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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

Characterization of MPs isolated from HUVEC culture medium.Size distribution of MPs in culture medium was calculated by flow cytometry and NanoSight particle-tracking analysis (NTA). (a) Changes in MP number following SW treatment over time were determined by NTA. Size distribution of MPs; below 100 nm (green), 100–200 nm (blue), over 200 nm (red). All experiments were performed in triplicate. *p < 0.05 versus the control group (no SW treatment). Error bars represent SD. (b and c) The distribution of MPs in culture medium over time following SW treatment was analyzed by flow cytometry. An MP count was performed for 100 s. Relative sizes were calculated using SPHERO Nano Polystyrene & Nano Fluorescent Size Standard kits. Sizes of 100–300 nm are shown: 220 nm (green), 400–600 nm as 440 nm (purple), 700–900 nm as 880 nm (blue), and 1,000–1,900 nm as 1340 nm (red). (d) Representative transmission electron micrographs of MPs obtained from the medium (upper panel) and cell pellets (lower panel). (e) The presence of MPs within larger vesicles in the cytoplasm is shown by transmission electron microscopy. Samples were collected 1 h post-SW treatment.
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f5: Characterization of MPs isolated from HUVEC culture medium.Size distribution of MPs in culture medium was calculated by flow cytometry and NanoSight particle-tracking analysis (NTA). (a) Changes in MP number following SW treatment over time were determined by NTA. Size distribution of MPs; below 100 nm (green), 100–200 nm (blue), over 200 nm (red). All experiments were performed in triplicate. *p < 0.05 versus the control group (no SW treatment). Error bars represent SD. (b and c) The distribution of MPs in culture medium over time following SW treatment was analyzed by flow cytometry. An MP count was performed for 100 s. Relative sizes were calculated using SPHERO Nano Polystyrene & Nano Fluorescent Size Standard kits. Sizes of 100–300 nm are shown: 220 nm (green), 400–600 nm as 440 nm (purple), 700–900 nm as 880 nm (blue), and 1,000–1,900 nm as 1340 nm (red). (d) Representative transmission electron micrographs of MPs obtained from the medium (upper panel) and cell pellets (lower panel). (e) The presence of MPs within larger vesicles in the cytoplasm is shown by transmission electron microscopy. Samples were collected 1 h post-SW treatment.

Mentions: To understand the mechanisms of SW-regulation of the secretion of various MPs from cells, Nanoparticle Tracking Analysis (NTA) was performed using a NanoSight NS300 (Malvern Instruments, Malvern, UK). The results showed an increase in secreted MPs at 30 min to 1 h after SW treatment, followed by a slow decrease. Following SW treatment, a greater number of large-sized particles (>200 nm) relative to smaller particles was revealed (Fig. 5a). FACS analysis was carried out to investigate the large particles using a BD FACS Canto II (Beckman Coulter, Brea, CA, USA); the particles were sized according to standard-sized beads of 220, 440, 880 and 1,340 nm. The beads were run through the flow cytometer using the default settings to collect MP data, and from this, the mean FSC measurement was calculated34. Similar to the NTA results, the number of MPs increased significantly at 30 min post-SW treatment and decreased slowly thereafter. These results suggest that SW treatment induces the secretion of various MPs within a short time frame. Furthermore, analysis of the distribution of larger MPs showed a dramatic increase in MPs 220 nm and 440 nm in diameter immediately following SW treatment (Fig. 5b,c and Supplementary Fig. 4). NanoSight and FACS results showed that SW dramatically induced MPs of 200–500 nm diameter within 30 min to 1 h of treatment. Additionally, transmission electron microscopy revealed higher numbers of MPs in the culture medium (Fig. 5d), along with budding of the plasma membrane and multi-vesicular body (MVB) in HUVECs (Fig. 5e). The incorporation of siGlo post-SW treatment was also examined using a NanoSight (Malvern Instruments). The absolute number of MPs with a diameter >200 nm was lower than those with a diameter <200 nm; however, the larger MPs had a siGlo uptake ratio more than 10-fold that of MPs sized ≤100 nm, and of those 100–200 nm (Fig. 6a,b). Taken together with the results shown in Figs 3, 5 and 6, the MPs secreted following SW treatment may interact with naked siRNA and can be transfected into cells not directly treated with SW.


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)

Characterization of MPs isolated from HUVEC culture medium.Size distribution of MPs in culture medium was calculated by flow cytometry and NanoSight particle-tracking analysis (NTA). (a) Changes in MP number following SW treatment over time were determined by NTA. Size distribution of MPs; below 100 nm (green), 100–200 nm (blue), over 200 nm (red). All experiments were performed in triplicate. *p < 0.05 versus the control group (no SW treatment). Error bars represent SD. (b and c) The distribution of MPs in culture medium over time following SW treatment was analyzed by flow cytometry. An MP count was performed for 100 s. Relative sizes were calculated using SPHERO Nano Polystyrene & Nano Fluorescent Size Standard kits. Sizes of 100–300 nm are shown: 220 nm (green), 400–600 nm as 440 nm (purple), 700–900 nm as 880 nm (blue), and 1,000–1,900 nm as 1340 nm (red). (d) Representative transmission electron micrographs of MPs obtained from the medium (upper panel) and cell pellets (lower panel). (e) The presence of MPs within larger vesicles in the cytoplasm is shown by transmission electron microscopy. Samples were collected 1 h post-SW treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Characterization of MPs isolated from HUVEC culture medium.Size distribution of MPs in culture medium was calculated by flow cytometry and NanoSight particle-tracking analysis (NTA). (a) Changes in MP number following SW treatment over time were determined by NTA. Size distribution of MPs; below 100 nm (green), 100–200 nm (blue), over 200 nm (red). All experiments were performed in triplicate. *p < 0.05 versus the control group (no SW treatment). Error bars represent SD. (b and c) The distribution of MPs in culture medium over time following SW treatment was analyzed by flow cytometry. An MP count was performed for 100 s. Relative sizes were calculated using SPHERO Nano Polystyrene & Nano Fluorescent Size Standard kits. Sizes of 100–300 nm are shown: 220 nm (green), 400–600 nm as 440 nm (purple), 700–900 nm as 880 nm (blue), and 1,000–1,900 nm as 1340 nm (red). (d) Representative transmission electron micrographs of MPs obtained from the medium (upper panel) and cell pellets (lower panel). (e) The presence of MPs within larger vesicles in the cytoplasm is shown by transmission electron microscopy. Samples were collected 1 h post-SW treatment.
Mentions: To understand the mechanisms of SW-regulation of the secretion of various MPs from cells, Nanoparticle Tracking Analysis (NTA) was performed using a NanoSight NS300 (Malvern Instruments, Malvern, UK). The results showed an increase in secreted MPs at 30 min to 1 h after SW treatment, followed by a slow decrease. Following SW treatment, a greater number of large-sized particles (>200 nm) relative to smaller particles was revealed (Fig. 5a). FACS analysis was carried out to investigate the large particles using a BD FACS Canto II (Beckman Coulter, Brea, CA, USA); the particles were sized according to standard-sized beads of 220, 440, 880 and 1,340 nm. The beads were run through the flow cytometer using the default settings to collect MP data, and from this, the mean FSC measurement was calculated34. Similar to the NTA results, the number of MPs increased significantly at 30 min post-SW treatment and decreased slowly thereafter. These results suggest that SW treatment induces the secretion of various MPs within a short time frame. Furthermore, analysis of the distribution of larger MPs showed a dramatic increase in MPs 220 nm and 440 nm in diameter immediately following SW treatment (Fig. 5b,c and Supplementary Fig. 4). NanoSight and FACS results showed that SW dramatically induced MPs of 200–500 nm diameter within 30 min to 1 h of treatment. Additionally, transmission electron microscopy revealed higher numbers of MPs in the culture medium (Fig. 5d), along with budding of the plasma membrane and multi-vesicular body (MVB) in HUVECs (Fig. 5e). The incorporation of siGlo post-SW treatment was also examined using a NanoSight (Malvern Instruments). The absolute number of MPs with a diameter >200 nm was lower than those with a diameter <200 nm; however, the larger MPs had a siGlo uptake ratio more than 10-fold that of MPs sized ≤100 nm, and of those 100–200 nm (Fig. 6a,b). Taken together with the results shown in Figs 3, 5 and 6, the MPs secreted following SW treatment may interact with naked siRNA and can be transfected into cells not directly treated with SW.

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