Protein-Cross-Linked Triple-Responsive Polymer Networks Based on

Letter pubs.acs.org/macroletters

Protein-Cross-Linked Triple-Responsive Polymer Networks Based on Molecular Recognition Dawei Chen,†,‡ Wangmeng Hou,†,‡ Dongxia Wu,§ Yunfang Wu,§ Guochen Cheng,§ and Hanying Zhao*,† †

Key Laboratory of Functional Polymer Materials, Ministry of Education, College of Chemistry, Nankai University; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China § The Institute of Seawater Desalination and Multipurpose Utilization, SOA, Tianjin 300192, China S Supporting Information *

ABSTRACT: Hydrogels containing protein components are a type of promising biomaterial. In this paper, we designed tripleresponsive polymer−protein networks based on molecular recognition. Reduced bovine serum albumin (BSA) was modified with multiple β-cyclodextrin (βCD) by thiol−disulfide exchange reaction. The βCD-modified BSA was added into the aqueous solution of acrylamide copolymer with pendant adamantyl groups, resulting in the formation of polymer− protein network structures. The assembled polymer networks show triple-responsive behaviors upon treatment with trypsin, reduced glutathione, or native βCD. The network structures may find applications in tissue engineering and drug controlled release.

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the addition or removal of Ca2+, resulting in responsive swelling/deswelling behaviors.17 Hydrogel networks based on molecular recognitions have been extensively studied.22−32 Due to the reversibility of the noncovalent inclusion, these networks exhibit shear-thinning23,30 and self-healing30,32 properties, are able to degrade in response to excess competing guest or host molecules,25 and have found applications in the detection of metal ions.33 In recent years, fabrications of hydrogels with multiresponsiveness have been attracting increasing attention.34−38 By integrating multiple responsive features into one system, structural diversity and better control over the desired functionalities can be achieved.35 For example, pH/temperature dual-responsive hydrogels used as injectable hydrogels avoid gel formation and clogging during injection.36,37 Also, hydrogels are designed to respond only when several external stimuli are present simultaneously.34,38 In addition, the introduction of bioresponsiveness, such as enzyme35,38 and antigen39 responsiveness, into multiresponsive hydrogels were also studied. The introduction of both protein cross-linkers and nature-inspired specific interactions into hydrogels will produce new hybrid materials with multibiofunctional/responsive features and promote the applications of the artificial materials in biomedical fields. One of the most important applications of hydrogels is in the field of tissue engineering. The hydrogels are used as scaffolds

ydrogels are three-dimensional polymer networks distinguished by high water content and diverse properties. Because of the hydrophilicity and the potential biocompatibility, the applications of hydrogels in biomedical fields have been widely investigated.1 In these years, with major breakthroughs in polymer chemistry and biomolecular research, rational designs of biomimetic hydrogels have been developed rapidly. To mimic biological structures, natural components such as proteins or peptides are introduced into synthetic hydrogels, which are endowed with nontoxicity, biodegradability, biocompatibility, and biofunctionalities. The protein hydrogels have found applications in tissue engineering, drug controlled release, and other related areas.2−4 A direct way of incorporating natural components into hydrogels is the use of biomacromolecules as cross-linkers. Previously, biomolecules including native,5−7 modified,8−10 and recombinant proteins and peptides2−4,11−14 have been used in hydrogel cross-linking. The protein components in the hydrogel networks act as carriers in the delivery of drugs,2,8 catalyze self-healing process,7 induce hydrogel motions by protein conformational changes,11,14 and present superabsorbent performance.5,6 Biomolecule-involved specific interactions3,15−20 or molecular recognitions21 have been used in the fabrication of hydrogel networks, leading to stimuli-responsive hydrogels. For example, hydrogel membranes cross-linked by antibody− antigen recognition are able to swell under antigen stimulus, which can be used to control the permeation of protein drugs.16 The binding between calmodulin and phenothiazine incorporated in the hydrogel networks can be reversibly controlled by © XXXX American Chemical Society

Received: October 1, 2016 Accepted: October 17, 2016

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DOI: 10.1021/acsmacrolett.6b00750 ACS Macro Lett. 2016, 5, 1222−1226

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groups on a polymer chain. On each BSA molecule, there are 17 disulfide bonds and a single free thiol group. By reducing part of disulfide bonds into thiol groups, BSA molecules with multiple thiol groups were obtained. As determined by Ellman’s assay (Figure S4), the average number of thiol groups on a reduced BSA molecule is 11. Pyridyl-disulfide-modified βCD (βCD-s-s-Py) was prepared by a reaction of 2,2′-dithiodipyridine and thiol-modified βCD. The synthetic details of βCD-ss-Py can be found in the Supporting Information. 1H NMR, 13C NMR, and mass spectrum of βCD-s-s-Py are shown in Figure S5. BSA-βCD was prepared by thiol−disulfide exchange reaction between the reduced BSA and βCD-s-s-Py. In the exchange reaction, pyridine-2-thione was produced as a byproduct (Scheme 1). UV−vis measurements based on absorption of pyridine-2-thione at 342 nm in aqueous solution (Figure S6) demonstrated that the average number of βCD connected to each BSA molecule is 8. The successful modification of BSA with βCD was also confirmed by matrixassisted laser desorption ionization time-of-flight (MALDITOF) spectrum (Figure S7). Cross-peaks in 2D 1H NOESY NMR spectrum identify protons undergoing “through space” dipolar interactions.40 2D 1 H NOESY NMR spectrum of BSA-βCD/PAM-co-PNADAM mixture prepared at an equal molar ratio of βCD to AD confirms the interaction between AD and βCD groups (Figure 1). As shown in Figure 1c, the observed cross-peaks indicate

to organize cells into three-dimensional architectures allowing the growth and the formation of tissues.4 The hydrogels as scaffolds should be degradable and can be prepared under mild conditions. Herein, we report a novel strategy to fabricate protein-cross-linked triple-responsive polymer networks. Due to the facile synthesis and the controlled biodegradability of the hydrogels, our strategy will be used in the preparation of scaffolds. The key step in the synthesis of the network structures is to modify protein cross-linkers with host molecules. Disulfide bonds of bovine serum albumin (BSA) were cleaved, leaving multiple thiol groups on the protein molecules. β-Cyclodextrin (βCD) groups were grafted to the protein molecules by thiol−disulfide exchange reaction. In the meanwhile, poly(acrylamide) (PAM) with pendant adamantyl (AD) groups was prepared by free-radical copolymerization. Due to the strong inclusion interaction between βCD and AD, the aqueous solution of the copolymer turns into gel upon addition of βCD-modified BSA (BSA-βCD). The network structure has triple responsiveness. The protein cross-linker is degradable in the presence of protease, the disulfide bonds can be cleaved by natural reductants such as reduced glutathione (GSH), and native βCDs can compete with the βCDs in the inclusion complexes, leading to the dissociation of the network structures. The triple-responsive polymer−protein hydrogels will find applications in tissue engineering. Scheme 1 illustrates the synthesis strategy for the fabrication of polymer−protein networks. The network structure is Scheme 1. Schematic Depiction for the Fabrication of the Protein-Cross-Linked Polymer Networksa

a

The magnified inset shows a crosslinking point in the network structure. BSA molecules were reduced with tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and thiols were produced on the protein molecules.

Figure 1. 2D 1H NOESY NMR spectrum of a 1.0 wt % mixture of PAM-co-PNADAM and BSA-βCD at a βCD/AD molar ratio of 1:1: (a) Numbering of the various hydrogen atoms in AD and βCD, and an illustration of their relative positions in the complex; (b) overall spectrum of the mixture; (c) the cross-peaks indicating the strong interaction between AD and βCD hydrogen atoms.

composed of PAM copolymer with pendant AD groups and BSA-βCD as the protein cross-linker. PAM was chosen for its good solubility in water. To graft AD groups to PAM, an ADbearing monomer (N-adamantyl acrylamide, NADAM) was synthesized and copolymerized with acrylamide. 1H NMR, 13C NMR, and mass spectrum of NADAM monomer are shown in the Supporting Information (Figure S1). As determined by gel permeation chromatography (GPC) equipped with light scattering detector, the absolute weight-average molecular weight and the dispersity of the copolymer, PAM-co-PNADAM, are 461.2k and 1.20, respectively. GPC curves of the copolymer obtained by using right angle laser light scattering (RALS) and refractive index (RI) detector are shown in Figure S2. Based on 1 H NMR result (Figure S3), the molar ratio of AM to NADAM in the copolymer is 83/1. In average, there are about 75 AD

strong interaction between the H3 protons of βCD and the Ha protons of AD in an inclusion complex, demonstrating a deep insertion of AD into the βCD ring. Figure 2a shows plots of viscosity vs shear rate for BSAβCD/PAM-co-PNADAM mixtures at different molar ratios of βCD/AD. Upon addition of BSA-βCD to PAM-co-PNADAM solutions, the viscosities of the solutions increase rapidly. At the same shear rate, the mixture solution prepared at an equal molar ratio of βCD to AD exhibits a viscosity value 2 orders of magnitude higher than the polymer solution. The rise in solution viscosity, together with 2D NMR analysis, demon1223

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reciprocal of the crossover frequency corresponds to the characteristic relaxation time of the network structure.27 As the molar ratio increases, the cross-linked network becomes more constrained, and the motion of the polymer chains is slowed down, which leads to longer relaxation time. It is expected that the highest cross-linking density is achieved at an equimolar ratio of βCD to AD, because the competition of the excess AD or βCD with the host−guest inclusion complexes will result in deficiency of the formed networks.24,25,31 As shown in Figure 2a, the network viscosity at the same shear rate increases with βCD/AD, even after the molar ratio exceeds 1:1. This can be explained by the steric hindrance effect. βCD groups are grafted to the protein molecules, and AD groups are attached to the polymer chains. Because of the steric hindrance between protein molecules and polymer chains, some βCD groups cannot reach AD groups, and as a result, not all the grafted βCDs are available to form inclusion complexes with AD groups. Under shear stress, the host−guest inclusion complexes of βCD and AD will be dissociated, which leads to shear-thinning behavior of the polymer networks.24,27 As shown in Figure 2a, the shear-thinning behaviors of the denser networks prepared at higher βCD/AD molar ratios appear at lower shear rates, and their viscosities decrease more drastically with shear rate. Hydrogel prepared at a low βCD/AD molar ratio has a low cross-linking density and loose network structure, which is capable of withstanding large shear force. Reversibility is one of the notable features of physical networks.23,30 After being broken by large deformation, the network structures are able to self-rebuild under gentle shear force. Rheological time sweeps were performed to investigate the speed and the reversibility of the network rebuilding process. Samples prepared at two different βCD/AD molar ratios were switched between low (0.1 s−1) and high (1000 s−1) shear rates, and the viscosity curves are shown in Figure 3a. It can be seen from the curves that, after shifting from a high shear rate to a low shear rate, the viscosities of the networks can be restored within short time, and the processes are totally reversible in three cycles without obvious changes in recovery speeds and steady-state viscosities. Comparing the two samples prepared at 1:1 and 1.5:1, the network structure with higher molar ratio shows slower shear-thinning and recovery behaviors. For the network prepared at equimolar ratio, it only takes about 30 s to regain 90% of its original viscosity, while for the network prepared at 1.5:1, the recovery process takes over 100 s (Figure 3b). The difference in the dynamic response is attributed to the higher cross-linking density and longer relaxation time of the networks with higher βCD/AD ratios. The hybrid polymer networks containing BSA molecules, disulfide bonds and the host−guest inclusion complexes exhibit triple responsiveness. Native βCD molecules compete with the βCD in the host−guest inclusion complexes, resulting in the cleavage of the cross-linking sites. The redox-responsive disulfides can be reduced to thiols by a reducing agent, leading to reduction-responsive degradation of the network structures, and BSA molecules in the network structures can be degraded in the presence of protease. Figure 4a shows the changes of viscosities of the hydrogels in the presence of different amounts of added βCD. Upon addition of 50% and 100% native βCD (both relative to the amount of βCD groups immobilized on protein molecules), the viscosities of the networks drop by one and 2 orders of magnitude, respectively, which indicate that the

Figure 2. Influences of βCD/AD molar ratio and polymer concentration on the rheological properties of the polymer networks at 20 °C: (a) steady shear viscosities at various βCD/AD molar ratios (polymer concentration: 2.5 wt %); (b) frequency sweeps at two different polymer concentrations (βCD/AD molar ratio: 1:1, γ = 0.1); (c) frequency sweeps at various βCD/AD molar ratios (polymer concentration: 2.5 wt %, γ = 0.1).

strates that AD on the copolymer and βCD on BSA are able to form inclusion complexes. Solid content has a significant impact on the network property. As shown in Figure 2b, an increase in the polymer concentration from 2.5 to 3.75 wt % results in a rise in the dynamic moduli of the network structures. The crossover of G′ and G″ is widely used as a qualitative indication of gelation.41 For the sample prepared at 3.75 wt % polymer concentration, a crossover of G′ and G″ is observed at a frequency about 7 Hz, which means the network structure behaves more like a solid than a fluid in response to external shear force at a frequency above 7 Hz.42 The βCD/AD molar ratio turns out to be a key factor in the control of the network structures. In Figure 2a, as the βCD/AD molar ratio goes from 1:3 all the way up to 3:1, a substantial increase covering 4 orders of magnitude in network viscosity is observed. The ascending trend of the dynamic moduli with βCD/AD molar ratio is observed in frequency sweeps (Figure 2c). An increase in βCD/AD molar ratio can gradually convert the solution from viscous fluid to hydrogel with solid-like behavior. In the frequency sweep of the sample prepared at the molar ratio of 1:1, the crossover of G′ and G″ is not observed. As the molar ratio increases to 1.5:1 and 3:1, the crossover appears at around 1 Hz, and 0.05 Hz, respectively. The 1224

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Figure 4. (a) Influence of added βCD on the viscosities of network structures. The steady shear measurements were conducted at 20 °C. (b) Changes of viscosities of network structures in the presence of GSH or trypsin at different time. Rheological time sweeps were carried out at a fixed shear rate of 1.0 s−1. The red and black curves show the decrease of viscosity under the treatments of 0.25% trypsin and 10 mM of GSH at 37 °C, respectively. The inset photographs were taken at 0 and 30 min after mixing with GSH (the upper two photos) and trypsin (the lower two photos). In all the experiments, the polymer concentrations were controlled at 2.5 wt %, and the molar ratios of βCD/AD were controlled at 1.5:1.

Figure 3. Shear-thinning and network restoring dynamics of two polymer networks prepared at different βCD/AD molar ratios (1.5:1 and 1:1). In the experiments, the polymer concentration is 2.5 wt %, and the temperature is kept at 20 °C. Shear rates were switched between 0.1 and 1000 s−1 every 300 s. (a) The changes of viscosities in three cycles; (b) A magnified section showing the time needed to restore 90% of the original viscosities.

network structures formed by BSA-βCD and PAM-coPNADAM are responsive to competing inclusion agents. Reduced GSH is a tripeptide, which naturally exists in the human body and is responsible for maintaining a reducing environment essential for many biophysical processes.43 Rheological time sweeps were conducted to monitor the GSH-induced degradation process of the network structures at body temperature (37 °C). The concentration of GSH was set as 10 mM, which is a common value in intracellular fluids.44 As shown in Figure 4b, the degradation process is quite fast, and the viscosity decreases by 1 order of magnitude within half an hour. The redox-responsive dissociation of the gel is also confirmed by comparing two photos shot before and after addition of GSH (inset of Figure 4b). BSA serving as the biological cross-linker can be degraded by protease. Protease cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, resulting in the degradation of the protein cross-linker and subsequent dissociation of the network structures. Herein, we choose trypsin as a typical protease to hydrolyze BSA.8 The rheological time sweep at 37 °C indicates that upon addition of trypsin the viscosity of the network structure decreases rapidly (Figure 4b). The dissociation of the network structure was also demonstrated by comparing photos obtained before and after addition of trypsin (inset in Figure 4b). In summary, hybrid polymer networks were fabricated with modified BSA as the cross-linker, and the molecular recognition

between AD and βCD groups as the cross-linking interaction. It was found that the mechanical properties of the networks were strongly dependent on the molar ratio of βCD to AD groups. Because of the protein cross-linkers, the disulfide linkages and the βCD/AD inclusion complexes in the structures, the networks present triple-responsive behaviors. The network viscosities can be greatly reduced by adding native βCD, and the polymer networks show rapid degradation behaviors upon treatment with GSH or trypsin at body temperature. Such multiresponsive smart material may find promising applications in tissue engineering. Fabrication of new hydrogels based on this method is being conducted in this laboratory, and the hydrogels will be used as scaffolds for the formation of a desired tissue.

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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/acsmacrolett.6b00750. Experimental details, 1H NMR spectra of NADAM monomer, βCD-s-s-Py and PAM-co-PNADAM, GPC curve of PAM-co-PNADAM, and UV−vis spectra 1225

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recorded in the measurement of the thiol contents of BSA and in the functionalization of βCD (PDF).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ‡

These authors contributed equally to this manuscript (D.C. and W.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National Natural Science Foundation of China (NSFC, 21074058 and 51473079), the National Basic Research Program of China (973 Program, 2012CB821500), and the key technologies R&D program of Tianjin (14ZCZDSF00007).

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