Delivery of Mature Cystic Fibrosis Transmembrane-Conductance Regulator Protein via Mechanically Generated Extracellular Vesicles

Introduction: : Cystic fibrosis (CF) is a genetic disorder caused by a defective cystic fibrosis transmembrane conductance regulator protein (CFTRp), leading to a hypersecretion and accumulation of thick, sticky mucus that impairs mucociliary clearance, increasing susceptibility to chronic infections, airway obstruction, and progressive lung damage Previous studies have explored the use of naturally secreted extracellular vesicles (sEVs) for CFTRp delivery; however, these efforts have been limited by low protein transfer efficiency, reported to be only around 10%. This inefficiency may result from two main factors: (1) CFTRp destined for sEV packaging is often recycled back to the plasma membrane of early endosomes before multivesicular body (MVB) formation, resulting in CFTRp-free sEVs, and (2) early fusion of sEVs with endosomes in recipient cells may occur before CFTRp is recycled to the plasma membrane. To overcome these limitations, this project proposes the use of mechanically generated EVs (mEVs) that are directly enriched with membrane-integrated CFTRp. We hypothesize that mEVs enable more efficient delivery of functional CFTRp to the PM of recipient cells compared to sEVs. This improved efficacy is attributed to a higher CFTRp loading efficiency in mEVs and the biophysical similarity between the mEV membrane and the recipient cell membrane, which may facilitate membrane fusion and protein integration.

Materials and

Methods: : To demonstrate the feasibility of the direct transfer of membrane-bound proteins by mechanically generated extracellular vesicles (mEVs). As compared to naturally secreted extracellular vesicles (sEVs), mEVs generated by mechanical extrusion through hydrophilic polycarbonate membranes with 5 µm pore size carry a high density of CFTR. mEVs were generated from CFTR-overexpressing 16HBE14o G542X human bronchial epithelial cells, transduced with lentiviral vectors encoding CFTR-GFP fusion proteins. Cells were cultured in Minimum Essential Media (MEM), and mEVs were generated through extrusion at varying pressures (2–10 psi). Particle concentration and size were assessed using nanoparticle tracking analysis (NTA), while morphology was confirmed via scanning electron microscopy (SEM). mEVs and sEVs were compared for protein yield and generation rates.

To evaluate stability, CFTRp-mEVs were incubated under three conditions (KBR buffer, MEM, and MEM + FBS) at 37 °C for 3 and 6 hours. Particle and protein stability were measured via NTA, BCA protein assays, and CFTR-specific Western blotting. Moreover, we used metabolic labelling and click chemistry to study the CFTR recycling back to the recipient cells' plasma membrane.

Results, Conclusions, and Discussions:: CFTRp-overexpressing epithelial cells successfully produced mEVs enriched with membrane-integrated CFTR-GFP (Fig. 1A and Fig. 2). Optimization of extrusion pressures revealed that both low (2 psi) and high (10 psi) pressures yielded the highest mEV particle counts (Fig. 1C), while SEM confirmed classical cup-shaped vesicle morphology (Fig. 1B). Across all pressures, mEV sizes remained consistent (~150 nm, Fig. 1D). Importantly, mEVs were generated approximately 100 times faster than sEVs, indicating the efficiency of this approach (Fig. 1E).

Stability analyses showed that mEV particles retained their integrity across all tested conditions (KBR, MEM, MEM+FBS, Fig. 3A), though protein concentration decreased over time (Fig. 3B). Notably, KBR buffer preserved the highest amount of intact CFTR protein, as confirmed by Western blot analysis (Fig. 3C). CFTRp aggregation increased with longer incubation times in MEM and MEM+FBS. Cell viability studies demonstrated that 16HBE14o G542X cells maintained over 70% viability in KBR for at least 4 hours, a timeframe sufficient for mEV uptake (Fig 3D).

These findings highlight the feasibility of mEV-based CFTRp delivery, overcoming limitations associated with sEVs, such as low protein incorporation and intracellular degradation. The plasma membrane origin of mEVs may facilitate more efficient membrane fusion and CFTR functional delivery in recipient cells. Furthermore, the rapid production rate and favorable stability of mEVs position them as a promising vehicle for therapeutic protein transfer. Ongoing efforts to enhance CFTRp stability on mEVs using CFTR modulators may further improve the efficacy of this system, especially for translational applications in cystic fibrosis therapy.

This study introduces mEVs as a novel and more effective platform for membrane protein delivery, addressing critical limitations of conventional sEV-based strategies. Future directions include functional assays of chloride channel activity and in vivo evaluation of therapeutic potential.

Acknowledgements and/or References (Optional):: We gratefully acknowledge the Cystic Fibrosis Foundation for their generous support of this research. Their contribution of CFTR-mutant cell lines and continued commitment to advancing cystic fibrosis therapeutics made this work possible.