Microtubule involvement in the intracellular dynamics for gene transfection mediated by cationic liposomes

Abstract

7 To improve the efficiency of the transfection, it is essential to elucidate the mechanism not only as to how exogenous DNA is taken up and transported to the nucleus, but also how DNA degrades, since inhibition of the degradation of exogenous DNA could contribute to the enhancement of transfection efficiency, as well as facilitation of the nuclear translocation. Liposome-DNA complex is taken up by endocytosis, and the internalized DNA is released from endosomes by their acidification. [8][9][10] However, the details of intracellular events during lipofection are still unknown. Although many lines of evidence suggest that microtubules are responsible for the intracellular trafficking of materials internalized by endocytosis, 11,12 no direct observation as to how microtubules are involved in the intracellular dynamics of exogenous liposome-DNA complex has been reported. In the present study, therefore, we undertook simultaneous visualization of lipo- some-DNA complexes and microtubules in living cultured cells, and showed that microtubules are involved in the intracellular transport of the exogenous liposome-DNA complex.In order to investigate the contribution of microtubules to the transport of exogenous DNA and liposomes, we tested the effects of nocodazole and taxol on transfection efficiency in COS-7 cells by luciferase assay. For transfection with luciferase plasmids using cationic lipids, pGL3 (Promega, Madison, WI, USA) were complexed with liposomes consisting of DOPE (dioleoylphosphatidylethanolamine; Avanti Polar Lipids, Alabaster, AL, USA) and HyC-Chol (DOPE:HyC-Chol = 2:3 (mol/mol)) to form liposome-DNA complexes. Liposome-DNA complexes were incubated with the cells for 4 h at 37°C, and then the cells were washed and cultured in DMEM for another 24 h at 37°C, followed by a luciferase assay. When cells were treated with nocodazole, which disrupts microtubules, the efficiency of transfection increased dose-dependently and about doubled at 10 m, as shown in Figure 1a. Taxol, which stabilizes microtubules, also enhanced transfection efficiency in a dose-dependent manner (Figure 1b). The concentrations at which these reagents had effects were within the range of their working concentrations to affect microtubules specifically. Therefore, the results suggest that modification of the polymerization states of microtubules changes the efficiency of gene transfection. To examine the possibility that these reagents increased the ability of cells to interact with exogenous liposome-DNA complexes, cells were incubated with the liposome-DNA complexes containing NBD-PE (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl )-1,2-dihexadecanoyl-sn-glycero-3-phospho-

Synergistic antitumoral effect of vinblastine and HSV-Tk/GCV gene therapy mediated by albumin-associated cationic liposomes

“…This observation is consistent with the reported role of actin rather than that of microtubules as the element responsible for the initial binding and uptake by endocytosis [34,35]. Vinblastine probably enhances transgene expression by decreasing the intracellular traffic of lipoplexes to lysosomes and/or by activating the nuclear factor NF-κB and consequently transgene transcription [23][24][25][26][27]. The lower accumulation of HSA-EPOPC:Chol/DNA lipoplexes observed in the endolysosomal pathway in the presence of vinblastine (Fig.…”

“…As the microtubules are crucial for the occurrence of chromosomal segregation during anaphase in mitosis, the resulting depolymerization of this cytoskeleton element results in a G2-M cell cycle arrest and consequently in cell death [23]. However, microtubules are not only involved in the chromosomal segregation, but also play important roles in intracellular dynamics, including the transport of lipoplexes to lysosomes after their uptake through endocytosis [24][25][26]. The compounds that affect the depolymerization state of microtubules may be used to enhance the transfection capacity of lipoplexes, by helping to overcome the endolysosomal entrapment, and consequently DNA degradation at the lysosomal level [24][25][26].…”

“…However, microtubules are not only involved in the chromosomal segregation, but also play important roles in intracellular dynamics, including the transport of lipoplexes to lysosomes after their uptake through endocytosis [24][25][26]. The compounds that affect the depolymerization state of microtubules may be used to enhance the transfection capacity of lipoplexes, by helping to overcome the endolysosomal entrapment, and consequently DNA degradation at the lysosomal level [24][25][26]. Moreover, it has been reported that these agents can increase transfection efficiency of cationic liposome/DNA complexes by activating the sequencespecific transcription factor NF-κB [23,24,27].…”

Intracellular trafficking of nonviral vectors

“…177 In this study, the microtubule disrupting drug, nocodazole, and the microtubule stabilizing drug, taxol, both increased transfection effiency. Nocodazole induced scattering and noncolocalization of liposomes and lysosomes, whereas taxol resulted in relocation of lysosomes only around the nucleus and noncolocalization with microtubules or liposomes.…”

Quantitating intracellular transport of polyplexes by spatio-temporal image correlation spectroscopy

“…We fit the temporal autocorrelation curve with both equations to see whether both a diffusive term and a directed velocity value could be determined for the polyplexes. These values can ideally be used to characterize a cell's microtubule dynamics, because the microtubules and their motors are instrumental for endosomal trafficking (33)(34)(35). The velocity component may provide a measure of the activity of the microtubule motors as the polyplex cargoes are transported through the cytoplasm.…”