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Electroporation is a commonly used gene delivery method with relatively high
efficiency in vivo[1]. The method has been
employed to transfect genes into various tissue types, including skeletal muscle, liver, skin, cornea, vasculature, and tumor
tissues[2]. High transfection efficiencies can be achieved relative to other nonviral delivery
methods[3]. It is possible that the electroporation delivery process can be further optimized to achieve higher transfection efficiency by varying the electric
pulse parameters, the plasmid DNA concentration, the ionic strength, or adding helper molecules such as
polymers[4_6]. In particular, it is known that ion composition and concentration in the DNA solution affects the efficiency of
electroporation-mediated transfection. Lee et
al examined the effect of a wide range of ionic strengths, and found that half saline (71 mmol/L)
was best for electroporation in skeletal
muscles[7]. In another study,
Ca2+ was identified as an important ionic component for
electroporation; transgene expressions were shown to be highest when
Ca2+ concentrations were in the range 20_100
mmol/L[8].
We reported here our findings using a different experimental protocol, which also suggest the importance of
Ca2+ in in vivo electroporation, but the effect we observed was quite the opposite of that observed in the previous study. We found
that the presence of Ca2+ in DNA solution significantly inhibited transgene expression after electroporation. By comparing
the effects of Ca2+ and various other ions at various concentrations in various experimental settings, we believe that the
inhibitory effect of Ca2+ in electro-gene transfer is a real and important phenomenon that has not been discussed before. An
in-depth understanding of such a phenomenon may provide some insights into the fundamental mechanism of
in vivo electro-gene transfer.
Materials and methods Plasmid and reagents The luciferase-encoding plasmid under the control of a SV40 promoter and a SV40 enhancer in the
pGL3 backbone (pGL3-Control Vector) was bought from Promega (Madison, USA). Plasmid batches were all purified using
the Qiagen GIGA kit. CaCl2 and all other reagents were analytical grade and were purchased from Shanghai Chemical Reagent
Co and Siji Reagent Co (Shanghai, China).
Animals and animal experiment protocols Female 4- to 6-week-old BALB/c mice were purchased from Fudan University
Animal Facility (Shanghai, China). At least 6 animals were included in each experimental group. The animal experiment
protocol in the present study was designed in accordance with the regulations of the Chinese Animal Welfare Agency. Before
the experiments, mice were anesthetized using 40 mg/mL chloral hydrate at 1% volume relative to body mass. After complete
sedation, the gastrocnemius muscle or ventral skin was shaved in each mouse and the mice were given injections of 20 µL
0.1 µg/µL plasmid DNA in various ionic solutions, followed by electroporation treatment using an electric pulse generator made
in our laboratory[9]. The electrodes (two silver acupuncture needles, 3 mm apart, each 1 cm long, and 0.3 mm in diameter) were
inserted into the muscle or skin covering the site of injection, and 650 ms square wave pulses were delivered at 1 s interval.
The field strength was set at 200 V/cm. Transgene expression was usually assayed after 2 d as described below.
Luciferase assay The mice were killed and the gastrocnemius muscles or ventral skin samples were collected for examination.
For the luciferase activity assay, tissues were homogenized in 1 mL Promega lysis reagent using a Bead-beater (Cole Pharma,
USA). The homogenates were then centrifuged at 2000×g for 6 min and the supernates were assayed using a TD20-20
luminometer (Turner BioSystems, CA), by integration of the light produced during 10 s, starting after the addition of 50 µL of
luciferase assay substrate (Promega) to 10 µL supernatant.
Statistical analysis Unless otherwise stated, we tested the significance of the differences between individual groups
using the one-tailed Student’s t-test for unpaired values. For statistical comparisons of several groups we used one-way
ANOVA. In the figures we report luciferase activity as mean relative light unit (RLU) ±SD.
Results
We examined the effect of different
Ca2+ concentrations in the plasmid DNA solution on luciferase expression after
electroporation delivery (Figure 1). With only 10 mmol/L of
Ca2+ included in the injection medium, transgene expression was
reduced to only 2% of the value without
Ca2+ (P<0.05,
t-test). Further increasing the
Ca2+ concentration to 100 mmol/L and 1 mol/L resulted in even lower gene expression. The
experiment was repeated a few times using
CaCl2 from different sources to avoid any effects of potential chemical impurities,
but the inhibitory effect was always large and always
reproducible.
To test whether other ions have similar inhibitory effects, we added
Mg2+, Sr2+, Na+, or
K+ into the DNA solution instead of
Ca2+ at comparable ionic concentrations and assayed for transgene expression after electroporation. Only
Ca2+ had a profound inhibitory effect; while all other cations had no effect. In addition, the substitution of the anion
Cl_ with NO3_ in
Ca2+-containing solutions did not alter the inhibitory effect (Figure 2).
Because the Ca2+ ion plays an important role in the physiological function of muscles, we wondered if the inhibitory effect
was unique to muscle tissues. Therefore, we examined
the effect of Ca2+ in another tissue type: skin. A mere 5
mmol/L of CaCl2 resulted in an approximately 50-fold reduction in luciferase expression in skin after electroporation (Figure 3).
We further examined the effects on transgene expression when the
Ca2+-containing buffer was administered separately
before or after plasmid DNA injection and electroporation. When the
Ca2+-containing buffer was administered by simple
needle injection 10 min before the plasmid DNA injection and electroporation, no inhibitory effect was observed. But when
the Ca2+-containing buffer was injected and immediately followed by an electroporation treatment, and 10 min later the DNA
solution was injected and electroporated, the resulting transgene expression was greatly reduced (Figure 4).
Simple intramuscular needle injection of
Ca2+-containing buffer 10 min after plasmid DNA injection and electroporation
had no inhibitory effect. But again, if the
Ca2+-containing buffers were delivered accompanied by an electroporation treatment,
transgene expression was greatly reduced. Even when
Ca2+ administration with electroporation was delayed for more than 24
h, the inhibitory effect on transgene expression was still evident (Figure 5).
Discussion
Ca2+ is one of the most common divalent cations found in biological fluids. For muscles, the extracellular calcium
concentration is estimated to be approximately 2
mmol/L[10]. In the present study, the addition of only 10
mmol/L Ca2+ to the DNA solution (which would be quickly diluted after injection) greatly interfered with transgene delivery and expression, not only
in muscles, but also in skin. The higher the
Ca2+ concentration was, the lower the transgene expression. Interestingly, other
studies on in vitro electroporation for cell transfection concluded that
Ca2+ is indispensable in the transfection medium to
maintain appropriate ion
concentrations[11]. But the results of the present study show that the situation is completely
different in vivo.
The mechanism responsible for this inhibitory effect
in vivo is unclear. It is possible that calcium might inhibit the activity
of luciferase, so to determine whether this was the case, we added 1_5 mol/L of
Ca2+ ions into a standard solution of
luciferase, but no decrease in luciferase activity was observed (data not shown). Therefore, calcium does not affect the
activity of luciferase. Another possibility is that the presence of
Ca2+ ions affects the ionic strength as well as the
conductivity of the plasmid DNA solution, and therefore alters the movement of DNA molecules in the tissue and into cells. In a recent
study by Lee and coworkers, it was suggested that ionic strength was the determining parameter of the effect of
Ca2+ on gene transfer. But data from the present
study indicated that the inhibitory effect was
Ca2+ specific[7]: none of
K+, Na+, Mg2+, or
Sr2+ at similar ionic strengths had any inhibitory effect. A similar argument could also be used to rule out the possibility that the
divalent Ca2+ interacted with plasmid DNA via a charge interaction, which altered the stability and physical properties of DNA
molecules. In addition, by injecting Ca2+
separately from the DNA solution, we also minimized the interaction between
Ca2+ ions and plasmid DNA, but the inhibitory effect was still
evident.
The data gathered during the separate injection experiment were quite striking. We found that
Ca2+-containing buffers could be delivered as late as 24 h after delivery of the plasmid to cause the inhibitory effect. Another important observation
is that the inhibitory effect was only evident when
Ca2+ was also delivered by electroporation, suggesting that
Ca2+ possibly needs to enter the cells in order to exert its inhibitory effect. When delivered by simple needle injection,
Ca2+ ions would be mostly present in the extracellular space, so the inhibitory effect would be limited.
The electroporation process would cause cell membrane disruption, which would allow
Ca2+ influx into the cells to result in damage to the muscle
cells[12]. During our experi-ments, we inspected the damage caused by electro-poration with or
without Ca2+. There was significant damage in the muscle fibers caused by electroporation. However, there was no noticeable
difference in the amount of damage with or without the addition of
Ca2+. However, it is still possible that the
Ca2+ caused damage that was not detected by us.
Another possibility is that Ca2+ influx exerted an effect on cell membrane stability. A recent report suggested that
the Ca2+ influx may initiate a membrane resealing
mechanism[13]. In that study, cell behaviors were studied after ultrasound treatment,
which is supposed to cause pores in the cell membrane. It was found that resealing of the cell membrane required extracellular
Ca2+ to enter cells through the pores. So in our experiments, it is possible that the administration of different concentrations
of Ca2+ ions effectively resulted in changes in cell membrane permeability to DNA molecules. The pores in the cell membrane
caused by electro-pulses resealed quickly after
Ca2+ influx to limit plasmid DNA entrance into the cells. Such a theory would
also explain the importance of extracellular
Ca2+ in in vitro electroporation mediums, because
in vitro electroporation conditions are harsher and are thought to result in more cell deaths. However, the exact mechanism for
Ca2+ influx-induced cell membrane pore resealing is not clear, and requires further investigation.
In summary, we hope that the present study will attract more attention on the complexity of optimizing
in vivo gene transfection by electroporation. Further studies to investigate the details of these effects will eventually build up a clearer
picture of the underlying mechanism of this promising technology.
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