Enhanced effect of nuclear localization signal peptide during ultrasound-targeted microbubble destruction-mediated gene transfection

Abstract

Ultrasound-targeted microbubble destruction (UTMD) can promote the entry of plasmid DNA (pDNA) into the cell cytoplasm, by increasing the permeability of the cell membrane. But the transfection efficiency remains low due to inability of the pDNA to enter the nucleus. Various methods have been explored to improve the UTMD transfection efficiency, but with little success. In cells, the classic nuclear localization signal (cNLS) peptide is an amino acid sequence that signals proteins that are due for nuclear transport. The present study aimed to investigate whether binding of a cNLS peptide to the pDNA may improve the transfection efficiency of UTMD. Four experimental groups were analyzed: Control group (UTMD + pDNA), group with cNLS (UTMD + pDNA + cNLS), group with mutated NLS (mNLS; UTMD + pDNA + mNLS), and group with cNLS and the nuclear import blocker, wheat germ agglutinin (WGA; UTMD + pDNA + cNLS + WGA). The NLS was labeled by fluorescein isothiocyanate, whereas pDNA was labeled with Cy3. Different molar ratios were tested for the NLS and pDNA combination in order to achieve optimal binding of the two molecules. Human umbilical vein endothelial cells were then transfected using the optimum ultrasonic irradiation parameters and NLS/pDNA molar ratio. At 6 h post-transfection, the rates of Cy3-labeled pDNA inside the cells and their nuclei were detected by flow cytometry and laser confocal microscopy, and the cellular vs. nuclear uptake of pDNA was calculated. In order to further evaluate the effect of NLS on UTMD-mediated gene transfection, the transfection efficiency and relative expression levels of mRNA and protein were detected at 48 h post-transfection. The results demonstrated that the optimal molar ratio of NLS with pDNA was 104:1. The rates of pDNA successful entry into the cell and nucleus were significantly higher in the cNLS group compared with the control group. The transfection efficiency, and relative expression levels of mRNA and protein from the plasmid were significantly increased in the cNLS group compared with the control group. The mNLS group displayed no significant difference compared with the control group, while the WGA group exhibited significant inhibition in most indicators of transfection efficiency compared to the cNLS group. These results suggest that combining a cNLS peptide with pDNA during UTMD-mediated transfection significantly improved transfection efficiency. Thus, a cNLS peptide may be an important mediator and a new strategy in enhancing the efficiency of UTMD-mediated gene transfection.

Enhancement of Angiogenesis by Ultrasound-Targeted Microbubble Destruction Combined with Nuclear Localization Signaling Peptides in Canine Myocardial Infarction

“…The constructed plasmid was transformed into E. coli strain DH5a for amplification and purified using the Wizard™ Maxiprep DNA Purification System (Promega Corporation, Madison, WI, USA). The NLS peptide was purified by high-performance liquid chromatography, and the optimal molar ratio of NLS and Ang-1-EGFP plasmid was 10 4 : 1, according to our previous experiments [ 16 ]. The suspension of NLS and Ang-1-EGFP plasmid was incubated at room temperature for 30 minutes and added to 1 ml of SonoVue microbubble suspension.…”

“…The cDNA synthesis was carried out with approximately 2 μ g of RNA using the PrimeScript™RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Real-time PCR was conducted using SYBR Green with the primers (5′-GAAGGAAACCGAGCCTATTCAC-3′ and 5′-CCACAAGCATCAAACCACCA-3′) synthesized by Invitrogen (Shanghai, China) according to the previous study [ 16 ]. The mRNA level was normalized by GAPDH.…”