Article Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Synthesis of Entirely Protein-Based Hydrogels by Enzymatic Oxidation Enabling Water-Resistant Bioadhesion and Stem Cell Encapsulation Jiren Luo, Xiaotian Liu, Zhongguang Yang, and Fei Sun*
ACS Appl. Bio Mater. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 11/08/18. For personal use only.
Department of Chemical and Biological Engineering, Center of Systems Biology & Human Health, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong S Supporting Information *
ABSTRACT: Growing complexity in modern surgery and guided tissue repair calls for new materials capable of dual functionswater-resistant adhesion to biological tissues and stem cell encapsulation/delivery. Here, we demonstrate the creation of entirely recombinant protein-based adhesive hydrogels by leveraging the sequence derived from natural adhesive moleculesmussel foot protein-3 (Mfp-3), in vitro enzymatic oxidation mediated by recombinant tyrosinase and genetically encoded SpyTag/SpyCatcher chemistry. The resulting materials exhibited varied stiffness dependent on the polymer concentration, strong water-resistant adhesion to porcine skin, and excellent compatibility with 3D stem cell culture. The presence of SpyCatcher domains within the protein networks also enabled postgelation decoration with SpyTagged proteins under mild physiological conditions. These results point to a new approach for designing genetically programmable materials that enable both water-resistant bioadhesion and stem cell encapsulation for biomedical applications. KEYWORDS: mussel foot protein, tyrosinase, SpyCatcher, underwater adhesion, protein hydrogel, cell encapsulation
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INTRODUCTION Compared with surgical sutures and staples, tissue bioadhesives are of great advantage for being able to simplify surgical procedures and minimize the risk of complications during treatment.1,2 Commercially available bioadhesives such as those based on fibrin, gelatin, or synthetic polymers (cyanoacrylate adhesives) are constrained by several drawbacks including poor degradability, toxicity, and weak adhesion to tissues. Although PEG hydrogel-based adhesives exhibit good biocompatibility, concerns remain over their excessive swelling and reduced mechanical properties in a moist environment.3−5 Mussel foot proteins, such as Mfp-3 and Mfp-5, originate from marine mussel Mytilus edulis and are noted for their strong underwater adhesion to a variety of substrates. Such an underwater adhesion phenomenon is largely attributed to the presence of abundant post-translationally modified amino acid residue, 3,4-dihydroxyphenyl-L-alanine (DOPA) within these proteins. Using natural mussel foot proteins as bioadhesives is hindered by their poor solubility, susceptibility to oxidation, and limited supply from natural sources.6,7 Nevertheless, mechanistic studies on these naturally occurring adhesives © XXXX American Chemical Society
have inspired the design and creation of numerous synthetic polymer-based materials and coatings that are capable of waterresistant adhesion.6,8−10 However, the ever-growing demand for adhesive materials in regenerative medicine requires a new level of craftsmanship from bioengineers who wish to obtain not only better biocompatibility and wet adhesion but also the ability to deliver stem cells. Recombinant Mfp’s have previously been produced using Escherichia coli expression systems in hope of exploring the potential of these proteins as bioadhesives.11−15 Since E. coli cells are not equipped with the cellular machinery needed to convert Tyr to DOPA, several methods have been employed to obtain adhesive proteins from bacterial synthesis, including in vivo residue-specific incorporation of DOPA by promiscuous E. coli tyrosyl-tRNA synthetase and in situ Tyr oxidation by a coexpressed bacterial tyrosinase.11−14 Nevertheless, these DOPA-containing proteins produced by cellular synthesis Received: September 17, 2018 Accepted: October 25, 2018
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DOI: 10.1021/acsabm.8b00541 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Article
ACS Applied Bio Materials
Figure 1. Synthesis of a recombinant Mfp-3-based hydrogel by enzymatic oxidation. (A) Genetic construct of the hydrogel precursor BMB. Two ELP domains are incorporated as spacers. An integrin-binding RGD sequence is included. Tyr and positively charged residues are colored in brown and red, respectively. (B) Schematic illustration of hydrogel formation and postgelation functionalization via SpyTag/SpyCatcher chemistry.
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RESULTS AND DISCUSSION Hydrogel Precursor Design. The DOPA-containing Mfp3 plays a key role in the underwater adhesion of marine mussels. The recombinant Mfp-3 produced by a simple E. coli expression system possesses rich Tyr residues instead of DOPA because of the absence of a cellular machinery for Tyr oxidation. In addition, overexpressing this intrinsically disordered protein, Mfp-3, in E. coli often led to the formation of inclusion bodies, which prevents us from using this protein and alike for material design. We previously reported the production of the telechelic protein construct, SpyCatcherELP-Mfp-3-ELP-SpyCatcher (BMB), in a soluble form using a heterologous E. coli expression, where the ELP domain consists of 15 repetitive pentapeptides, VPGXG (X is Val or Glu in a 4:1 ratio) (Figure 1A).15 This particular ELP composition exhibited good expression yield and solubility under physiological conditions.20 The soluble expression of this recombinant Mfp-3 construct offered us the possibility to examine the influence of in vitro enzymatic oxidation on protein polymers. In principle, the presence of SpyCatcher domains would also allow further decoration of the protein architecture by SpyTag-fusion proteins if they can survive the enzymatic oxidation conditions. The introduction of RGD cell binding ligands would promote cell adhesion within the protein polymers. Synthesis of Mfp-3 Hydrogels with Tunable Mechanical Properties. The recombinant BMB protein was produced in a satisfactory yield by E. coli (∼20 mg per liter cell culture in LB medium). Following Ni-NTA chromatography, dialysis against Milli-Q water, and lyophilization, white protein powders were obtained (Figure S3A). To mimic the natural enzymatic process for Tyr oxidation, the copper(II)-dependent oxidative enzyme, S. antibioticus tyrosinase, was cloned and produced in a recombinant form using E. coli expression followed by in vitro reconstitution as described in the Experimental Procedures section (Figure S2). The resulting enzyme showed the ability to oxidize the BMB protein; the BMB solutions (7 and 10 wt % in PBS) turned brownish gradually after addition of the enzyme at room temperature
exhibited strong aggregation and poor solubility due to overoxidation and thus posed significant challenges for purification and material fabrication. We previously reported the synthesis of protein hydrogels by photo-cross-linking recombinant Mfp’s, whereas abundant Tyr residues within the protein polymers were photo-oxidized in vitro to form di-Tyr cross-links by a Ru(II) catalyst.15 Despite the wide-ranging mechanical properties achieved, the resulting materials were not as adhesive as the naturally occurring Mfp’s because of the absence of DOPA. We envisioned that, with ease of control, an in vitro enzymatic oxidation process that mimics natural Tyr oxidation within the mussel and converts Tyr into DOPA would provide an alternative strategy for creating adhesive materials with tunable material properties including stiffness and adhesiveness. A previous study demonstrated that coexpressing Streptomyces antibioticus tyrosinase in E. coli led to in situ oxidation of Tyr residues within recombinant Mfp’s,12 which prompted us to examine the feasibility of using the same tyrosinase for in vitro oxidation to generate Mfp-based adhesive materials while circumventing the difficulty in isolating posttranslationally modified Mfp’s from in vivo oxidation systems. In this study, we created a multidomain fusion protein, SpyCatcher-ELP-Mfp-3-ELP-SpyCatcher, comprising one Mfp-3 domain, and two flanking ELP and SpyCatcher domains which can be produced heterologously by E. coli. Oxidation of the protein polymers by a reconstituted S. antibioticus tyrosinase in vitro led to the formation of a hydrogel that exhibited marked adhesion to biological tissues and excellent compatibility toward encapsulated stem cells. The presence of SpyCatcher domains within the protein network enabled postgelation decoration with SpyTagged globular proteins, showing its amenability toward further functionalization. This study illustrates a novel recombinant approach for creating multifunctional hydrogel bioadhesives using modular genetic designs, which is more tunable and advantageous than conventional polymer synthesis approaches.16−19 B
DOI: 10.1021/acsabm.8b00541 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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ACS Applied Bio Materials
Adhesion to Various Substrates. We performed shear adhesion tests on the Mfp-3 hydrogels formed in situ. The mixture of the BMB protein solution (10 wt %) and tyrosinase was applied to several substrates including glass slides, poly(methyl methacrylate) (PMMA) plates, and porcine skin. After 1 h of reaction, strong adhesions up to ∼33 and ∼35 N/cm2 were observed on dry glass and PMMA surfaces, respectively, while all control samples, the BMB solution without tyrosinase, the BSA solution with tyrosinase, and the SpyTag-ELP-SpyTag-ELP-SpyTag (AAA) solution (lacking Mfp-3 domains) with tyrosinase, exhibited very weak adhesion (Figure 3A). Although the oxidation of AAA by tyrosinase led
under atmospheric conditions, which is indicative of the occurrence of oxidation. The formation of a gel-like solid was observed within 1 h (Figure S3A). We speculated the formation of not only DOPA but also dopaquinone. The latter is an overoxidized product typical for prolonged oxidation mediated by tyrosinase. Given the abundance of Lys in Mfp-3, the primary amines from Lys and protein Ntermini further reacted with the dopaquinone moieties to Michael addition adducts or Schiff bases (Figure 1B). After 1 h of reaction, dynamic shear frequency sweep tests were performed to examine the viscoelastic properties of the products. The reaction products (7 and 10 wt %) both exhibited a steady storage modulus G′ and a substantially lower loss modulus G′′ over the shear frequency range 0.1−100 rad/ s, confirming the formation of covalently cross-linked polymer networks, which is likely attributed to the formation of amine− dopaquinone adducts (Figure 2A). Higher polymer concentration resulted in larger G′, suggesting that the stiffness of the material can be tuned by varying the protein concentration (Figure 2B).
Figure 3. Adhesion tests on Mfp-3 hydrogels. (A) Quantification of interfacial adhesion by tensile tests shows strong adhesion of the Mfp3 hydrogels to glass, PMMA, and porcine skin. PBS buffer, the BMB protein solution without tyrosinase, the BSA solution with tyrosinase, and the AAA solution (lacking Mfp-3 domains) with tyrosinase were used as control. TY, tyrosinase. Data are presented as mean ± SD (n = 3). (B) Shear adhesion strength of Mfp-3 hydrogels was changed from 12.82 ± 2.24 to 11.88 ± 0.73 N/cm2 (n = 3) after soaking the glued porcine skin in PBS for 6 h.
Figure 2. Rheological characterization of Mfp-3 hydrogels. (A) Frequency sweep tests on hydrogels with varied polymer concentration (7 and 10 wt %) over the frequency range 0.1−100 rad/s. G′, storage modulus. G′′, loss modulus. (B) Storage moduli at frequency of 1 rad/s showing that increased polymer concentrations led to higher stiffness. Data are presented as mean ± SD (n = 3). The strain was set constant at 5%. All the tests were performed at 25 °C.
to the formation of a gel-like material, which is presumably caused by the oxidative cross-linking of the Tyr residues within SpyTag domains, the product exhibited much weaker adhesion than oxidized BMB, suggesting that the oxidized Mfp-3 domain is essential for the adhesion observed. The material comprising oxidized BMB also exhibited rather strong adhesion (∼13 N/ cm2) to porcine skin (Figure 3A), which is comparable to most of the existing bioadhesives.21−25 More intriguingly, soaking the glued porcine skin in PBS for 6 h led to almost no loss of adhesion strength, demonstrating marked water resistance of the adhesion (Figure 3B). Decoration of Hydrogel Microstrips with Globular Proteins. SpyTag/SpyCatcher chemistry has emerged as a powerful technology for constructing advanced protein architectures by enabling facile covalent conjugation of protein
The resulting Mfp-3 hydrogel exhibited no significant swelling after being immersed in PBS for 1 week, a property which is important for in vivo applications (Figure S4). It is also noteworthy that although bovine serum album (BSA) possesses a number of Tyr and Lys residues, no gelation was observed upon treating BSA (10 wt % in PBS) with tyrosinase (Figure S3B), suggesting that the oxidation of Tyr by tyrosinase is sequence-dependent. Unlike unstructured Mfp-3, the globularly folded structure of BSA may render Tyr residues inaccessible to the catalyst because of steric hindrance. C
DOI: 10.1021/acsabm.8b00541 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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ACS Applied Bio Materials molecules under mild physiological conditions.26−30 The advancement in tissue engineering summons new materials with customizable biophysical properties for controlling cell fate decision. The presence of SpyCatcher in the Mfp-3 hydrogel provides the opportunity for specific biofunctionalization needed for regenerative purposes. The SpyTag/ SpyCatcher chemistry has proven effective in modifying protein hydrogels with globularly folded proteins even after photochemical cross-linking.15 It remains to be seen whether the SpyTag/SpyCatcher chemistry can tolerate the enzymatic oxidation involved in the gelation reaction. To test this, the microstrips composed of the Mfp-3 hydrogel were created and submerged in the PBS buffer containing two SpyTagged proteins, SpyTag-EGFP-SpyTag (A-EGFP-A) and SpyTagmCherry-SpyTag (A-mCherry-A), followed by an extensive wash with PBS to remove unreacted proteins. Both SpyTagged fluorescent proteins were successfully immobilized onto the hydrogels, while those lacking SpyTag were not (Figure 4). These results demonstrate the compatibility of SpyTag/ SpyCatcher chemistry with the tyrosinase oxidation and point to a robust postgelation modification strategy.
Figure 5. Encapsulation of human mesenchymal stem cells (hMSCs) by Mfp-3 hydrogels for (A) 1 day and (B) 7 days. Confocal fluorescence z-slice micrographs of live (green, stained with fluorescein diacetate) and dead (red, stained with ethidium homodimer) cells show high levels of cell viability (93.4% ± 5.9% and 92.1% ± 6.6%, respectively, n = 5). Scale bars, 100 μm.
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CONCLUSION In summary, we successfully created an entirely protein-based, wet-adhesive hydrogel comprising Mfp-3, ELP, and SpyCatcher domains using in vitro enzymatic oxidation. The resulting protein materials have exhibited multiple capabilities including water-resistant adhesion to biological tissues, postgelation modification with globular proteins, and stem cell encapsulation. This study illustrates a new strategy for designing genetically programmable hydrogel-based bioadhesives.
Figure 4. Postgelation decoration of Mfp-3 hydrogel microstrips by SpyTag/SpyCatcher chemistry. BF, bright field. A-EGFP-A, SpyTagEGFP-SpyTag. A-mCherry-A, SpyTag-mCherry-SpyTag. Scale bars, 200 μm.
Stem Cell Encapsulation by Mfp-3 Hydrogels. Adhesive hydrogels can have a variety of applications in regenerative medicine, one of which is to serve as stem cell carriers. To explore such a possibility, the Mfp-3 hydrogel was tested for stem cell encapsulation experiments. Human mesenchymal stem cells (hMSCs) were suspended in a solution of BMB dissolved in DMEM (7 wt %) followed by addition of tyrosinase to initiate the gelation. Cell growth within the hydrogels was monitored for 7 days, during which no cells migrated to the surrounding medium. Cell viability (∼93%) after encapsulation for 1 day and 7 days was determined by standard live/dead staining and laser scanning confocal microscopy (Figure 5). Some live cells exhibited spread morphologies after encapsulation for 7 days, indicating effective cell adhesion enabled by the RGD ligands within the gel network (Figure 5B). Although this hydrogel exhibited marked stability in vitro, this entirely protein-based material could be subject to proteolytic degradation in vivo. Moreover, through genetic engineering, matrix-metalloproteinase (MMP) cleavage sites can also be introduced to the material scaffold to promote cell migration and matrix remodeling.30 Together with its water-resistant adhesiveness, the Mfp-3 hydrogel could serve as a bifunctional tissue regeneration platform capable of stem cell delivery and biological adhesion simultaneously.
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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.26 The genes of tyrosinase, ORF438 and mf p-3, were purchased as gBlocks gene fragments from Integrated DNA Technologies. The gene of tyrosinase was cloned into plasmid pACYCDuet1 using BamHI and SalI cutting sites. The gene of ORF438 was cloned into plasmid pACYCDuet1 using NdeI and XhoI cutting sites. For the construction of SpyTag-ELP-mfp-3-ELP-SpyTag (AMA) and SpyTag-ELP-SpyTag (AA), the middle SpyTag between SacI and SpeI cutting sites in AAA was replaced by the gene encoding the sequence of mf p-3 and the gene encoding Glycine−Arginine− Glycine−Aspartate (GRGD) peptide, respectively. For the construction of SpyCatcher-ELP-mf p-3-ELP-SpyCatcher (BMB), the gene encoding the sequence of mf p-3 was inserted into the middle of BB using SacI and SpeI cutting sites. E. 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. For the expression and purification of BMB, E. coli strain BL21(DE3) harboring the corresponding BMB gene was grown at 37 °C, 220 rpm D
DOI: 10.1021/acsabm.8b00541 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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ACS Applied Bio Materials in LB medium until the optical density (600 nm) reached 0.6−0.8. Protein expression was induced with 500 μM isopropyl β-D-1thiogalactopyranoside (IPTG) at 30 °C. After 4 h, cells were harvested by centrifugation at 4 °C for 15 min. Cell pellets were frozen at −80 °C for 2 h before protein purification. After thawing at room temperature, the cell pellets were resuspended in the lysis buffer {300 mM NaCl, 10 mM Tris, pH 8.0, and 1 mM protease inhibitor [phenylmethylsulfonyl fluoride (PMSF)]}. Cell lysates were prepared using a sonication homogenizer and clarified by centrifugation at 4 °C for 30 min. Target proteins were purified by the HisTrap nickle (II) columns (GE Healthcare, Inc.) following the column manufacturer’s recommendations and eluted with the elution buffer (300 mM NaCl, 10 mM Tris, 500 mM imidazole, pH 8.0). Protein purity was assessed by SDS-PAGE. After the Ni-NTA purification, BMB was dialyzed against Milli-Q water (4.5 L × 7) at 4 °C, and then centrifuged to remove precipitants. The resulting protein solution was 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. For the production of tyrosinase, E. coli strain BL21(DE3) harboring the pACYCDuet1::tyrosinase/ORF438 was grown at 37 °C, 220 rpm in TB medium (tryptone 1.2%, yeast extract 2.4%, glycerol 0.4%, and potassium phosphate buffer 89 mM) until the optical density (600 nm) reached 0.6−0.8. Protein expression was induced by addition of 500 μM IPTG at 16 °C. After 20 h, cells were harvested by centrifugation at 4 °C for 15 min. Cell pellets were frozen at −80 °C for 2 h before protein purification. After thawing at room temperature, pellets were resuspended in the binding buffer (20 mM sodium phosphate buffer, 500 mM NaCl, pH 7.5, and 1 mM PMSF). Cell lysates were prepared using a sonication homogenizer and clarified by centrifugation at 18 000g, 4 °C for 30 min. Target proteins were purified by the HisTrap nickle (II) columns (GE Healthcare, Inc.) following the column manufacturer’s recommendations and eluted by elution buffer (20 mM sodium phosphate buffer, pH 7.5, 500 mM NaCl, and 500 mM imidazole). Protein purity was assessed by SDS/PAGE. After the Ni-NTA purification, proteins were dialyzed against dialysis buffer (50 mM sodium phosphate buffer, pH 6.5, and 0.01 mM CuSO4) at 4 °C, and then centrifuged at 5200g for 40 min to remove precipitants. The resulting protein solution was diluted to 1.0 mg/mL and snap-frozen in liquid nitrogen, stored at −80 °C before use. Hydrogel Formation. Lyophilized BMB protein was dissolved in sterilized phosphate-buffered saline (PBS, pH 7.4) to make a 10 wt % solution. The protein solution (30 μL) was mixed with 1 μg of tyrosinase in an Eppendorf tube to initiate gelation at room temperature. Dynamic Oscillatory Shear Rheology. Rheological measurements were performed on a TA Instruments ARES-RFS straincontrolled rheometer with a customized steel parallel-plate geometry (8 mm diameter for the upper fixture and 25 mm diameter for the bottom fixture). Test modes included dynamic time sweep, strain sweep, and frequency sweep. Lyophilized BMB was dissolved in PBS to make a 10 wt % solution. The protein solution (60 μL) was mixed with 2 μg of tyrosinase on Parafilm and let stand at room temperature for 48 h. The hydrogels were then mounted on the top of a 25 mm plate. An 8 mm plate was brought down, forming an 8 mm disk of gel with an average thickness of 0.90 mm. Gels were surrounded by silicone oil to prevent water evaporation. The gelation was monitored at room temperature (25.0 °C) by time sweep measurements while the strain was held constant at 5.0% and frequency at 5.0 rad/s. The storage modulus (G′) and loss modulus (G′′) were recorded over a period until both moduli reached a plateau. Frequency sweep tests were performed at room temperature while holding the strain at 10% and varying the oscillatory frequency from 100 to 0.01 rad/s. Strain sweep tests were performed with the strain amplitude varied from 0.1% to 100%. Shear Adhesion Tests. Shear adhesion tests on PMMA and glass were performed with an Instron tester. Protein solutions (30 μL) mixed with tyrosinase (1 μg) were spread on 25 mm × 25 mm surfaces of either 75 mm × 25 mm PMMA or glass strips. The ends of the strip were clamped in the jaws of the Instron tester and stretched
at a cross-head speed of 0.2 mm/min. The peak forces were recorded, as indicated by a following sharp drop of shear force resulting from the breakage of the hydrogel. Shear adhesion tests on biological tissues were performed on a TA Instruments ARES-RFS straincontrolled rheometer. Biological tissues were prepared by cutting precleaned porcine skin to 30 mm × 10 mm × 2 mm strips. The ends of the strip were clamped by a thin film fixture and stretched at a cross-head speed of 0.2 mm/min. The peak forces were recorded. Postgelation Decoration of Mfp Hydrogels. The hydrogel microstrips were created by flowing gelation solutions (30 μL of protein solution mixed with 1 μg of tyrosinase) into polydimethylsiloxane (PDMS) microchannels and then curing at room temperature. The microstrips were then immersed with PBS containing A-EGFP-A and A-mCherry-A at room temperature for 30 min. The microstrips were rinsed with PBS 5 times to wash away unreacted fluorescent proteins. Cell Encapsulation by Mfp Hydrogels. NIH/3T3 fibroblasts, Madin Darby canine kidney (MDCK) epithelial cells, and human mesenchymal stem cells (hMSCs) were used to test the feasibility of Mfp hydrogels for cell encapsulation. 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) penicillin-streptomycin (Sangon Biotech). At ∼75% confluence, cells were detached with 0.2 mL of 0.25% trypsin solution (Sangon Biotech) followed by addition of 2 mL of media to resuspend the cells and neutralize trypsin. The cell density was counted by a hemocytometer. The cells were then transferred to a centrifugal tube and harvested by spinning at 300g for 5 min at 37 °C. After removal of the medium, cell pellets were resuspended with the BMB solution in DMEM (10 wt %) to reach a final density of ∼2000 μL−1. Tyrosinase (1 μg) was added into the BMB solution (30 μL) to initiate the gelation on a microwell dish with a bottom coverslip at 37 °C. The mixture was cured for 30 min followed by addition of 2 mL of medium to immerse the gel. Cells encapsulated in the hydrogel were incubated at 37 °C with 5% CO2 for 24 h or 7 days before the assay. Cell viability was determined by live/dead staining. Briefly, after removing the medium, the cell culture was washed with 3 mL of PBS 3 times. The live/dead reagent in PBS (1 mL) composed of 4 μM ethidium homodimer (EthD-1) and 2 μM calcein AM was added and incubated for 45 min at room temperature. The cells were then washed by 3 mL of PBS to remove the dyes. Laser scanning confocal microscopy (Nikon C2) was used to visualize the stained cells.
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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/acsabm.8b00541. Sequences, genetic construct, SDS-PAGE analysis, Michaelis−Menten kinetics, and photographs (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Fei Sun: 0000-0002-3065-7471 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The funding support from Science, Technology and Innovation Commission of Shenzhen Municipality to F.S. (Basic Research Program Grant JCYJ20170818114000727), from PKU-HKUST ShenZhen-HongKong Institution to F.S. (SZIER18EG02), and from the Research Grants Council of Hong Kong SAR Government to F.S. (RGC-ECS Grant E
DOI: 10.1021/acsabm.8b00541 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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ACS Applied Bio Materials
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26103915 and CRF Grant C6004-17GF) are acknowledged. F.S. is grateful to the Department of Chemical and Biological Engineering, HKUST, for the faculty start-up fund. J.L. is a recipient of the Asian Future Leaders Scholar-ship with the support from the Bai Xian Education Foundation. X.L. is a recipient of the HKJEBN scholarship.
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DOI: 10.1021/acsabm.8b00541 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
