Ultrafast tyrosine-based cell membrane modification <i>via</i> diazonium salts: a new frontier for biomedical applications

Abstract In this study, we present an ultrafast, efficient, and broadly applicable strategy for cell membrane modification via tyrosine bioconjugation using diazonium salt derivatives. This chemical approach enables both one-step and two-step functionalization of adherent, suspension, and primary cells with diverse ligands, including imaging probes, carbohydrates, biotin, and proteins, without inducing cytotoxicity or immune activation. Membrane engineering through bioconjugation is emerging as a valuable tool in biomedical research, given the cell membrane’s central role in signaling, transport, and cell-cell interactions. Compared to traditional glyco-engineering methods that often require multiday incubations and can cause cellular stress, our approach achieves precise and high-density grafting in less than one hour, with improved reproducibility and biological compatibility. Importantly, we demonstrate that this strategy can be applied to a variety of cell types, encompassing both immortalized cell lines and primary cells, notably Peripheral Blood Mononuclear Cells (PBMCs) and human effector cells such as natural killer (NK) cells, to enhance their cytotoxic function against EGFR-positive cancer cells via surface-conjugated Nanofitin. Moreover, we show that the bioconjugated signal diminishes over time due to cell division, offering a self-limiting alternative to permanent genetic modifications such as CAR-T, thereby mitigating risks associated with overly prolonged immune activation. Finally, the feasibility of storing pre-functionalized cells at -80 °C expands the practicality of this platform for future ready-to-use applications. Together, these features make our method a compelling alternative to current technologies for applications in targeted therapy, diagnostics, and cell-based immunotherapy.