Chitosan Nanocomplexes for the Delivery of ENaC Antisense Oligonucleotides to Airway Epithelial Cells

Abstract

Nanoscale drug delivery systems exhibit a broad range of applications and promising treatment possibilities for various medical conditions. Nanomedicine is of great interest, particularly for rare diseases still lacking a curative treatment such as cystic fibrosis (CF). CF is defined by a lack of Cl− secretion through the cystic fibrosis transmembrane conductance regulator (CFTR) and an increased Na+ absorption mediated by the epithelial sodium channel (ENaC). The imbalanced ion and water transport leads to pathological changes in many organs, particularly in the lung. We developed a non-viral delivery system based on the natural aminopolysaccharide chitosan (CS) for the transport of antisense oligonucleotides (ASO) against ENaC to specifically address Na+ hyperabsorption. CS–ASO electrostatic self-assembled nanocomplexes were formed at varying positive/negative (P/N) charge ratios and characterized for their physicochemical properties. Most promising nanocomplexes (P/N 90) displayed an average size of ~150 nm and a zeta potential of ~+30 mV. Successful uptake of the nanocomplexes by the human airway epithelial cell line NCI-H441 was confirmed by fluorescence microscopy. Functional Ussing chamber measurements of transfected NCI-H441 cells showed significantly decreased Na+ currents, indicating successful downregulation of ENaC. The results obtained confirm the promising characteristics of CS as a non-viral and non-toxic delivery system and demonstrate the encouraging possibility to target ENaC with ASOs to treat abnormal ion transport in CF.

Capsaicin-Loaded Chitosan Nanocapsules for wtCFTR-mRNA Delivery to a Cystic Fibrosis Cell Line

“…The gel retardation assay shown in Figure 5 further enforces the successful surface-loading of wtCFTR-mRNA to the nanocapsules, as the nucleic acid was completely retained in the gel pocket by the CS-covered nanosystems. The outcome of the gel retardation assay corroborates previous reports showing overall strong binding efficiency of nucleic acids and CS [13,14,54,55].…”

“…Both systems remained stable over time ( Figure 6), as reported by previous studies [22,59]. However, the transfection medium supplemented with HEPES and mannitol was reported to display a very high osmolality, around 580 mOsmol/kg, having a shrinking effect on the cells [14]. Based on these data, transfection experiments should be conducted in medium without supplements.…”

“…Cytotoxicity of the nanocapsules was analyzed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously [ 14 ]. Briefly, CFBE41o- and 16HBE14o- cells were seeded in a 96-well microtiter plate (1 × 10 4 cells per well) and incubated for 24 h at 37 °C, at 5% CO 2 and 95% rH.…”

“…In our group, we have been working with wtCFTR-mRNA to improve chloride secretion of epithelial cells constantly optimizing delivery methods [10][11][12]. The most promising delivery systems of nucleic acids to epithelial cells proved to be self-assembled polyelectrolyte chitosan (CS)-based nanocomplexes due to their low cytotoxicity and high transfection efficiency [12][13][14]. CS itself, a family of pseudonatural polysaccharides composed of randomly distributed β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine units, is a promising candidate for non-viral gene delivery [15].…”

Biotechnology-Derived Chitosans with Non-Random Patterns of Acetylation Differ from Conventional Chitosans in Their Properties and Activities

“…5). The two commercial chitosans have been used in biomedical research, especially for the transfection of human cells [54][55][56] . We therefore prepared polyplexes by mixing these chitosans with plasmid DNA encoding eGFP at different molar charge chitosan/DNA ratios.…”