Carbon Dots with Tunable Charge as Mucus-Penetrating Gene Carriers

<b>Background/Objectives:</b> Local delivery of gene therapy products through the airways shows great promise for the treatment of a number of serious lung diseases, but its effectiveness is hampered by the mucus layer protecting the lung epithelium in the trachea and bronchi. <b>Methods:</b> To overcome this barrier, we engineered carbon dots (CDs) with mucus penetrating properties. <b>Results:</b> The CDs were synthesized by solvothermal treatment of citric acid and branched polyethyleneimine, and functionalized with maleamic acid groups to create cationic mucoinert nanoparticles with tunable charge. We characterized their interactions with a mucus model through turbidity and transport measurements, and assessed their impact on the viscoelastic properties of the biopolymer. We then demonstrated that the carriers are effective at delivering pDNA to a variety of cell models in vitro. In particular, mucus-producing Calu-3 cells cultured at the air-liquid interface (ALI) were used as a discriminating model to evaluate intracellular delivery of the genetic cargo through a thick layer of mucus at the cell surface. <b>Conclusions:</b> The functionalization of CDs with maleamic acid groups resulted in a 1000- to 10,000-fold increase in transfection efficiency in the mucus-producing model, offering new opportunities for lung gene therapy.