New nanoparticle-based systems for pulmonary drug and gene delivery

Advanced nanoparticle-based systems could dramatically improve the effectiveness and reduce the systemic side effects of pulmonary drug and gene therapies. However, the effectiveness of such systems has been hindered by cellular and extracellular barriers. To understand and begin to address the cellular barriers, we used a combination of live-cell confocal microscopy and flow cytometry to investigate the intracellular trafficking of highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with diblock copolymers of polyethylene glycol and poly-L-lysine (PEG-CK30). In addition, we investigated the intracellular trafficking of highly compacted, pH-responsive DNA nanoparticles, formulated with a triblock copolymer of polyethylene glycol, poly-L-histidine and poly-L-lysine (PEG-CH12K18). Surprisingly, we discovered that PEG-CK30 DNA nanoparticles enter human bronchial epithelial cells (BEAS-2B) via caveolae-mediated endocytosis and traffic to the nucleus via a non-degradative pathway, whereas PEG-CH12K 18 DNA nanoparticles enter BEAS-2B cells via clathrin-coated pits, where the poly-L-histidine moieties escape from acidic vesicles in a manner consistent with the proton sponge hypothesis. Despite trafficking within the degradative endolysosomal pathway, PEG-CH12K18 DNA nanoparticles improved the in vitro gene transfer by ∼ 20-fold, and in vivo pulmonary gene transfer in BALB/c mice by ∼ 3-fold, compared to PEG-CK30 DNA nanoparticles currently in human clinical trials, while maintaining a favorable toxicity profile.;Perhaps a more formidable barrier to effective drug and gene delivery in the lung airways of cystic fibrosis (CF) patients is the hyperviscoelastic and adhesive sputum layer that traps particles so that they may be removed prior to reaching the epithelium. Using a combination of multiple particle tracking analysis and rigorous physicochemical characterization techniques, we sought to better understand the nanoparticle design parameters responsible for poor sputum-penetration. We discovered that highly compacted PEG-CK 30 DNA nanoparticles, a promising non-viral gene carrier currently in clinical trials for CF, are immobilized in CF sputum due to inadequate surface PEG coatings. To overcome the formidable sputum barrier, we engineered DNA nanoparticles, formulated with a dense surface coating of PEG, that rapidly penetrate human CF sputum. In addition, we engineered a corticosteroid-loaded delivery system with reduced adhesivity to CF sputum, high drug loading and sustained drug release. Based on safety and efficacy studies in preclinical animal models, our results suggest that sputum-penetrating nanoparticle systems are promising for clinical development. In addition to treating pulmonary disease, sputum-penetrating therapeutic systems may find use for the local delivery of therapeutics at other mucosal sites in the body.