Cationic liposome-mediated gene delivery: Biophysical study and mechanism of internalization

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Abstract

To identify factors affecting cationic liposome-mediated gene delivery efficiency, we studied the relationship between the biophysical characteristics of liposome/DNA complexes (lipoplexes) at different (+/−) charge ratios, their structures as monitored by atomic force microscopy (AFM), and their mechanism(s) of internalization into the cells. Significant changes were observed in the particle size and zeta potential of liposomes and their structures assessed by AFM upon addition of DNA, which depended on (+/−) charge ratios. AFM images showed that lipoplexes were formed from extensively fused and apparently homogeneous lipid particles encapsulating DNA. Lipoplexes were found to internalize the cells through the endocytosis pathway. Lipoplex–cell fusion was found to occur mainly at the plasma membrane level; however, this lipoplex–cell membrane fusion was found to be essential for the uptake of the large particles. A new perspective for the internalization of large lipoplex particles into cytoplasm is discussed.

Section snippets

Materials

The cationic lipid DC-6-14 was a generous gift from Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan). DOPE was kindly donated by the Nippon Oil and Fats Co., Ltd. (Tokyo, Japan). Cholesterol was purchased from Wako Pure Chemicals (Osaka, Japan). The DNA expression plasmid pGL3-Control under SV40 promoter and enhancer and luciferase reporter gene were purchased from Promega (Madison, WI). Rh-PE and NBD-PE came from Avanti Polar Lipids (Alabster, AL). Antimycin A, NaF, NaN3, and Z-Phe-Phe-Gly were

Effect of (+/−) charge ratio on lipoplex physico chemical properties

Significant changes occur in the physico chemical features of cationic liposomes upon complexation with the negatively charged DNA [18], [19]. These changes are likely to alter the interaction of cationic liposomes with cell membranes. The zeta potential of the cationic liposomes and lipoplexes provides a measure of their net charge, while dynamic light scattering gives their mean particle size.

Fig. 1 shows the values obtained for mean particle size and zeta potential of lipoplexes at different

Discussion

Despite the growing body of research on the biophysical and biochemical factors involved in cationic liposome-mediated gene transfer, such as structural features [20], [21], cellular association [12], [21], and intracellular trafficking [13], the relationships between these factors and the mechanism(s) of lipoplex internalization into cells are not yet fully understood. Deep understanding of these factors is of great interest for developing efficient lipid-mediated gene delivery. In the present

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