Application of Direct Sonoporation from a Defined Surface Area of the Peritoneum: Evaluation of Transfection Characteristics in Mice

Abstract

In the present study, we developed a sonoporation system, namely "direct sonoporation", for transfecting the peritoneum from a defined surface area to avoid systematic side effects. Here, the transfection characteristics are explained because there is less information about direct sonoporation. Naked pDNA and nanobubbles were administered to diffusion cell attached to the visceral and parietal peritoneum from the liver and peritoneal wall surface, respectively. Then, ultrasound was irradiated. Direct sonoporation showed a higher transfection efficacy at the applied peritoneum site from the liver surface while other sites were not detected. Moreover, transgene expression was observed in the peritoneal mesothelial cells (PMCs) at the applied peritoneum site. No abnormality was observed in the inner part of the liver. Although transgene expression of the visceral peritoneum was tenfold higher than that of the parietal peritoneum, transgene expression was observed in the PMCs on both the applied peritoneum sites. These results suggest that direct sonoporation is a site-specific transfection method of the PMCs on the applied peritoneum site without transgene expression at other sites and show little toxicity in the inner tissues at the applied site via cavitation energy. This information is valuable for the development of an intraperitoneal sonoporation device for treatment of peritoneal diseases such as peritoneal fibrosis.

Keywords: gene transfection; nanobubbles; peritoneal mesothelial cells; sonoporation; ultrasound.

Figure 1

Scheme of the direct sonoporation to the defined area of the peritoneum from the peritoneal tissues. A glass-made cylindrical diffusion cell (in diameter: 9 mm) was attached to the peritoneal tissue surface by using surgical adhesive. A mixture consisting of pDNA and nanobubbles was directly added to the diffusion cell. Five min after administration, US (frequency, 2.04 MHz; duty, 50%; burst rate,10 Hz; intensity, 4.0 W/cm²) was irradiated using a Sonopore-4000 with a probe (diameter: 6 mm). Thus, direct sonoporation might be a site-specific transfection method into the peritoneal mesothelial cells (PMCs) on the applied peritoneum from the peritoneal tissue. In addition, under the PMCs, direct sonoporation showed little transgene expression and toxicity.

Figure 2

Effect of direct sonoporation to the visceral peritoneum from the liver surface. (a) Effects of the transfection conditions (applied volume, dose of nanobubbles, dose of pDNA, and duration of US irradiation). Administration of the mixture of CMV-Luciferase (the firefly luciferase gene expression vector driven by a cytomegalovirus (CMV) promoter) and nanobubbles followed by ultrasound (US) irradiation. Data is represented as mean + SE of 5–6 experiments. Statistical comparison was performed by Tukey’s multiple comparison test (* p < 0.05). (b–c): Transfection efficacy (b) and site-specificity (c) by direct sonoporation under the optimized conditions. Three-hundred microliters of the mixture of pCMV-Luciferase (30 µg) and nanobubbles (50 μg) was administrated to the applied site of the liver surface, then US were irradiated (4.0 W/cm²) for 3 min. Data is represented as mean + SD of 3–4 experiments. Statistical comparison was performed by Tukey’s multiple comparison test (b) and Dunnett’s multiple comparison test (c). (* p < 0.05, ** p < 0.01, *** p < 0.001)

Figure 2

Effect of direct sonoporation to the visceral peritoneum from the liver surface. (a) Effects of the transfection conditions (applied volume, dose of nanobubbles, dose of pDNA, and duration of US irradiation). Administration of the mixture of CMV-Luciferase (the firefly luciferase gene expression vector driven by a cytomegalovirus (CMV) promoter) and nanobubbles followed by ultrasound (US) irradiation. Data is represented as mean + SE of 5–6 experiments. Statistical comparison was performed by Tukey’s multiple comparison test (* p < 0.05). (b–c): Transfection efficacy (b) and site-specificity (c) by direct sonoporation under the optimized conditions. Three-hundred microliters of the mixture of pCMV-Luciferase (30 µg) and nanobubbles (50 μg) was administrated to the applied site of the liver surface, then US were irradiated (4.0 W/cm²) for 3 min. Data is represented as mean + SD of 3–4 experiments. Statistical comparison was performed by Tukey’s multiple comparison test (b) and Dunnett’s multiple comparison test (c). (* p < 0.05, ** p < 0.01, *** p < 0.001)

Figure 3

Evaluation of spatial distribution of transgene expression by direct sonoporation to the visceral peritoneum from the liver surface. Three-hundred microliters of a mixture containing pZsGreen1-N1 (30 µg) and nanobubbles (50 μg) was administrated to the applied site of the liver surface and US were irradiated (4.0 W/cm²) for 3 min. A view of the 3D observation at the non-applied site (a) and the applied site (b) after pZsGreen1-N1 transfection. Blue, green, and red signal show nuclei, transgene expression of pZsGreen1-N1 and DiI-stained surface cells. Objective lens used was 20x dry lens.

Figure 4

Identification of transgene-positive cell after pZsGreen1-N1 (green) transfection by direct sonoporation to the visceral peritoneum from the liver surface. Blue, green, and red signal show nuclei, transgene expression of pZsGreen1-N1 and Alexa-647^® (anti-mesothelin antibody). Observation was done using the 40× oil-immersion lens as 2D image (a), 3D image (b) and X-Z and Y-Z plane image (c).

Figure 5

Assessment of liver impairments after direct sonoporation to the visceral peritoneum from the liver surface. (a) Serum alanine aminotransferase (ALT) activities after transfection. ALT activities were determined at 3, 6, and 24 h after each treatment. Each value is represented as mean ± SD of 4 experiments. *** p < 0.01 vs sham (open surgery) on Tukey’s multiple test. (b) H&E staining of the liver. These evaluations were performed at 6 h after treatment and at 24 h after transfection. Scale bar represents 100 µm.

Figure 6

Characterization of the transgene expression by direct sonoporation of the parietal peritoneum from the peritoneal wall surface. a: transfection efficacy of the nanobubbles with US irradiation b: site-specificity. Thirty micrograms of pCMV-Luciferase and nanobubbles (50 µg) were administrated to applied site of the peritoneal wall surface, i.e., (a) total volume of 300 µL. Then, US was irradiated 4.0 W/cm² for 3 min. Six hours after transfection, the luciferase activity was measured. Data is represented as mean + SD of four experiments. Statistical comparison was performed by Tukey’s multiple comparison test (a) and Dunnett’s multiple comparison test (b) (*** p < 0.001). (c,d) the distribution of transgene expression on the parietal peritoneum by 3D observation. The depth of the transgene expression at the non-applied site (c) and the applied site (d) after pZsGreen1-N1 transfection. Blue, green, and red signal show nuclei, transgene expression of ZsGreen1-N1 and DiI-stained surface cells. The objective lens used was the 20× dry lens.