Enzymatic formation of a meta-stable supramolecular hydrogel for 3D cell culture

Abstract

We report the phosphatase-triggered formation of a meta-stable peptide-based supramolecular hydrogel that can easily change to a clear solution by mechanical forces. Cells cultured in the hydrogel can therefore be separated by pipetting followed by centrifugation.

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Peptide-based supramolecular hydrogels¹ are promising biomaterials because they mimic the extra-cellular matrix (ECM) and have been widely used for cell culture,² drug delivery,³ sensing,⁴ and regenerative medicine.⁵ Up to now, cell culture is probably the most successful one among those applications,^6,7 and peptide-based supramolecular hydrogels of RADARADARADARADA (RADA16), FEFKFEFK (EFK8) and Fmoc-FF are already commercially available. In order to facilitate their application in cell culture, especially in three dimensional (3D) cell culture, biocompatible methods allowing homogeneous encapsulation of cells have advantages.⁸ Extensive research efforts have helped to develop such methods including enzymatic triggers,⁹ ionic strength increase,¹⁰ photo irradiation,¹¹ redox control,¹² disulfide bond reduction,^13,14 ligand–receptor interactions,¹⁵ etc. These pioneering works provide useful methods to prepare hydrogels for 3D cell culture.

Supramolecular hydrogels made from peptides of EFK8 and RADA16 were first developed by Zhang and co-workers and are promising biomaterials for protein delivery and cell culture due to their excellent biocompatibility and degradability.⁶ However, these two peptides can only dissolve in acidic aqueous solution and form hydrogels after mixing with an equal volume of alkali buffer solution or cell culture medium. To overcome this shortcoming, derivatives of EFK8 and RADA16 that can form homogeneous solutions in neutral conditions have been developed.¹⁶ These peptides can form hydrogels in neutral conditions after mixing with buffer solutions. Recently, a method of disulfide bond reduction has also been applied to prepare EFK8 peptide hydrogels directly in a cell culture medium.¹³ Phosphatase has been used to catalyze the formation of supramolecular hydrogels for many biomedical applications.¹⁷ Stimulated by these pioneering works, we opted to develop a phosphatase catalyzed supramolecular hydrogel based on EFK8 for 3D cell culture.

As shown in Scheme 1, we designed a peptide of FEFKFEpYK. We believed that it could dissolve in neutral buffer solutions to form homogeneous solutions and then be converted to FEFKFEYK by phosphatase. The resulting peptide of FEFKFEYK might form supramolecular hydrogels because of its chemical similarity to the EFK8 peptide. We then used standard Fmoc-solid phase peptide synthesis (SPPS) to prepare the designed peptide directly. The pure peptide was obtained by high performance liquid chromatography (HPLC).

Scheme 1 Chemical structures of the peptides and schematic illustration of the transformation catalyzed by the enzyme of phosphatase.

After the synthesis, we first tested the solubility of FEFKFEpYK in aqueous solutions and its gelling ability after enzymatic conversion. The peptide could form homogeneous solutions in phosphate buffer solution (PBS, pH = 7.4) at concentrations up to 2.0 wt% (20 mg mL⁻¹). Addition of phosphatase enzyme to the solutions resulted in rapid hydrogelations. For instance, the addition of 15 U mL⁻¹ of phosphatase to the PBS solution of the peptide (0.8 wt%) led to a slightly opaque hydrogel formation within 5 minutes (Fig. 1A-II, 20–25 °C). The LC-MS trace indicated a rapid conversion from FEFKFEpYK to FEFKFEYK (Fig. 1B), and about 83% of the peptide had been converted after 2 h (70.2% had been converted at the gelling point). When using less enzyme, it took a longer time for hydrogel formation. For example, the gelation time was 20, 40, 120, and 270 minutes when using 10, 5, 2, and 1 U mL⁻¹ of the enzyme, respectively. The hydrogelations could also happen in the cell culture medium, suggesting it has a possible application in cell culture. The transmission electron microscopy (TEM) image revealed a dense network of nanofibers with a diameter of about 25 nm in the resulting hydrogel (Fig. 1C). As shown in Fig. 1D, the scanning electron microscopy (SEM) image indicated that these nanofibers entangled with each other and formed bundles of nanofibers with diameters of 30–40 μm in the dry gel.

Fig. 1 (A) Optical images of (I) the PBS buffer solution (pH = 7.4) of FEFKFEpYK (0.8 wt%, 8 mg mL⁻¹) and (II) the resulting gel formed following addition of 15 U mL⁻¹ of phosphatase to the solution in (I), (B) the percentage of conversion from FEFKFEpYK to FEFKFEYK in solution of A-II at different time points, and (C) a TEM image and (D) a SEM image of the gel in A-II at 24 h time point.

Interestingly, we found that the gels in both PBS and cell culture medium were meta-stable and would shrink after their formation when the concentration was lower than 1.2 wt%. As shown in Fig. S-4,† a large amount of water was excluded from the gel matrix and the colour changed to slightly yellowish after staying at room temperature for five days. Our recent study indicated that the phenol group on tyrosine (Y) could be oxidized to quinone by tyrosinase or oxygen in atmosphere.¹⁸ Such oxidation would led to the dissociation of the supramolecular network and shrinkage of the gel. Following addition of an equal volume of PBS to the gel on day 5 and then pipetting several times, the gel would change to a homogeneous solution. Since the gel was meta-stable, we were unable to characterize its mechanical properties using a rheometer. However, the unusual gelling property suggested its potential in cell culture because cells could be easily separated from it by simply adding culture medium and then pipetting.

We then tested application of the gel in a 3D cell culture model. The cell–gel constructs with low peptide concentration would be destroyed easily when replacing the culture medium, while the one with high peptide concentration would increase the difficulty of microscopic observation of cells within them. Taking into account both stability and clarity of the cell–gel constructs, we chose the peptide concentration to be 0.8 wt%. The hydrogelation could also happen within 5 minutes in Dulbecco’s Modified Eagle’s Medium (DMEM) cell culture (cell density = 1 400 000 mL⁻¹ and concentration of phosphatase was 15 U mL⁻¹). As shown in Fig. 2A, the confocal image revealed that cells were evenly distributed in the gels and most of them were alive (green and red dots showed live and dead cells, respectively), suggesting that the rapid enzymatic hydrogelation ensured a homogeneous encapsulation of the cells in gels and kept them alive. We used three kinds of cells (HeLa, HepG2, and A549) and the results obtained by the CCK-8 assay indicated that three kinds of cells kept proliferating within the cell–gel constructs during five days of culture (Fig. 2B). Similar to the gels formed in PBS solution, the cell–gel constructs kept shrinking during this time (Fig. 2C). We also found that the cells could be easily separated from the cell–gel constructs by pipetting and then centrifuging. These observations indicated that our meta-stable hydrogel was suitable for the 3D cell culture.

Fig. 2 (A) Confocal image of the cell–gel construct at 4 h time point (green dots reveal live cells), (B) cell proliferation curve measured by the CCK-8 assay, and (C) optical images of the cell–gel constructs in the incubator at different time points (from left to right: day 1, day 2, day 3, day 4, and day 5, respectively).

In summary, we have developed a peptide-based supramolecular hydrogel formed in a biocompatible and homogeneous way (enzymatic reaction). The resulting gel was meta-stable most probably due to the oxidation of the tyrosine residue on the peptide. Therefore, a gel–sol phase transition could be easily obtained by dilution and mechanical forces. Such properties rendered its application in 3D cell culture because cells could be homogeneously encapsulated in the gel and could also be easily separated post culture. However, one shortcoming of our system is that the oxidation of tyrosine may be affected by many factors especially the components in the system. We envision that the properties of meta-stable supramolecular hydrogels might be controlled by using different amounts of tyrosine residues or adding tyrosine-related enzymes such as tyrosinase, and then be applied in controlled delivery and tissue engineering.

Acknowledgements

This work is supported by National Natural Science Foundation of China (31271053, 51403105 and 31400858), Guangdong Province Natural Science Fund (S2013010015314), and Program for Changjiang Scholars and Innovative Research Team in University (IRT13023).

Notes and references

1. A. Tanaka, Y. Fukuoka, Y. Morimoto, T. Honjo, D. Koda, M. Goto and T. Maruyama, J. Am. Chem. Soc., 2014, 137, 770–775; J. H. Collier, J. S. Rudra, J. Z. Gasiorowski and J. P. Jung, Chem. Soc. Rev., 2010, 39, 3413–3424; Y. Gao, F. Zhao, Q. Wang, Y. Zhang and B. Xu, Chem. Soc. Rev., 2010, 39, 3425–3433; J. W. Steed, Chem. Soc. Rev., 2010, 39, 3686–3699.
2. H. Cho, S. Balaji, A. Q. Sheikh, J. R. Hurley, Y. F. Tian, J. H. Collier, T. M. Crombleholme and D. A. Narmoneva, Acta Biomater., 2012, 8, 154–164; V. Jayawarna, M. Ali, T. A. Jowitt, A. F. Miller, A. Saiani, J. E. Gough and R. V. Ulijn, Adv. Mater., 2006, 18, 611–614; V. Jayawarna, S. M. Richardson, A. R. Hirst, N. W. Hodson, A. Saiani, J. E. Gough and R. V. Ulijn, Acta Biomater., 2009, 5, 934–943; J. P. Jung, A. K. Nagaraj, E. K. Fox, J. S. Rudra, J. M. Devgun and J. H. Collier, Biomaterials, 2009, 30, 2400–2410; L. Lv, H. Liu, X. Chen and Z. Yang, Colloids Surf., B, 2013, 108, 352–357; Y. F. Tian, J. M. Devgun and J. H. Collier, Soft Matter, 2011, 7, 6005–6011; H. Wang, A. Han, Y. Cai, Y. Xie, H. Zhou, J. Long and Z. Yang, Chem. Commun., 2013, 49, 7448–7450.
3. A. G. Cheetham, P. Zhang, Y.-A. Lin, L. L. Lock and H. Cui, J. Am. Chem. Soc., 2013, 135, 2907–2910; J. Gao, W. Zheng, J. Zhang, D. Guan, Z. Yang, D. Kong and Q. Zhao, Chem. Commun., 2013, 49, 9173–9175; J. Li, X. Li, Y. Kuang, Y. Gao, X. Du, J. Shi and B. Xu, Adv. Healthcare Mater., 2013, 2, 1586–1590; X. Li, J. Li, Y. Gao, Y. Kuang, J. Shi and B. Xu, J. Am. Chem. Soc., 2010, 132, 17707–17709; Y.-A. Lin, A. G. Cheetham, P. Zhang, Y.-C. Ou, Y. Li, G. Liu, D. Hermida-Merino, I. W. Hamley and H. Cui, ACS Nano, 2014, 8, 12690–12700; H. Wang, C. Yang, L. Wang, D. Kong, Y. Zhang and Z. Yang, Chem. Commun., 2011, 47, 4439–4441; C. Yang, D. Li, Q. FengZhao, L. Wang, L. Wang and Z. Yang, Org. Biomol. Chem., 2013, 11, 6946–6951.
4. S. C. Bremmer, A. J. McNeil and M. B. Soellner, Chem. Commun., 2014, 50, 1691–1693; Y. Cai, Y. Shi, H. Wang, J. Wang, D. Ding, L. Wang and Z. Yang, Anal. Chem., 2014, 86, 2193–2199; J. Chen, W. Wu and A. J. McNeil, Chem. Commun., 2012, 48, 7310–7312; Y. Takaoka, T. Sakamoto, S. Tsukiji, M. Narazaki, T. Matsuda, H. Tochio, M. Shirakawa and I. Hamachi, Nat. Chem., 2009, 1, 557–561; X. D. Xu, B. B. Lin, J. Feng, Y. Wang, S. X. Cheng, X. Z. Zhang and R. X. Zhuo, Macromol. Rapid Commun., 2012, 33, 426–431; J. Zhang, C. Ou, Y. Shi, L. Wang, M. Chen and Z. Yang, Chem. Commun., 2014, 50, 12873–12876; D. M. Zurcher, Y. J. Adhia, J. D. Romero and A. J. McNeil, Chem. Commun., 2014, 50, 7813–7816.
5. J. Chen, R. R. Pompano, F. W. Santiago, L. Maillat, R. Sciammas, T. Sun, H. Han, D. J. Topham, A. S. Chong and J. H. Collier, Biomaterials, 2013, 34, 8776–8785; G. A. Hudalla, J. A. Modica, Y. F. Tian, J. S. Rudra, A. S. Chong, T. Sun, M. Mrksich and J. H. Collier, Adv. Healthcare Mater., 2013, 2, 1114–1119; R. R. Pompano, J. Chen, E. A. Verbus, H. Han, A. Fridman, T. McNeely, J. H. Collier and A. S. Chong, Adv. Healthcare Mater., 2014, 3, 1898–1908; W. Zheng, J. Gao, L. Song, C. Chen, D. Guan, Z. Wang, Z. Li, D. Kong and Z. Yang, J. Am. Chem. Soc., 2012, 135, 266–271.
6. D. M. Marini, W. Hwang, D. A. Lauffenburger, S. Zhang and R. D. Kamm, Nano Lett., 2002, 2, 295–299; H. Yokoi, T. Kinoshita and S. Zhang, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 8414–8419.
7. C. Ou, J. Zhang, Y. Shi, Z. Wang, L. Wang, Z. Yang and M. Chen, RSC Adv., 2014, 4, 9229–9232; A. M. Smith, R. J. Williams, C. Tang, P. Coppo, R. F. Collins, M. L. Turner, A. Saiani and R. V. Ulijn, Adv. Mater., 2008, 20, 37–41.
8. H. Wang, Z. Yang and D. J. Adams, Mater. Today, 2012, 15, 500–507.
9. Y. M. Abul-Haija, S. Roy, P. W. J. M. Frederix, N. Javid, V. Jayawarna and R. V. Ulijn, Small, 2014, 10, 973–979; S. Debnath, S. Roy and R. V. Ulijn, J. Am. Chem. Soc., 2013, 135, 16789–16792; J. Gao, Y. Shi, Y. Wang, Y. Cai, J. Shen, D. Kong and Z. Yang, Org. Biomol. Chem., 2014, 12, 1383–1386; J. Gao, H. Wang, L. Wang, J. Wang, D. Kong and Z. Yang, J. Am. Chem. Soc., 2009, 131, 11286–11287; Y. Kuang, J. Shi, J. Li, D. Yuan, K. A. Alberti, Q. Xu and B. Xu, Angew. Chem., Int. Ed. Engl., 2014, 53, 8104–8107; J. Li, Y. Gao, Y. Kuang, J. Shi, X. Du, J. Zhou, H. Wang, Z. Yang and B. Xu, J. Am. Chem. Soc., 2013, 135, 9907–9914; S. K. M. Nalluri, C. Berdugo, N. Javid, P. W. J. M. Frederix and R. V. Ulijn, Angew. Chem., Int. Ed. Engl., 2014, 53, 5882–5887; J. Shi, X. Du, D. Yuan, J. Zhou, N. Zhou, Y. Huang and B. Xu, Biomacromolecules, 2014, 15, 3559–3568; H. Wang, C. Yang, M. Tan, L. Wang, D. Kong and Z. Yang, Soft Matter, 2011, 7, 3897–3905.
10. J. D. Hartgerink, E. Beniash and S. I. Stupp, Science, 2001, 294, 1684–1688; A. Lomander, W. Hwang and S. Zhang, Nano Lett., 2005, 5, 1255–1260; A. Mershin, B. Cook, L. Kaiser and S. Zhang, Nat. Biotechnol., 2005, 23, 1379–1380; G. A. Silva, C. Czeisler, K. L. Niece, E. Beniash, D. A. Harrington, J. A. Kessler and S. I. Stupp, Science, 2004, 303, 1352–1355.
11. M. He, J. Li, S. Tan, R. Wang and Y. Zhang, J. Am. Chem. Soc., 2013, 135, 18718–18721; S. Matsumoto, S. Yamaguchi, S. Ueno, H. Komatsu, M. Ikeda, K. Ishizuka, Y. Iko, K. V. Tabata, H. Aoki, S. Ito, H. Noji and I. Hamachi, Chemistry, 2008, 14, 3977–3986; S. Matsumoto, S. Yamaguchi, A. Wada, T. Matsui, M. Ikeda and I. Hamachi, Chem. Commun., 2008, 1545–1547.
12. X. Miao, W. Cao, W. Zheng, J. Wang, X. Zhang, J. Gao, C. Yang, D. Kong, H. Xu, L. Wang and Z. Yang, Angew. Chem., Int. Ed. Engl., 2013, 52, 7781–7785; Z. Sun, Z. Li, Y. He, R. Shen, L. Deng, M. Yang, Y. Liang and Y. Zhang, J. Am. Chem. Soc., 2013, 135, 13379–13386; Y. Zhang, B. Zhang, Y. Kuang, Y. Gao, J. Shi, X. X. Zhang and B. Xu, J. Am. Chem. Soc., 2013, 135, 5008–5011.
13. C. J. Bowerman and B. L. Nilsson, J. Am. Chem. Soc., 2010, 132, 9526–9527.
14. C. Ren, Z. Song, W. Zheng, X. Chen, L. Wang, D. Kong and Z. Yang, Chem. Commun., 2011, 47, 1619–1621.
15. X. Zhang, X. Chu, L. Wang, H. Wang, G. Liang, J. Zhang, J. Long and Z. Yang, Angew. Chem., Int. Ed. Engl., 2012, 51, 4388–4392.
16. C. J. Bowerman, W. Liyanage, A. J. Federation and B. L. Nilsson, Biomacromolecules, 2011, 12, 2735–2745; C. J. Bowerman, D. M. Ryan, D. A. Nissan and B. L. Nilsson, Mol. BioSyst., 2009, 5, 1058–1069; M. R. Caplan, E. M. Schwartzfarb, S. Zhang, R. D. Kamm and D. A. Lauffenburger, Biomaterials, 2002, 23, 219–227; J.-B. Guilbaud, E. Vey, S. Boothroyd, A. M. Smith, R. V. Ulijn, A. Saiani and A. F. Miller, Langmuir, 2010, 26, 11297–11303.
17. Y. Gao, Y. Kuang, Z.-F. Guo, Z. Guo, I. J. Krauss and B. Xu, J. Am. Chem. Soc., 2009, 131, 13576–13577; Y. Gao, J. Shi, D. Yuan and B. Xu, Nat. Commun., 2012, 3, 1033; Q. Wang, Z. Yang, Y. Gao, W. Ge, L. Wang and B. Xu, Soft Matter, 2008, 4, 550–553; Z. Yang, H. Gu, D. Fu, P. Gao, J. K. Lam and B. Xu, Adv. Mater., 2004, 16, 1440–1444.
18. J. Gao, W. Zheng, D. Kong and Z. Yang, Soft Matter, 2011, 7, 10443–10448.

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Footnotes

† Electronic supplementary information (ESI) available: Synthesis and characterization of the compounds, optical images of the hydrogel being kept for 5 days, details experimental procedure of CCK-8 assay. See DOI: 10.1039/c5ra02449h

‡ The authors contributed equally to this work.

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