Functionalized Multi-Wall Carbon Nanotubes Enhance Transfection and Expression Efficiency of Plasmid DNA in Fish Cells.

Guanglu Liu,Gaoxue Wang,Xiaobo Yu,Yang Hu,Yuan Wang,Bin Zhu

doi:10.3390/ijms17030335

Abstract

DNA vaccines are considered to be the most promising method against infectious diseases in the aquaculture industry. In the present study, we investigated the potency of ammonium group-functionalized multi-walled carbon nanotubes (MWCNTs) in enhancing the transfection and expression efficiency of plasmid DNA (pEGFP-vp5) in Ctenopharyngodon idellus kidney (CIK) cells. Agarose gel shift assay results show that ammonium group-functionalized carbon nanotubes are able to condense DNA in varying degrees. Scanning electron microscope (SEM) images shows that CIK cells show a great affinity for MWCNTs-NH3+ and the CNTs covering the cell surface tend to orient their tips perpendicularly to the cell surface, and appear to be “needle-pricking the cells”. Transmission electron microscope (TEM) images confirmed that MWCNTs-NH3+ penetrate the cell membranes and are widely dispersed in the CIK cell. Real-time PCR was used to detect the transfection efficiency through the expression of the outer capsid protein (VP5). The results showed that the MWCNTs-NH3+:DNA complexes are able to transfect CIK cells effectively at different charge ratio than naked DNA. Subsequent studies confirmed that both functional groups and charge ratio are important factors that determine the transfection efficiency of plasmid DNA. All these results indicated that MWCNTs-NH3+:DNA complexes could be suitable for developing DNA vaccine for the control of virus infection in the aquaculture industry.

Highlights

During the past decades, a continuous growth in the aquaculture industry is emerging worldwide, with ever increasing cultured fish production from marine and freshwater environments
Our study provides a fundamental basis for the potential application of Carbon nanotubes (CNTs)-DNA vaccine delivery system in controlling infectious disease in the aquaculture industry
In order to determine whether it is possible to use f -multi-walled carbon nanotubes (MWCNTs) for intracellular delivery applications in fish cells we studied their interaction with Ctenopharyngodon idellus kidney (CIK) cells; the interaction of the f -MWCNTs with cells was studied using Scanning electron microscope (SEM) and Transmission electron microscope (TEM)

Summary

Introduction

A continuous growth in the aquaculture industry is emerging worldwide, with ever increasing cultured fish production from marine and freshwater environments. Among the problems that the aquaculture industry has to address, infectious pathogens are the most important [2,3,4]. In 1993, the economic losses caused by the infectious pathogens are more than ten percent of the total value of the fishery industry [5], and the loss ratio is increased gradually in the last decades. In 2010, aquaculture in China suffered production losses of 1.7 million tons caused by infectious pathogen diseases [6]. Economical and environmentally-friendly methods which can prevent or control infectious diseases are essential to the continued development of aquaculture

Methods

Results

Discussion

Conclusion