Identification of peptide coatings that enhance diffusive transport of nanoparticles through the tumor microenvironment

Rashmi Mohanty; Xinquan Liu; Jae You Kim; Xiujuan Peng; Sahil Bhandari; Jasmim Leal; Dhivya Arasappan; Dennis C. Wylie; Tony Dong; Debadyuti Ghosh

phage are biological nanoparticles that can multivalently display different "peptide-based" surface chemistries that can be combinatorially screened to identify peptide coatings. Here, we engineered random peptides to be displayed on T7 phage and screened the library against the selective pressure of ECM components found in solid tumors to collect peptide-presenting phage that permeated through the ECM. After successive rounds of selection, permeating phage were harvested, and using next generation sequencing and analysis, the most frequent peptides and motif sequences were identified and subsequently validated in transport studies. Diffusion assays were done to measure and identify peptide-presenting phage that exhibited most unhindered diffusivity through the ECM. Using one of the selected peptides, P4, as a model, we performed site-directed alanine mutagenesis to study the role of hydrophilicity, charge, and the order of the amino acid sequence on diffusive transport through the ECM. We next measured the ability of select peptide-presenting phage to achieve uptake in ex vivo ECM-rich pancreatic cancer xenografts. To demonstrate its feasibility as a surface coating for synthetic nanoparticles, the tumor penetrating peptide was conjugated onto polystyrene nanoparticles, and their diffusive transport in pancreatic tumor tissue was measured using multiple particle tracking and compared to PEG-functionalized and control uncoated nanoparticles. Phage display provides the molecular diversity that can be leveraged to screen and select peptides as surface coatings that facilitate diffusive transport and establish that charge, hydrophilicity, and their orientation govern transport through the tumor ECM. This work highlights the promise of peptides as alternative surface chemistries to improve transport and delivery of nanomedicines for solid tumors and potentially other disease microenvironments.