Engineering the Orientation, Density and Flexibility of Single Domain Antibodies on Nanoparticles to Improve Cell Targeting.

Nanoparticles targeted to specific cells have the potential to improve the delivery of therapeutics. The effectiveness of cell targeting can be significantly improved by optimizing how the targeting ligands are displayed on the nanoparticle surface. Crucial to optimizing the cell binding are the orientation, density and flexibility of the targeting ligand on the nanoparticle surface. In this paper, we used an anti-EGFR single domain antibody (sdAb or nanobody) to target fluorescent nanocrystals (Qdots) to EGFR positive cells. The sdAbs were expressed with a synthetic amino acid (azPhe), enabling site-specific conjugation to Qdots in an improved orientation. To optimize targeting effi-ciency, we engineered the point of attachment (orientation), controlled the density of targeting groups on the surface of the Qdot, and optimized the length of poly(ethylene glycol) (PEG) linker used to couple the sdAb to the Qdot surface. By optimizing orientation, density and flexibility, we improved cell targeting by more than an order of magnitude. This work highlights the importance of under-standing the structure of the nanoparticle surface to achieve the optimal interactions with the intended receptors, and how engineering the nanoparticle surface can significantly improve cell targeting.