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Electroporation by concentric-type needle electrodes and arrays
Bioelectrochemistry 118 (2017) 31–37
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Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem
Electroporation by concentric-type needle electrodes and arrays Yi Kung a,e, Alexey Lihachev b, Saulius Šatkauskas c, Keng-Li Lan d, Wen-Shiang Chen a,e,⁎ a
Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital, Taipei city, Taiwan Institute of Atomic Physics and Spectroscopy, University of Latvia, Riga, Latvia c Biology Department, Faculty of Natural Science, Vytautas Magnus University, Vileikos 8, Kaunas, Lithuania d Cancer Center, Taipei Veterans General Hospital, Taipei city, Taiwan e National Taiwan University College of Medicine, Taipei city, Taiwan b
a r t i c l e
i n f o
Article history: Received 2 March 2017 Received in revised form 29 June 2017 Accepted 29 June 2017 Available online 08 July 2017 Keywords: Electroporation Transfection Gene delivery Gene therapy Genomic medicine Adult zebrafish
a b s t r a c t The efficacy of genomic medicine depends on gene transfer efficiency. In this area, electroporation has been found to be a highly promising method for physical gene transfer. However, electroporation raises issues related to electrical safety, tissue damage, and the number of required wounds. Concentric-type needle electrodes seek to address these issues by using a lower bias (10 V), a single wound, fewer processing steps, and a smaller working area (≈10 mm3), thus offering greater accuracy and precision. Moreover, the needle can be arrayed to simultaneously treat several target regions. This paper proposes a novel method using concentric-type needle electrodes to improve the efficacy of genomic medicine in terms of electrical safety, human factor and usability engineering. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Gene therapy offers novel approaches for the prevention and treatment of a variety of diseases based on DNA sequencing and gene transfection techniques [1–4]. In vivo transfection methods play an important role not only in the therapy effect phase, but also directly help end users and patients in terms of the human factor and usability phases through the development of a wide spectrum of applications including virus-, microinjection-, ultrasound-, electroporation-, laser-, lipofection-, polycations-based gene transfection [5–11]. The most frequently used and efficient method for gene transfection is electroporation and its adjuncts (i.e. polycations-, lipofection-coated DNA), which show a high degree of clinical potential for stabilizing cells which are otherwise difficult to transfect in suspension, let alone in tissue. This approach is often used for treating primary isolated cells [12–15], but it requires specific parameter adaptation and optimization for particular cell types, and incurs extensive tissue damage from ohmic heating and puncture, while also raising serious concerns about electrical safety [16–20]. In electroporation, the gap between the gene injection site and the electrodes being triggered can raise precision issues that may be addressed by using a needle-array to increase the area, and to apply a ⁎ Corresponding author at: Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital, Taipei city, Taiwan. E-mail address: [email protected] (W.-S. Chen).
http://dx.doi.org/10.1016/j.bioelechem.2017.06.014 1567-5394/© 2017 Elsevier B.V. All rights reserved.
higher intensity of the electric field. Nonetheless, both approaches create additional wounds and produce additional tissue damage [21,22]. Due to the fact that the proposed electroporation electrodes and arrays were hand-made using concentric circle needle-electrodes, with a low cell constant, a key factor in measuring the intrinsic conductance of an electrode set [23], it was possible to reduce tissue damage along with the number of operational steps. This reduced wound damage as injecting and electro-pulsing were combined into a single step. This only requires around 5 mm2 of operating space, thus dramatically improving precision. 2. Materials and methods 2.1. Bio- and chemical materials The study proposal was approved by the ethics committee of the Laboratory Animal Center at National Taiwan University College of Medicine (approvals No. 20100137 and No. 20160326, respectively, for the use of mice and zebrafish). All mice (BALB/cByJNarl, 5 weeks) were purchased from the National Laboratory Animal Center (Taipei City, Taiwan), and were electro-transfected between 7 and 8 weeks of age. pCI-neo-Luc + (7187 bp) and pEGFP-C1 (4731 bp), purified by Qiagen Mega endotoxin free, were acquired from the Biomedical Resource Core of the First Core Laboratory, College of Medicine, National Taiwan University (Taipei city, Taiwan). D-Luciferin Firefly potassium salt was purchased from Biosynth AG (Lake Constance, Switzerland).
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Y. Kung et al. / Bioelectrochemistry 118 (2017) 31–37
was purchased from Thermo Fisher Scientific Inc. (Waltham, US). Forane (isoflurane) was purchased from Aesica Queenborough Ltd. (Queenborough, UK). Ethyl 3-aminobenzoate methanesulfonate (Tricaine, 98%) was purchased from Sigma-Aldrich Co. LLC. (St. Louis, US). All materials and their derivatives were processed at biotechnology grade.
2.2. Instruments & devices A square wave electroporation system (ECM 830, BTX) and 2 needle array electrodes (separated by approximately 5 mm, No. 533, BTX) were purchased from Harvard Apparatus Inc. (Holliston, US). A matrix isoflurane vaporizer (VIP 3000) was purchased from Midmark Corp. (Ohio, US). In vivo imaging system spectrum (IVIS) and XGI-8 gas anesthesia system (PerkinElmer, Waltham, US) were equipped in the Laboratory Animal Center at National Taiwan University College of Medicine. A fixed stage microscope (BX51) was purchased from Olympus Co. (Tokyo, Japan). A digital microscope camera system (RT-KE Color 3shot, SPOT) and its software (SPOT advanced) were purchased from Diagnostic Instruments, Inc. (Sterling Heights, US). A steel tube cutter (58692-U, Supelco) was purchased from Sigma-Aldrich Co. LLC. (St. Louis, US). Intramedic™ Polyethylene Tubing (PE10) was purchased from Becton, Dickinson (Franklin Lakes, US). An Omnican 50 insulin syringe (30 G with 12 mm needle) was purchased from B. Braun Melsungen AG (Melsungen, Germany). An AGANI needle (20 G) was purchased from Terumo Medical Corporation (Tokyo, Japan). ScotchWeld™ Epoxy Adhesive was purchased from 3M Company (Maplewood, US).
2.3. Fabrication of needle-electrode (NTUH 1) and needle-electrode array
Fig. 1. (a) Schematic diagram of electroporation experimental set-up; (b) structural diagram of the proposed needle-electrode. The length of 20 G needle was 5 mm (i.d. 0.58 mm; o.d. 0.88 mm); the length of 30 G needle was 10 mm (i.d. 0.13 mm; o.d. 0.31 mm); and the length of PE-10 polyethylene tube was approximately 2 mm (i.d. 0.28 mm; o.d. 0.61 mm).
150 mM NaCl, sterile-filtered by 0.22 μm PES membrane (Millipore syringe filter) was purchased from Polyplus-transfection (Illkirch, France). Dulbecco's phosphate-buffered saline (DPBS 10 ×, Gibco)
A hole was punched on the insulin needle hub in order to connect a wire to its needle part. A polyethylene tube (PE 10) was placed on the outer rim of the needle shaft as an insulating layer. A 20 G needle whose length was previously tuned by a tube cutter was then inserted into the shaft of the needle to form a concentric circle as shown in Fig. 1(b). A wire was next connected on the outside of the 20 G needle. Epoxy adhesive was applied to fix the wires in position and to serve as a protective layer. For array needle-electrodes, holes were punched on compressed foam for needle-electrode fixation. Needle-electrodes were then placed into the holes and connected with the wire to form a needle-electrode array.
Fig. 2. Comparison of electroporation performance between NTUH 1 and BTX 533. Exposure time was 1 s. Plasmid DNA was 40 μg of pCI-neo-Luc+. The substrate was 4 mg of D-Luciferin Firefly. Other conditions were the same as in Fig. 1, where * and # were respectively NTUH 1 at 10 V vs. 0 V of bias, and NTUH 1 vs. BTX 533 at 50 V of bias.
Y. Kung et al. / Bioelectrochemistry 118 (2017) 31–37
33
Fig. 3. Comparison of tissue damage by electroporation with NTUH 1, at 10 V and 100 V, and with BTX 533 at 50 V and 100 V. Other conditions were the same as in Fig. 1.
2.4. Electroporation on mice and IVIS analysis
2.6. Electroporation on zebrafishes
Once the mice were anesthetized with isoflurane, their thighs were fixed and the proposed needle-electrode was inserted into their quadriceps femoris muscle as shown in Fig. 1(a). 40 μg of pCI-neo-Luc+, predissolved in 50 μl of 150 mM NaCl, was then injected into the quadriceps femoris muscle. Electroporation was then performed 8 times with a 10 V square pulse (0.2 ms) at 1 s intervals. After 24 h, 50 μl of the substrate (40 mg/ml D-Luciferin Firefly) was injected into both sides of each mouse's lower abdomen and allowed to react for 10 min. The anesthetized mice were then placed into the IVIS scanning chamber to measure gene transfection performance. All IVIS data in the proposed research were analyzed using Living image 3.1 (Caliper Life Sciences, Waltham, US).
Zebrafish were restrained after being anesthetized with 0.2 mg/ml Tricaine, dissolved in distilled water. The needle-electrode was inserted into the fish abdomen and brain as described in Section 2.4. After 24 h, the zebrafish were analyzed using IVIS to obtain luminescent and fluorescent images (excitation at 465 nm, emission at 520 nm). 2.7. Statistics All data obtained were analyzed statistically using Student's t-test, and presented as mean ± SD. A probability value of p b 0.05 was considered indicative of a significant difference.
2.5. Histology section
3. Results & discussion
To evaluate the extent of tissue damage due to electroporation at various bias voltages, the experimental procedure described in Section 2.4 was modified using pork short fillets injected with 150 mM NaCl as a control solution instead of mice with plasmid DNA solution. The short fillets were then immersed in 10% formaldehyde solution for 24 h. Subsequently, the specimens were embedded in paraffin and H&E was applied to the short fillets. Slides were analyzed using an Olympus BX51 microscope, along with the Spot microscope camera system and its software, SPOT advanced.
3.1. Electroporation on mice
Fig. 4. Equivalent circuit for the proposed needle-electrodes. RT is the tissue resistance; CD is the interfacial capacitance of the electrode; RCT is the charge transfer resistance; WDiff is the impedance (a Warburg element) originating from the diffusion constraint.
To test the reusability and repeatability of the proposed needle-electrode, NTUH 1, electroporation was performed reusing a previously used NTUH 1 cleaned with distilled water along with unused NTUH electrodes. Both electroporation results show similar transfection outcomes (p ≫ 0.05). Therefore, all subsequent experiments were conducted with reused NTUH 1 electrodes, cleaned using distilled water between experiments. After confirming the reliability of the proposed needle-electrodes, NTUH 1 was compared with a commercial product, BTX 533. As shown in Fig. 2, the minimal detectable transgene expression was found when 10 and 50 V were applied using NTUH 1 and BTX 533 electrodes respectively. Moreover, comparing luminescent flux at 50 V of bias between NTUH 1 and BTX 533 shows that the electroporation efficiency of NTUH 1 was much higher than that of BTX 533. This efficiency difference may be caused by the cell-constant of the electrode structure, which was around 0.2 cm−1 and 7.06 cm−1, respectively, for NTUH 1 and BTX 533. Kaur and Singh found that conductivity is a key factor for tissue damage and cell membrane breakdown during electroporation [24]. Cell-
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Y. Kung et al. / Bioelectrochemistry 118 (2017) 31–37
Fig. 5. Timeline of electroporation bias dose with the proposed NTUH 1 needle-electrode. Exposure time was 1 s. Plasmid DNA was 40 μg of pCI-neo-Luc+. The substrate was 4 mg of DLuciferin Firefly. Other conditions were the same as in Fig. 1, where * was p b 0.05 vs. un-bias at the same time point in the t-test.
constancy is the key factor in evaluating electrode conductivity [23], where V ¼ EL
ð1Þ
I ¼ iA
ð2Þ
This suggests the proposed needle-electrode pair has an effective surface area (A), and given a parallel electric field, the electric field strength (E) will be linearly correlated with the voltage (V) by an effective distance (L) between the electrodes. Moreover, the current intensity (I) is the product of the current density (i) and the effective area (A)
Fig. 6. Luminescent image of electro-transfected mice with needle-electrode array. Mouse (L) was treated with a 2 needle-electrode array (one needle on either side, marked in red); mouse (R) was treated with a 3 needle-electrode array (two needles on the right side and one on the left, marked in red). Both were treated under 50 V of bias with an insertion depth of 5 mm. Exposure time was 1 s. Plasmid DNA was 20 μg of pCI-neo-Luc+. The substrate was 2 mg of D-Luciferin Firefly. Other conditions were the same as in Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
of the electrodes. So the conductance (G) by the proposed configuration will be: G¼
1 I iA 1 1 ¼ ¼ ¼ nFCu ¼ G0 R V EL k k
ð3Þ
k¼
L A
ð4Þ
where κ is the cell constant, which is determined by the geometry of the electrode set. G0 is conductivity, which is an intrinsic conductance dependent on the properties of the ionic conductor in the subject tissue, including ion concentration (C), charge (n) and mobility (μ). Referencing Bard and Faulkner, and Wang Fig. 4 shows the electrophysiological and electrochemical mechanism of the proposed needleelectrodes [25,26]. CD, RCT, and WDiff are respectively frequency, bias, and electrode material dependent, and RT is cell constant dependent. Under the working conditions for this study, CD is small, thus an electrode of lower cell constant, such as NTUH 1, can reduce tissue damage and operation bias. Furthermore, the ohmic drop is also reduced, along with impedance matching problems [27,28]. Fig. 3 shows that electroporation with NTUH 1 and BTX 533 at 100 V of bias produces similar tissue damage results (around 10,000 μm2). However to complete the electroporation, NTUH 1 only caused 1 wound while BTX 5333 produced 3 (1 for injecting the plasmid DNA, and 2 for inserting the electrodes). Tissue damage from ohmic heating can be clearly seen as dark shapes marked by arrows in the BTX 533 part of Fig. 3 and in RT of Fig. 4. This supports the proposed hypothesis that higher conductivity will produce less tissue damage [24]. The results of Figs. 2 and 3 suggest that NTUH 1 was not only effective under 10 V of bias, but can also decrease wound size. Pharmacokinetics is also a key factor in gene therapy. As shown in Fig. 5, gene expression achieved maximum performance after 1 week, and deteriorated thereafter because the plasmid DNA was degraded by lysosomes [29,30]. At higher bias values (e.g., 100, 50 V), the expression can last for 3 weeks before a second electroporation round was needed; however, for lower bias values (e.g., 25 and 10 V), the expression dropped to the baseline level after the first week. Unlike other commercial products, NTUH 1 was successfully used for array-based electroporation. As shown on the right mouse in Fig. 6, an electric field interaction exists between the two needles on the left side of the rat's back, thus extending the area of successful
Y. Kung et al. / Bioelectrochemistry 118 (2017) 31–37
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Fig. 7. (a) Luminescent image of electro-transfected zebrafish, where A and B were respectively the electro-transfected locations and non-electro-transfected body. (b). Comparison of luminescent flux between A and B in (a). All were treated under 50 V of bias with an insertion depth of 5 mm. Exposure time was 1 min. Plasmid DNA was 20 μg of pCI-neo-Luc+. The substrate was 2 mg of D-Luciferin Firefly. Other conditions were the same as in Fig. 1, where ** was p b 0.01 in the t-test.
electroporation [31]. However, for the left mouse in Fig. 6, the electric field formed by the left and right electrodes had difficulty passing the vertebral column (bone) due to the lack of an adequate mediator for transferring electrons, thus preventing electroporation in between them. Different combinations of needle electrodes in different shapes and sizes provide good opportunity for clinical gene therapy especially when dealing with pathological targets of various shapes and distributions [32,33]. 3.2. Electroporation on zebrafish Zebrafish serve as a common animal model in gene-related experiments for genes which are altered during the embryonic period [34, 35]. Despite the relative difficulty of using tissue and organs from mature fish, gene transfection experiments using mature fish are still important [36,37]. As depicted in Figs. 7 and 8, NTUH 1 can be used to transfect genes to abdomen muscle and the brain. Zou also used a selfmade electrode on zebrafish by modifying the two needle array
electrode of BTX533 [38]. However, the use of BTX 533 and other commercial products presented considerable difficulties in running this experiment, as the fish only measure 5 mm in width, with brains measuring approximately 10 mm2 in size, thus limiting electro-transfection precision, and also increasing the number of unnecessary wounds, experimental procedures, and the time required. 4. Conclusions The proposed concentric-type electroporation needle electrode and array can be used with a lower bias, fewer wounds, fewer processing steps, and a smaller working area to provide significantly improved accuracy and precision (Table 1). Moreover, the proposed solution can be arrayed to simultaneously treat several target regions, a novel innovation. Due to the limitation of customized resources, the proposed needleselectrodes were all made using commercial products. This imposes restrictions on prototype design (e.g., bevel size, needle shaft length). Nonetheless, once manufacturing issues are resolved, the proposed
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Fig. 8. (a) Fluorescent image of electro-transfected zebrafishes. A and B were respectively the electro-transfected brain and non-electro-transfected body. (b) Comparison of fluorescent flux between A and B in (a). All were treated under 50 V of bias with an insertion depth of around 1.5 mm. Plasmid DNA was 20 μg of pEGFP-C1. Other conditions were the same as in Fig. 1, where * was p b 0.05 in the t-test.
approach will provide a new tool for improving the efficacy of genomic medicine in terms of electrical safety, human factor and usability engineering. Acknowledgements This work was supported by Grant No. 100-2923-B-002-004-MY3, 103-2314-B-002-011-MY3 and 104-2325-B-010-007-CC2 from the Table 1 Comparison between NTUH 1 and BTX 533.
Process steps of electroporation Minimal transfer bias (V) [40 μg DNA] Arrayed-possibility Wound number Required working area (mm2) Accuracy Precision
NTUH 1
BTX 533
1 10 Yes 1 2.4 High High
2 50 No 3 113.1 Low Low
Ministry of Science and Technology of the Republic of China. We also would like to thank the staff of Laboratory of Developmental Signaling, National Taiwan University, for providing zebrafishes and technical support. As well as the staff of the Biomedical Resource Core at the First Core Labs, National Taiwan University College of Medicine, for technical support. References [1] M.R. Prausnitz, R. Langer, Transdermal drug delivery, Nat. Biotechnol. 26 (2008) 1261–1268. [2] Y. Taniyama, J. Azuma, Y. Kunugiza, K. Iekushi, H. Rakugiand, R. Morishita, Therapeutic option of plasmid-DNA based gene transfer, Curr. Top. Med. Chem. 14 (2012) 1630–1637. [3] J. Wang, C.M. Exline, J.J. DeClercq, G.N. Llewellyn, S.B. Hayward, P.W. Li, D.A. Shivak, R.T. Surosky, P.D. Gregory, M.C. Holmes, P.M. Cannon, Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors, Nat. Biotechnol. 33 (2015) 1256–1265. [4] P.A. Jones, J.P.J. Issa, S. Baylin, Targeting the cancer epigenome for therapy, Nat. Rev. Genet. 17 (2016) 630–641. [5] Y.W. Cho, J.D. Kim, K. Park, Polycation gene delivery systems: escape from endosomes to cytosol, J. Pharm. Pharmacol. 55 (2003) 721–734.
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Contents lists available at ScienceDirect
Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem
Electroporation by concentric-type needle electrodes and arrays Yi Kung a,e, Alexey Lihachev b, Saulius Šatkauskas c, Keng-Li Lan d, Wen-Shiang Chen a,e,⁎ a
Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital, Taipei city, Taiwan Institute of Atomic Physics and Spectroscopy, University of Latvia, Riga, Latvia c Biology Department, Faculty of Natural Science, Vytautas Magnus University, Vileikos 8, Kaunas, Lithuania d Cancer Center, Taipei Veterans General Hospital, Taipei city, Taiwan e National Taiwan University College of Medicine, Taipei city, Taiwan b
a r t i c l e
i n f o
Article history: Received 2 March 2017 Received in revised form 29 June 2017 Accepted 29 June 2017 Available online 08 July 2017 Keywords: Electroporation Transfection Gene delivery Gene therapy Genomic medicine Adult zebrafish
a b s t r a c t The efficacy of genomic medicine depends on gene transfer efficiency. In this area, electroporation has been found to be a highly promising method for physical gene transfer. However, electroporation raises issues related to electrical safety, tissue damage, and the number of required wounds. Concentric-type needle electrodes seek to address these issues by using a lower bias (10 V), a single wound, fewer processing steps, and a smaller working area (≈10 mm3), thus offering greater accuracy and precision. Moreover, the needle can be arrayed to simultaneously treat several target regions. This paper proposes a novel method using concentric-type needle electrodes to improve the efficacy of genomic medicine in terms of electrical safety, human factor and usability engineering. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Gene therapy offers novel approaches for the prevention and treatment of a variety of diseases based on DNA sequencing and gene transfection techniques [1–4]. In vivo transfection methods play an important role not only in the therapy effect phase, but also directly help end users and patients in terms of the human factor and usability phases through the development of a wide spectrum of applications including virus-, microinjection-, ultrasound-, electroporation-, laser-, lipofection-, polycations-based gene transfection [5–11]. The most frequently used and efficient method for gene transfection is electroporation and its adjuncts (i.e. polycations-, lipofection-coated DNA), which show a high degree of clinical potential for stabilizing cells which are otherwise difficult to transfect in suspension, let alone in tissue. This approach is often used for treating primary isolated cells [12–15], but it requires specific parameter adaptation and optimization for particular cell types, and incurs extensive tissue damage from ohmic heating and puncture, while also raising serious concerns about electrical safety [16–20]. In electroporation, the gap between the gene injection site and the electrodes being triggered can raise precision issues that may be addressed by using a needle-array to increase the area, and to apply a ⁎ Corresponding author at: Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital, Taipei city, Taiwan. E-mail address: [email protected] (W.-S. Chen).
http://dx.doi.org/10.1016/j.bioelechem.2017.06.014 1567-5394/© 2017 Elsevier B.V. All rights reserved.
higher intensity of the electric field. Nonetheless, both approaches create additional wounds and produce additional tissue damage [21,22]. Due to the fact that the proposed electroporation electrodes and arrays were hand-made using concentric circle needle-electrodes, with a low cell constant, a key factor in measuring the intrinsic conductance of an electrode set [23], it was possible to reduce tissue damage along with the number of operational steps. This reduced wound damage as injecting and electro-pulsing were combined into a single step. This only requires around 5 mm2 of operating space, thus dramatically improving precision. 2. Materials and methods 2.1. Bio- and chemical materials The study proposal was approved by the ethics committee of the Laboratory Animal Center at National Taiwan University College of Medicine (approvals No. 20100137 and No. 20160326, respectively, for the use of mice and zebrafish). All mice (BALB/cByJNarl, 5 weeks) were purchased from the National Laboratory Animal Center (Taipei City, Taiwan), and were electro-transfected between 7 and 8 weeks of age. pCI-neo-Luc + (7187 bp) and pEGFP-C1 (4731 bp), purified by Qiagen Mega endotoxin free, were acquired from the Biomedical Resource Core of the First Core Laboratory, College of Medicine, National Taiwan University (Taipei city, Taiwan). D-Luciferin Firefly potassium salt was purchased from Biosynth AG (Lake Constance, Switzerland).
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was purchased from Thermo Fisher Scientific Inc. (Waltham, US). Forane (isoflurane) was purchased from Aesica Queenborough Ltd. (Queenborough, UK). Ethyl 3-aminobenzoate methanesulfonate (Tricaine, 98%) was purchased from Sigma-Aldrich Co. LLC. (St. Louis, US). All materials and their derivatives were processed at biotechnology grade.
2.2. Instruments & devices A square wave electroporation system (ECM 830, BTX) and 2 needle array electrodes (separated by approximately 5 mm, No. 533, BTX) were purchased from Harvard Apparatus Inc. (Holliston, US). A matrix isoflurane vaporizer (VIP 3000) was purchased from Midmark Corp. (Ohio, US). In vivo imaging system spectrum (IVIS) and XGI-8 gas anesthesia system (PerkinElmer, Waltham, US) were equipped in the Laboratory Animal Center at National Taiwan University College of Medicine. A fixed stage microscope (BX51) was purchased from Olympus Co. (Tokyo, Japan). A digital microscope camera system (RT-KE Color 3shot, SPOT) and its software (SPOT advanced) were purchased from Diagnostic Instruments, Inc. (Sterling Heights, US). A steel tube cutter (58692-U, Supelco) was purchased from Sigma-Aldrich Co. LLC. (St. Louis, US). Intramedic™ Polyethylene Tubing (PE10) was purchased from Becton, Dickinson (Franklin Lakes, US). An Omnican 50 insulin syringe (30 G with 12 mm needle) was purchased from B. Braun Melsungen AG (Melsungen, Germany). An AGANI needle (20 G) was purchased from Terumo Medical Corporation (Tokyo, Japan). ScotchWeld™ Epoxy Adhesive was purchased from 3M Company (Maplewood, US).
2.3. Fabrication of needle-electrode (NTUH 1) and needle-electrode array
Fig. 1. (a) Schematic diagram of electroporation experimental set-up; (b) structural diagram of the proposed needle-electrode. The length of 20 G needle was 5 mm (i.d. 0.58 mm; o.d. 0.88 mm); the length of 30 G needle was 10 mm (i.d. 0.13 mm; o.d. 0.31 mm); and the length of PE-10 polyethylene tube was approximately 2 mm (i.d. 0.28 mm; o.d. 0.61 mm).
150 mM NaCl, sterile-filtered by 0.22 μm PES membrane (Millipore syringe filter) was purchased from Polyplus-transfection (Illkirch, France). Dulbecco's phosphate-buffered saline (DPBS 10 ×, Gibco)
A hole was punched on the insulin needle hub in order to connect a wire to its needle part. A polyethylene tube (PE 10) was placed on the outer rim of the needle shaft as an insulating layer. A 20 G needle whose length was previously tuned by a tube cutter was then inserted into the shaft of the needle to form a concentric circle as shown in Fig. 1(b). A wire was next connected on the outside of the 20 G needle. Epoxy adhesive was applied to fix the wires in position and to serve as a protective layer. For array needle-electrodes, holes were punched on compressed foam for needle-electrode fixation. Needle-electrodes were then placed into the holes and connected with the wire to form a needle-electrode array.
Fig. 2. Comparison of electroporation performance between NTUH 1 and BTX 533. Exposure time was 1 s. Plasmid DNA was 40 μg of pCI-neo-Luc+. The substrate was 4 mg of D-Luciferin Firefly. Other conditions were the same as in Fig. 1, where * and # were respectively NTUH 1 at 10 V vs. 0 V of bias, and NTUH 1 vs. BTX 533 at 50 V of bias.
Y. Kung et al. / Bioelectrochemistry 118 (2017) 31–37
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Fig. 3. Comparison of tissue damage by electroporation with NTUH 1, at 10 V and 100 V, and with BTX 533 at 50 V and 100 V. Other conditions were the same as in Fig. 1.
2.4. Electroporation on mice and IVIS analysis
2.6. Electroporation on zebrafishes
Once the mice were anesthetized with isoflurane, their thighs were fixed and the proposed needle-electrode was inserted into their quadriceps femoris muscle as shown in Fig. 1(a). 40 μg of pCI-neo-Luc+, predissolved in 50 μl of 150 mM NaCl, was then injected into the quadriceps femoris muscle. Electroporation was then performed 8 times with a 10 V square pulse (0.2 ms) at 1 s intervals. After 24 h, 50 μl of the substrate (40 mg/ml D-Luciferin Firefly) was injected into both sides of each mouse's lower abdomen and allowed to react for 10 min. The anesthetized mice were then placed into the IVIS scanning chamber to measure gene transfection performance. All IVIS data in the proposed research were analyzed using Living image 3.1 (Caliper Life Sciences, Waltham, US).
Zebrafish were restrained after being anesthetized with 0.2 mg/ml Tricaine, dissolved in distilled water. The needle-electrode was inserted into the fish abdomen and brain as described in Section 2.4. After 24 h, the zebrafish were analyzed using IVIS to obtain luminescent and fluorescent images (excitation at 465 nm, emission at 520 nm). 2.7. Statistics All data obtained were analyzed statistically using Student's t-test, and presented as mean ± SD. A probability value of p b 0.05 was considered indicative of a significant difference.
2.5. Histology section
3. Results & discussion
To evaluate the extent of tissue damage due to electroporation at various bias voltages, the experimental procedure described in Section 2.4 was modified using pork short fillets injected with 150 mM NaCl as a control solution instead of mice with plasmid DNA solution. The short fillets were then immersed in 10% formaldehyde solution for 24 h. Subsequently, the specimens were embedded in paraffin and H&E was applied to the short fillets. Slides were analyzed using an Olympus BX51 microscope, along with the Spot microscope camera system and its software, SPOT advanced.
3.1. Electroporation on mice
Fig. 4. Equivalent circuit for the proposed needle-electrodes. RT is the tissue resistance; CD is the interfacial capacitance of the electrode; RCT is the charge transfer resistance; WDiff is the impedance (a Warburg element) originating from the diffusion constraint.
To test the reusability and repeatability of the proposed needle-electrode, NTUH 1, electroporation was performed reusing a previously used NTUH 1 cleaned with distilled water along with unused NTUH electrodes. Both electroporation results show similar transfection outcomes (p ≫ 0.05). Therefore, all subsequent experiments were conducted with reused NTUH 1 electrodes, cleaned using distilled water between experiments. After confirming the reliability of the proposed needle-electrodes, NTUH 1 was compared with a commercial product, BTX 533. As shown in Fig. 2, the minimal detectable transgene expression was found when 10 and 50 V were applied using NTUH 1 and BTX 533 electrodes respectively. Moreover, comparing luminescent flux at 50 V of bias between NTUH 1 and BTX 533 shows that the electroporation efficiency of NTUH 1 was much higher than that of BTX 533. This efficiency difference may be caused by the cell-constant of the electrode structure, which was around 0.2 cm−1 and 7.06 cm−1, respectively, for NTUH 1 and BTX 533. Kaur and Singh found that conductivity is a key factor for tissue damage and cell membrane breakdown during electroporation [24]. Cell-
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Fig. 5. Timeline of electroporation bias dose with the proposed NTUH 1 needle-electrode. Exposure time was 1 s. Plasmid DNA was 40 μg of pCI-neo-Luc+. The substrate was 4 mg of DLuciferin Firefly. Other conditions were the same as in Fig. 1, where * was p b 0.05 vs. un-bias at the same time point in the t-test.
constancy is the key factor in evaluating electrode conductivity [23], where V ¼ EL
ð1Þ
I ¼ iA
ð2Þ
This suggests the proposed needle-electrode pair has an effective surface area (A), and given a parallel electric field, the electric field strength (E) will be linearly correlated with the voltage (V) by an effective distance (L) between the electrodes. Moreover, the current intensity (I) is the product of the current density (i) and the effective area (A)
Fig. 6. Luminescent image of electro-transfected mice with needle-electrode array. Mouse (L) was treated with a 2 needle-electrode array (one needle on either side, marked in red); mouse (R) was treated with a 3 needle-electrode array (two needles on the right side and one on the left, marked in red). Both were treated under 50 V of bias with an insertion depth of 5 mm. Exposure time was 1 s. Plasmid DNA was 20 μg of pCI-neo-Luc+. The substrate was 2 mg of D-Luciferin Firefly. Other conditions were the same as in Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
of the electrodes. So the conductance (G) by the proposed configuration will be: G¼
1 I iA 1 1 ¼ ¼ ¼ nFCu ¼ G0 R V EL k k
ð3Þ
k¼
L A
ð4Þ
where κ is the cell constant, which is determined by the geometry of the electrode set. G0 is conductivity, which is an intrinsic conductance dependent on the properties of the ionic conductor in the subject tissue, including ion concentration (C), charge (n) and mobility (μ). Referencing Bard and Faulkner, and Wang Fig. 4 shows the electrophysiological and electrochemical mechanism of the proposed needleelectrodes [25,26]. CD, RCT, and WDiff are respectively frequency, bias, and electrode material dependent, and RT is cell constant dependent. Under the working conditions for this study, CD is small, thus an electrode of lower cell constant, such as NTUH 1, can reduce tissue damage and operation bias. Furthermore, the ohmic drop is also reduced, along with impedance matching problems [27,28]. Fig. 3 shows that electroporation with NTUH 1 and BTX 533 at 100 V of bias produces similar tissue damage results (around 10,000 μm2). However to complete the electroporation, NTUH 1 only caused 1 wound while BTX 5333 produced 3 (1 for injecting the plasmid DNA, and 2 for inserting the electrodes). Tissue damage from ohmic heating can be clearly seen as dark shapes marked by arrows in the BTX 533 part of Fig. 3 and in RT of Fig. 4. This supports the proposed hypothesis that higher conductivity will produce less tissue damage [24]. The results of Figs. 2 and 3 suggest that NTUH 1 was not only effective under 10 V of bias, but can also decrease wound size. Pharmacokinetics is also a key factor in gene therapy. As shown in Fig. 5, gene expression achieved maximum performance after 1 week, and deteriorated thereafter because the plasmid DNA was degraded by lysosomes [29,30]. At higher bias values (e.g., 100, 50 V), the expression can last for 3 weeks before a second electroporation round was needed; however, for lower bias values (e.g., 25 and 10 V), the expression dropped to the baseline level after the first week. Unlike other commercial products, NTUH 1 was successfully used for array-based electroporation. As shown on the right mouse in Fig. 6, an electric field interaction exists between the two needles on the left side of the rat's back, thus extending the area of successful
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Fig. 7. (a) Luminescent image of electro-transfected zebrafish, where A and B were respectively the electro-transfected locations and non-electro-transfected body. (b). Comparison of luminescent flux between A and B in (a). All were treated under 50 V of bias with an insertion depth of 5 mm. Exposure time was 1 min. Plasmid DNA was 20 μg of pCI-neo-Luc+. The substrate was 2 mg of D-Luciferin Firefly. Other conditions were the same as in Fig. 1, where ** was p b 0.01 in the t-test.
electroporation [31]. However, for the left mouse in Fig. 6, the electric field formed by the left and right electrodes had difficulty passing the vertebral column (bone) due to the lack of an adequate mediator for transferring electrons, thus preventing electroporation in between them. Different combinations of needle electrodes in different shapes and sizes provide good opportunity for clinical gene therapy especially when dealing with pathological targets of various shapes and distributions [32,33]. 3.2. Electroporation on zebrafish Zebrafish serve as a common animal model in gene-related experiments for genes which are altered during the embryonic period [34, 35]. Despite the relative difficulty of using tissue and organs from mature fish, gene transfection experiments using mature fish are still important [36,37]. As depicted in Figs. 7 and 8, NTUH 1 can be used to transfect genes to abdomen muscle and the brain. Zou also used a selfmade electrode on zebrafish by modifying the two needle array
electrode of BTX533 [38]. However, the use of BTX 533 and other commercial products presented considerable difficulties in running this experiment, as the fish only measure 5 mm in width, with brains measuring approximately 10 mm2 in size, thus limiting electro-transfection precision, and also increasing the number of unnecessary wounds, experimental procedures, and the time required. 4. Conclusions The proposed concentric-type electroporation needle electrode and array can be used with a lower bias, fewer wounds, fewer processing steps, and a smaller working area to provide significantly improved accuracy and precision (Table 1). Moreover, the proposed solution can be arrayed to simultaneously treat several target regions, a novel innovation. Due to the limitation of customized resources, the proposed needleselectrodes were all made using commercial products. This imposes restrictions on prototype design (e.g., bevel size, needle shaft length). Nonetheless, once manufacturing issues are resolved, the proposed
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Fig. 8. (a) Fluorescent image of electro-transfected zebrafishes. A and B were respectively the electro-transfected brain and non-electro-transfected body. (b) Comparison of fluorescent flux between A and B in (a). All were treated under 50 V of bias with an insertion depth of around 1.5 mm. Plasmid DNA was 20 μg of pEGFP-C1. Other conditions were the same as in Fig. 1, where * was p b 0.05 in the t-test.
approach will provide a new tool for improving the efficacy of genomic medicine in terms of electrical safety, human factor and usability engineering. Acknowledgements This work was supported by Grant No. 100-2923-B-002-004-MY3, 103-2314-B-002-011-MY3 and 104-2325-B-010-007-CC2 from the Table 1 Comparison between NTUH 1 and BTX 533.
Process steps of electroporation Minimal transfer bias (V) [40 μg DNA] Arrayed-possibility Wound number Required working area (mm2) Accuracy Precision
NTUH 1
BTX 533
1 10 Yes 1 2.4 High High
2 50 No 3 113.1 Low Low
Ministry of Science and Technology of the Republic of China. We also would like to thank the staff of Laboratory of Developmental Signaling, National Taiwan University, for providing zebrafishes and technical support. As well as the staff of the Biomedical Resource Core at the First Core Labs, National Taiwan University College of Medicine, for technical support. References [1] M.R. Prausnitz, R. Langer, Transdermal drug delivery, Nat. Biotechnol. 26 (2008) 1261–1268. [2] Y. Taniyama, J. Azuma, Y. Kunugiza, K. Iekushi, H. Rakugiand, R. Morishita, Therapeutic option of plasmid-DNA based gene transfer, Curr. Top. Med. Chem. 14 (2012) 1630–1637. [3] J. Wang, C.M. Exline, J.J. DeClercq, G.N. Llewellyn, S.B. Hayward, P.W. Li, D.A. Shivak, R.T. Surosky, P.D. Gregory, M.C. Holmes, P.M. Cannon, Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors, Nat. Biotechnol. 33 (2015) 1256–1265. [4] P.A. Jones, J.P.J. Issa, S. Baylin, Targeting the cancer epigenome for therapy, Nat. Rev. Genet. 17 (2016) 630–641. [5] Y.W. Cho, J.D. Kim, K. Park, Polycation gene delivery systems: escape from endosomes to cytosol, J. Pharm. Pharmacol. 55 (2003) 721–734.
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