Glutathione and Bicarbonate Nanoparticles Improve Mucociliary Transport in Cystic Fibrosis Epithelia

Abstract

Introduction:

Cystic Fibrosis (CF) airway disease is characterized by thick mucus and impaired mucociliary transport (MCT). Loss of functional cystic fibrosis transmembrane receptor (CFTR) leads to acidification and oxidation of airway surface mucus. Replacing bicarbonate (HCO₃⁻) topically fails due to rapid reabsorption and neutralization, while the scavenging antioxidant, glutathione (GSH), is also rapidly degraded. The objective of this study is to investigate glutathione/NaHCO₃ nanoparticles as novel strategy for CF airway disease.

Methods:

GSH/NaHCO₃ poly (lactic-co-glycolic acid) nanoparticles were tested on primary CF (F508del/F508del) epithelial cultures to evaluate dose-release curves, surface pH, toxicity, and MCT indices using micro-optical coherence tomography. In vivo tests were performed in three rabbits to assess safety and toxicity. After 1 week of daily injections, histopathology, computed tomography (CT), and blood chemistries were performed and compared to three controls. Fluorescent nanoparticles were injected into a rabbit with maxillary sinusitis and explants visualized with confocal microscopy.

Results:

Sustained release of GSH and HCO₃⁻ with no cellular toxicity was observed over 2 weeks. Apical surface pH gradually increased from 6.54±0.13 (baseline) to 7.07±0.10 (24 hours) (p<0.001) and 6.87 +/−0.05 at 14 days (p<0.001). MCT, ciliary beat frequency, and periciliary liquid were significantly increased. When injected into the maxillary sinuses of rabbits, there were no changes to histology, CT, or blood chemistries. Nanoparticles penetrated rabbit sinusitis mucus on confocal microscopy.

Conclusion:

Findings suggest that GSH/NaHCO₃⁻ nanoparticles are a promising treatment option for viscous mucus in CF and other respiratory diseases of mucus obstruction such as chronic rhinosinusitis.

Introduction

Sinonasal mucociliary transport (MCT) is an airway defense mechanism crucial to maintaining a healthy respiratory system. MCT depends on the presence of intact respiratory epithelium with properly functioning cilia, as well as the biological properties of the airway surface liquid (ASL). The ASL is a thin layer of liquid lining the respiratory tract which is dependent on the coordinated action of epithelial ion transporters [i.e. epithelial sodium channels (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR)] to maintain airway pH balance, allow mucous clearance, and defend against pathogens.^1–3 In cystic fibrosis (CF) airway disease, the ASL is desiccated due to impaired flow of chloride (Cl⁻) ions through CFTR, but this is also associated with a decrease in bicarbonate (HCO₃⁻) secretion. This contributes to distinct issues with pH and mucus viscoelasticity.⁴ As a result, chronic stasis of mucus allows for bacterial infection resulting in chronic bronchitis and chronic rhinosinusitis (CRS) in the CF population.⁵

The development of CFTR modulator therapies (i.e. ivacaftor, tezacaftor etc.) has led to great improvement in clinical parameters of CF disease, including the sinuses, but has incompletely eliminated exacerbations or pulmonary decline.^6–11 While CFTR modulator therapy represents a partial solution for CF patients, novel strategies are needed to address residual disease and non-responders. The densely adherent mucus in the airways of CF patients underscores the crucial importance of addressing hyperviscous mucus with novel strategies.

In the setting of dysfunctional CFTR, decreased HCO₃⁻ secretion leads to the release of unexpanded, thick gel-forming mucins. Recent understanding regarding secretion from mucin granules is that O-glycosylated mucin multimers are packaged into secretory granules via the noncovalent, Ca²⁺-dependent interactions between N-terminal protein domains. The reversible connections between mucins in organized polymers are most active at the lower pH of the secretory granule, and form a “flower-like” structure (looped strands emanating from central nodes) observed by electron microscopy.^12,13 Recent data from CF newborn piglet airways prior to the onset of airway inflammation and infection suggest that extrusion of mucus from goblet cells and glands requires an environment rich in HCO₃⁻ to remove and displace Ca²⁺, which is ultimately replaced by an adequate supply of monovalent Na⁺ ions in the setting of an adequately hydrated epithelium. Mucins are unable to undergo normal posttranslational processing and expansion to their linear form in the acidified CF airway, and instead remain compact, contributing to mucus that is hyperviscous, cohesive, and adhesive to the surface epithelium.^14–17 As a whole, previous data demonstrates that mucins exist in a pathological state when secreted into the CF microenvironment, and this may be addressed through electrostatic correction.^13,18,19 Unfortunately topical delivery of HCO₃⁻ via irrigation or nebulization has been shown to be ineffective at therapeutically alkalizing the ASL in human patients due to rapid reabsorption and neutralization by carbonic anhydrase leading to a transient effect.²⁰

The highly oxidative environment of CF airways also contributes to viscous mucus. Previous studies have demonstrated that sputum collected from human CF airways exhibited a marked increase in elasticity, indicative of excessive cross-linking of mucin polymers.²¹ In the setting of CF, the high levels of reactive oxygen species in CF sputum produced by neutrophilic inflammation provide an oxidative environment for increasing disulfide (S-S) bond formation by converting cysteines to cystines. Indeed, tactics to utilize topical inhaled drugs to reduce S-S crosslinking have been introduced previously – in particular, the redox agent and free radical scavenger, N-acetyl cysteine, has been used to remove mucus plugs in CF and other respiratory diseases of mucus obstruction.^22,23 However, the long-term results with aerosolized formulations were disappointing due to the rapid absorption, high concentrations (as high as 1.27 M) necessary due to inherent instability of reducing potential, and side effect of bronchospasm.²⁴ This pathway has also been previously targeted by the use of glutathione sulfhydryl (GSH) (a free radical scavenger that cleaves S-S crosslinking) in clinical trials although with mixed results. Nebulized GSH was evaluated as therapy for CF lung disease, and despite early promise, randomized clinical trials demonstrated limited efficacy. ^25,26,22 GSH in biological fluids (e.g., plasma) is a very unstable tripeptide (half-life of ~20 min) with high degradation rate through bacterial and epithelial gamma-glutamyl transpeptidase catalysis.^27,28 Furthermore, these studies failed to first address the underlying electrostatic abnormalities in CF mucus from low HCO₃⁻ concentration, and in fact delivered an acidic fluid that may have exacerbated mucin unfolding, negating benefit.

The CF airway provides an environment where mucins incapable of unfolding due to an acidic airway surface are subjected to a highly oxidative environment. While improving HCO₃⁻ in the airway should release unfolded mucins, formation of S-S bonds in a tightly wound configuration may irreversibly prevent unfolding (Figure 1). Thus, a therapeutic strategy that delivers HCO₃⁻ and a redox agent, like GSH, to the airway and sinuses is critical to reverse both abnormalities. Indeed, GSH and HCO₃⁻ synergistically improved MCT during a previous investigation of a novel nebulized treatment of ascorbic acid, HCO₃⁻, and GSH (ARINA-1) in F508del/F508del primary human bronchial epithelial cells.²⁹ However, the observed effect waned past 6 hours, suggesting the need for very frequent dosing to achieve clinical effect.²⁹ HCO₃⁻ alone did not have the same effect and GSH alone had significant cellular toxicity from its inherently low pH of 3.0. Given the short half-life and transient effect of topical use, drug delivery remains a barrier to clinical implementation. This led us to create a novel strategy incorporating GSH/NaHCO₃ into nanoparticles for the treatment of mucus obstruction in CF. The purpose of this therapeutic approach is to provide sustained topical delivery of GSH/NaHCO₃ for prolonged epithelial surface contact to maintain alkalization of the ASL, while releasing GSH to reduce S-S crosslinking.

Figure 1. Two-step model of pathologic mucus formation.

Tightly packaged, gel-forming mucins electrostatically bound to divalent Ca²⁺ in globular form do not expand to their normal conformation upon secretion (required to maintain normal viscoelasticity and host defense properties). Generation of reactive oxygen species (ROS) causes subsequent irreversible covalent modifications of mucins (disulfide crosslinking of cysteines to cystines).

In this study, we investigate a novel approach using topical GSH/NaHCO₃ nanoparticles (NP) to address mucus obstruction in CF related CRS. We hypothesized that GSH/NaHCO₃ nanoparticles would allow sustained and synergistic improvement in MCT when tested on primary CF (F508del/F508del) bronchial epithelial cultures.

Materials and Methods

Preparation of GSH and NaHCO₃ poly(lactic-co-glycolic acid) (PLGA) nanoparticles

GSH-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles were synthesized utilizing an emulsion/solvent technique. Initially, 50 mg of GSH was dissolved in 400 μL of deionized water, followed by combination with a 2.5% (w/v) PLGA solution in dichloromethane (DCM). This amalgam was subjected to ultrasonic agitation within an ice-water bath to establish a homogeneous water-in-oil emulsion (w/o). For each batch, 500 μL of the resultant w/o emulsion was promptly introduced into 25 mL of a 1% (w/v) polyvinyl alcohol solution (PVA, hydrolyzed to 98.0–98.8%, Mw = 31,000–50,000) and vigorously stirred for 2 minutes utilizing a homogenizer. The suspended nanoparticles were subsequently exposed to a stir plate for overnight organic solvent evaporation in a chemical fume hood. The nanoparticles were harvested through centrifugation at 4000 rpm for 20 minutes at 4°C, followed by resuspension in 10 mL of distilled water, and the elimination of significant particulates and aggregates using a 40-μm cell strainer. Nanoparticles containing sodium bicarbonate (NaHCO₃) were also generated as follows: A solution of NaHCO₃ (400 μL, 70 mg/mL) was formulated in a 2.5% (w/v) PLGA solution in dichloromethane (DCM). After sonicating the NaHCO₃ solution, similar protocols as above were applied to manufacture NaHCO₃ loaded PLGA nanoparticles.

Morphology of fabricated PLGA nanoparticles

To assess the morphology of the produced PLGA nanoparticles, a field emission scanning electron microscope (SEM; QuantaTM FEG-650, ThermoFisher, Waltham, MA) was employed. Prior to scanning, a coating of Gold -Palladium was applied to the PLGA nanoparticles to improve surface conductivity and diminish charging artifacts. An accelerating voltage of 20 kV was utilized, and SEM images were processed within the SEM Laboratory at the University of Alabama at Birmingham.

Release assay

In order to assess in vitro release, 2 mg GSH nanoparticles and 20 mg NaHCO₃ nanoparticles were added to 1 mL deionized water. Cumulative release concentrations were measured at days 1–14 using Abcam Glutathione Fluorometric assay kit and Abcam Colorimetric Sodium assay kit. A 1:10 ratio of GSH:NaHCO₃was chosen to buffer the acidic pH of GSH while still achieving physiologic concentrations of GSH (reported at 1 to 10 mM intracellular and 1 to 6 μM in plasma).²⁷

Cell culture

Primary CF human bronchial epithelial (HBE) cell cultures were obtained from a single CF donor (F508del/F508del) under approval from the University of Alabama at Birmingham Institutional Review Board. Cells were cultured according to a previously described protocol.¹⁸ Wells were then treated with 1:10 ratio by mass of GSH:NaHCO₃ PLGA nanoparticles in phosphate buffered saline (PBS) to achieve in vitro concentrations of 0.5 mM GSH and 12 mM NaHCO₃ based on our release assay. These lower in vitro concentrations were chosen as to not overwhelm the cell culture surface with polymer. The negative control wells were treated with the same amount of bare PLGA nanoparticles in PBS. Micro optical coherence tomography (μOCT) (n=6), pH analysis (n=6), and lactate dehydrogenase (LDH) toxicity assay (n=3) were conducted at time points of baseline, 6 hours, 1 day, 2 days, 4 days, 7 days, 11 days, 14 days after nanoparticle application. Baseline (0h) data was collected prior to treatment initiation.

Epithelial surface pH and toxicity

The pH of the ASL layer was measured directly using the Seven Excellence pH meter (Mettler, Toledo). Medium was collected at each time point and LDH was measured as an indicator of cellular toxicity using CyQUANT^™ LDH Cytotoxicity Assay Kit (Cat#C20301, Invitrogen) as previously described.^30–32

μOCT

Micro optical coherence tomography (μOCT) is a technology that permits real-time evaluation of the MCT apparatus of airway cells in vitro, ex vivo, and in vivo.^33–38 Using this technique, we previously reported on the first in vivo imaging of the nasal airways in human subjects with CF, ³³ which showed delayed MCT, depletion of the periciliary liquid (PCL), and prevalent loss of ciliation. The μOCT imaging of HBE cultures was performed as previously described^18,38 to obtain cross sectional video rate imaging files which were analyzed to collect data on ASL, PCL, ciliary beat frequency (CBF) and MCT. A humidification chamber with conditions (37°C, 5% CO₂) was used throughout the experiments. The imaging optics axis was placed within 10 degrees of normal to the cell plane to decrease imprecisions in geometric measurements.³⁸ Three regions of interest were measured (one near the center of the well and 2 mm on either side) and averaged for a single value per well. ASL and PCL were determined by directly measuring the visible thickness of the respective layers in the image. CBF was evaluated using a time series of images and quantitatively measured by identifying peak amplitude frequency in the temporal Fourier transform of areas demonstrating oscillatory behavior. MCT rate was calculated by measuring the distance traveled of native mucus particles over multiple frames. MATLAB code (available by request) was used to derive quantitative parameters from μOCT images using ImageJ Software.^18,29

Animal safety and toxicity

Safety and toxicity testing was conducted on New Zealand white rabbits (n=3 experimental and 3 control) under approval of the institutional animal care and use committee (IACUC) at the University of Alabama at Birmingham. A mixture of 2mg GSH PLGA and 20 mg NaHCO₃ PLGA in 1 mL PBS was directly introduced into the left maxillary sinus using a 25-gauge needle daily for 7 days to achieve cumulative concentrations of 1 mM GSH and 20 mM NaHCO₃. All rabbits underwent micro computed tomography (micro CT) of the sinuses and lungs on day 7. Micro CT scanning was performed at the University of Alabama Birmingham small animal imaging shared facility using SPECT/CT (X-SPECT system, Gamma Medica, Northridge, CA). Images were reviewed by two independent clinicians for any evidence of sinus opacification, mucosal thickening, bronchial thickening, pneumonia, or interstitial pulmonary opacification. After scanning on day 7, blood samples were collected and sent for complete blood count (CBC) and complete metabolic panel (CMP) (Antech Superchem w/SDMA|Complete Blood Count) and rabbits were euthanized. Sinuses were harvested and ten representative sections of the maxillary sinus were selected and stained with hematoxylin & eosin (H&E). Slides were evaluated for inflammatory characteristics and examination of epithelial and subepithelial thickness.

Nanoparticle penetration of sinus mucus

The mucus layer of rabbit sinus explants from one rabbit with induced maxillary sinusitis using our previously described model^39,40 was stained with green fluorophores as previously described⁴¹ and nanoparticles coated with iron oxide for staining were injected at a concentration of 4mg/ml. Confocal imaging was obtained starting 15 minutes after injection and a time lapse 5 sec per frame.

Statistical analysis

Statistical analyses were performed using Graph Pad- Prism with 2-tailed, unpaired t tests for pH, toxicity and μOCT studies of HBE cultures. Paired t-tests were used to analyze lab values and weight in the animal studies. All values are reported as the mean ± standard deviation. A p value < 0.05 was considered statistically significant.

Results

Nanoparticles provided sustained release of GSH and NaHCO₃ over 14 days

Scanning electron microscopy images of the morphology of GSH and NaHCO₃ loaded PLGA are displayed in Figure 2A&B. Sustained cumulative release of GSH and NaHCO₃ was demonstrated over a 14-day period (Figure 2C) when nanoparticles were placed into 1 mL deionized water. When 2 mg of GSH NP were placed in 1 mL water, GSH cumulative concentrations reached 1.13 +/− 34 mM at day 14. Likewise, placement of 20 mg of NaHCO₃⁻ NP in 1 mL water eluted cumulative concentrations of 20.6 +/− 5.6 mM at day 14.

Figure 2. Morphology of GSH/NaHCO₃ nanoparticles and releasing concentrations in vitro.

Scanning electron microscopic images of A) GSH and B) NaHCO₃ poly lactic-co-glycolic acid nanoparticles. C) In vitro release profiles of 2 mg GSH and 20 mg NaHCO3 nanoparticles in 1 mL of deionized water were robust and sustained up to 14 days.

The apical surface pH had sustained improvement over 14 days with no cellular toxicity in vitro

A statistically significant increase in pH was observed at 6 hours and sustained until 14 days as compared to baseline (Figure 3A). The pH of the HBE apical surface peaked from baseline (6.54+/−0.13) to 7.29 +/− 0.22 at 6 hours of treatment and remained significantly elevated from baseline at 24 hours (7.07+/−0.10, p<0.001) until 14 days (6.87 +/−0.05, p<0.001). There was no significant increase in LDH levels in the cell culture medium from treatment day 1–14 when compared to negative controls (Figure 3B).

Figure 3. Apical surface pH changes and toxicity measurements of GSH/NaHCO₃ nanoparticles applied to CF human bronchial epithelial cells for 14 days.

A) The pH of the epithelial surface was measured directly via pH probe and significantly increased from 6h to 14 days after nanoparticle application when compared to baseline (0h). B) Cell medium was collected at days 1–14 of treatment with GSH/NaHCO₃ nanoparticles and lactate dehydrogenase (LDH) was measured as a surrogate for cellular toxicity. (LDH) levels were unchanged when compared to negative control wells. Positive control was measured after killing cells with cell lysis buffer.

Nanoparticles improve MCT, CBF, and PCL in CF airway cultures

All parameters of mucociliary function measured by μOCT (CBF, MCT, PCL) were significantly improved over negative controls except for depth of ASL which remained unchanged (Figure 4). In vitro treatment with GSH/NaHCO₃ nanoparticles significantly improved CBF from day 1–14 when compared to negative controls (Day 1: 7.48 +/− 0.31 Hz vs 7.04+/−0.27 Hz p<0.05, Day 14: 9.15 +/− 0.69 Hz vs 7.93 +/− 0.98 Hz p<0.05). MCT showed significant improvement from day 2–14 of treatment when compared to negative control (Day 2: 0.092 +/− 0.008 mm/min vs 0.066 +/−0.019 mm/min p<0.01, Day 14: 0.092 +/− 0.014 vs 0.061 +/− 0.008 mm/min p<0.001). Mean PCL depth was increased at days 1–14 of treatment (Day 1: 4.90 +/− 0.41 μm vs 4.09 +/−0.50 μm p<0.01, Day 14: 7.46 +/− 0.24 μm vs 6.63 +/− 0.25 μm p<0.001), although day 4 was not statistically significant (Day 4: 5.82 +/− 0.37 μm vs 5.21 +/−0.59 p=0.057). There was no appreciable change in the depth of ASL from days 1–14 with application of GSH/NaHCO₃⁻ nanoparticles.

Figure 4. The effect of GSH/NaHCO₃ nanoparticles on μOCT parameters of mucociliary function.

Representative μOCT images at 7 days of GSH/NaHCO₃ nanoparticle treated well and a control nanoparticle treated well (yellow line – airway surface liquid (ASL), red line – periciliary liquid (PCL)). A) PCL depth, B) ciliary beat frequency (CBF), and C) mucociliary transport (MCT) were significantly increased over negative controls after application of GSH/NaHCO₃ nanoparticles. D) Airway surface liquid (ASL) depth did not change. Negative control cells were treated with bare PLGA nanoparticles.

Nanoparticles are non-toxic, safe, and penetrate sinusitis mucus in vivo

Pre- and post-treatment bloodwork (CMP and CBC) from 3 rabbits undergoing 7 days of topical nanoparticle application to the maxillary sinus revealed no significant change in weight, total protein, albumin, AST, ALT, bilirubin, electrolytes, creatinine, BUN, hemoglobin, white blood cell count or differential (data not shown). Micro-CT imaging of the sinuses and lungs showed no evidence of sinus opacification, mucosal thickening, pneumonia, or inflammatory lung disease (Figure 5A&B). Histologic examination of the maxillary sinus mucosa revealed no evidence of inflammatory cell infiltrate, or mucosal thickening. Finally, nanoparticles injected into the infected maxillary sinus of a rabbit revealed intermingling and penetration of the nanoparticles within the thick mucus layer on confocal imaging 15 minutes after administration (Figure 5C & Supplementary Video 1).

Figure 5. Histology, micro CT scans, and penetration of sinusitis mucus.

In a preclinical safety study, the GSH/NaHCO₃ nanoparticle formulation was injected into the left maxillary sinus of 3 normal rabbits daily for 7 days. In comparison to non-injected negative controls (A), there were no demonstrable changes to the architecture of the epithelium (no inflammation, cilia damage, submucosal gland or goblet cell hypertrophy) in the sinuses, sinus opacification or lung damage on CT scans in the treatment group (B), or lab abnormalities (not shown). The mucus layer of rabbit sinusitis was stained with green fluorophores as previously described and nanoparticles coated with iron oxide (red) were injected at a concentration of 4mg/ml (C, Supplementary Video 1). Confocal imaging of explanted maxillary sinus mucosa at 15 minutes after injection shows complete penetration of the red nanoparticles with a propagating mucus raft (arrow – direction of moving raft, time lapse 1 sec per frame).

Discussion

Targeting both mucin polymer electrostatic and oxidative modifications using an extended-release nanoparticle formulation is a highly innovative therapeutic approach for CF that has not been investigated previously. Our study is the first of its kind showing GSH/NaHCO₃ in a nanoparticle formulation for use as a topical mucolytic. We demonstrated that topical delivery of GSH/NaHCO₃ in nanoparticle form overcomes the limitations of the short half-life of HCO₃⁻_, as well as the rapid degradation and direct cellular toxicity of GSH. We were able to achieve physiologic range concentrations of GSH (reported at 1 to 10 mM intracellular and 1 to 6 μM in plasma)²⁷ and significantly alkalinized the epithelial surface over a 2 week period without cellular toxicity. The increased pH also indicates that the acidity of GSH is overwhelmed by the release of HCO₃⁻. By addressing both the electrostatic and oxidative derangements of the mucins in the CF ASL, GSH/NaHCO₃ nanoparticles conferred synergistic and sustained improvement in MCT, PCL, and CBF.

Traditionally diseased ciliated epithelium has been reported to have decreased ASL and PCL depth as an indication of dehydration.^42–47 Given that the ASL is comprised of the PCL and mucous layers, this relationship is not as simple as direct correlation. Liu et al. showed dynamic increase of CBF in response to decreased PCL, until a point where the cilia were overwhelmed by an excessive mucus load.⁴⁸ Fernandez-Petty et al. demonstrated an increase in MCT rate in cells treated with an electrostatic glycopolymer, despite no effect on ASL depth.¹⁸ Leung et al. performed in vivo μOCT in human CF subjects demonstrating diminished PCL, CBF, and MCT in subjects with CF, but did not find a significant difference in ASL depth.³³ They proposed that a healthy physiologic range (at lower ASL depths <50um) exists in which ASL correlates with cilia hydration. However, at a certain point ASL depth only becomes an indicator of the presence of diseased and excessively thick CF mucus layer (depths near 50 um). Given the relatively high ASL depth (20–40 μm) in our cell culture, we believe ASL to be more of a marker of mucus accumulation over the 14 days, and not as sensitive to the hydration status of the cilia layer as PCL.

Nanoparticles have shown promise as a vehicle to enhance medication delivery to the sinuses and are capable of penetrating human mucus in the setting of CRS.^49–51 In a pilot rabbit study (n=1), we demonstrated effective penetration of the PLGA nanoparticles into a thick, viscous, and adhesive mucus layer in an infected rabbit maxillary sinus. The physical delivery of nanoparticles that penetrate the mucus layer is critical proof of concept that nanoparticle delivery is a sound strategy in future efficacy studies using the rabbit model of CRS.

There are several limitations to this study. We conducted our study using bronchial CF (F508del/ F508del) epithelial cultures. We anticipate that these findings would be consistent across CF genotypes and translate to donor sinus epithelium. This data sets the stage for therapeutic testing in animal models of CF sinus disease (i.e., the rabbit model)^39,52–54 or human studies where topical application to the sinuses is readily possible. Developing this further in sinus models provides the added advantage that direct intranasal delivery may be plausible as a first test of efficacy, while evaluation of sinus mucus would provide mechanistic confirmation, providing a path towards evaluation in the lower respiratory tract in the future. Finally, we cannot attribute the results of the drug to only the mucolytic effects without further evaluation of how this treatment affects the structure and function of mucins.

Conclusion

The findings of this study demonstrate that using nanoparticles eliminates the drawbacks of the short half-life of HCO₃⁻ (rapid absorption) and GSH (rapid conversion to glutathione disulfide) confirming sustained release over 14 days at levels sufficient to show bioactivity. The apical surface pH and MCT were increased over 14 days in CF airway cells in vitro. Importantly, nanoparticles demonstrated no toxicity in vitro or in vivo and penetrated the thick mucus layer of an infected rabbit maxillary sinus. Future studies using the preclinical rabbit CRS model are planned.