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Novel mechanism of gene transfection by low-energy shock wave.

Ha CH, Lee SC, Kim S, Chung J, Bae H, Kwon K - Sci Rep (2015)

Bottom Line: Furthermore SW-induced siRNA transfection was not mediated by SW-induced sonoporation, but by microparticles (MPs) secreted from the cells.Interestingly, the transfection effect of the siRNAs was transferable through the secreted MPs from human umbilical vein endothelial cell (HUVEC) culture medium after treatment with SW, into HUVECs in another culture plate without SW treatment.In this study, we suggest for the first time a mechanism of gene transfection induced by low-energy SW through secreted MPs, and show that it is an efficient physical gene transfection method in vitro and represents a safe therapeutic strategy for site-specific gene delivery in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul, 138-736, Korea.

ABSTRACT
Extracorporeal shock wave (SW) therapy has been studied in the transfection of naked nucleic acids into various cell lines through the process of sonoporation, a process that affects the permeation of cell membranes, which can be an effect of cavitation. In this study, siRNAs were efficiently transfected into primary cultured cells and mouse tumor tissue via SW treatment. Furthermore SW-induced siRNA transfection was not mediated by SW-induced sonoporation, but by microparticles (MPs) secreted from the cells. Interestingly, the transfection effect of the siRNAs was transferable through the secreted MPs from human umbilical vein endothelial cell (HUVEC) culture medium after treatment with SW, into HUVECs in another culture plate without SW treatment. In this study, we suggest for the first time a mechanism of gene transfection induced by low-energy SW through secreted MPs, and show that it is an efficient physical gene transfection method in vitro and represents a safe therapeutic strategy for site-specific gene delivery in vivo.

No MeSH data available.


Related in: MedlinePlus

Effects of low-energy level SW on in vitro gene transfection into endothelial cells.(a–c) siRNAs for VEGFR2 and VE-cadherin were added to HUVEC culture medium. Cells were treated with SW at 0.04 mJ/mm2 with 1,000 shots for 3 min (a) or at the energies indicated with 1,000 shots for 3 min (b and c). Representative immunoblots and quantitative data are shown (n = 3). *p < 0.05 versus the control group (no SW treatment). Error bars represent standard deviation (SD). (d) A fluorescent dye-labeled duplex was added to HUVEC culture medium followed by low-energy level SW treatment (0.04 mJ/mm2). (e and f) HUVECs were transfected with Cy3-labeled VEGFR-2 siRNA (e) or a vector encoding full-length enhanced green fluorescence protein (pEGFP-N1, 4.7 kbp) (f) by SW treatment (0.04 mJ/mm2). Lipofectamine served as the positive control. Cells were fixed, and transfection of siRNAs or the vector was visualized by fluorescence microscopy. Nuclei were stained with DAPI (blue).
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f1: Effects of low-energy level SW on in vitro gene transfection into endothelial cells.(a–c) siRNAs for VEGFR2 and VE-cadherin were added to HUVEC culture medium. Cells were treated with SW at 0.04 mJ/mm2 with 1,000 shots for 3 min (a) or at the energies indicated with 1,000 shots for 3 min (b and c). Representative immunoblots and quantitative data are shown (n = 3). *p < 0.05 versus the control group (no SW treatment). Error bars represent standard deviation (SD). (d) A fluorescent dye-labeled duplex was added to HUVEC culture medium followed by low-energy level SW treatment (0.04 mJ/mm2). (e and f) HUVECs were transfected with Cy3-labeled VEGFR-2 siRNA (e) or a vector encoding full-length enhanced green fluorescence protein (pEGFP-N1, 4.7 kbp) (f) by SW treatment (0.04 mJ/mm2). Lipofectamine served as the positive control. Cells were fixed, and transfection of siRNAs or the vector was visualized by fluorescence microscopy. Nuclei were stained with DAPI (blue).

Mentions: The first stage was to confirm the in vitro efficacy of SW-induced siRNA transfection into HUVECs (Fig. 1a). siRNAs for VEGFR2 and vascular endothelial cadherin (VE-cadherin) were added to HUVEC culture medium for transfection and treated with SW (0.04 mJ/mm2) with 1,000 shots for 3 min, and incubated at 37 °C. Transfection of siRNAs specifically targeting human VEGFR2 and VE-cadherin was dramatically reduced in HUVECs following SW treatment (0.04 mJ/mm2), indicating that siRNA transfection in primary cultured cells was induced by SW treatment (Fig. 1a). Next, the transfection efficiency of SW was compared with Lipofectamine. SW in the range of 0.02–0.06 mJ/mm2 enabled delivery of VEGFR2 siRNA into HUVECs, compared with Lipofectamine (Fig. 1b,c). The transfection efficiencies of Cy3-labeled VEGFR-2 siRNAs were comparable (Fig. 1b–e).


Novel mechanism of gene transfection by low-energy shock wave.

Ha CH, Lee SC, Kim S, Chung J, Bae H, Kwon K - Sci Rep (2015)

Effects of low-energy level SW on in vitro gene transfection into endothelial cells.(a–c) siRNAs for VEGFR2 and VE-cadherin were added to HUVEC culture medium. Cells were treated with SW at 0.04 mJ/mm2 with 1,000 shots for 3 min (a) or at the energies indicated with 1,000 shots for 3 min (b and c). Representative immunoblots and quantitative data are shown (n = 3). *p < 0.05 versus the control group (no SW treatment). Error bars represent standard deviation (SD). (d) A fluorescent dye-labeled duplex was added to HUVEC culture medium followed by low-energy level SW treatment (0.04 mJ/mm2). (e and f) HUVECs were transfected with Cy3-labeled VEGFR-2 siRNA (e) or a vector encoding full-length enhanced green fluorescence protein (pEGFP-N1, 4.7 kbp) (f) by SW treatment (0.04 mJ/mm2). Lipofectamine served as the positive control. Cells were fixed, and transfection of siRNAs or the vector was visualized by fluorescence microscopy. Nuclei were stained with DAPI (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525295&req=5

f1: Effects of low-energy level SW on in vitro gene transfection into endothelial cells.(a–c) siRNAs for VEGFR2 and VE-cadherin were added to HUVEC culture medium. Cells were treated with SW at 0.04 mJ/mm2 with 1,000 shots for 3 min (a) or at the energies indicated with 1,000 shots for 3 min (b and c). Representative immunoblots and quantitative data are shown (n = 3). *p < 0.05 versus the control group (no SW treatment). Error bars represent standard deviation (SD). (d) A fluorescent dye-labeled duplex was added to HUVEC culture medium followed by low-energy level SW treatment (0.04 mJ/mm2). (e and f) HUVECs were transfected with Cy3-labeled VEGFR-2 siRNA (e) or a vector encoding full-length enhanced green fluorescence protein (pEGFP-N1, 4.7 kbp) (f) by SW treatment (0.04 mJ/mm2). Lipofectamine served as the positive control. Cells were fixed, and transfection of siRNAs or the vector was visualized by fluorescence microscopy. Nuclei were stained with DAPI (blue).
Mentions: The first stage was to confirm the in vitro efficacy of SW-induced siRNA transfection into HUVECs (Fig. 1a). siRNAs for VEGFR2 and vascular endothelial cadherin (VE-cadherin) were added to HUVEC culture medium for transfection and treated with SW (0.04 mJ/mm2) with 1,000 shots for 3 min, and incubated at 37 °C. Transfection of siRNAs specifically targeting human VEGFR2 and VE-cadherin was dramatically reduced in HUVECs following SW treatment (0.04 mJ/mm2), indicating that siRNA transfection in primary cultured cells was induced by SW treatment (Fig. 1a). Next, the transfection efficiency of SW was compared with Lipofectamine. SW in the range of 0.02–0.06 mJ/mm2 enabled delivery of VEGFR2 siRNA into HUVECs, compared with Lipofectamine (Fig. 1b,c). The transfection efficiencies of Cy3-labeled VEGFR-2 siRNAs were comparable (Fig. 1b–e).

Bottom Line: Furthermore SW-induced siRNA transfection was not mediated by SW-induced sonoporation, but by microparticles (MPs) secreted from the cells.Interestingly, the transfection effect of the siRNAs was transferable through the secreted MPs from human umbilical vein endothelial cell (HUVEC) culture medium after treatment with SW, into HUVECs in another culture plate without SW treatment.In this study, we suggest for the first time a mechanism of gene transfection induced by low-energy SW through secreted MPs, and show that it is an efficient physical gene transfection method in vitro and represents a safe therapeutic strategy for site-specific gene delivery in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul, 138-736, Korea.

ABSTRACT
Extracorporeal shock wave (SW) therapy has been studied in the transfection of naked nucleic acids into various cell lines through the process of sonoporation, a process that affects the permeation of cell membranes, which can be an effect of cavitation. In this study, siRNAs were efficiently transfected into primary cultured cells and mouse tumor tissue via SW treatment. Furthermore SW-induced siRNA transfection was not mediated by SW-induced sonoporation, but by microparticles (MPs) secreted from the cells. Interestingly, the transfection effect of the siRNAs was transferable through the secreted MPs from human umbilical vein endothelial cell (HUVEC) culture medium after treatment with SW, into HUVECs in another culture plate without SW treatment. In this study, we suggest for the first time a mechanism of gene transfection induced by low-energy SW through secreted MPs, and show that it is an efficient physical gene transfection method in vitro and represents a safe therapeutic strategy for site-specific gene delivery in vivo.

No MeSH data available.


Related in: MedlinePlus