Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium.

The clinical impact of rhinovirus C (RV-C) is well-documented; yet, the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection. To further define RV-C15 replication sites, we analyzed the expression and colocalization of giantin, phosphatidylinositol-4-phosphate, and calnexin with dsRNA. Despite observing Golgi fragmentation by immunofluorescence during RV-C15 infection as previously reported for other RVs, a high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression and the induction of incomplete autophagy, a mechanism used by other RVs to facilitate non-lytic release of progeny virions. Notably, genetic depletion of STING in HAE attenuated RV-C15 and -A16 (but not -B14) replication, corroborating a previously proposed proviral role for STING in some RV infections. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality-aspects of infection that may contribute to pathogenesis in vivo.

Neutrophil Extracellular Traps Increase Airway Mucus Viscoelasticity and Slow Mucus Particle Transit.

Mucus obstruction is a key feature of many inflammatory airway diseases. Neutrophil extracellular traps (NETs) are released upon neutrophil stimulation and consist of extracellular chromatin networks studded with cytotoxic proteins. When released in the airways, these NETs can become part of the airway mucus. We hypothesized that the extracellular DNA and/or oxidative stress (e.g., by the release of reactive oxygen species and myeloperoxidase during NETs formation in the airways) would increase mucus viscoelasticity. We collected human airway mucus from endotracheal tubes of healthy patients admitted for elective surgery and coincubated these samples with NETs from phorbol 12-myristate 13-acetate-stimulated neutrophils. Unstimulated neutrophils served as controls, and blocking experiments were performed with dornase alfa for extracellular DNA and the free radical scavenger dimethylthiourea for oxidation. Compared with controls, the coincubation of mucus with NETs resulted in 1) significantly increased mucus viscoelasticity (macrorheology) and 2) significantly decreased mesh pore size of the mucus and decreased movement of muco-inert nanoparticles through the mucus (microrheology), but 3) NETs did not cause visible changes in the microstructure of the mucus by scanning EM. Incubation with either dornase alfa or dimethylthiourea attenuated the observed changes in macrorheology and microrheology. This suggests that the release of NETs may contribute to airway mucus obstruction by increasing mucus viscoelasticity and that this effect is not solely due to the release of DNA but may in part be due to oxidative stress.

Mucin Biopolymers and Their Barrier Function at Airway Surfaces.

In the lung, the airway epithelium produces secreted and tethered mucin biopolymers to form a mucus hydrogel layer and a surface-attached polymer brush layer. These layers work in concert to facilitate the cilia-mediated transport of mucus for the capture and clearance of inhaled materials to prevent lung damage. The mechanisms by which mucin biopolymers protect the lung from injury have been an intense area of study in airway biology for the past several decades. In this feature article, we will discuss how airway mucins achieve these protective barrier functions. We will present the key findings, rooted in polymer and surface science, that have aided in understanding mucin barrier function. In addition, we will describe how this work may influence the design of nanoparticles to overcome the mucus barrier to effective drug delivery.

Protein nanocages that penetrate airway mucus and tumor tissue.

Reports on drug delivery systems capable of overcoming multiple biological barriers are rare. We introduce a nanoparticle-based drug delivery technology capable of rapidly penetrating both lung tumor tissue and the mucus layer that protects airway tissues from nanoscale objects. Specifically, human ferritin heavy-chain nanocages (FTn) were functionalized with polyethylene glycol (PEG) in a unique manner that allows robust control over PEG location (nanoparticle surface only) and surface density. We varied PEG surface density and molecular weight to discover PEGylated FTn that rapidly penetrated both mucus barriers and tumor tissues in vitro and in vivo. Upon inhalation in mice, PEGylated FTn with optimized PEGylation rapidly penetrated the mucus gel layer and thus provided a uniform distribution throughout the airways. Subsequently, PEGylated FTn preferentially penetrated and distributed within orthotopic lung tumor tissue, and selectively entered cancer cells, in a transferrin receptor 1-dependent manner, which is up-regulated in most cancers. To test the potential therapeutic benefits, doxorubicin (DOX) was conjugated to PEGylated FTn via an acid-labile linker to facilitate intracellular release of DOX after cell entry. Inhalation of DOX-loaded PEGylated FTn led to 60% survival, compared with 10% survival in the group that inhaled DOX in solution at the maximally tolerated dose, in a murine model of malignant airway lung cancer. This approach may provide benefits as an adjuvant therapy combined with systemic chemo- or immunotherapy or as a stand-alone therapy for patients with tumors confined to the airways.

A rational approach to form disulfide linked mucin hydrogels.

We report the design of a mucin hydrogel created using a thiol-based cross-linking strategy. By using a cross-linking reagent capable of forming hydrogen bonds and disulfide linkages within the gel network, we were able to produce mucin-based hydrogels with viscoelastic properties similar to natural mucus as measured by bulk rheology. We confirmed disulfide cross-links strongly contribute to gel formation in our system using chemical treatments to block and reduce cysteines where we found mucin hydrogel network formation was inhibited and disrupted, respectively. Particle tracking microrheology was used to investigate the kinetics and evolution of microstructure and viscoelasticity within the hydrogel as it formed. We found that the rate of gel formation could be tuned by varying the mucin to crosslinker ratio, producing network pore sizes in the range measured previously in human mucus. The results of this work provide a new, simple method for creating mucin hydrogels with physiologically relevant properties using readily available reagents.

Interfacial and Confined Colloidal Rod Diffusion.

Optical microscopy is used to measure translational and rotational diffusion of colloidal rods near a single wall, confined between parallel walls, and within quasi-2D porous media as a function of rod aspect ratio and aqueous solution ionic strength. Translational and rotational diffusivities are obtained as rod particles experience positions closer to boundaries and for larger aspect ratios. Models based on position dependent hydrodynamic interactions quantitatively capture diffusivities in all geometries and indicate particle-wall separations in agreement with independent estimates based on electrostatic interactions. Short-time translational diffusion in quasi-2D porous media is insensitive to porous media area fraction, which appears to arise from a balance of hydrodynamic hindrance and enhanced translation due to parallel alignment along surfaces. Findings in this work provide a basis to interpret and predict interfacial and confined colloidal rod transport relevant to biological, environmental, and synthetic material systems.

The Mucus Barrier to Inhaled Gene Therapy.

Recent evidence suggests that the airway mucus gel layer may be impermeable to the viral and synthetic gene vectors used in past inhaled gene therapy clinical trials for diseases like cystic fibrosis. These findings support the logic that inhaled gene vectors that are incapable of penetrating the mucus barrier are unlikely to provide meaningful benefit to patients. In this review, we discuss the biochemical and biophysical features of mucus that contribute its barrier function, and how these barrier properties may be reinforced in patients with lung disease. We next review biophysical techniques used to assess the potential ability of gene vectors to penetrate airway mucus. Finally, we provide new data suggesting that fresh human airway mucus should be used to test the penetration rates of gene vectors. The physiological barrier properties of spontaneously expectorated CF sputum remained intact up to 24 hours after collection when refrigerated at 4 °C. Conversely, the barrier properties were significantly altered after freezing and thawing of sputum samples. Gene vectors capable of overcoming the airway mucus barrier hold promise as a means to provide the widespread gene transfer throughout the airway epithelium required to achieve meaningful patient outcomes in inhaled gene therapy clinical trials.

Diffusing Colloidal Probes of kT-Scale Biomaterial-Cell Interactions.

In the optimization of applied biomaterials, measurements of their interactions with cell surfaces are important to understand their influence on specific and nonspecific cell surface adhesion, internalization pathways, and toxicity. In this study, a novel approach using dark field video microscopy with combined real-time particle and cell tracking allows the trajectories of biomaterial-coated colloids to be monitored in relation to their distance from cell perimeters. Dynamic and statistical mechanical analyses enable direct measurement of colloid-cell surface association lifetimes and interaction potentials mediated by biomaterials. Our analyses of colloidal transport showed polyethylene glycol (PEG) and bovine serum albumin (BSA) lead to net repulsive interactions with cell surfaces, while dextran and hyaluronic acid (HA) lead to reversible and irreversible association to the cell surface, respectively. Our results demonstrate how diffusing colloidal probes can be used for nonobtrusive, sensitive measurements of biomaterial-cell surface interactions important to therapeutics, diagnostics, and tissue engineering.

Diffusing colloidal probes of cell surfaces.

Measurements and analyses are reported to quantify dynamic and equilibrium interactions between colloidal particles and live cell surfaces using dark field video microscopy. Two-dimensional trajectories of micron-sized polyethylene glycol (PEG)-coated silica colloids relative to adherent epithelial breast cancer cell perimeters are determined allowing measurement of position dependent diffusivities and interaction potentials. PEG was chosen as the material system of interest to assess non-specific interactions with cell surfaces and establishes a basis for investigation of specific interactions in future studies. Analysis of measured potential energies on cell surfaces reveals the spatial dependence in cell topography. With the measured cell topography and models for particle-cell surface hydrodynamic interactions, excellent agreement is obtained between theoretical and measured colloidal transport on cell surfaces. Quantitative analyses of association lifetimes showed that PEG coatings act to stabilize colloids above the cell surface through net repulsive, steric interactions. Our results demonstrate a self-consistent analysis of diffusing colloidal probe interactions due to conservative and non-conservative forces to characterize biophysical cell surface properties.

Tunable aggregation by competing biomolecular interactions.

Measurements and models are reported for Concanavalin A (ConA) mediated aggregation of dextran coated colloids that is tunable via a competing ConA-glucose interaction. Video and confocal scanning laser microscopy were used to characterize ConA adsorption to dextran colloids and quasi-2D dextran coated colloid aggregation kinetics vs [ConA] and [glucose]. ConA adsorption to, and aggregation rates of, dextran coated colloids increased from negligible values to high coverage and rapid rates for increasing [ConA] in the range 0.1-10 mM and decreasing [glucose] in the range 1-100 mM, consistent with dissociation constant estimates. Analysis of colloidal aggregation kinetics indicates ConA bridge formation is the rate-limiting step controlling the transition from slow to rapid aggregation. Our findings reveal a mechanism for tuning colloidal interactions and aggregation kinetics through specific, competitive biomolecular interactions, which lends insights into aggregation phenomena in mixed synthetic-biomaterial and biological systems.

Colloidal potentials mediated by specific biomolecular interactions.

We present a systematic study of receptor-ligand interactions with increasing complexity from interactions in bulk solution, on planar and colloid surfaces, and as part of mediating colloidal pair interactions. We report analytical models that relate receptor-ligand dissociation constants (KD), interactions potentials (U(r)), and adsorbed amounts (θ) for different ligand sizes, concentrations and immobilized receptor coverages. Analytical results are validated for hard core + harmonic well receptor-ligand interactions in bulk and interfacial systems using Monte Carlo (MC) simulations, although any biomolecular interaction can be incorporated into the reported analysis via a "B2 device". Results from analytical models are used to understand potentials of mean force (W(L)) for ligand mediated interactions between receptor decorated colloids. W(L) are generated using MC-umbrella sampling (MC-US) simulations with cluster moves, which provide self-consistent comparisons of net colloid scale interactions with receptor-ligand scale information. Our findings capture how receptor-ligand interactions mediate colloid scale interactions relevant to self-assembly, drug delivery, and biosensing.

Synthetic mucus barrier arrays as a nanoparticle formulation screening platform.

A mucus gel layer lines the luminal surface of tissues throughout the body to protect them from infectious agents and particulates. As a result, nanoparticle drug delivery systems delivered to these sites may become trapped in mucus and subsequently cleared before they can reach target cells. As such, optimizing the properties of nanoparticle delivery vehicles, such as their surface chemistry and size, is essential to improving their penetration through the mucus barrier. In previous work, we developed a mucin-based hydrogel that has viscoelastic properties like that of native mucus which can be further tailored to mimic specific mucosal tissues and disease states. Using this biomimetic hydrogel system, a 3D-printed array containing synthetic mucus barriers was created that is compatible with a 96-well plate enabling its use as a high-throughput screening platform for nanoparticle drug delivery applications. To validate this system, we evaluated several established design parameters to determine their impact on nanoparticle penetration through synthetic mucus barriers. Consistent with the literature, we found nanoparticles of smaller size and coated with a protective PEG layer more efficiently penetrated through synthetic mucus barriers. In addition, we evaluated a mucolytic (tris(2-carboxyethyl) phosphine, TCEP) for use as a permeation enhancer for mucosal drug delivery. In comparison to N-acetyl cysteine (NAC), we found TCEP significantly improved nanoparticle penetration through a disease-like synthetic mucus barrier. Overall, our results establish a new high-throughput screening approach using synthetic mucus barrier arrays to identify promising nanoparticle formulation strategies for drug delivery to mucosal tissues.

Anomalous silica colloid stability and gel layer mediated interactions.

Total internal reflection microscopy (TIRM) is used to measure SiO2 colloid ensembles over a glass microscope slide to simultaneously obtain interactions and stability as a function of pH (4-10) and NaCl concentration (0.1-100 mM). Analysis of SiO2 colloid Brownian height excursions yields kT-scale potential energy vs separation profiles, U(h), and diffusivity vs separation profiles, D(h), and determines whether particles are levitated or irreversibly deposited (i.e., stable). By including an impermeable SiO2 "gel layer" when fitting van der Waals, electrostatic, and steric potentials to measured net potentials, gel layers are estimated to be ~10 nm thick and display an ionic strength collapse. The D(h) results indicate consistent surface separation scales for potential energy profiles and hydrodynamic interactions. Our measurements and model indicate how SiO2 gel layers influence van der Waals (e.g., dielectric properties), electrostatics (e.g., shear plane), and steric (e.g., layer thickness) potentials to understand the anomalous high ionic strength and high pH stability of SiO2 colloids.

Synthetic Mucus Biomaterials for Antimicrobial Peptide Delivery.

Despite the promise of antimicrobial peptides (AMPs) as treatments for antibiotic-resistant infections, their therapeutic efficacy is limited due to the rapid degradation and low bioavailability of AMPs. To address this, we have developed and characterized a synthetic mucus (SM) biomaterial capable of delivering AMPs and enhancing their therapeutic effect. LL37 loaded SM hydrogels demonstrated controlled release of LL37 over 8 hours as a result of charge-mediated interactions between mucins and LL37 AMPs. Compared to treatment with LL37 alone where antimicrobial activity was reduced after 3 hours, LL37-SM hydrogels inhibited Pseudomonas aeruginosa PAO1 growth over 12 hours. LL37-SM hydrogel treatment reduced PAO1 viability over 6 hours whereas a rebound in bacterial growth was observed when treated with LL37 only. These data demonstrate LL37-SM hydrogels enhance antimicrobial activity by preserving LL37 AMP activity and bioavailability. Overall, this work establishes SM biomaterials as a platform for enhanced AMP delivery for antimicrobial applications.

Synthetic mucus biomaterials for antimicrobial peptide delivery.

Despite the promise of antimicrobial peptides (AMPs) as treatments for antibiotic-resistant infections, their therapeutic efficacy is limited due to the rapid degradation and low bioavailability of AMPs. To address this, we have developed and characterized a synthetic mucus (SM) biomaterial capable of delivering LL37 AMPs and enhancing their therapeutic effect. LL37 is an AMP that exhibits a wide range of antimicrobial activity against bacteria, including Pseudomonas aeruginosa. LL37 loaded SM hydrogels demonstrated controlled release with 70%-95% of loaded LL37 over 8 h due to charge-mediated interactions between mucins and LL37 AMPs. Compared to treatment with LL37 alone where antimicrobial activity was reduced after 3 h, LL37-SM hydrogels inhibited P. aeruginosa (PAO1) growth over 12 h. LL37-SM hydrogel treatment reduced PAO1 viability over 6 h whereas a rebound in bacterial growth was observed when treated with LL37 only. These data demonstrate LL37-SM hydrogels enhance antimicrobial activity by preserving LL37 AMP activity and bioavailability. Overall, this work establishes SM biomaterials as a platform for enhanced AMP delivery for antimicrobial applications.

Engineering in vitro models of cystic fibrosis lung disease using neutrophil extracellular trap inspired biomaterials.

Cystic fibrosis (CF) is a muco-obstructive lung disease where inflammatory responses due to chronic infection result in the accumulation of neutrophil extracellular traps (NETs) in the airways. NETs are web-like complexes comprised mainly of decondensed chromatin that function to capture and kill bacteria. Prior studies have established excess release of NETs in CF airways increases viscoelasticity of mucus secretions and reduces mucociliary clearance. Despite the pivotal role of NETs in CF disease pathogenesis, current in vitro models of this disease do not account for their contribution. Motivated by this, we developed a new approach to study the pathobiological effects of NETs in CF by combining synthetic NET-like biomaterials, composed of DNA and histones, with an in vitro human airway epithelial cell culture model. To determine the impact of synthetic NETs on airway clearance function, we incorporated synthetic NETs into mucin hydrogels and cell culture derived airway mucus to assess their rheological and transport properties. We found that the addition of synthetic NETs significantly increases mucin hydrogel and native mucus viscoelasticity. As a result, mucociliary transport in vitro was significantly reduced with the addition of mucus containing synthetic NETs. Given the prevalence of bacterial infection in the CF lung, we also evaluated the growth of Pseudomonas aeruginosa in mucus with or without synthetic NETs. We found mucus containing synthetic NETs promoted microcolony growth and prolonged bacterial survival. Together, this work establishes a new biomaterial enabled approach to study innate immunity mediated airway dysfunction in CF.

Inhaled drug delivery for the targeted treatment of asthma.

Asthma is a chronic lung disease affecting millions worldwide. While classically acknowledged to result from allergen-driven type 2 inflammatory responses leading to IgE and cytokine production and the influx of immune cells such as mast cells and eosinophils, the wide range in asthmatic pathobiological subtypes lead to highly variable responses to anti-inflammatory therapies. Thus, there is a need to develop patient-specific therapies capable of addressing the full spectrum of asthmatic lung disease. Moreover, delivery of targeted treatments for asthma directly to the lung may help to maximize therapeutic benefit, but challenges remain in design of effective formulations for the inhaled route. In this review, we discuss the current understanding of asthmatic disease progression as well as genetic and epigenetic disease modifiers associated with asthma severity and exacerbation of disease. We also overview the limitations of clinically available treatments for asthma and discuss pre-clinical models of asthma used to evaluate new therapies. Based on the shortcomings of existing treatments, we highlight recent advances and new approaches to treat asthma via inhalation for monoclonal antibody delivery, mucolytic therapy to target airway mucus hypersecretion and gene therapies to address underlying drivers of disease. Finally, we conclude with discussion on the prospects for an inhaled vaccine to prevent asthma.

Biomaterials for intranasal and inhaled vaccine delivery.

Delivery of vaccines by nasal sprays may enable more robust, protective mucosal immune responses against infectious diseases, such as COVID-19, compared with intramuscular injection. In this Comment, we highlight how biomaterials can be designed to allow intranasal and inhaled vaccination.

Engineering in vitro models of cystic fibrosis lung disease using neutrophil extracellular trap inspired biomaterials.

Cystic fibrosis (CF) is a muco-obstructive lung disease where inflammatory responses due to chronic infection result in the accumulation of neutrophil extracellular traps (NETs) in the airways. NETs are web-like complexes comprised mainly of decondensed chromatin that function to capture and kill bacteria. Prior studies have established excess release of NETs in CF airways increases viscoelasticity of mucus secretions and reduces mucociliary clearance. Despite the pivotal role of NETs in CF disease pathogenesis, current in vitro models of this disease do not account for their contribution. Motivated by this, we developed a new approach to study the pathobiological effects of NETs in CF by combining synthetic NET-like biomaterials, composed of DNA and histones, with an in vitro human airway epithelial cell culture model. To determine the impact of synthetic NETs on airway clearance function, we incorporated synthetic NETs into mucin hydrogels and cell culture derived airway mucus to assess their rheological and transport properties. We found that the addition of synthetic NETs significantly increases mucin hydrogel viscoelasticity. As a result, mucociliary transport in vitro was significantly reduced with the addition of mucus containing synthetic NETs. Given the prevalence of bacterial infection in the CF lung, we also evaluated the growth of Pseudomonas aeruginosa in mucus with or without synthetic NETs. We found mucus containing synthetic NETs promoted microcolony growth and prolonged bacterial survival. Together, this work establishes a new biomaterial enabled approach to study innate immunity mediated airway dysfunction in CF.