Ultrafast tyrosine-based cell membrane modification via diazonium salts: A new frontier for biomedical applications

In this study, we present an ultrafast, efficient, and broadly applicable strategy for cell membrane modification via tyrosine-selective bioconjugation using diazonium salt derivatives. This chemical approach enables both one-step and two-step functionalization of adherent, suspension, and primary cells with a wide range of ligands, including imaging probes, carbohydrates, biotin, and proteins, without inducing cytotoxicity or immune activation. Cell membrane engineering through bioconjugation has emerged as a powerful tool in biomedical research, given the central role of the membrane in signaling, transport, and cell-cell interactions. In contrast to conventional glyco-engineering methods, which typically require multiday incubations and can induce cellular stress, our approach achieves rapid, precise, and high-density surface functionalization in less than one hour, with improved reproducibility and biological compatibility. We further demonstrate the applicability of this strategy across diverse cell types, including immortalized cell lines and clinically relevant primary cells such as peripheral blood mononuclear cells (PBMCs) and human natural killer (NK) cells. Notably, surface grafting of an EGFR-targeting Nanofitin enhances the cytotoxic activity of NK cells against EGFR-positive cancer cells. In addition, we show that the bioconjugated signal progressively diminishes over successive cell divisions, providing a self-limiting and transient alternative to permanent genetic modifications such as CAR-based engineering, thereby potentially reducing the risk of prolonged immune activation. Finally, the ability to store pre-functionalized cells at -80 °C increases the practicality of this platform for future ready-to-use applications. Overall, this versatile and non-genetic bioconjugation strategy offers a compelling alternative to existing technologies for applications in targeted therapy, diagnostics, and cell-based immunotherapy.