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Sep 21, 2017 - hyaluronic acid-gallol hydrogels, allowing this hydrogel system to be injectable. ... tongue is the origin of the astringent taste.3−...
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Gallol-Rich Hyaluronic Acid Hydrogels: Shear-Thinning, Protein Accumulation against Concentration Gradients, and DegradationResistant Properties Mikyung Shin*,† and Haeshin Lee*,† †

Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), 291 University Road, Daejeon 34141, Republic of Korea S Supporting Information *

ABSTRACT: We report the multifunctionality of a small adhesive functional group called gallol (three hydroxyls attached to benzene), which is the ubiquitous moiety found in many vegetables and fruits. First, the chemical tethering of gallols to a polysaccharide backbone and the addition of another gallol-rich compound, oligo-epigallocatechin gallate, result in the spontaneous gelation of the hyaluronic acid-gallol, and the cross-linking is due to the extensive level of hydrogen bond formations from both gallol-to-gallol and gallol-tohyaluronic acid. Second, we found that the gallol-involved crosslinking is reversible, resulting in a shear-thinning effect of the hyaluronic acid-gallol hydrogels, allowing this hydrogel system to be injectable. Third, due to gallol’s superior ability to bind proteins via noncovalent interactions, the hyaluronic acid-gallol hydrogels exhibit spontaneous loading of proteins from a buffer solution to the hydrogel inside against the concentration gradient (i.e., active entrapment phenomenon). By simply dipping the gels into a protein-containing solution (270 μg/mL), approximately 93% of the total proteins is actively entrapped into the gels. Furthermore, the protein affinity of the gallols is useful for physically immobilizing the degradation enzyme, hyaluronidase, to prevent the rapid, uncontrolled degradation of the gallol-rich hyaluronic acid gels.

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INTRODUCTION Gallol, the well-known phenolic moiety with a chemical structure of three hydroxyls attached to benzene, is a nearly ubiquitous functional group found in flavonoids and polyphenols in plants.1,2 In research, the field with the longest history of gallol studies is food science. The lubrication failure caused by the coating of gallol-containing molecules on our tongue is the origin of the astringent taste.3−5 This failure results from the binding between surface-displayed lubricating macromolecules called mucins and gallol-containing polyphenols such as tannic acid.6,7 In general, the gallol moiety exhibits robust interactions, particularly with a variety of proteins via noncovalent bonds, such as hydrogen bonds and hydrophobic interactions. The study of these robust protein binding properties has been reported as the preparation of stable, self-assembled nanoparticles using the intermolecular interactions of oligo-epigallocatechin gallate (OEGCG) with herceptin and their capabilities to treat cancers.8 The gallolrich OEGCG’s protein binding showed nearly irreversible characteristics, resulting in stable and long-term in vivo circulation targeted to cancer tissues. In addition to proteins, the representative gallol-rich molecule, tannic acid, binds to the phosphodiester backbone of DNA by hydrogen bonds, producing DNA/tannic acid hydrogels.9 Thus, the gallol moiety shows unique characteristics which binds to nearly all biomacromolecules of peptides, proteins, and DNA (or RNA). © 2017 American Chemical Society

Another unique property of gallol moieties is their coordination with transition metals,10 particularly with Fe ions.11,12 In fact, most studies related to gallol-containing molecules have focused on gallol−metal interactions similarly with previous concepts of preparing reversible hydrogels utilizing boronate-catechol complexation13 and imidazolemetal binding.14 The landmark study by Caruso et al. showed pH-dependent gallol-Fe(III)-gallol coordination in which the adhesive coating properties of gallols in tannic acid connected by Fe(III) ions demonstrated virtually universal organic thinlayers now called a metal-phenol network formed on organic/ inorganic templates.11 Furthermore, the interactions between a gallol-tethered cationic polymer and metal ions resulted in selfhealing hydrogels.15 The self-healing characteristic originates from the pH-dependent, reversible interaction between gallol and Fe(III). Another transition metal, vanadyl ion V(III, IV, V), also strongly binds to a gallol functional group. Similarly, gallolV-gallol coordination has also resulted in self-healing hydrogels inspired by a tunicate vacuolar or cellular membrane.16−18 Until now, hydrogels prepared by gallol-containing polymers utilized ether by metal-gallol coordination or by direct gallol-togallol covalent linkages at a basic condition (∼pH 8).15−18 Thus, studies of hydrogels fabricated by utilizing only Received: June 11, 2017 Revised: September 20, 2017 Published: September 21, 2017 8211

DOI: 10.1021/acs.chemmater.7b02267 Chem. Mater. 2017, 29, 8211−8220

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Chemistry of Materials

the 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC; TCISU, Japan)/N-hydroxysuccinimide (NHS; Sigma-Aldrich, U.S.A.) coupling reaction. In detail, HA (0.5 wt %) was dissolved in deionized water (DDW). EDC (126 mg) was added to HA solution (10 mL) and activated for 10 min, after which NHS (152 mg) was also added to the HA/EDC solution. After 20 min, 5-hydroxydopamine hydrochloride (4-equivalent to carboxyl groups of HA) was added to the solution. The reaction was carried out overnight. The unreacted 5hydroxydopamine and EDC were removed by dialysis (MWCO = 3500 Da, regenerated cellulose membrane) in 1 M NaCl (pH 5.3) at 4 °C for 3 days. Finally, the sample was freeze-dried. To determine the degree of substitution (DOS %) of the conjugated gallol moieties on the HA backbone, ultraviolet−visible (UV−vis) spectroscopy (Hewlett-Packard 8453, U.S.A.), proton nuclear magnetic resonance spectroscopy (1H NMR; Bruker Avance 300 MHz, U.K.), and Fourier transform infrared (FT-IR; Agilent Technologies, Cary 600 series, U.S.A.) spectroscopy were used. In the UV−vis spectrum, the maximum absorption appeared at 275 nm for HA-Ga, from which the DOS (%) was determined using a pyrogallol standard curve established in the concentration range from 3.75 to 60 μg/mL. For the 1 H NMR analysis, the HA-Ga (10 mg/mL) was dissolved in deuterium oxide (D2O; Cambridge Isotope Lab. Inc., U.S.A.). Second, OEGCG was synthesized and characterized using a previously reported method.8 In detail, EGCG (46 mg) was dissolved in the mixture solvent of DDW/dimethyl sulfoxide (DMSO)/acetic acid (2.8/0.376/0.045 mL, total = 3.221 mL). The oligomerization of EGCG was started with the addition of acetaldehyde (0.5 mL, SigmaAldrich, U.S.A.). The reaction was performed for 48 h, and the sample was dialyzed using the regenerated cellulose membrane (MWCO = 1000 Da) in methanol (2 L) for 2 days and in DDW (1 L) for the subsequent 1 day. The residual solution was finally lyophilized. For the characterization of OEGCG, UV−vis, 1H NMR, and FT-IR spectroscopy and size-exclusion chromatography (SEC; Agilent 1200, U.S.A.) were also used, similarly to in the case of HA-Ga. For the UV−vis spectrum analysis, the change in the maximum absorption wavelength of OEGCG (10 mg/mL) was evaluated. In the 1H NMR spectrum, the proton peaks of OEGCG newly formed by the oligomerization were assigned in DMSO-d6. In addition, in the SEC study, 2-(Nmorpholino) ethanesulfonic acid (MES buffer, 10 mM, Sigma-Aldrich, U.S.A.) was used as an eluent at a flow rate of 0.5 mL/min. The retention time of OEGCG (250 μg) was analyzed using a refractive index (RI) detector, and the molecular weight of OEGCG was calculated using a standard curve established by mPEG-NH2 (5, 10, and 30 kDa, 500 μg): Log (MW) = −0.10556x + 6.3388 (x = retention time, MW = molecular weight (Da)). Preparation of Gallol-Rich Hydrogels Using HA-Ga and OEGCG. The gallol-rich hydrogels were prepared by simply mixing HA-Ga (2 wt % dissolved in DDW) and OEGCG solutions at a volume ratio of 3:1 (HA-Ga:OEGCG). As a function of the OEGCG concentration (0.5, 2, or 8 wt % in dissolved 10% ethanol), the gelation was evaluated using a rotating rheometer equipped with a temperature controller (Bohlin Advanced Rheometer, Malvern Instruments, U.K.). The samples were loaded on the 20 mm parallel plate, and the elastic/viscous moduli (G′ or G′′) were measured over the frequency range from 0.1 to 10 Hz under a constant stress of 10 Pa at room temperature. In addition, the gelation using HA-Ga (2 wt %) and other polyphenol compounds, tannic acid (8 wt %) and punicalagin (8 wt %), was performed. The volume ratio between HA-Ga and either tannic acid or punicalagin was 3:1. To demonstrate transparency of the HA-Ga/OEGCG gels, the bulk gels with thickness of approximately 3 mm (total volume = 400 μL) were layered with a background of “KAIST” text. The cross-sectional structure of the lyophilized gels were analyzed by scanning electron microscopy (SEM; Hitachi S-4800 FE-SEM, Japan). Rheological Characteristics for the Gallol-Rich Hydrogels and the Injectability Test. The rheological properties of the HA-Ga were analyzed varying the OEGCG concentration of 2 or 8 wt % for a gel state and 0.5 wt % for a sol sate at room temperature, using a rotating rheometer equipped with a temperature controller (Bohlin Advanced Rheometer, Malvern Instruments, U.K.). After the samples

noncovalent interactions mediated only by gallol groups have not been reported. Our main focus is to prepare a galloltethered polymer, which forms interpolymeric networks established by the gallol-mediated noncovalent interactions and uncovers unprecedented unique properties of the hydrogels. In a broad perspective considering reversible hydrogels, the use of a single species gallol-to-gallol cross-linking chemistry provides several advantages. Both shear-thickening as well as shear-thinning properties can be observed depending on a shear rate and local concentrations of components. The simplified cross-linking chemistry of gallol tethering can be performed with ease comparing other reversible pairings.19−21 In other words, most gels utilize two or more types of crosslinkers for complementary “pairing”. For example, metal/metal ion incorporated system metal−ligand pairing, such as zinc(II)/ copper-porthyrin or plantinum(Pt)-pyridyl complex.19 It is also true for supramolecular gelation20,21 by host−guest interactions, for example, cyclodextrin/cucurbit[8]uril or hydrophobic guest molecules. The gelation is observed only when the paring molecules bind each other. Herein, we report that the hydrogels established using only reversible gallol-mediated interactions between gallol-tethered hyaluronic acid (HA-Ga) and a gallol-rich oligomeric crosslinker (OEGCG). The introduction of the gallol moiety into an HA hydrogel led to the exhibition of interesting properties both during and after injections. First, the HA-Ga/OEGCG hydrogel exhibited a shear-thinning effect, which increased injectability. The gallol-mediated shear-thinning hydrogel is the first such material reported. Previous cross-linking chemistry for shearthinning hydrogels includes self-assembly, dynamic and reversible bonds (i.e., hydrogen bonding, electrostatic repulsion, or hydrophobic interactions), and entropy-driven polymeric network recovery.22−24 Even after the completion of hydrogel formation, the conjugated gallol moieties provide additional functions to the HA-Ga hydrogels. It was previously reported that gallol derivatives (i.e., tannic acid) show high affinity to biomacromolecules such as DNA9 or proteins.6 Thus, we hypothesized that hydrogels prepared by gallol-mediated interchain cross-linking might show two useful functions of (i) spontaneous accumulation of proteins within hydrogels by gallol-protein intermolecular hydrogen bonds and (ii) enzymatic degradation resistance by capturing HA-degrading enzymes. Thus, the conjugation of gallol moieties to a polymeric backbone influences not only the hydrogel injectability of hydrogels via shear-thinning mechanisms but also the protein binding and capturing properties.

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EXPERIMENTAL SECTION

Materials. Sodium hyaluronate (HA; MW 151−300 kDa) was purchased from Lifecore Biomedical (U.S.A.). (−)-Epigallocatechin gallate (EGCG; MW 458.37), 5-hydroxydopamine hydrochloride (MW 205.64), tannic acid (MW 1701.2), and punicalagin (MW 1084.72) were obtained from Sigma-Aldrich (St. Louis, U.S.A.). Methoxy poly(ethylene glycol)-amine (mPEG-NH2; Mn 5, 10, and 30 kDa) and PEG-diamine (H2N-PEG-NH2; MW 6 kDa) were obtained from Sunbio (U.S.A.). In addition, hyaluronidase from bovine testes (MW 55 kDa) for an enzyme-resistance test, albumin-fluorescein isothiocyanate conjugate (FITC-BSA) and alginic acid sodium salt from brown algae (low viscosity) for measuring protein loading efficiency were purchased from Sigma-Aldrich (St. Louis, U.S.A.). Rhodamine B isothiocyanate-dextran (Rho-Dex; MW ∼ 70 kDa) was also obtained from Sigma-Aldrich (St. Louis, U.S.A.). Preparation and Characterization of Gallol-Conjugated Hyaluronan (HA-Ga) and OEGCG. First, HA-Ga was prepared by 8212

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Chemistry of Materials were loaded on the 20 mm parallel plate (150 μm of the gap size), the shear viscosity (Pa·s) was evaluated at a shear rate from 0.001 to 10 s−1. Oscillating strain test was performed between 0.1% and 10% for total 1,300 s at a frequency of 1 Hz. The G′ and G′′ for each strain (0.1% or 10%) were measured for 230 s, and the strain changes were sequentially performed. The third cycle (0.1%) was performed for 840 s. In addition, the injectability of the hydrogels was confirmed using a commercial needle with the diameter of 26 G (Korea Vaccine, Republic of Korea). Gelation Mechanism Studies for the Hydrogels. To demonstrate the intermolecular interaction based on multiple hydrogen bonds between HA-Ga and OEGCG, the rheological properties dependent on pH and the addition of urea (2 M), which are hydrogen bonds inhibitors, were evaluated over the frequency range from 0.1 to 10 Hz under a constant stress of 10 Pa at room temperature. Additionally, the rheological properties of the gels prepared in phosphate-buffered saline (PBS; pH 7.4) and the gels preloaded with FITC-BSA or Rho-Dex (900 μg in HA-Ga) were measured in the same manner. FT-IR spectroscopy was also used for chemical analysis. The freeze-dried HA-Ga/OEGCG mixtures were dispersed in potassium bromide (KBr) and compressed into a disk pellet. All FT-IR spectra were obtained through 20 scans in the range of 400−4000 cm−1. Measurement for Protein Loading Efficiency (%) of the Hydrogels and Its Release Kinetics. For evaluating protein encapsulation efficiency in the hydrogels, FITC-BSA was used as a model protein. The gel (∼3 mg in dry weight) was preswollen for 20 min and then incubated in FITC-BSA solutions (30, 90, or 270 μg/mL dissolved in PBS (pH 7.4), each solution of 500 μL). After 20 min or 3 h, the gel was centrifuged, and the protein amounts in the supernatant were measured using a fluorescence microplate reader (Synergy Mx, Biotek, U.S.A.) and calculated from a FITC-BSA standard curve established over the range from 5.625 to 270 μg/mL (excitation = 460 nm, emission = 520 nm). As a control, alginate beads (∼3 mg) were prepared using an Encapsulator B-390 (Büchi, Germany). Alginate solution (1 wt %) was dropped in calcium chloride (1 M) solution with a 300 μm nozzle at a vibration frequency of 1200 Hz and an electrode tension of 1000 V under 10 mbar air pressure. Moreover, the morphology images of FITC-BSA-loaded gels were captured by optical and fluorescence microscopy (Nikon, Japan). To investigate the protein release kinetics, the FITC-BSA-loaded gels incubated in 270 μg/mL FITC-BSA solution were immediately transferred to PBS buffer (pH 7.4, 150 μL). At predetermined time intervals (0.5, 1, 2, 4, 7, 10, 24, 48, 103, 169, and 216 h (9 days)), the entire volume of the buffer solution was replaced with a fresh one. The released protein amount was measured using a fluorescence microplate reader (Synergy Mx, Biotek, U.S.A.) and was calculated from a FITC-BSA standard curve of the range from 30 to 270 μg/mL. Sequential Release Kinetics of Two Biomacromolecules Spatially Loaded in the Two Double-Layer Hydrogels of Dex@BSA and BSA@Dex. To encapsulate two different biomacromolecules, FITC-BSA and Rho-Dex, into the hydrogels, HA-Ga solution (2 wt %) containing FITC-BSA (900 μg) or Rho-Dex (900 μg) and OEGCG solution (8 wt %) were prepared. First, HA-Ga/ FITC-BSA solution (90 μL) and OEGCG solution (30 μL) were vigorously mixed for gelation. Second, the FITC-BSA containing HAGa gel was transferred into HA-Ga/Rho-Dex solution (90 μL), and then, the gels were taken from the tube after several minutes. At this step, nearly all outer HA-Ga/Rho-Dex solution should cover the core gel surface. Finally, OEGCG solution (30 μL) was spread onto the surface of the gel for secondary gelation. The three-step procedure prepared BSA@Dex double layer hydrogels. For the opposite double layer gel configuration (i.e., Dex@BSA), the aforementioned components were exchanged with each other. The release kinetics of the encapsulated FITC-BSA and Rho-Dex were evaluated in MES buffer (10 mM, 1 mL). At determined time intervals (30 min, 2, 4, 7, 10, 24, 96, 173, and 192 h), the entire volume of the buffer solution was freshly and entirely replaced. The released amount of FITC-BSA and Rho-Dex in the collected buffer solution (100 μL) was measured using a fluorescence microplate reader (Synergy Mx, Biotek, U.S.A.).

Each amount was calculated from a FITC-BSA standard curve established over the range from 0.625 to 5 μg (excitation = 460 nm, emission = 520 nm) or a Rho-Dex standard curve over the range from 0.131 to 4.2 μg (excitation = 550 nm, emission = 584 nm). Hyaluronidase degradation tests for evaluating enzymatic degradation resistance of the gallol-rich and HA/PEG-diamine hydrogels. To evaluate the enzymatic degradation resistance of the hydrogels, the enzyme inhibition properties of each polymer, HA-Ga and OEGCG, were analyzed using the microplate assay for hyaluronidase activity.25 HA, HA-Ga (8 mg/mL dissolved in DDW, 100 μL), and OEGCG solutions (13.3 mg/mL dissolved in 10% ethanol, 50 μL) were prepared and heated at 55 °C and then mixed with agarose solutions (9 mg/mL, 850 μL) liquefied in 0.3 M sodium phosphate buffer (pH 7.0) maintained at 60 °C. The final concentrations were 0.8 mg/mL for HA-Ga and HA, 0.67 mg/mL for OEGCG, and 7.6 mg/mL for agarose, and DDW was added to adjust the total sample volume to 1 mL. The mixed polymer/agarose solutions (100 μL) were set in a 96well microplate. After hyaluronidase (40−100 units/100 μL in 0.3 M sodium phosphate buffer) was added to each well, the samples were incubated at 37 °C. The hyaluronidase solutions were removed after 2 h, and cetylpyridinium chloride solutions (100 μL, 10 wt %) were subsequently added to each well. Finally, the absorbance at 595 nm was measured by a microplate reader (Varioskan Flash Multimode plate reader, Thermo Scientific, U.S.A.). Furthermore, to demonstrate the enzymatic resistance of the hydrogels, the gels (∼50 mg) were incubated in hyaluronidase solutions (100−250 units/2 mL) or PBS (pH 7.4) at 37 °C. For control groups, HA/poly(ethylene glycol) (PEG) gels were prepared by EDC/NHS coupling reaction between carboxyl groups of HA and amine groups of PEG-diamine (MW 6 kDa). In brief, HA solutions were prepared in DDW at a concentration of 2 wt %, and EDC 81 mg and NHS 64 mg (2-equivalent to carboxyl groups) were added to the HA solutions. After 30 min of activation, PEG-diamine (574 mg) was added. The HA/PEG gel formed overnight, and the residual EDC/NHS was removed by dialyzation in DDW 1 L for 1 day (MWCO = 3500 Da). At predetermined time intervals (30 min, 1, 2, 4, 7, 10, and 24 h), the weight change (%) was measured. To evaluate the long-term degradation of the gallol-rich HA gels, the weight change (%) in PBS alone or hyaluronidase treatment was continuously measured for 9 days. For evaluating the turbidity of the gel-incubated solution, the absorbance (A600) of the solutions (100 μL) was measured using a UV−vis detector (Hewlett-Packard 8453, U.S.A.). In Vitro GFP Release Profile from the Gallol-Rich Gels in the Presence of Exogenous Proteins and Its Captured Ability. The GFP-loaded gels (30.3 ± 2.4 mg) were prepared, and transferred to PBS alone, BSA-containing (150 μL, 0.5 mg/mL) or hyaluronidase (HAse)-containing (150 μL, 50−125 units/mL) PBS buffers. At predetermined time intervals of 1, 4, 24, 48, 72, 96, 120, 152, and 173 (7 days), the released solution (100 μL) was collected, and the fresh PBS, PBS/BSA, or PBS/HAse was supplemented. The GFP fluorescence emission in the released solution (excitationx= 450 nm, emission= 512 nm) was measured using a fluorescence microplate reader (Synergy Mx, Biotek, U.S.A.), and calculated by the GFP standard curve established in the range of 0.049 μg to 3.15 μg. The calculated amount was also multiplied by the OEGCG-induced intrinsic quenching constant, 4.77, at a stoichiometric ratio of [GFP] to [OEGCG] of 1. Moreover, to evaluate the adsorption degree of the exogenous proteins, the BSA-loaded gels (∼1 mg) were encountered in the exogenous GFP solution (0.63 mg/mL, 150 μL), and the green fluorescence of the gel was observed using fluorescence microscopy (Nikon, Japan).

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RESULTS AND DISCUSSION For the preparation of the gallol-rich hydrogels, we prepared HA-Ga and OEGCG (Figure S1). The gallol conjugation to HA was conducted by an EDC/NHS coupling reaction using 5hydroxydopamine as a reactant in a weak acidic condition (∼pH 5.5) (Figure S1a). The degree of substitution (DOS %) of the gallol moieties tethered to HA backbones was 8213

DOI: 10.1021/acs.chemmater.7b02267 Chem. Mater. 2017, 29, 8211−8220

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Figure 1. (a) Schematic illustration for preparing gallol-rich, shear-thinning hydrogels of HA-Ga/OEGCG. (b) Overlay of the HA-Ga/OEGCG bulk gel to demonstrate its transparency (400 μL) (top), and the cross-sectional SEM image of the lyophilized gel (bottom). (c to e) Frequency-sweep rheological analysis (elastic modulus, G′ (filled symbol) and viscous modulus, G′′ (empty symbol)) for the HA-Ga/OEGCG mixture with a [Dglucuronic acid-D-N-acetylglucosamine]/[gallol in OEGCG] stoichiometric ratio of (c) 7, (d) 2, and (e) 0.5. At a ratio of 2, the soft gels exhibit the G′ modulus of 341.6 ± 53.6 Pa at 1 Hz. At a ratio of 0.5, the gels show the G′ modulus of 1390.5 ± 128.0 Pa at 1 Hz. (f) Changes in viscosity as a function of shear rates for HA-Ga/OEGCG hydrogels with the [HA unit]/[Gallol in OEGCG] ratio of 7 (blue), 2 (black), or 0.5 (red). (g) The recovery measurement of G′ displaying the hydrogel structure under alternating strain from 0.1% to 10% back down to 0.1%. The red arrow shows the recovery of G′ back to the initial value. (h) A photograph showing the injectability of the HA-Ga/OEGCG hydrogel (the ratio = 0.5) using a 26 G needle (inner diameter = 0.26 mm).

equipped with an RI detector, the peak retention time of OEGCG was found to be 22.4 min, which corresponds to 9400 Da of the molecular weight determined by PEG standards (Figure S1g). The gallol-rich hydrogels spontaneously formed upon mixing HA-Ga and OEGCG (Figure 1a). The hydrogel was formed only when HA-Ga and OEGCG solutions are mixed. No other combinations, such as HA/EGCG, HA-Ga/EGCG, or HA/ OEGCG, showed any hydrogel formation (Figure S2a,b). We should emphasize the result of a particular control group mixing HA-Ga and EGCG. This mixture remained a sol state. The result is surprising because the only difference is oligo-EGCG vs EGCG, indicating that hydrogen bonds formed with unoligomerized EGCG is not sufficient for establishing 3D hydrogel networks. Another control group is the mixture between OEGCG and HA. It also resulted in a sol state. The only difference is HA vs HA-Ga. This result suggests that numerous hydrogen donor/acceptors existed along unmodified HA backbone are not sufficient for hydrogel network formations.

determined using a gallol standard curve by ultraviolet−visible (UV−vis) spectroscopy (A275). Overall, 9.7 ± 2.7% of the carboxylic acid groups in the HA backbone were conjugated with 5-hydroxyldopamine (Figure S1b). The conjugation degree of gallols was also confirmed using proton nuclear magnetic resonance spectroscopy (1H NMR). The DOS was 7.8% when calculated by the integration ratio between the aromatic proton peak adjacent to gallol groups (C2 and C6) (6.4 ppm (a)) and the peak of methyl protons on the HA backbone (1.9 ppm (c)) (Figure S1c). The four ethyl protons located between the amine and gallol groups also appeared at 2.8 ppm (b) (Figure S1c). In addition, OEGCG was synthesized by the polycondensation method with acetaldehyde (Figure S1d).8 Although the absorption of OEGCG in UV−vis spectrum was similar to that of EGCG (Figure S1e, black dashed line), the newly generated methyl peak (−CH3) resulting from the oligomerization reaction was observed as a broad peak and appeared at 1.0−2.1 ppm (the “d” peak indicated in Figure S1f, black) in the 1H NMR spectrum. The result showed noticeable difference when compared with that of EGCG alone (Figure S1f, red). As a result of SEC analysis 8214

DOI: 10.1021/acs.chemmater.7b02267 Chem. Mater. 2017, 29, 8211−8220

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Figure 2. (a) Changes in rheological properties of the HA-Ga/OEGCG gels depending on pH values (red for pH 2, blue for pH 4, and black for pH 10). (b) The conversion of rheological properties from gel to sol in the presence of urea (2 M in distilled water) (filled symbol for elastic modulus, G′, and empty symbol for viscous modulus, G′′). (c) FT-IR spectra of HA-Ga/OEGCG mixtures with the unit stoichiometric ratio of [D-glucuronic acid-D-N-acetylglucosamine] to [gallol in OEGCG] of 0.25 (red), 0.5 (blue), or 2 (purple); HA-Ga alone (green); or OEGCG itself (black). (d) The proposed multiple hydrogen bond formation (red dashed line) between the gallol-to-gallol moieties and gallol-to-HA backbone.

results indicate that the molecular weight of the polyphenolic cross-linker is critical for gelation of HA-Ga. In contrast to the previous report about the supramolecular tannic acid-metal gelation,10 our study showed no gelation despite adding tannic acid to HA-Ga. This result indicates the importance of bond strength. The catechol-Ti coordination force (∼800 pN)26 is far higher than that of hydrogen bonds ( G′ (black filled triangle)). Also, the physiological salts do not interfere with the gallol-togallol interactions. As shown in Figure S3a (pink), the elastic moduli of the hydrogels were 1601 ± 114.6 Pa for PBS and 1390.5 ± 128.0 Pa for deionized water. Second, the gels can be dissociated by adding the hydrogen bond inhibitor urea.32 The preformed HA-Ga/OEGCG hydrogels were transferred to distilled water with a 2 M concentration of urea. The addition of urea rapidly changed the gels to a sol state in which G′′ (green empty square) was higher than G′ (green filled square) in the overall rotational frequency scale (Figure 2b). Thus, the hydrogen bonds are thought to be the main mechanism for the HA-Ga/OEGCG gelation. Furthermore, for chemical analysis of the multiple hydrogen bonds, a Fourier-transform infrared spectroscopic (FT-IR) study was conducted by varying the [Dglucuronic acid-D-N-acetylglucosamine]/[gallol in OEGCG] stoichiometric ratio from 0.25 to 2 (Figure 2c). Regardless of the stoichiometric ratio, the O−H stretching vibrational bands centered at 3443 cm−1 obtained from the HA-Ga/OEGCG mixtures (blue, red, and purple) were significantly shifted from those of HA-Ga (3418 cm−1, green) alone and OEGCG (3392 cm−1, black) itself. In addition, the C−O stretching vibrational peak appearing at 1074 cm−1 (green) from the alcohol/ether groups in HA was gradually shifted to 1089 cm−1 (red) with increasing amount of OEGCG. These data suggest that the HA backbone participated in the formation of hydrogen bonds to OEGCG as well. Thus, in summary, the gallol (HA)-to-gallol (OEGCG) hydrogen bonds, as well as HA backbone-toOEGCG gallol hydrogen bonds, were formed, resulting in synergistic effects on the formation of the hydrogels (Figure 2d). As mentioned in the Introduction section, the gallol groups show a high affinity to proteins through noncovalent interactions, such as hydrogen bonding and van der Waals interactions.6 Thus, the residual gallols not participating in gelforming cross-linking may be involved with another useful function such as entrapment of proteins into the hydrogels. In general, the primary method for protein loading into hydrogels is direct encapsulation by adding it at an initial stage of hydrogel preparation.33,34 Recently, alternative ways focusing on affinity-based protein loading have been reported in which the gel exhibited delayed release kinetics of the encapsulated components due to the intermolecular affinity.35 Subsequently, affinity-based protein sequestration onto surfaces was reported.36 Although loading into hydrogels using a direct protein entrapment approach from surrounding environments can be performed (schematically described in Figure 3a), it is very rare because of an absence of protein-attracting functions in most hydrogels. For example, when alginate hydrogel beads were placed in the solution containing albumin-fluorescein isothiocyanate conjugate (FITC-BSA, 90 μg/mL, 500 μL), FITC-BSA molecules diffused into the alginate gels. However, the loading efficiency was very low at just 18.9 ± 0.9% for 20 min and 21.0 ± 10.2% for 3 h. Surprisingly, in contrast, the loading efficiency of the HA-Ga/OEGCG gels showed a very high value due to the protein binding capability of the gallol groups via intermolecular hydrogen bonds. For the first 20 min of incubation, 92.0 ± 0.8% of FITC-BSA entrapped into the gels (right light-green bar in Figure 3c). The amount of loading was further increased to 95.3 ± 0.3% after 3 h of incubation (right green bar in Figure 3). We further increased the initial protein concentration to 270 μg/mL (Figure 3c), which is 93.4 8217

DOI: 10.1021/acs.chemmater.7b02267 Chem. Mater. 2017, 29, 8211−8220

Article

Chemistry of Materials are customized by spatially and mechanically controlled loading drugs in the inner core or outer layers of the hydrogels. Finally, another useful characteristic of the gallol-rich hydrogels is the resistance to hydrogel degradation by enzymes. Loss of function by enzymatic degradation may result in rapid, uncontrolled release of an encapsulated content. Thus, in general, the controlled and expected biochemical functions of hydrogels, such as sustained long-term release of drugs, can be achieved by inhibiting enzymatic actions. If the gallols exposed to interfacial exterior surfaces of the hydrogels would capture enzymes, preventing free diffusion, the gels could be longlasting formulations tolerated even in the presence of adverse enzymatic degradation of the hydrogels in vivo. The enzymatic degradation resistance of the gels was evaluated using hyaluronidase (HAse). First, the degradation-resistant ability of the two gallol moieties, the backbone-conjugated gallol in HA and the one in OEGCG, was tested in solution states (Figure 4a). On the basis of a previously reported HAse assay,25 the remaining polymer amount was analyzed (details are described in the Experimental Section). As a control, HA was degraded up to 31.7 ± 2.2% during a 2-h incubation with HAse (40−100 units) (Figure 4b, the first blue bar). However, HAGa was degraded down to 23.7 ± 1.1% (the second blue bar). The result indicates that the gallol moieties conjugated onto the HA backbone resist the enzymatic degradation by capturing HAse. In particular, the enzyme became virtually inactive for the HA-Ga/OEGCG mixture, and most of the polymer remained (102.0 ± 4.0%) (the third blue bar). All these data suggest that the enzymatic degradation resistance resulted from the cooperative actions of the gallols tethered in HA as well as the gallols from OEGCG. Second, the degradation resistant ability of the gallols was further confirmed in gel states (Figure 4c). To quantitatively evaluate the enzymatic degradation, HA gels covalently cross-linked with PEG-diamine (the first photo of Figure 4c) were prepared as a control because the HA backbone itself is not gelated. Figure 4d shows the degradation kinetics of the HA/PEG gels or the gallol-rich HA-Ga/OEGCG gels by HAse. In PBS incubation (empty red/black graphs), both gels initially swelled during 4 h (up to 166.0 ± 7.4% for the control and 129.9 ± 10.4% for the gallol-rich gels) without the addition of HAse. After that, the weight changes remained nearly constant, approximately in the range between 120 and 160% after 4 h. However, nearly all mass of the HA/PEG gel was degraded within a 4 h incubation with HAse (filled black square), leaving only 4.0 ± 0.2% of the initial mass. In contrast, when the gallol-rich gels were incubated with HAse, the rapid and effective enzymatic degradation was not observed for even an extended period of 24-h incubation with HAse. Nearly all the swollen mass (116.9 ± 13.5%) remained intact in a gel state for 216 h (9 days) (filled red circle and top photo, Figure 4d), indicating the superior ability of the enzyme inhibition effect by the gallol moieties. In a case of the long-term PBS incubation, the gels exhibited slight physical disassemble at 9 days (74.7 ± 25.9%) (empty red circle and bottom photo). These results might be due to the enhanced mechanical stability by the adsorption of the exogenous HAse onto the remained gels. In particular, turbidity changes of the surrounding solutions for the gallol-rich gels were observed in the presence of HAse (100−400 units). Previously, gallol−protein interactions were easily detected by UV−vis spectroscopy because the protein/ gallol complexes often resulted in a certain degree of aggregation, resulting in increases in solution turbidity at A600.6 Thus, the observed turbidity in the surrounding solutions

Figure 4. (a) Schematic description for capturing active enzymes by HA-Ga and OEGCG mixtures. (b) In vitro degradation test of HA (1st group), HA-Ga (2nd group), or HA-Ga/OEGCG in a solution state (3rd group) by HAse action (gray bars for zero-unit addition of HAse and blue bars for 40−100 unit addition). (c) Experimental description of in vitro gel degradation test utilizing HA/PEG gels as a control and the gallol-rich gels, resulting from the activity of HAse. (d) In vitro gel degradation kinetics as a result of HAse action (red filled symbols for the gallol-rich gels and black filled symbol for controls) and the gel swelling properties in PBS incubation (red empty symbol for the gallol-rich gels and black empty symbol for controls). The inset photos show the remained gel at 9 days in the presence of hyaluronidase (top) or PBS alone (bottom). (e) Changes in turbidity of the gel-incubated solution as a function of enzymatic degradation time: 0 (black), 12 (green), 24 (red), and 48 (blue) h. The inset photos indicate the gel-incubated solutions corresponding to the degradation times. (f) GFP sustained release profiles in the presence of exogenous proteins, BSA (blue) and HAse (red). PBS (black) indicates no exogenous proteins in the release buffer.

indicates the intermolecular complexes between the dissociated OEGCG and HAse. As the enzymatic degradation time increased, the absorbance (A600) increased from 0.005 for 0 h (black) to 0.20 for 48 h (blue) (Figure 4e, black arrow). Furthermore, we performed the long-term GFP protein release experiments in the presence of exogenous BSA in the release buffer (blue, Figure 4f) or HAse (red). We hypothesized that exogenous proteins can infiltrate into the gel and subsequently bind to the gallol moieties, potentially affecting release kinetics. We found that the GFP release kinetics was varied depending on the type of the exogenous proteins. The release profiles of GFPs were nearly zero order, and the final amount after 7 days was 4.0 ± 0.01 μg in the presence of BSA, 2.5 ± 0.03 μg in the presence of HAse, and 3.2 ± 0.35 μg without exogenous proteins (i.e., PBS). For now, there are no available clear explanations for the observed differences in the GFP release amount. The variations can be batch-to-batch differences for 8218

DOI: 10.1021/acs.chemmater.7b02267 Chem. Mater. 2017, 29, 8211−8220

Article

Chemistry of Materials

Republic of Korea (1631060, H.L.) and the National Research Foundation of Republic of Korea (NRF) Grants: development program for convergence R&D over traditional culture and current technology (2016M3C1B5906485 H.L.), Basic Science Research Program (2016R1A6A3A11933589 M.S.), and Midcareer Researcher Program (2017R1A2A1A05001047, H.L.).

the prepared gels or pore size changes by infiltrations of exogenous proteins followed by mechanical properties changes of the gels. A clear evidence for the exogenous protein infiltration can be provided at this stage, which affect the gel mechanical properties. To visualize the infiltration of the exogenous proteins into the gels, we used GFP as an exogenous protein component (Figure S4), and nonfluorescent BSAloaded gallol-rich HA gel was placed. In an initial stage (1 h), the green fluorescence emission was slightly observed in the edge of the gel absorbed GFP (Figure S4, first photo). After 1 day, the emission area was further expanded to the depth of 418 μm (2nd photo). The exogenous GFP adsorption into the gel was continuously observed for 7 days. These results show that the gels would be highly effective as a drug-encapsulated platform capable of being protected from in vivo enzymatic degradation. In conclusion, we demonstrated the multifunctionality of gallol moieties in the hydrogel network and developed gallolrich hydrogels using HA-gallol conjugates and a gallol-rich cross-linker, OEGCG. The gallol-rich hydrogels exhibited three unique properties: (i) rapid and spontaneous gelation of gallolcontaining polymers by multiple hydrogen bonds of gallol-togallol and gallol-to-polymeric backbone; (ii) shear-thinning by breaking and reforming the gallol-mediated extensive hydrogen bonds, affording injectable hydrogels; and (iii) a large amount of protein loading and enzymatic degradation resistance resulting from the high affinity of gallol to proteins. Thus, our findings for the gallol moieties will be useful for easily achieving shear-thinning, injectable protein-encapsulated hydrogels showing enzymatic resistance for biomedical applications.

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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.chemmater.7b02267. Chemical characterization of HA-Ga and OEGCG and the gelation test for other mixtures (i.e., HA/EGCG, HAGa/other polyphenols); rheological properties of the HA-Ga/OEGCG gels depending on the gelation condition and the exogenous GFP infiltration images (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail for H.L.: [email protected]. *E-mail for M.S.: [email protected]. ORCID

Mikyung Shin: 0000-0002-7990-5210 Haeshin Lee: 0000-0003-3961-9727 Author Contributions

M.S conceived the project, planned and performed experiments, analyzed data, and wrote the manuscript. H.L. conceived this project, analyzed data, and wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the National R&D Program for Cancer Control, Ministry for Health and Welfare, 8219

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DOI: 10.1021/acs.chemmater.7b02267 Chem. Mater. 2017, 29, 8211−8220