Abstract Nano‐delivery systems have been applied to deliver various synthetic/botanical pesticides to increase the efficiency of pesticide use and reduce the volumes of pesticides applied. Previous studies have supported the hypothesis that the nanocarriers can help expand the insecticidal target of pesticides to include non‐target pests. However, the potential mechanism underlying this interesting phenomenon remains unclear. Herein, a widely applied star polycation (SPc) nanocarrier was synthesized to construct a thiamethoxam (TMX) nano‐delivery system. The SPc‐based delivery system could promote the translocation of exogenous substances across the membrane of Sf9 cells, increase the cytotoxicity of TMX against Sf9 cells by nearly 20%, and expand the insecticidal target of TMX to include Spodoptera frugiperda (the fall armyworm), with a 27.5% mortality increase at a concentration of 0.25 mg/mL. Moreover, the RNA‐seq analysis demonstrated that the SPc could upregulate various transport‐related genes, such as Rab, SORT1, CYTH, and PIKfyve, for the enhanced cellular uptake of TMX. Furthermore, enhanced cell death in larvae treated with the TMX–SPc complex was observed through changes in the expression levels of death‐related genes, such as Casp7, BIRC5, MSK1, and PGAM5. The SPc‐based nano‐delivery system improved the cellular uptake of TMX and expanded its insecticidal target by adjusting the expression levels of death‐related genes. The current study mainly identified the transport and cell death genes related to nanocarrier‐based insecticidal target expansion, which is beneficial for understanding the bioactivity enhancement of the nano‐delivery system.
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