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首页> 美国卫生研究院文献>Theranostics >Ultrasound-Mediated Gene Delivery with Cationic Versus Neutral Microbubbles: Effect of DNA and Microbubble Dose on In Vivo Transfection Efficiency
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Ultrasound-Mediated Gene Delivery with Cationic Versus Neutral Microbubbles: Effect of DNA and Microbubble Dose on In Vivo Transfection Efficiency

机译：阳离子介导的中性微泡与超声介导的基因传递：DNA和微泡剂量对体内转染效率的影响。

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>Objective: To assess the effect of varying microbubble (MB) and DNA doses on the overall and comparative efficiencies of ultrasound (US)-mediated gene delivery (UMGD) to murine hindlimb skeletal muscle using cationic versus neutral MBs.>Materials and Methods: Cationic and control neutral MBs were characterized for size, charge, plasmid DNA binding, and ability to protect DNA against endonuclease degradation. UMGD of a codon optimized firefly luciferase (Fluc) reporter plasmid to endothelial cells (1 MHz, 1 W/cm², 20% duty cycle, 1 min) was performed in cell culture using cationic, neutral, or no MBs. In vivo UMGD to mouse hindlimb muscle was performed by insonation (1 MHz, 2 W/cm², 50% duty cycle, 5 min) after intravenous administration of Fluc combined with cationic, neutral, or no MBs. Gene delivery efficiency was assessed by serial in vivo bioluminescence imaging. Efficiency of in vivo UMGD with cationic versus neutral MBs was systematically evaluated by varying plasmid DNA dose (10, 17.5, 25, 37.5, and 50 µg) while maintaining a constant MB dose of 1x108 MBs and by changing MB dose (1x107, 5x107, 1x108, or 5x108 MBs) while keeping a constant DNA dose of 50 µg.>Results: Cationic and size-matched control neutral MBs differed significantly in zeta potential with cationic MBs being able to bind plasmid DNA (binding capacity of 0.03 pg/MB) and partially protect DNA from nuclease degradation while neutral MBs could not. Cationic MBs enhanced UMGD compared to neutral MBs as well as no MB and no US controls both in cell culture (P < 0.001) and in vivo (P < 0.05). Regardless of MB type, in vivo UMGD efficiency increased dose-dependently with DNA dose and showed overall maximum transfection with 50 µg DNA. However, there was an inverse correlation (ρ = -0.90; P = 0.02) between DNA dose and the degree of enhanced UMGD efficiency observed with using cationic MBs instead of neutral MBs. The delivery efficiency advantage associated with cationic MBs was most prominent at the lowest investigated DNA dose (7.5-fold increase with cationic versus neutral MBs at a DNA dose of 10 µg; P = 0.02) compared to only a 1.4-fold increase at a DNA dose of 50 µg (P < 0.01). With increasing MB dose, overall in vivo UMGD efficiency increased dose-dependently with a maximum reached at a dose of 1x108 MBs with no further significant increase with 5x108 MBs (P = 0.97). However, compared to neutral MBs, cationic MBs enhanced UMGD efficiency the most at low MB doses. Relative enhancement of UMGD efficiency using cationic over neutral MBs decreased from a factor of 27 for 1x107 MBs (P = 0.02) to a factor of 1.4 for 1x108 MBs (P < 0.01) and no significant difference for 5x108 MBs.>Conclusions: Cationic MBs enhance UMGD to mouse skeletal muscle relative to neutral MBs but this is dependent on MB and DNA dose. The enhancement effect of cationic MBs on UMGD efficiency is more evident when lower doses of MBs or DNA are used, whereas the advantage of cationic MBs over neutral MBs is substantially reduced in the presence of excess MBs or DNA.
机译：>目的：使用阳离子与中性MBs评估不同微泡（MB）和DNA剂量对超声（US）介导的基因向小鼠后肢骨骼肌的基因传递（UMGD）总体和比较效率的影响>材料和方法：阳离子和对照中性MB的大小，电荷，质粒DNA结合以及保护DNA防止核酸内切酶降解的能力均得到了表征。使用阳离子，中性或无MBs在细胞培养中对内皮细胞进行密码子优化的萤火虫萤光素荧光素酶（Fluc）报告质粒的UMGD（1 MHz，1 W /cm²，20％占空比，1分钟）。静脉内施用Fluc与阳离子，中性或无MBs组合后，通过超声（1 MHz，2 W /cm²，50％占空比，5分钟）对小鼠后肢肌肉进行体内UMGD。通过连续体内生物发光成像评估基因递送效率。通过改变质粒DNA剂量（10、17.5、25、37.5和50 µg），同时保持1x10 8 MB的恒定MB剂量，通过系统地评估阳离子和中性MB的体内UMGD的效率。更改MB剂量（1x10 7 ，5x10 7 ，1x10 8 或5x10 8 MB），同时保持恒定DNA剂量为50 µg。>结果：阳离子型和大小匹配的对照中性MBs的Zeta电位差异显着，阳离子MBs能够结合质粒DNA（结合能力为0.03 pg / MB）并部分保护DNA核酸酶降解引起的，而中性MB则不能。与中性MB相比，阳离子MB增强了UMGD，并且在细胞培养（P <0.001）和体内（P <0.05）均没有MB和US对照。无论MB类型如何，体内UMGD效率均随DNA剂量的增加而呈剂量依赖性，并显示50μgDNA的总体最大转染率。但是，使用阳离子MB代替中性MB观察到的DNA剂量与UMGD效率提高之间存在负相关（ρ= -0.90; P = 0.02）。在最低的DNA剂量下，阳离子MB的传递效率优势最为明显（在10 mg的DNA剂量下，阳离子MB与中性MB相比增长7.5倍； P = 0.02），而在DNA剂量下仅为1.4倍剂量为50微克（P <0.01）。随着MB剂量的增加，总的体内UMGD效率随剂量的增加而增加，在1x10 8 MBs时达到最大值，而在5x10 8 MBs时没有进一步显着增加（P = 0.97）。但是，与中性MB相比，阳离子MB在低MB剂量下最大程度地提高了UMGD效率。与中性MB相比，使用阳离子的UMGD效率的相对提高从1x10 7 MB的27倍（P = 0.02）降低到1x10 8 MB的1.4倍（P <0.01），而5x10 8 MB则无显着差异。>结论：阳离子MB相对于中性MB增强了小鼠骨骼肌的UMGD，但这取决于MB和DNA剂量。当使用较低剂量的MBs或DNA时，阳离子MBs对UMGD效率的增强作用更加明显，而在存在过量MBs或DNA的情况下，阳离子MBs相对于中性MBs的优势将大大降低。

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