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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.


Time restricted effect of nsPEFs on gene electrotransfer (GET) with msEPs. Luciferase expression of HACAT cells after GET with comb-pulses and plasmid gWIZ-Luc. Ctr: plasmid alone; nsPEFs: pulse duration 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP: pulse duration 5 ms, applied electric field (a) 50 V or (b) 70 V, and 1 pulse; Comb-0, 5, 15, 60: pretreated with nsPEFs then followed by 1 msEP with time interval 0, 5, 15, and 60 minutes, respectively. n = 3 and error bar is represented as SD. *P < 0.001 for comb-groups versus msEP alone.
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fig6: Time restricted effect of nsPEFs on gene electrotransfer (GET) with msEPs. Luciferase expression of HACAT cells after GET with comb-pulses and plasmid gWIZ-Luc. Ctr: plasmid alone; nsPEFs: pulse duration 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP: pulse duration 5 ms, applied electric field (a) 50 V or (b) 70 V, and 1 pulse; Comb-0, 5, 15, 60: pretreated with nsPEFs then followed by 1 msEP with time interval 0, 5, 15, and 60 minutes, respectively. n = 3 and error bar is represented as SD. *P < 0.001 for comb-groups versus msEP alone.

Mentions: Besides the order of millisecond and nanosecond electric pulses having a significant impact on gene expression efficiency (Figure 2), we also discovered that the effect of nsPEFs on gene transfection was time restricted. The enhancement of gene expression occurred immediately after nsPEFs and reached the maximum at posttreatment 5 minutes, slightly reduced at 15 minutes and had a residual effect at 1 hour (Figure 6a). Under the parameters we adopted in this experiment, in contrast to 1 msEP alone, luciferase expression with combined pulses was increased to 20.3-fold, 43.5-fold, 37.9-fold, and 9.5-fold (all P < 0.001), respectively for time gap 0 minute, 5 minutes, 15 minutes, and 1 hour. For this reason, we performed GET after cells pulsed with nsPEFs 5 minutes for all experiments except with these time-restricted assays. The extent of gene expression increase after cells pretreated with nsPEFs may vary depending on the parameters of nsPEFs and msEPs. For example, under another set of parameters, luciferase expression with combined pulses was enhanced to 3.4-fold, 6.5-fold, and, 6.1-fold, (all P < 0.001), respectively for the same time gaps as above (Figure 6b).


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)

Time restricted effect of nsPEFs on gene electrotransfer (GET) with msEPs. Luciferase expression of HACAT cells after GET with comb-pulses and plasmid gWIZ-Luc. Ctr: plasmid alone; nsPEFs: pulse duration 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP: pulse duration 5 ms, applied electric field (a) 50 V or (b) 70 V, and 1 pulse; Comb-0, 5, 15, 60: pretreated with nsPEFs then followed by 1 msEP with time interval 0, 5, 15, and 60 minutes, respectively. n = 3 and error bar is represented as SD. *P < 0.001 for comb-groups versus msEP alone.
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Related In: Results  -  Collection

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fig6: Time restricted effect of nsPEFs on gene electrotransfer (GET) with msEPs. Luciferase expression of HACAT cells after GET with comb-pulses and plasmid gWIZ-Luc. Ctr: plasmid alone; nsPEFs: pulse duration 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP: pulse duration 5 ms, applied electric field (a) 50 V or (b) 70 V, and 1 pulse; Comb-0, 5, 15, 60: pretreated with nsPEFs then followed by 1 msEP with time interval 0, 5, 15, and 60 minutes, respectively. n = 3 and error bar is represented as SD. *P < 0.001 for comb-groups versus msEP alone.
Mentions: Besides the order of millisecond and nanosecond electric pulses having a significant impact on gene expression efficiency (Figure 2), we also discovered that the effect of nsPEFs on gene transfection was time restricted. The enhancement of gene expression occurred immediately after nsPEFs and reached the maximum at posttreatment 5 minutes, slightly reduced at 15 minutes and had a residual effect at 1 hour (Figure 6a). Under the parameters we adopted in this experiment, in contrast to 1 msEP alone, luciferase expression with combined pulses was increased to 20.3-fold, 43.5-fold, 37.9-fold, and 9.5-fold (all P < 0.001), respectively for time gap 0 minute, 5 minutes, 15 minutes, and 1 hour. For this reason, we performed GET after cells pulsed with nsPEFs 5 minutes for all experiments except with these time-restricted assays. The extent of gene expression increase after cells pretreated with nsPEFs may vary depending on the parameters of nsPEFs and msEPs. For example, under another set of parameters, luciferase expression with combined pulses was enhanced to 3.4-fold, 6.5-fold, and, 6.1-fold, (all P < 0.001), respectively for the same time gaps as above (Figure 6b).

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.