Chitosan/dsRNA polyplex nanoparticles advance environmental RNA interference efficiency through activating...

Area of Science:

• Biotechnology
• Nanomedicine
• Molecular Biology

Background:

• Chitosan is a promising nanocarrier for RNA interference (RNAi).
• The molecular mechanisms of chitosan/dsRNA polyplex nanoparticles (PNs) in enhancing dsRNA delivery remain unclear.
• Efficient dsRNA delivery is crucial for successful RNAi applications.

Purpose of the Study:

• To elucidate the molecular mechanism by which chitosan/dsRNA PNs enhance dsRNA delivery and RNAi efficiency.
• To investigate the role of endocytosis pathways in chitosan-mediated dsRNA delivery.
• To confirm the direct interaction between chitosan/dsRNA PNs and key proteins involved in cellular uptake.

Main Methods:

• Preparation of chitosan/dsRNA PNs via electrostatic attraction.
• Assessment of PNs' stability, cellular uptake, and RNAi efficiency.
• RNA-sequencing (RNA-Seq) and quantitative real-time PCR (qPCR) to identify gene expression changes.
• Inhibition of clathrin-dependent endocytosis (CDE) using chlorpromazine and an RNAi-of-RNAi strategy.
• Microscale thermophoresis assay to confirm protein binding.

Main Results:

• Chitosan/dsRNA PNs significantly improved dsRNA stability, cellular uptake, and RNAi efficiency.
• RNA-Seq and qPCR revealed upregulation of clathrin heavy chain (CHC) gene, indicating activation of the CDE pathway.
• Inhibiting CDE significantly reduced the RNAi response mediated by chitosan/dsRNA PNs.
• Microscale thermophoresis confirmed direct binding of chitosan/dsRNA PNs to CHC protein.

Conclusions:

• Chitosan/dsRNA PNs enhance RNAi efficiency by activating the CDE pathway through direct binding to CHC.
• This study elucidates the molecular mechanism of chitosan-based dsRNA delivery for RNAi.
• The findings support the broader application of nanocarrier-based RNAi in pest management and gene delivery.