General Characteristics of Microbubble-Adenovirus Vectors Carrying Genes

Abstract

Introduction: Transferring genes safely, targeting cells and achieving efficient transfection are urgent problems in gene therapy that need to be solved. Combining microbubbles (MBs) and viruses to construct double vectors has become a promising approach for gene delivery. Understanding the characteristic performance of MBs that carry genes is key to promoting effective gene transfer. Therefore, in this study, we constructed MB-adenovirus vectors and discussed their general characteristics.

Methods: We constructed MB-adenovirus vectors carrying the chemokine (C-X-C motif) ligand 12 (Cxcl12) and bone morphogenetic protein-2 (Bmp2) genes (pAd-Cxcl12 and pAd-Bmp2, respectively) to explore the general characteristics of double vectors carrying genes.

Results: The MB-adenovirus vectors had stable physical properties, and no significant differences in diameter, concentration, or pH were noted compared with naked MBs (p > 0.05). Flow cytometry and RT-PCR were used to detect the gene-loading capacity of MBs. The gene-loading efficiency of MBs increased with increasing virus amounts and was highest (91%) when 10.0 µL of virus was added. Beyond 10.0 µL of added virus, the gene-loading efficiency of MBs decreased with the continuous addition of virus. The maximum amounts of pAd-Cxcl12 and pAd-Bmp2 in 100 µL of MBs were approximately 14 and 10 µL, respectively.

Conclusions: This study indicates that addition of an inappropriate viral load will result in low MB loading efficiency, and the maximum amount of genes loaded by MBs may differ based on the genes carried by the virus.

Keywords: Gene vector; Gene-loading efficiency; Microbubbles; Ultrasound.

Figure 1

Binding of viruses to microbubbles (MBs). (a) Confocal laser scanning microscope bright-field images and the corresponding fluorescence image of MBs carrying the Cxcl12 or Bmp2 adenovirus after the addition of 0.625, 1.25, 2.5, 5, 10, 20 or 30 µL of virus. pAd-Cxcl12 was labeled with FITC, which emitted bright green fluorescence, and pAd-BMP2 was labeled with TRITC, which emitted red fluorescence. Bar, 20 µm. The proportions of MBs successfully carrying (b) pAd-Cxcl12 or (c) pAd-BMP2 among the total MBs when different volumes of virus were added are shown. n = 3; *: p < 0.05.

Figure 2

Physical characterization of microbubble (MB)-viral vectors. (a) The mean diameter, (b) concentration, and (c) pH value of MBs with or without virus were quantitatively measured for comparison. n = 3.

Figure 3

The capacity of microbubbles (MBs) loaded with Cxcl12 or Bmp2 adenovirus. (a) RT-PCR analysis of the gene-carrying capacity of MBs after addition of 0.625, 1.25, 2.5, 5, 10, 20 or 30 µL of adenoviral vectors carrying Cxcl12 or Bmp2 genes into 100 µL of MBs. The virus titer was 1 × 10⁹ pfu/mL. The bar graph shows the gene-loading amounts of the MBs. The line graph shows the gene-loading efficiency of the MBs, which was quantified by comparing the amount of DNA bound to MBs with the total amount of genes added. (b) Flow cytometry detection of the proportion of virus-carrying MBs, which is the proportion of MBs that successfully combined with adenovirus, and (c) a typical flow cytometry image of MBs carrying pAd-Cxcl12 (upper row) and pAd-Bmp2 (lower row). n = 3.