Mucus-penetrating phage-displayed peptides for improved transport across a mucus-like model

Abstract

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.

A combinatorial biomolecular strategy to identify peptides for improved transport across the sputum of cystic fibrosis patients and the underlying epithelia

“…Previously, using a different M13 phage library against CF mucus-like reconstituted mucin, we identified phage with up to 17.5-fold improved output ratios in mucus in the fourth round of screening, and phage-displayed peptides demonstrated up to 2.6-fold improved transport in mucus compared to wild-type control (i.e. non-recombinant, insertless phage) [66]. However, this low-throughput sample size (20-100 clones) from traditional Sanger sequencing, while providing valuable information, presents a limited repertoire of the complete sequence space (less than 0.01% of available sequences), and as a result, it is likely that better performing peptides are not identified [53].…”

Challenges and Recent Progress in Oral Drug Delivery Systems for Biopharmaceuticals

“…The mucus then discards such foreign moieties, acting as one of the main compartments of the immune system, known as mucosal immunity [76,77,78,79]. The mucus itself is mainly composed of water and mucin protein molecules coated with proteoglycans, giving the mucus a negative charge [80]. Carbohydrates, salts, bacteria, antibodies and cellular remnants are the other compounds found in mucus [81].…”

“…Materials developed for sustained delivery are either positively charged, in order to attach to mucus through electrostatic binding (e.g., cationic hydrogels made from chitosan), or thiol-functionalized hydrogels, to attach to the mucin glycoprotein through disulfide bonding [157,158]. Although these approaches have shown higher levels of attachment to mucus compared to the control samples, these particles can only slightly increase the delivery time (in the range of a few hours), due to the short turnover cycle of mucus in the intestine [67,80]. Furthermore, MPs employed for oral delivery are usually made of pH-responsive polymers, releasing their cargo only at pH values above the pKa of the hydrogel, which is usually >6 for oral applications.…”

Nanomedicine Approaches for the Pulmonary Treatment of Cystic Fibrosis

“…In the case of CF, the use of NPs could be a valid approach to overcome the thick mucus layer that is formed within the alveolar region as well as the bacterial biofilm (Ong et al, 2019). The CF mucus contains less water than normal mucus and presents mucin fibers (70-80%), DNA, actin and other macromolecules, which form a cross-linked network where the mesh size is reduced from above 500 nm (in healthy patients) to 300-100 nm in CF patients, depending on their conditions and on the disease stage (Ensign et al, 2012;Liu et al, 2014;Leal et al, 2018). This significant change in mucus structure and composition leads to an increased viscoelasticity (Ibrahim et al, 2011b;Ong et al, 2019), along with the formation of hydrophobic and electrostatic barriers that strongly inhibit NPs penetration.…”