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A Novel Ultrasound Microbubble Carrying Gene and Tat Peptide Preparation and Characterization

Posted Nov 30, 2009 Updated Apr 7, 2023
By Jianli Ren MD , Chuan Shan Xu PhD , Zhi Yi Zhou MD , Yong Zhang MM , Xing Sheng Li MD , Yuan Yi Zheng MD , Hai Tao Ran MD , Zhi Gang Wang MB
10 min read

Rationale and Objectives

Ultrasound-targeted microbubble destruction is a promising technology for the targeted gene delivery. The purpose of the present study is to prepare a novel lipid ultrasound microbubble-carrying gene and transactivating transcriptional activator (Tat) peptide and to investigate its transfection effect in vivo.

Methods and Materials

Lipid ultrasound microbubbles were prepared using mechanical vibration, and the appearance, distribution, concentration, diameter, and zeta potential of the lipid ultrasound microbubbles were measured. The efficiencies of the microbubble carrying gene and Tat peptide were investigated using the fluorospectrophotometer. Contrast-enhanced ultrasonography was performed on normal rabbits to observe the duration and intensity of enhancement in myocardium. Quantitative analysis was detected using the DFY Ultrasound Image Analyzer. Transfection in vivo was performed using the CGZZ ultrasound gene transfection instrument. The expression of enhanced green fluorescent protein in the organs was observed using confocal laser scanning microscope.

Results

The diameter of the lipid microbubbles carrying gene and Tat was (2.3 ± 0.4) μm, the concentration was (3.1 ± 0.4) ×10 9 /mL, and Zeta potential was (2.0 ± 0.1) mV. The gene encapsulation efficiency of the lipid ultrasound microbubbles was 32%, and the Tat encapsulation efficiency was 35%. In vivo experiment showed that lipid ultrasound microbubbles could enhance the echo intensity and transfection efficiency.

Conclusion

Lipid microbubbles containing gene and Tat peptide can be used as a new vehicle for gene transfection.

Gene therapy shows considerable promise as a novel treatment modality for human diseases . Gene delivery technique includes viral vector and nonviral vector, but the technique is limited by low safety of viral vector and poor efficiency of nonviral vector. As a negatively charged macromolecule, DNA can not enter cells actively and is rapidly degraded by nucleases . Therefore, developing an efficient gene delivery system is necessary.

Besides the well-known application of microbubbles as contrast agents for diagnostic ultrasound (US), microbubbles have also been demonstrated to be an effective technique for targeted gene delivery . Several studies investigated the mechanisms for US-targeted microbubble destruction (UTMD) enhancing gene delivery into cells. Electron microscopy demonstrated pore formation on cell membranes immediately after destruction of microbubbles. This phenomenon was transient and the pores disappeared after 24 hours. Such “sonoporation” effects may facilitate gene to entry into the cell. It was then postulated that these transient holes in the cell surface caused by UTMD resulted in a rapid translocation of gene from outside to cytoplasm . Christiansen et al showed that plasmid DNA remained intact after being loaded onto and subsequently released from microbubbles by US irradiation, and confirmed that UTMD was a novel and highly efficient gene delivery carrier. The technique involves intravenous or intraarterial injection of echogenic microbubbles combining with genetic materials, and then US is used to destroy microbubbles in the targeted tissue. Use of UTMD provides many desirable characteristics for gene therapy, including low toxicity, low immunogenicity, low invasiveness, the potential for repeated application, organ specificity, and broad applicability to sonographically accessible targets. However, gene material released from microbubbles destructed by US radiation only passively enters into targeted cells; therefore, the transfection efficiency is still limited.

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Materials and methods

Plasmid DNA

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Tat Peptide

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Cell Culture and Transfection

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Preparation of Microbubbles

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Characterization of the Microbubbles

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Confocal Laser Scanning Microscopy

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Encapsulation Efficiency of Plasmid DNA and Tat Peptide in Microbubbles

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Contrast-enhanced Imaging in vivo

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Quantitative Analysis

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Transfection in vivo

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Statistical Analysis

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Results

Tat Peptide Synthesis and Bioactivity Assay

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Figure 1, Assessment and bioactivity of Tat peptide. (a) High-performance liquid chromatograph (HPLC) image of Tat peptide; the Tat peptide was isolated at the detection wavelength of 210 nm using C18 pillar of HPLC; (b) chromatogram of Tat peptide, the collected main peak of Tat peptide as above was detected using matrix-assisted laser desorption/ionization time of flight mass spectrometry; (c) the image of EGFP expression in human umbilical vein endothelial cells (HUVEC) transfected with 1 μg plasmid internal ribosome entry site 2 (pIRES2)-enhanced green fluorescent protein (EGFP)-hepatocyte growth factor (HGF); (d) bioactivity image of Tat peptide in HUVEC transfected with Tat peptide (6 μg) and pIRES2-EGFP-HGF (1 μg). Cells were cultured for 24 h and EGFP expression was observed using fluorescent microscopy (×200).

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Physical Characterization of Microbubbles

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Table 1

Physical Characteristics of Lipid Ultrasound Contrast Agent (X¯¯¯±s) (

X

¯

±

s

)

Group Diameter (μm) Electric potential (mV) Concentration (×10 9 /mL) Blanked microbubbles 2.2 ± 0.7 −15.7 ± 0.9 3.3 ± 0.3 Microbubbles carrying gene 2.2 ± 0.7 −18.4 ± 2.0 2.9 ± 0.3 Microbubbles carrying

gene and Tat 2.3 ± 0.4 +2.0 ± 0.1 ∗ 3.1 ± 0.4

Compared with the blanked microbubbles.

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Confocal Laser Scanning Microscopy

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Figure 2, Confocal laser microscopy image of the microbubbles carrying gene and Tat peptide (×400). The microbubbles carrying gene and Tat peptide were prepared in the presence of propidium iodide (PI) to stain for plasmid DNA and FITC-labeled Tat peptide. (a) Fluorescence for PI; (b) fluorescence for FITC; (c) fluorescence for both.

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Encapsulation Efficiencies of Plasmid DNA and Tat Peptide in Microbubbles

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Figure 3, The standard curve. FITC-Tat and propidium iodide (PI)-DNA were diluted with phosphate-buffered saline (PBS) to a series of different concentration. PBS was used as the control. The optical density values of FITC-Tat and PI-DNA were determined using fluorospectrophotometer. (a) The standard curve of PI; (b) the standard curve of FITC.

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Heart Contrast Imaging In Vivo and Quantitative Analysis

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Figure 4, Heart contrast imaging and quantitative analysis image of the echo intensity in vivo. (a) Precontrast echo intensity of chambers heart; (b) postcontrast echo intensity of chambers heart; (c) precontrast echo intensity of myocardium; (d) postcontrast echo intensity of myocardium.

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Transfection in vivo

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Figure 5, Confocal laser microscopy image of the pIRES2-EGFP-HGF expression in the rat myocardium (×400). Frozen sections were observed using confocal laser microscope. (a) expression of pIRES2-EGFP-HGF in the rat myocardium of US + MB group; (b) transmission image in the rat myocardium of US + MB group; (c) expression of pIRES2-EGFP-HGF in the rat myocardium of US + HGF-MB group; (d) transmission image in the rat myocardium of US + HGF-MB group; (e) expression of pIRES2-EGFP-HGF in the rat myocardium of US + HGF-Tat-MB group; (f) transmission image in the rat myocardium of US + HGF-Tat-MB group. US, ultrasound; HGF, hepatocyte growth factor; Tat, Tat peptide; MB, microbubbles; pIRES2, plasmid internal ribosome entry site 2.

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Discussion

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Academic Radiology, Volume 16, Issue 12
Journals General Radiology
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