Safe and efficient intracellular delivery of functional antibodies into cultured neural cells shows a role for Alzheimer’s-relevant tau phosphorylation in cell integrity

IntroductionProtein proteoforms generated by post-translational modification are increasingly recognized as important regulators of neuronal function and neurodegenerative disease. However, methods for acutely interrogating specific intracellular proteoforms in living neuronal populations remain limited. We evaluated cationic liposomal delivery (CLD) as a strategy for introducing full-length functional antibodies into living neurons and used this approach to investigate the role of a phosphorylated tau proteoform recognized by the monoclonal antibody PHF-1.MethodsCationic liposomes composed of DOPC+ were complexed with antibodies or molecular probes and applied to cultured neuronal cell lines and primary mouse cortical neurons. Intracellular delivery, cellular localization, viability, and biological activity were assessed using fluorescence microscopy and live-cell imaging.ResultsCLD efficiently delivered full-length antibodies and molecular probes into living neuronal cells and primary neurons without detectable toxicity. Internalized antibodies retained target specificity, producing intracellular staining patterns consistent with the known localization of tau, vinculin, paxillin, and Fyn. Delivery was observed across essentially all cells in treated cultures and remained effective under standard culture conditions. Intracellular delivery of PHF-1 produced a rapid and striking morphological response characterized by neurite retraction and cell-body rounding within hours of treatment. These effects were not observed with heat-inactivated PHF-1, vehicle controls, or other intracellular antibodies, supporting a specific effect of intracellular engagement of PHF-1-reactive tau species.DiscussionThese findings establish CLD as a practical platform for the intracellular delivery of functional full-length antibodies into living neuronal populations. Beyond validating a delivery technology, the study demonstrates that acute intracellular engagement of PHF-1-reactive phosphorylated tau rapidly alters neuronal morphology, suggesting that this tau proteoform contributes to the maintenance of neurite integrity. CLD provides a versatile approach for probing the function of intracellular proteoforms that are difficult to study using conventional genetic or biochemical methods.