Uptake and Bioactivity of Chitosan/Double-Stranded RNA Polyplex Nanoparticles in Caenorhabditis elegans

Abstract

In this study, we investigated chitosan/dsRNA polyplex nanoparticles as RNAi agents in the nematode Caenorhabditis elegans. By measurement of an easily observed phenotype and uptake of fluorescently labeled dsRNA, we demonstrate that chitosan/dsRNA polyplex nanoparticles are considerably more effective at gene knockdown on a whole body concentration basis than naked dsRNA. Further, we show that chitosan/dsRNA polyplex nanoparticles introduce dsRNA into cells via a different mechanism than the canonical sid-1 and sid-2 pathway. Clathrin-mediated endocytosis is likely the main uptake mechanism. Finally, although largely reported as nontoxic, we have found that chitosan, as either polyplex nanoparticles or alone, is capable of downregulating the expression of myosin. Myosin is a critical component of growth and development in eukaryotes, and we have observed reductions in both growth rate and reproduction in chitosan exposed C. elegans. Given the increased potency, noncanonical uptake, and off-target effects that we identified, these findings highlight the need for a rigorous safety assessment of nano-RNAi products prior to deployment. Specifically, the potential adverse effects of the nanocarrier and its components need to be considered.

Comparison of strategies for enhancing RNA interference efficiency in Ostrinia nubilalis

“…Chitosan/dsRNA nanoparticles were first developed to facilitate oral RNAi in Anopheles gambiae larvae 19 . The nanoparticles have been used to facilitate oral RNAi in Aedes aegypti larvae, 32,33 Chilio suppressalis larvae, 30 Chironomus tentans larvae, 34 Penaeus vannamei shrimp, 35 and Caenorhabditis elegans nematodes 36 . In C. suppressalis , chitosan/dsRNA nanoparticles enhanced dsRNA stability and uptake, resulting in enhanced RNAi efficiency 30 .…”

“…In C. suppressalis , chitosan/dsRNA nanoparticles enhanced dsRNA stability and uptake, resulting in enhanced RNAi efficiency 30 . In addition, evidence from C. elegans indicates that uptake of chitosan/dsRNA nanoparticles occurs through clathrin‐mediated endocytosis 36 . Initial testing of chitosan/dsRNA nanoparticles on ECB larvae indicated that oral RNAi efficiency was not enhanced (unpublished).…”

Endocytosis: The Nanoparticle and Submicron Nanocompounds Gateway into the Cell

“…Chitosan Submicron NPs (CSNPs) Chitosan [(1, 4)-2-amino-2-deoxy-d-glucan] (CS) is a linear polyaminosaccharide that is obtained by N-deacetylation of chitin [24]. Most of the chitosan submicron NPs (CSNPs) mainly enter the cells by CME, independently of their size, as happens for 250 nm sized ones in macrophage murine cell line RAW 264.7 [25] or 15.6 ± 3.5 nm sized in Caenorhabditis elegans [26]. However, others with a size close to or below 200 nm can enter the cells by macropinocytosis and CVME, this one to a lesser extent in human cervical carcinoma HeLa cells [27].…”

Control of postharvest fungal diseases in fruits using external application of RNAi

“…Theerawanitchpan et al (2012) demonstrated the nanoencapsulation of long dsRNA (>100 bp) with chitosan, and its quarternized derivative (QCH4) were efficient in improving the stability of dsRNA and in controlling the shrimp virus in Spodoptera frugiperda 9 cells. In another study, Lichtenberg et al (2019) found that chitosan/dsRNA polyplex nanoparticles were efficient in reducing the growth rate and reproduction of the nematode Caenorhabditis elegans.…”