Polysaccharide Gels Based on Chitosan and Modified Starch

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Biomacromolecules 2010, 11, 1534–1543

Polysaccharide Gels Based on Chitosan and Modified Starch: Structural Characterization and Linear Viscoelastic Behavior Aure´lie Serrero,†,‡ Ste´phane Trombotto,† Philippe Cassagnau,† Yves Bayon,‡ Philippe Gravagna,‡ Suzelei Montanari,*,‡ and Laurent David*,† Laboratoire des Mate´riaux Polyme`res et Biomate´riaux (IMP/LMPB), Inge´nierie des Mate´riaux Polyme`res, UMR CNRS 5223, Universite´ de Lyon, Universite´ Lyon 1, F-69622 Villeurbanne Cedex, France, and Research and Development, Covidien, F-01600 Tre´voux, France Received February 18, 2010; Revised Manuscript Received April 29, 2010

We investigated the properties of polymeric systems formed by cross-linking chitosan with modified starch (oxidized maltodextrin). Such a macromolecular cross-linker proved to be efficient to react with chitosan with potentially minimal toxicity. The structural characterization of modified starch alone and of the two-polysaccharide reactive systems was performed using 1H NMR and FTIR. The rheological behaviors of all systems, from solutions to gels, were also characterized. Depending on experimental parameters, such as chitosan concentration, crosslinking pH, degree of oxidation of starch, and molar ratio of reactive groups, different kinds of systems ranging from pure viscoelastic solutions to stiff hydrogels were formed. These versatile systems could be used in biomedical applications because of the good biocompatibility of their constituents.

Introduction Polysaccharides are a large source of biomass-based materials useful for various applications.1 They can be processed in different ways but their ability to form gels under specific conditions is particularly interesting: polysaccharide hydrogels have been proposed for food, cosmetic, biomedical, or pharmaceutical applications.2 In life science hydrogels have drawn particular interest because of their suitability for drug delivery, implant coating, tissue engineering, and wound healing applications.3,4 Chitosan is a linear polysaccharide composed of β-(1f4) linked D-glucosamine and N-acetyl D-glucosamine residues. This polysaccharide has received considerable attention, particularly in the biomedical field, due to its biocompatibility, bioresorbability, and bioactivity, in addition to its bacteriostatic and fungistatic properties.5 Chitosan, though absent from mammalian organisms, can be assimilated to glycosaminoglycans, which are present in the extracellular matrix of mammalian tissues. The processing of materials from pure chitosan is particularly important because the chemical modification of chitosan, by altering its chemical structure, may also alter its biological properties. Hydrogels are well-suited materials for biomedical applications in the context of tissue engineering due to their high water content, close to natural tissue composition. In particular, chitosan hydrogels are able to mimic the physical and chemical structure of native tissues. Different strategies have been developed to form hydrogels from native chitosan. Chemical gels can be obtained by reacting the amine group of chitosan with a cross-linker.6 Physical gels can be obtained without crosslinkers by acting on the balance between hydrophilic and hydrophobic interactions.7 However, control of the final rheological properties of the gel is easier when a cross-linker is used. Indeed gel stiffness is linked to the cross-linker functionality * To whom correspondence should be addressed. E-mail: suzelei.montanari@ covidien.com (S.M.); [email protected] (L.D.). † Universite´ de Lyon. ‡ Covidien.

and cross-link density. Moreover, physical hydrogels require a coagulating agent (sodium hydroxide or ammonia), which is hardly compatible with an in situ application in contact with living media. Small molecules and, more particularly, glutaraldehyde have been proposed to cross-link chitosan. However, applications of such systems are limited due to the potential toxicity of resulting hydrogels.6 Another strategy illustrated here consists of using functionalized macromolecules from biocompatible polymers, in particular, oxidized polysaccharides, to react with chitosan via a Schiff reaction.8 In this work, we focused on this last strategy and chose to functionalize starch because this polysaccharide is abundant, inexpensive, and biocompatible. Starch is composed of amylose, a linear polymer of R-(1f4) glucose units, and amylopectin, a linear polymer of R-(1f4) glucose units with periodic branches of R-(1f6) linkages. The R-(1f4) glucose units bring mobility to the starch chains. Starch can be easily functionalized due to its various hydroxyl groups. In particular, oxidation of starch has been abundantly described. Among the various oxidation reactions, periodate-mediated oxidation has drawn a lot of interest.9-12 This reaction causes the cleavage of the C2-C3 bonds and the formation of dialdehydes. Oxidized starch is, thus, likely to react with chitosan to form an imine linkage (Schiff Base).8 Imine groups between chitosan and a small molecule bearing aldehyde groups can be easily characterized.13,14 However, this is not the case when macromolecular cross-linkers are used. Indeed, to our knowledge, no such system has been characterized with both NMR and FTIR.8,15-18 In this work, we analyzed chitosan/oxidized starch systems with both FTIR and NMR, along with rheology, which proved to be an efficient tool for the characterization of these types of complex multipolysaccharidic associations.8,19 This enabled us to interpret the influence of experimental parameters (chitosan concentration, pH, amine-to-aldehyde molar ratio (MR), degree of oxidation (DO) of starch) on the gelation of chitosan and oxidized starch solutions.

10.1021/bm1001813  2010 American Chemical Society Published on Web 05/27/2010

Polysaccharide Gels Based on Chitosan and Starch

Material and Methods Materials. Chitosan (degree of acetylation (DA) of 2%, Mw ) 450000 g/mol, PDI ) 1.5, monomodal distribution), produced from squid pens, was purchased from Mahtani Chitosan (batch 113), purified, and characterized as previously described.20,21 Starch (Glucidex 1, potato-based maltodextrin with multimodal distribution, Mw ) 80000 g/mol, PDI ) 3.2) was purchased from Roquette. All other chemicals were purchased from Acros Organics or Sigma Aldrich. Preparation of Oxidized Starch and Assessment of the Degree of Oxidation. Starch was solubilized in water and residual proteins were removed by using cellulose-based ion-exchange resin. Oxidation protocol was inspired from Veelaert et al.22 Briefly, oxidation was carried out on a 6% (w/w) starch solution and different amounts of sodium metaperiodate were added (sodium metaperiodate concentration in reactive medium: 0.18, 0.09, and 0.023 M). Temperature was fixed at 22 °C, and the reaction lasted 2 h. Resulting solutions were dialyzed against water (14000 MW cutoff). Once oxidized, the starch was kept in solution because freeze-drying lowers the product solubility in water. The DO of starch, which is defined as the number of oxidized units per 100 elementary units, was determined by reacting an excess of hydroxylammonium chloride with oxidized starch (OS). This reaction produced an oxime moiety and hydrochloric acid.23 Released HCl was titrated with NaOH and related to the amount of aldehyde in starch.24 The oxidized starch products were named by using their DO as a suffix to the abbreviation. For example, an OS with a DO of 30 will be referred to as OS30. The molecular weight of starch was determined by size exclusion chromatography coupled with a differential refractometer (Waters R 410, from Waters-Milipore) and a multiangle laser-light scattering detector operating at 632.8 nm (Wyatt Dawn DSP). A 0.15 M NaCl/0.01 M Na2HPO4 buffer was used as eluent. The molecular weight appeared higher after oxidation (160000 vs 80000 g/mol for OS30). This was due to the last step of the oxidation process, which encompasses a dialysis that removes all small molecules including the oligomers potentially formed during oxidation. The decrease in the polydispersity index from 3.2 to 2 can be explained by the same reason. The mass profile as well as the average molecular weight of OS was similar whatever the DO. In some cases, the OS aldehyde moieties were reduced to alcohols using NaBH4. These samples are referred to as reduced OS. Preparation of CTS/OS Solutions and Hydrogels. To solubilize chitosan in water at a 5% (w/w) concentration, stochiometric amounts of HCl were added to the water to protonate amine residues. The pH of the solution was then adjusted to desired levels using NaOH. In every case, the pH of OS and CTS solutions were adjusted to the same value before mixing. Concentrations of OS solutions were calculated to obtain a given MR of amines to aldehydes such that MR ) [NH2]/ [CHO]. Structural Characterization of Chitosan, Oxidized Starch, CTS/OS Solutions, and Hydrogels. Infrared spectroscopy was performed on a Perkin-Elmer Spectrum 100 spectrometer using ATR mode (diamond/ZnSe crystal). An average of eight scans was taken with a resolution of 4 cm-1. Oxidized starch and chitosan hydrochloride were analyzed as dry films, nonoxidized starch as a powder and the chitosan/ oxidized starch mix as a dried hydrogel. Starch and its oxidized derivatives were analyzed by NMR spectroscopy at room temperature either on an ALS 300 Bruker spectrometer (300 MHz) or on a DRX 400 Bruker spectrometer (400 MHz). Three different sample preparation and analysis methods were used. In the first method, the samples were freeze-dried and then dissolved in DMSO-d6 at 1 mg/mL prior to analysis. Five mm tubes were used for this measurement. In the second method, the analysis was conducted directly on a 3% (w/w) aqueous solution without altering the physical state of the sample. For this method, 10 mm tubes were used with coaxial inserts containing DMSO-d6 as the reference. These samples were analyzed using the DRX 400 Bruker spectrometer. In the last method samples were analyzed in the gel state. Gel NMR was carried out using the DRX 400 Bruker spectrometer with a 4 mm high

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Scheme 1. Chemical Structures of (a) Chitosan and (b) Oxidized Starch

resolution magic angle spinning proton (HR-MAS) probe. Briefly, hydrogels were dried at room temperature and then swollen in D2O before analysis. All NMR measurements were performed at 25 °C, unless otherwise stated. Rheological Measurements. Rheological experiments were conducted on a controlled stress rheometer (AR 2000, TA Instruments). The linear viscoelastic behavior (more easily referred to as the “rheological behavior” herein) of solutions and gels was assessed in dynamic mode. A 25 mm parallel plate geometry was used with a gap of 500 µm. Prior to each measurement, the limits of linear viscoelasticity were measured by performing a stress sweep test. The applied shear stress required to remain in the linear viscoelastic regime ranged from 1 to 10 Pa depending on the sample. The temperature was fixed at 25 °C for all experiments and silicon oil was used to prevent evaporation during the experiment. Gelation kinetics measurements were performed at 1, 5.5, and 10 rad · s-1. For these measurements, the chitosan solution was placed in the rheometer and then the oxidized starch solution was applied on top of the chitosan solution, with no additional mechanical mixing. Concerning the final rheological behavior, it was assessed 24 h after mixing chitosan hydrochloride and oxidized starch solutions. These experiments were performed in the oscillation mode, with angular frequency sweep measurements from 100 to 10-1 rad · s-1.

Results and Discussion Structural Characterization of Starch and Oxidized Starch. Throughout the entire study, maltodextrin rather than native starch was used. Maltodextrin is a polysaccharide consisting of R-(1f4)-D-glucose units, produced from starch by partial enzymatic hydrolysis. Compared to native starch, maltodextrin is easier to dissolve in water and its smaller molar mass makes the control of mobility and reactivity easier. However, because maltodextrin and starch have a similar chemical structure, we will hereafter keep the term “starch” to name this polysaccharide. The reaction of periodate with starch results in the formation of dialdehyde moieties, as shown in Scheme 1. The 1H NMR spectrum of starch before and after oxidation was compared (Figure 1). The samples were dissolved in DMSO because after freeze-drying oxidized starch exhibited poor water solubility. We obtained a characteristic spectrum of unoxidized starch and attributed the different signals, according to Falk et al.25 Several additional peaks were detectable on the 1 H NMR spectrum of oxidized starch in the 8.5-10 ppm region. Only one peak at 9.39 ppm was really well-defined and was attributed to an aldehyde form.12 Because the local chemical

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Figure 1. 1H NMR (300 MHz) spectra of (a) starch and (b) oxidized starch OS30 in DMSO-d6; the inset gives a magnified view of the 8.5-10 ppm region of spectrum (b). New peaks appeared in the oxidized starch spectrum and were attributed to aldehydic (*) or hemiacetal (+) forms. Scheme 2. Structure of Dialdehyde Starch: Equilibrium of the Aldehydic Form through the Formation of Hemiacetalsa26

a This structure is obtained with the reaction between an aldehyde and an alcohol, C2-C6 and C3-C6, or between two aldehydes, C2-C3 (intracycle) and C2-C3′ (inter-cycle).

environment of both aldehydes (HC2dO and HC3dO) is different, multiple signals were expected and detectable. However, the signals in this region have a low intensity. We explained this result by invoking that aldehydes exhibit an equilibrium with hemiacetals (reaction between an aldehyde and an hydroxyl or reaction between two aldehyde moieties, see Scheme 2).22,26,27 Indeed, new peaks appeared between 6.3 and 7.3 ppm and between 4 and 4.6 ppm. They were attributed to, respectively, the protons of the hydroxyl groups and the protons of the -CH groups of the hemiacetal structures, as previously proposed by Heidelberg et al.28 for similar chemical structures. Zhang et al.12 did not suggest the presence of this equilibrium, although they did observe a new peak at 4.2 ppm for the oxidized starch. Unfortunately, their data did not include spectra between 6 and 9 ppm. Alternatively, one could argue that the signals between 6.3 and 7.3 ppm could also correspond to unsaturated carbons due to degradation products resulting from elimination reactions. However, such unsaturated carbons should result in peaks between 110 and 150 ppm in 13C NMR, and no such signal was observed in that ppm range (results not shown). The existence of an equilibrium between aldehydes and hemiacetals was further evidenced with 13C NMR (see Figure 2). Figure 2a represents the 13C NMR spectrum of unoxidized starch. Again, carbon shifts were attributed following Falk et al.25 Figure 2b represents the 13C NMR spectrum of an OS30 oxidized starch in DMSO-d6. Although a long acquisition time was performed (with over 25000 scans), the signal was not welldefined. This was probably due to the low solubility of oxidized starch in DMSO, which was crucial in 13C NMR due to the lower sensitivity of this technique. Increasing the analysis temperature to 70 °C did not result in a more well-defined signal. To overcome this issue, we analyzed oxidized starch in water,

after oxidation and dialysis, without any freeze-drying step. To perform these analyses, DMSO-d6 was used as the reference in a coaxial insert. This method led to a better resolved spectrum (Figure 2c). No signal was observed in the range 190-205 ppm, where aldehydes usually resonate. However, we observed new peaks at 85-89 and 93 ppm that we attributed to hemiacetal carbons. This shows that the equilibrium between aldehyde and hemiacetals was displaced toward the formation of hemiacetals. Similar results were reported by Dai et al.29 for periodateoxidized dextran. The chemical structure of oxidized starch was further investigated using FTIR. The typical structure of starch was identified and correlated with previous studies30,31 (see Table 1 for details). IR spectrum of oxidized starch (Figure 3b) showed two new vibrational bands: one at 1730 cm-1, due to aldehyde stretching vibrations and another at 1100 cm-1, typical of hemiacetal stretching vibrations. The relatively lower intensity of the aldehyde band was due to the equilibrium between aldehydes and hemiacetals. FTIR results were consistent with 1 H NMR results and confirmed the coexistence of a free aldehyde form as well as its stabilized hemiacetal form. However, due to the low intensity of these bands and due to the ATR mode used, quantitative estimation of the ratio of each moiety was not possible. Characterization of CTS/OS System. In addition to the FTIR characterization of OS, we assessed the chemical structure of chitosan alone (Scheme 1) by FTIR (Figure 3c), and the assignment of all characteristic bands is summarized in Table 1. The analysis was performed on a non-neutralized chitosan film made by evaporation of a chitosan hydrochloride solution. The most characteristic bands of chitosan hydrochloride were visible at 1524 and 1624 cm-1 and were assigned to NH3+ deformation vibrations. Chitosan and oxidized starch OS65 mix, at pH ) 5 and with [NH2]/[CHO] ) 1, was also analyzed. Figure 3d shows the disappearance of both aldehydes (1730 cm-1) and hemiacetals (1100 cm-1), consistent with the consumption of aldehydes most likely by a reaction with amines. However, the resulting imine functions should have resulted in a band close to 1665 cm-1.14 No such band was detectable in the IR spectra, even when the second and forth derivative of the FTIR spectra were plotted. These peaks may have been masked by the multiple absorbance



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Figure 2. 13C NMR (400 MHz) spectra of (a) starch in DMSO-d6 (2048 scans), (b) oxidized starch OS30 in DMSO-d6 (over 25000 scans), and (c) oxidized starch OS30 in water with DMSO-d6 as the reference in a coaxial insert (over 15000 scans). New peaks appeared at 93 ppm and in the 85-89 ppm region and were attributed to hemiacetals. Table 1. FTIR Assignments for Native Chitosan and Starch wave number (cm-1) 3700-3100 2880-2930 1655 (shoulder) 1640 1624 and 1524 1455 1420-1320 1413 1385 1240,1202 1190-960

chitosan assignment32 O-H and N-H stretching C-H stretching amide I O-H bending of water NH3+

starch assignment30,31 O-H stretching C-H stretching O-H bending of water CH2 bending C-H bending and wagging

CH2 bending C-H bending and wagging O-H bending C-O stretching

O-H bending C-O stretching

peaks at this wavenumber. Indeed, both NH3+ and amide groups of chitosan and water adsorbed on the polysaccharide give broad peaks at this wavenumber. Tang et al.16 also studied a chitosan/ oxidized starch system using FTIR. They reported a signal at 1653 cm-1, attributable to the imine moiety; however, the analysis was performed on neutralized films, that is, without the NH3+ moiety. In this work, when ammonia vapor neutralized films were analyzed, the imine band still could not be resolved (results not shown). To further investigate the chemical structure of CTS/OS systems, additional NMR analyses were carried out. Conventional liquid NMR was not feasible due to the insolubility of these systems. Gel phase HR-MAS NMR proved to be an alternative. As shown in Figure 4b, OS30 alone was characterized with this gel NMR spectroscopy. Indeed, OS30, after freeze-drying, is only partly soluble in water, but it does swell in contact with water (or D2O) so that gel-phase NMR is feasible. Two signals appeared at 9.75 and 8.5 (Figure 4b); another slight signal appeared at 9.35 ppm. All these resonance peaks were assigned to aldehydes, which is consistent with the

Figure 3. FTIR spectra of (a) nonoxidized starch powder, (b) freezedried oxidized starch (OS65), (c) chitosan film, and (d) oxidized starch (OS65) and chitosan mix after evaporation of the water. For the CTS/ OS hydrogel, pH was fixed at 5 and [NH2]/[CHO] was fixed at 1. Oxidation led to new vibration bands (I) at 1730 and 1100 cm-1 in the oxidized starch spectrum. They were attributed to aldehyde and hemiacetal groups, respectively. These two bands disappeared (X) when oxidized starch was mixed with chitosan.

results obtained with liquid NMR. However, the signal obtained with HR-MAS was ill-defined, probably because the swelling of the material is not optimal. Figure 4a gives the usual 1H NMR spectrum of chitosan hydrochloride. The singlet around 2 ppm is attributable for the -CH3 of the N-acetamide moiety. Signals in the range 3-4.2 ppm accounted for all of the protons of the glucosamine backbone carbons.33 On the spectrum of the CTS/OS system, both polysaccharides can be identified (Figure 4c). Chitosan gave specific signals at 2 and 3.2 ppm, which are still identifiable in Figure 4c. The contribution of oxidized starch could be identified with signals between 5 and 5.6 ppm. Peaks due to the oxidation were still visible at 9.75 and 8.5 ppm. A new signal appeared at 10.1 ppm, and the signal at 9.35 ppm had a more defined shape for the gel (Figure 4c) compared with the precursor

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Figure 4. 1H HR-MAS NMR (400 MHz) spectra of (a) chitosan hydrochloride D2O solution, (b) oxidized starch OS30 D2O solution, and (c) oxidized starch and chitosan system swollen in D2O. Peak assignments: cts ) characteristic peaks of chitosan; os ) characteristic peaks peaks of oxidized starch; * represents the signal associated to aldehyde in oxidized starch; and i represents the signal associated to imine in the association of oxidized starch with chitosan.

oxidized starch solution (Figure 4b). We attributed these peaks to the imine group because the chemical shifts of such moieties are usually found in this ppm range.34 13C NMR did not provide any additional insight. The difficulties characterizing the presence of the imine moieties may be explained by reversibility of the imine formation, especially in aqueous media. To conclude, we tried to investigate precisely the covalent linkage existing between chitosan and oxidized starch. Both FTIR and 1 H NMR spectrum of the CTS/OS system showed modifications (disappearance or appearance of specific bands or peaks) in the region characteristic of aldehydes. Moreover, the hydrogel formed is insoluble whatever the solvent or the pH used. These observations tend to suggest that the structure of the gel is, at least partly, governed by covalent bonds. However, additional interactions such as H-bonds and hydrophobic interactions may contribute to form a network between both polysaccharides (i.e., CTS and OS). Indeed, such interactions were reported in rheological studies of nonisothermal gelation of a similar polysaccharide-based system.8 Rheological Characterization. Kinetics of Gelation. Determining the chemical structure of the association chitosan/ oxidized starch was a first step. Rheological studies can provide additional insight into the structure of the association. For this series of experiments no mechanical premixing was performed between chitosan and oxidized starch. Figure 5 depicts the evolution of the real part (G′, i.e., the storage modulus) and the imaginary part (G′′, i.e., the loss modulus) of the dynamic shear modulus G* during gelation for an association of chitosan and oxidized starch. As expected, at the beginning G′, the storage modulus was lower than G′′, the loss modulus. This is characteristic of a liquid-like behavior. For longer reaction times, both moduli increased, but the buildup rate of G′ was higher than that of G′′, which resulted in a crossover between both

Figure 5. Evolution of G′ and G′′ with cross-linking reaction time for a chitosan/oxidized starch OS15 system. All solutions were fixed at pH ) 5 and were mixed to obtain a ratio [NH2]/[CHO] ) 5. In the final system, chitosan concentration was 1% (w/w) and oxidized starch 0.66%. Measurement was performed at 1 rad · s-1.

moduli at t1 ) 780 s for the CTS/OS15 system (pH ) 5, [NH2]/ [CHO] ) 5, Ccts ) 1%). After this point, G′ kept increasing, whereas G′′ reached a plateau. This corresponds to a solid-like behavior with a high elastic part (G′) compared to the viscous contribution (G′′). This crossover thus defines a characteristic time for reactive systems. Some authors have defined this point as the gel point.35 However, it is better to consider it only as a first approximation of the gel point. Indeed, this criterion is not satisfying enough because it depends on the frequency at which the measurement is performed. To overcome this issue, Winter and Chambon proposed a model to describe the rheological behavior around the gel point.36,37 The relation between the rheological moduli at the gel point is described by eq 1


G ∼ G ∼ ωn


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(1)

where n is a relaxation exponent linked to the architecture of the gel. This results in eq 2, which defines a gel point independent of frequency.

tan(δ) )

( )

G nπ ) tan 2 G

( )

(2)

To determine the factor n, the kinetics were performed at different frequencies and the value of the loss tangent, tan(δ), was plotted for different frequencies (Figure 6). The gel point occurred at t2 ) 680 s, with n ) 0.67, as deduced from eq 2 applied at the gel point. The value of n can be related to the percolation theory. This theory describes gelation: polymer chains form bonds randomly between each other with the probability p. At the critical value, pc, an infinite cluster appears and p ) pc marks the transition from sol to gel.38 For systems with a low amount of cross-linker a value of n ) 2/3 is often observed. Thus, the value of n ) 0.67 found in our work is in good agreement with such theory. Weng et al.8 found similar results (n ) 0.61) for an oxidized dextran/N-carboxyethyl chitosan system. The fractal dimension df given by the eq 3 was also evaluated.

n)

d(d + 2 - 2df) 2(d + 2 - df)

Figure 6. Evolution of tan(δ) with time at three different frequencies for a chitosan/oxidized starch OS15 system. All solutions were fixed at pH ) 5 and were mixed to obtain a ratio [NH2]/[CHO] ) 5. In the final system, chitosan concentration was 1% (w/w) and oxidized starch was 0.7%.

(3)

where d is the space dimension (d ) 3 in this case). The fractal dimension gives insight into how compact or open a structure is (1.25 < df < 2.5). A low df is characteristic of open structures with low cross-link density. We found df ) 1.78 in the studied system (pH ) 5, Ccts 1%, [NH2]/[CHO] ) 5). Such a structure is likely to depend both on the oxidation degree and concentration of oxidized starch. Rheological BehaVior of Chitosan/Starch Solutions. The interactions between chitosan and oxidized starch were further studied in relation with the rheological properties of a wide range of systems. For these studies, samples were evaluated 24 h after mixing. Before investigating the properties of such complex systems, we studied the rheological behavior of a chitosan solution and a system comprising chitosan and nonoxidized starch. Pure chitosan in solution (Figure 7) exhibited a rheological behavior typical of a viscoelastic solution,7,39 whereby G′ > G′′ at high frequencies (ω > 15 rad · s-1) and G′ < G′′ at low frequencies (ω < 10 rad · s-1). This latter phenomenon is indicative of flow associated with chain disentanglement. It is also possible to observe the characteristic slope of viscoelastic fluids, that is, G′ ∼ ω2 and G′′ ∼ ω at low frequencies, which is in agreement with the Maxwell model. When nonoxidized starch was added to the chitosan solution (Figure 7), no difference was observed between the rheological behavior of the chitosan solution and the chitosan/starch system, suggesting that there was no chemical reaction or physical association between chitosan and nonoxidized starch under these conditions. Therefore, the aldehyde groups of oxidized starch are essential to the formation of the network of both polysaccharides. Further, when reduced OS (oxidized starch treated with NaBH4 to reduce aldehyde to hydroxyls) was mixed with chitosan, there was no observed change in the rheological behavior of the mixture compared to chitosan solution alone (results not shown). This observation further evidence that one key to generate the

Figure 7. Rheological behavior of a chitosan (2.3%)/starch (1.4%) system vs chitosan alone (2.3%). All solutions were adjusted to pH ) 4. Chitosan and chitosan/starch system exhibited the same rheological behavior: s representing a slope of 1 (G′′ ∼ ω at low frequencies) and --- representing a slope of 2 (G′ ∼ ω2 at low frequencies).

network is the aldehyde groups of the oxidized starch reacting with the amine moiety in chitosan. Influence of Chitosan Concentration on the Rheological BehaVior of Chitosan Solutions and CTS/OS Systems. The influence of the concentration of chitosan was studied. The increase in chitosan concentration (from 1.9 to 2.3% (w/w), Figure 8a) resulted in an increase in both loss and storage moduli, which corresponded to an increase in solution viscosity. Moreover, the crossover between G′ and G′′, which is related to a disentanglement time, occurred at a lower frequency for the 2.3% solution than for the 1.9% solution (15 rad · s-1 vs 23 rad · s-1). This is due to a higher entanglement density in the 2.3% solution compared to the 1.9% solution. When these solutions were mixed with oxidized starch (Figure 8b) and allowed to react for 24 h, an increase in the moduli for both concentrations was observed. The rheological behavior in the G′-G′′ crossover frequency range exhibited a two-decade drop (from 25 to 4 rad · s-1) for the 1.9% chitosan-based system. Additionally, the G′-G′′ crossover was not detectable anymore for the 2.3% chitosan-based system in the frequency range investigated.

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Figure 8. Influence of the concentration of chitosan on the rheological behavior on (a) G′ and G′′ of chitosan solutions, (b) G′ and G′′ of the chitosan/oxidized starch OS15 system (24 h after mixing), and (c) tan δ of the different systems. All solutions were adjusted to pH ) 4. The ratio was fixed at [NH2]/[CHO] ) 20 for CTS/OS systems. In the final system, chitosan concentration was 1.9 (w/w) and 2.3%, which correspond to concentrations of oxidized starch of 0.3 and 0.4%, respectively. The addition of oxidized starch led to an increase in the viscosity and the elasticity.

Figure 9. Influence of the pH on the rheological behavior of chitosan/oxidized starch OS15 association. (a) G′ and G′′ moduli and (b) tan δ. The MR was fixed at [NH2]/[CHO] ) 20 for CTS/OS systems. In the final system, chitosan concentration was 1.9% (w/w), which corresponded to a concentration of oxidized starch of 0.3%. The increase in the pH favored the formation of the gel.

Before the addition of oxidized starch, both 1.9 and 2.3% chitosan solutions exhibited a viscoelastic fluid behavior (with tan δ ∼ 1/ω at low frequencies). After mixing and sitting for 24 h, the 1.9% chitosan-based system was still a viscoelastic fluid (Figure 8b), but the relaxation mechanisms were greatly affected. The modification in the rheological behavior after addition of oxidized starch was even more marked for the 2.3% chitosan system, where G′ was higher than G′′ for the entire frequency range investigated (Figure 8b) and tan δ was constant at low frequencies (Figure 8c). This criterion is characteristic of a gel. However, G′ in an ideal gel should exhibit a pronounced plateau extending to times at least on the order of seconds and G′′ should be significantly smaller than G′ in this region,40 which

is not the case. Therefore, our material is not really a solution anymore but does not exhibit all the characteristics of a gel and, thus, should be described as being between a solution and a gel, that is, in a transition state. Associating chitosan and oxidized starch resulted in modifying the rheological behavior, which suggests covalent interactions between both polysaccharides, although physical interactions consecutive to the formation of the chemical gel can not be ruled out. Influence of the pH. The influence of pH on the reaction between chitosan and starch was also investigated (Figure 9). The intrinsic rheological effect of pH on pure 1.9% chitosan solutions was not significant in the investigated pH range (4-5; results not shown). On the contrary, the chitosan/oxidized starch



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Figure 10. Influence of the MR ) [NH2]/[CHO] on the rheological behavior of the chitosan/oxidized starch OS15 system. (a) G′ and G′′ moduli and (b) tan δ. All solutions were adjusted to pH ) 4. In the final system, chitosan concentration was 1.9% (w/w), which corresponded to concentrations of oxidized starch of 0.3% for MR20 and 6% for MR1. The increase in the amount of aldehyde (MR decrease) led to a modification of the rheological behavior of the system with the formation of a complex chemical/physical hydrogel.

systems exhibited a different rheological behavior depending on the pH; the rheological behavior being evaluated after reaction, that is, 24 h after mixing the two polysaccharides. At pH ) 5, the material (CTS/OS) exhibited a gel-like behavior, with G′ > G′′ (or tan δ < 1, see Figure 9b) in the entire frequency range. This difference indicates that the cross-linking reaction between chitosan and oxidized starch is pH-dependent, in relation with the amine moieties present in chitosan.41 At low pH, the NH2 free amine form is negligible and the amine groups are mainly protonated. This prevents the imine formation resulting from the reaction between -NH2 and -CHO moieties. The reaction is slowed considerably, and the final state assessed at 24 h exhibited a viscoelastic solution behavior. Measurements performed after 1 week did not show any further modification of the reacted system (results not shown). At pH ) 5, the pH is still lower than the pKa of chitosan (∼6 at low DA42); however, there are significantly more -NH2 moieties in the solution than at pH ) 4. Thus, when the pH increases, the equilibrium is gradually displaced toward the formation of -NH2, which can further react with aldehydes to form a covalent network. This leads to the formation of a cross-linked gel, which can be considered “weak” because G′ exhibited no plateau zone43 in the investigated frequency range. Besides, we noted a maximum in G′′, which is discussed below. Influence of the Molar Ratio. The role of the MR, MR ) [NH2]/[CHO], on the cross-linking reaction between chitosan and oxidized starch was also investigated (Figure 10). To study the role of MR, the concentration of the oxidized starch solution was changed while holding all other parameters constant. It is particularly important to maintain the concentration of chitosan constant because chitosan highly contributes to the viscosity and the cohesion of the system. Indeed, only a slight increase in its concentration would impact the rheological behavior and would complicate the dicussion.8 The MR ratio is linked to the final cross-link density. We observed that the behavior of the material depended greatly on this parameter. At MR ) 20, we observed a solution-like behavior (tan δ > 1 for ω < 4 rad · s-1) with higher viscosity compared to the initial chitosan solution. For MR ) 1, that is, for a stoichiometric ratio of NH2 to CHO moieties, the material is considered a gel, as defined by Almad et al.40 Indeed, the storage moduli exhibited a plateau zone extending to times at least on the order of seconds (0.4-100 rad · s-1 range) and was significantly higher than G′′ in the plateau zone. Using a stoichiometric amount of reactive groups, as well as increasing the pH (Figure 9a), enabled a high crosslink density. More precisely, in such systems, the loss modulus

Scheme 3. Schematic Networks Involved between Chitosan and Oxidized Starch: (a) Heterogeneous Network Model and (b) Physical/Covalent Network Model

exhibited a particular behavior with a maximum at low frequencies. A first possible interpretation of this result is related to disentanglements in the poorly cross-linked zones. In the highly cross-linked zones (harder microgel domains), the viscoelastic behavior is that of a gel, but in the lower crosslink density zone (interdomain regions), a disentanglement is still possible and could yield a dissipative effect, mainly by the reorganization of neighboring microgels (Scheme 3a). A second interpretation relies on the existence of different cross-link types, for example, physical and covalent cross-links. Such a complex network organization is apparently favored at high covalent cross-link density, which favors interchain interactions. The disruption of low energy physical interactions (H-bonds, hydrophobic junctions) could induce a specific relaxation process of chains that were trapped by physical bonds (Scheme 3b). The difference in storage moduli across this relaxation process thus represents the physical contribution of the gel, whereas the relaxed modulus is due to the covalent network. Influence of the Degree of Oxidation. The DO was also an important parameter governing the association of the two polysaccharides (Figure 11). To study this parameter, both the aldehyde concentration and MR (MR ) 20) remained constant

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Figure 11. Influence of the DO of oxidized starch on the rheological behavior of the chitosan/oxidized starch system: (a) represents the G′ and G′′ moduli and (b) tan δ. All solutions were adjusted to pH ) 4. The ratio was fixed at [NH2]/[CHO] ) 20 for CTS/OS systems. In the final system, chitosan concentration was 2.3% (w/w), which corresponded to concentrations of oxidized starch of 1.4% for OS4, 0.4% for OS15, and 0.2% for OS30. The increase in the DO favored the formation of the gel.

in all samples. Therefore, we varied only the oxidized starch concentration. At DO4, the rheological behavior was still similar to the chitosan solution and chitosan/starch solution, that is, the material was still a viscoelastic solution. This indicates that the amount of aldehyde on the oxidized starch chain was too low to generate a significant interaction with chitosan. At DO15, the amount of aldehyde on the starch chain is clearly impacting the rheological response of the system. As shown in Figure 11, the material can be considered at an intermediate state between the solution and the gel (tan δ ∼ 0.9 at low frequency). At DO30, the material behaved like a gel with higher moduli and a significant difference between G′ and G′′. G′ exhibited a slightly sloped plateau but can still be considered as a gel. Because the total amount of aldehyde remained the same in all the samples, the only difference between each formulation is the density of aldehyde moiety per starch chain. When the DO increases, there are more aldehyde groups on one chain. Hence, the distance between cross-links decreases, which leads to an increase in the cohesiveness of the system: the system evolves from a chitosan-graft-oxidized starch solution to a networked system. As a general result, we processed a wide range of systems with controlled rheological behaviors, from viscoelastic liquids (low density of cross-links) to stiff gels (high density of cross-linked) via intermediate states between the solution and the gel that do not exhibit the ideal extreme behaviors. The mechanism of formation of the hydrogels is complex. Chemical cross-link and physical interactions appear to be intimately related. Nevertheless, all network contributions are related to the covalent cross-links, because without aldehyde moieties, no physical interaction seems to occur between chitosan and starch. Even though the spectroscopic signatures of covalent cross-linking were low, the rheological behavior was drastically modified as a result of both chemical and physical complex interactions.

Conclusion The association of native chitosan and oxidized starch was studied. The systems were characterized precisely with HRMAS NMR and FTIR. The equilibrium of oxidized starch in aqueous solution was also characterized and we showed the formation of hemiacetal moieties. Evidence of covalent bonds between both polysaccharides were found, however, expected imine moieties were difficult to identify with certainty. The rheological behavior of the system was also thoroughly studied. We studied the gelation kinetics, but also the final state of the

Figure 12. Scheme of the different physical states of the chitosan/ oxidized starch systems at pH ) 4 and for a 2.3% chitosan based system. For example, at MR 20 and OS15, the material is in the transition zone (between solution and gel). Plus sign (+) corresponds to experimental characterizations of rheological behavior.

material 24 h after mixing, and found that gelation of our materials can be described by percolation theory, although different interaction types are implied. In conclusion, we succeeded in forming a versatile family of biomaterials with rheological properties that can be adjusted easily by modulating combinations of the different experimental parameters such as the concentration of chitosan, pH, DO, and MR (see Figure 12). One outcome is the possibility to separate our material into three domains depending on the rheological behavior: the viscoelastic solution, the gel, and the intermediate state. Each type of material has its own field of application. For example, some of the material presented in this paper showed significant adhesive properties, which are currently under investigation. The investigated systems could be considered as a model association and open the way to the interpretation of the behavior of polysaccharide networks obtained by varying the nature of the oxidized macromolecular cross-linker. Acknowledgment. This work was financially supported by Sofradim Production and the ANRT. The authors also thank F. Boisson from la plateforme de RMN des Polyme´ristes Lyonnais (Institut de Chimie de Lyon) for helping with the NMR measurements and for fruitful discussions and I. Royaud for advice concerning the FTIR analyses. The authors also thank S. S. Gleiman and R. A. Hadba for their thorough reading of the manuscript.

References and Notes (1) Dumitriu, S. Polysaccharides: structural diVersity and functional Versatility, 2nd ed.; Marcel Dekker: New York, 2004.

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Polysaccharide Gels Based on Chitosan and Modified Starch

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