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Effect of Cryogenic Treatment on the Rheological Properties of Chitosan/Poly(vinyl alcohol) Hydrogels Simona Morariu,* Maria Bercea, and Cristina-Eliza Brunchi “Petru Poni” Institute of Macromolecular Chemistry, 41-A Grigore Ghica Voda Alley, 700487, Iasi, Romania ABSTRACT: Chitosan (CS) and poly(vinyl alcohol) (PVA) mixtures in acetic acid solution (AcAc) were investigated in order to determine the optimal composition required to design hydrogels in well-defined conditions. The main interest was to obtain materials which are in a liquid-like state at room temperature (injectable form) and transform into a gel at physiological temperature. CS/PVA mixtures with different compositions in 0.2% and 2% AcAc were investigated in dilute solution at 37 °C by viscometry. The intrinsic viscosity was evaluated by a classical method (Huggins equation) as well as by using a recent approach reported in literature (Wolf model). On the basis of miscibility parameter (Δb) and refractive index values, the miscibility between CS and PVA was discussed. Mixtures with total polymer concentration between 3% and 10% and optimum polymer composition have been submitted to freezing/thawing cycles, and their rheological behavior was investigated. The viscoelastic properties of CS/PVA hydrogels depend on the polymer total concentration and the freezing/thawing cycles number. The gel state is reached at lower temperatures as the number of freezing/thawing cycles increases. engineering.17 Porous structure can be obtained from chitosan solutions by cooling them at temperatures below −20 °C.18 The control of pore structure can be achieved by controlling the freezing conditions, chitosan concentration, and acid acetic concentration in the solution used as solvent.19 Recently, a new “dynamic gel” (dynamic on both the molecular and supramolecular levels) with potential applications in chemical separations, drug delivery, and sensors was obtained by condensation of cinnamaldehyde and chitosan.20 These gels are restructured on the micrometric scale leading to the formation of highly ordered microporous morphologies. However, pure chitosan hydrogel is fragile and presents poor mechanical properties, and its application is limited. A possibility to avoid these disadvantages is the use of mixtures of natural macromolecules with synthetic polymers, such as PVA, which is of high interest for preparing new stimulisensitive materials. The composites based on CS/PVA mixtures present improved thermal and mechanical properties as compared with pure polymers and thus they can meet the requirements for applications as biomaterials.21,22 CS/PVA hydrogels obtained by using different chemical cross-linkers (e.g., glutaraldehyde, formaldehyde, borate, tripolyphosphate, tetraethoxysilane) have a tridimensional network which can negatively affect the biocompatibility.23 Moreover, some chemical cross-linkers are not desirable in the hydrogel structure because they increase the material toxicity. For these reasons, for biomedical applications, it is preferable to design hydrogels by physical interactions between CS and PVA chains, which combine advantageously the properties of both polymers. The physical hydrogels presented in the literature exhibit a higher swelling sensitivity toward pH changes

1. INTRODUCTION Chitosan/poly(vinyl alcohol) hydrogels, either chemically or physically cross-linked, have received much attention during the last years.1−4 Because of the biocompatibility, biodegradability, and antimicrobial activity of chitosan (CS), on the one hand, and the excellent mechanical strength, biocompatibility, and nontoxicity of poly(vinyl alcohol) (PVA), on the other hand, hydrogels based on the two polymers can have applicability mainly in biomedical and pharmaceutical industries.5,6 In our previous studies, we have shown that entangled polymer solutions are able to form stable physical gels with a change in temperature, as for example poly(acrylonitrile) in dimethylformamide,7,8 xanthan/Pluronic F127 and poly(vinyl alcohol)/Pluronic F127 mixtures in water,9,10 or after several freezing/thawing cycles applied to PVA entangled solutions.11 The final properties of hydrogels are influenced by the freezing/thawing conditions (number of cryogenic cycles, freezing time, rest time, and temperature), solution composition (concentration and molar mass of the polymer), and gelation conditions (thawing rate or aging temperature).11−13 PVA forms aggregates in aqueous solution due to the hydrogen bonds that occur between −OH groups along polymer chains. The freezing of PVA aqueous solution determines the formation of ice crystals in the amorphous region and the polymer crystallites which lead to a porous structure after thawing.11,14 By gradual thawing, the polymerenriched microphases and associations of polymer chains appear. The low thawing rates lead to a sufficient time for crystallization and intermolecular physical bonding occurs, while rapid heating rates can even prevent the network stabilization. Gupta et al.15 have shown that the transparency, crystallinity degree, wettability, swelling, and mechanical properties of PVA hydrogel film depend on the solution concentration, on the one hand, and the number of freeze− thaw cycles, on the other hand.15,16 The freeze-dry method was applied to a CS solution in order to obtain porous chitosan scaffolds for applications in tissue © 2015 American Chemical Society

Received: Revised: Accepted: Published: 11475

August 21, 2015 October 29, 2015 October 29, 2015 November 6, 2015 DOI: 10.1021/acs.iecr.5b03088 Ind. Eng. Chem. Res. 2015, 54, 11475−11482

Article

Industrial & Engineering Chemistry Research

freezing/thawing cycles (up to 10 cycles) in order to obtain the hydrogels which were investigated by rheological measurements. After each freezing cycle the samples were kept at 4 °C until their complete thawing. 2.3. Measurements. Viscometric measurements were performed at 37 °C ± 0.01 °C using an Ubbelohde capillary viscometer (type 0a with a capillary diameter of 0.53 mm) in combination with an automatic viscosity measurement system (Lauda Instrument, Germany). Upon dilution, each polymer solution was kept about 15 min prior the measurements for thermal equilibration. Flow times were obtained with good reproducibility; each measurement was repeated at least six times and the errors were less than 1%. Rheological investigations were carried out by using a Bohlin CVO rheometer equipped with Peltier device for temperature control and parallel-plane geometry (diameter of 60 mm, gap of 0.5 mm). The water evaporation was limited by using an antievaporation device which created a saturated atmosphere near the sample. Amplitude sweep tests were carried out at constant frequency (ω = 1 rad·s−1), in order to establish the linear viscoelastic range for each sample. Oscillatory frequency sweep experiments were performed in the frequency range of 10−1 rad·s−1 to 2 × 102 rad·s−1 at 37 °C and 1 Pa (within the linear viscoelastic regime). Temperature sweep tests were conducted at 1 rad·s−1 and 1 Pa in order to follow the evolution of the viscoelastic parameters of CS/PVA mixtures after 1, 2, and 3 freezing/ thawing cycles. The samples were subjected to heating by increasing the temperature from 4 to 70 °C with a heating rate of 1 °C·min−1. Refractive index of the solutions containing CS and PVA in different ratios was measured at 37 °C ± 0.01 °C by using an Abbe refractometer NAR-1T liquid with a thermostat watercirculation system (Lauda Eco Silver).

compared to those chemically cross-linked. Thus, physical CS/ PVA hydrogels could be an alternative for preparing biocompatible drug delivery systems if pH-controlled release is not required.24 Abdel-Mohsen et al.25 reported the preparation of the physical CS/PVA hydrogels by freezing/ thawing in order to deliver sparfloxacin antibiotic. The release of antibiotic depends on the membrane thickness, pH, and temperature of the medium. Hydrogels based on CS/PVA as vascular tissue engineering scaffolds were prepared by freeze− thaw and further cross-linking with KOH/Na2SO4. The increase of the number of freeze−thaw cycles causes the change of the surface morphology, hydrophilicity, and protein adsorption.26 CS/PVA hydrogel membranes have shown promise for cell culture and tissue engineering due to their mechanical and biological properties resulting from the combination of the two polymers.27 Many investigations have been focused on CS/PVA mixtures due to their ability to change the properties as a response to external stimuli, such as pH or electrical changes.28,29 The ratio of polymers influences the interactions between them and therefore the structure and properties of the gels.30 Moreover, the gelling process of hydrogels formed by the freeze−thaw method is favored when PVA concentration increases and the thawing rate decreases.24 The gel structure becomes less regular by increasing the CS concentration into CS/PVA mixtures.31 The aim of this work is to design hydrogels by cryogenic treatment of the CS/PVA solutions with an optimum composition for which the interactions between the two polymers are more favorable. The rheological properties of the hydrogels were followed in order to get information concerning the network formation.

2. EXPERIMENTAL SECTION 2.1. Materials. Poly(vinyl alcohol) (PVA) (98−99% hydrolyzed) with Mw = 1.66 × 105 g·mol−1 and chitosan (CS) were purchased from Aldrich and Fluka, respectively. The polymers were used without any further purification. The viscometric molecular weight and the degree of acetylation of chitosan sample were previously established as being 7.14 × 105 g·mol−1 and 26%, respectively.32 Acetic acid (AcAc) with a purity of 99.8% was acquired from Sigma-Aldrich; 0.2% and 2% (v/v) AcAc solutions used as solvents were prepared by mixing AcAc with Millipore water (obtained with a Millipore-Q water purification system). 2.2. Samples Preparation. PVA was dissolved in water at 80 °C under magnetic stirring for 8 h and the solution with 0.06 g·dL−1 PVA was equilibrated overnight at room temperature. CS solutions (0.06 g·dL−1) in 0.2% and 2% AcAc were obtained under magnetic stirring for 4 h at room temperature. To prevent degradation of the chitosan macromolecules, the solutions prepared 1 day before their use were kept at a temperature of 4 °C. CS/PVA/solvent ternary mixtures with different polymer compositions were prepared by mixing the homogeneous binary solutions of chitosan and PVA. For each sample, the final AcAc concentration of 0.2% and 2% was reached by further addition of acid. In the present paper, the index 1 corresponds to CS, whereas the index 2 corresponds to PVA. The CS/PVA mixture composition is expressed as weight fractions, thus w2 represents PVA weight fraction in the polymer mixtures. CS/PVA mixtures with total polymer concentration (% wt/wt) between 3% and 10% and w2 = 0.9 in 0.2% AcAc were subjected to

3. RESULTS AND DISCUSSION 3.1. Viscometric Behavior of Dilute Solutions of CS/ PVA Mixtures. Viscometric investigations were carried out as a function of total polymer concentration (c) for different weight fractions of PVA in the binary polymer mixture and experimental results were compared with those corresponding to parental CS and PVA solutions. It is well-known that for polyelectrolytes in solution, the deviation from the linear dependence of the reduced viscosity with the concentration at low polymer concentration makes difficult the evaluation of the intrinsic viscosity by the classical methods (such as Huggins method). For this reason, a new method reported in the literature by Wolf33 was also used in the present study. Although it was initially developed to be applied for the polyelectrolytes,33−38 this method has also proven its validity for neutral (co)polymers or polymer mixtures in solution.39−42 According to the Wolf method, the intrinsic viscosity, [η], can be evaluated from the initial slope of the dependence of ln ηr as a function of polymer concentration, c, at sufficient low shear rates: ln ηr =

c[η]W + B W c 2[η]W [η]• 1 + BW c[η]W

(1)

where ηr is the relative viscosity, BW represents the hydrodynamic interaction parameter and [η]• is the characteristic specific hydrodynamic volume. 11476

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Industrial & Engineering Chemistry Research Table 1. Viscometric Parameters of CS/PVA Mixtures with Different Compositions in 0.2% and 2% AcAc Solutions Evaluated by Huggins and Wolf Methods Huggins method w2

−1

kH

[η]H (dL·g )

0 0.0725 0.250 0.500 0.625 0.750 0.850 0.900 0.920 1

42.403 39.299 32.026 21.653 16.484 13.285 10.959 9.159 5.641 0.920

0 0.0725 0.150 0.250 0.350 0.500 0.625 0.750 0.900 1

25.787 26.553 26.834 23.631 20.527 14.413 12.157 7.452 3.653 0.758

Wolf method

AcAc (%) = 0.2 0.702 0.656 0.551 0.602 0.598 0.703 0.859 1.758 1.108 1.150 AcAc (%) = 2 0.693 0.629 0.536 0.759 0.5742 1.046 0.946 1.446 3.545 9.930

[η]W (dL·g−1)

BW

44.369 41.770 33.495 21.835 16.640 12.962 11.020 9.590 5.928 0.920

0.100 0.085 0.120 0 0 −0.240 0.557 −0.520 −0.380 −0.654

26.513 26.898 26.837 23.378 20.980 14.424 11.495 7.492 3.747 0.729

0 0 0 −0.142 −0.100 −0.347 −0.616 −0.707 −1.972 −9.550

Figure 2. Plot of Δb (eq 3) vs weight fraction of PVA (w2) for CS/ PVA mixtures in 0.2% and 2% AcAc solutions at 37 °C.

Figure 3. Variation of refractive index with composition of CS/PVA mixture in 0.2%AcAc and 2%AcAc at 37 °C.

the intrinsic viscosity were found for pure PVA in 2% AcAc: 5.66% by using Huggins method and 3.43% with the Wolf approach. This can be explained by the worsening of the solvent (water) quality for PVA in the presence of AcAc. According to literature data,43,44 the association of PVA chains in water occurs as a result of the intermolecular interactions between PVA chains through hydrogen bonds formed between hydroxyl groups along PVA chains. For CS solution in acidic medium, the protonation of amine groups located along of the chains takes place and the strong electrostatic repulsions between the NH3+ groups lead to an expansion of chitosan chains.32 Rinaudo et al.45 reported that the intrinsic viscosity of chitosan solutions presents a maximum value in its dependence on initial AcAc concentration and the viscosity decreases at an excess of ionic strength (high AcAc concentration). At high acid concentrations, the viscosity of chitosan solution remains nearly constant due to the complete protonation and the low ionic concentration in relation with the pK of acetic acid.32,45 According to Figure 1, the expansion of chitosan chains, which influences the intrinsic viscosity, is higher in the solution with lower AcAc concentration. The viscosity of PVA solutions is less sensitive to changes of AcAc concentration. The BW parameter from eq 1 reflects the curvature of the plot of ln ηr as a function of c indicating that the viscosity increases slow down with an increase in the polymer concentration.46 Generally, BW is positive for uncharged polymers, and it indicates favorable polymer−solvent interactions typical for a good solvent.40,42

Figure 1. Variation of [η]W with w2 for CS/PVA mixtures in 0.2% and 2% AcAc at 37 °C. The dotted lines represent the additive rule.

The parameter [η]• can be considered zero for weak polyelectrolytes (such as chitosan) or neutral polymers, without affecting the accuracy of the experimental data evaluation with eq 1. Table 1 gives the viscometric parameters, [η]W and BW, used for the modeling of the experimental data with eq 1, and also it includes the intrinsic viscosity ([η]H) and kH constant values obtained by applying the Huggins equation (eq 2). ηsp /c = [η]H + kH[η]2H c

(2)

where ηsp/c is the reduced viscosity (ηsp = ηr − 1), and c is the polymer concentration in solution. From Table 1, it can be observed that the intrinsic viscosity values determined with the Wolf model were slightly different than those calculated by the Huggins method, especially for high chitosan content. The maximum errors in the evaluation of 11477

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Figure 6. Schematic representation of polymer−polymer interactions in CS/PVA hydrogels obtained by freezing/thawing method: (gray oval) crystallite junction zone between PVA chains, (gray line) CS chain, (black line) PVA chain, and (dotted line) hydrogen bond.

According to the results given in Table 1, it can be observed that the polymer−solvent interactions are more favorable (BW > 0) in less acidic solution (0.2% AcAc) and for small amounts of PVA in the polymer mixtures (w2 < 0.25). The polymer−solvent interactions are diminished by increasing PVA quantity in polymer mixture (BW ≤ 0) in favor of the formation of the interactions between polymers chains. Some CS-PVA interactions are not fully excluded even in conditions of low concentrations of PVA and AcAc in aqueous solution. The CS-PVA interactions are favored in more acidic solution (2% AcAc) which represents a poor solvent for PVA but which allows the NH2 groups along CS chains to be completely protonated. In 2% AcAc solution, the BW parameter is zero for the mixtures with very low quantity of PVA, and it becomes negative for the mixtures with w2 > 0.25. Wolf and co-workers47,48 have compared the BW parameter (from eq 1) with the Huggins constant (kH from eq 2), establishing that kH = 1/2 − BW for the neutral polymers. Both hydrodynamic interaction parameters offer information about the polymer interactions and the solvent quality. For the investigated systems, kH values higher than 0.5 suggest the formation of the aggregates as a result of the polymer−polymer interactions in solution. 3.2. Polymer−Polymer Miscibility. The miscibility between CS and PVA in solution at 37 °C was first evaluated considering the miscibility parameter, Δb, calculated with the relation proposed by Krigbaum and Wall:49

Figure 4. Evolution of the viscoelastic parameters as a function of temperature for CS/PVA mixtures (w2 = 0.9) with (a) 3% total polymer and (b) 10% total polymer in 0.2% AcAc and subjected to 1, 2, and 3 freezing/thawing cycles. The solid lines represent the fitting of the experimental data with the Hill relationship (eq 6).

exp id Δb = b12 − b12

(3)

where b12 is a parameter that reflects the interactions between different polymer molecules. The experimental value bexp 12 can be obtained from the following relationship: exp bmexp = b11·w12 + b22 ·w22 + 2b12 ·w1·w2

(4)

where b11 and b22 are self-interaction parameters obtained from the slopes of ηsp/c vs c for the pure polymers in solution. The indices 1, 2, and m refer to CS, PVA, and their mixture, respectively. bexp m is the slope of ηsp/c vs c for the polymers mixtures in solution and it reflects the binary interactions between polymer segments. bid12 is given by the following equation:

Figure 5. Dependence of tan δ as a function of temperature for CS/ PVA mixtures (w2 = 0.9) with (a) 3% polymer and (b) 10% polymer in 0.2% AcAc subjected to different number of freezing/thawing cycles. 11478

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Figure 7. Effect of c on: (a) G′ and G″; (b) tan δ for CS/PVA hydrogels with w2 = 0.9 after 10 freezing/thawing cycles. The viscoelastic parameters were obtained at 37 °C, 1 Pa, and 1 rad·s−1. The dashed line corresponds to tan δ = 1, and below this limit the samples show gel-like properties (G′ > G″). id b12 =

b11·b22

purpose, CS/PVA mixtures with different total polymer concentrations in 0.2% AcAc solution were investigated. The mixture composition was set at w2 = 0.9, appropriately to the CS/PVA ratio at which the two polymers show high miscibility (Figure 2). All mixtures have been subjected to 10 freezing/ thawing cycles. The evolution of the viscoelastic parameters, elastic (G′) and viscous (G″) moduli, was followed after 1, 2, and 3 cycles for samples with 3% and 10% total polymer in order to evidence the structural evolution and the transition temperature. Also, for all hydrogels obtained after 10 freezing/ thawing cycles, the viscoelastic properties were determined at 37 °C. The experimental data obtained in temperature sweep tests from 4 to 70 °C with a heating rate of 1 °C·min−1 were fitted with a modified Hill equation53 in order to estimate the final viscoelastic moduli (the viscoelastic moduli show plateau values, Gmax ′ and Gmax ″ ):

(5)

Two polymers are considered miscible if Δb > 0 (attractive interactions) and immiscible if Δb < 0.49 Considering the Δb parameter calculated with the viscometric data obtained in the present study (Figure 2), CS and PVA in 0.2% AcAc solution are not miscible for w2 < 0.68 and the interactions between different macromolecules are favorable at high PVA content. Δb criterion indicates that the two polymers are miscible in 2% AcAc solution for a whole range of studied compositions (Δb > 0), as it was also reported previously by Naveen Kumar et al.50 The highest Δb values observed in 2% AcAc for w2 < 0.3 and in 0.2% AcAc around w2 = 0.9 suggest more favorable interactions between unlike macromolecules. Another possibility to predict the miscibility of two polymers supposes the determination of the refractive index of their solution. The refractive index as a function of the mixture composition is linear for miscible polymer mixture and nonlinear for nonmiscible polymers.50 In Figure 3 is shown the refractive index for CS/PVA solutions vs w2 at 37 °C. The variation of refractive index is linear for CS/PVA mixtures with w2 > 0.58 in 0.2% AcAc solution indicating the miscibility of the two polymers in this composition domain. The refractive index shows a nonlinear dependence for the CS/ PVA mixture with high content of CS in 0.2% AcAc solution confirming that CS and PVA are not miscible for w2 < 0.58. For PVA content up to w2 = 0.35, the refractive index of CS/ PVA mixture in 2% AcAc solution shows a linear dependence on polymer composition, indicating that the two polymers are miscible. For w2 > 0.35, the miscibility of CS with PVA in 2% AcAc solution decreases, and the refractive index values deviate slightly from linearity. The miscibility results obtained using the refractive index measurements were in agreement with those from viscometry investigations (Figures 1 and 2). 3.3. Effect of Polymer Concentration and Freezing/ Thawing Cycles Number on Viscoelastic Properties of CS/PVA Physical Hydrogels. The effect of total polymer concentration and the number of freezing/thawing cycles on the viscoelastic properties of CS/PVA mixtures was investigated in order to establish the optimal conditions to elaborate stimuliresponsive hydrogels with potential applications in medicine. The thermosensitive systems are in the liquid state prior to injection and the liquid changes into a solid-like state (gel) under physiological conditions postinjection.51,52 For this

G(T ) = Gmax

Tn Tn + θm

(6)

where θ represents the temperature (T) for which G(θ) = Gmax/2. The sample with 3% polymer remains in the liquid state (G′ < G″) after the first three freezing/thawing cycles. The evolution of the rheological parameters during heating depends on the number of freezing/thawing cycles (Figure 4a). After the first freezing/thawing cycle, the mixture with 3% polymer shows no significant variations of G′ and G″ with the temperature, and it remains in the liquid state on whole studied temperature range. A strong increase of the viscoelastic moduli at the transition temperature (which decreases with the increase of the number of freezing/thawing cycles) was evidenced for the same sample after two and three cryogenic cycles. The gelation temperatures (Tgel) at which the mixture with 3% polymer starts to have gel-like properties (G′ exceeds G″) were 56.6 and 43.8 °C for two and three freezing/thawing cycles, respectively. The sol−gel transition reflected in the viscoelastic behavior of the CS/PVA mixtures subjected to the freezing/thawing cycles depends also on polymer concentration. Thereby, the mixture with 10% total polymer shows the gelation at 48.5 °C after one freezing/thawing cycle unlike the mixture with 3% polymer which has retained the liquid-like properties up to 60 °C (Figure 4b). The mixture with 10% polymer which has been frozen and thawed twice has shown a sol−gel transition at 34 °C. The 11479

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temperature, the intermolecular hydrogen bonding interactions are weakened and the polymer chains become more mobile. The high temperature determines also the movement of free water molecules which increase the entropy in the system. The water surrounding the CS chains is removed in order to decrease the entropy change and the hydrophobic chitosan chains associate leading to gelation.55 The gelling mechanism of PVA in aqueous solution by freezing/thawing involves the formation of the crystallite junction zones between PVA chains consisting of 2 or 3 ordered chains having 42−120 similar segments.56 During the first freezing cycle, ice crystals appear inside of the sample and the interactions between the polymer chains, concentrated in the still liquid regions, lead to stable physical connections. During the thawing with controlled heating rate, the polymer enriched microphases which alternate with amorphous ones are formed. The physical bonds formed after the first freezing/thawing cycle act as a building block to form a network structure inside the polymer-rich regions.11 The formation of the gel structure by successive freezing/thawing cycles of CS/PVA mixture in aqueous solution is due to the hydrophobic interactions between CS chains, on the one hand, and to the crystallite junction zones between PVA chains, on the other hand (Figure 6). The hydrogen bonds which appear between different groups of CS and PVA chains can also contribute to the formation of the physical network structure. After ten freezing/thawing cycles, all CS/PVA mixtures with c between 3% and 10% have revealed solid-like properties; that is, G′ > G″ and tan δ < 1 (Figure 7). One can observe that the G′ value shows an increase with an order of magnitude by increasing the polymer concentration from 3% to 10% unlike the G″ value which remains nearly constant. It was observed that tan δ decreases with an increase in the polymer content following a linear dependence in the investigated concentration range. The increase of G′ more quickly than G″ with increasing polymer concentration could be due to the increase of crystallites size between PVA chains and the hydrophobic interactions between CS chains which determine the formation of a stronger network with more pronounced elastic properties.

mixture presents a different behavior if it is subjected to three freezing/thawing cycles. The sample has solid-like properties (G′ > G″) at low temperature and it becomes more structured by increasing the temperature (the difference between G′ and G″ increases). The variation of loss tangent (tan δ = G″/G′) with the temperature can provide information about the change of the ratio between the viscous and the elastic response of the sample; tan δ is higher than unity for liquid-like materials and it becomes very small (≪ 1) for solid-like materials. Figure 5 shows the evolution of tan δ during heating with a controlled rate for CS/PVA mixtures with 3% and 10% total polymer subjected to 1, 2, and 3 freezing/thawing cycles. CS/PVA mixture with 3% polymer and w2 = 0.9 has shown a slight increase of tan δ up to about 27 °C irrespective of the number of freezing/thawing cycles (Figure 5a). The mixture subjected to one cycle of freezing/thawing keeps a liquid-like behavior, and it becomes less viscous by further increasing the temperature up to about 60 °C. After two and three freezing/ thawing cycles, the mixture acquires solid-like properties at 56.6 and 43.8 °C, respectively (Figures 4a and 5a). One can observe that after two and three freezing/thawing cycles the structure of the gels remains unchanged by increasing the temperature above about 60 °C (tan δ is nearly constant, Figure 5a). The increase of polymer concentration at 10% induces changes in the evolution of viscoelastic properties with the temperature as it was observed in temperature sweep tests (Figure 5b). The network structure is formed above 44 or 28 °C when the sample was subjected to 1 or 2 freezing/ thawing cycles, respectively. After the third freezing/thawing cycle, the week gel structure is preserved up to 22 °C, when it starts to form a stronger network, as evidenced by an abrupt decrease of the loss tangent. At further increases in temperature, the samples reach an equilibrium structure for which tan δ becomes constant. After one freezing/thawing cycle, the CS/PVA mixture with 10% polymer shows a final value of tan δ much smaller than that corresponding to 3% polymer, proving a stronger structuring (Figure 5). The further freezing/thawing treatments of CS/PVA mixtures with 3% and 10% polymer lead to hydrogels with the viscoelastic moduli having higher values as the polymer concentration increases. By analyzing the rheological data obtained for CS/PVA mixtures with w2= 0.9 in 0.2% AcAc solution, we can conclude that the number of freezing/thawing cycles required to obtain thermosensitive polymer systems with potential biomedical applications (which are liquid-like at room temperature and became gel-like at 37 °C) decreases with increasing polymer concentration from 3% to 10%. Thereby, for the mixture with 3% polymer more than three cycles of the freezing/thawing are necessary unlike the mixture with 10% polymer for which are sufficient only two cycles. The formation of hydrogel structures by freezing and thawing of CS/PVA mixtures in aqueous solution is due to the hydrogen bonds, electrostatic, or hydrophobic interactions.24 The main interactions formed at low temperature in CS/PVA hydrogel are hydrogen bonds established between the same type of macromolecular chains or different ones through the hydroxyl groups along PVA chains and hydroxyl or amino groups of CS.30 The polymers with hydroxyl groups along chains promote the formation of a shield of water around macromolecules in aqueous solutions.54 By increasing the

4. CONCLUSIONS Chitosan/poly(vinyl alcohol) mixtures with different compositions in 0.2% and 2% acetic acid solutions were investigated at 37 °C by means of viscometry and refractive index measurements in order to establish the optimum conditions (acetic acid concentration, ratio between polymers) at which polymer− polymer interactions occur. A new method proposed by Wolf was applied to evaluate the viscometric parameters of the polymer mixtures in solution, and the estimated values were compared with those obtained by Huggins method. According to the obtained results, the two polymers are miscible in 2% acetic acid solution irrespective of poly(vinyl alcohol) content in the mixture, and in 0.2% acetic acid solution, the polymers show a high miscibility for the mixtures with a large amount of PVA. Mixtures with different polymer concentration and optimum CS/PVA composition (PVA weight fraction of 0.9) in 0.2% acetic acid solution have been subjected up to 10 freezing/ thawing cycles. The temperature effect on the viscoelastic properties of the CS/PVA mixture depends on the total polymer concentration and the number of freezing/thawing cycles. An increase of viscoelastic moduli of CS/PVA mixtures with polymer concentrations of 3% and 10% was evidenced at 11480

DOI: 10.1021/acs.iecr.5b03088 Ind. Eng. Chem. Res. 2015, 54, 11475−11482

Article

Industrial & Engineering Chemistry Research

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the gelation temperature which decreases as the number of cycles of freezing/thawing increases. CS/PVA mixture in 0.2% acetic acid solution with the PVA weight fraction of 0.9 can be considered as a potential candidate for developing injectable materials in medical applications. The number of freezing/ thawing cycles necessary to obtain the sol−gel transition at physiological temperature depends on the polymer total concentration in the polymers mixture. Thus, the mixture with 3% polymer requires more than three freeze/thaw cycles in contrast to the mixture with 10% polymer for which two cryogenic cycles are enough to obtain a material with sol−gel transition near 37 °C. After 10 freezing/thawing cycles, the CS/PVA mixtures revealed gel-like behavior. The viscous modulus was independent of polymer concentration, whereas the elastic modulus increased with an order of magnitude by increasing the total polymer concentration from 3% to 10%.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +40 232 217454. Fax: +40 232 211299. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS-UEFISCDI, project number PN-II-ID-PCE-2011-3-0199 (contract 300/2011).

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DOI: 10.1021/acs.iecr.5b03088 Ind. Eng. Chem. Res. 2015, 54, 11475−11482

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DOI: 10.1021/acs.iecr.5b03088 Ind. Eng. Chem. Res. 2015, 54, 11475−11482