Inhalable pH-responsive charge-reversal polymer-small interfering RNA polyplexes for directed gene therapy of anaplastic lymphoma kinase fusion-positive lung cancer

Anaplastic lymphoma kinase (ALK) oncogenic fusions are an infamously evolving target in ALK fusion-positive (ALK + ) non-small cell lung cancer (NSCLC). RNA interference (RNAi) is a potential alternative to small-molecule ALK inhibitors but suffers from poor cellular delivery and low stability, restricting sustained on-target therapeutic efficacy. To attain non-invasive direct RNAi at target sites in the lungs, inhalable vehicles are demanded to shield unstable siRNA from degradation in alkaline pleural fluid and to shuttle siRNA in the acidic tumor microenvironment. Here, we chemically synthesize a universal polyethylene glycol-poly(β-amino esters)-histidine(PEG-PBAE-His, PPH) system that enables siRNA-directed ALK gene silencing in ALK + NSCLC with controlled release. The rational design of PEGylated polycationic PPH integrates the non-ionic nature, inertness, stealth effect, and low immunogenicity of PEG coronae with dual positively-charged PBAE and His to encapsulate siRNA payloads through enhanced electrostatic complexations. The assembled polyplexes with low polydispersity are overall anionic in alkaline pleural fluid and reversed into cationic charges at acidic pH, facilitating cellular uptake and lysosomal pH-governed release. The PPH-directed delivery significantly improves siRNA stability and ALK sequence-specific knockdown in H3122 cells, with a transfection efficiency comparable to commercial polyethylenimine but with lower cytotoxicity. The ALK-centric RNAi therapy prohibits cancer clonogenicity, migration, and invasion and induces potent growth inhibition in ALK + NSCLC xenografts through inhalation. The inhalable polyplexes represent a transformative delivery platform that combines pH-responsive targeting, enhanced stability, and low toxicity, addressing critical limitations of existing siRNA delivery systems.