The objective of this work is to use phage display libraries as a screening tool to identify peptides that facilitate transport across the mucus barrier. Mucus is a complex selective barrier to particles and molecules, limiting penetration to the epithelial surface of mucosal tissues. In mucus-associated diseases such as cystic fibrosis (CF), mucus has increased viscoelasticity and a higher concentration of covalent and non-covalent physical entanglements compared to healthy tissues, which greatly hinders permeability and transport of drugs and particles across the mucosae for therapeutic delivery. Treatment of CF lung diseases and associated infections must overcome this abnormal mucosal barrier. Critical bottlenecks hindering effective drug penetration remain and while recent studies have shown hydrophilic, net-neutral charge polymers can improve the transport of nanoparticles and minimize interactions with mucus, there is a dearth of alternative carriers available. We hypothesized that the screening of a phage peptide library against a CF mucus model would lead to the identification of phage-displayed peptide sequences able to improve transport in mucus. These combinatorial libraries possess a large diversity of peptide-based formulations (108 - 109) to achieve unprecedented screening for potential mucus-penetrating peptides. Here, phage clones displaying discovered peptides were shown to have up to 2.6-fold enhanced diffusivity in the CF mucus model. In addition, we demonstrate reduced binding affinities to mucin compared to wild-type control. These findings suggest that phage display libraries can be used as a strategy to improve transmucosal delivery.
High throughput phage display screening to identify peptides that enhances diffusive transport of nanoparticles through tumor microenvironment.
Preparation in Materials and Methods) in the apical chamber of a 12 mm diameter (12 well) 109 polyester Transwell chamber with 3 micrometer pore size (Corning, Corning, NY). Before 110 addition of phage and mucin to the apical chamber, the basolateral chamber (i.e. donor 111 compartment) was filled with phosphate buffered saline without calcium and magnesium (PBS, 112 Corning, Corning, NY), as recommended by manufacturer. After 1 hour, peptide-presenting 113
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 All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/659524 doi: bioRxiv preprint 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. Quantification Amplification Phage libraryScreening of phage-presenting peptide library through a tumor ECM model Identification and selection of potential peptide using nextgeneration sequencingSelection of a faster diffusing clone through in vitro validation using Transwell and multichannel diffusion assay Ex vivo uptake validation of the selected clone followed by comparison of diffusivity of peptide-coated nanoparticle with gold standards using multiple particle tracking All rights reserved. No reuse allowed without permission.
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