Poster O11 - Multiscale Characterization of Airway Surface Layer to Optimize Inhaled Drug Delivery

Introduction: Pulmonary mucus protects the airways from airborne pathogens and particulates. However, in muco-obstructive pulmonary diseases (MOPD), the penetration of inhaled therapies through mucus faces challenges absent in healthy individuals. Hyperconcentration of the polymeric, high molecular weight mucin glycoproteins can lead to physical entrapment of therapeutic molecules. Physical entrapment depends on the relative sizes of the inhaled molecule and mesh size of mucus, which is defined by the concentration and size of the mucins. In patients with MOPDs, the mesh size will be smaller than in health, physically trapping progressively smaller molecules. Additionally, chemical interactions between therapeutic molecules and mucus may impede successful drug delivery. Therefore, designing drugs that are capable of penetrating through the mucus barrier requires a comprehensive characterization of mucus at length scales relevant to therapeutic molecules. In this study, we characterize the physical and chemical properties of mucus relevant to disease progression across scales ranging from 1nm to 2μm, with a variety of surface chemistries. By integrating our mucus characterization with separate techniques, we can span the nano- to macroscopic length scales and determine the efficacy at which inhaled therapeutics traverse pathological mucus layers.

Materials and

Methods: Mucus is harvested from primary human bronchial epithelial cells and prepared to concentrations of 2% (healthy), 4% (moderate airway disease), and 6% (severe airway disease). Mucus mesh structure and chemical interactions are examined across length scales ranging from 1 nm to 2μm, covering a spectrum both below and above the polymeric mesh size of mucus. Larger length scales, ranging from 500nm to 2μm, are measured with particle tracking micro-rheology (PTMR). Surface chemistries, including carboxylated, amine, and polyethylene glycol (PEG) formulations, are utilized to study the effect of chemical interactions on diffusion through mucus. For nano-scale characterization of mucus, a one-directional migration assay(1-DMA) is utilized to quantify the diffusion of probes ranging in size from 1nm to 500nm through mucus. Finally, for physiological relevance, we compare nano-sized probes with commercially available lipid nanoparticles (LNP), a popular inhaled drug delivery vehicle, and NL-63, a coronavirus.

Results, Conclusions, and Discussions: Our PTMR data reveal that when probes exceed the mucus mesh size, they measure the bulk rheology of mucus, are dependent on mucus concentration, and are largely independent of surface chemistry. However, our data also show the inherent risk of only using one outcome measure to determine the effects of size and surface chemistry on diffusion in mucus. When particles with an amine surface chemistry are added to mucus, the measured viscosity is slightly lower than when measured with PEG or carboxylic acid. Interestingly, the positively charged amine beads cause mucus to clump, drawing beads in such proximity that automated tracking analysis will reject these particles, and only beads in areas where mucus did not clump are measured. We additionally find that, for particles larger than the mucus mesh size, the size of the probes does not significantly influence the measurements. On the nanoscopic length scale, diffusion of nanoprobes that are below the mesh size in our 1-DMA is independent of mucus concentration, indicating these probes avoid the polymeric network. Our data show that the diffusion of 100nm LNPs become highly restricted at 4% mucus and immobile at 6%. Conversely, while the diffusion of NL-63 (75 -115nm in diameter) is impeded by mucus, it is not immobilized, highlighting the importance of surface chemistry to the diffusion of objects near or below the mesh size. Finally, our data indicate that treating mucus with a thiol reducing agent or a surfactant will increase the ability of molecules with hindered diffusion to penetrate even pathological mucus layers.

Our multi-tool approach enables comprehensive characterization of mucus and the diffusion of molecules across various length scales, providing valuable insights into drug delivery mechanisms. Our observations indicate that probe size plays a crucial role in improving drug delivery efficiency, with smaller probes exhibiting faster diffusion in mucus. Furthermore, our findings highlight that at length scales comparable to the mesh size, factors beyond size and zeta potential, such as specific interactions between mucus components and therapeutic agents, significantly influence penetration efficacy.

Acknowledgements (Optional): This work was funding by the NIH (P01HL164320, P30DK065988, and P01HL108808) and Cystic Fibrosis Foundation (HILL20Y2-OUT, CAUGHE23G0, 004489G222, and Bouche19R0).