Development of ZmT-PEG hydrogels through Michael addition reaction and protein self-assembly for 3D cell culture

Bioactive protein-derived hydrogels are highly attractive three-dimensional (3D) platforms for in vitro cell culture. However, most protein and polypeptide hydrogels are extracted from animal tissues or chemically synthesized, with many drawbacks. Herein, we fabricated an optically transparent ZmT-PEG hydrogel via a facile one-pot strategy. The modified Z1Z2 (Zm) was obtained by introducing cysteine at the C-terminus of Z1Z2 (ZC) and inserting the RGD sequence into the low conserved (CD) loop (ZR). A Michael addition reaction occurred between Zm and 4-arm PEG-MAL, and Zm-PEG self-assembled with truncated Telethonin (Tm) to form the hydrogel. We expressed the Zm and Tm proteins in Escherichia coli. CD spectroscopy showed that genetic modification and the reaction with 4-arm PEG-MAL had no effect on the secondary structure of the Zm protein. When Zm was at 10 wt% and the ratio of Zm : 4-arm PEG-MAL : Tm was 2 : 1 : 1, the gelation time was 6-8 hours. SEM results revealed that the hydrogels had an interconnected porous structure with pore diameters of 20-150 mu m. Cell experiments showed that MCF-7 cells could grow and proliferate significantly on the hydrogel after 7 days of culture. Immunofluorescence results suggested that MCF-7 cells on the ZmT hydrogel had a spherical structure similar to that on Matrigel. These results indicate that the ZmT-PEG hydrogel can be used for cell culture in vitro and is promising for large-scale production. Schematic diagram of ZmT-PEG hydrogel preparation. ZC indicates Zm with Cysteine at the C-terminal, while ZR indicates ZC with a RGD sequence in the CD loop. Purple: Zm protein. Blue: 4 arm PEG-MAL. Green: Tm protein.