Protein-conjugated nanoparticles have the potential to precisely deliver therapeutics to target sites in the body by specifically binding to cell surface receptors. To maximize targeting efficiency, the threedimensional presentation of ligands toward these receptors is crucial. Herein, we demonstrate significantly enhanced targeting of nanoparticles to cancer cells by controlling the protein orientation on the nanoparticle surface. To engineer the point of attachment, we used amber codon reassignment to incorporate a synthetic amino acid, p-azidophenylalanine (azPhe), at specific locations within a single domain antibody (sdAb or nanobody) that recognizes the human epidermal growth factor receptor (EGFR). The azPhe modified sdAb can be tethered to the nanoparticle in a specific orientation using a bioorthogonal click reaction with a strained cyclooctyne. The crystal structure of the sdAb bound to EGFR was used to rationally select sites likely to optimally display the sdAb upon conjugation to a fluorescent nanocrystal (Qdot). Qdots with sdAb attached at the azPhe13 position showed 6 times greater binding affinity to EGFR expressing A549 cells, compared to Qdots with conventionally (succinimidyl ester) conjugated sdAb. As ligand-targeted delivery systems move toward clinical application, this work shows that nanoparticle targeting can be optimized by engineering the site of protein conjugation.
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 epidermal growth factor receptor (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 the targeting efficiency, we engineered the point of attachment (orientation), controlled the density of targeting groups on the surface of the Qdot, and optimized the length of the poly(ethylene glycol) 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 understanding 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.
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