Carbon dots boost dsRNA delivery in plants and increase local and systemic siRNA production

Carbon dots (CDs) have been proposed for nucleic acid delivery in many biological applications. In this work we have obtained carbon dots by a hydrothermal synthesis method for developing nanocomposites with dsRNA. These CDs were produced using glucose or saccharose as the nucleation source and passivated with branched polyethyleneimines for conferring positive charges. Hydrodynamic analyses and transmission electron microscopy TEM showed that they sized on average 4 and 5 nm, depending on the sugar. The CDs were fluorescent and showed a peak at 468 nm when excited with UV light. Physicochemical characteristics of their surfaces were revealed by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FT-IR). The {zeta} potential determined that both types of CDs had positive charges as well as good electrophoretic mobility and conductivity. Coating of the CDs to dsRNA was efficient but did not protect from nucleases. DsRNA naked or coated with the CDs were delivered to leaves of cucumber plants by spraying at 2.5 bar. Quantitation of the dsRNA that entered the leaves showed that when coated with the CDs, 50-fold more dsRNA was detected than when naked dsRNA was applied. Moreover, specific siRNAs derived from the sprayed dsRNAs were 130 times more abundant when the dsRNA was coated with the CDs. Systemic dsRNAs were determined in distal leaves showing a dramatic increase in concentration when delivered as a nanocomposite. Similarly, systemic siRNAs were significantly more abundant in distal leaves when spraying with the CD-dsRNA nanocomposite. Furthermore, FITC-labeled dsRNA was shown to accumulate in the apoplast and increase its entry in the plant when coated with CDs. These results indicate that CDs obtained by hydrothermal synthesis are suitable for dsRNA foliar delivery in RNAi plant applications and provide insight into CD-dsRNA translocations.