Morphological and functional features of the colonic mucus barrier in patients with symptomatic uncomplicated diverticular disease and acute uncomplicated diverticulitis.

OBJECTIVE: Aim: The purpose was to identify the morphological and functional features of the colonic mucus barrier in patients with symptomatic uncomplicated diverticular disease and acute uncomplicated diverticulitis. PATIENTS AND METHODS: Materials and Methods: In the research, three groups were formed. Group 1 included fragments of the mucous membrane of the large intestine, which were collected from 12 people during autopsies. The results of autopsies and histological examination of the material did not reveal any gastrointestinal pathology. Group 2 included biopsies of the mucous membrane of the large intestine from the area of the diverticulum of 34 patients with symptomatic uncomplicated diverticular disease. Group 3 included biopsies of the mucous membrane of the large intestine of 26 patients with acute uncomplicated diverticulitis. Histological (hematoxylin and eosin staining), histochemical (PAS reaction) and immunohistochemical (mouse monoclonal antibodies to Mucin 2 (MUC2) and Mucin 4 (MUC4)) staining methods were used. A morphometric study was also carried out. RESULTS: Results: In patients with diverticular disease, the authors identified disturbances in the morphofunctional state of the mucus barrier of the colon, the structure and function of goblet cells contained in its mucous membrane, characterized by a decrease in the thickness of the mucus layer covering the surface of the mucous membrane; a decrease in the size and number of goblet cells with a decrease in their mucus-producing ability; a change in the mucin profile, characterized by a violation of the content of MUC2 and MUC4. These changes were greatest in patients with acute uncomplicated diverticulitis compared with patients with symptomatic uncomplicated diverticular disease. CONCLUSION: Conclusions: The identified disturbances in the morphofunctional state of the mucus barrier of the colon, structural and functional changes in goblet cells may be one of the mechanisms for the development of acute uncomplicated diverticulitis and symptomatic uncomplicated diverticular disease.

Inflammation promotes stomach epithelial defense by stimulating the secretion of antimicrobial peptides in the mucus.

The mucus serves as a protective barrier in the gastrointestinal tract against microbial attacks. While its role extends beyond merely being a physical barrier, the extent of its active bactericidal properties remains unclear, and the mechanisms regulating these properties are not yet understood. We propose that inflammation induces epithelial cells to secrete antimicrobial peptides, transforming mucus into an active bactericidal agent. To investigate the properties of mucus, we previously developed mucosoid culture models that mimic the healthy human stomach epithelium. Similar to organoids, mucosoids are stem cell-driven cultures; however, the cells are cultivated on transwells at air-liquid interface. The epithelial cells of mucosoids form a polarized monolayer, allowing differentiation into all stomach lineages, including mucus-secreting cells. This setup facilitates the secretion and accumulation of mucus on the apical side of the mucosoids, enabling analysis of its bactericidal effects and protein composition, including antimicrobial peptides. Our findings show that TNFα, IL1ß, and IFNγ induce the secretion of antimicrobials such as lactotransferrin, lipocalin2, complement component 3, and CXCL9 into the mucus. This antimicrobial-enriched mucus can partially eliminate Helicobacter pylori, a key stomach pathogen. The bactericidal activity depends on the concentration of each antimicrobial and their gene expression is higher in patients with inflammation and H.pylori-associated chronic gastritis. However, we also find that H. pylori infection can reduce the expression of antimicrobial encoding genes promoted by inflammation. These findings suggest that controlling antimicrobial secretion in the mucus is a critical component of epithelial immunity. However, pathogens like H. pylori can overcome these defenses and survive in the mucosa.

Synthesis and characterization of fluorinated graphene oxide nanosheets derived from Lissachatina fulica snail mucus and their biomedical applications.

The modern nanomedicine incorporates the multimodal treatments into a single formulation, offering innovative cancer therapy options. Nanosheets function as carriers, altering the solubility, biodistribution, and effectiveness of medicinal compounds, resulting in more efficient cancer treatments and reduced side effects. The non-toxic nature of fluorinated graphene oxide (FGO) nanosheets and their potential applications in medication delivery, medical diagnostics, and biomedicine distinguish them from others. Leveraging the unique properties of Lissachatina fulica snail mucus (LfSM), FGO nanosheets were developed to reveal the novel characteristics. Consequently, LfSM was utilized to create non-toxic, environmentally friendly, and long-lasting FGO nanosheets. Ultraviolet-visible (UV-vis) spectroscopy revealed a prominent absorbance peak at 235 nm. The characterization of the synthesized FGO nanosheets involved X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), high-resolution transmission electron microscopy (HR-TEM), and atomic force microscopy (AFM) analyses. The antimicrobial activity data demonstrated a broad spectrum of antibacterial effects against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The cytotoxicity efficacy of LfSM-FGO nanosheets against pancreatic cancer cell line (PANC1) showed promising results at low concentrations. The study suggests that FGO nanosheets made from LfSM could serve as alternate factors for in biomedical applications in the future.

Design of Auto-Adaptive Drug Delivery System for Effective Delivery of Peptide Drugs to Overcoming Mucus and Epithelial Barriers.

Oral administration of peptide represents a promising delivery route, however, it is hindered by the harsh gastrointestinal environment, leading to low in vivo absorption. In this study, auto-adaptive protein corona-AT 1002-cationic liposomes (Pc-AT-CLs) are constructed with the characteristic of hydrophilic and electrically neutral surface properties for the encapsulation of liraglutide. BSA protein corona is used to coat AT-CLs reducing the adherence of mucus, and may fall off after penetrating the mucus layer. Transmucus transport experiment demonstrated that the mucus penetration amount of Pc-AT-CLs are 1.45 times that of AT-CLs. After penetrating the mucus layer, AT-CLs complete transmembrane transport by the dual action of AT and cationic surface properties. Transmembrane transport experiment demonstrated that the apparent permeability coefficient (Papp) of AT-CLs is 2.03 times that of CLs. In vivo tests demonstrated that Pc-AT-CLs exhibited a significant hypoglycemic effect and enhanced the relative bioavailability comparing to free liraglutide. Pc-AT-CLs protect liraglutide from degradation, facilitate its absorption, and ultimately improve its oral bioavailability.

Antibiotics damage the colonic mucus barrier in a microbiota-independent manner.

Antibiotic use is a risk factor for development of inflammatory bowel diseases (IBDs). IBDs are characterized by a damaged mucus layer, which does not separate the intestinal epithelium from the microbiota. Here, we hypothesized that antibiotics affect the integrity of the mucus barrier, which allows bacterial penetrance and predisposes to intestinal inflammation. We found that antibiotic treatment led to breakdown of the colonic mucus barrier and penetration of bacteria into the mucus layer. Using fecal microbiota transplant, RNA sequencing followed by machine learning, ex vivo mucus secretion measurements, and antibiotic treatment of germ-free mice, we determined that antibiotics induce endoplasmic reticulum stress in the colon that inhibits colonic mucus secretion in a microbiota-independent manner. This antibiotic-induced mucus secretion flaw led to penetration of bacteria into the colonic mucus layer, translocation of microbial antigens into circulation, and exacerbation of ulcerations in a mouse model of IBD. Thus, antibiotic use might predispose to intestinal inflammation by impeding mucus production.

Normal Saline Versus Hypertonic Saline for Airway STENT Maintenance: SALTY STENT Study.

BACKGROUND: Mucus plugging is a common complication of airway stenting. There is no data or guidance on the best airway hygiene regimen and consequently wide practice variation exists. METHODS: This single-center, nonblinded, randomized, pilot study aims to evaluate the effectiveness and safety of nebulized 3% saline (3%S) versus normal saline (NS) in reducing the incidence of mucus plugging in adult patients that undergo central airway stent placement. Patients were enrolled immediately after stent placement and randomized to nebulized 3%S or NS (3 mL) 3 times a day. Patients were scheduled for surveillance bronchoscopy in 4 to 6 weeks. Unscheduled bronchoscopies due to symptomatic mucus plugging were recorded. RESULTS: From December 2022 to March 2024, 37 patients were screened, and 35 were enrolled. Four in the 3%S and 8 in the NS group did not undergo a surveillance bronchoscopy and were excluded from the final analysis. During surveillance bronchoscopy for the 3%S (n=13) and NS (n=10) groups, obstructive mucus plugging was noted in 7.7% versus 40%, granulation requiring intervention in 7.7% versus 10%, and >25% circumferential biofilm in 0% versus 30%, respectively. In the 3%S versus NS groups, 0% versus 20% of patients required an unscheduled bronchoscopy due to mucus plugging. There were no side effects reported with the daily use of 3%S or NS. CONCLUSION: Nebulized 3%S is safe and may be equally or more effective than NS in preventing obstructive mucus plugging in patients who undergo airway stenting. A larger blinded randomized controlled trial is necessary to confirm this finding.

The Use of Bronchial Rheoplasty in Emphysema Patients Previously Treated with Endoscopic Lung Volume Reduction: A Case Series.

Endoscopic lung volume reduction (ELVR) is an established treatment option for patients with severe emphysema. Not all patients are candidates for this type of intervention, and in the context of significant airway secretions, they may be excluded from treatment. Bronchial Rheoplasty (BR) was developed to treat mucus hypersecretion by delivering nonthermal pulsed electric fields to the airway epithelium and submucosa. The literature to date demonstrates that patients treated with BR in clinical studies have a reduction in airway goblet cell hyperplasia as well as substantive clinical improvement in the setting of chronic bronchitis (CB). In this case series, we present four patients treated at three different institutions who had previously undergone ELVR with beneficial outcome. However, over time, these patients subsequently developed worsening clinical issues, including complaints of increased and thickened mucus, along with exacerbations in the setting of a loss of some ELVR-associated benefits. These patients then underwent exploratory treatment with BR with the intent of reducing their secretion burden and potentially restoring the efficacy associated with the initial placement of the airway valves. All BR procedures were well tolerated, and three of the four patients showed substantial improvement in their symptom burden. Airway examinations during the second of the two BR procedures also revealed what appeared to be less airway mucosal inflammation and a decrease in the quantity of airway secretions. Therefore, treatment with BR may have the potential to improve and restore the initial benefits associated with ELVR, thus enhancing long-term outcomes. Further clinical studies with sufficient follow-up are warranted to assess this in a larger cohort of patients, and to determine whether treatment with BR prior to ELVR may make more patients eligible for this treatment through reduction in their secretions and/or symptoms.

In vitro fish mucosal surfaces producing mucin as a model for studying host-pathogen interactions.

Current prophylactic and disease control measures in aquaculture highlight the need of alternative strategies to prevent disease and reduce antibiotic use. Mucus covered mucosal surfaces are the first barriers pathogens encounter. Mucus, which is mainly composed of highly glycosylated mucins, has the potential to contribute to disease prevention if we can strengthen this barrier. Therefore, aim of this study was to develop and characterize fish in vitro mucosal surface models based on commercially available cell lines that are functionally relevant for studies on mucin regulation and host-pathogen interactions. The rainbow trout (Oncorhynchus mykiss) gill epithelial cell line RTgill-W1 and the embryonic cell line from Chinook salmon (Oncorhynchus tshawytscha) CHSE-214 were grown on polycarbonate membrane inserts and chemically treated to differentiate the cells into mucus producing cells. RTGill-W1 and CHSE-214 formed an adherent layer at two weeks post-confluence, which further responded to treatment with the γ-secretase inhibitor DAPT and prolonged culture by increasing the mucin production. Mucins were metabolically labelled with N-azidoacetylgalactosamine 6 h post addition to the in vitro membranes. The level of incorporated label was relatively similar between membranes based on RTgill-W1, while larger interindividual variation was observed among the CHSE in vitro membranes. Furthermore, O-glycomics of RTgill-W1 cell lysates identified three sialylated O-glycans, namely Galß1-3(NeuAcα2-6)GalNAcol, NeuAcα-Galß1-3GalNAcol and NeuAcα-Galß1-3(NeuAcα2-6)GalNAcol, resembling the glycosylation present in rainbow trout gill mucin. These glycans were also present in CHSE-214. Additionally, we demonstrated binding of the fish pathogen A. salmonicida to RTgill-W1 and CHSE-214 cell lysates. Thus, these models have similarities to in vivo mucosal surfaces and can be used to investigate the effect of pathogens and modulatory components on mucin production.

Allometric comparison of viral dynamics in the nasal cavity-nasopharyngeal mucus layer of human and rhesus monkey by CFD-HCD approach.

BACKGROUND AND OBJECTIVE: Viral respiratory infections stand as a considerable global health concern, presenting significant risks to the health of both humans and animals. This study aims to conduct a preliminary analysis of the time series of viral load in the nasal cavity-nasopharynx (NC-NP) of the human and rhesus macaque (RM). METHODS: Taking into account the random uniform distribution of virus-laden droplets with a diameter of 10 µm in the mucus layer, this study applies the computational fluid dynamics-host cell dynamics (CFD-HCD) method to 3D-shell NC-NP models of human and RM, analyzing the impact of initial distribution of droplets on the viral dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), estimating parameters in the HCD model based on experimental data, integrating them into simulations to predict the time series of viral load and cell counts, and being visualized. The reproductive number (R0) are calculated to determine the occurrence of infection. The study also considers cross-parameter combinations and cross-experimental datasets to explore potential correlations between the human and RM. RESULTS: The research findings indicate that the uniform distribution of virus-laden droplets throughout the whole NC-NP models of human and RM is reasonable for simulating and predicting viral dynamics. The visualization results offer dynamic insights into virus infection over a period of 20 days. Studies involving parameter and dataset exchanges between the two species underscore certain similarities in predicting virus infections between the human and RM. CONCLUSIONS: This study lays the groundwork for further exploration into the parallels and distinctions in respiratory virus dynamics between humans and RMs, thus aiding in making more informed decisions in research and experimentation.

Mucus Mimic Hydrogel Coating for Lubricous, Antibiofouling, and Anti-Inflammatory Urinary Catheters.

Silicone is a common elastomer used in indwelling urinary catheters, and catheters are widely used in various medical applications due to their exceptional biocompatibility, hypoallergenic properties, and flexibility. However, silicones exhibit hydrophobic characteristics, lack inherent biolubrication, and are susceptible to nonspecific biosubstance adsorption, resulting in complications including but not limited to tissue trauma, postoperative pain, and urinary tract infections (UTIs). The development of effective surface designs for biomedical catheters to mitigate invasive damage and UITs has been a longstanding challenge. Herein, we present a novel approach to prepare a mucus mimic hydrogel coating. A thin layer of hydrogel containing xylitol is fabricated via photopolymerization. The surface modification technique and the interface-initiated hydrogel polymerization method ensure robust interfacial coherence. The resultant coating exhibits a low friction coefficient (CoF ≈ 0.1) for urinary catheter applications. Benefiting from the hydration layer and the antifouling of the xylitol unit, the xylitol hydrogel-coated surfaces (pAAAMXA) demonstrate outstanding antibiofouling properties against proteins (98.9% reduction relative to pristine polydimethylsiloxane (PDMS)). Furthermore, the pAAAMXA shows general adhesion resistance against bacteria primarily responsible for UITs (Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Methicillin-resistant strains of Staphylococcus aureus (MRSA), and Staphylococcus epidermidis (S. epidermidis)) without compromising biotoxicity (cell viability 98%). In vivo, catheters coated with the mucus mimic hydrogel displayed excellent biocompatibility, resistance to adhesion of bio substance, and anti-inflammatory characteristics. This work describes a promising alternative to conventional silicone catheters, offering potential for clinical interventional procedures with minimized complications.

CD11c+ dendritic cells PlexinD1 deficiency exacerbates airway hyperresponsiveness, IgE and mucus production in a mouse model of allergic asthma.

Dendritic cells (DCs) are pivotal in regulating allergic asthma. Our research has shown that the absence of Sema3E worsens asthma symptoms in acute and chronic asthma models. However, the specific role of PlexinD1 in these processes, particularly in DCs, remains unclear. This study investigates the role of PlexinD1 in CD11c+ DCs using a house dust mite (HDM) model of asthma. We generated CD11c+ DC-specific PlexinD1 knockout (CD11cPLXND1 KO) mice and subjected them, alongside wild-type controls (PLXND1fl/fl), to an HDM allergen protocol. Airway hyperresponsiveness (AHR) was measured using FlexiVent, and immune cell populations were analyzed via flow cytometry. Cytokine levels and immunoglobulin concentrations were assessed using mesoscale and ELISA, while collagen deposition and mucus production were examined through Sirius-red and periodic acid Schiff (PAS) staining respectively. Our results indicate that CD11cPLXND1 KO mice exhibit significantly exacerbated AHR, characterized by increased airway resistance and tissue elastance. Enhanced mucus production and collagen gene expression were observed in these mice compared to wild-type counterparts. Flow cytometry revealed higher CD11c+ MHCIIhigh CD11b+ cell recruitment into the lungs, and elevated total and HDM-specific serum IgE levels in CD11cPLXND1 KO mice. Mechanistically, co-cultures of B cells with DCs from CD11cPLXND1 KO mice showed significantly increased IgE production compared to wild-type mice.These findings highlight the critical regulatory role of the plexinD1 signaling pathway in CD11c+ DCs in modulating asthma features.

Gilthead seabream mucus glycosylation is complex, differs between epithelial sites and carries unusual poly N-acetylhexosamine motifs.

Gilthead seabream (Sparus aurata) is a marine finfish of economic importance in aquaculture. Despite its adaptability to varying culture conditions, gilthead seabream culture can be affected by viral, bacterial or parasitic diseases. The main route of entry of pathogens is through mucosal surfaces. Teleost external and internal surfaces are covered by mucus, mainly comprised of highly glycosylated proteins called mucins. The mucin glycans regulate pathogen growth, adhesion, virulence and inter and intra species communication. Here, we characterized the gilthead seabream mucus glycosylation, compared it to previously described species and investigated associations with microbiota. 214 glycans were identified. The majority of the glycans were found at more than one epithelial surface, but 27, 22 and 89 O-glycan structures were unique to skin, gill and intestinal sample groups, respectively. Six O-glycan core types were observed. The majority of the seabream skin and gill O-glycans were neutral with unusual poly HexNAc motifs. In contrast, seabream intestinal O-glycans were highly acidic and not of the 'poly HexNAc' type observed in skin and gill. Furthermore, gilthead seabream gill mucosa had less oligomannose and more complex N-glycans compared to skin and intestine. The concentration and diversity of bacteria was similar in skin, gill and intestine, but the bacterial species differed between epithelia and co-varied with glycan epitopes. The presence of a complex mucus glycosylation with plenty of glycan epitopes for bacterial foraging, suggest that the skin mucosal defense in seabream includes an abundant resident microbiota. This large library of structures provides a platform for further studies, for example aiming to identifying glycans to use for diagnostic purposes, to study host-microbe interactions or disease intervention therapies.

Dysfunctional mucus structure in cystic fibrosis increases vulnerability to colibactin-mediated DNA adducts in the colon mucosa.

Colibactin is a recently characterized pro-carcinogenic genotoxin produced by pks+ Escherichia coli. We hypothesized that cystic fibrosis (CF)-associated dysfunctional mucus structure increases the vulnerability of host mucosa to colibactin-induced DNA damage. In this pilot study, we tested healthy-appearing mucosal biopsy samples obtained during screening and surveillance colonoscopies of adult CF and non-CF patients for the presence of pks+ E. coli, and we investigated the possibility of detecting a novel colibactin-specific DNA adduct that has not been yet been demonstrated in humans. While CF patients had a lower incidence of pks+ E. coli carriage (~8% vs 29%, p = 0.0015), colibactin-induced DNA adduct formation was detected, but only in CF patients and only in those who were not taking CFTR modulator medications. Moreover, the only patient found to have colon cancer during this study had CF, harbored pks+ E. coli, and had colibactin-induced DNA adducts in the mucosal samples. Larger studies with longitudinal follow-up should be done to extend these initial results and further support the development of colibactin-derived DNA adducts to stratify patients and their risk.

Artificial mucus layer formed in response to ROS for the oral treatment of inflammatory bowel disease.

The artificial mucus layer, such as hydrogels, used to repair the damaged intestinal barrier, is a promising treatment for inflammatory bowel disease (IBD). However, the currently reported hydrogel-based artificial barriers are administered via rectal injection, causing unnecessary discomfort to patients. Herein, we report an oral hydrogel precursor solution based on thiol-modified hyaluronic acid (HASH). Owing to the reactive oxygen species (ROS)-responsive gelling behavior, our precursor solution formed an artificial mucus coating over the inflamed regions of the intestines, blocking microbial invasion and reducing abnormally activated immune responses. Notably, HASH also modulated the gut microbiota, including increasing the diversity and enhancing the abundance of short-chain fatty acid-associated bacteria, which play a key role in gut homeostasis. We believe that the ROS-responsive artificial mucus layer is a promising strategy for the oral treatment of IBD.

Reduced sialylation of airway mucin impairs mucus transport by altering the biophysical properties of mucin.

Mucus stasis is a pathologic hallmark of muco-obstructive diseases, including cystic fibrosis (CF). Mucins, the principal component of mucus, are extensively modified with hydroxyl (O)-linked glycans, which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however, the consequences of reduced sialylation on mucus clearance have not been fully determined. Here, we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin, MUC5B, and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation de novo impaired MCT in primary human bronchial epithelial cells and rat airways, and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally, we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall, this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases.

A history of repeated antibiotic usage leads to microbiota-dependent mucus defects.

Recent evidence indicates that repeated antibiotic usage lowers microbial diversity and ultimately changes the gut microbiota community. However, the physiological effects of repeated - but not recent - antibiotic usage on microbiota-mediated mucosal barrier function are largely unknown. By selecting human individuals from the deeply phenotyped Estonian Microbiome Cohort (EstMB), we here utilized human-to-mouse fecal microbiota transplantation to explore long-term impacts of repeated antibiotic use on intestinal mucus function. While a healthy mucus layer protects the intestinal epithelium against infection and inflammation, using ex vivo mucus function analyses of viable colonic tissue explants, we show that microbiota from humans with a history of repeated antibiotic use causes reduced mucus growth rate and increased mucus penetrability compared to healthy controls in the transplanted mice. Moreover, shotgun metagenomic sequencing identified a significantly altered microbiota composition in the antibiotic-shaped microbial community, with known mucus-utilizing bacteria, including Akkermansia muciniphila and Bacteroides fragilis, dominating in the gut. The altered microbiota composition was further characterized by a distinct metabolite profile, which may be caused by differential mucus degradation capacity. Consequently, our proof-of-concept study suggests that long-term antibiotic use in humans can result in an altered microbial community that has reduced capacity to maintain proper mucus function in the gut.

Creation of a spatially complex mucus bilayer on an in vitro colon model.

The colonic epithelium is comprised of three-dimensional crypts (3D) lined with mucus secreted by a heterogeneous population of goblet cells. In this study, we report the formation of a long-lived, and self-renewing replica of human 3D crypts with a mucus layer patterned in the X-Y-Z dimensions. Primary colon cells were cultured on a shaped scaffold under an air-liquid interface to yield architecturally accurate crypts with a mucus bilayer (605 ± 180 µm thick) possessing an inner (149 ± 50 µm) and outer (435 ± 111 µm) region. Lectins with distinct carbohydrate-binding preferences demonstrated that the mucus in the intercrypt regions was chemically distinct from that above and within the crypts replicating in vivo chemical patterning. Constitutive mucus secretion ejected beads from crypt lumens in 8-10 days, while agonist-stimulated secretion increased mucus thickness by 17-fold in 8 h. The tissue was long-lived, > 50 days, the longest time assessed. In conclusion, the in vitro mucus replicated key physiology of the human mucus, including the bilayer (Z) structure and intercrypt-crypt (X-Y) zones, constitutive mucus flow, spatially complex chemical attributes, and mucus secretion response to stimulation, with the potential to reveal local and global determinants of mucus function and its breakdown in disease.

Integration of mucus and its impact within in vitro setups for inhaled drugs and formulations: Identifying the limits of simple vs. complex methodologies when studying drug dissolution and permeability.

Traditionally, developing inhaled drug formulations relied on trial and error, yet recent technological advancements have deepened the understanding of 'inhalation biopharmaceutics' i.e. the processes that occur to influence the rate and extent of drug exposure in the lungs. This knowledge has led to the development of new in vitro models that predict the in vivo behavior of drugs, facilitating the enhancement of existing formulation and the development of novel ones. Our prior research examined how simulated lung fluid (SLF) affects the solubility of inhaled drugs. Building on this, we aimed to explore drug dissolution and permeability in lung mucosa models containing mucus. Thus, the permeation of four active pharmaceutical ingredients (APIs), salbutamol sulphate (SS), tiotropium bromide (TioBr), formoterol fumarate (FF) and budesonide (BUD), was assayed in porcine mucus covered Calu-3 cell layers, cultivated at an air liquid interface (ALI) or submerged in a liquid covered (LC) culture system. Further analysis on BUD and FF involved their transport in a mucus-covered PAMPA system. Finally, their dissolution post-aerosolization from Symbicort® was compared using 'simple' Transwell and complex DissolvIt® apparatuses, alone or in presence of porcine mucus or polymer-lipid mucus simulant. The presence of porcine mucus impacted both permeability and dissolution of inhaled drugs. For instance, permeability of SS was reduced by a factor of ten in the Calu-3 ALI model while the permeability of BUD was reduced by factor of two in LC and ALI setups. The comparison of dissolution methodologies indicated that drug dissolution performance was highly dependent on the setup, observing decreased release efficiency and higher variability in Transwell system compared to DissolvIt®. Overall, results demonstrate that relatively simple methodologies can be used to discriminate between formulations in early phase drug product development. However, for more advanced stages complex methods are required. Crucially, it was clear that the impact of mucus and selection of its composition in in vitro testing of dissolution and permeability should not be neglected when developing drugs and formulations intended for inhalation.