and 30 mL for the 0.2 n+/n- ratio, after which particle size variations could reach 20-30%. In fact, sizes of anionic PEC particles are less accurate with regard to their large corona of dextran sulfate. Particles have been concentrated to 3% and 0.7% solids for respectively the cationic and anionic ones, lower solid content for anionic particles being due to their more swollen structure. After four months of storage at room temperature, with no or gentle stirring, no alteration of the particle mean size was observed. 4. Conclusion The influence of the molecular weight of components on the formation of chitosan/dextran sulfate PEC particles obtained by a dropwise addition was investigated for both cationic and anionic colloids. First, when the component in excess could act as host and accommodate several molecules of the titrant, the segregation of hydrophobic segments led to particles electrostatically stabilized by a large hydrophilic corona of excess component. Second, when the component in excess was smaller than the titrant, the outcome of the process depended on the capacity of the excess polymer at

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for both modes of addition (slow and fast). This one-shot process, simple to setup, easy to perform, insensitive to the mixing order of reactants, led to highly stable charged biopolymer-based colloids, which could be obtained in fair amount and at high solid contents. Further work is underway to exemplify this approach toward colloids based on biopolymer polyelectrolyte complexes. Acknowledgment. This work was financially supported by a grant from the French Ministry of Research and Technologies. C.S thanks P. Sorlier for the preparation of the high molar mass chitosan. Note Added after ASAP Posting. Minor changes were made to sentences in the first and second paragraphs of the Introduction for the sake of clarity. A change was also made to the caption of Table 1. The manuscript was originally posted on 8/6/2004. The corrected manuscript was reposted on 8/23/2004. References and Notes

Figure 9. Hydrodynamic diameters (a) and electrophoretic mobilities (b) of chitosan/dextran sulfate PEC particles (see experimental conditions in Figure 7) in 10-2 M acetic acid solution at pH ) 4.0 (µ ) 1.5 × 10-3 M) as a function of added sodium chloride concentration. (4) n+/n- ) 2, (2) n+/n- ) 0.2.

Figure 10. Stability factor (W) as a function of the electrolyte concentration (NaCl) at pH 4.0 for two compositions of chitosan/ dextran sulfate particles (see experimental conditions in Figure 7): (2) n+/n- ) 2, (4) n+/n- ) 0.2.

performing charge neutralization. When charge neutralization could be optimum, flocculation occurred for low amounts of added titrant (case of chitosan into DS 5k or DS 10k in excess). When charge neutralization could not be optimum, the formation of dense particles, stabilized by unpaired groups was observed (case of DS 500k added to low molecular weight chitosan). The one-shot addition of polyelectrolyte was developed to overcome the flocculation occurring in the dropwise approach when the mixing ratios were close to unity. This method proved very versatile for the elaboration of colloidal PECs. Indeed, the process was far less sensitive to the order of mixing than the previous one, as the excess polymer could be used either as titrant or as starting solution. Variations of the particle mean size on varying n+/n- ratios were similar

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Versatile and Efficient Formation of Colloids of Biopolymer-Based

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