Poster W20 - Amelioration of Chronic Airway Inflammation by Highly Mucopenetrative Polymer Nanoparticles

Introduction: Chronic inflammation of the airways is a common hallmark of several chronic lung diseases (e.g.- COPD, asthma), restricting lung function and reducing both exercise capacity and general quality of life. Besides the constant need for the administration of anti-inflammatory drugs, this persistent state of inflammation within the airway mucosa sets up what is now known as Cole’s “vicious cycle”. A key driver of the mucosal dysfunction and attendant structural damage herein is the trapping of inhaled pathogens, their toxins and other irritants within the highly viscous mucus over-produced by the diseased airway lining. This requires a separate administration of mucolytics and expectorants. In this work, we aim to formulate a dual-action, inhalable nanoparticle (NP) that can administer both mucolytics and anti-inflammatories. The NP consists of a dipalmitoylphosphatidylcholine (DPPC) lipid outer layer containing N-acetylcysteine (NAC; a well-characterized disulfide-reducing mucolytic), and a poly(lactic-co-glycolic) acid (PLGA) inner core containing the anti-inflammatory all-trans retinoic acid (ATRA). This is expected to effect bulk mucus thinning, local mucus thinning to prevent NP entrapment and clearance, and anti-inflammatory effects upon cellular uptake by the diseased airway epithelium.

Materials and

Methods: NPs were synthesized using acid-terminated PLGA (50:50) in an oil-in-water single-emulsion process and characterized via dynamic light scattering and SEM. ATRA was added to the oil phase directly during synthesis. Thin film hydration using DPPC was employed to form the outer layer; NAC was entrapped in this layer using a cyclical freeze-thaw method. Synthesized particles were characterized by FT-IR, thermogravimetric analysis and the Stewart assay to confirm coating and drug encapsulation as well as quantify lipid coating. NP cytocompatibility was assessed using the MTT assay against both A549 lung epithelial cells and MRC-5 lung fibroblasts. In vitro uptake (qualitative and quantitative) of fluorescently-tagged NPs was assessed in A549 cells, which were inflamed using IL-1β prior to treatment; non-inflamed controls were included. Release kinetics of both drugs were assessed in phosphate-buffered saline (37°C, pH 7.4), and quantified using UV-vis spectrophotometry. In addition, mucolytic properties were assessed using bulk rheology and mucus penetration studies. Anti-inflammatory effects were characterized by measurement of markers such as IL-6, IL-8 and reactive oxygen species. in inflamed A549 cells.

Results, Conclusions, and Discussions: DPPC-PLGA NPs of approx. 300 nm with a surface charge of 6-8 mV were synthesized successfully, with an ATRA loading of 5 ug/mg NPs and a NAC loading of 73 ug/mg NPs. Formulated NPs were found to be cytocompatible (>80% viability) over 24 h upto 2 mg/mL. DPPC-coatedcoumarin-6 (C6)-tagged NPs exhibited higher uptake by A549s regardless of inflammatory status, indicating enhanced uptake as a result of coating (Fig. 1A). These NAC-DPPC-PLGA NPs showed significantly higher penetration through a 1 mm mucus plug on a Transwell setup (Fig. 1B), demonstrating the efficacy of the encapsulated NAC. ATRA-encapsulating NPs were shown to be capable of reducing levels of IL-6 and IL-8 in A549 cells, indicating promise for the amelioration of epithelial inflammation.

In conclusion, stable and cytocompatible DPPC-PLGA NPs were synthesized with an average diameter of about 300 nm. NAC-containing NPs demonstrated greater mucopenetration than NPs without NAC. Dose-dependent increase in NP uptake was observed in A549 cells. Preliminary results confirmed decrease in IL-6 and IL-8 expressions in inflamed A549 cells following NP treatment. Ongoing studies are assessing the combinatorial effects of NP-delivered NAC and ATRA on an air-liquid interface model in vitro using Western blotting, ELISA and immunocytochemistry.

Acknowledgements (Optional): We acknowledge funding support from NIH grant R37CA283937 to JUM.