Heuristics for the Optimal Presentation of Bioactive Peptides on Polypeptide Micelles Jing Wang, Soumen Saha, Jeffrey L. Schaal, Parisa Yousefpour, Xinghai Li, and Ashutosh Chilkoti* ,† † Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708, United States * S Supporting Information ABSTRACT: Bioactive peptides describe a very large group of compounds with diverse functions and wide applications, and their multivalent display by nanoparticles can maximize their activities. However, the lack of a universal nanoparticle platform and design rules for their optimal presentation limits the development and application of peptide ligand-decorated nanoparticles. To address this need, we developed a multivalent nanoparticle platform to study the impact of nanoparticle surface hydrophilicity and charge on peptide targeting and internalization by tumor cells. This system consists of micelles of a recombinant elastin-like polypeptide diblock copolymer (ELP BC ) that present genetically encoded peptides at the micelle surface without perturbing the size, shape, stability, or peptide valency of the micelle, regardless of the peptide type. We created the largest extant set of 98 combinations of 15 tumor-homing peptides that are presented on the corona of this ELP BC micelle via 8 different peptide linkers that vary in their length and charge and also created control micelles that present the linker only. Analysis of the structure-function relationship of tumor cell targeting by this set of peptide-decorated nanoparticles enabled us to derive heuristics to optimize the delivery of peptides based on their physicochemical properties and to identify a peptide that is likely to be a widely useful ligand for targeting across nanoparticle types. This study shows that ELP BC micelles are a robust and convenient system for the presentation of diverse peptides and provides useful insights into the appropriate presentation of structurally diverse peptide ligands on nanoparticles based on their physicochemical properties. KEYWORDS: Bioactive peptide, nanoparticle, cell uptake, multivalency, tumor targeting ligand, optimization N anoparticles have several useful attributes for the delivery of therapeutic or targeting peptides. 1-3 Because most peptides have a very short half-life ranging from a few minutes to a few hours, 2,3 the presentation of a targeting peptide on a nanoparticle can greatly extend the plasma circulation time of a peptide drug by impeding renal clearance. The display of multiple copies of a targeting peptide on the surface of a drug- loaded nanoparticle can also enhance the effective binding affinity (the avidity) of the peptide for its cell-surface target due to multivalency and thereby maximize the delivery of the drug payload to the target cell of interest. 4-7 Despite the conceptual advantages offered by a multivalent display of peptides on nanoparticles, very few therapeutic or targeting peptides have been successfully delivered in a clinical setting using nanoparticles, 8 likely due to two factors. First, there is no nanoparticle that serves as a universal platform to experimentally compare the affinity and specificity of different bioactive peptides in their multivalent form. The availability of a consistent nanoparticle platform would allow direct comparison between different peptides that target the same receptor or different therapeutic peptides that act by a similar mechanism 9,10 and allow selection of the optimal peptide for a specific therapeutic indication. Second, we lack general guidelines to optimize the presentation of peptides on a nanoparticle surface to achieve optimal targeting. This is a critical issue, as the suboptimal display of peptides at the nanoparticle surface will compromise their targeting. For example, when hydrophobic peptides are tethered to the hydrophilic end of diblock copolymers, they tend to be buried into the hydrophobic core of polymer micelles and hence lose accessibility to their Received: July 31, 2019 Revised: October 18, 2019 Published: October 23, 2019 Letter pubs.acs.org/NanoLett Cite This: Nano Lett. XXXX, XXX, XXX-XXX © XXXX American Chemical Society A DOI: 10.1021/acs.nanolett.9b03141 Nano Lett. XXXX, XXX, XXX-XXX Downloaded via DUKE UNIV on October 30, 2019 at 15:22:26 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.