Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung.

INTRODUCTION: Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. METHODS: We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air-liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. RESULTS: Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and ßENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. CONCLUSION: Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.

Mucus penetrating properties of soft, distensible lipid nanocapsules.

Designing nanomaterials to release their drug pay-load upon exposure to an exogenous trigger can help to direct drug delivery, but how the triggered release, which often modifies the nanomaterial properties, influences the biological fate of these systems is currently unknown. The aim of this study was to investigate how the triggered drug release from PEG coated, soft, 50 nm distensible lipid nanocapsules (LNC) influenced their diffusion across a mucus barrier. The translocation speed of the non-triggered LNC across a 35 µm thick purified gastric mucin (PGM) barrier was 3 times faster (30.08 ±â€¯2.49 × 10-10 cm2 s-1) compared to equivalent-sized negatively charged polystyrene particles (9.87 ±â€¯0.61 × 10-10 cm2 s-1, p < 0.05). In cystic fibrosis mucus (CFM), harvested from patient primary cells, the non-triggered LNC translocation speed was similar to the PGM, but the polystyrene particle diffusion was so slow it could not be measured. The trigger induced LNC distension process had no effect on the particle diffusion rate in both PGM and CFM (p > 0.05) in a static mucus barrier, but when shear was applied to the barrier the distended LNCs diffused more slowly (3.97 ±â€¯1.38 × 10-8 cm2 s-1, p < 0.05) compared to the non-distended materials (4.94 ±â€¯0.04 × 10-8 cm2 s-1). This data suggested the rapid mucus penetration of the distended LNCs, despite their increased size, was a consequence of their capacity to take a less tortuous path through the barrier, i.e., they experienced less steric hinderance, compared to the non-distended LNC.

Controlled drug release from lung-targeted nanocarriers via chemically mediated shell permeabilisation.

Nanocarriers can aid therapeutic agent administration to the lung, but controlling drug delivery from these systems after deposition in the airways can be problematic. The aim of this study was to evaluate if chemically mediated shell permeabilisation could help manipulate the rate and extent of nanocarrier drug release. Rifampicin was loaded into lipid shell (loading efficiency 41.0±11.4%, size 50nm) and polymer shell nanocarriers (loading efficiency 25.9±2.3%, size 250nm). The drug release at pH 7.4 (lung epithelial pH) and 4.2 (macrophage endosomal pH) with and without the chemical permeabilisers (Pluronic L62D - lipid nanocarriers; H(+)- polymer nanocarriers) was then tested. At pH 7.4 the presence of the permeabilisers increased nanocarrier drug release rate (from 3.2µg/h to 6.8µg/h for lipid shell nanocarriers, 2.3µg/h to 3.4µg/h for polymer shell nanocarriers) and drug release extent (from 50% to 80% for lipid shell nanocarriers, from 45% to 76% for polymer shell nanocarriers). These effects were accompanied by lipid nanocarrier distension (from 50 to 240nm) and polymer shell hydrolysis. At pH 4.2 the polymer nanocarriers did not respond to the permeabiliser, but the lipid nanocarrier maintained a robust drug release enhancement response and hence they demonstrated that the manipulation of controlled drug release from lung-targeted nanocarriers was possible through chemically mediated shell permeabilisation.