期刊
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
卷 68, 期 25, 页码 6811-6818出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.0c02182
关键词
hydroxyapatite nanoparticles; RNAi; dsRNA; polyarginine; layer-by-layer; SF9 cells; gene knockdown; endosomal escape
资金
- National Institute of Allergy and Infectious Diseases of the National Institutes of Health [R21AI131427]
Conventional synthetic insecticides have limited success due to insect resistance and negative effects on off-target biota and the environment. Although RNA interference (RNAi) is a tool that is becoming more widely utilized in pest control products, naked dsRNA has limited success in most taxa. Nanocarriers have shown promising results in enhancing the efficacy of this tool. In this study, we used a layer-by-layer electrostatic assembly where we synthesized poly(acrylic acid) (PAA)-coated hydroxyapatite (HA) nanoparticles (PAA-HA NPs) as inorganic nanocarriers, which were then coated with a layer of a cationic poly(amino acid), 10 kDa poly-L-arginine (PLR10), to allow for binding of a layer of negatively charged dsRNA. Binding of PLR,PAA-HA NPs to dsRNA was found to increase as the mass ratio of NPs to dsRNA increased. In vitro studies with transgenic SF9 cells (from Spodoptera frugiperda) expressing the firefly luciferase gene showed a significant gene silencing (35% decrease) at a 5:1 NP-to-dsRNA ratio, while naked dsRNA was ineffective at gene silencing. There was a significant concentration-response relationship in knockdown; however, cytotoxicity was observed at higher concentrations. Confocal microscopy studies showed that dsRNA from PLR10-PAA-HA NPs was not localized within endosomes, while naked dsRNA appeared to be entrapped within the endosomes. Overall, polymer-functionalized HA nanocarriers enabled dsRNA to elicit gene knockdown in cells, whereas naked dsRNA was not effective in causing gene knockdown.
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