Delivery of short hairpin RNA in the neotropical brown stink bug, Euschistus heros, using a composite nanomaterial.

The response of insects to orally delivered double-stranded RNA ranges widely among taxa studied to date. Long dsRNA does elicit a response in stink bugs but the dose required to achieve an effect is relatively high compared to other insects such Colorado potato beetle or western corn rootworm. Improving the delivery of dsRNA to stink bugs will improve the likelihood of using RNA-based biocontrols for the management of these economically important pests. Short hairpin RNA (shRNA) is a useful molecule with which to test improvements in the delivery of double stranded RNA in the neotropical brown stink bug, Euschistus heros, since shRNA alone does not elicit a clear effect like that for long dsRNA. Here, we show for the first time the oral delivery of shRNA triggering RNA interference (RNAi) in E. heros using 4 nm cerium oxide nanoparticles (CeO2 NPs) coated with diethylamioethyl dextran (Dextran-DEAE) as a carrier. We identified particle properties (coating composition and degree of substitution, hydrodynamic diameter, and zeta potential) and shRNA loading rates (Ce:shRNA mass ratio) that resulted in successful transcript reduction or RNAi. When the Z-average diameter of CeO2 Dextran-DEAE-shRNA NP complex was less than 250 nm and the zeta potential was in the 15-25 mV range (Ce:shRNA mass ratio of 0.7:1), significant mortality attributed to RNAi was observed with a shRNA concentration in feeding solution of 250 ng/µl. The degradation of the targeted troponin transcript by NP-delivered shRNA was equivalent to that observed with long dsRNA, while naked shRNA transcript reduction was not statistically significant. Elemental mapping by synchrotron X-ray fluorescence microprobe confirmed uptake and distribution of Ce throughout the body with the highest concentrations found in gut tissue. Taken together, our results suggest that a nanoparticle delivery system can improve the delivery of RNA-based biocontrols to E. heros, and therefore its attractiveness as an application in the management of this important pest in soybean production.

Efficient Binding, Protection, and Self-Release of dsRNA in Soil by Linear and Star Cationic Polymers.

Double stranded RNA (dsRNA) exhibits severe degradation within 3 days in live soil, limiting its potential application in crop protection. Herein we report the efficient binding, protection, and self-release of dsRNA in live soil through the usage of a cationic polymer. Soil stability assays show that linear poly(2-(dimethylamino)ethyl acrylate) can delay the degradation of dsRNA by up to 1 week while the star shaped analogue showed an increased stabilization of dsRNA by up to 3 weeks. Thus, the architecture of the polymer can significantly affect the lifetime of dsRNA in soil. In addition, the hydrolysis and dsRNA binding and release profiles of these polymers were carefully evaluated and discussed. Importantly, hydrolysis could occur independently of environmental conditions (e.g., different pH, different temperature) showing the potential for many opportunities in agrochemicals where protection and subsequent self-release of dsRNA in live soil is required.

Phosphonium Polymethacrylates for Short Interfering RNA Delivery: Effect of Polymer and RNA Structural Parameters on Polyplex Assembly and Gene Knockdown.

Synthetic polymers containing quaternary phosphonium salts are an emerging class of materials for the delivery of oligo/polynucleotides. In this work, cationic phosphonium salt-containing polymethacrylates and their corresponding ammonium analogues were synthesized by reversible addition-fragmentation chain transfer polymerization. Both the nature of the charged heteroatom (N vs P) and the length of the spacer separating the cationic units along the polymer backbone (oxyethylene vs trioxyethylene) were systematically varied. Polymers efficiently bound short interfering RNA (siRNA) at N(+)/P(-) or P(+)/P(-) ratios of 2 and above. At a 20:1 ratio, small polyplexes (Rh: 4-15 nm) suitable for cellular uptake were formed that displayed low cytotoxicity. While siRNA polyplexes from both ammonium and phosphonium polymers were efficiently internalized by green fluorescent protein (GFP)-expressing 3T3 cells, no knockdown of GFP expression was observed. However, 65% Survivin gene knockdown was observed when siRNA was replaced with novel, multimerized long interfering RNA in HeLa cells, demonstrating the importance of RNA macromolecular architecture on RNA-mediated gene silencing.