Effective intraperitoneal gene transfection system using nanobubbles and ultrasound irradiation

Abstract

In this study, we demonstrate the low toxicity and highly efficient and spatially improved transfection of plasmid DNA (pDNA) with liposomal nanobubbles (bubble liposomes [BLs]) using ultrasound (US) irradiation in mice. Naked pDNA with BLs was intraperitoneally injected, followed by US irradiation. The injection volume, the duration of US irradiation, and the dose of BLs were optimized. Both BLs and US irradiation were essential to achieve high transgene expression from naked pDNA. We observed transgene expression in the entire peritoneal tissues, including the peritoneal wall, liver, spleen, stomach and small and large intestines. The area of transfection could be controlled with focused US irradiation. There were few changes in the morphology of the peritoneum, the peritoneal function or serum alanine aminotransferase levels, suggesting the safety of BLs with US irradiation. Using a tissue-clearing method, the spatial distribution of transgene expression was evaluated. BLs with US irradiation delivered pDNA to the submesothelial layer in the peritoneal wall, whereas transgene expression was restricted to the surface layer in the liver and stomach. Therefore, BLs with US irradiation could be an effective and safe method of gene transfection to the peritoneum.

Advances in Ex Situ Tissue Optical Clearing

“…Additional examination by injection of two plasmids, encoding ZsGreen and tdTomato fluorescent proteins revealed that ZsGreen can be imaged deeper into the tissue (1500 µm vs 1200 µm) with the latter being also less photostable, when placed in CUBIC reagent 2. These results are corroborated by a further study of the same group led by Nishimura et al . who also performed SeeDB and Clear T2 ‐based liver clearing post gene transfection using bubble liposomes with ultrasound irradiation, achieving only modest transparency allowing for ≈100 µm in depth examination of the peritoneal tissues.…”

Evaluation of the targeted delivery of 5-fluorouracil and ascorbic acid into the brain with ultrasound-responsive nanobubbles

“…L-Ascorbic acid was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and dissolved in water at 10 mg/mL. BLs composed of 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) (Avanti Polar Lipids, Inc., Alabaster, Alabama, USA) and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolaminemethoxy-polyethyleneglycol (DSPE-PEG (2k)-OMe) (NOF Corporation, Tokyo, Japan) (94:6 (m/m)) were prepared according to previous reports at 1 mg/mL in terms of their lipids [16,17,20,21]. To encapsulate the perfluoropropane gas into BLs, vials were vortexed in a sonicated bath (BRANSONIC B1200, Emerson Electric Co., St. Louis, MO, USA).…”

“…Our group has been developing DDS for liver [13], spleen [14], solid tumours [15,16], and peritoneal tissues [17] using ultrasound-responsive bubble formulations.…”

Determining Transgene Expression Characteristics Using a Suction Device with Multiple Hole Adjusting a Left Lateral Lobe of the Mouse Liver

“…We developed 3-dimensional multicolor deep imaging, applied with a combination of the tissue clearing methods, with observation by confocal laser microscopy. 16,18,19) This 3-dimensional multicolor deep imaging was then applied to analyze the spatial distribution of transgene expression in the suctioned liver. We evaluated the selectivity and depth of transgene expression using CUBIC reagents 20) that have high sensitivity for transgene expression.…”