Ultrasound-mediated gene delivery into suspended plant cells using polyethyleneimine-coated mesoporous silica nanoparticles

Abstract

Sonoporation, ultrasound-mediated membrane perforation can potentially puncture plasma membrane and rigid cell wall on presumably reversible basis which benefit gene transfection and plant biotechnology. Herein, positively charged poly-ethyleneimine (PEI)-coated mesoporous silica nanoparticles (MSNs) with an average diameter of 100 ± 8.7 nm was synthesized for GUS-encoding plasmid delivery into the suspended tobacco cells using the ultrasound treatment. The overall potential of PEI-MSN for DNA adsorption was measured at 43.43 μg DNA mg^-1 PEI-MSNs. It was shown that high level of sonoporation may adversely upset the cell viability. Optimal conditions of ultrasonic treatment are obtained as 8 min at 3 various intensities of 160, 320 and 640 W. Histochemical staining assay was used to follow the protein expression. It was shown that PEI-coated MSNs efficiently transfer the GUS-encoding plasmid DNA into the tobacco cells. The results of this study showed that ultrasonic treatment provides an economical and straightforward approach for gene transferring into the plant cells without any need to complicated devices and concerns about safety issues.

Keywords: GUS-encoding plasmid DNA; Gene transfection; Mesoporous silica nanoparticles; Ultrasonic treatment.

Graphical abstract

Fig. 1

Preparation of suspension cultures from callus. a) Picking up ~6 g callus of tobacco, transfering the callus part in 100 mL Erlenmeyer flask containing MS medium and covering the flask with sterile parafilm. b) Growing the cultures in shaking incubator under optimum conditions. c) First culture of suspended cells in a liquid medium. d) Last subculture of cell suspensions.

Fig. 2

A. Scanning transmission electron microscopy (STEM) images of MSNs. 2B Particle size distribution of MSNs.

Fig. 3

Adsorption/desorption isotherms of MSNs and PEI-MSNs.

Fig. 4

FTIR spectra of a bare MSNs b AAS-MSNs c carboxyl-MSNs d PEI-MSNs samples.

Fig. 5

Adsorption isotherm of hctDNA on PEI-MSNs. Adsorption tests were performed using 2 mg PEI-MSNs in 1.5 mL Tris-HCl buffer (0.1 M, pH 8) for 90 min.

Fig. 6

A Light microscopy images of tobacco cells after ultrasonic treatment at various intensities and times, a Untreated cells b 160 W, 5 min. c 160 W, 8 min. d 160 W, 10 min. e 320 W, 5 min. f 320 W, 8 min. g 320 W, 10 min. h 640 W, 5 min. i 640 W, 8 min. j 640 W, 10 min. (Dashed circles indicate the damaged regions of cells). 6B The results of cellular viability assessment in tobacco cells following ultrasound treatment with different ultrasonic intensities and times. Error bars represent standard error of sample mean in plot and * Indicates statistically significant difference between different treatment intensities (p < 0.01).

Fig. 7

A Gel electrophoresis test indicate the binding pBI221 plasmid onto PEI-MSNs. Lane 1, DNA marker; Lane 2, free pBI221 plasmid with 10 µg mL⁻¹ concentration; Lane 3, pBI221 plasmid solution with an estimated concentration of 0.8 µg mL⁻¹ (the supernatant resulted from plasmid adsorption samples); Lane 4, pDNA-PEI-MSNs conjugate before enzymatic digestion. Lane 5, the unloaded PEI-MSNs. 7B Agarose gel electrophoresis assay of pDNA and pDNA-PEI-MSNs conjugate; Lane 1, DNA marker; Lane 2, free pDNA (6 kbp); Lane 3, pDNA-PEI-MSNs conjugate after enzymatic digestion with faint electrophoresis shift.

Fig. 8

GUS gene transfection efficiency of tobacco cells in a suspended culture in terms of GUS enzyme specific activity. GUS enzyme activity difference is significant for samples a, b, c and d.* indicates statistically significant difference between various treatment groups (p < 0.01).