Continuum of Structural Organization from Chitosan Solutions to

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Biomacromolecules 2010, 11, 6–12

Continuum of Structural Organization from Chitosan Solutions to Derived Physical Forms Simina Popa-Nita,† Pierre Alcouffe,† Cyrille Rochas,‡ Laurent David,† and Alain Domard*,† Universite´ de Lyon, Universite´ Lyon 1, UMR CNRS 5223 IMP, Laboratoire des Mate´riaux Polyme`res et Biomate´riaux, Baˆt. ISTIL, 15, bd. Andre´ Latarjet, F-69622 Villeurbanne Cedex, France, and Universite´ Joseph Fourier de Grenoble, UMR CNRS 5588, Laboratoire de Spectrome´trie Physique, Boıˆte Postale 87, F-38402 St. Martin d’He`res, France Received April 27, 2009

The structural organization of chitosan, a cationic polyelectrolyte, in aqueous solutions of high ionic strength, is investigated by quasi-elastic light scattering and wet scanning transmission electron microscopy. The formation of submicrometric chain aggregates driven by hydrophobic interactions is evidenced. These heterogeneities are at the core of the multiscale morphology of physical hydrogels processed from this polysaccharide. Therefore, a close structural relationship exists between the initial solution and the final hydrogel.

1. Introduction Polysaccharides have been widely studied for their interesting properties in solution1,2 and their diversified applications.3-5 Polysaccharide polyelectrolytes are potentially water-soluble in the ionized state. In this family, chitosan, the only cationic form, knows a growing interest especially due to its outstanding biological properties.6 The organization in solution of such an amphiphilic polymer is rather complex and depends essentially on the balance between hydrophilic and hydrophobic interactions in relation with the presence of different types of contributions: electrostatic, van der Waals (hydrophobic), and hydrogen bonding. Therefore, heterogeneities at different length scales exist in solution,7,8 and the solution microstructure depends on numerous physicochemical parameters9,10 influencing these interactions, such as the linear charge density, the distribution of repeating units along the chains, pH, ionic strength, temperature, and so on. Intermolecular associations formed by hydrogen bonding and hydrophobic interactions are classically observed in polyelectrolyte solutions. In the case of chitosan, Anthosen et al.11 showed by static light scattering (SLS) measurements, the presence of aggregates dependent on the polymer concentration. These large scale heterogeneities were more pronounced for polymers with a high content of acetylated residues, that is, a high degree of acetylation (DA). These authors suggested the involvement of intermolecular hydrophobic interactions between acetyl groups. This interpretation was confirmed using fluorescence probe spectroscopy.12,13 More recently, Sorlier et al.7 evidenced the phenomenon of self-association of chitosan chains in semidilute solutions arising mainly from hydrogen bonding in the case of low DA samples (0-20%) and essentially from hydrophobic interactions for higher DA values. Quasi-elastic light scattering (QELS) experiments showed the presence of supramolecular structures in chitosan dilute solutions depending on DA and the neutralization degree.14 Moreover, a thorough study of a large series of samples having different DAs and molecular weights allowed the development of a new elaboration * To whom correspondence should be addressed. E-mail: alain.domard@ gmail.com. † Universite´ de Lyon. ‡ Universite´ Joseph Fourier de Grenoble.

process of chitin nanoparticles by a simple dissolution of the polymer in a specific solvent containing an acid and a salt.15 Aggregates in polysaccharide solutions are highly significant for practical reasons such as the clarification of solutions for accurate polymer characterizations like by means of viscosity or light scattering measurements, but also because they might affect biological properties. Furthermore, the control of the solution morphology, and more precisely the presence of heterogeneities, appears essential in the formation of nanoparticles,16 the elaboration of physical hydrogels17 or the processing of solid forms.18 The main experimental techniques used to investigate the formation of such aggregates in chitosan solutions are light scattering (static and dynamic) and, less frequently, the transmission electron microscopy (TEM). In the latter case, a staining agent15,16 or a metal shadowing11 are necessary as chitosan particles have a very low scattering contrast. A new innovative technique allowing the direct observation of such particles in the hydrated and unperturbed state is the wet scanning transmission electron microscopy (wet-STEM).19 In the present work, chain aggregates in chitosan solutions at high ionic strength are visualized using this technique and the results are compared with the corresponding QELS investigations. Furthermore, the importance of such heterogeneities in the processing of other physical forms is evidenced by illustrating the relation between the structure of chitosan physical hydrogels and the initial solution morphology.

2. Materials and Methods Purification and Acetylation of Chitosan. Chitosan, produced by chemical heterogeneous N-deacetylation of chitin from squid pens, was kindly donated by Mahtani Chitosan (Veraval, India). This linear copolymer is constituted of randomly distributed 2-acetamido-2-deoxyD-glucan (GlcNAc) and 2-amino-2-deoxy-D-glucan (GlcN) residues linked together via β-(1f4) glycosidic bonds. The initial chitosan was subjected to a step of purification consisting in dissolution at 0.5% (w/v) in a stoichiometric amount of aqueous acetic acid. The solution obtained was filtered successively on membranes (Millipore) of porosity 3, 1.2, 0.8, and 0.45 µm. Then chitosan was precipitated using aqueous ammonia. After repeated washings with deionized water, the precipitate was freeze-dried.

10.1021/bm9012138  2010 American Chemical Society Published on Web 12/03/2009

Structural Organization of Chitosan

Biomacromolecules, Vol. 11, No. 1, 2010

Table 1. Degrees of Acetylation (DA), Weight-Average Degrees of Polymerization (DPw), and Polydispersity Indexes (Ip) of the Different Reacetylated Chitosansa DA (%) 1.5 ( 0.1 14.5 ( 0.5 37 ( 0.4 69 ( 0.9 a

DPw 3100 2890 2680 2360

( ( ( (

Ip 60 60 70 80

1.6 1.6 1.5 1.7

( ( ( (

0.1 0.1 0.1 0.2

Average values from at least three independent measurements.

The resulting chitosan, of low content in GlcNAc units (DA ) 1.5%) was reacetylated to obtain a homogeneous series of samples with the same average DP but different DAs. The reaction took place under soft conditions in a fresh solution of acetic anhydride in a water/ propanediol mixture (50/50% w/w), thus allowing the preservation of a statistical distribution of residues within the chains and the only acylation of amine functions.7 The products were isolated by precipitation on adding aqueous ammonia followed by repeated washings with deionized water. The reacetylated chitosan of DA ) 69% was soluble in water, whatever the pH. Therefore, acetone was used to precipitate and wash this sample. In this case, the acetic acid and the acetate salt were removed by dialysis. The obtained DAs were determined by 1H NMR spectroscopy on a Bruker DRX 300 spectrometer using the method developed by Hirai et al.20 The weight-average degree of polymerization (DPw) and polydispersity index (Ip) of the samples were determined by size exclusion chromatography (SEC) using two columns: Protein Pack Glass 200SW (Waters) and TSKgel G6000PW (Tosohaas) coupled on line with a differential refractometer (Waters R 410, from Waters-Milipore) and a multiangle laser-light scattering detector (Wyatt Dawn DSP). A 0.15 M ammonium acetate/0.2 M acetic acid buffer (pH ) 4.5) was used as eluent. For each chitosan, the refractive index increment dn/dc was evaluated from previous studies.14 Results are listed in Table 1. Preparation of Solutions. A 0.15 M ammonium acetate/0.2 M acetic acid buffer (pH ) 4.5) was used as solvent. Mother solutions of concentration 0.1% (w/w) were prepared with the different chitosan samples (Table 1) and then diluted to achieve the best experimental conditions for QELS and wet-STEM analyses. Solutions were studied without any prior clarification, i.e. no filtration or centrifugation was carried out. The only exception was the solution used in the study of the role of filtration, which was filtered on a 0.45 µm pore size membrane (Millipore). Processing of Chitosan Physical Hydrogels. To form physical hydrogels containing only chitosan and water, it is necessary to fulfill three major conditions:21 (i) the initial polymer concentration must stay above the critical concentration of chain entanglement, c*, to favor the formation of interchain junctions; (ii) a change of the balance between hydrophilic and hydrophobic interactions must occur in favor of hydrophobic interactions and hydrogen bonding to induce the phase separation corresponding to the gel point; and (iii) gelation must not occur locally, but has to correspond to the formation of a bidimensional sol-gel transition. Homogenous conditions can be found by forming a gel front usually displacing from the surface to the bottom of the initial solution sample. In our case, the concentration of chitosan solutions was 2.2% (w/w), a value largely over the polymer c*. Two types of gels were prepared by processing an aqueous21 and a hydroalcoholic22 solution, respectively. In the first case, chitosan was dispersed in deionized water, and dilute hydrochloric acid was added to achieve the stoichiometric protonation of NH2 sites. The solution was placed on a glass slide to form a thin and flat layer that was put in contact with aqueous ammonia. The resulting gel was washed thoroughly with deionized water to eliminate ammonium chloride and the excess ammonia. The washed hydrogel was then stored in deionized water. Thin gels with a flat surface were necessary for small angle light scattering (SALS) experiments to avoid the scattering from the surface asperities. In the second gelation process, the chitosan aqueous solution (same protocol and concentration as above) was mixed with 1,2-propanediol

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(50% w/w). After homogenization, the solution was put in Petri dishes and left in an oven at 50 °C until the water was evaporated and an alcohol gel was formed. To regenerate the free amine form of the polymer, the gel was neutralized with a solution of 2 M NaOH. It was then washed thoroughly with deionized water to eliminate the propanediol, the hydrochloric acid, and the excess sodium hydroxide. The resulting hydrogel was then stored in deionized water. Quasi-Elastic Light Scattering Experiments. QELS measurements were performed at a fixed angle θ ) 90° using an experimental setup equipped with an argon laser (Malvern, France) working at a wavelength of 488 nm and a correlator ALV-5000 (ALV, Germany). The normalized time autocorrelation function of the scattered intensity given by G(τ) ) 〈I(0)I(τ)〉/〈I(0)〉2 was measured. An ALV software was used for the data treatment and the constrained regularization method (Contin) developed by Provencher23 allowed us to deduce the distribution A(τ) of decay times. Small Angle Laser Light Scattering Experiments. SALS measurements were performed using an experimental setup equipped with a helium-neon laser (Spectra-Physic, U.S.A.) working at a wavelength of 633 nm. Images were obtained with a two-dimensional detector (CCD camera Micam VHR 1000). Hydrogels were placed into quartz cells filled with water allowing the refractive index matching and avoiding the dehydration of the hydrogel during measurements. Wet Scanning Transmission Electron Microscopy. Observations were performed with an environmental scanning electron microscope (ESEM XL 30 FEG, FEI Company, U.S.A.). A complete description of the experimental device can be found elsewhere.19 A total of 2-3 µL of chitosan solution was deposited on a holey carbon film-coated TEM copper grid (HC300-Cu, Delta Microscopies, France). The grid was placed with the carbon layer down so that the copper squares could be used as retention basins. A purge sequence19,24 was carefully optimized to prevent the total evaporation and the water condensation on the sample. The sample temperature and the partial water vapor pressure in the microscope chamber were then adjusted to obtain and keep a solvent layer thin enough (∼hundreds of nm) so that the scattered electrons pass through. This step is crucial for the following sample analysis. The samples were observed using an incident electron beam accelerated with a 25 or 30 kV voltage. The scattered electrons were collected by a dipolar detector placed under the sample in such a way that it occults the transmitted electron beam. This specific geometry allows the detection of a more important part of the scattered electrons available and, thus, more contrasted images can be obtained.19 Scanning Electron Microscopy. Pieces of hydrogels of about 2-3 mm3 were placed on a copper sample holder, which was then rapidly immersed in slushy nitrogen (T ≈ -210 °C, prepared under vacuum). This allows an optimal thermal conductivity by avoiding calefaction. This sample preparation is then well adapted for hydrogels, as the water crystallization is avoided on larger thicknesses, and the gel network is not broken. The frozen samples were introduced into the microscope chamber and the residual ice formed on their surface was completely eliminated by sublimation: the sample temperature was increased from -160 to -80 °C, followed by a 10 min isotherm at -80 °C, under reduced pressure. Hydrogels were cooled again at -160 °C and then gold-coated to increase their surface conductivity. The samples were observed on a Hitachi S800 SEM with a voltage between 5 and 10 KV.

3. Results and Discussion 3.1. QELS Study of the Self-Association of Chitosans of Different Apparent Charge Densities in Dilute Aqueous Solutions of High Ionic Strength. Chitosan is an amphiphilic copolysaccharide for which the molar fraction of acetylated residues (DA) is a crucial parameter influencing various aspects of the balance between hydrophilic and hydrophobic interactions.7,9,10,25 Several solutions of chitosan varying in DA (Table 1), at a constant polymer concentration (cp ) 0.01% (w/w)),

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Figure 1. Normalized distributions of decay times A(τ) for a scattering angle θ ) 90° using the Contin method for solutions of chitosan of different DAs and a 0.01% (w/w) polymer concentration in an AcNH4 0.15 M/AcOH 0.2 M buffer (pH ) 4.5). The abscissa gives both the decay times τ and the corresponding hydrodynamic radii R.

below c*, as deduced from the intrinsic viscosity, were studied by QELS. Due to the presence of salt (AcNH4 0.15 M/AcOH 0.2 M), the protonated amine functions were screened and therefore, electrostatic repulsions reduced. The normalized distributions of decay times obtained by the Contin method23 are presented in Figure 1 for chitosans of three different DAs. For a chitosan of high apparent charge density (DA ) 1.5% at full ionization), we observe a single distribution related to a unique although broad population of objects corresponding to individual polymer coils.14 Their hydrodynamic radius is of about 40 nm at the peak maximum. Furthermore, when DA increases, the formation and stabilization of chain aggregates seem to be favored. Indeed, for intermediate and high DA values (37 and 69% in Figure 1), a second peak corresponding to a slower relaxation mode appears in the distribution of decay times. A rough evaluation of the position of this peak maximum gives a hydrodynamic radius of aggregates of about 300 nm. Moreover, we observe that the two populations are better defined and become narrower when DA increases. A third population of an even slower relaxation mode can also be observed on the distribution of decay times (solid triangles in Figure 1). The latter corresponds to very few agglomerates of sizes beyond the micrometer range. When the polymer concentration increases, this population becomes more important and cannot be totally removed even by repeated filtrations of the solution (results not shown). It might represent a specific organization in semidilute and concentrated solutions at higher length scales, but the QELS analysis of these systems is rather delicate. According to DA, a general law of behavior of chitosan in aqueous solutions was evidenced,25 pointing out the existence of three distinct domains: (i) for DAs below 28%, chitosan behaves as a polyelectrolyte of high charge density and then, the ionic condensation occurs.26 In this region, the long-range intra- and intermacromolecular electrostatic interactions are responsible for a high solubility of the polymer chains. (ii) for DAs between 28 and 50%, constituting a transition range, hydrophilic and hydrophobic interactions are progressively counterbalanced, and (iii) for DAs over 50%, the polymer becomes hydrophobic with a low charge density. The three chitosans studied were deliberately chosen to represent each of these domains. Then, we noticed that, for intermediate and high DAs, when short-range hydrophobic interactions become progressively predominant,10,25 polymer chain associations are favored.

Popa-Nita et al.

Figure 2. Normalized distributions of decay times A(τ) for a scattering angle θ ) 90° using the Contin method for solutions of high DA (69%) chitosan at a 0.01% (w/w) polymer concentration in an AcNH4 0.15 M/AcOH 0.2 M buffer (pH ) 4.5): (black square) nonfiltered and (gray triangle) filtered solutions. The abscissa gives both the decay times τ and the corresponding hydrodynamic radii R.

The formation of chain aggregates in the dilute regime was not observed in previous studies14 as authors thoroughly clarified the samples before analysis (filtered twice through 0.8 and 0.45 µm pore size membranes). We carried out a filtration test on the chitosan solution of DA ) 69%. As seen in Figure 2, chain aggregates initially present in the solution could be fully removed by filtration (for cp ) 0.01% (w/w)), and the first peak, only present, was displaced toward lower hydrodynamic radii, with a maximum at about 25 nm. At higher concentrations, filtration only had little impact, as chain aggregates were either not fully eliminated, or, more probably, regenerated very quickly, as if a dynamic equilibrium existed between isolated and aggregated chains. These results together with the above ones confirm that these heterogeneities are not the result of an initial incomplete dissolution of the polymer. Therefore, at high concentration, the solution is heterogeneous at different length scales. As little is known about the nature of these aggregates, a direct estimation of their size and shape from the determination of the hydrodynamic radius and the diffusion coefficient is not straightforward. We then gathered complementary information using electron microscopy techniques. 3.2. Imaging of Submicrometric Chain Aggregates in Dilute Chitosan Solutions. Classical TEM can be used to evidence nanoheterogeneities or nanoparticles thanks to its high resolution and the possibility of obtaining a detailed description of objects under the sample surface. Nevertheless, for species of low scattering contrast (like polysaccharide aggregates), a staining agent is necessary. It consists of a solution of heavy metals added to the sample. The conditions and protocols of this procedure are rather restrictive as the reaction between the sample and the staining agent should be prevented at all means. The presence of salt in the buffer used, the property of chitosan to bind heavy metals,27,28 the solution pH are factors interfering during the sample preparation and can perturb the investigated solution. Moreover, classical TEM only allows the study of dried samples. To overcome these difficulties and image hydrated samples, wet scanning transmission electron microscopy (wet-STEM) was used. This technique uses low voltage to accelerate the incident electrons, and the effective cross sections of electron-sample interactions are high and thus induce a better contrast than with the classical TEM.29,30 Another advantage of wet-STEM is that chromatic aberrations are reduced by the



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Figure 3. Wet scanning transmission electron micrograph of (a) nanoparticles formed by self-association of chitosan chains of high DA (69%) and high DPw (∼2400) in an AcNH4 0.15 M/AcOH 0.2 M buffer. Micrograph (b) represents the control, that is, the buffer solution alone. Both images were obtained at 30 kV in annular dark-field conditions with a magnification of 12939. The sample temperature was 2.2 °C and the chamber pressure was (a) 5.9 and (b) 4.5 Torr, respectively.

Figure 4. TEM micrographs, obtained in bright field, of nanoparticles formed by self-association of chitosan chains of high DA (69%) and high DPw (∼2400) in an AcNH4 0.15 M/AcOH 0.2 M buffer. Samples were prepared by very slow and controlled evaporation in an ESEM chamber. Scale bar lengths are (a) 1 µm and (b) 100 nm. Magnifications: (a) ×19500 and (b) ×140000.

absence of the projection lens, found in front of the detector in a transmission electron microscope.31 An example of micrograph obtained for a chitosan solution of DA ) 69% at an initial polymer concentration cp ) 0.01% (w/w) is shown in Figure 3a. For a sample temperature of 2.2 °C and a water vapor pressure in the microscope chamber of 5.9 Torr, chitosan chain aggregates were imaged while they were kept in a thin solvent film at equilibrium. Aggregates formed by self-association are almost spherical with a diameter of about 150-300 nm. Their size is comparable with the hydrodynamic radius estimated from the normalized distributions of decay times from QELS analysis (§3.1). A variation in contrast can be clearly observed between the periphery (brighter) and the core (darker) of the particles. Taking into account that micrographs are registered under annular darkfield conditions, this illustrates that particles are less concentrated at their core. During the partial drying of the solution, a more or less hydrated film can be formed and, thus, the periphery of the polymer aggregates may dry more easily while their core

remains more hydrated. The contrast may then simply be related to the observation technique but may also be a proof of a core/ shell organization in the structural or chemical constitution of the aggregates. At this point, the gathered information does not allow an unquestionable explanation. Because of the complexity of the numerous scattering mechanisms involved, it is difficult to theoretically explain every observed contrast. This aspect needs further investigations consisting for example in a Monte Carlo simulation of the number of scattered electrons in the solid angle of the detector in the case of a more or less hydrated particle. After the wet-STEM analysis, the grids were totally dried by slowly reducing the vapor pressure in the microscope chamber to 4.1 Torr. They were subsequently transferred to a classical TEM (Philips CM120, accelerating voltage ) 80 kV) and examples of the obtained micrographs are presented in Figure 4. Micrographs in Figure 4 let appear interesting details concerning the morphology of dried particles. Their sizes stay

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in good agreement with the wet-STEM result (150-300 nm diameter). As explained in the Materials and Methods section, the drying procedure (using well-defined purge sequences19 and very small and controlled pressure variations) is a key element in the sample preparation. Thus, Figure 4 shows that the gradual evaporation of the samples allows us to obtain a high enough contrast so that no staining agent is necessary for classical TEM observations. These results are necessarily different from those described above (Figure 3a). Indeed, in the above case, although the polymer is relatively hydrophobic, it is at its maximum hydration due to the full ionization of the glucosamine residues. On the contrary, in the present case, all the structure is at its maximum hydrophobicity. Indeed, during evaporation, both acetic acid and ammonium acetate present in the buffer are eliminated32 and the polymer is converted under its free amine form. Tight filaments between aggregates, favored both by hydrophobic interactions and hydrogen bonding are clearly observed. In the first case, electrostatic repulsions between the shells limit this agglomeration phenomenon. No contrast between the periphery and the center of the particles is observed in the second case. This could be explained by more homogeneous particles but could also represent a detail which is not visible by the classical TEM technique due to the use of a more energetic electron beam. 3.3. Structure Relationship between Chitosan Solutions and Derived Physical Hydrogels. Due to the nonaccessibility (in most cases) of the glass transition temperature before thermal decomposition, aqueous solutions are obviously essential for the processing of almost all materials based on natural polymers. Knowing and controlling the morphology of the solutions is then a key step in the development of physical hydrogels.17 Chitosan physical hydrogels were obtained following two methods reminded in Materials and Methods. The initial solution concentration (cp ) 2.2% (w/w)) was chosen to have a structural organization governed by hydrophobic interactions.10,17,22 Ideally, at the nanoscale and in the framework of the pearl necklace model33,34 the chains adopt a conformation controlled by hydrophobic nanodomains (“pearls”) connected by more elongated polymer chain portions (“strings”). At a higher length scale of organization in solution over c*, this hydrophobic regime is characterized by the presence of spherical submicrometric aggregates of chains, as shown above (Figure 3a). The morphology is therefore that of a dispersion of hydrophobic aggregates in a more or less hydrophilic solution of isolated polyelectrolyte chains. The structural organization of physical hydrogels elaborated from such solutions was first observed by scanning electron microscopy (Figure 5). The micrograph in Figure 5 displays a structure of gel at two levels of organization consisting in submicrometric spherical particles interconnected or agglomerated into micrometric clusters entrapping the solvent. In the case presented here, the average diameter of the spherical particles (aggregates) is close to 200-300 nm. Previous cryo-microscopy studies carried out in our team35,36 demonstrated that the aggregate size is influenced by the polymer concentration, the nature and the concentration of the neutralizing agent but always stays in the range between 200 and 400 nm. At a higher length scale of organization, the hydrogel morphology was investigated by SALS (Figure 6). The well-defined correlation halo on the SALS diagrams evidence the existence of a structure autocorrelation over micrometric domains (clusters) in hydrogels. When DA increases, this correlation peak shifts to smaller q values in relation

Popa-Nita et al.

Figure 5. Scanning electron micrograph of a physical hydrogel of chitosan processed from a hydro-alcoholic solution (DA ) 37%; DPw ∼ 2700; cp ) 2.2% (w/w); initial solvent: water/ 1,2-propanediol 50/ 50 (w/w); neutralizing agent: 2 M NaOH). Magnification: ×10000.

Figure 6. Log-log plots of scattered intensity I as a function of the scattering vector q for chitosan physical hydrogels of different DAs. Gels were processed from aqueous solutions of polymer concentration cp ) 2.2% (w/w). The neutralizing agent was 7 M NH4OH except for plot (b), for which 1 M NH4OH was used. The scattering curves are shifted vertically for visual convenience.

with an increase of the interparticle distance (ξ ∼ 2π/q). In this example, the characteristic average size ξ varies from 1.9 up to about 3.1 µm for DA ) 1.5-37%, respectively. Moreover, the micrometric domains are larger when the neutralizing solution concentration is lower (see curves for DA ) 14.5% in Figure 6). This is consistent with previous results showing that during neutralization with increased concentration base solutions, the shrinkage of polymer chains leads to the formation of smaller domains.37 The micrometric domains can be observed in Figure 5, where the gel resembles a packing of raspberries which size between 2-3 µm coincides with those mentioned above. These analyses allow us to have an overview picture of the multiscale structural organization of chitosan physical hydrogels. Thus, they are constituted of interconnected submicrometric aggregates organized in micrometric clusters where the solvent is entrapped at different length scales. The aggregate and cluster sizes depend strongly on polymer characteristics (DA, DP), initial solution properties (concentration, solvent constitution),



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4. Conclusion

Figure 7. Schematic view of the continuum of structural organization from chitosan solutions to physical hydrogels.

and processing conditions (nature and concentration of the neutralizing agent). Nevertheless, this morphology seems to correspond to a general situation in chitosan physical hydrogels.37,38 Turning back to the structural organization of the initial solution, a parallel with that of hydrogels seems appropriate, exhibiting a continuum of structure between the two physical forms. Previous studies14,39 showed that, during the neutralization of the amine functions, the population of free polymer coils in solution decreases to the benefit of the formation of chain aggregates. In this view, when physical hydrogels are elaborated from aqueous solutions,21 the submicrometric aggregates present are initiators of gelation and during the sol-gel transition they collapse to form a nanostructured gel thanks to the condensation on their surface of isolated chains becoming more hydrophobic. In the case of the processing of physical hydrogels from a hydroalcoholic solution,22 the critical value of the hydrophilic/ hydrophobic interaction balance inducing gelation is achieved during the water evaporation stage. The solvent then becomes poorer (lower dielectric constant), the interactions polymer/ polymer are favored and the individual polymer chains condense on the already existing nanoaggregates. Hence, these chain aggregates in solution are essential in the processing of physical hydrogels and constitute precursors of the submicrometric objects observed in the final material. Their organization during the gelation process allows entrapping the solvent both in submicrometric and micrometric domains. This continuum of structural organization can be schematized as in Figure 7. Thus, two situations can be encountered in solution (1). In the first (1-a), which constitutes a reference state, polymer chains are in the dilute regime, in highly hydrophilic conditions corresponding to: a maximum electrostatic potential (especially for a low DA), a full ionization, a low ionic strength. Then, the solution is a dispersion of isolated hydrophilic chains. Their hydrodynamic sizes are close to 20-40 nm. On making the chains more hydrophobic by means of numerous parameters contributing to a decrease in the electrostatic potential, such as an increase of DA, the neutralization degree, the ionic strength, or an increase of the concentration to overcome c*, then c**, we contribute to the formation of submicrometric aggregates increasing in number but not in size, which remains between 200-400 nm (1-b). As described above, the gel point (2) corresponds to the collapse of these aggregates by condensation on their surface of the isolated hydrophilic chains becoming more hydrophobic. This also induces the formation of raspberrylike clusters of solvent (3), with a size between 2-3 µm. This multiscale organization certainly plays a particular role on the mechanical and biological properties of these physical hydrogels.

A continuum of structure from the solution to the physical hydrogel was proposed in the case of chitosan, a cationic polyelectrolyte. The submicrometric heterogeneities due to the self-aggregation of chains present in solution play a major role during the gelation process and represent the basement of the multiscale morphology of physical hydrogels. We first studied by dynamic light scattering and electron microscopy techniques these submicrometric particles (aggregates). We concluded that their formation is driven by hydrophobic interactions. To our knowledge, wet-STEM was used for the first time to visualize chitosan submicrometric aggregates without any perturbation of the sample and in a hydrated state. Moreover, the gradual total evaporation of the samples was used as preparation technique for classical TEM observations without any staining agent. Starting from a “homogenous” solution of solvated polyelectrolyte chains, we can progressively displace the hydrophilic/ hydrophobic interaction balance in favor of a more hydrophobic regime, where chain aggregates form in a more or less hydrophilic polyelectrolyte solution. This transition was illustrated by varying the polymer charge density through the change in DA. In addition, chain aggregates in the gel state were visualized and both light scattering and electron microscopy showed their organization in micrometric raspberry-like clusters of solvent. Acknowledgment. We are grateful to A. Perrat (CTµ, UCB, Lyon, France) and G. Thollet (CLYME, INSA, Lyon, France) for their technical assistance during wet-STEM experiments. We thank Dr. G. Lamarque for valuable discussions and lecture of the manuscript. These studies are part of the NanoBioSaccharides project from the 6th European Framework Program, “Nanotechnologies and nanosciences, knowledge-based multifunctional materials, and new production processes and devices”.

References and Notes (1) Braccini, I.; Grasso, R. P.; Pe´rez, S. Conformational and Configurational Features of Acidic Polysaccharides and their Interactions with Calcium Ions: A Molecular Modeling Investigation. Carbohydr. Res. 1999, 317, 119–130. (2) Rinaudo, M.; Milas, M.; Jouon, N.; Borsali, R. On Some Original Properties of Dilute Polyelectrolyte Solutions at Low Salt Content: Sodium Hyaluronate Example. Polymer 1993, 34 (17), 3710–3715. (3) Dumitriu, S. Polysaccharides as Biomaterials. In Polymeric Biomaterials; Dumitriu, S., Ed.; Marcel Dekker: New York, 2002; pp 161. (4) Francis Suh, J. K.; Matthew, H. W. T. Application of Chitosan-Based Polysaccharide Biomaterials in Cartilage Tissue Engineering: A Review. Biomaterials 2000, 21 (24), 2589–2598. (5) Vander, P.; Varum, K. M.; Domard, A.; El Gueddari, N. E.; Moerschbacher, B. M. Comparison of the Ability of Partially NAcetylated Chitosans and Chitooligosaccharides To Elicit Resistance Reactions in Wheat Leaves. Plant Physiol. 1998, 118, 1353–1359. (6) Domard, A.; Domard, M. Chitosan: Structure-Properties Relationship and Biomedical Applications. In Polymeric Biomaterials, 2nd ed.; Dumitriu, S., Ed.; Marcel Dekker: New York, 2002; pp 187-212. (7) Sorlier, P.; Denuzie`re, A.; Viton, C.; Domard, A. Relation between the Degree of Acetylation and the Electrostatic Properties of Chitin and Chitosan. Biomacromolecules 2001, 2, 765–772. (8) Popa-Nita, S.; David, L.; Rochas, C.; Domard, A. Analysis of the Structure of Solutions of Chitosan with Controlled Degrees of Acetylation and Polymerization. In AdVances in Chitin Science; Domard, A., Guibal, E., Varum, K. M., Eds.; Ecole des Mines d’Ale`s: Montpellier, France, 2006; Vol. IX, pp 261-267. (9) Lamarque, G.; Lucas, J.-M.; Viton, C.; Domard, A. Physicochemical Behavior of Homogeneous Series of Acetylated Chitosans in Aqueous Solution: Role of Various Structural Parameters. Biomacromolecules 2005, 6, 131–142.

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Continuum of Structural Organization from Chitosan Solutions to

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