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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.


Viability of HACAT cells after gene electrotransfer (GET). msEP: 1 msEP with applied electric field 40–130 V. Comb: treated with nsPEFs then 5 minutes later with 1 msEP. nsPEF parameters: 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP parameters: 5 ms, 1 pulse, applied electric field as indicated. Error bars represent SD.
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fig1: Viability of HACAT cells after gene electrotransfer (GET). msEP: 1 msEP with applied electric field 40–130 V. Comb: treated with nsPEFs then 5 minutes later with 1 msEP. nsPEF parameters: 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP parameters: 5 ms, 1 pulse, applied electric field as indicated. Error bars represent SD.

Mentions: One important issue we wanted to address was whether cells pretreated with nsPEFs could result in additional cell death. Cell viability was decreased proportionally after applied electric field was above 70 V for 1 msEP alone (Figure 1). Cells pretreated with nsPEFs amplified this influence. However, no additional cell death would be induced if no cell death occurred with 1 msEP alone with applied electric field below 70 V (Figure 1). This result was associated with particular parameters of nsPEFs, 24 KV/cm of applied electric field, 60 ns of pulse duration, 1 Hz of frequency, and 23 pulses. If the applied electric field was increased to 32 KV/cm without change of other parameters of nsPEFs, cell death would be observed even combined with 1 msEP at 50 V of applied electric field. Obviously, both parameters of msEPs and nsPEFs were critical to determine cell viability. Cell death could occur or increase if pulse number and/or electric field strength of either electric pulse(s) reached certain threshold or above. To minimize cell death and maximize gene expression, both parameters of msEPs and nsPEFs should be optimized.


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)

Viability of HACAT cells after gene electrotransfer (GET). msEP: 1 msEP with applied electric field 40–130 V. Comb: treated with nsPEFs then 5 minutes later with 1 msEP. nsPEF parameters: 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP parameters: 5 ms, 1 pulse, applied electric field as indicated. Error bars represent SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362372&req=5

fig1: Viability of HACAT cells after gene electrotransfer (GET). msEP: 1 msEP with applied electric field 40–130 V. Comb: treated with nsPEFs then 5 minutes later with 1 msEP. nsPEF parameters: 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP parameters: 5 ms, 1 pulse, applied electric field as indicated. Error bars represent SD.
Mentions: One important issue we wanted to address was whether cells pretreated with nsPEFs could result in additional cell death. Cell viability was decreased proportionally after applied electric field was above 70 V for 1 msEP alone (Figure 1). Cells pretreated with nsPEFs amplified this influence. However, no additional cell death would be induced if no cell death occurred with 1 msEP alone with applied electric field below 70 V (Figure 1). This result was associated with particular parameters of nsPEFs, 24 KV/cm of applied electric field, 60 ns of pulse duration, 1 Hz of frequency, and 23 pulses. If the applied electric field was increased to 32 KV/cm without change of other parameters of nsPEFs, cell death would be observed even combined with 1 msEP at 50 V of applied electric field. Obviously, both parameters of msEPs and nsPEFs were critical to determine cell viability. Cell death could occur or increase if pulse number and/or electric field strength of either electric pulse(s) reached certain threshold or above. To minimize cell death and maximize gene expression, both parameters of msEPs and nsPEFs should be optimized.

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.