Optimization of Lung Surfactant Coating of siRNA Polyplexes for Pulmonary Delivery

Abstract

Purpose The aim of this study was to understand how coating with a pulmonary surfactant, namely Alveofact, affects the physicochemical parameters as well as in vitro behavior of polyethylenimine (PEI) polyplexes for pulmonary siRNA delivery. Methods Alveofact-coated polyplexes were prepared at different Alveofact:PEI coating ratios and analyzed in terms of size, PDI and zeta potential as well as morphology by transmission electron microscopy. The biological behavior was evaluated in a lung epithelial cell line regarding cell viability, cellular uptake via flow cytometry and gene downregulation by qRT-PCR. Furthermore, a 3D ALI culture model was established to test the mucus diffusion and cellular uptake by confocal microscopy as well as gene silencing activity by qRT-PCR. Results After optimizing the coating process by testing different Alveofact:PEI coating ratios, a formulation with suitable parameters for lung delivery was obtained. In lung epithelial cells, Alveofact-coated polyplexes were well tolerated and internalized. Furthermore, the coating improved the siRNA-mediated gene silencing efficiency. Alveofact-coated polyplexes were then tested on a 3D air-liquid interface (ALI) culture model that, by expressing tight junctions and secreting mucus, resembles important traits of the lung epithelium. Here, we identified the optimal Alveofact:PEI coating ratio to achieve diffusion through the mucus layer while retaining gene silencing activity. Interestingly, the latter underlined the importance of establishing appropriate in vitro models to achieve more consistent results that better predict the in vivo activity. Conclusion The addition of a coating with pulmonary surfactant to polymeric cationic polyplexes represents a valuable formulation strategy to improve local delivery of siRNA to the lungs. Graphical Abstract

Role of Hydrophobic Modification in Spermine-Based Poly(β-amino ester)s for siRNA Delivery and Their Spray-Dried Powders for Inhalation and Improved Storage

“…It is therefore hypothesized that the oleic acid analogues in the polymers promoted efficient endosomal escape, reflected by a stronger impact of hydrophobic content on gene silencing than on cellular uptake. To further assess the suitability of the most promising polymer for pulmonary siRNA delivery, gene silencing in an air–liquid interface model of mucus-producing 16HBE14o-cells on Transwells was quantified as described before, 42 where significant endogenous gene silencing was observed ( Figure S5 ).…”

Targeting Intracellular Bacteria with Dual Drug-loaded Lactoferrin Nanoparticles

“…Given the high basal phagocytic activity coupled with the targeting ability, pulmonary surfactant is expected to increase particle uptake by macrophages, while for epithelial cells, particle aggregation may rather reduce the uptake. Previously, pulmonary surfactant has been demonstrated to play a foe in nanoparticle uptake by alveolar epithelial cells, 61−63 while other studies 64,65 show the contrary. The difference between these studies is that the former used nanocarriers fabricated with materials that are not native to the pulmonary system, while the latter used native pulmonary surfactant to modify the nanocarriers.…”

State-of-the-Art Review on Inhalable Lipid and Polymer Nanocarriers: Design and Development Perspectives

“…As a result, polymeric materials with polyamine groups, such as polyethyleneimine (PEI), polylysine (PLL), or poly-(amidoamine) PAMAM, have been extensively studied for nucleic acid delivery [ 249 ]. For instance, PEI-based polyplexes have demonstrated significant potential in pulmonary delivery, and their ease of modification enables the attachment of shielding agents, targeting moieties, or lytic peptides to enhance their efficacy [ 250 , 251 , 252 ].…”