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.