Thermoresponsive Behavior of Chitosan-g-N-isopropylacrylamide

Biomacromolecules 2009, 10, 1633–1641

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Thermoresponsive Behavior of Chitosan-g-N-isopropylacrylamide Copolymer Solutions Maricarmen Recillas,† Luisa L. Silva,† Carlos Peniche,‡ Francisco M. Goycoolea,# Marguerite Rinaudo,§ and Waldo M. Arguëlles-Monal*,† CIAD - Guaymas, Carr. Varadero Nac. Km 6.6, Guaymas, Sonora 85480, Mexico, Centro de Biomateriales, Universidad de La Habana, La Habana 10400, Cuba, CIAD - Laboratory of Biopolymers, Carret. a la Victoria Km 0.6, Hermosillo, Sonora, Mexico, and CERMAV-CNRS, affiliated with Joseph Fourier University, BP53, 38041 Grenoble Cedex 9, France Received February 23, 2009; Revised Manuscript Received March 20, 2009

Chitosan-g-N-isopropylacrylamide (NIPAm) water-soluble copolymers were synthesized and characterized by FTIR and 1H NMR spectroscopies combined with conductometric and potentiometric titrations. Their thermoresponsive, fully reversible, behavior in aqueous solutions was characterized by means of microcalorimetry and rheology. During heating of copolymer solutions there is a well-known endothermic effect, which coincides with a marked increase in G′ and a moderate decrement in G′′ due to the formation of a hydrophobic network at the expense of the net amount of sol fraction. It was also found that a straight dependence between the values of G′ above the LCST and the enthalpies associated with the transition reflecting that the connectivity in the gel network is governed by the net number of formed enthalpic-hydrophobic driven-junctions. Both the LCST and the enthalpy change vary with the ionic strength of copolymer solutions, but no dependence was found with the neutralization of the polyelectrolyte chain.

Introduction Smart polymers are usually defined as macromolecules able of undergoing fast and reversible phase transitions from a hydrophilic to a hydrophobic microstructure. These transitions are triggered by small shifts in their microenvironment such as variations in temperature, pH, ionic strength, and electric or magnetic field. Smart materials have found extensive applications in biotechnology, medicine, agriculture, and cosmetic fields, among others.1 pH-responsive polymers contain weak ionizable groups and exhibit a reversible swelling-collapse transition by decreasing the net charge, while the thermosensitive ones usually display a change in hydrophobicity or efficiency of hydrogen bonding when temperature increases. Soluble thermoresponsive polymers show a reversible phase transition associated with phase separation. It has been reported that there is mutual influence between pH and temperature stimuli on the hydrophilicity of copolymers, besides the individual influence of these parameters.2 Chitosan is a linear polysaccharide obtained by deacetylation of chitin. It is mainly composed of two kinds of structural units: 2-amino-2-deoxy-D-glucose and N-acetyl-2-amino-2-deoxy-Dglucose linked by a β(1f4) bond. In acidic solutions, chitosan amino groups become protonated, which render chitosan watersoluble. Chitosan is a very interesting polymer for biomedical applications because of its biocompatibility, biodegradability, and low toxicity.3,4 Poly N-isopropylacrylamide (PNIPAm) is a well-known water-soluble thermoresponsive polymer whose properties have * To whom correspondence should be addressed. Fax: +52 (622) 221 65 33, ext, 220. E-mail: [email protected]. † CIAD - Guaymas. ‡ Centro de Biomateriales. # CIAD - Laboratory of Biopolymers. § CERMAV-CNRS.

been widely studied by many groups.5 PNIPAm undergoes a sharp hydrophilic to hydrophobic phase transition when temperature increases and, hence, exhibits lower critical solution temperature (LSCT) around 32-33 °C. Below the LCST, the polymer chains are soluble in water due to the formation of hydrogen bonds between the water molecules and the amide side chains. When temperature increases, the polymer experiences a volume phase transition. The abrupt nature of this transition and the fact that this change is reversible make it exploitable in specific and novel technological applications such drug delivery systems. It is well-known that chitosan can be modified by graft copolymerization, via its amino or hydroxyl groups, to achieve versatile molecular designs. Grafting small chains of Nisopropylacrylamide onto chitosan gives possibilities to develop materials exhibiting both temperature and pH dependence. Kim et al. have obtained insoluble chitosan-g-NIPAm materials, which are thermosensitive.6 The aim of this contribution was to obtain soluble chitosang-NIPAm copolymers and to evaluate the physicochemical behavior of their solutions by means of differential scanning microcalorimetry and dynamic oscillatory high-sensitive rheological measurements. The influence of the pH and the ionic strength of the medium on the phase transition was also considered.

Experimental Section Materials. Low-molecular weight chitosan was purchased from Fluka. Its viscosity-average molecular weight was 1.3 × 105, estimated at 25 °C in 0.3 M acetic acid/0.2 M sodium acetate,7 and the degree of N-acetylation (DA ) 0.26) was determined by 1H NMR, potentiometry, and conductimetry. Prior to use, chitosan was dissolved in 0.2 M acetic acid solution (5 g L-1) and successively filtered through sintered glass filters (pore diameters: up to 16 µm) and membranes (3, 1.2, and 0.8 µm). Then it

10.1021/bm9002317 CCC: $40.75  2009 American Chemical Society Published on Web 04/13/2009

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Table 1. Feed Composition Used in Preparation of Chitosan-g-NIPAm Copolymers copolymer

chitosan (%)

NIPAm/NH2 molar ratio

CAN/NH2 molar ratio × 103

C1 C2 C3 C4

2 1 1 1

2.5 2.5 2.5 2.5

10 10 7.5 5

was precipitated by dropwise addition of 1 M ammonium hydroxide until pH ≈ 9, carefully washed with water until no change in conductivity was detected, and further washed with ethanol at concentrations of 70, 80, 90, and 100% (v/v). The purified polymer was finally dried under vacuum at room temperature. N-Isopropylacrylamide (NIPAm, Sigma-Aldrich) was recrystallized from hexane at 40 °C and suddenly cooled down to 0 °C. Ceric ammonium nitrate (CAN, Sigma-Aldrich) was used as initiator without further purification. All experiments were carried out with distilled water (conductivity lower than 3 µS cm-1). Preparation of Chitosan-g-NIPAm Copolymer. Graft polymerization of NIPAm onto chitosan was carried out using CAN as initiator under nitrogen atmosphere at room temperature following the method published by Kim et al. with some modifications.6 Chitosan was dissolved in 10% (w/w) aqueous acetic acid solution. After bubbling nitrogen gas during 30 min, NIPAm and CAN were successively added into the chitosan solution and the reaction mixture was stirred at 25 °C for 2 h. Chitosan and CAN concentration were varied as described in Table 1. After graft polymerization, the products were precipitated in excess of acetone and separated by centrifugation. Remaining homopolymer was removed by Soxhlet extraction with methanol during 48 h. The resulting product was then dried under vacuum at room temperature until constant weight was attained. Characterization of Copolymers. High resolution liquid 1H NMR spectroscopy was carried out on a Bruker AV 300 (300 MHz) at 300 K. Spectra were recorded by accumulation of 32 scans. The samples were previously depolymerized as follows: 100 mg of copolymer were suspended in 10 mL 0.07 M HCl at room temperature overnight. Then, 10 mg of NaNO2 were added to the solution and left to react for 4 h.8,9 The solution was then freeze-dried three times to exchange labile protons for deuterium atoms and passed through a cotton fiber filter to remove any insoluble particle. Conductimetric titrations were carried out in a glass cell at 10 ( 0.2 °C. A digital equipment CDB-430 Omega conductimeter and pHmeter was employed. Fourier transformed infrared (FT-IR) transmission spectra were recorded on a Nicolet Prote´ge´ 460 ESP (Madison, WI) by accumulation of 64 scans, with a resolution of 2 cm-1. Samples were prepared in KBr pellets. Differential Scanning Microcalorimetry. Calorimetric measurements were carried out in a micro-DSC-IIIa (Setaram, France). All experiments were carried out between 5 and 45 °C with a scanning rate of 0.6 °C/min. Standard Hastelloy vessels were used with an average 600 µL sample volume. Water was used as reference. The same mass of sample and reference were weighted to minimize the differences in heat capacities between them. The samples were equilibrated at 5 °C for 30 min before each scan. Rheological Measurements. The rheological behavior of chitosang-NIPAm copolymers was studied using a high sensitive straincontrolled rheometer (Rheometrics Mod. RFII Fluids Spectrometer, Piscattaway, NJ) equipped with a stainless steel cone-plate geometry (cone angle: 0.0397 rad, diameter 50 mm, gap 53 µm) and a circulating environmental system for temperature control. To prevent drying of the samples during experiments, a plastic ring of diameter 60 mm was fitted around the measuring geometry and the annulus was filled with low viscosity silicone oil. The phase transition was studied by differential frequency-temperature sweeps measuring the storage, G′(ω), and loss, G′′(ω), moduli at

Figure 1. FTIR spectra of (i) chitosan and (ii) chitosan-g-NIPAm (sample C3).

frequencies 1, 2.15, 4.64, and 10 rad s-1 (γ ) 5%) between 5 and 45 °C, with a temperature rate of 0.3 °C min-1. At both, initial and final temperatures, a mechanical spectrum was recorded (ω ) 0.1-100 rad s-1; γ ) 5%). To make sure that we were working within the linear viscoelastic regime, strain sweep experiments were carried out at fixed angular frequency (ω ) 10 rad s-1). The thermoreversible behavior of a copolymer solution during three heating-cooling cycles was monitored by evolution of mechanical moduli with stepwise changes in temperature between 10 and 30 °C (ω ) 1 rad s-1; γ) 5%) in an AR-G2 rheometer (TA Instruments) equipped with a stainless steel cone-plate geometry (cone angle: 0.0353 rad, diameter 60 mm, gap 65 µm) and a Peltier system for temperature control.

Results and Discussion To prepare chitosan-g-NIPAm copolymers, chitosan was submitted to the reaction procedure reported by Kim et al.,6 but some synthesis conditions were changed to achieve watersoluble derivatives (Table 1). As it can be noticed in Table 1, two series of experimental conditions were used. In the first one (C1, C2), the chitosan concentration was different, while in the other one (C2, C3, C4) chitosan concentration remains constant, but the amount of initiator varied. The reaction was performed at 25 °C. Even when the reaction was carried out under homogeneous phase condition which gives a more regular distribution of the grafted chains, during the reaction time a white opalescence gradually appeared, indicating the formation of a nonsoluble product at this temperature. Nevertheless, if cooled below 10 °C, the mixture becomes transparent. All the products obtained by the above-outlined procedure were soluble in acidic aqueous media at low temperatures, having a transparent liquid appearance. When temperature increased up to LCST, solutions changed to a white opaque apparently elastic gel. This result differs from the insoluble products yielded by Kim et al.6 and is a consequence of the modifications introduced in the reaction conditions. Figure 1 shows the FTIR spectra of chitosan and C3 copolymer. It could be observed in the spectrum of the copolymer the appearance of NIPAm characteristic bands at 2970 and 1460 cm-1 (C-H stretching and CH3 bending deformation, respectively), as well as a band at 1386 cm-1, which corresponds to -CH(CH3)2 bending. Amide I and II bands (at 1655 and 1548 cm-1) are also strengthened in the copolymer spectrum, while the band at 1596 cm-1 (-NH2 scissoring) is weakened.

Thermal-Responsive Behavior of Chitosan-g-NIPAm

Figure 2. 1H NMR spectra of (i) chitosan and (ii) chitosan-g-NIPAm copolymer (sample C3). 1

H NMR spectra also confirm the grafting of NIPAm onto chitosan chain (Figure 2). The presence in the copolymer spectrum of signals at 1.05, 1.49, and 1.63 ppm corresponding to protons in NIPAm moiety are annotated in the Figure 2. The peak centered at 1.97 ppm corresponds to CH3 protons in acetyl group (d, Figure 2). These results clearly indicate the introduction of NIPAm units onto the chitosan chain without clear modification of its NMR spectrum. Conductimetric and potentiometric titrations were carried out with NaOH at 10 °C (Figure 3a and b, respectively). For this purpose, solutions of chitosan and copolymers were prepared in a slight excess of HCl. Traces from conductometric titrations (Figure 3a) show three different steps: the decrease in conductivity during the first part is due to the neutralization of the excess of HCl, followed by a monotonic increase in conductivity of the medium as a consequence of the consumption of protonated amino groups. The high slope observed during the last step is associated with the appearance in the solution of an excess of OH- ions. Similar processes occur during potentiometric titrations (Figure 3b). In both cases, it is obvious the reduction in the content of amino groups not only due to the higher monomeric mass associated in copolymers samples but also as a result of the consumption of amino groups during the grafting of chitosan. In Table 2 the values of molar mass per free amino group estimated by each of these techniques are summarized. In general, similar figures were calculated for each sample. The average value of this parameter was used in forthcoming calculations. An estimation of the composition of the copolymers was achieved, taking into account the integration of 1H NMR signals and the average equivalent mass per free amino group. At this point it is convenient to recall that there are two opinions in the literature regarding the anchored points for grafted chains when copolymerization is conducted using cerium as initiator. On the one hand, Jenkins and Hudson suggest that a similar mechanism to that of cellulose could take place;10 according to their point of view, there is an opening of the glucosidic ring giving rise to the radicals responsible for the initiation of grafted polymer on carbon C3.11 Another possibility is the allocation of the radical on carbon C2, but in this case an aldehyde is formed on carbon C3 after the C2-C3 bond cleavage induced by Ce4+ ions.12 Nevertheless, this possibility should be discarded


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on the grounds that no CdO absorption band appears around 1720-1740 cm-1 (Figure 1). On the other hand, other authors point out that copolymerization of chitosan in the presence of CAN could directly occur onto amino groups,13,14 producing a structure resembling that shown in Figure 4. Assuming the structure given in Figure 4, it is simple to estimate the composition of the copolymer samples using the data from titrations and NMR spectroscopy. Because the amount of N-acetyl groups is not expected to be affected during the grafting reaction, it is possible to estimate how much NIPAm monomers per chitosan unit have been grafted; then one can calculate the weight fraction and, from the number of free NH2 groups (data from conductometric and potentiometric titrations, Table 2), the fraction of grafted units; DPn of NIPAm grafted chains could be also evaluated. Data for grafting percentage have also been determined by this way and expressed as the weight of NIPAm per total copolymer weight. The results are presented in Table 3. The influence of reaction conditions on the chemical composition of copolymers is evident. A decrease in chitosan concentration (C1fC2) causes a great increment in the amount of substituted amino groups, probably due to a better accessibility to chitosan functional groups with dilution. Furthermore, the lower the amount of initiator (C2 > C3 > C4), the lower is the degree of substitution. The higher degree of substitution obtained for sample C2 corresponds with the fact that it was more difficult to dissolve this sample. An increment in the degree of polymerization of NIPAm chains would be also expected, but no tendency could be appreciated, probably as a consequence of the partial phase separation occurring during the copolymerization causing a progressively stopping of the reaction. Calorimetric traces during heating of 1% (w/w) solutions in 10% aqueous acetic acid of copolymers are shown in Figure 5. In all cases, it could be appreciated sharp endothermic peaks between 19 and 20 °C with different calorimetric enthalpies associated, which are similar to those previously reported by other authors.2,15-17 Comparable exothermic peaks were obtained for each sample during cooling (curves not shown), giving rise to fully reversible transition. The transition temperatures of copolymers and the enthalpy change associated decreased in the sequence C2 > C3 > C4. It is well-known that the hydrophilic and hydrophobic contributions are important factors governing thermosensitive properties of NIPAm polymers. NIPAm molecule presents a hydrophilic character given by NH and CdO groups, as well as a hydrophobic one with methyl groups substituents. Water molecules form regular ice-like structures around hydrophobic methyl groups. An increase in temperature results in a reduction of the total number of water molecules structured around hydrophobic groups, which are expelled out from these groups. As a result, hydrophobic interactions between methyl groups from different NIPAm grafted blocks are promoted, giving rise to a polymer network. Water-polymer interactions and hydrogen bonding contribute as well to the phase transition. From a thermodynamic point of view, such a phase transition should generate a conformational entropy-loss upon polymer association, which should be compensated by the translational-entropy gain of expelled water molecules. Therefore there is a total entropy increment upon phase transition that overcomes the observed endothermic enthalpy, thus giving rise to a decrement in Gibbs free energy.2,16,18,19

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Figure 3. Conductimetric (a) and potentiometric (b) titration curves corresponding to the addition of 0.1 M NaOH solution to chitosan (filled squares) and C1 copolymer (open squares) dissolved in an excess of 0.01 M HCl. The values of specific conductivity have been corrected to take into account the dilution during the gradual addition of the solution. In both cases, the same mass of polymer was used. Table 2. Molar Mass per Amino Group Estimated for Chitosan and Chitosan-g-NIPAm Copolymers from Conductimetric and Potentiometric Titrations

sample

Meq (g/mol NH2) cond.

Meq (g/mol NH2) potent.

Meq (g/mol NH2) avg

chitosan C1 C2 C3 C4

235.92 439.79 506.52 375.24 312.91

231.80 423.03 527.20 392.06 309.79

233.86 431.41 516.86 383.65 311.35

It has been found that the temperatures at the maxima of the DSC endotherms of NIPAm containing polymers correspond closely to the cloud point temperatures and could be referred to as the LCST’s of these polymers.2 From Figure 5 it could be appreciated that observed LCST are lower than those reported

for other NIPAm materials.2,20 This behavior is a consequence of the high ionic strength in these solutions as it will be shown below. A small hysteresis was evidenced when comparing both heating and cooling curves in micro-DSC experiments. The influence of the heating rate on hysteresis was also evaluated. Additional calorimetric cycles at different heating/cooling rates between 0.2 and 1 °C/min were conducted (curves not shown). It was observed that both transition temperatures become closer with decreasing the heating/cooling rates, which coincides with previous reports.18,21 Extrapolation to zero-rate gave a ∆T equal to 1.2 °C. At the same time, variation of viscoelastic G′ and G′′ moduli during heating at four different frequencies (Figure 6) confirms the existence of a thermal transition at similar temperatures as those obtained by micro-DSC. This phenomenon is reflected by a marked increase in storage modulus and a moderate



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Figure 4. Schematic structure of chitosan-g-NIPAm copolymers. For more information, see Table 3 and its footnote. Table 3. Composition of Synthesized Chitosan-g-NIPAm Copolymersa sample

A

D-x

x

DPn

grafting (%)

chitosan C1 C2 C3 C4

0.263 0.263 0.263 0.263 0.263

0.737 0.675 0.49 0.66 0.69

0 0.061 0.247 0.071 0.047

10.5 2.1 6.8 3.1

29.6 25.7 23.9 8.7

a A, acetylated units; D - x, deacetylated units; x, NIPAm-grafted deacetylated units; DPn, number-average degree of polymerization of NIPAm grafted chains; grafting percentage, weight of NIPAm per 100 g of copolymer.

decrement in loss modulus. The reduction in G′′ observed for samples C1 and C2 can be interpreted as the result of the formation of hydrophobic junctions at the expense of the net amount of sol fraction (i.e., lower tan δ values). This could be due to formation of more elastic networks produced by the derivatives of greater grafting percent. In sample C3 weaker gels are formed without apparent change in G′′. Furthermore, the transition temperature estimated by calorimetry and rheology is almost the same in all samples. It is evident that the thermal transition is concomitant with the formation of a weak gel above the LCST in almost all copolymer samples. On the one hand, it should be noted that sample C2 behaves like a very weak gel even at temperatures below LCST (in fact G′ ∼ G′′), which is the direct consequence of the high degree of substitution obtained in this copolymer. On the other hand, the change in viscoelastic properties is roughly negligible for sample C4 in correspondence with the very low amount of grafting exhibited by this copolymer (Table 3). When comparing mechanical spectra registered beyond the gel transition temperature, at 45 °C, (Figure 7) it is apparent that samples C1 and C2 form hydrogels with stronger mechanical properties (G′ of C2 larger than G′ of C1 as indicated by the degree of substitution). For these two samples, G′ is greater than G′′ in the tested frequency window (between 0.1 and 100 rad s-1) and G′ is independent of frequency over all the frequency range. Nevertheless, for sample C3, the overall mechanical strength of the system is weaker than those of the former samples, while the mechanical spectrum of copolymer C4 indicates the presence of only a very weakly structured network of self-associated chitosan stabilized by hydrophobic associations of NIPAm methyl groups. This last copolymer shows restricted chain mobility by few physical cross-linking points, thus effectively behaving as an entangled solution. It is worth noting that sample C1, having a greater grafting degree than sample C2 exhibited lower LCST and ∆H values than C2 (Figure 5). It also had a lower elastic modulus than C2. This must be the result of the much greater lateral interaction

of neighboring NIPAm grafted chains in sample C2 as compared to C1 copolymer (given by a larger value of degree of substitution).22 It is interesting to point out the straight dependence observed between the values of storage modulus at 30 °C (ω ) 1 rad s-1) and the enthalpies associated with the phase transition (Figure 8). This dependence could indicate that there is direct relation between the energy necessary to form the physical gel network and its mechanical properties, reflecting that the connectivity in the gel network is directly governed by the net number of formed enthalpic-hydrophobic driven-junctions. To evaluate the influence of pH on the thermal transition of copolymer solutions, 1% (w/w) solutions of sample C1 in 10% (w/w) acetic acid were neutralized with concentrated NaOH solution. In these conditions, the balance between electrostatic repulsions and hydrophobic attraction is perturbed. Microcalorimetric traces of these solutions are shown in Figure 9. It is evident the strong dependence of the transition temperature upon neutralization. There is a notable decrease in LSCT from 19.4 down to 13.4 °C, as the pH was increased from pH 2.0, fully protonated chitosan copolymer (no neutralized solution) up to pH 4.70. At the same time, there is a slight increase on the enthalpy change associated with the thermal transition. The viscoelastic properties of these neutralized solutions were also studied. The variation of the storage modulus (at ω ) 1 rad s-1) with temperature of three representative solutions are plotted in Figure 10. There is an increase in G′ at the phase transition, and the transition temperature shows a similar tendency as manifested in micro-DSC experiments. It should be noticed that the increment in the pH of the solution produces a fall in the storage modulus in sol and gel states. This phenomenon is more significant in the sol state and it could be interpreted as a consequence of the decrease of the charge density of the copolymer, thus, decreasing the electrostatic expansion of the polymer chain. Even when it would be expected that the reduction in the charge density will favor greater hydrophobic association, the fact that the dependence of the values of G′ above the LCST on the pH is similar to that observed in the sol state, argues in favor of the existence of an additional mechanism besides hydrophobic association. The influence of an increment of the ionic strength, which is unavoidable during the neutralization of the solution, should not be discarded as well. The relative increment in storage modulus -after and before the phase transition- increases with the neutralization of the polymer chain, when the hydrophobic interactions are favored, resulting in a more densely cross-linked polymer network. The associated enthalpy change also increases with the neutralization of the copolymer, and there is a good relationship between this parameter and the ratio G′25/G′10 (Figure 11).

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Figure 5. Micro-DSC curves of 1% (w/w) solutions in aqueous acetic acid of (a) copolymers C1 and C2 (samples synthesized varying chitosan concentration) and (b) copolymers C2, C3, and C4 (samples synthesized varying the amount of initiator). Heating rate: 0.6 °C min-1.

Figure 6. Dynamic temperature-frequency sweeps of 1% (w/w) solutions of copolymers in 10% acetic acid, showing G′ (filled symbols) and G′′ (open symbols) at 1 (9), 2.15 (b), 4.64 (2) and 10 (1) rad s-1; γ ) 5%.

Previous works on gels and solutions of linear copolymers of NIPAm with weakly ionizable comonomers have shown that the transition temperature is shifted to higher temperatures as the ionization of functional groups increases, giving the possibility to tailor a thermosensitive material to meet specific technological requirements by changing the pH of the solution.2,23

This behavior has been explained in terms of the increased hydrophobicity of the polymer chain when its charge density is decreased. In preceding experiments there is a concurrent influence of a reduction in polyelectrolyte charge density and an increase of ionic strength on the phase transition with the neutralization of


Figure 7. Mechanical spectra of 1% C1(9), C2(b), C3(2) and C4(1) solutions in 10% acetic acid at 45 °C; γ ) 5%. G′ (filled symbols) and G′′ (open symbols).

Figure 8. Dependence of storage modulus, G′, (ω ) 1 rad s-1, γ ) 5%. T ) 30 °C) with enthalpy change associated with transition, ∆H, of 1% copolymer solutions in 10% acetic acid. Data obtained during heating processes.

the polymer solution. In order to elucidate the specific effect of each of these two parameters the following experiments were carried out. On the one hand, an aqueous solution of C3 copolymer in the form of hydrochloric salt was prepared by dissolving the copolymer sample under stoichiometric conditions. Solutions with different degrees of neutralization between 0 and 0.34 were obtained by NaOH progressive additions to the former solution. There is no apparent difference in the features of the phase transition neither in terms of the transition temperature, nor in the enthalpy associated with it, nor in the viscoelastic properties of these solutions (Figure 12). In addition, the LCST is very near that of PNIPAm homopolymer in absence of external salt. On the other hand, different amounts of NaCl were added to the same hydrochloric salt solution of C3 sample without any neutralization. In this case, however, it is possible to appreciate a variation in the calorimetric behavior of the copolymer. Figure 13 shows that a raise in NaCl concentration leads to a decrease in LCST and an increase in the amount of enthalpy change, which is similar to the overall effect observed in 10% acetic


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Figure 9. Micro-DSC curves of 1% (w/w) C1 copolymer solutions in 10% aqueous acetic acid neutralized up to different pH values with concentrated NaOH solution; heating rate 0.6 °C min-1.

Figure 10. Temperature dependence of G′ measured at 1 rad s-1 and γ ) 5% for 1% (w/w) C1 solutions in 10% aqueous acetic acid during heating at 0.3 °C min-1. The pH of solutions was adjusted with concentrated NaOH solution.

acid copolymer solutions (Figure 9). Analogous behavior has been reported in NIPAm homopolymers aqueous solutions.24,25 Eeckman et al. have described that, under similar experimental conditions, the regular ice-like structure formed by water molecules around NIPAm moieties is disrupted by the addition of salt, which results in an increase of the hydrophobic character of NIPAm chains giving raise to a decrement in the transition temperature.24 The large difference in the LCSTs in 10% acetic acid as compared with stoichiometric hydrochloric copolymer solutions, as well as the low transition temperatures exhibited in the former solutions (Figure 5) are obviously explained by this effect. The increment in the amount of enthalpy associated with the phase transition (Figure 13) could also be understood by the higher hydrophobicity of NIPAm moieties in the presence of salt. The enthalpy remains of the same order of magnitude than in acetic medium. The fully reversible behavior of the phase transition of solutions of this copolymer was also confirmed. On the one hand, from microcalorimetric data (Table 4) it is obvious that no variation in either the temperature or the enthalpy of the

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Figure 11. Ratio between the storage modulus at 25 and 10 °C, G′25/ G′10, measured at 1 rad s-1 and γ ) 5% for 1% (w/w) C1 solutions in 10% aqueous acetic acid as a function of the enthalpy change associated with the phase transition, ∆H. The pH of solutions was adjusted with concentrated NaOH solution.

Recillas et al.

Figure 13. Influence of the salt concentration on the LCST (9) and enthalpy (b) for a 1% (w/w) aqueous solution of hydrochloric salt of copolymer (sample C3). Table 4. Transition Temperatures and Enthalpy Change Associated with Ita cycle

T (°C)

∆H (J g-1 polymer)

1 2 3 4

20.12 20.12 20.12 20.12

16.93 17.03 16.99 16.99

a From micro-DSC experiments during four heating/cooling cycles at 0.6°C min-1 of 1% (w/w) solution in 10% (w/w) aqueous acetic acid (sample C3).

Figure 12. Dependence of enthalpy (2), LCST (b), and G′45/G′10 ratio (9) on the degree of neutralization, R′, for a 1% (w/w) aqueous solution of hydrochloric salt of copolymer (sample C3).

phase transition is apparent after four heating-cooling cycles. On the other hand, a similar conclusion is also noticeable from the fast and reversible variation of the viscoelastic moduli during three heating-cooling cycles between 10 and 30 °C (Figure 14). It is worth noting that the storage modulus undergoes the most important variation in comparison with the smaller, scarcely appreciable change in the loss modulus; this behavior is consistent with above-discussed results (Figure 6). The behavior of chitosan-g-NIPAm copolymer hydrogels exhibiting a marked dual thermal and salt concentration sensitivity, addressed throughout this work, bears great potential in the development of innovative advanced materials that combine the advocated advantages of chitosan with those of poly-NIPAm. Such materials may include devices such as biosensors, actuators, controllable membranes for separations, and programmed drug delivery systems for use in medicine, biotechnology, and other fields.

Figure 14. Variations in mechanical moduli, G′ (b) and G′′ (O), for 1% (w/w) solution in 10% aqueous acetic acid (sample C3) to stepwise periodic changes in temperature between 10 and 30 °C (ω ) 1 rad s-1; γ ) 5%). Experiment carried out in AR-G2 rheometer.

Conclusions Soluble chitosan-g-NIPAm copolymers with various compositions have been synthesized by free radical copolymerization of NIPAm in aqueous acetic acid chitosan solutions. The composition of copolymers was strongly dependent on the reaction conditions. The degree of substitution and grafting percent decreased as the initiator concentration decreased for copolymers prepared in 1% chitosan solution (C2 > C3 > C4).


Copolymer C1, prepared in 2% chitosan solution, but using the same initiator concentration as for C2, had a higher degree of grafting percent (29.6 vs 25.7) but a much lower degree of substitution (0.061 vs 0.247). This must be due to the different reaction conditions: in C1 the reaction solution was much more viscous than the others, and as the reaction proceeded it became highly heterogeneous. The copolymers prepared were fully thermoreversible, with LCST between 19 and 20 °C, when dissolved in 10% aqueous acetic acid. The transition temperatures of copolymers and the enthalpy change associated decreased in the sequence C2 > C3 > C4. The mechanical spectra of 1% copolymers solutions above the LCST were also dependent on composition. While copolymers with higher grafting degrees (C1 and C2) behaved as moderately strong gels, C3 exhibited the pattern of a weakly structured network and C4 behaved as an entangled solution. For all copolymer samples, it was observed a marked increase in G′ and a moderate decrement in G′′, due to the formation of hydrophobic junctions at the expense of the net amount of sol fraction. It was also found a straight dependence between the values of G′ above the LCST and the enthalpies associated with the transition reflecting that the connectivity in the gel network is governed by the net number of formed enthalpic-hydrophobic driven-junctions. Sample C1, having a greater grafting degree than sample C2, exhibited lower LCST and ∆H values than C2. It also had a lower elastic modulus than C2. This must be the result of the much greater lateral interaction of NIPAm grafted chains in C2 as compared to C1 copolymer. With the addition of NaCl to hydrochloric stoichiometric solutions of chitosan-g-NIPAm the LCST decreased and the enthalpy change increased. The marked thermal and salt concentration sensitivity of chitosan-g-NIPAm copolymer hydrogels anticipate their great potential in the development of innovative advanced materials that combine the advocated advantages of chitosan with those of poly-NIPAm. Acknowledgment. This work was financed by SEPCONACYT Project 2006-C01-61252, Mexico. Part of this study was also supported by the European Union - Latin America ALFA Project Polylife (II-0259-FA-FDC). M. Recillas thanks CONACYT for her Ph.D. grant.


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References and Notes (1) Galaev, I., Mattiasson, B., Eds. Smart Polymers. Applications in Biotechnology and Biomedicine, 2nd ed.; CRC Press, Taylor & Francis Group: Boca Raton, 2008. (2) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. Macromolecules 1993, 26, 2496–2500. (3) Peniche, C.; Arguëlles-Monal, W.; Goycoolea, F. M., Chitin and Chitosan: Major Sources, Properties and Applications. In Monomers, Polymers and Composites from Renewable Resources, 1st ed.; Belgacem, M. N., Gandini, A., Eds.; Elsevier Science: Amsterdam, 2008; pp 517-542. (4) Rinaudo, M. Prog. Polym. Sci. 2006, 31, 603–632. (5) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163–249. (6) Kim, S. Y.; Cho, S. M.; Lee, Y. M.; Kim, S. J. J. Appl. Polym. Sci. 2000, 78, 1381–1391. (7) Rinaudo, M.; Milas, M.; Le Dung, P. Int. J. Biol. Macromol. 1993, 15, 281–285. (8) Allan, G. G.; Peyron, M. Carbohydr. Res. 1995, 277, 257–272. (9) Allan, G. G.; Peyron, M. Carbohydr. Res. 1995, 277, 273–282. (10) Mcdowall, D. J.; Gupta, B. S.; Stannett, V. T. Prog. Polym. Sci. 1984, 10, 1–50. (11) Jenkins, D. W.; Hudson, S. M. Chem. ReV. 2001, 101, 3245–3273. (12) Pourjavadi, A.; Mahdavinia, G. R.; Zohuriaan-Mehr, M. J.; Omidian, H. J. Appl. Polym. Sci. 2003, 88, 2048–2054. (13) Caner, H.; Yilmaz, E.; Yilmaz, O. Carbohydr. Polym. 2007, 69, 318– 325. (14) Joshi, J. M.; Sinha, V. K. Polymer 2006, 47, 2198–2204. (15) Badiger, M. V.; Lele, A. K.; Bhalerao, V. S.; Varghese, S.; Mashelkar, R. A. J. Chem. Phys. 1998, 109, 1175–1184. (16) Maeda, Y.; Yamamoto, H.; Ikeda, I. Langmuir 2001, 17, 6855– 6859. (17) Otake, K.; Inomata, H.; Konno, M.; Saito, S. Macromolecules 1990, 23, 283–289. (18) Ding, Y.; Ye, X.; Zhang, G. Macromolecules 2005, 38, 904–908. (19) Tokuhiro, T.; Amiya, T.; Mamada, A.; Tanaka, T. Macromolecules 1991, 24, 2936–2943. (20) Aseyev, V. O.; Tenhu, H.; Winnik, F. M. AdV. Polym. Sci. 2006, 195, 1–85. (21) Grinberg, N. V.; Dubovik, A. S.; Grinberg, V. Y.; Kuznetsov, D. V.; Makhaeva, E. E.; Grosberg, A. Y.; Tanaka, T. Macromolecules 1999, 32, 1471–1475. (22) Zhu, X.; Yan, C.; Winnik, F. M.; Leckband, D. Langmuir 2007, 23, 162–169. (23) Salgado-Rodrı´guez, R.; Licea-Claverıé, A.; Arndt, K. F. Eur. Polym. J. 2004, 40, 1931–1946. (24) Eeckman, F.; Amighi, K.; Moe¨s, A. J. Int. J. Pharm. 2001, 222, 259– 270. (25) Geever, L. M.; Nugent, M. J. D.; Higginbotham, C. L. J. Mater. Sci. 2007, 42, 9845–9854.

BM9002317

Thermoresponsive Behavior of Chitosan-g-N-isopropylacrylamide

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