Mucin-coated nanoparticles for modulating neutrophilic inflammation

Saturday, October 11, 2025

9:20 AM - 9:25 AM PDT

Location: Room 29B

Presenting Author(s)

Alexis Johnson, Undergraduate

Bioengineering Senior Student
University of Maryland, College Park
Bowie, Maryland, United States

Co-Author(s)

Allison Boboltz

Graduate Student
University of Maryland
College Park, Maryland, United States
OT

Owen Tabah

PhD Student
University of Maryland, United States

Last Author(s)

GD

Gregg Duncan, Ph.D.

Associate Professor
University of Maryland
Columbia, Maryland, United States

Introduction: : Chronic respiratory diseases (CRDs) are incurable, require long-term symptom management, and are a leading cause of morbidity and mortality worldwide, affecting hundreds of millions of people.1 Patients suffering from CRDs (e.g., cystic fibrosis, chronic obstructive pulmonary disease, asthma) often experience mucus overproduction and inflammation in the airways. Mucus is a gel composed of large glycoproteins called mucins, which protects against inhaled pathogens. However, the mucus barrier also prevents therapeutics from reaching the airway epithelium.2,3 Neutrophils release neutrophil extracellular traps (NETs) that perpetuate inflammation and contribute to epithelial barrier dysfunction in CRDs. The NET formation process, referred to as NETosis, involves the decondensation of chromatin and the disintegration of membranes. NETosis results in the extracellular release of a web-like complex of DNA, histones, and granular proteins from the neutrophil.4 Elevated NET levels are associated with worsened lung function and increased respiratory disease severity.5 Prior work has shown that mucins purified from bovine cervical mucus are able to suppress NETosis in bovine neutrophils via the mucin glycans. Mucin nanoparticles could therefore be a potential therapy with anti-inflammatory function against NETs. We complexed mucins with a synthetic polymer, poly-lactic co-glycolic acid (PLGA), to form a mucin coating. We found that mucin-coated PLGA nanoparticles were able to modulate NETosis successfully in comparison to the PLGA particles alone.

Materials and

Methods: : A neutrophil-model cell line, HL-60, was used to evaluate the effects of the nanoparticles on NET formation. To differentiate the cells to be neutrophil-like, 1.3% dimethyl sulfoxide (DMSO) was added to the media for 5-7 days. Commercially available porcine gastric mucin (PGM) was dissolved in water to create four solutions (5, 2.5, 1, 0 mg/mL) and syringe-filtered to filter out large bacteria. To develop the nanoparticles, PLGA was added to each mucin solution, and solutions were emulsified to conjugate the polymers, evaporated to remove excess moisture, centrifuged to separate unconjugated mucin, and then the supernatant containing mucin-coated nanoparticles was collected for characterization and cell treatment. Particle size and charge were characterized using a nanoparticle tracking analyzer. Mucin conjugation was quantified using 2-cyanoacetamide, which fluoresces in the presence of the O-linked glycans on mucins. The HL-60 cells were seeded in 96-well plates (105 cells/well) and incubated with the mucin-coated nanoparticles for 30 minutes. Half of the HL-60 cells were induced to undergo NETosis using phorbol-12-myristate-13-acetate (PMA), and the remainder were treated with a vehicle to evaluate spontaneous NETosis over the course of 3-4 hours. NET formation was quantified using the fluorescence of the extracellular DNA stain Sytox Green. The background fluorescence from the nanoparticles and cell culture media was subtracted from the values of the experimental wells.

Results, Conclusions, and Discussions:: We tested mucin-PLGA particles synthesized using nano-precipitation versus nano-emulsion techniques, and found that nano-emulsion particles seemed to more effectively prevent NETosis. Characterization of nano-emulsion particles revealed that increasing mucin concentrations led to larger particle diameters and a shift in zeta potential, with the mucin coating resulting in a less negative surface charge compared to uncoated PLGA particles (Figure 1). Although not all mucin from the initial solution adhered to PLGA, fluorescence quantification showed that mucin incorporation remained proportional to the starting concentration.
In a follow-up experiment using only nano-emulsion particles, we expanded the range of mucin concentrations (0, 1, 2.5, 5 mg/mL mucins). Higher mucin concentrations on the PLGA particles correlated with greater suppression of NETosis both in cells induced to undergo NETosis with PMA and spontaneous NETosis (Figure 2). Notably, significantly higher levels of NETosis occurred in HL-60 cells treated with PLGA particles (0 mg/mL mucin) and stimulated with PMA than in the baseline PMA-stimulated cells with no nanoparticle treatments applied, suggesting that PLGA may exacerbate PMA-induced NETosis. The 2.5 and 5 mg/mL mucin-coated particles were able to dramatically reduce the very high levels of PMA and PLGA-induced NETosis.
These findings support the potential of porcine gastric mucin-coated nanoparticles, particularly those synthesized via nano-emulsion and loaded with higher mucin concentrations, as modulators of NET-driven inflammation. While PLGA provides particle structural integrity, its pro-inflammatory properties may limit therapeutic efficacy. Fortunately, the mucin coating appears to counteract NETosis caused by PLGA in a concentration-dependent manner, likely due to its anti-inflammatory glycoproteins. With improvements such as increasing mucin concentration or eliminating PLGA, these particles may be a promising vehicle to reduce neutrophil-driven airway inflammation in respiratory diseases.

Acknowledgements and/or References (Optional): : References
1. World Health Organization. (2025). Chronic respiratory diseases. World Health Organization. https://www.who.int/health-topics/chronic-respiratory-diseases#tab=tab_1
2. Brightling, C. & Greening, N. (2019). Airway inflammation in COPD: progress to precision medicine. Eur Respir J. 54: 1900651. https://doi.org/10.1183/13993003.00651-2019
3. Ray, A. and Kolls, J. K. (2017). Neutrophilic Inflammation in Asthma and Association with Disease Severity. Trends Immunol, 38(12): 942-954. DOI: 10.1016/j.it.2017.07.003
4. Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D. S., Weinrauch, Y., & Zychlinsky, A. (2004). Neutrophil Extracellular Traps Kill Bacteria. Science, 303:5663. DOI: 10.1126/science.1092385
5. Zhu, S., Ying, Y., Ren, Y., Xu, L., Wang, H., Ling, X., Jin, L., Hu, Y., Zhang, H., Miao, C., & Guo, K. (2021). The emerging roles of neutrophil extracellular traps in wound healing. Cell Death and Disease, 12:984. DOI: 10.1038/s41419-021-04294-3