Embedding Well-Defined Responsive Hydrogels with Nanocontainers

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Article Cite This: ACS Omega 2017, 2, 6658-6667

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Embedding Well-Defined Responsive Hydrogels with Nanocontainers: Tunable Materials from Telechelic Polymers and Cyclodextrins Mehmet Arslan,† Duygu Aydin,† Aysun Degirmenci,† Amitav Sanyal,*,†,‡ and Rana Sanyal*,†,‡ †

Department of Chemistry, Bogazici University, Bebek, Istanbul 34342, Turkey Center for Life Sciences and Technologies, Bogazici University, Istanbul 34342, Turkey

‡

S Supporting Information *

ABSTRACT: Design, synthesis, and application of cyclodextrin (CD) containing thermoresponsive hydrogels fabricated from thiol-reactive telechelic polymers are reported. Hydrophilic polymers containing 2hydroxyethyl methacrylate and/or di(ethylene glycol)methylether methacrylate monomers as side chains and thiol-reactive groups at chain ends were synthesized. A series of hydrogels was fabricated using thiol−ene conjugation of these thiol-reactive polymers with multivalent thiolcontaining CDs as crosslinkers. Clear and transparent hydrogels were obtained with good conversion (79−89%) by utilizing the “nucleophilic” and “radical” thiol−ene “click” reactions. Analysis of the amount of residual thiol groups in these hydrogels using Ellman’s reagent suggested that gels with a moderately well-defined network structure were obtained. Hydrogels fabricated using different telechelic polymers were examined for their properties such as morphology, equilibrium water uptake, and rheological characteristics. Cytocompatibility of these hydrogels was ascertained by a cell viability assay that demonstrated low toxicity toward fibroblast cells. Thereafter, the CD-containing hydrogels were evaluated for the loading and controlled release of puerarin, an antiglaucoma drug. Utilization of thermoresponsive polymers as the matrix for these hydrogels allows use of temperature as a stimulus to modulate the drug release. A slower and more sustained drug release was observed at physiological temperatures compared to ambient conditions. The effect of temperature on the elasticity of the hydrogel was investigated rheologically to demonstrate that the collapse of the network structure occurs near physiological temperatures. The increased hydrophobicity and compactness of the gel matrix at higher temperatures results in a slower drug release. The strategy employed here demonstrates that tuning the matrix composition of hydrogels with well-defined network structures through appropriate choice of responsive copolymers allows design of materials with control of their physical properties and drug-release behavior.

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INTRODUCTION Cross-linked hydrophilic polymeric networks, often referred to as hydrogels, have drawn widespread interest in recent years as potential candidates for applications in areas such as fabrication of drug-release devices, tissue engineering scaffolds, implant materials, and bioimmobilization platforms.1−11 Over the past decade, a variety of different synthetic strategies have been employed for the preparation of hydrogels. Besides the synthesis of covalently cross-linked hydrogels via free-radical cross-linking of hydrophilic polymerizable units, various “click chemistry”-based methodologies including thiol−ene, azide− alkyne cycloaddition, and Diels−Alder reactions were effectively utilized in cross-linking of monomers and polymers.12−16 Along with the development of efficient cross-linking strategies, an important concept that has evolved in recent years relates to their synthesis with a well-defined network structure. Such structurally well-defined hydrogels are obtained when efficient network formation is accurately controlled by using building blocks containing precisely positioned cross-linking points.17−19 © 2017 American Chemical Society

Hydrogels with a near-ideal network structure enable fabrication of materials with a well-defined structure−property relationship.20 Near-ideal network structure hydrogels imply that polymer chain connectivity is homogenous throughout the gel. This is in contrast to hydrogels where cross-linking between polymeric chains is random, for example, often encountered in photopolymerized hydrogels. A simple approach to obtain near-well-defined network materials involves effective cross-linking of polymers bearing reactive groups at their chain ends, with multivalent cross-linkers. Polymers bearing reactive functional groups at their chain ends are referred to as telechelics and are important functional macromolecules widely used as building blocks, cross-linkers, and chain extenders.21−24 Telechelic polymers with precise Received: June 14, 2017 Accepted: September 27, 2017 Published: October 12, 2017 6658

DOI: 10.1021/acsomega.7b00787 ACS Omega 2017, 2, 6658−6667

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Scheme 1. Illustration of Thermoresponsive Hydrogel Fabrication via Thiol−Ene Addition Reactions and Their Drug-Loaded States

structures will enable rational improvement and tunability of such materials. We recently reported the fabrication of CD-containing hydrogels with well-defined network structures.41,42 Hydrogels were synthesized using nucleophilic and radical thiol−ene “click” reactions by utilizing the telechelic linear PEG-based polymers. Although the study demonstrated that multivalent CDs act as a versatile cross-linker, the utilization of linear PEGs can only allow tailoring of the properties of these hydrogels to a certain extent, and it lacks stimuli-responsiveness. To expand the properties of these materials to include aspects such as stimuli-responsiveness, the methodology should be adaptable to polymers other than linear PEGs. It is well-established that PEG-based graft polymers containing oligo(ethylene glycol) side chains exhibit lower critical solution temperature (LCST) in aqueous solution, and this behavior of DEGMA-containing copolymers has been exploited to obtain thermoresponsive hydrogel materials.43−48 Herein, we report the fabrication of CD-containing thermoresponsive hydrogels with near-well-defined network structures by employing copolymers containing PEG-based side chains. Thiol−ene click reactions between homotelechelic maleimide and vinyl group-functionalized (co)polymers with a heptathiol-functionalized β-CD crosslinker were utilized (Scheme 1). Efficient reactions between maleimide and vinyl groups with thiols through the nucleophilic and radical thiol− ene reactions, respectively, provided hydrogels with good yields. By varying the composition of the polymers, characteristics such as their water uptake capacity, surface morphologies, and rheological behaviors can be tuned. Importantly, utilization of thermoresponsive copolymers provides a handle for varying the properties of these hydrogels as a response to temperature. Temperature-responsive modulation of drug release from these CD-containing hydrogels was evaluated using puerarin, a poorly water-soluble drug employed in treatment of glaucoma. It can be envisioned that the reported hydrogels with discrete CD units and a temperature-responsive polymer matrix can be of potential interest in drug delivery devices such as soft contact lenses. The demonstrated UV-initiated curing of hydrogels, as well as catalyst-free thiol−maleimide addition can also enable fabrication of injectable hydrogels for tissue engineering and regenerative medicine.

end-group reactive functionalities provide ideal building blocks for fabrication of well-defined network structures. Utilization of hydrogels in drug delivery is widespread, and it is well-established that the release of drugs can be controlled by varying the chemical and physical properties of these materials.25−27 Depending on the drug and the intended application, the loading and release of the drug can be tuned using electrostatic and/or hydrophobic interactions with the hydrogel matrix. While most hydrophobic interactions are nonspecific, utilization of precise building blocks like cyclodextrins (CDs) provide an element of control because the interaction of the guest molecules depends on the size of the CD cavity. It has been demonstrated that β-CD, along with other CDs, has a remarkable ability of forming inclusion complexes with hydrophobic molecules.28−30 Because of this unique behavior, β-CD and its derivatives have been extensively investigated in the design of sustained drug delivery systems.31−34 The multivalent construct of β-CD also allows its use as a cross-linking agent for obtaining hydrogels, and several hydrogels have been prepared using this approach.35,36 In a recent study, Shih et al. utilized the multivalent thiol- and alkene-functionalized β-CDs for fabrication of polyethylene glycol (PEG)-based hydrogels in applications of drug delivery and cell encapsulation. The approach was to modify the CD hydroxyl groups with various degrees of substitution that the resulting CD derivatives enable the tuning of gelation kinetics and gel properties.37 Several significant studies combine the molecular recognition abilities of CDs with temperatureresponsive polymers to fabricate responsive hydrogels.38−40 However, importantly, none of these reports utilizes a strategy that would yield near-well-defined hydrogel structures, so the CDs are located in a heterogeneous environment instead of a homogenous one. This usually arises from employing CD building blocks where the number of reactive units is not precisely controlled. In addition to this, current CD-based hydrogel platforms utilize mostly N-isopropylacrylamide and related monomers as temperature-responsive units. New materials employing di(ethylene glycol)methylether methacrylate (DEGMA) and other PEG-based building blocks as temperature-responsive handles may provide a wider perspective of the material design by exploiting their important features, such as high biocompatibility and antibiofouling nature. Envisaging materials which combine discrete welldefined building blocks to produce well-defined network 6659


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RESULTS AND DISCUSSION Telechelic polymers with thiol-reactive end groups and 2hydroxyethyl methacrylate (HEMA)/DEGMA side chain groups were evaluated in the fabrication of CD-containing stimuli-responsive hydrogel networks (Scheme 2). Bismalei-

Subsequently, radical-induced cross-coupling reactions with functionalized azo-initiators were performed to introduce thiolreactive functional groups to the polymer chain ends. The dithiobenzoate end groups of the copolymers were replaced with reactive maleimide or unactivated alkene moieties by using 20-fold excess of appropriately functionalized azo compounds per chain end group to provide cross-coupling products in a quantitative manner. After extensive purification of the modified polymers to remove the small molecule impurities, successful end-group transformation was established by 1H NMR analysis (Figures S8 and S9). Hydrogels were prepared via thiol−ene reactions of bisalkene-functionalized (co)polymers with a heptavalent thiol-functionalized β-CD cross-linker (β-CD(SH)7). Relatively small molecular weight polymers were chosen to promote high coupling efficiency during gelation. Hydrogels via nucleophilic thiol−ene reactions were obtained by combining the solutions of maleimide-functionalized homotelechelic polymers and βCD(SH)7. Through multiple Michael additions of thiol groups onto maleimides, rapid cross-linking occurs, and in approximately 1 min, no flow of sample was observed. To ensure complete network formation, gelation was continued for 12 h. As expected for the nucleophilic thiol−maleimide reaction, it was observed that gelation was promoted in the presence of a catalytic amount of Et3N. For the bisvinyl-functionalized RAFT polymers, hydrogels were obtained via UV-induced thiol−ene reaction of vinyl functionalities with thiol groups of the crosslinker β-CD(SH)7. The reactions were conducted under UV light (365 nm) in the presence of 2,2-dimethoxy-2-phenylacetophenone (DMPA) as a photoinitiator. To compare the effect of side chain groups on the physical and morphological properties, a library of hydrogels was prepared using HEMA-/ DEGMA-based homopolymers and copolymers. The nomenclature indicating composition of polymers and hydrogels utilized is as follows: P-BM-D refers to polymer (P)-containing bismaleimide (BM) and DEGMA (D), whereas H-CM-D suggests hydrogel (H) obtained from conjugation of CD with a maleimide polymer (CM) composed of DEGMA. The abbreviation HD refers to polymers and hydrogels with an equimolar amount of HEMA and DEGMA. Table 1 summarizes the compositions, feed ratios, and gel conversions of thus obtained hydrogels. Thiol−maleimide Michael addition and radical thiol−ene reactions have been widely employed to synthesize and functionalize polymeric materials because the reactions proceed with high conversions under metal-free conditions.49−54 Polymers with maleimide or alkene end functionalities were reacted with a β-CD(SH)7 cross-linker through nucleophilic and radical thiol−ene reactions to prepare hydrogels H-CMs and H-CVs (Table 1, entry 1−6). HEMA-/DEGMA-based

Scheme 2. Synthesis of Thermoresponsive Hydrogels via Thiol−Ene Addition Reactions

mide and bisvinyl end-functional polymers (P-BMs and P-BVs, Scheme 2) were obtained by utilization of reversible addition− fragmentation chain transfer (RAFT) polymerization, with subsequent postpolymerization modification (synthetic details provided in Supporting Information). Chain transfer agents carrying furan-protected maleimide and vinyl groups were used in the polymerization of HEMA and DEGMA monomers (Scheme S1 and Figures S1−S6). Analyses of these polymers using proton nuclear magnetic resonance (1H NMR) and size exclusion chromatography revealed that polymers were obtained with good control over molecular weight and relatively narrow distributions (Table S1 and Figure S7).

Table 1. Gel Conversions, Total Thiol Contents, and Drug Loading Amounts of Synthesized Hydrogels entry

hydrogel

polymer

feed ratio [−SH]/[alkene]

% gel conv.

1 2 3 4 5 6

H-CM-D H-CM-HD H-CM-H H-CV-D H-CV-HD H-CV-H

P-BM-D P-BM-HD P-BM-H P-BV-D P-BV-HD P-BV-H

1:1 1:1 1:1 1:1 1:1 1:1

84.0 87.0 89.0 81.0 79.0 83.0

thiol contenta (mmol × 10−4) 21.7/3.95 22.9/3.82 23.8/3.38 24.5/5.75 25.0/6.35 25.3/4.98

(±0.41) (±0.38) (±0.32) (±0.53) (±0.47) (±0.43)

% thiol consumed 82.0 83.0 86.0 77.0 75.0 80.0

drug loadb (mg/g dry gel) 19.07 23.84 27.59 15.31 20.29 22.17

± ± ± ± ± ±

3.41 2.73 4.31 2.42 3.56 3.19

a

Amount of thiol (molar) used/amount of thiol (molar) determined in the hydrogel (triplicate runs). bDrug loaded in hydrogels from 0.80 mg/mL puerarin solution. 6660


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Figure 1. ESEM micrographs of freeze-dried gel samples: (a) H-CM-D, (b) H-CM-HD, (c) H-CM-H, (d) H-CV-D, (e) H-CV-HD, and (f) H-CVH. Large image scale bar: 100 μm; small images: 20 μm.

Figure 2. Equilibrium swelling of hydrogels prepared using (a) bismaleimide-functionalized polymers and (b) bisvinyl-functionalized polymers.

proceeded with efficiencies between 80 and 90%, and a slightly higher conversion was observed for the hydrogels obtained using thiol−maleimide addition. The most likely reason for nonquantitative thiol consumption is the high steric crowding around the heptavalent cross-linker that diminishes the efficient coupling of complementary groups. Thus, to test the effect of cross-linker functional group crowding on conjugation efficiency of maleimide and vinyl groups with thiols, a series of hydrogels were synthesized using tetra-thiol-functionalized cross-linker and analyzed for thiol-group consumption (Figure S10). Hydrogels synthesized by using P-BM-H and P-BV-H polymers with a sterically noncrowded tetra-thiol-functionalized cross-linker pentaerythritol tetrakis(3-mercaptopropionate) resulted in higher gel conversions (97 and 91%, respectively) as well as increased thiol consumptions (91.6 and 86.3%, respectively, Table S2, Supporting Information), which suggests the effect of steric crowding at the cross-linking points on conjugation efficiency. The hydrogels obtained using nucleophilic and radical thiol− ene reactions were clear and transparent in their wet state. On the basis of the environmental scanning electron microscope (ESEM) analysis of freeze-dried hydrogels, continuous gel structures with low porosities were observed for DEGMAbased hydrogels (Figure 1). Notably, for both of the hydrogel series prepared by bismaleimide- and bisvinyl-functionalized polymers, relatively higher porous structures were observed for

polymers with similar molecular weights were used in gel fabrication. In each case, the amount of cross-linker and polymer was adjusted to obtain molar-equivalence coupling of alkene and thiol functionality. In ideal circumstances, it can be assumed that complete pairing of maleimide or vinyl groups with thiols would render well-defined network structures. On the other hand, the steric bulk of polymer chains, chain entanglements, loop formations, steric crowding, and undesirable side reactions at cross-linking nodes will compromise the ideal network formation. Hydrogels were obtained with good gel conversions, yet attempts of further increase in yields were unsuccessful, and this was attributed to abovementioned reasons. In general, slightly higher gel conversions were obtained for the hydrogels prepared by thiol−maleimide conjugation. To gather further information about the cross-linking efficiency of the processes, residual thiol content in hydrogels were obtained via Ellman’s thiol analysis. Hydrogels prepared using various telechelic polymers were reacted with 5,5-dithiobis-2-nitrobenzoic acid (DTNB, Ellman’s reagent), and the resulting 2-nitro-5-thiobenzoic acid (TNB) fragment were quantified spectrophotometrically. To keep the disulfide formation minimal, hydrogels were analyzed directly after their preparation. The values of the amount of thiol used and the percent thiol consumed in hydrogel formation are reported in Table 1. According to the results, thiol consumptions 6661


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Figure 3. The frequency dependence of storage (G′, solid symbols) and loss (G″, open symbols) moduli of hydrogels prepared by (a) bismaleimidefunctionalized polymers and (b) bisvinyl-functionalized polymers.

Figure 4. Thermoreversible nature of hydrogels monitored by heating the swollen gel. Transition temperature was measured by monitoring the change of transmittance upon heating.

pH, and magnetic field) have been extensively investigated as important materials in drug delivery.56 These materials are often referred as intelligent/smart hydrogels because they respond to physical or chemical stimuli by phase transition or volume collapse. Especially, changes in swelling/deswelling kinetics allow the controlled release of cargo entities. Temperature-responsive hydrogels are by far the most extensively studied materials as smart delivery platforms. Among the widely employed and most extensively studied polymers for the preparation of stimuli-responsive drug delivery platforms are poly(N-isopropylacrylamide) (PNIPAAm)-based systems that exhibit thermally reversible phase changes above ∼32 °C. Far less studied polymers based on oligoethylene glycol groups that possess an LCST of ∼26 °C and above, depending on the number of ethylene glycol groups, might provide a convenient tool to control the temperature responsiveness over a wider temperature range. The presence of diethylene glycol side chains leads to a thermoresponsive behavior because such polymers possess an LCST of ∼26 °C.57,58 Thus, in this study, the hydrogel H-CV-D, prepared from a bisvinyl-functionalized DEGMA homopolymer was investigated in terms of temperature-responsive gel properties. According to the turbidimetry measurements of H-CV-D, a transition temperature near 28 °C was observed when monitoring the loss of transmittance with increasing temperature (Figure 4). The sample displayed a reversible phase transition as a transparent gel was obtained upon cooling. The variation of the swelling ratio of the hydrogel below and above the LCST temperature (25 and 35 °C) also indicated the

HEMA-based gel networks. Hydrogels were investigated in terms of their swelling behaviors by monitoring their water uptake with time until a constant weight was reached. All hydrogels showed high swelling capacity and fast swelling kinetics because of hydrophilic side chains (Figure 2). Higher swelling was obtained with HEMA-based hydrogels, which can be attributed to the higher hydrophilic character of the HEMA monomer. The viscoelastic gel properties were examined via dynamic frequency scan analysis of samples at equilibrium swelling. The measurements revealed that the storage and loss moduli of networks exhibit low oscillation frequency dependence suggesting the viscoelastic gel behavior (Figure 3).55 The storage (G′) and loss (G″) moduli values ranged from 101 to 104 Pa, and for the all samples tested, the storage modulus was found to be greater than loss modulus which is a distinguishing rheological property of elastic solid materials. It was observed that the feed of DEGMA in hydrogel composition affects the rheological properties, as stronger viscoelastic materials were obtained upon increasing the DEGMA content. The potential applications of hydrogels are mostly related to the composition of the network structure, whereby the structural and chemical compositions of the hydrogel define their physical and chemical characteristics. To further demonstrate the utility of the polymeric building blocks chosen in this study, hydrogels obtained from HEMA- and DEGMAbased hydrophilic polymers were evaluated as temperatureresponsive materials. Hydrogels undergoing conformational changes in response to changing stimulus (i.e., temperature, 6662


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Figure 5. Temperature-dependent swelling of hydrogels (a) H-CV-D and (b) H-CV-H.

Figure 6. Temperature dependence of storage (G′, solid symbols) and loss (G″, open symbols) moduli of hydrogels (a) H-CV-D and (b) H-CV-H.

tunability of the gel behavior. Above the LCST, the equilibrium water uptake capacities of these gels were appreciably reduced (Figure 5). Rheological studies performed at temperatures below and above the LCST revealed increased storage modulus (G′) values at higher temperatures (Figure 6). Such a behavior can be expected based on the collapsed network structure due to loss of hydration of polymer chains. As expected, the change in transparency, water uptake, and rheological studies performed using a bisvinyl-functionalized HEMA polymer demonstrated no significant temperature responsive behavior (Figures 4−6). The in vitro cytotoxicity of nucleophilic and radical thiol− ene hydrogels was evaluated on L929 fibroblast cell line using CCK-8 cell viability assay (Figure 7). The results demonstrate

no significant cytotoxicity in case of both types of hydrogels, and thus suggest that the biocompatibility of the fabricated materials makes them suitable for various biomedical applications. Release of therapeutically relevant materials such as drugs and biomolecules from hydrogels holds immense potential to realize pragmatic administration systems. Most of the strategies to load and release drugs from noneroding hydrogels are based on the diffusion of encapsulated drug molecules into and out of the network. Thus, the matrix composition plays a very important role. The drug uptake and release properties of obtained hydrogels were studied using a poorly water-soluble drug, puerarin, a traditional Chinese medicine used in the treatment of glaucoma (solubility as 0.011 M at 25 °C).59 It is been established that β-CD can form inclusion complexation with puerarin, and the stability constant of the inclusion complex between puerarin and β-CD is 266.26 M−1.60 Hydrogels prepared from HEMA-/DEGMA-based polymers and β-CD(SH)7 were loaded by puerarin using the solution absorption method. Hydrogel disks swollen in water were soaked in drug solution (0.80 mg/mL), and the drug loading was checked spectrophotometrically until it reached equilibrium, usually after incubating for a period of 24 h (Table 1). Hydrogel drug loading capacities were found to be dependent on the nature of the copolymer used. For all hydrogels, increasing the HEMA content caused an increase in drug loading. This result could be attributed to the fact that the highly hydrophilic nature of the HEMA-based matrix allows better diffusion of the drug into the hydrogel. For the release

Figure 7. Cytotoxicity of hydrogels as determined using CCK-8 cell viability assay. 6663


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Figure 8. Cumulative release of puerarin from hydrogels (a) H-CMs and (b) H-CVs.

Figure 9. Temperature-dependent release of puerarin from hydrogels (a) H-CV-D and (b) H-CV-H.

expected, for the HEMA-based hydrogel (H-CV-H), a higher initial burst release was observed, as temperature was increased from 25 to 37 °C (Figure 9b). These results demonstrate the judicious choice of synthetic polymers as the hydrogel matrix to obtain near-well-defined structured materials which can provide responsive materials with tunable properties.

studies, hydrogels loaded with puerarin were gently rinsed with distilled water and placed in a release medium. To mimic sink conditions, the drug-released solution was switched with a fresh one after regular time intervals. The drug release was monitored by collecting the release samples and analyzing via UV spectroscopy. As shown in Figure 8, all hydrogels exhibited an initial burst release which could be ascribed to the removal of the free drug near the surface of the hydrogel.61 An increase in the amount of burst release was observed for hydrogels with a higher HEMA content, while all DEGMA-based hydrogels showed a lower initial burst and more sustained release. Among the hydrogel series prepared by nucleophilic and radical thiol− ene reactions, radical thiol−ene-based hydrogels exhibited a relatively low initial burst and a more sustained release. This discrepancy may originate from the more compact network structure of UV-fabricated gels, as well as the difference in polarity of chemical composition at cross-linking points because these gels also exhibited lower water retention. The cumulative release of puerarin from CD-incorporated hydrogels and release profiles are comparable with other systems reported in literature and may be altered by using different concentrations of puerarin in the soaking solution.62 The temperature-dependent release studies of the drug from hydrogels demonstrated stimuli-responsive modulation of release profiles. The release of the drug from DEGMA-based hydrogel (H-CV-D) decreased upon increasing the temperature from 25 to 37 °C (Figure 9a). The lower initial burst release above LCST was attributed to the entrapment of the drug molecules within the collapsed hydrogel matrix. However, as

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CONCLUSIONS Utilization of telechelic polymers in the fabrication of β-CDcontaining hydrogels was demonstrated. HEMA- and/or di(ethylene glycol) methyl ether methacrylate (DEGMA)based hydrophilic polymers were synthesized using RAFT polymerization. Thiol-reactive maleimide and vinyl functional groups were placed at polymer end groups by using a functional group containing chain transfer agents and postpolymerization modification of existing polymers. The end-group functional polymers were utilized in hydrogel fabrication. Obtained hydrogels comprising different polymers were characterized in terms of properties like equilibrium water uptake, rheological behavior, and morphology. These β-CD-containing hydrogels have been tested on drug sorption and controlled release by employing an antiglaucoma drug puerarin. According to the release studies, radical thiol−ene-based hydrogels exhibited a relatively lower initial burst and more sustained release on puerarin instance. In addition, temperature-responsive release profiles were observed in the DEGMA-based network. It can be envisioned that the methodology outlined here can be extended to obtain near-well-defined CD-containing hydrogels that 6664


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reaction medium. A solution of Ellman’s reagent was obtained using DTNB (4 mg) in 1 mL of reaction buffer. A fresh hydrogel sample (5 mg) was placed in a vial and 2.5 mL of reaction buffer and Ellman’s reagent solution was added onto the sample. After incubation for 2 h at 37 °C, the total thiol content was calculated by measuring the absorbance at 412 nm and using the extinction coefficient of TNB2− species (14 150 M−1 cm−1).65 Swelling Studies. A cylindrical sample of a hydrogel with a size of ∼1 cm in diameter and ∼0.2 cm in height was placed in a vial of deionized water, and the change in mass of the sample was measured at different times till the hydrogel reached constant weight. The percentage change in weight was calculated using the equation

would be responsive toward other physical, chemical, or biological triggers through judicious choice of the polymeric building blocks.

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EXPERIMENTAL SECTION Materials and Characterization. All chemicals used were of reagent grade (Sigma-Aldrich Co., USA) and were used as received. The solvents were obtained from Merck Co. (Germany) and used as received. The detailed synthesis of thiol-reactive telechelic polymers is described in Supporting Information. Synthesis of β-CD thiol (β-CD(SH)7) was undertaken according to reported literature.63 The puerarin used was supplied by TCI Chemicals (Japan). Thiol−ene reactions of hydrogel synthesis were performed at 365 nm using a handheld UV lamp (Blak-Ray UVP model B-100AP/R high intensity UV lamp with a 100 W spot bulb and 7° beam width). Morphologies of dried gels were investigated without sputtering with an ESEM XL-30 (Philips, Eindhoven, The Netherlands) SEM operating under high vacuum, with a 10 kV accelerating voltage at a working distance around 10 mm. The rheological behaviors of hydrogel samples were characterized in terms of loss (G″) and storage (G′) moduli of hydrogel samples equilibrated in water by measuring the angular frequency dependence of modulus values. Measurements were conducted out at 25 °C using 0.5% strain between 0.05 and 100 rad/s using an Anton Paar MCR 302 rheometer. A parallel plate of 8 mm diameter was used for analysis and a gap of 2.0 mm between plates was used. During measurements, a solvent trap was used to reduce solvent evaporation. Representative Hydrogel Formation via Nucleophilic Thiol−Ene Reaction. Hydrogels were obtained through multiple thiol−ene reactions between maleimide groups of polymers and β-CD(SH)7. For a representative example, the bismaleimide-functionalized polymer P-BM-HD (50.0 mg, 14.0 × 10−3 mmol) was added to a vial containing dimethylformamide (DMF) (100 μL). A solution of β-CD(SH)7 (5.0 mg, 4.0 × 10−3 mmol) and triethylamine (0.39 μL, 2.8 × 10−3 mmol) in DMF (100 μL) was then added to this solution. To achieve homogenous gelation, the mixture was sonicated briefly. In about 1 min, no flow of sample was observed. To ensure complete conjugation, gelation was continued for 12 h. After gelation, unreacted reagents were removed by washing the gel with DMF followed by distilled water several times. The gel sample was frozen and lyophilized to yield a dried sample. Representative Hydrogel Formation via Radical Thiol−Ene Reaction. Hydrogel synthesis was accomplished through multiple thiol−ene reactions between vinyl groups of telechelic polymers and thiol groups of β-CD(SH)7. The bisvinyl-functionalized polymer P-BV-HD (50.0 mg, 0.013 mmol) was placed in a vial and dissolved in DMF (100 μL). A mixture of β-CD(SH)7 (4.72 mg, 3.8 × 10−3 mmol) and DMPA (0.2 equiv per thiols) in DMF (100 μL) was then added and irradiated with UV light for 30 min at 365 nm. After gelation, residual chemicals were removed by rinsing the gel with DMF and distilled water several times. The dried gel was obtained by freeze-drying. Determination of Thiol Content. The total thiol amount left in the gels were obtained using Ellman’s sulfhydryl assay.64 After reacting free thiol groups of hydrogels with DTNB, they were obtained with reference to the extinction coefficient of TNB2− species. The experimental procedure is briefly as follows: 0.1 M sodium phosphate, containing ethylenediaminetetraacetic acid (1 mM, pH 8.0) buffer was prepared as a

% swelling = (Wwet − Wdry )/Wdry × 100

(1)

where Wwet and Wdry refer to the wet and dry hydrogel weights, respectively. The swelling curves were plotted by taking the average of three measurements. Loading and Release of Drug. Loading of drug into the gels was accomplished by the solution absorption method. Gel samples (∼50 mg) were incubated in a puerarin-containing aqueous solution (25 mL 0.80 mg/mL) with gentle stirring at 100 rpm. Change in the puerarin concentration in the solution was analyzed using a UV−vis spectrophotometer at 250 nm until a constant concentration was achieved (usually about 24 h). The drug loading amount was determined from the difference between the initial and the final concentration of the drug in the incubation medium. The percent drug loading was calculated as % loading = Wdrug /(Wdrug + Whydrogel) × 100

(2)

where Wdrug and Whydrogel refer to the weight of the drug and the weight of the hydrogel sample, respectively. The drug loaded hydrogels were rinsed with distilled water (2 mL) and then immersed in distilled water (15 mL) as the release medium. The release tests were carried out at 37 °C by shaking the medium at 100 rpm. The release medium (5 mL) was sampled at predetermined time intervals and replaced with the same volume. The amount of the drug in the collected media was determined using UV−vis spectroscopy. The results were expressed in terms of cumulative release as a function of time. Cytotoxicity Experiments. Cytotoxic activity of the hydrogels was tested using the L929 mouse fibroblast cell line. Cells were seeded in a 96-well plate, 100 μL of appropriate culture medium at a density of 5000 cells/well, and incubated at 37 °C for 24 h. Cell-adhered plates were treated with hydrogels H-CM-HD and H-CV-HD (5 mg) at 37 °C for 48 h. After the incubation, sample solutions were removed, and wells were washed twice with PBS (100 μL). Cell viability was determined by using CCK-8 assay. By adding CCK-8 (10%) to every well (total 70 μL) and incubation for 4 h, absorbance at 450 nm was measured using a microplate reader. These experiments were repeated three times.

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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/acsomega.7b00787. Synthesis details of telechelic polymers (PDF) 6665


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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +90-212-3597613. Fax: +90-212-287-2467 (A.S.). *E-mail: [email protected]. Phone: +90-212-359-4793. Fax: +90-212-287-2467 (R.S.). ORCID

Amitav Sanyal: 0000-0001-5122-8329 Rana Sanyal: 0000-0003-4803-5811 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors thank The Scientific and Technological Research Council of Turkey (TUBITAK, project no. 211T036) and the Ministry of Development of Turkey (DPT, 2009K120520 and 2012K120480) for financial support.

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