Versatile Engineered Protein Hydrogels Enabling Decoupled

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Article Cite This: ACS Appl. Nano Mater. 2018, 1, 1579−1585

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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*,†

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Department of Chemical and Biological Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong ‡ Division of Life Science, State Key Laboratory of Molecular Neuroscience, and Center of Systems Biology and Human Health, School of Science and Institute for Advanced Study, Hong Kong University of Science and Technology, Hong Kong S Supporting Information *

ABSTRACT: Development of engineered protein materials with wide-ranging mechanical strength and stiffness while maintaining the biofunctionality of protein molecules within remains a big challenge. Here we demonstrate the synthesis of protein hydrogels by photochemically cross-linking recombinant mussel foot protein-3 (Mfp3). The hydrogels’ stiffness can be broadly tuned by adjusting the concentration of protein polymers or co-oxidants, or light intensity needed for the chemical crosslinking. The protein polymers were also designed to contain SpyCatcher domains, which enabled postgelation 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 they were also able to activate the JAK/STAT3 pathway and induce neurite growth via the covalently immobilized leukemia inhibitory factor (LIF). These results illustrate a new strategy for designing bioactive materials for regenerative neurobiology. KEYWORDS: protein hydrogel, mussel foot protein, SpyTag/SpyCatcher, neurite growth, LIF signaling

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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 cross-linking chemistry, cross-linking 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 subsequent function© 2018 American Chemical Society

alization 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 post-translationally 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 Received: January 16, 2018 Accepted: March 26, 2018 Published: March 26, 2018 1579

DOI: 10.1021/acsanm.8b00077 ACS Appl. Nano Mater. 2018, 1, 1579−1585

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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-cleavage (MMP-cleavage) site that enables matrix remodeling by encapsulated cells. (B) Schematic illustration of the creation of a hydrogel by photochemically cross-linking 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.

entirely engineered protein-based hydrogels, 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 cross-linking 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 wideranging mechanical properties and diverse biological functionality through the combined use of photo-cross-linkable Mfp3 proteins and SpyTag/SpyCatcher chemistry. We also examined the neurotrophic properties of the leukemia inhibitory factor (LIF) immobilized onto the hydrogel and proved the feasibility of using this protein material to direct neuronal signaling and neurite outgrowth.

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 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 mussel foot proteins 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 coexpressed bacterial tyrosinase inside cells, or in vivo residue-specific incorporation of Dopa enabled by promiscuous E. coli tyrosyl-tRNA synthetase.10−13 However, 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 the post-translational modification (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 cross-linking 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, di-Tyr cross-linking reactions, especially those induced photochemically, have received significant attention from materials scientists, and have 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, the SpyTag/SpyCatcher chemistry has emerged as one of the most exciting tools for constructing advanced protein architectures and biomaterials because of its marked efficiency, selectivity, and full genetic encodability.19−21 The synthesis of diverse,

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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, because of 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-ELPSpyCatcher (BMB), which encodes a multidomain protein harboring an Mfp3, flanked by two elastin-like polypeptides (ELPs) and SpyCatcher domains (Figures 1 and S1). 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 Arg-Gly-Asp (RGD) cell-binding domains and matrix-metalloproteinase-cleavage 1580

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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) Frequencysweep 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.

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. 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. Ru(II)-mediated photo-cross-linking of Tyr residues undergoes a radical mechanism and can be detrimental to marginally stable globular proteins. Thus, a milder chemistry that is independent of the gelation reaction is needed for the biofunctionalization of the hydrogel. We leveraged the preexisting SpyCatcher domains within the BMB protein. The microbeads and microstrips were immersed in the PBS solution containing both SpyTag-mCherry-SpyTag (A-mCherry-A) and SpyTag-EGFP-SpyTag (A-EGFP-A) by extensive washing with PBS to remove unreacted proteins. It turned out that both AmCherry-A and A-EGFP-A were efficiently immobilized onto the microbeads and microstrips, as made evident by fluorescence micrographs of these materials, while the two proteins, mCherry and EGFP, lacking SpyTag failed to decorate the hydrogels, suggesting that the SpyTag/SpyCatcher chemistry was responsible for the observed postgelation modification (Figure 3). The SpyCatcher domains survived the photochemical gelation and remained reactive, showcasing the robustness of the 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. To determine whether the tuning processes over gel mechanics and biochemical aspects are truly decoupled, we

(MMP-cleavage) sites, both of which are expected to facilitate the interactions between cells and the substrates. 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 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 visiblelight-harvesting compound with a λmax of 452 nm in H2O28 and ammonium persulfate (APS) was adopted to covalently cross-link 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 the 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 cross-linked hydrogel (Figure S4). The photo-cross-linked product emitted blue fluorescence (excitation, 350 nm; emission, 426 nm), consistent with the spectral feature of the dityrosine moiety (Figure S5). Mechanical properties of the hydrogels can be adjusted by varying the concentration of the co-oxidant APS or the protein polymers (Figure 2A,C). Reduced amounts of either APS or BMB led to significantly softened hydrogels (Figure 2A,C), 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 (Figure 2B,D). The gelation also depends on light intensity, and the reaction rates were much slower under 1581

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Cell Adhesion and Proliferation on Mfp Hydrogel Substrates. Examination of the Mfp3 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 Mfp3 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 cross-links.

Figure 3. Microfabrication and functionalization of Mfp hydrogels. (A, B) Micrographs of Mfp hydrogel microbeads and microstrips decorated with SpyTag-mCherry-SpyTag (A-mCherry-A) and SpyTag-EGFP-SpyTag (A-EGFP-A). The mCherry and EGFP proteins that do not possess SpyTag were used as controls. BF, bright field. The blue fluorescence arises from the dityrosine cross-links. (C, D) Comparison of the fluorescence of microbeads decorated with mCherry, A-mCherry-A, EGFP, and A-EGFP-A. (E, F) Comparison of the fluorescence of microstrips decorated with mCherry, AmCherry-A, EGFP, and A-EGFP-A. Data are presented as mean ± SD (n = 10).

The Mfp hydrogel also enabled facile micropatterning of cell cultures. When a microstrip gel was used, cells preferably adhered to the gel areas, and significant cell proliferation was observed on day 3, while few cells were found in the exposed areas (Figure 5). Taken together, these results showed the feasibility of using Mfp hydrogels as a general substrate for cell culturing and micropatterning. 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 The leukemia inhibitory factor (LIF), a 20 kDa cytokine, has

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 cross-linking density and stiffness, suggesting that their mechanical properties have negligible effects on the postgelation modification. To examine the influence of postgelation 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 postgelation modification by SpyTag/SpyCatcher chemistry per se did not modulate the gel network structure within as well as its cross-linking density. Taken together, these results show that the mechanical and chemical tuning over the material properties are indeed decoupled.

Figure 5. Adhesion and proliferation of 3T3 fibroblasts on microstrips. 1582

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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 A-LIF-A after culturing merely for 1 day (Figure 6A,B). For confirmation of the influence of immobilized LIF on JAK/STAT3 signaling, Western blot analysis was performed to determine the level of Tyr705phosphorylated 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 equiv) was used for the gelation reaction in this study. In addition, 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 made evident by the efficient postgelation modification with the SpyTagged proteins (Figures 3A,B and 6). The activities of these proteins were also preserved after immobilization, suggesting that this photochemical gelation strategy is by and large compatible with the engineered protein hydrogels. Moreover, these photocross-linked 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-cross-linkable Mfp3 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 materials neurobiology.

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 exists 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 postgelation modification with 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. 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-ELP-SpyTag (A-LIFA) 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, 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

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EXPERIMENTAL PROCEDURES

Gene Construction. The creation of the gene constructs, SpyTagELP-SpyTag-ELP-SpyTag (AAA), SpyTag-ELP-mCherry-ELP-SpyTag (A-mCherry-A), SpyTag-ELP-LIF-ELP-SpyTag (A-LIF-A), SpyTagELP-EGFP-ELP-SpyTag (A-EGFP-A), and SpyCatcher-ELP-SpyCatcher (BB), has been described previously.22 The Mf p3 gene was purchased as a gBlocks gene fragment from Integrated DNA Technologies (IDT). For the creation of SpyCatcher-ELP-Mf p3-ELP-SpyCatcher (BMB), the Mf p3 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 until optical density reached 0.6−0.8 at 600 nm. Protein expression was induced by addition of 500 μM isopropyl β-D-1-

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 Tyr705phosphorylated 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). 1583

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ACS Applied Nano Materials thiogalactopyranoside (IPTG) at 32 °C. After 4 h, cells were harvested by centrifugation at 4200g, 4 °C for 15 min. Cell pellets were frozen at −80 °C for 2 h before protein purification. The pellets were suspended in the lysis buffer containing 300 mM NaCl, 10 mM Tris-HCl (pH 8), and 1 mM phenylmethylsulfonyl fluoride (PMSF), lysed by a sonication homogenizer, and clarified by centrifugation at 18 000g, 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 5200g for 40 min to remove insoluble fractions. The supernatants were snap-frozen in liquid nitrogen, stored at −80 °C overnight, and lyophilized for 72 h. 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-sweep 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-shaped” droplet-generating microfluidic device.29 Microstrips were created by flowing gelation solutions into PDMS microchannels and then curing under white LED light illumination. For covalent immobilization of fluorescent proteins, the microbeads/microstrips were immersed in the solutions containing A-EGFP-A and A-mCherry-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. 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’s medium (DMEM; Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco), and 1% (v/v) penicillinstreptomycin (Sangon Biotech). The cells were passaged at ∼75% confluence. Trypsin (0.25%) solution (Sangon Biotech) was used to digest and suspend cells. Approximately 2000 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 was 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 of 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 were dissected in cool Hank’s balanced salt solution (HBSS). Cortices were collected and digested by the digestion medium containing 10 mg/mL papain for 30 min. After resuspension, isolated cortical neurons were plated on the poly-Dlysine (PDL) or Mfp-gel-coated 6-well plates. Immobilization of ALIF-A was accomplished by immersing the Mfp-gel films in the A-LIFA (1 mg/mL in PBS) solution for 1 h, followed by 3× PBS wash to remove unreacted A-LIF-A. The culture medium comprising neuronalA and 2% B-27 supplement (Gibco) was added to cover the cells. After 1 day of 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 PBS with Tween (PBST) 4 times, the membranes were further incubated with goat anti-HRP (Life Technologies) for 1 h. Images were taken with the Bio-Rad ChemiDoc gel imaging system and analyzed by ImageJ software. For examination of the influence of LIF on neurite growth, the other half of the cells were fixed by paraformaldehyde (4% in PBS) for 10 min, blocked with 4% normal goat serum (NGS), and stained with mouse anti-beta3-tubulin antibody (BioLegend) at room temperature for 2 h. Then, the cells were washed with PBS 3 times and incubated with goat anti mouse 555 (Life Technologies) for 1 h. 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 software.

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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/acsanm.8b00077. Amino acid sequences and additional characterization data including time-sweep results, photographs, SEM micrograph, excitation and emission spectra, G′ vs time plot, fluorescence intensity comparison, and frequencysweep results (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Songzi Kou: 0000-0003-4662-3652 Fei Sun: 0000-0002-3065-7471 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS F.S. is thankful for the funding support from the Research Grants Council of Hong Kong SAR Government (Grant 26103915 and AoE/M-09/12). F.S. is grateful to the Department of Chemical and Biological Engineering, HKUST, for the faculty startup fund. X.L. is a recipient of the HKJEBN scholarship.

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REFERENCES

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DOI: 10.1021/acsanm.8b00077 ACS Appl. Nano Mater. 2018, 1, 1579−1585

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DOI: 10.1021/acsanm.8b00077 ACS Appl. Nano Mater. 2018, 1, 1579−1585