(A-27) Evaluating receptor-ligand communication between programmed cells with synthetic transmembrane receptors and orthogonal peptide ligands conjugated to hyaluronic acid hydrogels

Introduction:: In mammalian tissue, cells influence their external environment and in turn respond to signals in that environment. Advances in synthetic biology have brought about the ability to program cells with transmembrane receptors that respond to soluble and cell-laden ligands; however, there is limited research investigating synthetic receptors capable of responding to material-laden ligands. Hydrogels are soft materials that can be designed to enable receptor-ligand interactions among cells atop (2D) or within (3D) peptide-functionalized hydrogels. This research focuses on the development of platforms to control the two-way communication between cells and engineered materials by using synthetic transmembrane receptors and peptide conjugated biomaterials to better mimic the dynamic interaction between cells and the ECM. Towards this, this abstract reports the programming of HEK293 cells with novel transmembrane cell receptors which utilize a phosphorylation-based post translational circuit. This circuit is used to activate the transcription of matrix modifying molecules upon binding with peptide ligands not native to the extracellular environment that can be patterned onto hydrogels using a thiol-norbornene click chemistry. This work has the potential to merge the technological advances in synthetic biology and biomaterials science and aid the development of cell therapies and model systems with advanced capabilities.


Materials and Methods:: The intracellular signaling platform is highly modular and consists of a reversible leucine zipper dimerization domain to recruit a CD3z substrate (S) towards one receptor half. The intracellular region of the other receptor has a kinase specific to CD3z. Once phosphorylated, the substrate recruits a Zap70 SH2 domain (Figure 1A). This platform has been recently used to reconstitute two split mNeonGreen proteins connected to the substrate and SH2 domains resulting in green fluorescence, which can be measured by flow cytometry (Figure1B) or 3D confocal microscopy (Figure 1C).

Receptors were designed de novo using protein modeling software to bind to two orthogonal epitope tag peptides (HA for the leucine zipper or FLAG for the kinase). Configurations were tested in biological quadruplet in HEK293 cells by transfecting plasmids containing the genes for each receptor half, the split mNeonGreen halves connected to the substrate, and SH2 domains. After a 12-hour incubation, media was replaced with 25 nmol of soluble peptides. After 24 hours, the mean mNeonGreen expression levels were measured.

Hydrogel networks were formed of hyaluronic acid macromers, modified with norbornene (NorHA) and reacted with di-thiol crosslinkers. Unreacted norbornene groups in the hydrogels then underwent a second reaction to pattern thiolated peptide ligands of interest. As a demonstration, cells transfected with the full transmembrane receptor platform were encapsulated in these gels, after 2 days 1mM of peptide was conjugated, and after an additional one-day incubation the cell were stained with red phalloidin, and the red and green fluorescence was visualized using confocal microscopy.


Results, Conclusions, and Discussions:: Results: The soluble peptides receptors activated the intracellular domain of programmed HEK293 cells as evidenced by a significantly higher mNeonGreen expression in the peptide/receptor matched groups over the no ligand groups (One tailed T-test, α=0.05, n=4, * p-value=0.005) (Figure 1B). When these cells were encapsulated in hydrogel, there was visibly higher expression of green within the cells imaged for the peptide ligand group vs. the no peptide group (Figure 1C). Visualization of fluorescent molecules attached to these epitope tag peptides suggests that these ligands can be patterned on the gel using a photomask allowing for the localized control over platform activation.

Discussion: This data suggests that these transmembrane receptors selectively respond to soluble FLAG-HA peptide in HEK293 cells in media as well as when the cells are encapsulated in, and the peptides are conjugated to a hydrogel. Future work will focus on developing an additional epitope tag receptor containing a phosphatase domain capable of inactivating the platform in response to that signal, thus allowing for negative feedback regulation. Additionally, the split fluorescence molecules will be replaced with split transcriptional activation platforms to allow for transcription of genes of interest in response to these peptide signals. This transcriptional activation will allow for the release of matrix modifying proteases which could be used to degrade the hydrogel or release the peptides depending on the potential application.

Conclusion: This platform is the first of its kind and bridges the fields of cellular engineering and biomaterials. It expands the control and avenues that investigators have in their research and has the potential to be implemented for use in model systems for monitoring cell behavior, tissue engineering and cell therapies.


Acknowledgements (Optional): : The authors would like to acknowledge The Pantazes group and Dr. M. Benmassaoud for their contributions to this work and funding (NSF EAGER 2037055).


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