Mapping Antibody Domain Exposure on Nanoparticle Surfaces Using DNA-PAINT

[Image: see text] Decorating nanoparticles with antibodies (Ab) is a key strategy for targeted drug delivery and imaging. For this purpose, the orientation of the antibody on the nanoparticle is crucial to maximize fragment antibody-binding (Fab) exposure and thus antigen binding. Moreover, the exposure of the fragment crystallizable (Fc) domain may lead to the engagement of immune cells through one of the Fc receptors. Therefore, the choice of the chemistry for nanoparticle-antibody conjugation is key for the biological performance, and methods have been developed for orientation-selective functionalization. Despite the importance of this issue, there is a lack of direct methods to quantify the antibodies’ orientation on the nanoparticle’s surface. Here, we present a generic methodology that enables for multiplexed, simultaneous imaging of both Fab and Fc exposure on the surface of nanoparticles, based on super-resolution microscopy. Fab-specific Protein M and Fc-specific Protein G probes were conjugated to single stranded DNAs and two-color DNA-PAINT imaging was performed. Hereby, we quantitatively addressed the number of sites per particle and highlight the heterogeneity in the Ab orientation and compared the results with a geometrical computational model to validate data interpretation. Moreover, super-resolution microscopy can resolve particle size, allowing the study of how particle dimensions affect antibody coverage. We show that different conjugation strategies modulate the Fab and Fc exposure which can be tuned depending on the application of choice. Finally, we explored the biomedical importance of antibody domain exposure in antibody dependent cell mediated phagocytosis (ADCP). This method can be used universally to characterize antibody-conjugated nanoparticles, improving the understanding of relationships between structure and targeting capacities in targeted nanomedicine.