Efficient gene transfection to the brain with ultrasound irradiation in mice using stabilized bubble lipopolyplexes prepared by the surface charge regulation method

Abstract

Introduction: We previously developed anionic ternary bubble lipopolyplexes, an ultrasound-responsive carrier, expecting safe and efficient gene transfection. However, bubble lipopolyplexes have a low capacity for echo gas (C₃F₈) encapsulation (EGE) in nonionic solution such as 5% glucose. On the other hand, we were able to prepare bubble lipopolyplexes by inserting phosphate-buffered saline before C₃F₈ encapsulation. Surface charge regulation (SCR) by electrolytes stabilizes liposome/plasmid DNA (pDNA) complexes by accelerated membrane fusion. Considering these facts, we hypothesized that SCR by electrolytes such as NaCl would promote C₃F₈ encapsulation in bubble lipopolyplexes mediated by accelerated membrane fusion. We defined this hypothesis as SCR-based EGE (SCR-EGE). Bubble lipopolyplexes prepared by the SCR-EGE method (SCR-EGE bubble lipopolyplexes) are expected to facilitate the gene transfection because of the high amount of C₃F₈. Therefore, we applied these methods for gene delivery to the brain and evaluated the characteristics of transgene expression in the brain.

Methods: First, we measured the encapsulation efficiency of C₃F₈ in SCR-EGE bubble lipopolyplexes. Next, we applied these bubble lipopolyplexes to the mouse brain; then, we evaluated the transfection efficiency. Furthermore, three-dimensional transgene distribution was observed using multicolor deep imaging.

Results: SCR-EGE bubble lipopolyplexes had a higher C₃F₈ content than conventional bubble lipopolyplexes. In terms of safety, SCR-EGE bubble lipopolyplexes possessed an anionic potential and showed no aggregation with erythrocytes. After applying SCR-EGE bubble lipopolyplexes to the brain, high transgene expression was observed by combining with ultrasound irradiation. As a result, transgene expression mediated by SCR-EGE bubble lipopolyplexes was observed mainly on blood vessels and partially outside of blood vessels.

Conclusion: The SCR-EGE method may promote C₃F₈ encapsulation in bubble lipopolyplexes, and SCR-EGE bubble lipopolyplexes may be potent carriers for efficient and safe gene transfection in the brain, especially to the blood vessels.

Keywords: brain; bubble lipopolyplex; echo gas; gene delivery; spatial distribution.

Figure 1

Physicochemical property of SCR-EGE bubble lipopolyplexes. Notes: (A) Effect of NaCl concentration on the size and zeta potential of bubble lipopolyplexes. Bubble lipopolyplexes were prepared with pDNA, protamine, and ALs in a 5% glucose solution. Then, varying amounts of NaCl were added, followed by C₃F₈ encapsulation. The size and zeta potential were measured at both pre- and post-gas encapsulation (n = 3). Data are represented as mean ± SD. ^*P < 0.05 and ^**P < 0.01 compared with the size of bubble lipopolyplexes without NaCl (0 mM NaCl; Tukey’s multiple comparison test). (B) Effect of NaCl concentration on C₃F₈ encapsulation by bubble lipopolyplexes. The amount of C₃F₈ in bubble suspensions was measured by GC–MS (n = 3). Data are represented as mean ± SD. ^*P < 0.05, Tukey’s multiple comparison test. (C) Effect of NaCl concentration on the appearance of bubble lipopolyplexes. Abbreviations: ALs, anionic liposomes; EGE, echo gas encapsulation; GC–MS, gas chromatography–mass spectroscopy; pDNA, plasmid DNA; SCR, surface charge regulation; SCR-EGE, SCR-based EGE; SD, standard deviation.

Figure 2

Evaluation of membrane fusion between liposomes. Notes: FRET analysis to evaluate membrane fusion among ALs during C₃F₈ encapsulation. Mean emission spectra of (A) pre- and (B) post-C₃F₈ encapsulation are shown. The excitation wavelength in both conditions was 484 nm. Abbreviations: ALs, anionic liposomes; FRET, fluorescent resonance energy transfer.

Figure 3

Transfection efficiency of bubble lipopolyplexes. Notes: Transfection efficiency of bubble lipopolyplexes in the brain (A) and other organs (B). Mice were intravenously administered with bubble lipopolyplexes with or without NaCl (pDNA, 50 μg), followed by ultrasound irradiation (duration, 120 s; intensity, 1 W/cm²) of the brain. Six hours after transfection, luciferase activities were analyzed in the brain and other organs (n = 3 or 4). Data are represented as mean ± SD. ^**P < 0.01, Tukey’s multiple comparison test. Abbreviations: pDNA, plasmid DNA; RLU, relative light units; US, ultrasound.

Figure 4

Optimization of transfection condition. Notes: Luciferase activities under different transfection conditions, ultrasound duration (A), ultrasound intensity (B), and dose of administered pDNA (C) were analyzed 6 h after transfection. Mice were intravenously administered with SCR-EGE bubble lipopolyplexes containing 6 mM NaCl, followed by ultrasound irradiation of the brain. (A) pDNA (50 μg) was administered, and ultrasound irradiation was applied at 1 W/cm² at different durations. (B) pDNA (50 μg) was administered, and ultrasound irradiation was applied at various intensities for 10 s. (C) Various doses of pDNA were administered, and ultrasound irradiation was applied at 1 W/cm² for 10 s (n = 3). Data are represented as mean ± SD. Abbreviations: EGE, echo gas encapsulation; pDNA, plasmid DNA; SCR, surface charge regulation; SCR-EGE, SCR-based EGE; SD, standard deviation.

Figure 5

Distribution of transgene expression in the brain. Notes: Observation of the ZsGreen1 distribution in the brain using tissue clearing. Mice were transfected with conventional or SCR-EGE bubble lipopolyplexes carrying pZsGreen1-N1 under the following conditions: ultrasound duration, 10 s; intensity, 1 W/cm²; pDNA dose; 50 μg. Twenty-four hours after transfection, mice were fixed and cleared using CUBIC (A–G), ScaleSQ (H–J), or Clear^T2 (K–M). Transmission color images of the brain after tissue clearing using CUBIC (A), ScaleSQ (H), or Clear^T2 (K) are shown. Confocal microscopy images of brains transfected with conventional (B, I, L) or SCR-EGE bubble lipopolyplexes (C, J, M) are shown. The transmission color images and confocal microscopy image of the lungs and liver cleared with CUBIC are shown as control organs (D–G). Green, red, and blue signals indicate ZsGreen1 expression, DiI-labeled blood vessels, and DAPI staining, respectively. Arrows denote ZsGreen1 expression outside of blood vessels. Abbreviations: CUBIC, clear, unobstructed brain imaging cocktails; DAPI, 4′,6-diamidino-2-phenylindole; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; EGE, echo gas encapsulation; pDNA, plasmid DNA; SCR, surface charge regulation; SCR-EGE, SCR-based EGE.

Figure 5

Distribution of transgene expression in the brain. Notes: Observation of the ZsGreen1 distribution in the brain using tissue clearing. Mice were transfected with conventional or SCR-EGE bubble lipopolyplexes carrying pZsGreen1-N1 under the following conditions: ultrasound duration, 10 s; intensity, 1 W/cm²; pDNA dose; 50 μg. Twenty-four hours after transfection, mice were fixed and cleared using CUBIC (A–G), ScaleSQ (H–J), or Clear^T2 (K–M). Transmission color images of the brain after tissue clearing using CUBIC (A), ScaleSQ (H), or Clear^T2 (K) are shown. Confocal microscopy images of brains transfected with conventional (B, I, L) or SCR-EGE bubble lipopolyplexes (C, J, M) are shown. The transmission color images and confocal microscopy image of the lungs and liver cleared with CUBIC are shown as control organs (D–G). Green, red, and blue signals indicate ZsGreen1 expression, DiI-labeled blood vessels, and DAPI staining, respectively. Arrows denote ZsGreen1 expression outside of blood vessels. Abbreviations: CUBIC, clear, unobstructed brain imaging cocktails; DAPI, 4′,6-diamidino-2-phenylindole; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; EGE, echo gas encapsulation; pDNA, plasmid DNA; SCR, surface charge regulation; SCR-EGE, SCR-based EGE.

Figure 6

Sustained transgene expression mediated by CpG-depleted vector. Notes: Sustained transgene expression in the brain using conventional or SCR-EGE bubble lipopolyplexes carrying pCMV-Luc or CpG-depleted vectors. Each vector was transfected into the brain under the following conditions: ultrasound duration, 10 s; intensity, 1 W/cm²; pDNA dose; 50 μg. At 1, 7, 14, and 28 days after administration, mice were sacrificed and luciferase activity was measured in their brains (n = 4). Data are represented as mean ± SD. ^*P < 0.05 and ^**P < 0.01, Tukey’s multiple comparison test. Abbreviations: EGE, echo gas encapsulation; pCMV-Luc, vector coding firefly luciferase under cytomegalovirus promoter; pDNA, plasmid DNA; RLU, relative light units; SCR, surface charge regulation; SCR-EGE, SCR-based EGE.