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

Small interfering RNAs (siRNAs) 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 chronic obstructive pulmonary disease are highly appealing. However, such delivery systems must be nontoxic, 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. In addition, 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, whereas 40 G/P showed significant but transient changes, with barrier integrity restored in ∼6 h. Furthermore, 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.NEW & NOTEWORTHY siRNA delivery to target cellular processes is an appealing area in lung diseases such as asthma. To realize this potential requires efficient, targeted cytosolic delivery without cellular toxicity or siRNA degradation. We demonstrate the efficacy of PONI-Guan nanoparticles in accessing airway epithelial and smooth muscle cells with specific siRNA targeting of mRNAs of interest while maintaining barrier integrity and avoiding cellular toxicity.