New chitosan nanobubbles for ultrasound-mediated gene delivery: preparation and in vitro characterization

Abstract

Background: The development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release.

Methods and results: Chitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions.

Conclusion: Based on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery.

Keywords: DNA; chitosan; gene carrier; nanobubbles; transfection; ultrasound.

Figure 1

Schematic apparatus used for transfection experiment in the presence of ultrasound.

Figure 2

Transmission electron microscopy image of (A) unloaded and (B) DNA-loaded chitosan nanobubbles.

Figure 3

Differential scanning calorimetry profile of chitosan solution and chitosan nanobubbles.

Figure 4

Electrophoresis of DNA-loaded chitosan nanobubbles on agarose gel.

Figure 5

In vitro pDNA release from DNA-loaded chitosan nanobubbles after exposure to ultrasound.

Figure 6

(A) COS7 cells exposed to 30 seconds of insonation in presence of pDNA-loaded nanobubbles carrying 10 μg/mL of pDNA and examined 24 hours post transfection by confocal laser scanning microscopy without fixation. (B) COS7 cells treated as in (A) but not sonified. (C) COS7 cells neither exposed to ultrasound nor to DNA-loaded nanobubbles. Note: The upper panels show fluorescence images while the lower panels show merged phase-contrast and fluorescence images.

Figure 7

Effect of ultrasound on COS7 viability.

Figure 8

COS7 viability assay after incubation with DNA-loaded chitosan nanobubbles.