Dendrimer-coated carbon nanotubes deliver dsRNA and increase the efficacy of gene knockdown in the red flour beetle Tribolium castaneum

Abstract

In this study, the use of dendrimer-coated carbon nanotubes (CNTs) as a delivery vehicle for dsRNA was assessed in Tribolium castaneum. Exposure to low dosages of polyamidoamine dendrimer carbon nanotubes (PAMAM-CNTs) did not affect T. castaneum larval mortality. Expression of key apoptotic factors, Dronc (Tc12580), Dredd (Tcn-like, Tc014026) and Buffy, (Tcinhib apop1), which can act as toxicity indicators, were not altered in T. castaneum larvae following injection of PAMAM-CNTs. The level of knockdown of two target genes, α-tubulin and mitochondrial RNA polymerase (mtpol), were significantly increased when larvae were injected with double-stranded RNA bound to CNTs (PAMAM-CNT-dsRNA), compared to those injected with target dsRNA alone. PAMAM-CNTs were visualised in cellular vacuoles and in the cell nucleus. Increase occurrence of a blistered wing phenotype was found in a subset of PAMAM-CNT-dsRNA αtub injected larvae, relative to the level seen in larvae injected with naked dsRnA αtub alone. These results suggest that the use of functionalised CNTs for dsRNA delivery could increase the efficacy of RNA interference in insect pest species. RNA interference (RNAi) is a promising tool for the control of insect pests. Introduction of exogenous doublestranded RNA (dsRNA) is effective at triggering gene knockdown and associated phenotypes in many problem pest species such as the Western Corn Rootworm 1 , the Colorado potato beetle 2 and the Varroa mite 3. New developments in dsRNA delivery methods to crop pests, such as dsRNA expression in transgenic plants 4 and foliar application 5 , offer the possibility of dsRNA-based insecticides 6. Despite this promise, there is notable variation in RNAi response between insect species 7 , with some, such as many lepidopterans 8 , unable to mount a strong RNAi response to dsRNA. Ingestion of dsRNA can be a valid route for delivery, yet many insects secrete enzymes in their gut that breakdown dsRNA before the latter can elicit an effect on target gene transcripts 9-11. When dsRNA enters into the cytoplasm, it is generally very effective in mounting an RNAi response 12,13. However, the route to the cytoplasm can act as a barrier to effective RNAi, with stability and uptake of dsRNA within the insect thought to be the significant limiting factor 10,11,14,15. Therefore, the protection of dsRNA from degradation coupled with an efficient cellular uptake is key to RNAi success. The development of delivery vehicles or carriers that enable these two processes is now necessary to fully realise the potential for dsRNA as an effective control measure. Combining dsRNA with other substances that act as efficient carriers, such as chitosan 16,17 , perfluorocarbon nanoparticles 18-20 or ribonuceloparticles 21 , can stabilise dsRNA and increase the chances of successful gene knockdown. Encapsulation of dsRNA within carbon quantum dots 22 and liposomes 23,24 has also shown some promise in increasing efficacy in more challenging insect species. In the last decade, carbon nanotubes (CNTs) have emerg...

Nanomaterials for Postharvest Management of Insect Pests: Current State and Future Perspectives

“…In addition, no effect of environmental variation was noticed. Delivering dsRNA by the utilization of polyamidoamine dendrimer-coated carbon nanotubes (PAMAM-CNT-dsRNA) help in raising the level of RNA interference in T. castaneum (Edwards et al, 2020). Thereby, PAMAM-CNT-dsRNA increases the efficacy of gene knockdown in red flour beetle.…”

Nanotechnology‐Assisted RNA Delivery: From Nucleic Acid Therapeutics to COVID‐19 Vaccines

“…[ 93 ] A similar approach was used in the study conducted by Edwards et al. [ 94 ] In their report, polyamidoamine dendrimer and CNT suspension were placed under sonication to modify the surface of CNTs and improve their functional properties. On the other hand, GO is a sheet of oxidized carbon atoms with a hexagonal conformation resembling a honeycomb, which has a higher specific surface area, suspension stability, and biocompatibility than CNTs.…”

Green Approaches to Carbon Nanostructure-Based Biomaterials

“…Use of ultrasounds promoted the wrapping of CNTs by polyethylene glycol-pyrene polymer to achieve stable dispersions to induce apoptosis of colon cancer cells by applying nanosecond electric pulses, which affected calcium flux within cells [185]. Simple mixing of CNTs with cationic polyamidoamine polymer and anionic dsRNA, with ultrasonication at room temperature, provided coated CNTs for gene knockdown interventions [186]. Similarly, sonication of CNTs with DNA in saline solutions, followed by purification using polymers, was effective to yield dispersible CNTs that showed very good short-term and long-term biocompatibility in vivo [187].…”