Engineered carbon dots for mucosal gene delivery

Although lung gene therapy holds promise for treating various life-threatening lung diseases, its efficacy is hindered by the mucus layer covering the airways, whose role is to protect the lung epithelium from airborne threats. For efficient gene delivery to the epithelial cells, it is necessary to ensure rapid passage of the transfection particles through the mucus layer before they are eliminated by mucociliary clearance. We developed mucus-penetrating gene carriers using carbon dots (CDs) synthesized from citric acid and bPEI600. Various strategies were investigated to convert these CDs into muco-inert nanoparticles, including PEGylation and decoration with zwitterionic or mucolytic species. After thorough characterization, we assessed their interactions with a mucus model through turbidimetry and transport measurements, as well as their effects on mucus rheology. The efficacy of the carriers to deliver DNA to various cell models was established. Particularly, Calu-3 cells, cultured at the air-liquid interface to obtain abundant mucus production, were used as a discriminating model to evaluate the potency of CDs to deliver their DNA cargo through mucus. While zwitterion-coated CDs failed to induce significant transgene expression, those with PEG decorations yielded moderate results, and CDs designed as thiol reservoirs for local mucolytic action achieved high transfection rates.