Polymeric nanoparticle-mediated siRNA delivery into primary human bronchial airway cells

Small interfering RNAs (siRNA) are powerful tools to target cellular protein expression, making them promising candidates for therapeutic applications. siRNA-based approaches to target detrimental mechanisms in airway diseases such as asthma and COPD are highly appealing. However, such delivery systems must be non-toxic, protect siRNAs from degradation and enable intracellular uptake that accesses cytosolic RNA machinery. The present study examines the mechanisms by which guanidinium-functionalized poly(oxanorbornene)imide polymer (PONI-Guan) nanoparticles can effectively and safely deliver siRNA in human bronchial epithelial (BEC) and airway smooth muscle cells (ASM). PONI-Guan polymers were engineered to self-assemble with siRNA through electrostatic interactions. Primary BEC and ASM cells were preincubated with methyl-β-cyclodextrin, dynasore, dansylcadaverine chlorpromazine, latrunculin B or cytochalasin D followed by incubation with nanoparticles at a single concentration but different guanidinium/phosphate ratios (G/P). BEC and ASM treated with methyl-β-cyclodextrin and dynasore demonstrated significant decrease in nanoparticle uptake. Additionally, BEC showed decreased uptake with latrunculin B. Minimal BEC toxicity was observed with 20, 30 and 40 G/P ratios; ASM showed some toxicity with 40 G/P. Transepithelial electrical resistance readings were stable with 20 and 30 G/P while 40 G/P showed significant but transient changes, with barrier integrity restored in ~6h. 30 G/P did not induce markers of necrosis, apoptosis or inflammation in BEC or ASM. Transfection of BDNF siRNA in ASM and Arginase 1 and 2 siRNA in BEC showed significant decrease in corresponding mRNA and protein expression. Overall, these data indicate that PONI-Guan polymers can deliver siRNA safely and effectively in bronchial cells, primarily through caveolar or macropinocytosis uptake, offering a promising tool for future siRNA based therapies.