Pointing in the Right Direction: Controlling the Orientation of Proteins on Nanoparticles Improves Targeting Efficiency

Abstract

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.

Next‐Generation Polymeric Nanomedicines for Oncology: Perspectives and Future Directions

“…Recent studies have demonstrated that site-specific approaches to generate optimal biomolecule presentation can drastically enhance cell binding affinities. [17] Such approaches are greatly benefited by advances in molecular biology, allowing site-specific incorporation of unnatural amino acids [18] (UAA; often azido/alkynyl species) capable of undergoing rapid and selective bioorthogonal reactions. While initially requiring significant in-house expertise, the commercial availability of many such UAAs now allows more broad uptake of this approach, as evidenced by the progression of PEG-protein conjugates taking advantage of UAAs to clinical trials.…”

Refined construction of antibody-targeted nanoparticles leads to superior antigen binding and enhanced delivery of an entrapped payload to pancreatic cancer cells

“…Other common strategies for the site-specific functionalisation of nanoparticles include maleimide-based coupling, or engineering of antibodies with enzyme recognition motifs or non-canonical amino acids. [22][23][24][25][26] Whilst these can markedly improve the homogeneity and antigen binding ability of targeted nanoparticles, multiple factors often limit their success, such as the instability of the resultant conjugate, disruption of the native structure of antibodies and the need for a significant investment of time and expense. In contrast, our approach generates a highly stable triazole linkage, preserves the structural integrity of the F(ab) through re-bridging of the reduced disulfide and circumvents the need for engineering, ensuring facile translation to other platforms as we have previously demonstrated.…”

Understanding Cell Interactions Using Modular Nanoparticle Libraries

“…Qdots with optimally oriented sdAbs showed .5-fold improvement in cell targeting compared with randomly oriented sdAbs. [21] To design an effective nanoparticle delivery system, many biological roadblocks must be overcome. However, very little is understood about how nanoparticle structure impacts on these roadblocks.…”