A novel UTMD system facilitating nucleic acid delivery into MDA-MB-231 cells.

Hui Zhang,Zhiyi Chen,Yue Li,Yi Wang,Fang Rao,Chun Liufu

doi:10.1042/bsr20192573

Abstract

Gene therapy is emerging as a promising method for the treatment of various diseases. The safe and efficient delivery of therapeutic nucleic acids is a gene therapy prerequisite. Ultrasound, particularly in combination with microbubbles composed of biocompatible materials such as lipid, PLGA and chitosan, is a novel non-viral tool for gene transportation. Under ultrasound irradiation, microbubbles explode and generate pores in the cell membrane. Hence, genes can enter cells more easily. In order to transfect nucleic acids into MDA-MB-231 cells in a low-cost and non-viral manner for further breast cancer gene therapy studies, we explored ultrasound targeted microbubble destruction (UTMD) technology and evaluated the efficiency and safety of the delivery of plasmid encoding enhanced green fluorescent protein (pEGFP) and a microRNA-34a (miR-34a) mimic by UTMD. Sonovitro ultrasonic apparatus was employed to generate ultrasonic field, which was developed by our group. Ultrasonic parameters, including acoustic intensity (AI), exposure time (ET) and duty cycle (DC), were optimized at 0.6 W/cm2 AI, 20 s ET and 20% DC, the cell viability was not obviously impaired. Under these conditions, the UTMD-mediated transfection efficiency of pEGFP was greater than 40%. In addition to plasmid DNA, an miR-34a mimic was also successfully introduced into the cytoplasm by UTMD and found to inhibit proliferation, induce apoptosis of MDA-MB-231 cells and regulate downstream molecules. The present study indicates that further in vivo UTMD-mediated gene therapy studies are warranted.

Highlights

With the rapid development of molecular biology, increasing number of gene mutations and alterations associated with diseases, such as cancer and inherited diseases, have been discovered
When acoustic intensity (AI) was fixed at 0.6 W/cm2, duty cycle (DC) was fixed at 20% and 10 μl of cationic microbubble (CMB) were added, exposure time (ET) had no obvious effect on cell viability (Figure 2A)
When AI was fixed at 0.6 W/cm2, and ET was fixed at 20 s, DC over 20% impaired obviously cell viability (Figure 2C)

Summary

Introduction

With the rapid development of molecular biology, increasing number of gene mutations and alterations associated with diseases, such as cancer and inherited diseases, have been discovered. Gene therapy represents a promising means by which therapeutic genetic materials can be introduced into target cells to cure diseases [1]. Several preclinical or clinical gene therapy studies for the treatment of breast cancer have been launched [2,3]. Despite the great promise of gene therapy, the safe and efficient delivery of therapeutic genetic materials remains the foremost challenge to be overcome. Viral vectors and non-viral vectors are available to transfect foreign nucleic acids into target cells in vitro or in vivo. Non-viral vectors, with the advantages of low cytotoxicity and immunogenicity, large gene payloads and ease of preparation, are increasingly being recognized as promising gene vehicles. Low transfection efficiency poses one of the primary challenges for the extensive application of non-viral vectors [4,5]

Methods

Results

Conclusion