Freeze–Thaw-Induced Gelation of Hyaluronan: Physical

Article pubs.acs.org/Macromolecules

Freeze−Thaw-Induced Gelation of Hyaluronan: Physical Cryostructuration Correlated with Intermolecular Associations and Molecular Conformation Zhixiang Cai, Fei Zhang, Yue Wei, and Hongbin Zhang* Advanced Rheology Institute, Department of Polymer Science and Engineering, School of Chemistry and Chemical Technology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China S Supporting Information *

ABSTRACT: Physically cross-linked hydrogels from hyaluronan (hyaluronic acid, HA) were prepared by a freeze−thaw technique at low pH. The effect of the freezing−thawing of HA solutions on the formation of physical cryogels is typical for the processes of noncovalent cryostructuration that takes the advantages of mild fabrication conditions and the absence of organic solvents and toxically cross-linking agents. The effects of processing steps (freezing time and number of freeze−thaw cycles), HA molecular weight (Mw), and the addition of typical polycarboxylic and polyhydric small molecules such as dicarboxylic acids and polyols on the formation of HA cryotropic hydrogels were investigated. Results verified that long freezing time and repeated freeze−thaw cycles benefited the alignment of polymer chains in the unfrozen liquid microphase, thereby promoting the formation of intermolecular aggregations and dense fibrillar network structures. High Mw of HA endowed the cryogel with strong mechanical strength. The influences of various small molecules on the cryogelation of HA revealed the different intermolecular association patterns in the gel network. Both succinic and glutaric acids participated in HA cryogelation, whereas oxalic, malic, and tartaric acids as well as some polyols (glycol, butanediol, and glycerol) inhibited the cryostructuration of HA. Hydrogen bonding and intermolecular interactions in acidic cryogels and in neutral cryogels obtained by in situ neutralizing the acidic cryogel were discussed at the molecular level in correlation with intermolecular associations and molecular conformation. A gelation mechanism for HA cryogel was proposed. In addition, experimental findings showed that the neutral HA cryogels possessed enhanced thermostability, resistance to acid decomposition, and enzyme degradation which are essentially important properties for biomaterials.

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treatment,6 postoperative adhesion prevention,7 wound dressing,8,9 scaffolding materials,10−13 drug delivery,14−16 and tissue repair and regeneration.17−20 However, most HA-based hydrogels are fabricated by chemical cross-linking, and in addition to native HA, various HA derivatives such as thiol-,21 dihydrazide-,22 and aldehydemodified HA23 have also been developed to prepare such chemically cross-linked hydrogels. In the chemical synthesis of HA hydrogels, the use of a cross-linking agent or organic solvent and the existence of reacting byproducts in the final hydrogels are inevitable. These problems likely impair biological compatibility in both short-term and long-term applications of HA, especially in the biomedical area. Physically cross-linked hydrogels, constructed by noncovalent interactions, such as hydrogen bonding, hydrophobic interactions, and ionic interactions,24−27 can be used to address existing problems of chemical gels. Although HA is known to be a nongelling polysaccharide, physical gels of HA can be

INTRODUCTION Hyaluronic acid (HA) or hyaluronan is one of the most important and ubiquitous glycosaminoglycans in vertebrate bodies. HA consists of unbranched molecular chains comprising repeating disaccharide units of (1 → 4)-β-D-glucuronic acid and (1 → 3)-β-D-N-acetylglucosamine. HA plays an important role in various biological processes, such as cellular signaling, wound repair, morphogenesis, and matrix organization.1 Recently HA has been used to modify various nanomaterials such as gold, silver, and magnetic nanoparticles, carbon, and mesoporous materials to enhance their distribution in specific cells and tissues, and these HA-functionalized nanomaterials are used for biotechnological and biomedical applications and as nanocarriers in drug delivery, contrast agents in molecular imaging, and diagnostic agents in cancer therapy.2,3 Besides its important physiological functions, the viscoelasticity of HA aqueous solution is also of equal importance.4 Given the inherent biocompatibility, biodegradability, and nonimmunogenicity of HA, it is a very promising material for the construction of hydrogels with desired morphology, stiffness, and bioactivity.5 HA-based hydrogels hold realized and potential applications in many fields, such as in osteoarthritis © XXXX American Chemical Society

Received: June 14, 2017 Revised: July 28, 2017

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DOI: 10.1021/acs.macromol.7b01264 Macromolecules XXXX, XXX, XXX−XXX

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Hyaluronidase (300 U/mg) and malic acid were obtained from Aladdin Reagent Co., Ltd. (China). All other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (China), and of analytical grade. Preparation of Acidic and Neutral HA Cryogels. Preparation of Acidic HA Cryogels. Acidic HA cryogels were prepared as previously described.24 HA solutions (10 mg/mL) were prepared by weighing appropriate amounts of polysaccharide in vials and adding distilled water. The vials were immediately sealed and gently stirred on a roller mixer until the polymer was completely solubilized. Various small-molecule substances were added at 0.05 M to some HA solutions and stirred to form 10 mg/mL HA. The pH of the solutions with or without additives was adjusted to 1.5 using 1 M HCl, and the acidified solutions were centrifuged at 10 000 rpm for 30 min to remove possibly trapped air bubbles. About 5 mL of the resulting acidified solutions was then transferred into 10 mL beakers. These beakers were tightly sealed with parafilms and subsequently stored at −20 °C for a required duration. No sublimation was found during the freezing process, and the weight remained constant for all frozen samples. The frozen acidified solutions were then allowed to thaw at 5 °C for 6 h until weak cryotropic gels were obtained. Typically, one freeze−thaw cycle consisted of 3 days of freezing and 6 h of thawing. Preparation of Neutral HA Cryogels. Acidic cryogels were extracted from acidic water by suction filtering the gel using a sintered glass filter. The sponge-like cryotropic gels were immersed in PBS solution (pH 7) for 6 h to neutralize the acid, taken out, and immersed in a large amount of distilled water for 30 min. The neutralized hydrogels were first frozen at −20 °C and then transferred to a freezedryer (LGJ-10D, Beijing Sihuan Scientific Ltd., China) for drying at −60 °C at least 24 h. The obtained dried gel samples were used in subsequent XRD and FTIR experiments. Dynamic Rheometry. The rheological measurements were performed on a rotational rheometer ARG2 (TA Instruments, USA) with a parallel plate geometry (40 mm in diameter and 1 mm in gap). The temperature was regulated by a circulating water bath and a Peltier system. For the acidic cryogels, the clear liquid phase was removed and water was wiped off from the surface using a filter paper, and then the in situ gelled samples were used for rheological measurements. For the neutral gels, dried samples were accurately weighed, mixed with distilled water, and kept in swollen state for at least 48 h at 25 °C to obtain hydrogels at certain concentrations. Before measument, a thin layer of low-viscosity silicone oil was placed on the peripheral surface of the sample held between the plates to avoid the evaporation of water from the sample. The measurements of the dynamic rheological properties of the samples were carried out within the linear viscoelastic region at 25 °C. Determination of the Gel-Fraction Yield. The formed acidic cryogel was insoluble and can thus be separated from the solution by filtration. The aliquot of the left transparent liquid layer was neutralized using several microliters of 1 M NaOH solution. The neutral solution was passed through a membrane filter of 0.22 μm and injected into a GPC system to determine the HA concentration with an RI detector. The gel-fraction yield was calculated according to Damshkaln et al.44 following the formula [(C0 − Ct)/C0] × 100%, where C0 is the initial HA concentration in the solution before cryogenic treatment and Ct is the polysaccharide concentration in the clear liquid layer of the sample taken after freeze−thaw treatment. Optical Microscopy. Optical microscopy observations were made with a Leica DMLP polarizing optical microscope (Leica Microsystems GmbH, Germany). An automatic hot stage (Linkam TH960, Linkam Scientific Instruments Ltd., UK) with a precision of ±0.1 °C was used to observe the samples at elevated temperatures at a rate of 5 °C/min. Samples were sandwiched between precleaned glasses during observation. X-ray Diffraction (XRD). XRD patterns were obtained using a D/ max-2200/PC X-ray diffractometer (Rigaku Corporation, Japan) with Cu Kα rays. The voltage and current were 40 kV and 20 mA, respectively. The scan rate was 4°/min, and the 2θ scan range was from 5° to 50°.

prepared by a freeze−thaw method at low pH. Such a cryotropic HA gel (cryogel) resulting from cryogelation that occurs upon cryogenic treatment of the initial solution was first reported by Okamoto and Miyoshi28 and is produced by onetime or repeated freeze−thaw cycles. It is known that as the polymeric concentration is increased by conversion of the solvent of water to ice resulting in the occurrence of a phase separation, the forced alignment of polymer chains in polymerrich phases provides a mechanism for the formation of side-byside associations that remain intact on thawing, acting as the junction zones of the hydrogel.29,30 HA cryogel has since been used in a broad range of medical applications.28,31,32 Apart from HA, many other natural polymers such as xanthan gum, βglucan, locust bean gum, starch, carboxymethyl cellulose, and carboxymethylated Curdlan,27,33−37 as well as some synthetic polymers, typically poly(vinyl alcohol) (PVA),38 can reportedly form cryogels by freezing and thawing their aqueous solutions. Initial factors affecting cryogel formation (such as the molecular size of the material) and those related to processing steps (such as freezing time, number of freeze−thaw cycles, freezing or thawing temperatures, etc.) have been investigated, especially for PVA cryogels.39,40 However, despite published works on HA cryogels,24,28,41 cryogelation of HA and some key factors influencing the gelation and physicochemical properties of HA cryogels (e.g., molecular size, processing steps, and molecular conformation) have not been fully described. Moreover, few studies have explored the effect of small molecules rich in the same groups of −COOH and −OH as in HA chains (such as dicarboxylic acids and polyols) on the gelation and viscoelasticity of HA cryogel. HA differs from PVA in that PVA only has hydroxyl groups on the backbone of the polymer chain. To further understand the gelation mechanism of HA and manipulate the physicochemical properties of HA cryogels, the effects of freezing time, number of freeze−thaw cycles, and molecular size of HA, as well as some small-molecule additives of dicarboxylic acids and polyols, on the cryogelation and the final properties of HA cryogels were investigated. A gelling mechanism and a molecular interaction pattern were also proposed for HA cryogel.

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EXPERIMENTAL SECTION

Materials. HA in the form of sodium salt that originated from bacterial fermentation were purchased from Freda Biochem Co. Ltd. (China). By using size exclusion chromatography (SEC)−light scattering, molecular characterization was performed based on our previous work. 42 In SEC measurements, the samples were characterized using a Viscotek TDA 305 instrument (Malvern Instruments, USA) equipped with two Viscotek A6000 M columns. The sample concentration was 0.5 mg/mL. The experimental conditions consisted of a mobile phase of 0.15 M NaNO3, a temperature of 30 °C, a flow rate of 0.7 mL/min, and an injection volume of 100 μL. The refractive index increment dn/dc was set as 0.144 mL/g. Data were collected and analyzed using OmniSEC software. Based on the SEC results, the average molecular weight (Mw), polydispersity index, intrinsic viscosity [η], hydrodynamic radius (Rh), and z-average radius of gyration (Rg) of the HA sample were 1.16 × 106, 1.96, 23.11 dL/g, 73.40, and 130.43 nm, respectively. HA with different molecular weights were prepared by ultrasonic degradation according to Chabreček et al.43 In a typical procedure, vials containing 10 mL of HA solution (10 mg/mL) in distilled water were ultrasonicated at 25 °C. Samples were withdrawn at defined periods and dried by lyophilization. Their molecular characteristics were determined by the same SEC method as described above. B

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Macromolecules FTIR Spectroscopy. FTIR spectra were recorded from 4400 to 400 cm−1 on a Spectrum 100 FTIR spectrometer (PerkinElmer, Inc., USA). The samples were fixed in KBr discs.

resulting in an inhomogeneous microstructure with polymerpoor and polymer-rich regions. With increased freezing time, the fibrillar structures and corresponding formed network became dense. Upon heating the gel from 25 to 90 °C at 5 °C/ min, the network became gradually sparse at elevated temperatures. However, the morphology differed at comparatively high temperatures for samples obtained by freezing for different days. The gel formed by freezing for 3 days melted and gradually changed to a homogeneous solution at 80 °C (Figure 1A), whereas the gels formed by freezing for 6 days (Figure 1B) and 12 days (Figure 1C) maintained their network architectures even at 90 °C until most of the water evaporated. This phenomenon was easily observed in the left network of the gel obtained by freezing for 12 days, which had a denser structure than that of the gel obtained by freezing for 6 days. Thus, the cryogel obtained by a longer freezing duration had a higher “melting” temperature and thus presented higher thermotolerance similar to physical cryogels obtained from PVA.48 This finding accorded with our previous optical microscopy and scanning electron microscopy (SEM) results.24 Therefore, the long freezing time positively contributed to the alignment of polymer chains in the unfrozen liquid microphase, thereby promoting the formation of aggregation that led to high thermotolerant cryogels. The drastic change for HA from a solution state to an intermolecular associated network by freeze−thaw treatment was also clearly identified by dynamic rheological measurements (Figure 2A,B). The mechanical behavior of the solution before freezing was typical of a polymer solution, i.e., apparent loss modulus G″ > apparent storage modulus G′ at lower frequencies, and both moduli increased with increasing frequency with a cross at a certain frequency. After freeze− thaw treatments for different freezing times or cycles, gradual transition of rheological behavior was observed from liquid-like to weak gel behavior (G′ > G″ and both apparent moduli almost independent of frequency, with a small difference of less than one logarithmic cycle). Both G′ and G″ of the resulting cryogel increased with increasing the freezing time at a fixed cycle number or, conversely, with increasing freeze−thaw cycles at a fixed freezing time. Only a slight difference in gel strength was found in the cryogels obtained by freezing for the same total time but with different freeze−thaw cycles within the freezing time (e.g., the strength of the gel obtained by 12 days of freezing before thawing was equal to that of the gel subjected to four cycles of freezing and thawing for 3 days). The gelfraction yields for the different freezing times are shown in Figure 2C, and the yields were found to increase gradually with increased freezing time. Over 80% of the HA solution changed to gel after 12 days of freezing. Either longer freezing duration or more freeze−thaw cycles led to a more concentrated unfrozen liquid microphase that benefited the formation of gel network and enhancement of gel mechanical property.24,36,48 A long freezing time or repeated freeze−thaw procedure was apparently conducive to the formation of associations among the alignment of HA chains, which remained intact upon thawing and acted as junction zones in the gel network as revealed by the microscopic images (Figure 1) and SEM images of our previous work.24 A similar influence of freezing time and number of freeze− thaw cycles on the cryogel strength was also observed in other physical cryogels, such as PVA,49 locust bean gum,36 and cereal β-glucan.33 Our data also agreed well with our previous preliminary work on HA24 and carboxymethylated Curdlan37

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RESULTS AND DISCUSSION Formation of Physically Cross-Linked Network in HA Cryogel with Various Process Parameters. HA cryogel was prepared by three main steps: (1) HA aqueous solution acidification, (2) freezing, and (3) thawing. In the precursorcontaining systems, the formation of ice crystals accompanying phase separation in the frozen state of the aqueous system has a good effect on gelling development. Therefore, related process parameters such as freezing time and freeze−thaw cycles are important in the formation and modulation of HA cryogel properties. In this study, the effects of freezing time, freeze− thaw cycle, and HA molecular size on the construction of gel network and further related enhanced properties involving the intermolecular associations in the in situ formed acidic HA cryogel were investigated. A fundamental feature of the cryogels is their heterogeneous structures that arise from the formation of ice crystals and the presence of unfrozen concentrated liquid microphase upon freezing.45 Optical microscopy has been approved as a useful method for the identification of microstructural features in a polymer hydrogel.24,46,47 Microscopic images of HA cryogel prepared by freezing for different days (Figure 1) indicated that many fibril-like aggregations were present in the cryogel,

Figure 1. Images of HA cryogels (prepared by freezing for 3 (A), 6 (B), and 12 days (C)) obtained by optical microscopy observation at different heating temperatures. Scale bar: 50 μm. C

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Figure 2. Dynamic rheological properties of HA obtained by different freezing days (0 to 12 days) with one final thawing (A) and different numbers of freeze−thaw cycle (one cycle refers to 3 days of freezing and then thawing) (B), gel-fraction yields of HA cryogels obtained by different freezing days (C), and polarizing microscopy image of HA cryogel obtained by 12 days of freezing or four freeze−thaw cycles (D).

cryogels. To identify possible ordered structures such as crystallines in HA cryogel, polarization was used to observe the network in detail. However, nothing was found in the polarizing microscope image even for cryogels frozen for 12 days or subjected to four freeze−thaw cycles (Figure 2D). This phenomenon of lack of crystalline structures was also confirmed by our following XRD experiments. This finding indicated that the crystalline structures in HA cryogel were tiny and also possibly lacked regularity and perfection, consistent with our previous work.24 The molecular size of polymer significantly affected the rheological properties of the solution, which reflected the state of entanglement or aggregation of the polymer chains in the solution.50,51 The relevant molecular parameters of the HA prepared by ultrasonic degradation are listed in Table S1 and Figure S1A. The steady shear viscosities of 10 mg/mL HA solutions with different molecular weights are shown in Figure S1B, which demonstrated shear-thinning behavior. However, the shear-thinning behavior of HA solutions gradually became insignificant with decreased Mw. Shear thinning is a well-known property of polymer solutions that feature a typical entangled network.52 The low viscosity and insignificant shear-thinning behavior of the HA (sample 5) with the lowest Mw indicated that these molecular chains were insufficient to entangle together. The aggregation or entanglement of polymer chains was generally accepted as a critical step in cryogel formation through the freeze−thaw process.45 From this viewpoint, the formation of the cryogels is closely related to the molecular size of HA in the initial solutions. Figure 3 shows the relationship between the molecular size and the gel-fraction yield at the

Figure 3. Gel-fraction yields of HA prepared by freezing for 6 and 12 days with different Mw.

same concentration of 10 mg/mL. The results indicate the existence of a critical Mw below which cryogels cannot be formed at the concentration of 10 mg/mL HA. Above this value, a higher Mw of HA appeared to be conducive to a higher gel-fraction yield, suggesting that at the same concentration a larger molecular size promoted the association of molecular chains as junction zones of network in the gel. However, the yields for higher molecular weights exhibited little difference from each other, indicating that polymer chain with high Mw has high contribution for network formation, but the increase of the contribution with Mw becomes small at the high-Mw region. This phenomenon indicated that long polymer chains had a limited contribution to construction of gel network, which has D

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The frequency dependences of viscoelasticity for acidified 10 mg/mL HA solutions with and without additional dicarboxylic acids were then compared, and the results are shown in Figure 4. Apart from a very slight decrease in shear modulus and viscosity, the rheological behavior of acidified HA solution in the presence of 0.05 M oxalic acid, succinic acid, and glutaric acid was similar to that of acidified HA solution without the additives. This observation confirmed that the addition of small-molecule additives had a negligible effect on the essential transient network of acidic HA solutions featured by its rheological properties. However, significant effects of various dicarboxylic acids on the strength of the cryogels were observed when they were prepared by freeze−thaw treatment. Figure 5A shows that in the presence of oxalic acid the cryogels were unable to form after freezing for 3 days (G′ < G″). On the other hand, Figure 5B shows that succinic acid and glutaric acid did not affect HA cryogel formation and the corresponding values of moduli and complex viscosities of the gels. These results indicated that while the addition of oxalic acid hindered the cryogelation of HA, both succinic acid and glutaric acid did not interfere with the formation of the HA network. Succinic acid and glutaric acid are dicarboxylic acids with two carboxylic groups on each end of a molecule and can form dimers through hydrogen bonding between the two carboxyl groups at pH values below pKa. The acidified HA solution had a pH of 1.5; thus, both carboxylate groups in succinic acid or glutaric acid were nearly protonated. Protonation of the carboxylate groups allowed them to participate in the formation of the threedimensional network of the gel through hydrogen bonding with the other protonated carboxylate groups in the HA chains. Therefore, the cryogel network was formed and stabilized either by the binding of HA chains with one another or by the binding of the two ends of small molecules with HA chains in the presence of either succinic acid or glutaric acid. By contrast, the pKa values of the two carboxyl groups in oxalic acid were 1.19 and 4.21, respectively. At pH 1.5, only one carboxylate group of oxalic acid was protonated and can bind to HA through hydrogen bonding. The carboxylate group on the other end of oxalic acid hindered the formation of such an association. Therefore, the impairing effect of the addition of oxalic acid may occur in a similar mechanism with salts NaCl on the cryogel of HA24 and locust bean gum.34 Association between HA chains was blocked when the binding sites were occupied by the unprotonated end of the oxalic acid molecules. In addition, the rigidity of the small molecules also influenced the three-dimensional architecture of the network. The rodlike and short oxalic acid molecules may not be conducive to the formation of the network. To further demonstrate the active role of carboxyl groups in the formation of HA cryogels, malic acid and tartaric acid were added to acidified 10 mg/mL HA solutions, and their corresponding effects on the cryogelation of HA were observed. Table 2 shows the molecular structures of malic acid and tartaric acid, which have one and two additional hydroxyl groups in the backbone, respectively, compared with succinic acid. The differences in their molecular structures led to distinct behaviors of HA solutions after freeze−thaw treatment. Figure 6A shows the frequency dependence of apparent G′ and G″ of the acidified HA solutions after freezing and thawing. The aqueous system containing 0.05 M succinic acid or 0.05 M glutaric acid formed gels with rheological behavior similar to cryogels formed from HA alone (Figure 5). However, 10 mg/ mL HA solutions containing 0.05 M malic acid or tartaric acid

also been observed for locust bean gum cryogels whose chain length insignificantly affected mechanical properties within a certain high Mw range.53 The longer molecular chains with more entangled points in starting solution can also benefit to construct denser network after freeze−thaw treatment reflected by the stronger mechanical properties of the formed gels with the similar trend of yields (Figure S2). The same trend of gel strength and yields with Mw revealed that a high molecular size resulted in better association of molecular chains, implying that the formation of HA cryogels proceeded in a manner similar to that of physical PVA cryogels.38 For a HA cryogel, its formation and physical property depend on the regimes in the cryogenic treatment (temperature and duration of freezing, temperature, time and rate of thawing, the number of refreezing cycles) as well as pH, size, and concentration of HA and so on. The main procedures of the preparation of HA cryogels include acidifying HA solution and then freeze−thaw of the acid HA solution at proper subzero temperature by once or repeating freeze−thaw. It should be noted that concerning the effect of process factors on the cryogelation of HA solution, apart from the above-mentioned freezing time and number of freeze−thaw cycles, the influence of cooling rate and defrosting rate should also play an important role in the cryogelation of HA solutions and the properties of the resulting HA cryogels, similar to the physical cryogels obtained from PVA.54 Participation and Perturbation of Probing Small Molecules in HA Physical Cryogel. Hydrogen bonding among −COOH and NHCOCH3 groups in HA chains instead of −OH groups was previously proposed to play a crucial role in cryogelation.24 To gain further understanding on the intermolecular interactions in the cryogels, small molecules such as dicarboxylic acids and polyols (shown in Table 2) were chosen as probes, and their corresponding influences on the gelation and viscoelasticity of the in situ prepared acidic HA cryogel were investigated. Table 2. Molecular Formulas and pKa Values of SmallMolecule Additives

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Figure 4. Frequency dependence of G′ and G″ (A) and steady shear viscosity (B) for acidified 10 mg/mL HA solution (pH 1.5) with and without 0.05 M oxalic acid, succinic acid, and glutaric acid.

Figure 5. Frequency dependence of G′ and G″ (A) and complex viscosity (B) at 25 °C for the HA cryogels obtained by freezing the acidified 10 mg/ mL HA solution with and without 0.05 M oxalic acid, succinic acid, and glutaric acid for 3 days.

Figure 6. Frequency dependence of G′ and G″ for HA cryotropic gels obtained by freezing acidified 10 mg/mL HA solution with and without 0.05 M malic acid and tartaric acid (A) and 0.05 M polyols (glycol, butanediol, and glycerol) (B) for 3 days freezing.

neutral conditions, which signified that the −OH groups did not significantly contribute to the gelation process. Intramolecular hydrogen bonding among −COOH and −OH groups in the small molecules may have also prevented the −COOH groups from contributing to the formation of the gel network. The role of −OH groups in the formation of HA cryogel were evaluated by the influence of various polyols on the formation of HA cryogel. Figure 6B shows the rheological behaviors of acidified aqueous systems with three kinds of polyol additives (glycol, butanediol, and glycerol) after freeze− thaw treatment. The solution-like behaviors of these systems

after the same freeze−thaw process resulted in solution-like rheological behavior, with G″ > G′ and both apparent moduli values increasing with increased frequency. Therefore, malic acid or tartaric acid interfered with the formation of the gel network by inhibiting the effective association of HA chains, which can be explained by the presence of hydroxyl groups. This phenomenon also confirmed the previous conclusion that the −OH groups did not significantly affect the formation of intermolecular hydrogen bonds in the formation of HA cryogel24 or carboxymethylated Curdlan cryogel.37 Although some polysaccharides with carboxylated groups were structurally rich in −OH groups, they cannot gel like PVA under F

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Figure 7. (A) FTIR spectra, (B) frequency dependence of G′ and G″, (C) optical microscopy images for the acidic HA cryogel, and (D) and the corresponding neutral HA cryogel.

stretching of −COOH, which indicated the existence of −COOH groups in acidic HA and acidic HA gel. The two bands at 1651 and 1558 cm−1 were assigned to amides I and II, respectively, which cannot be observed for native HA powders. In the protonated form of HA, the amide I and II bands were clearly resolved, in which the asymmetric stretching of carboxylate group greatly decreased.55 This observation confirmed that most of the carboxyl groups were protonated in the acidic HA gel. The peak at 1743 cm−1 corresponding to the asymmetrical CO stretching of −COOH disappeared in the neutral HA cryogel, indicating that most of the carboxyl groups were unprotonated in the neutral gel. In addition, the absorption peaks around at about 600 cm−1 were due to residual water in HA and HA cryogels.55,56 Figure 7B compares the rheological properties of acidic and neutral cryogels. The neutral gel displayed typical characteristics of a weak gel. The acidic HA cryogel showed stronger mechanical properties than the neutral gel, and its apparent moduli parameters showed little dependence on frequency, indicating a denser gel network. Figures 7C and 7D are microscopic images showing changes in microstructures. The density of the network composed of entangled bundle-like strands decreased as the acidic cryogel was neutralized, which provided clues to elucidate the weaker mechanical strength of the neutral gel. The neutralization of −COOH groups disrupted hydrogen bonds between protonated carboxyl groups in HA chains, which led to a dramatic reduction of the network density. These results demonstrated that hydrogen bonding between −COOH groups indeed played an important role in stabilizing the gel structure of acidic cryogels. The experimental results in Figure 7 also implied that other interactions that can maintain the weak gel network and even the hydrogen bonds between −COOH groups were broken after neutralization. To investigate the likelihood of interaction model of HA molecules in neutral weak gels, FTIR and XRD experiments

indicated that small amounts of these molecules containing multiple hydroxyl groups blocked the formation of gel network. Considering the low concentration of these polyols in the systems (only 0.2−0.5% w/w), their effects as cosolvent on the aqueous system can be ignored. Therefore, the inhibiting influence on the formation of HA cryogel may be due to the hindering effect of hydroxyl groups in these small molecules. The polyols may block hydrogen bonding between −COOH groups. The disruptive action of polyols, as well as malic acid and tartaric acid, on the formation of cryogel suggested that hydrogen bond interactions among −OH groups in the HA cryogel were weaker than interactions involving −COOH or −NHCOCH3 groups. These results were consistent with our previous work on HA24 and carboxymethylated Curdlan cryogels.37 Gelation Mechanism with Association Patterns of HA Chains within Acidic and Neutral Cryogels. Although acidification of HA solution to low pH was required for the gelation of HA solution, the sample from neutralizing the in situ formed HA cryogel still showed weak gel characteristic. The neutralization may break the proposed hydrogen bonding among −COOH groups, which is regarded to be a dominant driving force for formation of acidic HA cryogels. Thus, comparing the properties of acidic and neutral cryogels by in situ neutralization of the acidic cryogel was necessary and provided deeper understanding of the molecular interaction patterns in the gel. FTIR measurements for the acidic and neutral gels were performed to monitor changes in the protonation or dissociation states of the carboxyl groups as well as other functional groups in the HA chain. Figure 7A compares the FTIR spectra of native HA, acidic HA, acidic cryogels, and neutral cryogels. The acidic HA and acidic HA gel exhibited different features compared to native HA and neutral HA gel. The peak at 1743 cm−1 was assigned to the asymmetrical CO G

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Figure 8. FTIR spectra of native HA powders and freeze-dried neutral HA cryogels (A) prepared by freezing for different days (B) and partial spectra at low wavenumbers.

the lack of crystalline structure. However, the gel frozen for 3 days had a very broad peak from ca. 10° to 25°, and the other two gels frozen for 6 and 12 days both showed one broad peak at ca. 20°. In addition, several additional sharp peaks at 2θ of ca. 22.3, 23.1, 26.0, 31.02, and 31.87 were found in the spectra of the gels, which well agreed with the typical XRD peaks for Na2HPO4. This observation was due to the neutralization of the acidic cryogel in PBS that contained a higher amount of Na2HPO4. Some phosphates were left in the final cryogel. XRD patterns of HA in previous reports reflect various helical conformations, including 2-, 3-, and 4-fold single and double helices depending on conditions, such as hydration, pH, and cation.66,67 Differences in XRD patterns between native HA powder and HA neutral cryogel (Figure 10) may reflect conformational changes in HA molecules. According to molecular dynamics modeling,58,68,69 the rotational angle of the β(1 → 3) bond in HA chain had only one energetic minimum. The β(1 → 4) bond had two comparable energy minima (two favored dynamic states) and easily adopted one or the other dynamic state depending on conditions and time. If all β(1 → 4) bonds in the chain resided in the first minimum, the chain would adopt a flat-strand conformation. If all β(1 → 4) bonds resided in the second minimum, a helical chain conformation (left-handed for folded helix) would form. The circular dichroism spectra of HA solution at neutral pH suggested that the polymer chains existed in two main conformations: the left-handed multiple-fold helix and the flat strand with a 2-fold helix70,71 The two distinct diffraction peaks for native HA powder observed in Figure 10 may correspond to these two dominant conformations. In acidic HA solution especially at a low pH of ca. 1−2, the HA chain preferred the flat strand with 2-fold helix conformation.67 Scott and Heatley59 also revealed that the flat-strand conformation forms aggregates in antiparallel manner and is mainly stabilized by hydrogen bonding between −COOH and −NHCOCH3 groups on the HA backbone. The concentrated HA in the unfrozen phase may promote the formation of flat strands that are probably stacked and stabilized with one another, thereby forcing more chains to assume a flat-strand conformation. Therefore, the peak at 10° gradually disappeared, and only one at peak 20° was observed for the cryogel by freezing for 6 days or longer, indicating the flat-strand conformation existed in the gel. The flat-strand conformation transition promoted the formation of the three-dimensional network.

were performed. The FTIR spectra of neutral HA cryogels with different freezing times are shown in Figure 8. The wide peak at 3419 cm−1 corresponded to the stretching vibration of −OH groups. No obvious shift in this peak was observed for all neutral HA cryogels, indicating that the hydroxyl groups participated less in hydrogen bonding. However, obvious differences in bands from 1420 to 1380 cm−1 were observed between native HA powder and neutral gels. These bands were assigned to the symmetrical C−O stretching of −COO−.24,55,56 Two peaks at 1415 and 1385 cm−1, which showed similar absorbance intensities, were observed for native HA powder. However, the spectra of neutral gels showed a sharp peak at around 1385 cm−1, and the intensity of this single peak strengthened with prolonged freezing time (Figure 8B). Previous reports have shown that in the HA infrared spectrum the symmetrical C−O stretching of −COO− at 1415 cm−1 indicates a carboxyl −COO− group participating in zero or only one hydrogen bond and that the peak at around 1385 cm−1 indicates a carboxyl −COO− group involved in one or more hydrogen bonds.56 Our results indicated that the hydrogen bonding caused by −COO− groups may be important for stabilizing the network in the neutral gel. The little, if any, contribution of hydroxyl groups to the formation of the gel network24,37 may suggest that other functional groups such as −NHCOCH3 can form hydrogen bonds with −COO− groups and thus be involved in stabilizing the gel network. In fact, a series of studies on the conformation and intermolecular interactions of HA have revealed that hydrogen bonding between −COO− and −NHCOCH3, whether intramolecular or intermolecular, is one of the factors affecting the helical conformation of HA.57−62 Therefore, intermolecular hydrogen bonding among −COO− and −NHCOCH3 groups may be an important driving force for the formation of HA neutral gel. Figure 9 shows the proposed hydrogen-bonding interaction mechanisms. XRD experiments were also carried out to identify changes in conformation or crystallinity of polymer chains in native HA powder and neutral HA cryogels. Over the past decades, a number of publications made clear that there were no microcrystalline zones in HA.63,64 Figure 10 shows the XRD pattern of native HA powder of typically poor crystalline polymer with broad peaks at 2θ of ca. 10° and 20°, which well accorded with the XRD pattern of Lee et al.65 in which the low crystallinity is indicated by a low-intensity peak. The peaks of all three gels were also very broad and flat, strongly indicating H

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Figure 9. Proposed intermolecular interaction models in HA: (A) acidic cryogels, (B) neutral cryogels, and (C) cryogels with dicarboxylic acid additives.

by stronger the mechanical strength (Figure 2 and Figure S2) and better antiacid and antienzyme degradation properties (Figures S3 and S4). The driving force for the association was the hydrogen bonding among −COOH and −NHCOCH3 groups located in the HA backbone. After thawing at a positive temperature, the acidic HA cryogel was formed, in which the hydrogen bonds between protonated carboxylic groups were dominated by intermolecular interactions stabilizing the network. Upon neutralization, the carboxyl groups became ionized, and the hydrogen bonds between protonated carboxylate groups were broken because of the ionization of carboxyl groups. Therefore, in neutral cryogels, hydrogen bonds involving −COO− and −NHCOCH3 groups became the main force stabilizing the network. Considering the occurrence of very broad peaks in the XRD pattern and noncrystalline feature

Figure 11 shows the proposed scheme of the formation of HA cryogel with possible intermolecular interactions (Figure 9) for either acidic or neutral gels. For native HA in solution, the polyanionic chains behaved like “stiff” random coils. Martens et al.70 suggested that two kinds of segments mainly coexisted with different conformations: multiple-fold helical segment and flat-strand segment. Upon acidification, and subsequent screening of intermolecular and intramolecular electrostatic repulsion by discharging carboxylate groups in HA, the HA chains were found to adopt the flat-strand conformation and formed junction knots. In the freezing process for cryogel fabrication, more helical parts of the chain were forced to assume the flatstrand conformation by associating with one another in the concentrated unfrozen phase. Longer freezing time or more freeze−thaw cycles as well as larger molecular size can benefit the process, giving the stronger and more stable gels reflected I

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and the subsequent formation of the three-dimensional network. The prepared HA cryogels formed a fibrillar network in which HA chains were associated into bundles. Although the contribution of the crystallization of HA was not obvious in the cryogel, gel formation was essentially related to the HA conformation changes and the molecular aggregations driven by the hydrogen bonding among −COOH and −NHCOCH3 groups. Hydrogen bonding between −COOH dominated the intermolecular interactions stabilizing the network of HA acidic cryogels, whereas hydrogen bonding involving −COO− and −NHCOCH3 groups was the main driving force stabilizing the network in neutral cryogels. All experimental results strongly suggested that the hydroxyl groups of HA did not significantly contribute to hydrogen bonding in the gel network in both acidic and neutral cryogels.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b01264. Figures S1−S4 and Table S1 (PDF)

Figure 10. X-ray diffraction spectra of native HA powder and freezedried neutral HA cryogels with different freezing times.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel +86 21 54745005 (H.Z.). ORCID

Hongbin Zhang: 0000-0002-4419-4818 Notes

The authors declare no competing financial interest.

Figure 11. Scheme of the molecular states of HA chains during the preparation of HA cryogel.

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ACKNOWLEDGMENTS The authors are thankful for the financial support for this work from the National Natural Science Foundation of China (Grants 21074071 and 21274090).

of HA molecules, very few ordered crystalline structures, if any, contributed to gel formation. Compared with PVA cryogels, visibly no regular and perfect crystalline structures formed in the HA cryogel, as evidenced by the polarizing microscopic image in Figure 1D and XRD pattern (Figure 10). This observation was due to the existence of diversified, dispersed hydrogen bonding among many HA chains (Figure 9) in the HA cryogel, whereas only regular hydrogen bonding between −OH groups existed in the PVA cryogel, resulting in obvious crystallization of PVA chains with high regularity and perfection.

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REFERENCES

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CONCLUSIONS HA cryogelation is a physical cryostructuration process. In this work, it was shown that the physicochemical properties of the resulting cryogels were significantly influenced by freezing time, number of freeze−thaw cycles, Mw of HA, and addition of small molecules such as dicarboxylic acids and polyols. Long freezing times, repeated freeze−thaw cycles, and high Mw of HA and dicarboxylic acids (succinic acid or glutaric acid) enhanced the mechanical strength of the cryogels whereas the addition of polyols such as glycol, butanediol, and glycerol inhibited gelation. The existence of physical cross-links provided considerable thermostability to the cryogels and high resistance to acid decomposition and enzyme degradation, which are important properties of biomaterials. Protonation of the HA polyanion at appropriately low pH values is a prerequisite for the association of multiple interchain hydrogen bonding among −COOH and −NHCOCH3 groups J

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