Multivalent Polyaspartamide Cross-Linker for Engineering Cell

Jan 30, 2018 - (1-3) Hydrogels possess a unique blend of interesting physical properties, such as elasticity, hydrophilicity and permeability, which m...
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Article Cite This: Biomacromolecules 2018, 19, 691−700

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Multivalent Polyaspartamide Cross-Linker for Engineering CellResponsive Hydrogels with Degradation Behavior and Tunable Physical Properties Jinhyeong Jang† and Chaenyung Cha*,‡ †

Department of Chemistry, School of Natural Science and ‡School of Materials Science and Engineering, Ulsan National Institute of Science and Technology, Ulsan 44919, South Korea S Supporting Information *

ABSTRACT: Hydrogels possess favorable physical properties ideally suited for various biotechnology applications. To tailor to specific needs, a number of modification strategies have been employed to tune their properties. Herein, a multifunctional polymeric cross-linker based on polyaspartamide is developed, which allows for the facile adjustment of the type and number of reactive functional groups to fit different reaction schemes and control the physical properties of the hydrogels. The amine-based nucleophiles containing desired functional groups are reacted with polysuccinimide to synthesize polyaspartamide cross-linkers. The cross-linking density and the concurrent change in mechanical properties of the resulting hydrogels are controlled in a wide range only with the degree of substitution. This multivalency of the polyaspartamide linkers also induced the degradation of hydrogels by the unreacted functional groups on polyaspartamide involved in the hydrolysis. Furthermore, the polyaspartamide cross-linker conjugated with cell-recognition molecules via the same conjugation mechanism (i.e., nucleophilic substitution) render the hydrogels cell-responsive without the need of additional processing steps. This versatility of polyaspartamide-based cross-linker is expected to provide an efficient and versatile route to engineer hydrogels with controllable properties for biomedical applications. functions, ECM proteins (e.g., collagen, fibronectin) or cellresponsive peptides (e.g., RGD peptides) are separately conjugated.11,12 Although these methods have been widely used to engineer hydrogels with varying degrees of properties, it is still desirable to attain the controllability of multiple hydrogel properties by utilizing a multifunctional and tunable component. Polysuccinimide, commonly synthesized by acid-catalyzed polycondensation of aspartic acid, is a unique polymer which undergoes nucleophilic substitution with amine-based molecules due to the labile succinimidyl ring groups in the backbone, resulting in polyaspartamide formation.13−16 Polyaspartamide with various functional groups have been successfully used in biomedical applications. For example, Jeong et al. demonstrated the fabrication of self-assembled polymersomes using alkyl chain-grafted amphiphilic polyaspartamide for theranostic applications.17,18 Cha et al. also demonstrated that polyaspartamide can be used to link proteins to various materials, as the proteins naturally contain amine groups for the attachment to the polymer backbone, while providing functional groups that can participate in the chemical reaction with a particular material.19 In addition, hydrogels

1. INTRODUCTION Hydrogels made from biocompatible polymers, both natural and synthetic, have become a powerful platform for various biomedical applications such as tissue engineering and drug delivery.1−3 Hydrogels possess a unique blend of interesting physical properties, such as elasticity, hydrophilicity and permeability, which make them highly suitable for those applications. Various fabrication strategies are often employed to impart modalities designed to elicit controlled biological responses. Furthermore, these physical and biological properties can be tuned in a wide range to tailor to specific needs. The strategies for controlling various properties of hydrogels often entail several processing steps, as most gel-forming polymers do not possess those properties in their native forms. As such, engineering the hydrogels with tunable properties are generally accomplished by the assembly of various modalities serving different purposes. For example, the rigidity of a hydrogel is controlled by the amount of cross-linking molecules used for the fabrications.3−5 This same method is also commonly used to control the hydrogel porosity for drug delivery applications, as the cross-linking density influences both factors inversely.2,6,7 Increasing fracture toughness and structural durability as well as imparting novel functionalities of a hydrogel is often accomplished by creating interpenetrating networks or composite formation using various nanomaterials as fillers.8−10 To induce and control cell adhesion and © 2018 American Chemical Society

Received: January 16, 2018 Published: January 30, 2018 691

DOI: 10.1021/acs.biomac.8b00068 Biomacromolecules 2018, 19, 691−700

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by gel permeation chromatography (Model 1200S, Agilent) was 22000 g mol−1. 2.2. Synthesis of Poly(2-hydroxyethyl-co-hydrazidoadipoyl aspartamide) (PHHZA) and Poly(2-hydroxyethyl-co-ethylenediaminoethyl aspartamide) (PHEDA). Poly(2-hydroxyethyl-cohydrazidoadipoyl aspartamide) (PHHZA), a polyaspartamide presenting hydrazide groups, was synthesized by derivatizing PSI with a varying amount of dihydrazide-based molecules via ring opening nucleophilic reaction, following a previously published procedure with modifications.17,19 Briefly, PSI (0.8 g) was dissolved in 10 mL of dimethylformamide (DMF). Ethanolamine (Sigma-Aldrich) was first added and stirred overnight at 70 °C under dry N2 to first develop polyaspartamide with hydroxyl groups (i.e., poly(2-hydroxyethyl aspartamide)). Then, adipic acid dihydazide (AAD, Sigma-Aldrich) was added to the mixture and continued to react at 70 °C for 48 h. The amount of AAD was 2 mol equiv of the remaining unopened succinimidyl rings in order to make sure only one hydrazide group of AAD reacted with the succinimidyl groups in the polyaspartamide chain and leave the other hydrazide group intact. The mixture was extensively dialyzed against DI water, and the product was obtained by lyophilization. The chemical structure of PHHZA was analyzed with 1 H NMR, 13C NMR, and FT-IR (Figures S2−S4). The degree of substitution (DS) of hydrazide on PHHZA was controlled by changing the amounts of reactants; ethanolamine and adipic acid dihydazide. The amounts of ethanolamine and adipic acid dihydazide used per 0.8 g of PSI were 0.398 mL and 0.575 g (DS1), 0.348 mL and 0.862 g (DS2), 0.299 mL and 1.149 g (DS3), and 0.248 mL and 1.437 g (DS4). The DS of hydrazide groups on PHHZA was confirmed by comparing the peak integration ratios from 13C NMR spectra and identifying characteristic peaks in FT-IR spectra (Figures S3a and S4a). The 13C NMR was performed using the inverse gated decoupling sequence without Nuclear Overhauser Effect (NOE; C13IG sequence from Bruker Standard Library), which allows quantitative analysis of peak integration values.26 13C NMR was used instead of 1H NMR to calculate the DS, due to the many overlapping peaks in 1H NMR spectra prevented accurate identification and quantification of characteristic peaks. The synthesis of poly(2-hydroxyethyl-co-ethylenediaminoethyl aspartamide) (PHEDA) was carried out following the same procedure as PHHZA, except diethylenetriamine (Sigma-Aldrich) and DMSO were used as the reactant and the solvent to present amine groups on the polyaspartamide instead of adipic acid dihydrazide and DMF, respectively. The DS of amine groups on PHEDA was similarly confirmed by 13C NMR spectra and FT-IR spectra (Figures S3b and S4b). The changes in DS of PHHZA and PHEDA were also verified using a colorimetric trinitrobenzenesulfonic acid (TNBS) assay, which measures the amount of amine and related functional groups (Figure S5).27,28 Briefly, 0.300 mL of TNBS working solution (0.1% TNBS, 4% NaHCO3, pH 8.5) was added to 0.300 mL of a PHHZA or PHEDA sample and allowed to react for 2 h at 37 °C. After the reaction, 0.300 mL of 1 M HCl was added. The absorbance at 335 nm to four 0.200 mL samples was measured using a spectrophotometer (Multiskan GO, Thermo Fisher Scientific) and averaged. Peptide-conjugated PHHZA was accomplished by first reacting GRGDS pentapeptide (“RGD peptide”), which has been widely used to promote cell adhesion, with PSI at 70 °C for 6 h before reacting with adipic acid dihydrazide and ethanolamine as described above. Here, the molar feed ratio of RGD peptide to the succinimidyl rings of PSI was 0.03. The presence of RGD peptide on PHHZA was analyzed by 13C NMR spectra (Figure S6). 2.3. Fabrication of PHHZA- and PHEDA-Linked Hydrogels. Poly(ethylene glycol) diacrylate (PEGDA, Mw 700, Sigma-Aldrich) or oxidized alginate (OAlg) were reacted with PHHZA or PHEDA to form the hydrogels. The OAlg was prepared by partial oxidation of alginate with sodium periodate (Sigma-Aldrich) to present aldehyde groups. A detailed procedure for the synthesis of OAlg is described elsewhere.5,29,30 Stock solutions of PEGDA or OAlg and PHHZA or PHEDA dissolved in 0.1 M sodium phosphate buffer (pH 8.0) are mixed in various ratios, and the mixture was cast into a custom mold

prepared by cross-linking polyaspartamide via various chemical reaction schemes have been proposed.20−23 In this study, we have expanded the previous scope of polyaspartamide utilization by taking advantage of the versatile chemical platform provided by polysuccinimide. There have been several previous studies on polyaspartamide-based hydrogels in recent years, but they mostly utilized polyaspartamide as the gel-forming macromers with cross-linkable functional groups, such as catechol and methacrylate,24 or utilize bis-amino molecules to cross-link to polyaspartamide chains via unopened succinimidyl groups.25 In addition, rather than conjugating the functional groups directly using aminebased functional groups on PSI, additional synthetic procedures are employed to conjugate functional groups to poly(2hydroxyethyl polyaspartamide) or poly(aspartic acid), widely explored polyaspartamide derivatives.23,25 However, in this study, the polyaspartamide capable of adjusting the type and number of reactive functional groups by direct ring-opening nucleophilic reaction with PSI was synthesized for use as a cross-linker to fabricate hydrogels via in situ cross-linking reaction with various gel-forming polymers under physiological condition. Using the polyaspartamide with varying number of amine-based functional groups as a cross-linker would allow for the broad applicability to different macromer systems for engineering hydrogels with controllable mechanical properties. It would be also possible to concurrently control the number of remaining unreacted functional groups for further modifications. Herein, the polyaspartamide cross-linkers developed in this study were applied to two different types of macromers, oxidized alginate and poly(ethylene glycol) diacrylate, to induce Schiff base formation and Michael addition, respectively. The hydrogels engineered by the polyaspartamide cross-linkers demonstrated versatility by allowing (1) the control of their mechanical properties, (2) the biodegradation, and (3) the attachment of bioactive molecules for cell adhesion in a combinatorial manner. Using this multivalent polyaspartamide cross-linker, hydrogels with varying mechanical, diffusional, and degradation properties could be fabricated only by changing the degree of substitution of functional groups on a polyaspartamide molecule, while independently conferring cell adhesion by conjugating cell-binding peptide motifs onto the same polymer chain. It was also shown that the unreacted nucleophilic functional groups on the polyaspartamide facilitated the degradation of hydrogels under physiological condition. The combination of tunable mechanical and degradation properties were applied to controlled drug release. To further test the versatility of this cross-linker, the type of functional groups was varied to cross-link different types of polymers to prepare hydrogels.

2. MATERIALS AND METHODS 2.1. Synthesis of Polysuccinimide (PSI). Polysuccinimide (PSI) was synthesized by polycondensation of aspartic acid under acidic condition, following a procedure described in a previously published report.13,14,19 Briefly, aspartic acid (25 g, Sigma-Aldrich) and orthophophoric acid (9.4 mmol, Sigma-Aldrich) were mixed in sulfolane (80 mL, Junsei Chemical) and refluxed at 180 °C with mechanical stirring for 7 h under dry N2. Water being formed during the reaction was continuously removed with a Dean−Stark trap. After the reaction, the product was precipitated in methanol. This crude product was filtered and washed several times with deionized (DI) water, and dried under vacuum to obtain the final product. The molecular structure of the product was analyzed with 1H NMR (Figure S1). The number-average (Mn) molecular weight of PSI determined 692

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Figure 1. (a) Synthesis of polyaspartamide cross-linkers via ring-opening nucleophilic addition of polysuccinimide (PSI). (b) Fabrication of polyaspartamide-linked hydrogels with varying DS of functional groups to control the mechanical properties. (c) Synthesis of polyaspartamide crosslinkers presenting amine (PHEDA) or hydrazide (PHHZA) with controllable DS (the ratio of x and y). and allowed to react for 2 h at 37 °C to form hydrogels. The hydrogel disks (5 mm diameter, 1 mm thickness) were cut out and incubated in PBS for 1 day before characterization. 2.4. Mechanical and Swelling Characterizations of Hydrogels. Mechanical properties of hydrogels were evaluated by calculating elastic moduli from stress−strain relationship obtained from uniaxial compression experiments.31,32 Briefly, the hydrogel disk was subjected to uniaxial compressed at 1 mm min−1 (Model 3343, Instron), and a stress−strain curve was obtained. The elastic moduli were then calculated as the slope of the stress−strain curve at the first 10% strain where the curve remained linear (the elastic region). Hydrogel degradation was monitored by measuring the change in elastic moduli over time.5,33 Hydrogel disks were incubated in PBS at 37 °C, and at various time points the moduli of the hydrogels were measured by uniaxial compression as described above until complete dissolution. The degradation rate (kd) of a hydrogel was determined by fitting the fractional change in moduli vs time profile with the following exponential decay model, Et = e−kd·t E0

hydrogel by dissolving in the precursor solution prior to hydrogel fabrication. The hydrogel (8 mm diameter, 1 mm thickness) was incubated in PBS at 37 °C, and the amount of released FITC-BSA was measured at various times by detecting the fluorescence intensity using a microplate spectrofluorometer (Synergy HTX, BioTek). The cumulative drug release profile was fitted to the following two models,

Mt = k1·t n M∞

(2)

b Mt = 1 − e−k 2(t − T ) M∞

(3)

where Mt is the amount of drug released at a time, t, M∞ is the total amount of drug in the hydrogel, k1 and k2 are the kinetic rate constants, T is the lag time constant, and n and b are the exponents related to the release mechanism.34−37 The Ritger-Peppas model (eq 2) was used for the power-law time dependence, while the Weibull model (eq 3) was used for the sigmoidal time dependence. 2.6. Cell Culture on Hydrogels. RGD-linked PHHZA and PHEDA were conjugated to alginate hydrogel and PEGDA hydrogel, respectively, and 3T3 fibroblasts were cultured on the hydrogel to confirm validate the conjugation of RGD peptide via polyaspartamide linker. Briefly, 200 μL aqueous solution of RGD-PHHZA (3 wt %) was coated on a glass slide and dried overnight. Oxidized alginate (10%) and adipic dihydrazide (10%) were mixed in 2:1 ratio, and the solution was placed on the RGD-PHHZA coated glass and reacted at 37 °C for overnight for the hydrogel formation. The hydrogel (8 mm diameter, 1 mm thickness) was washed several times with PBS, and the cell suspension in the culture medium (Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptymycin, all purchased from Thermo Fisher) was placed on the hydrogel and incubated at 37 °C with 5% atmospheric CO2 for 1 day to allow cell adhesion. After washing with PBS, a fresh medium was supplied and the cell culture was continued. To visualize

(1)

where Et was the modulus measured at time, t, and E0 was the initial modulus.5 For hydrogels linked via Schiff base, the hydrogels treated with 0.1 M sodium cyanoborohydride (Sigma-Aldrich) to reduce the Schiff base to nonhydrolytic form were used as a control.28 The swelling ratio of a hydrogel was calculated as the mass ratio of a swollen hydrogel (WS) to its dried polymeric mesh (WD), which was obtained by lyophilization. WS was measured after incubating the bead in PBS for 24 h at room temperature, and WD was measured after drying the bead by lyophilization. 2.5. Drug Release Kinetics of Hydrogels. Drug release profiles from various hydrogels were measured to evaluate their permeability. Briefly, 5 mg mL−1 of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA, Thermo Fisher) were encapsulated in a 693

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Figure 2. (a) Fabrication of alginate-PHEDA or alginate-PHHZA hydrogels via Schiff base formation. Elastic moduli (E) of (b) alginate-PHHZA hydrogels and (c) alginate-PHEDA hydrogels controlled with the concentration and DS of the cross-linkers. *p < 0.05 at the same DS in (b) and (c). the cells and evaluate their viability, the cells were treated with calceinAM and ethidium homodimer-1 to label live (green fluorescence) and dead (red fluorescence) cells, respectively (LIVE/DEAD Viability/ Cytotoxicity Kit, Thermo Fisher). A fluorescence microscope was then used to visualize the cells (XDS-3FL, Optika).

available for cross-linking with other macromers. The insufficient amount of AAD would lead to reaction of both functional groups with polyaspartamide chains, resulting in cross-linked networks without the available functional groups for cross-linking. All PHHZA samples dissolved readily in aqueous solutions. In addition, the degree of substitution (DS), defined as the percentage of succinimidyl rings conjugated with the functional groups, for dihydrazide was controlled from 5.5 to 18% which was confirmed with the peak integration ratio of hydroxyl to hydrazide in 13C NMR spectra (Figure S3a).19 These results clearly demonstrated that the AAD molecules predominantly reacted with polyaspartamide chains via one hydrazide leaving the other hydrazide in an unreacted state. Similarly, PHEDA was synthesized from the PSI with diethylenetriamine (DETA) as a nucleophile to present amine groups to the polyaspartamide (Figure 2a). The DS of amine groups on PHEDA, also obtained from 13C NMR spectra, was controlled from 6 to 25% (Figure S3b). The changes in DS of PHHZA and PHEDA were further analyzed with a colorimetric TNBS assay, which measures the amount of chromogenic complex formed by the reaction of amine and related functional groups with TNBS (Figure S5). The results were in line with the DS of PHHZA and PHEDA obtained from 13C NMR, further verifying the controllability. It should be noted that the percentage of unreacted succinimidyl groups, calculated from 13C NMR spectra, were much lower for PHEDA (approximately 10% or less) than PHHZA (from 11 to 22% with the DS; Figure S3). This result suggested that the primary amine groups in DETA was more reactive toward succinimidyl groups in PSI than hydrazide groups in AAD. Since the DS of PHHZA was well controlled, the presence of the unreacted succinimidyl groups did not lead to unwanted cross-linking of polyaspartamide chains, and the conjugation of AAD to polyaspartamide likely reached the end point. 3.2. Mechanical Properties of PolyaspartamideLinked Hydrogels. Hydrazide and amine-based cross-linkers have been commonly used to cross-link aldehyde-, acrylic-, or epoxy-based resins for industrial applications.40,41 The same

3. RESULTS AND DISCUSSION 3.1. Synthesis of Polyaspartamide Cross-Linker. Polysuccinimide (PSI) was synthesized by thermal polycondensation of aspartic acid, with phosphoric acid as a catalyst and sulfolane as a solvent (Figures 1a and S1). The PSI backbone consisted of succinimidyl ring structures that could undergo nucleophilic substitution reaction with amine-based molecules, resulting in polyaspartamide formation. Therefore, the polyaspartamide backbone conjugated with the amine-based nucleophiles containing functional groups that can react with a particular gel-forming monomer or macromer could be utilized as the polymeric cross-linker to engineer hydrogels. Furthermore, the number of reactive functional groups on a polyaspartamide backbone could be controlled simply by adjusting the molar feed ratio of the nucleophile to the PSI. With this adjustable multivalency of a polyaspartamide crosslinker, it would be possible to control the cross-linking density of a polymeric network without having to change the concentration (Figure 1b). In this study, two different types of polyaspartamide crosslinkers were explored; hydrazide-containing poly(2-hydroxyethyl-co-hydrazidoadipoyl aspartamide) (PHHZA), and amine-containing poly(2-hydroxyethyl-co-ethylenediaminoethyl aspartamide) (PHEDA). Both functional groups are capable of undergoing nucleophilic reactions at physiological conditions to form hydrogels, such as Schiff base formation and Michael addition.38,39 To synthesize PHHZA, PSI was first reacted with 2-ethanolamine to present hydroxyl groups for aqueous solubility, followed by adipic acid dihydrazide (AAD) to present hydrazide groups (Figure 1c). The excess amount of AAD was reacted with the remaining succinimidyl groups in polyaspartamide to ensure that only one hydrazide group in each AAD molecule reacted, leaving the other hydrazide group 694

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Figure 3. (a) Fabrication of PEG-PHEDA or PEG-PHHZA hydrogels via Michael addition. (b) Elastic moduli (E) and (c) swelling ratios (Q) of PEG-PHEDA hydrogels controlled with the concentration and DS of the cross-linkers. *p < 0.05 at the same DS in (b) and (c).

range of concentration and DS (Figure 2c). Third, the PHEDA with the lowest DS (DS1) was able to form hydrogels at the same range of concentration, whereas the lowest critical DS for gelation became higher with decreasing concentrations for PHHZA. These results suggested that the Schiff base reaction by primary amine groups of PHEDA were more facile than hydrazide groups of PHEDA, due to the higher nucleophilicity. As a result, more rigid hydrogels were formed at the same DS and concentration. Taken together, these results demonstrated that the mechanical properties of the hydrogels cross-linked by the polyaspartamide cross-linkers via Schiff base formation could be controlled in a wide range. 3.2.2. Michael Addition. Michael addition involving amine or thiol groups as Michael donor and vinyl or (meth)acrylate groups as Michael acceptor has been successfully utilized to engineer hydrogels at physiological conditions for biomedical applications.42 In this study, the feasibility of PHHZA or PHEDA as a cross-linker for hydrogels via Michael addition was further explored, with poly(ethylene glycol) diacrylate (PEGDA), a widely used polymer for hydrogel fabrication, as the macromer system (Figure 3a).43 The PEGDA concentration was kept constant at 20% (w/v) while varying the concentration of PHEDA and PHHZA. Similar to the alginate hydrogels via Schiff base formation, PEGDA was also cross-linked by PHEDA. The reaction also occurred readily, with the gelation occurring within 5 min. The moduli also increased with DS and concentration of PHEDA: 2−565 kPa for 10% PHEDA, 0.3−70 kPa for 7.5% PHEDA, and 2 kPa for 5% PHEDA (Figure 3b). The swelling ratios also decreased with DS and concentration of PHEDA (Figure 3c). It is interesting to note that the reduction in moduli of PEGPHEDA hydrogels with decreasing PHEDA concentration was much steeper than alginate-PHEDA hydrogels. Aside from the obvious difference in reaction kinetics and physical properties between oxidized alginate and PEGDA, the cross-linking of PEGDA, having only two functional groups, may have been more susceptible to the change in the concentration of PHEDA, as compared to the oxidized alginate which, having multiple functional groups, could undergo cross-linking reaction at lower concentration.

chemistry has been widely applied to cross-link hydrogels for biomedical applications. Herein, PHEDA and PHHZA were used to engineer hydrogels by cross-linking macromers having acrylate and aldehyde via Michael addition and Schiff base formation, respectively, which are generally mild reactions that could occur in aqueous media with physiological pH and ionic strength.38,39 3.2.1. Schiff Base Formation. Oxidized alginate, which contains multiple aldehyde groups in the polymer backbone, was first used as the macromer system for hydrogel fabrication via Schiff base formation (Figure 2a).28 The concentration of oxidized alginate was kept constant at 5% (w/v), and the concentration of PHHZA was varied from 5 to 10% to fabricate the hydrogels. The gelation occurred approximately 5 min after the mixing of two stock solutions, demonstrating the extent of Schiff base formation in the given concentration range was sufficient for hydrogel fabrication. The moduli of the alginatePHHZA hydrogels increased with DS of PHHZA, and the range of moduli became greater with PHHZA concentration, as expected: 0.5−72 kPa for 10% PHHZA, 0.3−24 kPa for 7.5% PHHZA, and 2−12 kPa for 5% PHHZA (Figure 2b). On the other hand, the swelling ratios expectedly followed the opposite trend to the moduli, decreasing with DS and concentration of PHHZA; increasing cross-linking density limits the permeability of the hydrogels as outlined by the rubber-elasticity theory (Figure S7). This result clearly demonstrated the increased number of reactive functional groups on PHHZA were able to sufficiently participate in the cross-linking of oxidized alginate, leading to enhanced hydrogel rigidity. Also, the lowest DS of PHHZA for hydrogel became higher with decreasing PHHZA concentration (e.g., DS1 for 10% PHHZA, DS2 for 7.5% PHHZA, DS3 for 5% PHHZA), highlighting the critical number of reactive functional groups needed for sufficient cross-linking reaction. PHEDA was also used to cross-link the oxidized alginate to form hydrogels (Figure 2a). There were several notable differences from the PHHZA-linked hydrogels. First, the rate of gelation for the alginate-PHEDA hydrogels was much higher (within 5 s at room temperature) than that of alginate-PHHZA hydrogels. Second, their moduli were also larger at the same 695

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Biomacromolecules Unlike PHEDA reaction with PEGDA, the reaction between PHHZA and PEGDA did not result in hydrogel formation at the same range of concentration (5−10%). When the concentration was increased up to 20%, there was a small increase in the viscosity, but it did not lead to hydrogel formation. The extent of Michael addition between hydrazide in PHHZA and acrylate in PEGDA may not have been substantial enough at the given condition hydrogel formation. Combined with the lower range of elastic moduli for PEGPHHZA hydrogels over PEG-PHEDA hydrogels shown in Figure 2, it is likely that the reactivity of primary amine in PHEDA toward both Schiff base formation and Michael addition was greater than hydrazide groups in PHHZA. Though in different contexts, several previous studies have similarly demonstrated the greater nucleophilicity of primary amine over hydrazide.44,45 The reduced nucleophilicity of hydrazide over primary amine and hydrazine has been attributed to the electron-withdrawing effect of carbonyl group on the hydrazide. 3.3. Degradation of Polyaspartamide-Linked Hydrogels. It has been demonstrated that the polyamine-based hydrogels containing hydrolyzable groups could undergo accelerated degradation process possibly due to the increased local hydroxide ions with the protonation of amine groups and/ or the nucleophilic attack of the unreacted amine groups on the ester groups.46 Since PHEDA and PHHZA contain multiple nucleophilic amine and hydrazide groups that may stay unreacted after hydrogel formation, and the hydrogels containing hydrolyzable groups, it was thus postulated that the hydrogels could undergo degradation via hydrolysis. First, the hydrogels were incubated in PBS (pH 7.4) at 37 °C and their degradation was monitored by measuring the change in moduli over time until complete dissolution. The degradation rate constants (kd) were obtained by fitting the profile with eq 1. Alginate hydrogels linked by either PHHZA or PHEDA contain Schiff base linkages that could undergo hydrolysis especially under basic environment by unreacted hydrazide or amine.47 For alginate-PHHZA hydrogels, those with higher DS and concentration of PHHZA showed lower degradation rate and delayed dissolution, indicating the hydrogels with greater mechanical strength showed greater resistance toward degradation (Figure 4a,b). Between two conditions, 10% (DS3) and 7.5% (DS4) that had similar initial moduli, the degradation rate was higher for 10% (DS3). Similarly, the degradation rate of 7.5% (DS3) was higher than that of 5% (DS4). These results alluded to the greater presence of unreacted hydrazide groups at higher PHHZA concentrations likely facilitating the hydrogel degradation via increased hydrolysis. Surprisingly, on the other hand, the degradation of alginate-PHEDA hydrogels were extremely fast, regardless of the conditions, with the complete dissolution within an hour (data not shown). Unreacted primary amine groups in PHEDA likely induced much faster hydrolytic process of Schiff base than hydrazide in PHEDA. As a control, the hydrogels were treated with sodium cyanoborohydride to reduce the Schiff base to nonhydrolytic secondary amine groups and monitored the degradation.28 As expected, all hydrogels remained stable in the same aqueous media, demonstrating the degradation did occur via hydrolysis of the Schiff base linkages. The degradation of PEG-PHEDA hydrogels followed the similar trend as alginate-PHHZA hydrogels; lower degradation rates at higher PHEDA concentrations at a given DS. Unlike

Figure 4. Degradation of (a, b) alginate-PHHZA and (c, d) PEGPHEDA hydrogels were determined by measuring the change in moduli (E/E0) over time and calculating degradation rate constants (kd). The moduli (E) measured at various time points were normalized with the initial value (E0); kd were calculated by fitting with eq 1. *p < 0.05 in (b) and (d).

alginate-PHHZA hydrogels, the degradation rate was not as significantly affected by the difference in DS, despite the wide range of initial moduli. In addition, the degradation rates of the PEG-PHEDA hydrogels were generally higher than those of alginate-PHHZA hydrogels at their respective DS and concentrations, even though their initial moduli were much larger. Combined with the extremely fast degradation of PEGPHEDA hydrogels, it could be concluded that the unreacted primary amine groups in PHEDA could more rapidly facilitate the hydrolysis of Schiff base or ester groups than the hydrazide groups in PHHZA. 3.4. Drug Release Kinetics of Polyaspartamide-Linked Hydrogels. With the ability to control the mechanical and diffusional properties as well as induce degradation, the polyaspartamide-linked hydrogels were evaluated for controlled drug delivery applications. Bovine serum albumin (BSA) as a model protein drug was encapsulated in PHHZA- or PHEDAlinked hydrogels with varying DS and concentration, and the time-dependent release profiles were obtained. The release profiles from alginate-PHHZA hydrogels showed a typical diffusion-controlled release, with a power-law dependence on time (Figure 5a).31,36 The kinetic rate constants (k1), obtained by fitting the profiles with Ritger-Peppas model (eq 2), closely followed the trend of swelling ratios shown in Figure S7, where the values became smaller with DS and concentration of PHHZA as expected (Figure 5b). This result demonstrated that the drug release was further expedited in a more swellable hydrogel. In addition, the exponent values (n) were in between 0.3 and 0.4 for all conditions, confirming that the release followed a Fickian diffusion mechanism (Figure 5c).36,48 In contrast to the alginate-PHHZA hydrogels, the drug release from PEG-PHEDA hydrogels displayed a sigmoidal release profile, suggesting there was a more gradual increase in drug release rate than a typical initial burst release commonly found in diffusion-controlled systems (Figure 5d). Since PEGPHEDA hydrogels demonstrated faster degradation rate than alginate-PHHZA hydrogels, it was suggested that the degradation was a more significant factor for the drug release 696

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Figure 5. Drug release studies of (a−c) alginate-PHHZA and (d−f) PEG-PHEDA hydrogels. The release profiles (cumulative release over time) in (a) and (d) were fitted with eqs 2 and 3, respectively. From the release profiles of alginate-PHHZA hydrogels in (a), (b) kinetic rate constants (k1) and (c) exponents (n) were obtained. From the release profiles of PEG-PHEDA hydrogels in (d), (e) kinetic rate constants (k2) and (f) lag time constants (T) were obtained. *p < 0.05 in (b), (e), and (f).

Figure 6. (a) Synthesis of RGD peptide-linked polyaspartamide cross-linker, and the fabrication of peptide-linked hydrogel and subsequent cell culture. (b) The live (green) and dead (red) cells cultured on alginate hydrogel (left), alginate hydrogel linked with PHHZA (middle), and alginate hydrogel linked with RGD-PHHZA (right), are fluorescently labeled (scale bar: 100 μm). The cells were imaged after 1 (upper) and 3 days (lower) of culture.

the PEG-PHEDA hydrogels, which produced release rate constant (k2) and lag time constant (T).34,35 k2 values, similar to k1 values for alginate-PHHZA hydrogels, became smaller

in PEG-PHEDA hydrogels than alginate-PHHZA hydrogels. Therefore, a Weibull model (eq 3) that is better suited for fitting sigmoidal profiles, was used to fit the release profiles of 697

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degree of substitution (DS) of reactive functional groups on the polyaspartamide also could be controlled efficiently by varying the amount of nucleophilic reactant. Here, two different polyaspartamide cross-linkers having amine and hydrazide as nucleophilic functional groups were prepared, namely, PHEDA and PHHZA, which were capable of in situ cross-linking reaction under physiological conditions and showed different reactivities toward gel-forming polymers. The mechanical properties of the resulting hydrogels could be controlled in a wide range simply by changing the DS, while maintaining the overall polymer concentration. In addition, the hydrogels crosslinked by PHEDA and PHHZA were all shown to undergo degradation under physiological conditions, likely due to unreacted functional groups involved in hydrolysis. Adjusting the mechanical properties, combined with the degradation behavior, of the hydrogels allowed the controlled release of encapsulated drug molecules. Furthermore, by using a polyaspartamide cross-linker conjugated with cell-responsive peptides, cell-adhesive hydrogels as cell-culture scaffolds could also be prepared. With these collection of tunable physical and biological properties, it is expected that the strategy of utilizing the multivalency of polyaspartamide-based cross-linkers to create hydrogels with diverse functionalities could be successfully applied to biomedical engineering, including but not limited to drug delivery and tissue engineering.

with DS and concentration of PHEDA (Figure 5e). The trend in T values, which represent the amount of time required for the 50% release and thus signify the delay in release, was expectedly opposite to the k2 values, becoming larger with DS and concentration of PHEDA (Figure 5f). Taken together, the drug release rate and release pattern could be controlled using the polyaspartamide-linked hydrogels with varying cross-linking density. 3.5. In Vitro Cell Culture on Cell-Adhesive Peptide Linked Polyaspartamide Hydrogels. The ability to incorporate various amine-based molecules onto the polymer backbone via facile ring-opening nucleophilic addition is a hallmark of polyaspartamide. This aspect could open the door to accommodating moieties other than those involved with cross-linking reactions. This versatility of the polyaspartamide linker was further explored by conjugating cell adhesion peptide (i.e., RGD peptide) onto the PHHZA or PHEDA. Using this linker, the resulting peptide-conjugated hydrogels could be used as a cell culture platform. The RGD peptide-conjugated polyaspartamide was conveniently synthesized by first reacting the peptide with PSI prior to the conjugation of the hydroxyl and reactive functional groups (Figures 6a and S6). The RGD peptide-linked PHHZA (“RGD-PHHZA”) was conjugated onto alginate hydrogel to assess its capability of allowing cell adhesion to the hydrogel (Figure 6a). The alginate hydrogel was fabricated on top of a RGD-PHHZA coated surface in order to conjugate the RGD-PHHZA to the hydrogel surface. The alginate hydrogel was fabricated by cross-linking OAlg with AAD. Then, 3T3 fibroblasts were placed on top of the hydrogels and allowed to adhere after hydrogel fabrication. Pure alginate hydrogel and PHHZA-linked alginate hydrogel (without peptide) were used as controls. Only a few cells were adhered to the alginate hydrogel via nonspecific interaction, judging from their round morphology, which was expected since the pure alginate does not contain cell-specific moieties (Figure 6b). There was more cell adhesion and spreading on the PHHZA-alginate hydrogels than the pure alginate hydrogels possibly due to some cells adhered to unreacted amine groups of PHHZA by electrostatic interaction, a similar mechanism to poly(L-lysine) as a cell adhesion material.49 But compared to those two conditions, the number of viable cells was much higher on RGD-PHHZA-alginate hydrogels, indicating the RGD-specific cell adhesion further promoted the cell viability. In addition, after 3 days of culture, there were far more cells on the RGD-PHHZA-alginate hydrogels than other conditions, which also demonstrated that specific cell adhesion resulted in greater cell proliferation (Figure 6b). The same in vitro experiments were performed on the alginate hydrogels linked with RGD-PHEDA, instead of RGD-PHEDA. As expected, a similar result was obtained in which more cells were adhered on the RGD-PHEDA-alginate hydrogels over pure alginate hydrogel and PHEDA-alginate hydrogel (Figure S8). These results highlighted the versatility of the polyaspartamide crosslinker by providing cell-responsive moieties in conjunction with controlling the mechanical properties of the hydrogels.

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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.biomac.8b00068. Supporting figures, Figures S1−S8 (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +82-52-217-5328. Fax: +8252-217-2019. ORCID

Chaenyung Cha: 0000-0002-3615-0145 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Civil-Military Technology Cooperation Program (15-CM-SS-03), and Bio & Medical Technology Development Program and Leading Foreign Research Institute Recruitment Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017M3A9C6033875, 2017K1A4A3015437).

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REFERENCES

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