Abstract Small distal airways differ from proximal large airways in structure, airflow dynamics, and epithelial composition, and represent a central site of muco-obstructive lung disease pathogenesis. However, due in part to their inaccessibility, the molecular mechanisms that establish regional epithelial identity and govern mucociliary defense in distal airway epithelia remain poorly defined. Here, we integrate transcriptomic, secretomic, and chromatin accessibility analyses of matched primary human large and small airway epithelial cultures to define region-specific regulatory networks. We identify distal airway-specific transcriptional and chromatin programs required for maintaining epithelial identity and mucus homeostasis. Loss of NKX2-1 impairs distal airway secretory cell (DASC) differentiation and shifts mucus properties toward a disease-associated state. Lineage-resolved organoid assays identify an NKX2-1-high distal airway basal cell population with hybrid basal-secretory features as a selective progenitor for DASCs. Collectively, these findings establish a molecular framework for distal airway epithelial biology and define mechanisms regulating region-specific mucociliary host defense.

Airway mucus clearance from the lungs occurs by 2 widely recognized mechanisms: cilia-mediated clearance and high-velocity airflow-mediated cough clearance. However, a potentially important third mechanism of mucus clearance, referred to as cilia-independent gas-liquid transport (GLT), was proposed based on in vitro model systems to occur during normal tidal breathing but has largely been overlooked. We conducted in vitro and in vivo studies to investigate the role of tidal breathing airflow rates in mucus clearance. An in vitro airway culture bead-tracking model demonstrated airflow-dependent mucus transport at tidal breathing flow rates. As with other modes of mucus clearance, GLT was critically dependent on mucus concentration. In vivo studies in cilial beat-deficient mice demonstrated that GLT-mediated mucus clearance occurs during tidal breathing in the absence of cough, and the rate of GLT mucus clearance was dependent on breathing frequency and body orientation. These studies demonstrated that GLT is a third mechanism of mucus clearance and likely represents a significant mode of clearance in persons with cilial dysfunction. These findings indicate that increasing breathing rates through exercise, using mucus rehydrating agents or mucolytics, or combining these approaches may restore clinically and physiologically meaningful airway clearance in these patients.

Heat wave burden is rapidly rising relative to pre-industrial levels and exacerbating human respiratory illnesses. Yet, how to protect human airways, short of deep and sustained reductions in greenhouse gas emissions, remains unclear. Here, we find by climate modeling, continuum mechanics analysis, in vitro cell culture models, and human clinical data, that heat waves increase risks of cough hypersensitivity, which can be reduced by alkaline aerosols of endogenous salt ions. We predict that the mouth breathing of indoor and outdoor air during heat waves, by exposing human airways to aridity, causes mucosal collapse onto cilia and inflammatory compression of airway epithelial cells. Using airway lining interface cultures of human bronchial epithelial cells, we find that mucosal collapse alters the expression of genes associated with airway hypersensitivity, including TRPV4, PIEZO1, SCNN1A, SCNN1B, ANO1, and CFTR. Our theoretical and experimental models indicate that mucosal collapse can be reversed by inhalation of alkaline (pH > 8) hypertonic divalent salts for 4-6 hours by globular protein translocation toward the airway epithelium. We find, on analysis of cough recordings in a recently published clinical trial, that inhaling alkaline hypertonic divalent salt aerosols with pH > 8 every 4-6 hours significantly reduced placebo-adjusted cough bouts (34%) (n = 8) (p = 0.01) among refractory chronic cough patients. Inhaled aerosols of the same composition with pH < 8 had no effect on cough bouts (-12%) (n = 4). Aerosols with endogenous ions may help reduce global warming threats to human respiratory health.

Global warming increases water evaporation rates from planetary ecosystems. Here, we show that evaporation rates encountered during human breathing in dehydrating atmospheres promote airway inflammation and potentially exacerbate lung diseases. Continuum mathematical analysis predicts that water evaporation thins airway mucus layers and compresses epithelial cells during tidal breathing. Experiments using human tracheal-bronchial cells confirm that exposure to air with progressive degrees of dryness (relative humidities of 95%, 60%, and 30% at 37 °C) causes the mucus layer to progressively thin (by 5%, 35%, and 58%). Associated compression of epithelial cells elevates secretion of inflammatory cytokines (TNF-α, IL-33, and IL-6). Exposing mice with a muco-inflammatory phenotype to intermittent dry air for 14 days results in histopathological changes and alteration of inflammatory infiltrates. Together with climate model simulations, these findings suggest that most of the United States will be at elevated risk of airway inflammation by the latter half of this century. Exposure of human upper respiratory tracts to dry atmospheres promotes airway inflammation and aggravates lung diseases, according to an analysis of water efflux in human breathing, epithelial cell and mouse experiments, and climate model simulations

Rationale: Hyper-IgE syndrome (STAT3-HIES), also known as Job's syndrome, is a rare immunodeficiency disease typically caused by dominant-negative STAT3 mutations. STAT3-HIES is characterized by chronic pulmonary infection and inflammation, suggesting impaired innate host defense. Objectives: To identify airway epithelial host defense defects caused by STAT3 mutations that, together with immune dysfunction, contribute to recurrent pulmonary infections in STAT3-HIES. Methods: STAT3-HIES sputum was analyzed for biochemical and biophysical properties. STAT3-HIES excised lungs were harvested for histology, and bronchial brush samples were collected for RNA sequencing and in vitro culture. A STAT3-HIES-specific R382W mutation, expressed via lentivirus, and STAT3 knockout (CRISPR/Cas9) were studied in normal human bronchial epithelial cells under basal or inflammatory (IL1β)-stimulated conditions. Effects of STAT3 deficiency on transcriptomics, epithelial ion channel, secretory, antimicrobial, and ciliary functions were assessed. Measurements and Main Results: STAT3-HIES sputum showed increased mucus concentration and viscoelasticity. STAT3-HIES excised lungs exhibited mucus obstruction and elevated IL1β expression. STAT3 mutations reduced CFTR mRNA and protein amounts, impaired CFTR-dependent fluid and mucin secretion, suppressed antimicrobial peptide, cytokine, and chemokine expression, and acidified airway surface liquid at baseline and after IL1β exposure. Notably, mutant STAT3 suppressed IL1R1 expression. Furthermore, STAT3 mutations impaired multiciliogenesis by blocking commitment to ciliated cell lineages through inhibition of HES6, leading to defective mucociliary transport. Administration of a γ-secretase inhibitor restored HES6 expression and improved ciliogenesis in STAT3 R382W mutant cells. Conclusions: STAT3 dysfunction leads to multicomponent defects in airway epithelial innate defense, which, in conjunction with immune deficiency, contributes to chronic pulmonary infection in STAT3-HIES.

AbstractRepetitive bouts of coughing expose the large airways to significant cycles of shear stress. This leads to the release of alarmins and the tussive agent adenosine triphosphate (ATP) which may be modulated by the activity of ion channels present in the human airway. This study aimed to investigate the role of the transient receptor potential subfamily vanilloid member 2 (TRPV2) channel in mechanically induced ATP release from primary bronchial epithelial cells (PBECs).PBECs were obtained from individuals undergoing bronchoscopy. They were cultured in vitro and exposed to mechanical stress in the form of compressive and fluid shear stress (CFSS) or fluid shear stress (FSS) alone at various intensities. ATP release was measured using a luciferin–luciferase assay. Functional TRPV2 protein expression in human PBECs was investigated by confocal calcium imaging. The role of TRPV2 inhibition on FSS-induced ATP release was investigated using the TRPV2 inhibitor tranilast or siRNA knockdown of TRPV2. TRPV2 protein expression in human lung tissue was also determined by immunohistochemistry.ATP release was significantly increased in PBECs subjected to CFSS compared with control (unstimulated) PBECs (N = 3, ***P < 0.001). PBECs expressed functional TRPV2 channels. TRPV2 protein was also detected in fixed human lung tissue. ATP release from FFS stimulated PBECs was decreased by the TRPV2 inhibitor tranilast (N = 3, **P < 0.01) (vehicle: 159 ± 17.49 nM, tranilast: 25.08 ± 5.1 nM) or by TRPV2 siRNA knockdown (N = 3, *P < 0.05) (vehicle: 197 ± 24.52 nM, siRNA: 119 ± 26.85 nM).In conclusion, TRPV2 is expressed in the human airway and modulates ATP release from mechanically stimulated PBECs.

Rationale:Hyper IgE syndrome (STAT3-HIES), also known as Job's syndrome, is a rare immunodeficiency disease typically caused by dominant-negative STAT3 mutations. STAT3-HIES syndrome is characterized by chronic pulmonary infection and inflammation, suggesting impairment of pulmonary innate host defense. Objectives:To identify airway epithelial host defense defects consequent to STAT3 mutations that, in addition to reported mutant STAT3 immunologic abnormalities, produce pulmonary infection. Methods:STAT3-HIES sputum was evaluated for biochemical/biophysical properties. STAT3-HIES excised lungs were harvested for histology; bronchial brush samples were collected for RNA sequencing and in vitro culture. A STAT3-HIES-specific mutation (R382W), expressed by lentiviruses, and a STAT3 knockout, generated by CRISPR/Cas9, were maintained in normal human bronchial epithelia under basal or inflammatory (IL1β) conditions. Effects of STAT3 deficiency on transcriptomics, and epithelial ion channel, secretory, antimicrobial, and ciliary functions were assessed. Measurements and Main Results:Mucus concentrations and viscoelasticity were increased in STAT3-HIES sputum. STAT3-HIES excised lungs exhibited mucus obstruction and elevated IL1β expression. STAT3 deficiency impaired CFTR-dependent fluid and mucin secretion, inhibited expression of antimicrobial peptides, cytokines, and chemokines, and acidified airway surface liquid at baseline and post-IL1β exposure in vitro. Notably, mutant STAT3 suppressed IL1R1 expression. STAT3 mutations also inhibited ciliogenesis in vivo and impaired mucociliary transport in vitro, a process mediated via HES6 suppression. Administration of a γ-secretase inhibitor increased HES6 expression and improved ciliogenesis in STAT3 R382W mutant cells. Conclusions:STAT3 dysfunction leads to multi-component defects in airway epithelial innate defense, which, in conjunction with STAT3-HIES immune deficiency, contributes to chronic pulmonary infection.

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O 2 , airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na + and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of β and γENaC (epithelial Na + channel) subunit expression. The combination of increased Na + absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA–in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

Background: CF gene therapies that seek to provide functional CFTR to human airway epithelial cells are a promising approach to improving lung function.Targeting human airway epithelial cells with aerosolized particles is an attractive strategy for CF gene therapy because inhaled particles can be easily delivered to airway cells in a highly targeted manner.One of the challenges associated with aerosolized particle delivery is that gene therapy vectors must penetrate protective barriers that prevent particles and microbes in the airway from entering the body.One component of these barriers is made up of highly glycosylated, cell membrane-spanning mucin proteins.These long (up to 3 μm) mucins overlap to form a dense brushwork at the cell surface that prevents particles from freely accessing the cell membrane.Methods: The goal of this study was to determine if transiently cleaving airway cell-surface mucins improves gene therapy transfection efficiency.Although nonspecific proteinases (e.g., trypsin) can cleave mucins, they also cleave most other proteins, potentially causing damaging off-target effects.Therefore, we studied the use of StcE, a quasi-mucin-selective protease from enterohemorrhagic Escherichia coli and investigated its effects on cell surface mucins expressed on polarized air-liquid interface cultures from human airway bronchial epithelial cells.Initial studies focused on assessing the effect of StcE on cell-surface mucins.We then studied the effect of StcE treatment on transfection efficiency.After treatment with StcE, cells were washed and treated with lipid nanoparticles containing mCherry mRNA or AAV2-GFP viral vectors.Comparable cultures treated with a vehicle ( phosphate-buffered saline) served as a control.After the transfection, fluorescence microscopy was used to assess the percentage of reporter-positive cells.Results: Treatment of airway cell cultures with StcE decreased the abundance of cell-surface mucins, as evidenced by a decrease in fluorescently labeled mucins in a time-dependent manner.Studies were also used to measure turnover of tethered mucins, which is approximately 24 to 48 hours after cleavage.To demonstrate a change in the barrier properties of the tethered mucins, we show that StcE treatment increased the permeability of the cell surface glycocalyx, as measured by penetration of fluorescently labeled dextrans of various sizes.Using two independent gene therapy vectors, adeno-associated virus-2 and lipid nanoparticles, both expressing a fluorophore transgene, we demonstrate that StcE treatment results in greater transfection efficiency than vehicle-treated cultures.Conclusions: We hypothesize that the physical barrier function of airway cell-surface mucins is a major challenge for the delivery of viral and nonviral vectors for gene therapy in CF.Our studies demonstrate that treatment of human air-liquid interface cell cultures with a mucin-specific protease can increase the transfection efficiency of gene therapy delivery vectors.Given the rapid turnover of tethered mucins after StcE-mediated removal, this approach may increase transfection efficiency while only transiently increasing permeability of airway cell-surface mucin barriers.

ABSTRACTAirway mucociliary clearance (MCC) is required for host defense and often diminished in chronic lung diseases. Effective clearance depends upon coordinated actions of the airway epithelium and a mobile mucus layer. Dysregulation of the primary secreted airway mucin proteins, MUC5B and MUC5AC, is associated with a reduction in the rate of MCC; however, how other secreted proteins impact the integrity of the mucus layer and MCC remains unclear. We previously identified the geneBpifb1/Lplunc1as a regulator of airway MUC5B levels using genetic approaches. Here, we show that BPIFB1 is required for normal mucociliary clearancein vivousingBpifb1knockout (KO) mice. Reduced MCC inBpifb1KO mice occurred in the absence of defects in sodium or chloride ion transport or reduced ciliary beat frequency. BPIFB1 loss resulted in airway mucus flakes with significantly increased complex viscosity, a key biophysical property of mucus known to impact MCC. Finally, we detected colocalization of BPIFB1 and MUC5B in secretory granules in mice and in the protein mesh of secreted mucus in human airway cultures. Collectively, our findings demonstrate that BPIFB1 is an important component of the mucociliary apparatus in mice and a key component of the mucus protein network.

The convergent positioning of functional groups in biomacromolecules leads to good binding, catalytic and transport capabilities. Synthetic frameworks capable of convergently locking functional groups with minimized conformational uncertainty-leading to similar properties-are highly desirable but rare. Here we report C5-symmetric aromatic pentaamide macrocycles synthesized in one pot from the corresponding monomers. Their crystal structures reveal a star-shaped, fully constrained backbone that causes ten alternating NH/CH hydrogen-bond donors and five large amide dipoles to orient towards the centre of the macrocycle. With a highly electropositive cavity in a high-energy unbound state, the macrocycles bind anions in a 1:1 stoichiometry in solution, with high affinity for halides and very high affinity for oxoanions. We demonstrate that such macrocycles are able to transport anions across lipid bilayers with a high chloride selectivity and restore the depleted airway surface liquid of cystic fibrosis airway cell cultures.

In the lungs, defective CFTR associated with cystic fibrosis (CF) represents the nidus for abnormal mucus clearance in the airways and consequently a progressive lung disease. Defective CFTR-mediated Cl- secretion results in altered mucus properties, including concentration, viscoelasticity, and the ratio of the two mucins, MUC5B and MUC5AC. In the past decades, therapies targeting the CF mucus defect, directly or indirectly, have been developed; nevertheless, better treatments to prevent the disease progression are still needed. This review summarizes the existing knowledge on the defective mucus in CF disease and highlights it as a barrier to the development of future inhaled genetic therapies. The use of new mucus-targeting treatments is also discussed, focusing on their potential role to halt the progress of CF lung disease.

As the nasal cavity is the portal of entry for inspired air in mammals, this region is exposed to the highest concentration of inhaled particulate matter and pathogens, which must be removed to keep the lower airways sterile. Thus, one might expect vigorous removal of these substances via mucociliary clearance (MCC) in this region. We have investigated the rate of MCC in the murine nasal cavity compared to the more distal airways (trachea). The rate of MCC in the nasal cavity (posterior nasopharynx, PNP) was ∼3–4× greater than on the tracheal wall. This appeared to be due to a more abundant population of ciliated cells in the nasal cavity (∼80%) compared to the more sparsely ciliated trachea (∼40%). Interestingly, the tracheal ventral wall exhibited a significantly lower rate of MCC than the tracheal posterior membrane. The trachealis muscle underlying the ciliated epithelium on the posterior membrane appeared to control the surface architecture and likely in part the rate of MCC in this tracheal region. In one of our mouse models (Bpifb1 KO) exhibiting a 3-fold increase in MUC5B protein in lavage fluid, MCC particle transport on the tracheal walls was severely compromised, yet normal MCC occurred on the tracheal posterior membrane. While a blanket of mucus covered the surface of both the PNP and trachea, this mucus appeared to be transported as a blanket by MCC only in the PNP. In contrast, particles appeared to be transported as discrete patches or streams of mucus in the trachea. In addition, particle transport in the PNP was fairly linear, in contrast transport of particles in the trachea often followed a more non-linear route. The thick, viscoelastic mucus blanket that covered the PNP, which exhibited ∼10-fold greater mass of mucus than did the blanket covering the surface of the trachea, could be transported over large areas completely devoid of cells (made by a breach in the epithelial layer). In contrast, particles could not be transported over even a small epithelial breach in the trachea. The thick mucus blanket in the PNP likely aids in particle transport over the non-ciliated olfactory cells in the nasal cavity and likely contributes to humidification and more efficient particle trapping in this upper airway region.

Background: Elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) modulator therapy for people with cystic fibrosis (PwCF) carrying at least one copy of the F508del mutation of the CF transmembrane conductance regulator (CFTR) protein has reduced clinical exacerbations and increased forced expiratory volume in 1 second and general wellbeing.ELX/TEZ/IVA rescues the misfolded CFTR protein and traffics it to the surface membrane of airway cells, potentially restoring normal airway physiology such as airway secretions, pH balance, and cilia movement, but it is unknown whether ELX/TEZ/IVA will also help reduce pathogenic infections and prevalence in PwCF.One such deadly pathogen is Burkholderia cenocepacia, which can cause variable lung infections, ranging from asymptomatic to chronic, or 'cepacia syndrome,' characterized by a rapid decline in lung function.Because PwCF carrying B. cenocepacia are not included in ELX/TEZ/IVA clinical trials, we proposed using in vitro primary CF human bronchial epithelial (CF-HBE) 3D cultures to investigate the effects of ELX/TEZ/IVA on B. cenocepacia infections.Methods: Fully differentiated primary CF-HBE cells (3-4 donors) at the airliquid interface (ALI) were treated with ELX/TEZ/IVA before B. cenocepacia (lab strain K56-2 labeled with DsRed; MOI 0.1-1) infection on the apical surface.Response of CF-HBE cultures to ELX/TEZ/IVA was confirmed by measuring CFTR protein expression and CFTR ion channel function.Airway physiology, including ciliary beat frequency, pH of the airway-surface liquid (ASL), and expression of antimicrobial proteins such as SPLUNC1 (activity against gram-negative bacteria) were recorded after ELX/TEZ/IVA treatment.B. cenocepacia growth was measured using immunofluorescence in non-CF HBE (NHBE) and CF-HBE ± ELX/TEZ/IVA-treated cultures.Results: ELX/TEZ/IVA treatment in CF-HBE cultures significantly restored CFTR protein expression at the apical membrane and CFTR ion channel function.Downstream physiology, including ciliary beat frequency and pH of the ASL, was significantly restored to levels observed in NHBE cultures.SPLUNC1 antimicrobial expression was lower in the ASL secretions of CF-HBE than in those of NHBE cultures but increased significantly after ELX/ TEZ/IVA treatment.Furthermore, B. cenocepacia (K56-2) infections on CF-HBE cultures were significantly reduced with pre-treatment with ELX/TEZ/ IVA, despite greater burden in CF-HBE than NHBE cultures (Figure 1).B. cenocepacia infection also appeared to interfere with ELX/TEZ/IVA efficacy by reducing CFTR expression and function compared to non-infected CF-HBE+ELX/TEZ/IVA cultures, although not to the extent of non-ELX/TEZ/IVA treated CF-HBE cultures.Conclusions: The risk of B. cenocepacia infections in PwCF may be reduced with modulator therapy such as ELX/TEZ/IVA, although B. cenocepacia may also interfere with ELX/TEZ/IVA efficacy.Whether by ELX/TEZ/IVA treatment reduces B. cenocepacia infection in PwCF already colonized with B. cenocepacia remains to be determined.Starting ELX/TEZ/IVA treatment could be beneficial to PwCF to prevent infections with pathogens such as B. cenocepacia.

The mucus clearance system is the dominant mechanical host defense system of the human lung. Mucus is cleared from the lung by cilia and airflow, including both two-phase gas-liquid pumping and cough-dependent mechanisms, and mucus transport rates are heavily dependent on mucus concentration. Importantly, mucus transport rates are accurately predicted by the gel-on-brush model of the mucociliary apparatus from the relative osmotic moduli of the mucus and periciliary-glycocalyceal (PCL-G) layers. The fluid available to hydrate mucus is generated by transepithelial fluid transport. Feedback interactions between mucus concentrations and cilia beating, via purinergic signaling, coordinate Na+ absorptive vs Cl- secretory rates to maintain mucus hydration in health. In disease, mucus becomes hyperconcentrated (dehydrated). Multiple mechanisms derange the ion transport pathways that normally hydrate mucus in muco-obstructive lung diseases, e.g., cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), non-CF bronchiectasis (NCFB), and primary ciliary dyskinesia (PCD). A key step in muco-obstructive disease pathogenesis is the osmotic compression of the mucus layer onto the airway surface with the formation of adherent mucus plaques and plugs, particularly in distal airways. Mucus plaques create locally hypoxic conditions and produce airflow obstruction, inflammation, infection, and, ultimately, airway wall damage. Therapies to clear adherent mucus with hydrating and mucolytic agents are rational, and strategies to develop these agents are reviewed.

Vestibulodynia (VBD), an idiopathic pain disorder characterized by erythema and pain of the vulvar vestibule (the inner aspect of the labia minora and vaginal opening), is the most common cause of sexual pain for women of reproductive age. Women also feel discomfort with contact with clothing and tampon use. As most women with this disorder only have pain with provocation of the tissue, topical anesthetics applied to the vestibule are the current first line treatment for temporary pain relief. Treatment options are limited due to anatomical constraints of the vestibular region, poor drug retention time, imprecise dosing, leakage, and overall product messiness. In this study we report a novel approach to treatment of VBD using thin film designed to fit the vulvar vestibule and deliver lidocaine locally. Two use cases for VBD treatment were identified 1) rapid drug release (<5 min), for use prior to intercourse and 2) long-acting release (>= 120 min) for prolonged use and relief throughout the day. Cellulose-based mucoadhesive thin films were fabricated using a solvent casting method. Three polymers including hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), and hydroxypropylmethycellulose (HMPC), were selected owing to their biocompatibility and ideal properties for film casting. Films casted with HEC, HPC, and HPMC exhibited mucoadhesive properties relative to a control, with the highest mucoadhesive force recorded for films casted with HPC. Effect of media volume, pH, presence of mucin and presence of drug on film dissolution rates were investigated. Dissolution rates were independent of media volume, media pH or drug presence, whereas faster dissolution rates were obtained for all films in presence of mucin. In vitro lidocaine release kinetics were influenced by polymer type, percent drug loading and film casting thickness. Lidocaine release was based on a diffusion mechanism rather than through film dissolution and faster release (similar to 5 min) was observed for HEC films compared HPC films (similar to 120 min). Higher drug loading and film thickness resulted in slower and more prolonged release kinetics of lidocaine. All films were biocompatible and exhibited good mechanical properties. Two film formulations (9% w/w HPC with 12% w/w LHC, 5% w/w HEC with 6% w/w LHC) were optimized to meet the two use case scenarios for VBD treatment and moved into in vivo testing. In vivo testing demonstrated the safety of the films in BALB/c mice, and the pharmacokinetic analysis demonstrated the delivery of lidocaine primarily to the vaginal tissue. We demonstrate the ability to develop a mucoadhesive, biodissolvable thin film and fine-tune drug release kinetics to optimize local delivery of lidocaine to the vulva.