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Versatile Engineered Protein Hydrogels Enabling Decoupled Mechanical and Biochemical Tuning for Cell Adhesion and Neurite Growth Xiaotian Liu, Xin YANG, Zhongguang Yang, Jiren Luo, Xiaozhen Tian, Kai Liu, Songzi Kou, and Fei Sun ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00077 • Publication Date (Web): 26 Mar 2018 Downloaded from http://pubs.acs.org on March 26, 2018

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ACS Applied Nano Materials

Versatile Engineered Protein Hydrogels Enabling Decoupled Mechanical and Biochemical Tuning for Cell Adhesion and Neurite Growth Xiaotian Liu,1 Xin Yang,2 Zhongguang Yang,1 Jiren Luo,1 Xiaozhen Tian,2 Kai Liu,2 Songzi Kou,1* and Fei Sun1*

1

Department of Chemical and Biological Engineering, Hong Kong University of Science

and Technology, Clear Water Bay, Kowloon, Hong Kong 2

Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong

University of Science and Technology, Hong Kong; Center of Systems Biology and Human Health, School of Science and Institute for Advanced Study, Hong Kong University of Science and Technology, Hong Kong

*To whom correspondence should be addressed: [email protected]; [email protected].

KEYWORDS: protein hydrogel, mussel foot protein, SpyTag/SpyCatcher, neurite growth, LIF signaling

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ABSTRACT Development of engineered protein materials with wide-ranging mechanical strength and stiffness while maintaining the bio-functionality of protein molecules within remains a big challenge.

Here we demonstrate the synthesis of protein hydrogels by

photochemically crosslinking recombinant mussel foot protein-3 (Mfp3). The hydrogels’ stiffness can be broadly tuned by adjusting the concentration of protein polymers or cooxidants, or light intensity needed for the chemical crosslinking. The protein polymers were also designed to contain SpyCatcher domains, which enabled post-gelation decoration with diverse folded globular proteins under mild physiological conditions. Not only did the resulting hydrogels support the adhesion and proliferation of a variety of cell lines, but also were able to activate JAK/STAT3 pathway and induce neurite growth via covalently immobilized leukemia inhibitory factor (LIF). These results illustrate a new strategy for designing bioactive materials for regenerative neurobiology.


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INTRODUCTION Mechanical cues play a key part in directing cell behavior in development and regeneration. A material’s mechanical properties in general can be modulated by changing the crosslinking chemistry, crosslinking density, polymer composition, or the ratios of different chemical reactants.1-4 One caveat is that the discrete biochemical composition is likely to confound the effects of mechanical cues on cellular signaling. Hence it is desirable to develop a material system with mechanical properties decoupled from other biophysical properties so that their respective influence on cell behavior can be determined unambiguously.5 In this regard the ideal strategy for creating a protein material should consist of two separate processes: covalent assembly of structural proteins into a material scaffold with desirable mechanical strength (likely under nonphysiological conditions) and subsequently functionalization with labile molecules for targeted biological activities under mild conditions. Mussel foot proteins such as Mfp3 and Mfp5 play primary roles in the adhesive interactions between the marine mussel Mytilus and solid surfaces in the sea.6 Their exceptional underwater adhesiveness is attributed to the abundance of the posttranslationally modified amino acid residue, 3, 4-dihydroxyphenyl-L-alanine (Dopa), within the proteins.7 The adhesive protein obtained from natural extraction is scarce,8 and exhibits poor solubility and inherent oxidation sensitivity under physiological conditions (pH 6.5-8.5), which restrains its wider use for biotechnological applications.6 Nevertheless, this naturally occurring underwater adhesiveness has inspired the development of various catechol-functionalized synthetic polymers for diverse coating, anchoring, adhesive and sealant applications. Notably, a simple, robust dip-coating



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technology based on dopamine self-polymerization has been widely used to functionalize organic and inorganic surfaces for a variety of purposes such as electroless metallization, antifouling and cell adhesion.9 Dopa-containing recombinant Mfps have recently been synthesized using heterologous Escherichia coli expression, where Dopa was introduced via several methods, including Tyr oxidation by mushroom tyrosinase in vitro or by a co-expressed bacterial tyrosinase inside cells, or in vivo residue-specific incorporation of Dopa enabled by promiscuous E. coli tyrosyl-tRNA synthetase.10-13 Yet, the inherent difficulties of handling these adhesive proteins arising from solubility and oxidation sensitivity remain, especially for material engineering. As E. coli cells are not equipped with the machinery needed for PTM of Tyr, the use of a simple E. coli expression system would give rise to the production of unmodified proteins possessing abundant Tyr (e.g., 20 mol% for Mfp3) rather than Dopa.6 It is noteworthy that Tyr can readily form dityrosine (di-Tyr) adducts via a radical-based crosslinking mechanism, which is often employed by living organisms to stabilize naturally occurring materials such as resilin, silks and other structural proteins.14 Because of its high efficiency as well as the prevalence of Tyr, diTyr crosslinking reactions, especially those induced photochemically, have received significant attention from materials scientists, and served as an intriguing strategy for the creation of protein materials enabling a wide range of mechanical properties.15-18 The finding of stabilizing isopeptide bonds within bacterial adhesins has led to the creation of several peptide/protein pairs, also known as “molecular superglues” that can spontaneously

form

covalent

bonds

under

mild

conditions.

Among

them,

SpyTag/SpyCatcher chemistry has emerged as one of the most exciting tools for


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constructing advanced protein architectures and biomaterials because of its marked efficiency, selectivity and full genetic encodability.19-21 The synthesis of diverse, entirely engineered protein-based hydrogels, as known as “Spy networks”, further demonstrated the power of SpyTag/SpyCatcher chemistry in designing functional materials.22-26 However, despite their versatile functionality and superior programmability, the Spy network hydrogels are mechanically weak because of low crosslinking density and thus unsuitable for the applications where high stiffness and mechanical strength are needed. In this study, we demonstrate the creation of an engineered protein hydrogel system that enables wide-ranging mechanical properties and diverse biological functionality through the combined use of photo-crosslinkable Mfp3 proteins and SpyTag/SpyCatcher chemistry. We also examined the neurotrophic properties of LIF immobilized onto the hydrogel and proved the feasibility of using this protein material to direct neuronal signaling and neurite outgrowth. RESULTS AND DISCUSSION Protein Construct Design. Naturally occurring Mfp3 contains abundant Dopa and is known to play a key role in the underwater adhesion of mussels. Recombinant Mfp3 produced by E. coli possesses a large quantity of Tyr (21 mol %) instead because of the absence of the enzymatic machinery for tyrosine oxidation in bacterial cells. In addition, due to high Arg (17 mol %) and Lys (11 mol %) content, Mfp3 is a largely unstructured and highly positively charged polymer, of which poor solubility and low expression yield pose a big challenge for bacterial expression (Figure 1A). To address this, we designed and cloned the genetic construct, SpyCatcher-ELP-Mfp3-ELP-SpyCatcher (BMB), which encodes a multi-domain protein harboring an Mfp3, flanked by two elastin-like



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polypeptides (ELPs) and SpyCatcher domains (Figure 1A). The ELP consists of (VPGXG)15 [X = Val or Glu at a 4:1 ratio], a composition that should lead to enhanced expression yield and solubility under physiological conditions.27 The introduction of SpyCatcher would enable us to leverage powerful SpyTag/SpyCatcher chemistry for further functionalization of the material (Figure 1B). In addition, the BMB construct contains the DNA sequences that encode putative RGD cell binding domains and MMPcleavage sites, both of which are expected to facilitate the interactions between cells and the substrates.

Figure 1. Two-step synthesis of an Mfp3-based bioactive hydrogel with mechanical tuning decoupled from biochemical functionalization. (A) Genetic construct for the protein polymer SpyCatcher-ELP-Mfp3-ELP-SpyCatcher (BMB). The construct also contains an RGD cell-binding domain and a matrix metalloproteinase (MMP) cleavage site that enables matrix remodeling by encapsulated cells. (B) Schematic illustration of the creation of a hydrogel by photochemically crosslinking Tyr residues within Mfp3. The resulting protein hydrogel is subject to a subsequent modification by a globular protein [e.g., leukemia inhibitory factor (LIF)] via SpyTag/SpyCatcher chemistry under mild physiological conditions. For clarity, only Mfp3 and SpyCatcher domains are shown within the gel network.

Synthesis of Protein Hydrogels with Wide-Ranging Mechanical Properties. The BMB protein can be produced and purified in a considerable yield using an E. coli


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expression system and Ni-NTA chromatography (~20 mg per liter of E. coli culture in LB medium). After extensive dialysis against distilled water and freeze-drying, white protein powder was obtained. The protein turned out to be highly soluble and can be dissolved in PBS up to 10 wt%. In light of their high Tyr content, the photo-oxidation method involving tris(bipyridine)ruthenium(II) chloride {[Ru(bpy)3]Cl2}—an efficient visible light-harvesting compound with a λmax of 452 nm in H2O28—and ammonium persulfate (APS) was adopted to covalently crosslink the protein polymers to make a hydrogel. Rapid gelation occurred upon addition of APS and [Ru(bpy)3]Cl2 into the BMB solution under white LED illumination (400-800 nm, 90 klx) (Figure S2). The reaction product (8 wt%, 10 mM APS and 0.2 mM Ru) exhibited a steady storage modulus G’ (8.7-9.2 kPa) over a range of shear frequency (0.04-100 rad/s), substantially higher than loss modulus G” (0.22-0.26 kPa), which suggests the formation of an elastic solid (Figure 2A). The hydrogel showed minimal swelling in water (Figure S3). The scanning electron microscopy (SEM) image revealed a highly porous microstructure, as expected for a sufficiently covalently crosslinked hydrogel (Figure S4). The photo-crosslinked product emitted blue fluorescence (excitation, 350 nm; emission, 426 nm), consistent with the spectral feature of the dityrosine moiety (Figure S5).



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Figure 2. Broadened mechanical properties of Mfp hydrogels. (A) Frequency sweep tests on Mfp hydrogels (8 wt%) formed in the presence of varied amounts of ammonium persulfate (APS). (B) Storage moduli G’ at a frequency of 10 rad/s at different APS concentrations. (C) Frequency sweep tests on Mfp hydrogels that comprise varied concentrations of BMB protein polymers (4, 6 and 8 wt%). The concentration of APS was fixed at 10 mM for all the gelation reactions. (D) Storage moduli G’ at a frequency of 10 rad/s for three hydrogels (4, 6 and 8 wt%). Data are presented as mean ± SD (n = 3). The shear frequency varied from 0.04 to 100 rad/s. The strain was set constant at 5%. All tests were performed under white LED illumination (90 klx) at room temperature. Mechanical properties of the hydrogels can be adjusted by varying the concentration of the co-oxidant APS or the protein polymers (Figure 2A and 2C). Reduced amounts of either APS or BMB led to significantly softened hydrogels (Figure 2A and 2C), yielding G’ of 1.3 and 0.6 kPa at a frequency of 10 rad/s for 1 mM APS + 8 wt% BMB and 10 mM APS + 4 wt% BMB, respectively (Figures 2B and 2D). The gelation also depends on light intensity, and the reaction rates were much slower under weaker illumination (30 klx and dark) conditions (Figure S6). It is noteworthy that this wide-range mechanical tuning was done by altering the reaction parameters like the concentration of oxidants and light intensity while maintaining nearly identical biochemical composition of the materials.


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Decoration of Hydrogel Microbeads and Microstrips with Globular Proteins. Monodisperse microbeads were readily created using a two-channel droplet-generating microfluidic

device

(Figure

3A).29

Microstrips

were

generated

using

a

polydimethylsiloxane (PDMS) microchannel mold in a similar manner (Figure 3B). The droplets and microstrips containing BMB, Ru (II) and APS solidified after exposure to white LED light (90 klx) for 1 h.



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Figure 3. Microfabrication and functionalization of Mfp hydrogels. (A and B) Micrographs of Mfp hydrogel microbeads and microstrips decorated with SpyTag-mCherry-SpyTag (A-mCherry-A) and SpyTag-EGFP-SpyTag (A-EGFPA). The mCherry and EGFP proteins that do not possess SpyTag were used as controls. BF, bright field. The blue fluorescence arises from the dityrosine crosslinks. (C and D) Comparison of the fluorescence of microbeads decorated with mCherry, A-mCherry-A, EGFP and A-EGFP-A. (E and F) Comparison of the fluorescence of microstrips decorated with mCherry, A-mCherry-A, EGFP and A-EGFP-A. Data are presented as mean ± SD (n = 10). Ru (II) mediated photo-crosslinking of Tyr residues undergoes a radical mechanism and can be detrimental to marginally stable globular proteins. So a milder chemistry that is independent of the gelation reaction is needed for the biofunctionalization of the hydrogel. We leveraged the pre-existing SpyCatcher domains within the BMB protein. The microbeads and microstrips were immersed in the PBS


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solution containing both SpyTag-mCherry-SpyTag (A-mCherry-A) and SpyTag-EGFPSpyTag (A-EGFP-A) by extensive washing with PBS to remove unreacted proteins. It turned out that both A-mCherry-A and A-EGFP-A were efficiently immobilized onto the microbeads and microstrips, as evidenced by fluorescence micrographs of these materials, while the two proteins, mCherry and EGFP, lacking SpyTag failed to decorate the hydrogels, suggesting that SpyTag/SpyCatcher chemistry was responsible for the observed post-gelation modification (Figure 3). The SpyCatcher domains survived the photochemical gelation and remained reactive, showcasing the robustness of SpyTag/SpyCatcher chemistry. With the emergence of orthogonal peptide/protein superglues like SnoopTag/SnoopCatcher,30 we envision that the creation of more complicated, information-rich protein materials will be possible. In order to determine whether the tuning processes over gel mechanics and biochemical aspects are truly decoupled, we first compared the efficiency of EGFP immobilization onto two sets of hydrogel microstrips of the same size but differing in stiffness (Figure S7). After the reaction and rinsing with PBS, the resulting strips exhibited similar fluorescence intensity regardless of their difference in crosslinking density and stiffness, suggesting that their mechanical properties have negligible effects on the post-gelation modification. To examine the influence of post-gelation modification on the gel’s mechanical properties, we performed frequency-sweep tests on two sets of hydrogels with and without EGFP decoration (Figure S8). It turned out that the hydrogels’ viscoelasticity, especially the storage modulus G’, was not significantly altered by the EGFP immobilization, which is not surprising given that the post-gelation modification by SpyTag/SpyCatcher chemistry per se did not modulate the gel network structure



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within as well as its crosslinking density. Taken together, these results show that the mechanical and chemical tuning over the material properties are indeed decoupled. Cell Adhesion and Proliferation on Mfp Hydrogel Substrates. Examination of the Mfp-3 sequence revealed high Arg (17 mol %) and Lys (11 mol %) content, which is reminiscent of poly-lysine, a coating material widely used to enhance cell attachment to plastic and glass surfaces. We envisioned that Mfp hydrogels could also be used to facilitate cell adhesion and proliferation because of high positive-charge density of Mfp-3 and the presence of RGD cell-binding ligands. Using an 8 wt% Mfp hydrogel as a model substrate, a variety of cell lines including 3T3 fibroblasts, COS-7, Hela and MDCK epithelial cells were tested. It turned out that these cells not only attached well to the gel surfaces but also displayed high viability (> 90%) and extended morphology after culturing for 5 days (Figure 4). In addition, marked cell proliferation was observed, particularly for MDCK epithelial cells that formed a highly dense population on day 5 (Figure 4).

Figure 4. Mfp hydrogels support cell adhesion and proliferation. Representative merged micrographs (blue, red and green) of 3T3, COS-7, Hela and MDCK cells grown on the Mfp hydrogel films after the live/dead staining. The blue background originates from the fluorescence of dityrosine crosslinks.


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The Mfp hydrogel also enabled facile micropatterning of cell cultures. When microstrip coating was used, cells preferably adhered to the coated areas and significant cell proliferation was observed on day 3, while few cells were found in uncoated areas (Figure 5). Taken together, these results showed the feasibility of using Mfp hydrogels as a general coating material for cell culturing and micropatterning.

Figure 5. Adhesion and proliferation of 3T3 fibroblasts on microstrips. LIF Immobilization for STAT3 Activation and Neurite Outgrowth. Development of materials with bound protein ligand presentation is central to many regenerative tasks such as treating acute neural injuries or chronic neuro-disorders.31-32 Leukemia inhibitory factor (LIF), a 20-kDa cytokine, have been well recognized for its potential in neuroprotection and nerve regeneration.33-34 This important signaling protein can activate the JAK/STAT3 pathway and lead to Tyr705 phosphorylation in STAT3, a key transcriptional regulator for neuroprotection and repair after nerve injury.35 In vivo, endogenous LIF exist in two forms, a diffusible form and a form bound with the extracellular matrix (ECM),36 which may play distinct roles in biological signaling.37-38 Since the neurotrophic properties of LIF have been established mainly through the use of genetic studies and/or exogenous diffusible LIF, it remains to be seen whether an alternative mode of presentation (immobilized) still exerts such neurotrophic effects. Since Mfp hydrogels allowed for both cell adhesion and post-gelation modification with



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globular proteins, we envisioned that the materials, once covalently decorated with LIF, can be used to examine the influence of immobilized LIF on neuronal signaling.

Figure 6. Functionalization of Mfp hydrogels with LIF for cortical neuron culturing and neurite growth. (A) Cortical neurons grown on Mfp hydrogel films with and without immobilized SpyTag-LIF-SpyTag (A-LIF-A) for 1 day. Cells were stained using mouse anti-beta3-tubulin antibody. (B) Length of the three longest neurites of cortical neurons in the absence and presence of A-LIF-A. Data are presented as mean ± SEM (n = 3, p < 0.01). (C) Immobilized A-LIF-A induces Tyr705 phosphorylation of STAT3. Western blots were performed on whole cell lysates with the antibodies against STAT3 and Tyr705-phosphorylated STAT3. (D) Ratios of p-STAT3 (Y705) to total STAT3 in the absence and presence of A-LIF-A. Data are presented as mean ± SD (n = 3, p < 0.0001). We used MH35-LIF, a chimeric LIF variant, which can be functionally expressed in E. coli and recognized by both mouse and human cells.39 The SpyTag-ELP-LIF-ELPSpyTag (A-LIF-A) protein was obtained from E. coli expression as reported previously.22 Immobilization of A-LIF-A was achieved by immersing Mfp-gel coated polystyrene plates in the A-LIF-A solution at room temperature for 1 h. Unreacted A-LIF-A was removed by extensive washing with PBS. Cortical neurons from late embryonic stage (E17) mice have been known to respond robustly to LIF33 and therefore were used to examine the activity of immobilized LIF. It turned out that Mfp-gel coated substrates, 14 ACS Paragon Plus Environment

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regardless of LIF immobilization, were able to support the attachment of cortical neurons, presumably reflecting the contributions from highly positively charged Mfp3 domains and RGD cell-binding ligands (Figure 6A). The presence of immobilized LIF turned out to be beneficial to neurite extension, as the mean length of the longest neurites (~45 µm) on the substrate modified by A-LIF-A was 1.7 times that (~26 µm) in the absence of ALIF-A after culturing merely for 1 day (Figure 6A-B). To confirm the influence of immobilized LIF on JAK/STAT3 signaling, western blot analysis was performed to determine the level of Tyr705-phosphorylated STAT3 (p-STAT3), a key effector for neuronal survival and regeneration. As a result, the presence of A-LIF-A indeed led to significantly increased Tyr750-phosphorylation, 13 times higher than that in the absence of LIF (Figure 6C-D), confirming the functionality of the immobilized LIF in activating the JAK/STAT3 pathway. In principle, other neurotrophic factors apart from LIF can also be used to functionalize the Mfp hydrogels via SpyTag/SpyCatcher chemistry, and a versatile platform may therefore be established for enhanced neuronal survival and neurite regeneration. Although it has been shown that the presence of Ru(II)bpy32+ may destabilize folded globular proteins,40 only a catalytic amount of Ru(II) (~0.1 eq.) was used for the gelation reaction in this study. Besides, the BMB protein mainly consists of two unstructured domains, ELP and Mfp3, and the folded SpyCatcher domains. The previous study has shown the exceptional stability and robustness of SpyCatcher under various conditions.41 .SpyCatcher was indeed able to survive the gelation reaction, as evidenced by the efficient post-gelation modification with the SpyTagged proteins (Figures 3A-B and 6). The activities of these proteins were also preserved after immobilization,



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suggesting that this photochemical gelation strategy is by and large compatible with the engineered protein hydrogels. Moreover, these photocrosslinked hydrogels, once the remaining APS and Ru(II) are removed by washing with PBS, have proven to be versatile substrates for a variety of cell lines. In summary, using a combination of photo-crosslinkable Mfp-3 domains and SpyTag/SpyCatcher chemistry, we have created a versatile protein hydrogel system that not only decouples wide-ranging mechanical tuning from biochemical functionalization but also enables the adhesion and proliferation of various cell lines. Moreover, the material, upon decoration with neurotrophic factors like LIF, exhibited the ability to activate STAT3 signaling and induce neurite outgrowth of cortical neurons. Featuring a robust photochemical gelation medicated by structural protein polymers and a facile postgelation functionalization with folded protein domains, this design strategy for protein materials opens up new opportunities for future material neurobiology. EXPERIMENTAL PROCEDURES Gene Construction. The creation of the gene constructs, SpyTag-ELP-SpyTag-ELPSpyTag (AAA), SpyTag-ELP-mCherry-ELP-SpyTag (A-mCherry-A), SpyTag-ELP-LIFELP-SpyTag (A-LIF-A), SpyTag-ELP-EGFP-ELP-SpyTag (A-EGFP-A) and SpyCatcherELP-SpyCatcher (BB), has been described previously.22 The mfp3 gene was purchased as a gBlocksTM gene fragment from Integrated DNA Technologies (IDT). To create SpyCatcher-ELP-Mfp3-ELP-SpyCatcher (BMB), the mfp3 gene was inserted into the middle of the BB construct using SacI and SpeI cutting sites. Escherichia coli strain DH5α and the pQE80l plasmid were used for molecular cloning. All constructs were verified by sequencing (Beijing Genomics Institute). Protein Expression, Purification and Characterization. E. coli strain BL21(DE3) harboring pQE80l was grown in LB medium at 37°C, 220 rpm till optical density reached 0.6-0.8 at 600 nm. Protein expression was induced by addition of 500 µM isopropyl β-D16 ACS Paragon Plus Environment

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1-thiogalactopyranoside (IPTG) at 32°C. After 4 hours, cells were harvested by centrifugation at 4,200 g, 4°C for 15 min. Cell pellets were frozen at −80°C for 2 hours before protein purification. The pellets were suspended in the lysis buffer containing 300 mM NaCl, 10 mM Tris-HCl (pH 8), and 1mM phenylmethylsulfonyl fluoride (PMSF), lysed by a sonication homogenizer, and clarified by centrifugation at 18,000 g, 4°C for 30 min. Target proteins were purified using the HisTrap Nickel(II) columns (GE Healthcare, Inc.). Protein purity was assessed by SDS-PAGE. After the Ni-NTA purification, the proteins were further dialyzed against Milli-Q water (4.5 L × 5) at 4°C, then centrifuged at 5,200 g for 40 min to remove insoluble fractions. The supernatants were snap-frozen in liquid nitrogen, stored at -80°C overnight and lyophilized for 72 hours. Lyophilized protein powders were stored at -80°C before use. Mfp Hydrogel Formation. Lyophilized BMB protein powders were dissolved in phosphate-buffered saline (PBS, pH 7.4), followed by the addition of ammonium persulfate and tris(bipyridine)ruthenium(II) chloride. The final Ru (II) concentration was fixed at 0.2 mM, and APS varied from 1 to 10 mM. Gelation was achieved by exposing the solutions to white LED light (90 klx) for 10 min. Dynamic Shear Rheology. Rheological measurements were performed on a TA Instruments ARES-RFS strain-controlled rheometer with a standard steel parallel-plate geometry (8-mm). Test modes included time and frequency sweep. Time sweep tests were conducted by holding the strain at 5% and frequency at 1 rad/s. Frequency weep tests were conducted by holding the strain at 10% while varying the oscillatory frequency from 100 to 0.01 rad/s. For the reactions in the dark, aluminum foil was used to block light during the rheological measurements. Microfabrication and Functionalization of Mfp Hydrogels. Microbeads were created using a “Y”-shape droplet generating microfluidic device.29 Microstrips were created by flowing gelation solutions into PDMS microchannels and then cured under white LED light

illumination.

To

covalently

immobilize

fluorescent

proteins,

the

microbeads/microstrips were immersed with the solutions containing A-EGFP-A and AmCherry-A (50 µM in PBS) at room temperature for 30 min. Then the microbeads and microstrips were rinsed with PBS 5 times to remove the excess of fluorescent proteins. 17 ACS Paragon Plus Environment


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Cell Adhesion and Proliferation on Mfp Hydrogels. NIH/3T3 fibroblasts, HEK293T, African green monkey kidney fibroblast-like COS-7 cells, and Madin Darby canine kidney (MDCK) epithelial cells were used to test the suitability of Mfp hydrogels for cell culturing. Cells were grown at 37°C with 5% CO2 in high glucose Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Gibco), and 1% (vol/vol) penicillin-streptomycin (Sangon Biotech). The cells were passaged at ~75% confluence. Trypsin (0.25%) solution (Sangon Biotech) was used to digest and suspend cells. Approximately 2,000 cells were used for each experiment. Thin Mfp-gel films were prepared on 35 mm (in diameter) plastic petri dishes and washed with sterilized PBS three times after gelation. Cells were added onto the top of gel films and grown at 37°C with 5% CO2.16 The viability of cultured cells were assessed by LIVE/DEAD staining assay (Invitrogen). After removing culture media, the cultured cells were washed three times with PBS, followed by treating with ethidium homodimer (4 µM) and calcein AM (2 µM). After 10-min incubation at room temperature, the cells were visualized using a confocal fluorescence microscope (Nikon). LIF-Decorated Mfp Hydrogels for Cortical Neuron Culturing. Cortical neurons were cultured using the method previously described with slight modifications.42 Briefly, embryonic 17 days (E17) mice were sacrificed, and brains was dissected in cool HBSS. Cortices were collected and digested by the digestion medium containing 10 mg/ml papain for 30 min. After re-suspension, isolated cortical neurons were plated on the PolyD-Lysine (PDL) or Mfp-gel coated 6-well plates. Immobilization of A-LIF-A was accomplished by immersing the Mfp-gel films with the A-LIF-A (1 mg/mL in PBS) solution for 1 h, followed by 3 x PBS wash to remove unreacted A-LIF-A. The culture medium comprising neuronal-A and 2% B-27 supplement (Gibco) was added to cover the cells. After one day culturing, half of the cells were harvested for western blot analysis. The whole cell lysates were separated using SDS-PAGE. After transfer, nitrocellulose membranes were incubated with antibodies against p-STAT3 (Tyr705) and total STAT3 for overnight. After washing with PBST 4 times, the membranes were further incubated with goat anti-HRP (Life Technologies) for 1 hour. Images were taken with the Bio-Rad ChemiDoc gel imaging system and analyzed by ImageJ. In order to examine the influence of LIF on neurite growth, the other half of the cells were fixed by 18 ACS Paragon Plus Environment

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paraformaldehyde (4% in PBS) for 10 minutes, blocked with 4% normal goat serum (NGS), and stained with mouse anti-beta3-tubulin antibody (BioLegend) at room temperature for 2 hours. Then the cells were washed with PBS for 3 times and incubated with goat anti mouse 555 (Life Technologies) for 1 hour. The cells were further washed with PBS 5 times before imaging. Images were taken by a Carl Zeiss LSM 880 confocal microscope. The length of neurites was quantified with ImageJ. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Amino acid sequences (Figure S1) and additional characterization data for Mfp hydrogels (Figures S2-S6) (PDF). AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected], [email protected] ORCID Xiaotian Liu: 0000-0001-5009-1287 Xin Yang: 0000-0003-2153-256X Zhongguang Yang: 0000-0002-4069-6693 Jiren Luo: 0000-0001-8157-2745 Xiaozhen Tian: 0000-0003-0891-2509 Kai Liu: 0000-0001-7956-6098



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Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS Fei Sun thanks the funding support from the Research Grants Council of Hong Kong SAR Government (Grant 26103915 and AoE/M-09/12). Fei Sun is grateful to the Department of Chemical and Biological Engineering, HKUST for the faculty startup fund. Xiaotian Liu is a recipient of the HKJEBN scholarship. REFERENCES 1. Cameron, A. R.; Frith, J. E.; Cooper-White, J. J., The influence of substrate creep on mesenchymal stem cell behaviour and phenotype. Biomaterials 2011, 32 (26), 5979-5993. 2. Cameron, A. R.; Frith, J. E.; Gomez, G. A.; Yap, A. S.; Cooper-White, J. J., The effect of time-dependent deformation of viscoelastic hydrogels on myogenic induction and Rac1 activity in mesenchymal stem cells. Biomaterials 2014, 35 (6), 1857-1868. 3. McKinnon, D. D.; Domaille, D. W.; Cha, J. N.; Anseth, K. S., Biophysically defined and cytocompatible covalently adaptable networks as viscoelastic 3D cell culture systems. Adv Mater 2014, 26 (6), 865-872. 4. Zhao, X.; Huebsch, N.; Mooney, D. J.; Suo, Z., Stress-relaxation behavior in gels with ionic and covalent crosslinks. J Appl Phys 2010, 107 (6), 63509. 5. Vining, K. H.; Mooney, D. J., Mechanical forces direct stem cell behaviour in development and regeneration. Nat Rev Mol Cell Biol 2017, 18 (12), 728-742. 6. Lee, B. P.; Messersmith, P. B.; Israelachvili, J. N.; Waite, J. H., Mussel-Inspired Adhesives and Coatings. Annu Rev Mater Res 2011, 41, 99-132. 7. Lin, Q.; Gourdon, D.; Sun, C.; Holten-Andersen, N.; Anderson, T. H.; Waite, J. H.; Israelachvili, J. N., Adhesion mechanisms of the mussel foot proteins mfp-1 and mfp-3. Proc Natl Acad Sci U S A 2007, 104 (10), 3782-3786. 8. Hwang, D. S.; Yoo, H. J.; Jun, J. H.; Moon, W. K.; Cha, H. J., Expression of functional recombinant mussel adhesive protein Mgfp-5 in Escherichia coli. Appl Environ Microbiol 2004, 70 (6), 3352-3359. 9. Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B., Mussel-inspired surface chemistry for multifunctional coatings. Science 2007, 318 (5849), 426-430. 10. Gim, Y.; Hwang, D. S.; Cha, H. J., Expression of functional recombinant mussel adhesive protein Type 3A in Escherichia coli. J Biotechnol 2005, 118, S169-S169. 11. Choi, Y. S.; Yang, Y. J.; Yang, B.; Cha, H. J., In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli. Microb Cell Fact 2012, 11, 139. 12. Zhong, C.; Gurry, T.; Cheng, A. A.; Downey, J.; Deng, Z. T.; Stultz, C. M.; Lu, T. K., Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nat Nanotechnol 2014, 9 (10), 858-866. 20 ACS Paragon Plus Environment

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