Abstract
Mosquito-borne diseases are a major threat to human health and are responsible for millions of deaths globally each year. Vector control is one of the most important approaches used in reducing the incidence of these diseases. However, increasing mosquito resistance to chemical insecticides presents challenges to this approach. Therefore, new strategies are necessary to develop the next generation vector control methods. Because of the target specificity of dsRNA, RNAi-based control measures are an attractive alternative to current insecticides used to control disease vectors. In this study, Chitosan (CS) was cross-linked to sodium tripolyphosphate (TPP) to produce nano-sized polyelectrolyte complexes with dsRNA. CS-TPP-dsRNA nanoparticles were prepared by ionic gelation method. The encapsulation efficiency, protection of dsRNA from nucleases, cellular uptake, in vivo biodistribution, larval mortality and gene knockdown efficiency of CS-TPP-dsRNA nanoparticles were determined. The results showed that at a 5:1 weight ratio of CS-TPP to dsRNA, nanoparticles of less than 200 nm mean diameter and a positive surface charge were formed. Confocal microscopy revealed the distribution of the fed CS-TPP-dsRNA nanoparticles in midgut, fat body and epidermis of yellow fever mosquito, Aedes aegypti larvae. Bioassays showed significant mortality of larvae fed on CS-TPP-dsRNA nanoparticles. These assays also showed knockdown of a target gene in CS-TPP-dsRNA nanoparticle fed larvae. These data suggest that CS-TPP nanoparticles may be used for delivery of dsRNA to mosquito larvae.
Highlights
Mosquitoes affect human health by transmitting deadly diseases such as malaria, Chikungunya, dengue, and yellow fever
The CS-TPP-double-stranded RNA (dsRNA) nanoparticles were prepared by ionic gelation method by cross-linking the negatively charged phosphate groups of TPP with the positively charged amino groups of CS
The size of the particles increased from CS-TPP ratio of 1:1 to 7:1 except at a ratio of 5:1 where the particle size was lower than those formed by all other ratios tested
Summary
Mosquitoes affect human health by transmitting deadly diseases such as malaria, Chikungunya, dengue, and yellow fever. The efficacy of these methods has been decreasing over time due to the widespread development of resistance by mosquitoes to chemical insecticides and growing environmental concerns of residual toxicity[2] In this context, RNA interference (RNAi), a sequence-specific, post-transcriptional gene silencing method that is triggered by the introduction of double-stranded RNA (dsRNA) may help[3]. DsRNA must overcome specific obstacles such as poor cellular internalization, rapid degradation by nucleases, and limited blood stability[11] Another limitation for successful RNAi in insects is the lack of conserved dsRNA transporter genes, resulting in a poor systemic RNAi response[12]. Developing a safe and effective dsRNA carrier system for delivery into target cells of the cytoplasm is critical in order to investigate its potential application for control of insect pests and disease vectors. Positively charged CS-TPP-dsRNA nanoparticles were prepared to deliver dsRNA to induce RNAi in the yellow fever mosquito, Aedes aegypti
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