Zhang Li, Liu Yingying, Xiang Guangya, Lv Qing, Huang Gui, Yang Yali, Zhang Yanrong, Song Yue, Zhou Huan, Xie Mingxing
Key Laboratory of Molecular Imaging of Hubei Province, Department of Ultrasound, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
J Huazhong Univ Sci Technolog Med Sci. 2011 Feb;31(1):39-45. doi: 10.1007/s11596-011-0147-3. Epub 2011 Feb 19.
This study aimed to examine the preparation of cationic lipid microbubble (CLM), and evaluate its physical and chemical properties and toxicity, measure the gene transfection efficiency by ultrasound triggered microbobble destruction (UTMD) in combination with CLM. The CLM was prepared by the method of the thin film hydration, and its morphology was observed under the electron microscopy at 1st, 3rd, 7th, 10th, and 14th day after preparation, respectively. The size, Zeta potential and stability of CLM were tested. The acute toxicity of CLM was assessed. The green fluorescent protein gene (EGFP) transfection efficiency was evaluated. The experiment grouping was as follows: naked plasmid group (P group), ultrasonic irradiation plus naked plasmid group (P-US group), naked plasmid plus CLM group (P-CLM group), naked plasmid plus ultrasound and CLM group (UTMD group). The expression of EGFP was detected by fluorescent microscopy and flow cytometry. The results showed that CLMs were spherical in shape, with the similar size and good distribution degree under the light and electron microscopies. The size of CLMs was varied from 250.4±88.3 to 399.0±99.8 nm and the Zeta potential of CLMs from 18.80±4.97 to 20.1±3.1 mV. The EGFP expression was the strongest in the UTMD group, followed by the P-CLM group, P-US group and P group. Flow cytometry results were consistent with those of fluorescent microscopy. The transfection efficiency was substantially increased in the P-US group, P-CLM group and UTMD group as compared with that in the P group, almost 7 times, 10 times and 30 times higher than that in the P group respectively. It is suggested that CLMs prepared by the method of thin film hydration are uniform in diameter, and proved non-toxic. UTMD combined with CLM can significantly increase the transfection efficiency of EGFP to targeted cells.
本研究旨在制备阳离子脂质微泡（CLM），评估其理化性质和毒性，通过超声触发微泡破坏（UTMD）联合CLM来测定基因转染效率。采用薄膜水化法制备CLM，分别在制备后的第1天、第3天、第7天、第10天和第14天在电子显微镜下观察其形态。检测CLM的大小、Zeta电位和稳定性。评估CLM的急性毒性。评估绿色荧光蛋白基因（EGFP）的转染效率。实验分组如下：裸质粒组（P组）、超声照射加裸质粒组（P-US组）、裸质粒加CLM组（P-CLM组）、裸质粒加超声和CLM组（UTMD组）。通过荧光显微镜和流式细胞术检测EGFP的表达。结果显示，CLM呈球形，在光学显微镜和电子显微镜下大小相似且分布均匀。CLM的大小在250.4±88.3至399.0±99.8 nm之间，Zeta电位在18.80±4.97至20.1±3.1 mV之间。UTMD组中EGFP表达最强，其次是P-CLM组、P-US组和P组。流式细胞术结果与荧光显微镜结果一致。与P组相比，P-US组、P-CLM组和UTMD组的转染效率显著提高，分别比P组高近7倍、10倍和30倍。提示采用薄膜水化法制备的CLM直径均匀，且无毒。UTMD联合CLM可显著提高EGFP对靶细胞的转染效率。
