Letter Cite This: ACS Macro Lett. 2018, 7, 1468−1474
pubs.acs.org/macroletters
Genetically Programming Stress-Relaxation Behavior in Entirely Protein-Based Molecular Networks Zhongguang Yang,†,△ Songzi Kou,‡,△ Xi Wei,# Fengjie Zhang,§,∥ Fei Li,¶ Xiao-Wei Wang,⊥ Yuan Lin,# Chao Wan,*,§,∥ Wen-Bin Zhang,*,⊥ and Fei Sun*,†,‡
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†
Department of Chemical and Biological Engineering and Center of Systems Biology & Human Health, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China ‡ Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen 518036, China § Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China ∥ School of Biomedical Sciences Core Laboratory, Institute of Stem Cell, Genomics and Translational Research, Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen 518057, China ⊥ Key Laboratory of Polymer Chemistry & Physics of Ministry of Education, Center for Soft Matter Science and Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China # Department of Mechanical Engineering, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR, China ¶ National Engineering Research Center of Industrial Crystallization Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China S Supporting Information *
ABSTRACT: We report the synthesis of a series of elastinlike polypeptide (ELP)-based molecular networks through the combined use of the covalent bond-forming SpyTag/ SpyCatcher chemistry, physically entangled p53dim domains (Xs), and site-directed mutagenesis. The resulting networks shared similar chemical composition but differed significantly in their viscoelasticity. These materials exhibited excellent compatibility toward encapsulated fibroblasts and stem cells. These results point to a versatile strategy for designing viscoelastic materials by tapping into diverse protein−protein interactions.
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(covalent or ionic) as well as the length of the polymers. Grindy and co-workers reported the creation of 4-arm poly(ethylene glycol) hydrogels with decoupled spatial structure and viscoelastic properties using multiple, kinetically distinct dynamic metal−ligand cross-links.12 Dooling and coworkers demonstrated the feasibility of programming the viscoelastic behavior of recombinant protein-based hydrogels through the combined use of engineered coiled-coil domains and 4-arm poly(ethylene glycol) vinyl sulfone-based covalent cross-links.13 A caveat is that the chemistry involved in these materials, such as metal ions, chemical cross-linkers, and polymer structure/length, may confound our efforts to elucidate the influence of viscoelasticity and stress relaxation on cell behavior unambiguously. Therefore, it is desirable to have an artificial ECM system with programmable stress-
onventional wisdom on how extracellular matrices (ECMs) influence cell behavior largely focuses on the roles of the physicochemical properties such as biochemical composition, stiffness, degradability, porosity, and ligand tethering. 1−3 On the other hand, native ECMs are viscoelasticany traction force cells exert quickly dissipates with rapid mechanical remodelling of the matrix as well as the rearrangement of the cell-binding ligands within. The role of stress-relaxation behavior in regulating cell behavior has not been fully appreciated until recently; it has been shown that the stress relaxation rates of 2D substrates and 3D matrices affect cell behavior in vitro, including spreading, proliferation, and differentiation,4−6 as well as scaffold remodelling and bone regeneration in vivo, highlighting the importance of stress relaxation as a material design parameter for tissue engineering.7 A material’s viscoelasticity can be modulated by altering cross-linking mechanisms among polymer chains.8−11 For instance, the viscoelastic properties of the commonly used alginate hydrogels, stress relaxation rates in particular,4,5 can be readily adjusted by varying the cross-linking chemistry © XXXX American Chemical Society
Received: November 1, 2018 Accepted: November 27, 2018
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DOI: 10.1021/acsmacrolett.8b00845 ACS Macro Lett. 2018, 7, 1468−1474
Letter
ACS Macro Letters relaxation properties while avoiding extensive chemical modification. The emergence of SpyTag/SpyCatcher chemistrya peptide/protein conjugation technology inspired by naturally occurring isopeptide bonds within bacterial adhesin proteins14,15has enabled the direct assembly of recombinant proteins into macroscopic hydrogel materials, also known as Spy networks.15−20 Various motifs such as elastin-like polypeptides (ELPs), RGD cell-binding ligands, MMP sites, and globular protein growth factors have been introduced to modulate the biochemical properties of the materials.16 However, despite recent progress in designing various protein hydrogels,21−28 genetically programming their mechanical propertiesthe stress-relaxation behavior in particularhas rarely been demonstrated.21,22 Mechanically interlocked structures can be found in both natural and engineered proteins;29−31 engineered p53 domains that form physically entangled dimers have been employed to create a variety of protein catenanes (Figure 1B).30−33 We envisioned that integrating these p53dim domains with the Spy network may lead to a new protein network, namely, Spy-X network, with genetically programmable viscoelasticity, where the p53dim domains would facilitate stress relaxation, while rigid covalent cross-links by SpyTag/SpyCatcher could slow it down. In this study, we designed and synthesized a series of protein networks that possess abundant RGD cell-binding ligands and MMP cleavage sites but differ in their stress-relaxation behavior. The study demonstrated the feasibility of creating cyto-compatible protein materials with well-defined molecular association and delicately controlled mechanical properties for potential biomedical applications. To create protein networks that possess both types of interchain interactions, five genetic constructs, including SpyTag-ELP-RGD-ELP-SpyTag (AA), SpyTag-ELP-p53dimELP-SpyTag (AXA), SpyTag-ELP-SpyTag-ELP-SpyTag (AAA), SpyCatcher-ELP-p53dim-ELP-SpyCatcher (BXB), and SpyCatcher-ELP-SpyCatcher-ELP-SpyCatcher (BBB), were designed and subsequently cloned into Escherichia coli (Figure 1C and Table S1). The ELP domains comprising repetitive pentapeptides (VPGXG)15, where X can be either Val or Glu at a ratio of 4:1,34 serve as spacers that connect the functional domains within the constructs.35,36Both AA and BBB constructs were designed to contain an RGD cell-binding ligand in the middle. The matrix metalloproteinase cleavage (MMP) sequence,37,38GPQG↓IWGQ, that facilitates matrix remodelling by cells was also introduced into BXB and BBB, adjacent to the first SpyCatcher domain. Using these five building blocks, we envisioned that four types of protein networks can be synthesized (Figure 1D). Specifically, the reaction of AA +BBB would lead to the formation of a covalently cross-linked protein network, while that of AAA+BBB would give rise to a second covalent network with increased cross-linking density. Replacing the middle A in AAA with X would result in the protein network AXA+BBB that contains both physical entanglements and covalent cross-links. The protein network consisting of AXA+BXB was expected to be the most dynamic one due to the absence of interchain covalent cross-links. All proteins including AA, AAA, AXA, BBB, and BXB were produced by heterologous E. coli expression and purified using standard Ni-NTA affinity chromatography (Figure S1). The lyophilized protein powders were dissolved in phosphatebuffered saline (PBS, pH 7.4) to make 8 wt % solutions for the gelation reactions. Lower critical solution temperatures
Figure 1. Design of entirely recombinant protein-based hydrogels with programmable viscoelasticity and stress relaxation. (A) Covalent SpyTag/SpyCatcher complex containing an isopeptide bond formed between the side chains of Lys and Asp residues. (B) Physically entangled p53dim domains. (C) Genetic constructs, AA, AXA, AAA, BXB, and BBB, encoding the building blocks for the synthesis of protein hydrogels. A, SpyTag. B, SpyCatcher. X, p53dim. RGD, ArgGly-Asp-based cell-binding domain. ELP, elastin-like polypeptide. (D) Schematic showing the cross-linking mechanisms for four types of protein hydrogels, including (i) AA+BBB and (ii) AAA+BBB that possess covalent interchain cross-linking, (iii) AXA+BBB that integrates both covalent and physically entangled interchain interactions, and (iv) AXA+BXB that contains physical entanglements only.
(LCSTs) of these proteins were obtained by light transmittance measurements using a UV−vis spectrometer. The temperature at 50% of the initial transmittance is defined as LCST.39 It turned out that all five proteins exhibited LCSTs within the range of 45−60 °C (Figure S2). Since all experiments described later in the study were performed either at room temperature or 37 °C, far below the LCST, the possible influence of the LCST phase transition behavior of ELPs on the materials’ mechanical properties can be ruled out. All four reactions, AA+BBB and AXA+BBB at a 3:2 molar ratio and AAA+BBB and AXA+BXB at an equimolar ratio, led to the formation of gel-like materials at room temperature within 10 min. After 12 h reaction, dynamic strain and frequency-sweep tests were performed on the resulting materials. The strain-sweep tests revealed a linear viscoelastic range (1%−40% strain) for all the protein hydrogels tested in this study (Figures S3, S4, and S5). The material formed by the AA+BBB reaction exhibited steady storage (G′, 0.31−0.59 kPa) and loss moduli (G″, 54−60 Pa) over a frequency range 1469
DOI: 10.1021/acsmacrolett.8b00845 ACS Macro Lett. 2018, 7, 1468−1474
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ACS Macro Letters
Figure 2. Rheological characterizations of engineered protein hydrogels. (A−D) Frequency sweep tests on the hydrogels (8 wt %) composed of AA +BBB (molar ratio, 3:2), AAA+BBB (molar ratio, 1:1), AXA+BBB (molar ratio, 3:2), and AXA+BXB (molar ratio, 1:1) at 23 °C. The best-fit lines of the experimentally measured G′(ω) and G′′(ω) were generated using linear interpolation. The continuous relaxation spectra H(τ) were calculated using eqs 1 and 2. The experiments were performed with fixed strain (5%) at 23 °C. (E) Stress relaxation tests with an initial strain of 10%. (F) Quantification of time scale, τ1/2, needed for the stress to be relaxed to half its initial value based on the stress relaxation tests in (E). Data are presented as mean ± SD (n = 3).
of 0.01−100 rad/s and a fixed strain of 5%, indicative of the formation of an elastic hydrogel (Figure 2A). The reaction of AAA+BBB led to a material that exhibited G′ (0.9−1.4 kPa) substantially higher than that of AA+BBBand G″ (104−68 Pa) over the same frequency range. The increased stiffness reflects a higher cross-linking density within the AAA+BBB network (Figure 2B). The G’s of both hydrogels were largely frequency independent, as expected for elastic, covalently cross-linked molecular networks. On the contrary, G′ and G″ of the product of AXA+BBB displayed greater frequency dependence, where the low-frequency G′min (0.28 kPa) reflects the contributions from interchain covalent cross-links involving the middle SpyCatcher in BBB, and the high-frequency G′max (1.3 kPa) corresponds to combined effects of p53dimmediated physical entanglements and covalent cross-links (Figure 2C). A local maximum of G″ for AXA+BBB appeared at a frequency of ∼0.4 rad/s. G′ was consistently larger than G″, and no crossover of G′ and G″ was observed, suggesting the formation of a viscoelastic solid by AXA+BBB. As expected, the reaction of AXA+BXB led to the formation of a viscoelastic liquid due to the absence of covalent cross-links, which exhibited a crossover point of G′ and G″ at a frequency of 0.1 rad/s (Figure 2D). The product exhibited liquid (G′ < G″) and solid (G′ > G″) like properties at low and high frequency, respectively, consistent with the behavior of a physical network. Swelling tests further confirmed the nature of these four protein networks; unlike the other three covalently cross-linked networks, AA+BBB, AAA+BBB, and AXA+BBB, which remained stable after 48 h swelling, the product of AXA +BXB, a physical network, was completely dissolved in water and PBS within 4−6 h (Figure S6).
Stress relaxation tests revealed distinct relaxation behavior of the four protein networks (Figures 2E,F and S7). The relaxation rates of the AXA+BBB [τ1/2 = ∼3 s] and AXA +BXB [τ1/2 = ∼2 s] networks turned out to be much faster than that of AA+BBB and AAA+BBB (τ1/2 = ∼15 min), showing that the p53dim domains relax stress more effectively than covalent SpyTag/SpyCatcher complexes within the molecular networks. These experimentally measured single characteristic relaxation time scales τ1/2 represent naive approximations of the dynamic material properties. In order to correlate the macroscopic stress-relaxation behavior with specific interactions at the molecular level, we converted the frequency sweep data into a series of continuous relaxation spectra, H(τ) (units Pa), which allowed us to quantify the full distribution of relaxation times.12 The relaxation spectrum, H(τ), and complex modulus, G′(ω) and G″(ω), are mathematically linked by ∞
2 2
G′(ω) =
∫−∞ H(τ) 1 +w wτ 2τ 2 d ln(τ)
G″(ω) =
∫−∞ H(τ) 1 +wwτ 2τ 2 d ln(τ)
(1)
∞
(2)
H(τ) is proportional to the amount of energy dissipated from an infinite number of parallel Maxwell elements at dynamic loading frequency ω(1/τ). The relaxation spectra of the networks comprising AA+BBB and AAA+BBB revealed no significant dissipation peak (Figure 2A,B), as expected for covalently cross-linked networks, while the network consisting of AXA+BBB exhibited a major 1470
DOI: 10.1021/acsmacrolett.8b00845 ACS Macro Lett. 2018, 7, 1468−1474
Letter
ACS Macro Letters
Figure 3. Tuning stress relaxation by adjusting the amount of physical entanglements. (A) Frequency sweep tests on the networks composed of AA +AXA+BBB with the molar ratios of AA to AXA varied from 100:0, 75:25, 50:50, 25:75, to 0:100. The best-fit lines of the experimentally measured G′(ω) and G′′(ω) were generated using linear interpolation. The continuous relaxation spectra H(τ) were calculated using eqs 1 and 2. The strain was held at 5%. (B) Stress relaxation tests on the hydrogel networks containing varied amounts of p53dim domains. The initial strain was set at 10%. (C) Quantification of time scale (τ1/2) needed for the stress to be relaxed to half its initial value based on the stress relaxation tests in (B). Data are presented as mean ± SD (n = 3).
dissipation peak at ω ∼ 0.5 rad/s (τ ∼ 1.4 s) (Figure 2C), which may reflect the contribution from the dimerization interactions between the X domains within the network. More interestingly, the network composed of AXA+BXB that possesses more X domains and lacks covalent interchain cross-links exhibited three distinct relaxations: one slow dissipation mode at ω ∼ 0.19 rad/s (τ ∼ 5.2 s), one fast, dominant mode at ω ∼ 0.7 rad/s (τ ∼ 1.4 s), and one faster mode at ω ∼ 36 rad/s (τ ∼ 0.028 s). These three mechanically distinct relaxation modes may reflect the contributions from different types of molecular association33aggregation (
