Enhanced Delivery of Nucleic Acids to Insect Cells by Star Polycation Formulation

Gene delivery to target cells is required for bioengineering or medical/agricultural applications. However, the hydrophobicity of the cell membrane always makes it resistant to polar nucleic acids. This physicochemical barrier is usually overcome by a nano-formulation to hide the polarity. This study evaluated a specific formulation called star polycation (SPc) regarding its efficacy in DNA/RNA delivery to insect cells. The delivery efficiency of the SPc formulation was assessed by transient expression of green fluorescence protein (GFP) in Sf9 cells, in which SPc formulation significantly enhanced the gene expression compared with an unformulated vector. In vivo transient expression (IVTE) was performed by injection of the expression construct with the SPc formulation into larvae of S. exigua. Fluorescence was detected in all four tissues, namely, epidermis, midgut, hemocyte, and fat body, where the SPc formulation enhanced the expression in most tissues except epidermis. Under this IVTE condition, an additional injection of SPc-formulated dsRNA specific to GFP suppressed the gene expression significantly more than the unformulated vector. The enhanced RNA interference (RNAi) efficiency caused by the SPc formulation was confirmed against four endogenous genes of S. exigua, namely, Snf7, PSMB5, vATPase, and α-tubulin, by either injection or feeding of dsRNA. These RNAi treatments were lethal to S. exigua, in which dsRNA specific to vATPase formulated with SPc resulted in almost 80% mortality through oral delivery. A similar oral toxicity by SPc-formulated dsRNA was demonstrated in another lepidopteran Plutella xylostella. The oral delivery of dsRNA was applied to control sucking insects such as aphids and thrips by spraying SPc-formulated dsRNA onto plant surfaces. The sprayed dsRNA labeled with Cy3 fluorescence was detected in the internal tissues of plant leaves, in which the penetration of dsRNA into the plant tissues was further accelerated by SPc formulation. The SPc formulation of dsRNA specific to vATPase was effective at killing the sucking insects by spraying on plant surfaces. These results suggest the application of an SPc formulation to deliver DNA/RNA to insect cells.