Efficient Delivery of dsRNA and DNA in Cultured Silkworm Cells for Gene Function Analysis Using PAMAM Dendrimers System.

Chenchen Lu,Zhaoming Dong,Zhiqing Li,Pengchao Guo,Guanwang Shen,Ping Zhao,Li Chang,Qingyou Xia

doi:10.3390/insects11010012

Abstract

Polyamidoamine (PAMAM) dendrimers are emerging as intriguing nanovectors for nucleic acid delivery because of their unique well-defined architecture and high binding capacity, which have been broadly applied in DNA- and RNA-based therapeutics. The low-cost and high-efficiency of PAMAM dendrimers relative to traditional liposomal transfection reagents also promote their application in gene function analysis. In this study, we first investigated the potential use of a PAMAM system in the silkworm model insect. We determined the binding property of G5-PAMAM using dsRNA and DNA in vitro, and substantially achieved the delivery of dsRNA and DNA from culture medium to both silkworm BmN and BmE cells, thus leading to efficient knockdown and expression of target genes. Under treatments with different concentrations of G5-PAMAM, we evaluated its cellular cytotoxicity on silkworm cells, and the results show that G5-PAMAM had no obvious toxicity to cells. The presence of serum in the culture medium did not affect the delivery performance of DNA and dsRNA by G5-PAMAM, revealing its convenient use for various purposes. In conclusion, our data demonstrate that the PAMAM system provides a promising strategy for delivering dsRNA and DNA in cultured silkworm cells and promote its further application in individuals.

Highlights

A variety of carriers have been developed for the delivery of nucleic acids into cells to achieve gene regulation
To evaluate the ability of G5-PAMAM dendrimer to deliver nucleic acids in cultured silkworm cells, we first studied the complex formation between our target molecules and G5-PAMAM in vitro using gel mobility-shift assays
Consistent with a previous report that PAMAM could form stable complexes with small interfering RNA (siRNA) [23], G5-PAMAM was able to interact with the double-stranded RNA (dsRNA) of translationally controlled tumor protein (TCTP), which led to retarded dsRNA migration in agarose gel (Figure 1A)

Summary

Introduction

A variety of carriers have been developed for the delivery of nucleic acids into cells to achieve gene regulation. Two kinds of carriers exist: viral and non-viral vectors [1]. Viral vectors have been widely used due to their high efficiency in transferring nucleic acids to cells. The risk of inducing immune responses and mutagenic effects, has limited their application in vivo [2,3]. Non-viral vectors have received increased attention [4,5]. Various types of non-viral vectors have emerged, such as cationic molecular, liposomes, polymeric nanoparticles, polymeric micelles, polyamidoamine (PAMAM) dendrimers, solid-lipid nanoparticles, silica nanoparticles, and carbon nanotubes, which have demonstrated different abilities in delivering DNA, small interfering RNA (siRNA), and double-stranded RNA (dsRNA) [6,7,8,9]

Methods

Results

Conclusion