Generating Multilayer Polyelectrolyte Coating Libraries on Protein Nanoparticles for Mucosal Delivery

The efficacy of nanoparticle therapeutics is greatly limited by biological barriers that hinder access to intended sites. One biological barrier that has yet to be overcome is the mucus layer, which covers epithelial surfaces throughout the body. Mucus is a viscoelastic gel that restricts movement of foreign matter in the body, including beneficial material such as nanoparticles. Conventional therapeutic nanoparticles have adhesive interactions within the mucus layer that hinder mobility. To enable mucus-penetration, polyethylene glycol (PEG) is commonly used to achieve a hydrophilic and neutral surface coating for improved diffusion across the mucus barrier. However, we have demonstrated that PEG coatings on protein nanoparticles elucidate off-target immune responses through the production of anti-PEG antibodies in a murine model upon intranasal administration. In this study, we developed a library of protein nanoparticles with different multilayer-polyelectrolyte coatings that modulate diffusion in mucus as an alternative to PEG coatings. We formulated over 30 unique nanoparticles and screened them for enhanced diffusion coefficient using Nanoparticle Tracking Analysis (NTA) in mucus. NTA revealed that certain formulations had higher diffusion coefficients. In addition, we screened four different cell lines derived from colorectal, lung, cervical epithelial tissue, and dendritic cells using flow cytometry to assess their uptake profile with a mucosal barrier. We identified formulations with improved interaction with different cell lines, linking unique polyelectrolyte coatings with specific cell types. These experiments indicate that certain multilayer polyelectrolytes formulations have the potential for improved diffusion and increase cellular uptake delivery in mucus enriched cells.