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Here, we describe a combinatorial approach to identify peptide shuttles that successfully traverse the multiple barriers to systemic drug delivery to the brain--1. the blood-brain barrier (BBB) and the 2. extracellular space (ECS) of the brain. Leveraging the excellent molecular diversity of M13 phage libraries, we identified peptides that actively transport across the BBB and diffuse through the extracellular matrix of the ECS in cell culture and in mice. These peptides demonstrate higher accumulation in the brain than gold standard peptides used in clinical trials. Importantly, these findings suggest that different sized therapeutics that were previous poorly permeable into the brain can accumulate at higher concentrations, thereby improving the therapeutic index and ultimate efficacy of drugs into the brain.

In solid tumors, increasing drug penetration promotes their regression and improves the therapeutic index of compounds. However, the heterogeneous extracellular matrix (ECM) acts a steric and interaction barrier that hinders effective transport of therapeutics, including nanomedicines. Specifically, the interactions between the ECM and surface physicochemical properties of nanomedicines (e.g. charge, hydrophobicity) impedes their diffusion and penetration. To address the challenges using existing surface chemistries, we used peptide-presenting phage libraries as a high-throughput approach to screen and identify peptides as coatings with desired physicochemical properties that improve diffusive transport through the tumor microenvironment. Through iterative screening against the ECM and identification by next-generation DNA sequencing and analysis, we selected individual clones and measured their transport by diffusion assays. Here, we identified a net-neutral charge, hydrophilic peptide P4 that facilitates significantly higher diffusive transport of phage than negative control through in vitro tumor ECM. Through alanine mutagenesis, we confirmed that the hydrophilicity, charge, and their spatial ordering impact diffusive transport. P4 phage clone exhibited almost 200-fold improved uptake in ex vivo pancreatic tumor xenografts compared to the negative control. Nanoparticles coated with P4 exhibited ∼40-fold improvement in diffusivity in pancreatic tumor tissues, and P4-coated particles demonstrated less hindered diffusivity through the ECM compared to particles functionalized with gold standard poly(ethylene) glycol or iRGD peptide ligand. By leveraging the power of molecular diversity using phage display, we can greatly expand the chemical space of surface chemistries that can improve the transport of nanomedicines through the complex tumor microenvironment to ultimately improve their efficacy.Abstract Figure