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Gene electrotransfer enhanced by nanosecond pulsed electric fields.

Guo S, Jackson DL, Burcus NI, Chen YJ, Xiao S, Heller R - Mol Ther Methods Clin Dev (2014)

Bottom Line: We demonstrated that the effect of nsPEFs on gene transfection was time restricted.The effect of nsPEFs on cell viability is also dependent on the specific pulse parameters.We also determined that both calcium independent and dependent mechanisms are involved in nsPEF effects on gene electrotransfer.

View Article: PubMed Central - PubMed

Affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University , Norfolk, Virginia, USA.

ABSTRACT
The impact of nanosecond pulsed electric fields (nsPEFs) on gene electrotransfer has not been clearly demonstrated in previous studies. This study was conducted to evaluate the influence of nsPEFs on the delivery of plasmids encoding luciferase or green fluorescent protein and subsequent expression in HACAT keratinocyte cells. Delivery was performed using millisecond electric pulses (msEPs) with or without nsPEFs. In contrast to reports in the literature, we discovered that gene expression was significantly increased up to 40-fold by applying nsPEFs to cells first followed by one msEP but not in the opposite order. We demonstrated that the effect of nsPEFs on gene transfection was time restricted. The enhancement of gene expression occurred by applying one msEP immediately after nsPEFs and reached the maximum at posttreatment 5 minutes, slightly decreased at 15 minutes and had a residual effect at 1 hour. It appears that nsPEFs play a role as an amplifier without changing the trend of gene expression kinetics due to msEPs. The effect of nsPEFs on cell viability is also dependent on the specific pulse parameters. We also determined that both calcium independent and dependent mechanisms are involved in nsPEF effects on gene electrotransfer.

No MeSH data available.


Luciferase expression after combination pulses. (a) luciferase expression of HACAT cells 1 day after gene electrotransfer (GET) with gWIZ-luc. Ctr: plasmid alone; nsPEFs: nsPEFs with pulse duration 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP: 1 msEP with pulse duration 5 ms and applied electric field 70 V; Comb-0, 5, 15, 60: treated with 1 msEP then followed by nsPEFs with time interval 0, 5, 15, and 60 minutes, respectively. (b) luciferase expression (pg/million cells) of HACAT cells 1 day after GET with gWIZ-luc. Ctr: plasmid alone; nsPEFs: nsPEFs with pulse duration 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP: 1 msEP with pulse duration 5 ms and applied electric field 50 V; Comb-12, 16, and 24 KV: pretreated with nsPEFs with electric field 12, 16, and 24 KV/cm, respectively then 5 minutes later followed by 1 msEP. Error bars represent SD. n = 8–12, *P < 0.001 for Combination groups versus msEP.
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fig2: Luciferase expression after combination pulses. (a) luciferase expression of HACAT cells 1 day after gene electrotransfer (GET) with gWIZ-luc. Ctr: plasmid alone; nsPEFs: nsPEFs with pulse duration 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP: 1 msEP with pulse duration 5 ms and applied electric field 70 V; Comb-0, 5, 15, 60: treated with 1 msEP then followed by nsPEFs with time interval 0, 5, 15, and 60 minutes, respectively. (b) luciferase expression (pg/million cells) of HACAT cells 1 day after GET with gWIZ-luc. Ctr: plasmid alone; nsPEFs: nsPEFs with pulse duration 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP: 1 msEP with pulse duration 5 ms and applied electric field 50 V; Comb-12, 16, and 24 KV: pretreated with nsPEFs with electric field 12, 16, and 24 KV/cm, respectively then 5 minutes later followed by 1 msEP. Error bars represent SD. n = 8–12, *P < 0.001 for Combination groups versus msEP.

Mentions: Although it was reported that GFP expression was enhanced when nsPEFs was applied after msEPs in previous reports,34,36 we have not observed any significant increase of luciferase expression of HACAT cells while similar parameters and the same order of two electroporations were adopted (Figure 2a). On the contrary, significant increase of gene expression was obtained while nsPEFs were applied prior to msEPs (Figures 2b and 3). The luciferase expression resulting from the combined electric pulses was enhanced two- to fourfold compared to 1 msEP alone dependent on the electric field strength of nsPEFs (Figure 2b, 12–24 KV/cm). The increase of gene expression was positively correlated with both electric field strength (Figures 2b and 3d) and pulse number (Figure 3c) of nsPEFs. Under the same parameters of nsPEFs, the percentage of GFP expression could be enhanced three- to tenfold dependent on the applied electric field strength of 1 msEP from 50 to 100 V (Figure 3). The fluorescence intensity of GFP was two- to threefold higher for the combined pulses than for one millisecond pulse alone. However, those results did not mean the total expression was increased to the same extent, because more cell death was observed along with higher electric field of millisecond pulses (Figure 1).


Gene electrotransfer enhanced by nanosecond pulsed electric fields.

Guo S, Jackson DL, Burcus NI, Chen YJ, Xiao S, Heller R - Mol Ther Methods Clin Dev (2014)

Luciferase expression after combination pulses. (a) luciferase expression of HACAT cells 1 day after gene electrotransfer (GET) with gWIZ-luc. Ctr: plasmid alone; nsPEFs: nsPEFs with pulse duration 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP: 1 msEP with pulse duration 5 ms and applied electric field 70 V; Comb-0, 5, 15, 60: treated with 1 msEP then followed by nsPEFs with time interval 0, 5, 15, and 60 minutes, respectively. (b) luciferase expression (pg/million cells) of HACAT cells 1 day after GET with gWIZ-luc. Ctr: plasmid alone; nsPEFs: nsPEFs with pulse duration 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP: 1 msEP with pulse duration 5 ms and applied electric field 50 V; Comb-12, 16, and 24 KV: pretreated with nsPEFs with electric field 12, 16, and 24 KV/cm, respectively then 5 minutes later followed by 1 msEP. Error bars represent SD. n = 8–12, *P < 0.001 for Combination groups versus msEP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Luciferase expression after combination pulses. (a) luciferase expression of HACAT cells 1 day after gene electrotransfer (GET) with gWIZ-luc. Ctr: plasmid alone; nsPEFs: nsPEFs with pulse duration 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP: 1 msEP with pulse duration 5 ms and applied electric field 70 V; Comb-0, 5, 15, 60: treated with 1 msEP then followed by nsPEFs with time interval 0, 5, 15, and 60 minutes, respectively. (b) luciferase expression (pg/million cells) of HACAT cells 1 day after GET with gWIZ-luc. Ctr: plasmid alone; nsPEFs: nsPEFs with pulse duration 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP: 1 msEP with pulse duration 5 ms and applied electric field 50 V; Comb-12, 16, and 24 KV: pretreated with nsPEFs with electric field 12, 16, and 24 KV/cm, respectively then 5 minutes later followed by 1 msEP. Error bars represent SD. n = 8–12, *P < 0.001 for Combination groups versus msEP.
Mentions: Although it was reported that GFP expression was enhanced when nsPEFs was applied after msEPs in previous reports,34,36 we have not observed any significant increase of luciferase expression of HACAT cells while similar parameters and the same order of two electroporations were adopted (Figure 2a). On the contrary, significant increase of gene expression was obtained while nsPEFs were applied prior to msEPs (Figures 2b and 3). The luciferase expression resulting from the combined electric pulses was enhanced two- to fourfold compared to 1 msEP alone dependent on the electric field strength of nsPEFs (Figure 2b, 12–24 KV/cm). The increase of gene expression was positively correlated with both electric field strength (Figures 2b and 3d) and pulse number (Figure 3c) of nsPEFs. Under the same parameters of nsPEFs, the percentage of GFP expression could be enhanced three- to tenfold dependent on the applied electric field strength of 1 msEP from 50 to 100 V (Figure 3). The fluorescence intensity of GFP was two- to threefold higher for the combined pulses than for one millisecond pulse alone. However, those results did not mean the total expression was increased to the same extent, because more cell death was observed along with higher electric field of millisecond pulses (Figure 1).

Bottom Line: We demonstrated that the effect of nsPEFs on gene transfection was time restricted.The effect of nsPEFs on cell viability is also dependent on the specific pulse parameters.We also determined that both calcium independent and dependent mechanisms are involved in nsPEF effects on gene electrotransfer.

View Article: PubMed Central - PubMed

Affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University , Norfolk, Virginia, USA.

ABSTRACT
The impact of nanosecond pulsed electric fields (nsPEFs) on gene electrotransfer has not been clearly demonstrated in previous studies. This study was conducted to evaluate the influence of nsPEFs on the delivery of plasmids encoding luciferase or green fluorescent protein and subsequent expression in HACAT keratinocyte cells. Delivery was performed using millisecond electric pulses (msEPs) with or without nsPEFs. In contrast to reports in the literature, we discovered that gene expression was significantly increased up to 40-fold by applying nsPEFs to cells first followed by one msEP but not in the opposite order. We demonstrated that the effect of nsPEFs on gene transfection was time restricted. The enhancement of gene expression occurred by applying one msEP immediately after nsPEFs and reached the maximum at posttreatment 5 minutes, slightly decreased at 15 minutes and had a residual effect at 1 hour. It appears that nsPEFs play a role as an amplifier without changing the trend of gene expression kinetics due to msEPs. The effect of nsPEFs on cell viability is also dependent on the specific pulse parameters. We also determined that both calcium independent and dependent mechanisms are involved in nsPEF effects on gene electrotransfer.

No MeSH data available.