Kwon Seok Min, Nam Hae Yun, Nam Taehwan, Park Kyeongsoon, Lee Seulki, Kim Kwangmeyung, Kwon Ick Chan, Kim Joon, Kang Dongmin, Park Jae Hyung, Jeong Seo Young
Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, South Korea.
J Control Release. 2008 May 22;128(1):89-97. doi: 10.1016/j.jconrel.2008.02.004. Epub 2008 Feb 19.
To make stable and biocompatible non-viral gene carriers for therapeutic gene therapy, we developed a cationic lipid-based emulsion (CLE) prepared by an oil-in-water (O/W) emulsion method, wherein squalene oil was used as an oil core and the cationic lipid, 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP), was employed as an emulsifier. To evaluate in vivo characteristics such as toxicity and time-dependent gene expression, a bioluminescence reporter gene in pCMV-luc plasmid DNA was simply mixed with CLE in aqueous condition, resulting in a CLE/DNA complex. The CLE/DNA complex was optimized to form a compact and stable nano-sized particle by adding different amounts of plasmid DNA, and an optimal cationic lipid-to-DNA (C/D) weight ratio of 4 was identified. Freshly prepared CLE/DNA complex, with a C/D of 4, showed a high transfection efficiency and minimal cytotoxicity in vitro, compared to controls of a liposome (DOTAP)/DNA complex and a branched poly(ethyleneimine) (Mw=25 kDa) (bPEI)/DNA complex, respectively. The in vivo characteristics of the CLE/DNA complex were evaluated after intravenous injection into Balb/c mice. Time-dependent gene expression data in vivo were obtained using a non-invasive, whole animal bioluminescence imaging system. These data showed that the CLE/DNA complex offered prolonged high-level gene expression for 1 week, particularly in the liver and spleen. On the other hand, the controls of DOTAP/DNA complex and bPEI/DNA complex showed a relatively lower gene expression, because of the unstable and toxic properties of the control carriers. Our in vivo gene expression data demonstrate the potential of the CLE/DNA complex as a non-viral gene carrier for in vivo gene delivery.
为制备用于治疗性基因治疗的稳定且生物相容的非病毒基因载体，我们开发了一种基于阳离子脂质的乳液（CLE），它通过水包油（O/W）乳液法制备，其中角鲨烯油用作油核，阳离子脂质1,2 - 二油酰基 - sn - 甘油 - 3 - 三甲基氯化铵（DOTAP）用作乳化剂。为评估其体内特性，如毒性和时间依赖性基因表达，将pCMV - luc质粒DNA中的生物发光报告基因在水性条件下简单地与CLE混合，形成CLE/DNA复合物。通过添加不同量的质粒DNA，对CLE/DNA复合物进行优化以形成紧凑且稳定的纳米级颗粒，并确定了最佳的阳离子脂质与DNA（C/D）重量比为4。与脂质体（DOTAP）/DNA复合物和支链聚（乙烯亚胺）（Mw = 25 kDa）（bPEI）/DNA复合物的对照相比，新制备的C/D为4的CLE/DNA复合物在体外显示出高转染效率和最小细胞毒性。将CLE/DNA复合物静脉注射到Balb/c小鼠体内后评估其体内特性。使用非侵入性的全动物生物发光成像系统获得体内时间依赖性基因表达数据。这些数据表明，CLE/DNA复合物可提供长达1周的延长的高水平基因表达，特别是在肝脏和脾脏中。另一方面，DOTAP/DNA复合物和bPEI/DNA复合物的对照显示出相对较低的基因表达，因为对照载体具有不稳定和有毒的特性。我们的体内基因表达数据证明了CLE/DNA复合物作为用于体内基因递送的非病毒基因载体的潜力。
