Engineering cell fate through redox modulation, matrix mechanics and scaffold geometry

Cell fate is governed by a complex interplay of biochemical and biophysical cues. This thesis explores three complementary strategies for engineering cell behavior: intracellular redox modulation, extracellular matrix (ECM) mechanics, and scaffold geometry. Together, these approaches provide a framework for understanding and controlling cellular responses in health, disease, and tissue engineering applications. The dysregulation of the intracellular redox homeostasis and the accumulation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), serve as primary drivers for the progression of severe pathologies, such as cancer and fibrosis. ROS are most detrimental when they accumulate inside cells as they are in direct contact with the cellular protein machinery and genetic information. Although synthetic biology strategies, such as nanozymes, have been proposed to mitigate oxidative stress, these methods lack the precision and sustainability required to effectively safeguard cells from within. To overcome existing limitations, we engineered artificial nano-organelles (AnOs) as a specialized solution for neutralizing high ROS levels directly at the site of damage in cells. Our AnOs are polymersome-based nano-compartments that encapsulate lactoperoxidase for H₂O₂ degradation, incorporate melittin pores in their membranes for molecular exchange with the environment, and are functionalized with cell-penetrating peptides for efficient cellular uptake. AnOs significantly reduced H₂O₂ in human K562 leukemia cells and attenuated the activation of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway, the master regulator of the cell’s antioxidant defense. NRF2 pathway modulation was reported using an engineered cell line that expresses mCherry under NRF2‑responsive promoter elements. Further, AnOs conferred resilience even when endogenous defenses were compromised and even extended protection to primary human T lymphocytes. These findings establish AnOs as a powerful tool to tune cell survival and stress responses. Beyond the role of ROS in cancer, dysregulated redox homeostasis is a critical driver of fibrotic progression. Idiopathic pulmonary fibrosis (IPF) is an irreversible and fatal disease characterized by excessive ECM deposition, which ultimately leads to loss of organ function. While mechanical tissue properties, such as stiffness, are known to change during fibrotic progression, the mechanical alterations of individual ECM fibers, such as fibronectin (FN) fibers, remain largely underexplored. As the established gold standard for pulmonary fibrosis in vivo research, the bleomycin mouse model served as our starting point to investigate the tensional state of FN fibers during disease onset and progression. By probing mouse lung cryosections with the peptide FnBPA5, which preferentially binds to relaxed FN fibers, we demonstrate that fibrotic progression is characterized by a loss of FN fiber tension. To extend our findings to a more human‑relevant context, we aimed to reproduce these results in an in vitro model using fibroblasts derived from IPF patients. Probing the FN fiber tension in this system revealed that FN fibers are more relaxed following treatment with transforming growth factor beta 1 (TGF-β1), the central pro-fibrotic cytokine. As a proof-of-concept, we demonstrated that FN fiber tension can be partially restored upon treatment with antifibrotic agents, suggesting that this assay could serve as a useful starting point for drug screening. Crucially, the ability of FnBPA5 to bind pathologically remodeled tissue highlights its potential as a vehicle for targeted drug delivery if conjugated to a therapeutic agent. While further refinement is needed, our study lays the foundation for the development of first‑in‑class mechano‑therapeutics aimed at improving IPF patient health. To assess whether relaxed FN fibers represent a broader hallmark of fibrosing diseases, even beyond the lung, we extended ou…