The generalized phase contrast method is employed as an efficient "phase-only" laser beam-shaping technique in an optical setup built for catapulting microspheres through simple mucus models. The influence of the laser power and mucin concentration on the motion of the microspheres is investigated in terms of instant and average velocities on a 250-μm trajectory, corresponding to the mucus thickness in the human gastrointestinal tract. Increasing the laser power leads to higher velocities in all the tested samples, while increasing the mucin concentration leads to significant velocity decrease for similar laser input power. However, velocities of up to 95  μm  ·  s  -  1 are demonstrated in a 5% mucin simple mucus model using our catapulting system. This study contributes to understanding and overcoming the challenges of optical manipulation in mucus models. This paves the way for efficient optical manipulation of three-dimensional-printed light-controlled microtools with the ability to penetrate the mucus biobarrier for in vitro drug-delivery studies.

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.

Tracheobronchial secretions are a complex mixture of secretory fluids derived from sources within the lung. Important constituents include the mucous glycoproteins, other secretory proteins, serum proteins, lipids, salts; water makes up 95% of mucus by weight. These secretions form two phases at the epithelial surface: a mucous gel and an aqueous layer (periciliary fluid). Polymerization and aggregation of mucous glycoproteins create the gel matrix. Other macromolecules such as lysozyme, albumin, and immunoglobulin A also may participate in the process of gelation. Intermolecular forces contributing to gelation include disulfide bonding, sugar-sugar interactions between adjacent glycoproteins, and ionic interactions between the glycoprotein anionic groups (sialic acid carboxyl and sulfate) and cationic components in the secretions. Respiratory tract mucous glycoproteins are large, extended molecules, which have a high carbohydrate content. They are polydisperse, with variation occurring largely in the content of sulfated sugars and sialic acid. Factors such as cell of origin, chronic lung disease, and pharmacologic effects influence the density of these anionic (acidic) groups. Variation in acidic properties may influence the physical and virus-binding properties of mucus. Little information is available concerning the biosynthetic mechanisms in airway epithelium through which these variations are effected.

The mucus covering of epithelial tissues presents one significant biological barrier to the uptake and absorption of particulate carriers. Improved understanding of the mechanisms mediating the transport of nanoparticles across such mucus layers would accelerate their development as optimised mucosal drug delivery formulations (e.g. via oral and rectal routes). Herein, an in vitro mucus model ("Mucus-on-Chip") was developed to enable the interaction and transport of functionalised nanoparticles and reconstituted mucus to be quantitatively investigated in real-time. We verified that the diffusion of nanoparticles into mucus is highly dependent on their biointerfacial properties. Muco-inert modification (PEGylation) significantly enhanced the mucopenetration of 50 nm and 200 nm nanoparticles, whereas limited mucopenetration was observed for pectin coated mucoadhesive nanoparticles. Furthermore, this model can be easily adapted to mimic specific physiological mucus environments. Mucus pre-treated with a mucolytic agent displayed reduced barrier function and therefore significantly accelerated mucopenetration of nanoparticles, which was independent of their size and biointerfacial properties. This new "Mucus-on-Chip" methodology provides detailed insight into the dynamics of nanoparticle-mucus interaction, which can be applied to refine the design of particulate formulations for more efficient mucosal drug delivery.

The colonic mucus layer is organized as a two-layered system providing a physical barrier against pathogens and simultaneously harboring the commensal flora. The factors contributing to the organization of this gel network are not well understood. In this study, the impact of transglutaminase activity on this architecture was analyzed. Here, we show that transglutaminase TGM3 is the major transglutaminase-isoform expressed and synthesized in the colon. Furthermore, intrinsic extracellular transglutaminase activity in the secreted mucus was demonstrated in vitro and ex vivo. Absence of this acyl-transferase activity resulted in faster degradation of the major mucus component the MUC2 mucin and changed the biochemical properties of mucus. Finally, TGM3-deficient mice showed an early increased susceptibility to Dextran Sodium Sulfate-induced colitis. Here, we report that natural isopeptide cross-linking by TGM3 is important for mucus homeostasis and protection of the colon from inflammation, reducing the risk of colitis.

Fish skin mucus is composed of insoluble components, which form the physical barrier, and soluble components, which are key for interrelationship functions. Mucus is continuously secreted, but rates of production and exudation are still unknown, as are the underlying mechanisms. Using stable isotope analysis, here, we evaluate skin mucus turnover and renewal in gilthead sea bream, separating raw mucus and its soluble and insoluble fractions. Isotopic abundance analysis reveals no differences between mucus and white muscle, thus confirming mucus samples as reliable non-invasive biomarkers. Mucus production was evaluated using a single labelled meal packaged in a gelatine capsule, with both 13C and 15N, via a time-course trial. 13C was gradually allocated to skin mucus fractions over the first 12 h and was significantly (4-fold) higher in the soluble fraction, indicating a higher turnover of soluble mucus components that are continuously produced and supplied. 15N was also gradually allocated to mucus, indicating incorporation of new proteins containing the labelled dietary amino acids, but with no differences between fractions. When existent mucus was removed, dietary stable isotopes revealed stimulated mucus neoformation dependent on the components. All this is novel knowledge concerning skin mucus dynamics and turnover in fish and could offer interesting non-invasive approaches to the use of skin mucus production in ecological or applied biological studies such as climate change effects, human impact, alterations in trophic networks or habitat degradation, especially of wild-captured species or protected species.

Self-emulsifying drug delivery systems (SEDDS) have potential applications in the delivery of hydrophobic components. Oral drugs are readily captured and cleared by intestinal mucus, a natural barrier that covers the mucosal epithelium and prevents the entry of foreign substances. In this study, we investigated for the first time the ability of SEDDS to deliver the lipophilic aldehyde cinnamaldehyde (CA-SEDDS) in rat mucus, mucin solution, Caco-2 and Caco-2/HT29 co-culture monolayer systems. CA-SEDDS was characterized by particle size, Zeta potential and the logDSEDDS/release medium. The capacity of CA-SEDDS to enhance mucus permeability was investigated in rat intestinal mucus gel and mucin solution with the period of in 12 h by Transwell® diffusion. We evaluated the potential of CA-SEDDS delivery of CA in a co-culture system of absorptive Caco-2 and mucus-secreting HT29 cells. CA-SEDDS exhibited excellent mucus permeability in mucus and mucin solutions, 5.1- and 2.8-fold higher than the free CA group, respectively. CA-SEDDS penetration increased by 2.5-fold compared with free CA when using the mucus-secreting co-culture cell model as a barrier. The relative oral bioavailability of CA-SEDDS was 242% compared to CA without formulation. These findings suggest that SEDDS exhibited good release and superior mucus permeability, displaying great potential for the future of hydrophobic oral applications.

Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are associated with acute and chronic bacterial infections of the lung. Excessive differentiation of basal cells to mucus-producing goblet cells can result in mucus hyperproduction and loss of mucociliary clearance in the airways of CF and COPD patients. Here, we aimed to investigate the effect of pathogen-associated molecular patterns (PAMPs) on the differentiation of human 3D bronchospheres. Primary human bronchial epithelial cells (HBECs) were differentiated to bronchospheres in the presence of bacterial flagellin and LPS and the synthetic Toll-like receptor (TLR) ligands Pam3CSK4 (TLR-2) and polyinosinic:polycytidylic acid (pIC, TLR-3). Electron and fluorescence microscopy showed that the differentiation of bronchospheres associated with the formation of lumina and appearance of cilia within 30 days after seeding. Incubation with flagellin resulted in a decreased formation of lumina and loss of cilia formation. Incubation with Pam3CSK, pIC, and LPS did not significantly affect formation of lumina and ciliation. Mucus production was strongly increased in response to flagellin and, to a lesser degree, in response to Pam3CSK4. Our results indicate that bacterial factors, such as flagellin, drive the differentiation of the respiratory epithelium towards mucus hyperproduction.

Fish skin mucus serves as a first line of defense against pathogens and external stressors. In this study the proteomic profile of lumpsucker skin mucus was characterized using 2D gels coupled with tandem mass spectrometry. Mucosal proteins were identified by homology searches across the databases SwissProt, NCBInr and vertebrate EST. The identified proteins were clustered into ten groups based on their gene ontology biological process in PANTHER (www.patherdb.org). Calmodulin, cystatin-B, histone H2B, peroxiredoxin1, apolipoprotein A1, natterin-2, 14-3-3 protein, alfa enolase, pentraxin, warm temperature acclimation 65 kDa (WAP65kDa) and heat shock proteins were identified. Several of the proteins are known to be involved in immune and/or stress responses. Proteomic profile established in this study could be a benchmark for differential proteomics studies.

Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation. Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD.

Antibodies have been shown to hinder the movement of herpes simplex virus virions in cervicovaginal mucus, as well as other viruses in other mucus secretions. However, it has not been possible to directly observe the mechanisms underlying this phenomenon, so the nature of virion-antibody-mucin interactions remain poorly understood. In this work, we analyzed thousands of virion traces from single particle tracking experiments to explicate how antibodies must cooperate to immobilize virions for relatively long time periods. First, using a clustering analysis, we observed a clear separation between two classes of virion behavior: freely diffusing and immobilized. While the proportion of freely diffusing virions decreased with antibody concentration, the magnitude of their diffusivity did not, implying an all-or-nothing dichotomy in the pathwise effect of the antibodies. Proceeding under the assumption that all binding events are reversible, we used a novel switch-point detection method to conclude that there are very few, if any, state switches on the experimental timescale of 20 s. To understand this slow state switching, we analyzed a recently proposed continuous-time Markov chain model for binding kinetics and virion movement. Model analysis implied that virion immobilization requires cooperation by multiple antibodies that are simultaneously bound to the virion and mucin matrix and that there is an entanglement phenomenon that accelerates antibody-mucin binding when a virion is immobilized. In addition to developing a widely applicable framework for analyzing multistate particle behavior, this work substantially enhances our mechanistic understanding of how antibodies can reinforce a mucus barrier against passive invasive species.

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.

Mucosal drug delivery nanotechnologies are limited by the mucus barrier that protects nearly all epithelial surfaces not covered with skin. Most polymeric nanoparticles, including polystyrene nanoparticles (PS), strongly adhere to mucus, thereby limiting penetration and facilitating rapid clearance from the body. Here, we demonstrate that PS rapidly penetrate human cervicovaginal mucus (CVM), if the CVM has been pretreated with sufficient concentrations of Pluronic F127. Importantly, the diffusion rate of large polyethylene glycol (PEG)-coated, nonmucoadhesive nanoparticles (PS-PEG) did not change in F127-pretreated CVM, implying that F127 did not significantly alter the native pore structure of CVM. Additionally, herpes simplex virus type 1 (HSV-1) remains adherent in F127-pretreated CVM, indicating that the presence of F127 did not reduce adhesive interactions between CVM and the virions. In contrast to treatment with a surfactant that has been approved for vaginal use as a spermicide (nonoxynol-9 or N9), there was no increase in inflammatory cytokine release in the vaginal tract of mice after daily application of 1% F127 for 1 week. Pluronic F127 pretreatment holds potential as a method to safely improve the distribution, retention, and efficacy of nanoparticle formulations without compromising CVM barrier properties to pathogens.

No abstract available

Luminal accumulation of viscous, poorly hydrated, and less transportable mucus has been associated with altered mucus rheology and reduced mucociliary clearance. These symptoms are some of the cardinal clinical manifestations found throughout major respiratory diseases as well as gastrointestinal and digestive disorders. Applications of current mucolytics may yield short-term improvements but are continuously challenged by undesirable side-effects. While nanoparticles (NPs) can interact with mucin polymers,whether functionalized NPs can rectify mucus rheology is unknown. Herein, we report that carboxyl-functionalized NPs (24 nm and 120 nm) dramatically reduced mucin gel size and accelerated mucin matrix hydration rate (diffusivity). Our results suggest that carboxyl-functionalized NPs disperse mucin gels possibly by enhancing network hydration. This report highlights the prospective usages of carboxyl-functionalized NPs as a novel mucus dispersant or mucolytic agent in adjusting mucus rheological properties and improving mucociliary transport to relieve clinical symptoms of patients suffering from relevant diseases.

The gut mucosal environment is key in host health; protecting against pathogens and providing a niche for beneficial bacteria, thereby facilitating a mutualistic balance between host and microbiome. Lack of dietary fiber results in erosion of the mucosal layer, suggested to be a result of increased mucus-degrading gut bacteria. This study aimed to use quantitative analyses to investigate the diet-induced imbalance of mucosal homeostasis. Seven days of fiber-deficiency affected intestinal anatomy and physiology, seen by reduced intestinal length and loss of the colonic crypt-structure. Moreover, the mucus layer was diminished, muc2 expression decreased, and impaired mucus secretion was detected by stable isotope probing. Quantitative microbiome profiling of the gut microbiota showed a diet-induced reduction in bacterial load and decreased diversity across the intestinal tract, including taxa with fiber-degrading and butyrate-producing capabilities. Most importantly, there was little change in the absolute abundance of known mucus-degrading bacteria, although, due to the general loss of taxa, relative abundance would erroneously indicate an increase in mucus degraders. These findings underscore the importance of using quantitative methods in microbiome research, suggesting erosion of the mucus layer during fiber deprivation is due to diminished mucus production rather than overgrowth of mucus degraders.

Mucus in the lung plays an essential role as a barrier to infection by viral pathogens such as influenza A virus (IAV). Previous work determined mucin-associated sialic acid acts as a decoy receptor for IAV hemagglutinin (HA) binding and the sialic-acid cleaving enzyme, neuraminidase (NA), facilitates virus passage through mucus. However, it has yet to be fully addressed how the physical structure of the mucus gel influences its barrier function and its ability to trap viruses via glycan mediated interactions to prevent infection. To address this, IAV and nanoparticle diffusion in human airway mucus and mucin-based hydrogels is quantified using fluorescence video microscopy. We find the mobility of IAV in mucus is significantly influenced by the mesh structure of the gel and in contrast to prior reports, these effects likely influence virus passage through mucus gels to a greater extent than HA and NA activity. In addition, an analytical approach is developed to estimate the binding affinity of IAV to the mucus meshwork, yielding dissociation constants in the mM range, indicative of weak IAV-mucus binding. Our results provide important insights on how the adhesive and physical barrier properties of mucus influence the dissemination of IAV within the lung microenvironment.

Airway mucus obstruction is the main cause of morbidity in cystic fibrosis, a disease caused by mutations in the CFTR Cl- channel. Activation of non-CFTR Cl- channels such as TMEM16A can likely compensate for defective CFTR. However, TMEM16A was recently described as a key driver in mucus production/secretion. Here, we have examined whether indeed there is a causal relationship between TMEM16A and MUC5AC production, the main component of respiratory mucus. Our data show that TMEM16A and MUC5AC are inversely correlated during differentiation of human airway cells. Furthermore, we show for the first time that the IL-4-induced TMEM16A up-regulation is proliferation-dependent, which is supported by the correlation found between TMEM16A and Ki-67 proliferation marker during wound healing. Consistently, the notch signaling activator DLL4 increases MUC5AC levels without inducing changes neither in TMEM16A nor in Ki-67 expression. Moreover, TMEM16A inhibition decreased airway surface liquid height. Altogether, our findings demonstrate that up-regulation of TMEM16A and MUC5AC is only circumstantial under cell proliferation, but with no causal relationship between them. Thus, although essential for airway hydration, TMEM16A is not required for MUC5AC production.

Efficient mucosal delivery remains a major challenge for the reason of the respiratory tract mucus act as a formidable barrier to nanocarriers by trapping and clearing foreign particulates. The surface property of nanoparticles determines their retention and penetration ability within the respiratory tract mucus. However, the interaction between nanoparticles and mucus, and how these interactions impact distribution has not been extensively investigated. In this study, polymeric nanoparticles loaded with a baicalein-phospholipid complex were modified with two kinds of polymers, mucoadhesive and mucus-penetrative polymer. Systematic investigations on the physicochemical property, mucus penetration, transepithelial transport, and tissue distribution were performed to evaluate the interaction of nanoparticles with the respiratory tract. Both nanoparticles had a similar particle size and good biocompatibility, exhibited a sustained-release profile, but showed a considerable difference in zeta potential. Interestingly, mucus-penetrative nanoparticles exhibited a higher diffusion rate in mucus, deeper penetration across the mucus layer, enhanced in vitro cellular uptake, increased drug distribution in airways, and superior local distribution and bioavailability as compared to mucoadhesive nanoparticles. These results indicate the potential of mucus-penetrative nanoparticles in design of a rational delivery system to improve the efficiency of inhaled therapy by promoting mucus penetration and increasing local distribution and bioavailability.

To examine the in vivo effects of the 15-member macrolide, azithromycin (AZM), on mucus hypersecretion, we induced hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium by intranasal instillation of ovalbumin (OVA) in OVA-sensitized rats, or by intranasal lipopolysaccharides (LPS) instillation. Oral administration of AZM (5-10 mg/kg) or clarithromycin (CAM, 5-10 mg/kg) significantly inhibited OVA- and LPS-induced mucus production, whereas josamycin (JM) or ampicillin (ABPC) showed no effect. In vitro effects of AZM on airway epithelial cells were examined using NCI-H292 cells and human nasal epithelial cells cultured in air-liquid interface. Mucus secretion was evaluated by enzyme-linked immunosorbent assay using an anti-MUC5AC monoclonal antibody. AZM or CAM significantly inhibited tumor necrosis factor-α (TNF-α) (20 ng/mL)-induced MUC5AC secretion from NCI-H292 cells at 10⁻⁶-10⁻⁷ M, whereas JM or ABPC showed no effect. AZM significantly inhibited TNF-α (20 ng/mL)-induced MUC5AC secretion from human nasal epithelial cells at 10⁻⁴ M. MUC5AC mRNA expression was also significantly inhibited. These results indicate that the 15-member macrolide, AZM, exerts direct inhibitory effects on mucus secretion from airway epithelial cells and that it may be useful for the treatment of mucus hypersecretion caused by allergic inflammation and LPS stimulation.