Invasive electrodes require electrode penetration into a tissue. The electrode is usually needle-shaped, with a sharp tip. Therefore, most invasive electrodes deliver the electric pulses through typically stainless-steel needles of different length. Currently, fixed-position as well as adjustable position (electrodes, IGEA Medical; Electrodes for in vivo electroporation, 2021a) or needle composition (Adjustable Electrodes, 2022) electrode pairs or arrays are commercially available. Fixed-position electrodes are further categorized into two-needle electrodes (Isobe et al., 2004; Chen et al., 2015; Langus et al., 2016; Zager et al., 2016; Yao et al., 2017) and longitudinal or hexagonal electrode arrays. Basically, in this category, the electric field distribution is dependent on the number of needles (Adeyanju et al., 2012), length of the needles, gap spacing, and diameter of the needle tip (Davalos et al., 2005). The electric field distribution of the two-needle electrode configuration, including variation of gap size and length, is shown in Figures 3A–C.

Free position electrodes are advantageous when the tumor or tissue composition is not predetermined. Usually, this type of electrode is combined with an adjustable handle to fix the position after insertion into the target. Nevertheless, the needles are very thin; therefore, the distance between electrodes in deeper tissues may vary, which means the effects of non-parallelism should be considered since it affects the spatial electric field distribution (Figures 3G–I).

As can be seen in the aforementioned figures, the visible gap size in each case is the same—5 mm; therefore, the top view does not change (Figures 3G–I). However, non-parallelism may occur in deeper tissues, especially when using longer needles. Typically, this occurs due to skin surface curvature and composition (Wei et al., 2017; Kopcewicz et al., 2020). Therefore, non-parallelism leads to electric field inhomogeneity and non-uniform treatment, as potentially healthy tissue is affected and target tissues (white dashed lines representing the tumor) remain unaffected or treated insufficiently. It is clear from Figure 3 that using two-needle electrode configurations may involve inhomogeneity of the PEF distribution. This limitation can be minimized using an array of adjustable needles as shown in Figure 4.

Needle arrays or repositioning of needles can be used to ensure more homogeneous deep-seated tumor electroporation. Placement of needle electrodes of variable geometry is adjusted to the individual size and shape of the tumor. To obtain an above-threshold electric field and cover the entire tumor volume, multiple needles are placed at the tumor margins and/or within the tumor. At the same time, the number of needles should be limited to reduce treatment invasiveness and complexity. The electrical pulses are, therefore, subsequently delivered between predetermined needle pairs. Such an electroporation procedure requires very precise pretreatment planning and PEF parameter evaluation (Miklavcic et al., 2010; Pavliha et al., 2012a; Blazevski et al., 2020). However, it enables efficient treatment of tumors with multiple nodules (Figure 4B). Nevertheless, non-parallelism is still a problem and is usually solved by x-ray imaging during the operation after electrode positioning and fixation (Moreta-Martínez et al., 2022). If non-parallelism is detected, adjustment of the treatment parameters and/or repositioning of the electrodes can be performed.

When the target tissue or tumor is subcutaneous, the electroporation procedure requires access to deep-seated cancer lesions without making a large incision in the skin. Such treatment is performed using an open laparoscopy approach or trans-oral and trans-anal endoscopy through a catheter (Lee et al., 2019; Li et al., 2021). Therefore, the requirements for the electrodes become more complex: electrodes have to be placed strictly parallel in order to ensure homogeneous PEF; the procedure must be performed on a relatively small probe area; and at the same time, the operating area has to cover the whole tumor volume.

An electrode prototype considering the aforementioned features was presented by Izzo et al. (2020). The study shows the evaluation of the effectiveness and suitability of deployable and expandable 4-needle or 5-needle electrode configurations for IRE via laparoscopy and open surgery in the liver of a pig. The electrodes were also tested with trans-anal and trans-oral endoscopic approaches using different electrode configurations. All procedures were performed under ultrasound guidance. The authors state that the electrodes and their mechanical functionality are suitable for the listed procedures, and the electrodes are compatible with the 5-mm laparoscopic trocar and other surgical instruments. Laparoscopic and endoscopic approaches to deep-seated tumors could potentially minimize the risk of bleeding and infection. The FEM model of such an electrode array is presented in Figures 5A, B. Colored needle parts represent the non-conductive adjustable 2-, 3-, or 4-cm-length electrodes, positioned at 0°, 10°, 20°, and 30° angles. The deployable electrodes connected to high and ground potentials are shown in red and blue, respectively, with a fixed length of 2 cm.

The expected spatial electric field distribution at different insertion angles of the electrodes (0° 10°, 20°, and 30°) is shown in Figures 5C–J. The computations were performed with 2-cm needle extension. Diagonal and semi-diagonal black arrows represent the pairs of electrodes where the voltage is applied. It can be seen that by changing the active electrode pairs, the volume of the electroporated tissue can be controlled. If required, the whole volume could be ablated and/or reversibly electroporated due to overlapping of the high-intensity PEF regions. The capability to increase the length of each needle independently also allows for controlling the depth of the electroporated volume. Essentially, these electrodes are a specific case of the applicators presented in Figure 4, but when non-parallelism is intentional. The problems of precise needle positioning are still applicable.

At the same time, fixed-position electrodes are advantageous for minimizing non-parallelism during needle insertion. Electric field distribution analysis of a hexagonal array of four-needle pair fixed electrodes is represented in Figures 6A–F. In the first case (Figures 6D–F), the applied voltage is 600 V, which induces a relatively homogeneous electric field between the positive and negative electrode pairs of 2- and 10-mm needle lengths.

Such electrodes are commercially available and used for intradermal (ID) electroporation, featuring 2- and 10-mm electrode lengths (Needle Array Electrodes for BTX AgilePulse In Vivo, 2021; Fixed Electrodes, 2022b). Intradermal electrodes are used when the penetration of outer skin layers, i.e., the stratum corneum, dermis, and epidermis, is sufficient. The spatial electric field distribution generated by an ID electrode with two rows of four needles is presented in Figures 6D–F. There is a notable difference in spatial electric field distribution in the tissue; 10-mm needles inserted at approximately 7 mm depth provide a uniform electric field; on the contrary, 2-mm electrodes feature a less homogeneous electric field distribution in the effective volume of effect. Nevertheless, when the limitations are taken into account, electrodes can be successfully utilized in practice (Roos et al., 2006; 2009; Lladser et al., 2010).

In the case of hexagonal electrodes (Figures 6A–C), the electric field is located around the positively charged electrode; therefore, the electric field is highest in the central part of the target tissue, while potentially healthy tissue on the edges remains intact. These electrodes are suitable for bigger tumors in the ECT context (Pichi et al., 2018), and different similar configurations can be used for gene therapy (Gilbert et al., 1997).

To summarize, fixed-position electrodes are likely to produce a more uniform electric field due to better control of tissue penetration—the chances of non-parallelism of electrodes are minimized. However, fixed-position electrodes are suitable only for tumors of predetermined size and, therefore, are less flexible for cancer treatment, especially when the tumor size is significantly smaller or bigger than the gap between the fixed electrodes. ID electrodes are appropriate for gene therapy; however, the penetration depth should be considered to ensure sufficient homogeneity of the electric field.

One of the solutions to minimize the challenges of electrode positioning is the use of single-needle electrode configuration (Neal et al., 2010; Garcia et al., 2014). Such an electrode type consists of an electrode body, cathode, insulator, and anode on the sharp tip of the needle, where each part has a predetermined length and width (Figure 7A). Figure 7B shows the spatial electric field distribution. It can be seen that the design solves the problem of non-parallelism; however, as a trade-off, the electric field distribution is relatively non-homogeneous. Moreover, the diameter of this electrode is relatively large; therefore, it is applicable mainly for bigger tumors.

The new prototype of invasive electrodes, called curved electrodes, was proposed by Ritter et al. (2018). Curved electrodes are minimally invasive electrodes consisting of a penetrating central needle and four thin hollow expandable electrodes for pulse application and injection of chemotherapeutic agents. For simulation, the electrode terminal voltage was set to 1500 V. Furthermore, electric field distribution analysis was performed and is presented in Figure 8. Cut plane Cp1 shows the field strength on the surface when the penetration depth is shallow. As it can be seen, the highest electric field is around the central needle and at the positively charged satellite electrode tips. As a consequence, other areas will be treated with a lower PEF. However, penetration into deeper tissue layers results in a more homogeneous treatment (Cp2), which is also supported by the side view simulation (Cp3 and Cp4). Changing the number of active electrodes allows for controlling of the treatment volume. Moreover, the position of satellite needles can be adjusted with the movable part, which introduces additional flexibility.

To conclude, needle electrodes are advantageous for deep-seated tumors and intramuscular or intradermal GET targets. Both fixed-position and independent needle arrays show acceptable performance and are applied in clinical treatment. Fixed-position electrodes are easier to apply, but they are mostly suitable for specific size targets, i.e., the target should be of similar size as the gap distance. Otherwise, the healthy tissue will receive unnecessary pulsing. On the contrary, if the target exceeds the space between the electrodes, the treatment will result in partial response due to only a fraction of the tumor being affected. To overcome this problem, manual needle repositioning or more accurate multiple needle application along with a brachytherapy grid (van den Bos et al., 2016) may be considered. In addition, such fixing equipment and non-conductive ring nuts or “stoppers” help minimize non-parallelism after needle penetration. When the tumor is deep-seated, the requirements for treatment and electroporation electrodes are even more intricate; thus, tumor boundaries cannot be seen with the naked eye. Real-time imaging, such as ultrasound (van den Bos et al., 2016; Hsiao and Huang, 2017) or fluoroscopy (Pavliha et al., 2012b) guidance, is a solution. The combination of invasive electrodes with an imaging procedure gives the possibility for more accurate target boundary assessment, minimizing the possibility of multiple pulsing on the same area of the tissue since the field strength for each electrode pair is predetermined. Real-time imaging is also advantageous when tumors can be reached through the skin without incision (Lee et al., 2007).

Various minimally invasive (Choi et al., 2010; Yan et al., 2010; Xia et al., 2021) and non-invasive (Heller et al., 2010; Guo et al., 2011) microneedle array electrodes are mainly designed for transdermal drug delivery. The aim of microneedles is to affect the outer skin layers or muscle and ensure distribution of sufficient electric field for reversible electroporation, which is usually employed for electroporation-based gene delivery (also called gene vaccination). An example of such an array of electrodes is presented in Figures 9A–F.

The model consists of 900 (30 per row and 30 per column) 0.2-mm-length microneedles placed at 0.1 mm distance between needle tips. Terminal and ground potential electrodes are distributed in each row as shown in Figures 9A, C, and field analysis was performed using 100 V terminal voltage. The results are summarized in Figures 9E, F.

It can be seen that the depth of high-intensity electric field penetration is limited; however, it is still sufficient for transdermal gene delivery. The most significant disadvantage of most gene therapy electrodes is the relatively small operating area. In order to increase the effective area of the minimally invasive electrodes, roller type electrodes can be used as proposed by Yang et al. (2021). In Figure 9G, the model of such an electrode structure is shown, and the expected electric field distribution is presented in Figure 9H.

The FEM analysis results indicated that the highest value of electric field strength is located around the microneedle tips; however, it decreases drastically in the gap between the negatively and positively charged needle pairs (Figure 9H). A similar electrode type was analyzed by Huang et al. (2018). In order to improve the non-homogeneity, it was proposed to combine the structure with conductive gel microchannels, which are formed by applying gel on the skin and rolling the needles on the skin ten times before the pulsing. As an approximation, our simulation of this treatment covers the electric field distribution with one layer of microchannels filled with conductive gel (Figure 9I). The results indicated that the conductive gel channels can improve electric field homogeneity.

As previously mentioned, non-invasive electrodes interact through the skin interface. Non-invasive electrodes are less suitable for deep subcutaneous tumors; however, they may be advantageous on exophytic tumors or melanoma, which appears on the skin surface. Several configurations of such electrodes are presented in the following.

Plate electrodes are most commonly used as a non-invasive electrode type (Al-Sakere et al., 2007; Sedlar et al., 2012; Novickij et al., 2021). The configuration consists of two rectangular stainless steel plates with fixed gap size [or adjustable with clippers (Caliper Electrodes for Electroporation Applications, 2022; Wang et al., 2008)] placed in parallel representing the anode and cathode (Fixed Electrodes, 2022a). Figures 10D-F show the application of plate electrodes for electroporation of skin (Figure 10D) and small skin lump (Figure 10E).

Tweezer-type electrodes are also a sub-population of parallel plate electrodes that are comfortable to be used when the gap between electrodes need to be adjustable. An example of round tweezer electrodes with adjustable 1–20 mm gap size is shown in Figures 10G–I (Tweezertrodes Electrodes for In Vivo and In Utero Electroporation Applications, 2022). For prediction of electric field distribution, we used a specific case with 5-mm-diameter and 4.5-mm-gap electrodes covering a tissue lump. As expected, due to limited contact area, the electric field was not homogeneous. Proper and maximum contact of the electrode surface with the tissue should be ensured for the electrodes to be applicable in cancer treatment. Nevertheless, the requirements for electric field homogeneity during gene therapy are lower; thus, this type of tweezer electrodes is sometimes favorable due to the ease of use and compactness (Maiorano and Mallamaci, 2009; Shi et al., 2010; Zhang et al., 2022). Tweezer electrodes are available in a variety of tip shapes and sizes (Tweezertrodes Electrodes for In Vivo and In Utero Electroporation Applications, 2022).

In the case of superficial tumors, good contact between the electrodes and the tissue is essential, as it may dramatically affect the electric field distribution (Figures 10A, B). Also, forming a lump can be sometimes advantageous. Nevertheless, it can be clearly seen that the top and bottom of the tumor are covered by a significantly lower electric field, especially when the tumor is embraced insufficiently (Figure 10B). If not taken into account during the treatment planning step, it may result in re-occurrence of the tumor.

L-shaped electrodes are another commonly used applicator for electroporation-based treatments. This type of electrodes is mostly used for large cutaneous margins and is designed for the treatment of skin tumors of all sizes. An example of such a commercially available electrode arrangement is shown in Figure 11A (Accessories ElectroVet, 2021). For the simulation, the electrodes were placed on the tissue boundary and pushed into the skin; furthermore, 1300 V voltage was applied. Figures 11B, C show that the electric field of the L-shaped electrodes is inhomogeneous, i.e., the highest dose of PEF is expected at the skin surface, while deeper tissue layers are affected by a significantly lower electric field.

Similar L-shaped electrode configurations have been employed for in vivo electroporation for both gene therapy and electrochemotherapy by Mazères et al. (2008). In their study, electrodes were repositioned by 90° after each pulse train to compensate electric field inhomogeneity. The results showed 93% of complete response on treated animals, and the electrodes were efficient on tumors of up to 5 cm diameter. Gene therapy was also successful when performed using 4-mm-gap electrodes. However, ablation of the skin was observed.

Another option of non-invasive electrodes is the 4-plate electrode (4PE), where pulses are delivered by two parallel electrode pairs instead of rotating two plates by 90°. The 4PE was developed by Heller et al. initially for gene electro-transfer procedures (Heller et al., 2007). The electrodes operate as follows: metal plates are placed on the target to “grab” the desirable skin fold, and the non-conductive ring-shaped nut is tightened to establish a constant gap size. Two different gap sizes were analyzed—6 mm, without plate expansion, and 8 mm, with 8° expansion (Figures 11D,G, respectively). As can be seen from Figures 11E, F, the spatial electric field distribution of the 4PE is similar to that of two-plate electrodes. However, since the pulsing is performed between 90° shifted electrode pairs, during the second pulse train, the non-homogeneity of the treated volume can be better compensated.

According to the spatial electric field distribution presented in Figure 11F, the value of the electric field decreases at the top and the bottom of the electroporated tissue. The problem is even more apparent when the plates are expanded (Figure 11I). However, when the target tissue is larger in size, such expansion may be advantageous, which ensures a wider contact area by squeezing the tissue between all the plates.

Another type of superficial electrode currently being developed is the pliable electroporation patch with thin flexible electrodes. Currently, such electrodes are successfully employed for gene delivery. Flexible electrodes adapt to the skin surface and, therefore, ensure good contact. Figure 12 illustrates the micromachined pliable electroporation patch (ep-Patch), which consists of rectangular parallel gold electrodes presented by Wei et al. (2015). We analyzed a model with 0.2 mm width and 0.5 mm spacing between the electrodes (Figure 12A). The electric field distribution was evaluated with 50 V terminal voltage (Figures 12B, C). It can be seen that the electrodes ensure acceptable transdermal electric field distribution, while the flexibility to adapt to the skin surface is advantageous for practical applications.

As previously pointed out, electroporation success strongly depends on the electric field distribution in the tissue (Miklavcic et al., 2006). In order to guarantee effective treatment, homogeneous PEF must be spread through the whole target; otherwise, only partial treatment of the target will be achieved. This problem is especially apparent for non-invasive electrodes in ECT, resulting in tumor re-occurrence (Shankayi and Firoozabadi, 2012). Therefore, either alternative electrode configurations should be considered or a repositioning strategy developed during the treatment planning step.

Simulation results showed that non-invasive applicators are not capable of reaching subcutaneous or deeper tissues with sufficient PEF value. A higher pulse amplitude could be considered, but it would trigger IRE of the tumor in close proximity with the electrode terminals, which is not always desired. Increasing the amount of conductive gel between the electrodes and tissue fold improves the PEF distribution (Ivorra et al., 2008); however, the parts of the tumor without direct contact with the electrodes are likely to be affected by the insufficient electric field (Novickij et al., 2021). Additionally, too much conductive gel, especially in the top part of the tumor, can short-circuit the generator.

Plate-and-fork-type electrodes are commercially available electrodes (Electrodes for in vivo electroporation, 2021b) that are effective for gene therapy (Maruyama et al., 2001).

The analyzed model consists of a pair of tweezers, one with a stainless steel fork consisting of 3-mm needles and the other with a 5 × 8-mm plate, and a spherical tumor (Figure 13). The fork with needles is inserted into the tissue, and simultaneously, the rectangular plate embraces the target. This electrode also contains a fixing part to keep the gap size stable between the fork and the plate while applying the pulses. Clearly, this is a convenient way for tight and accurate grasp of the target tissue; however, it does not solve the field homogeneity problem; thus, the application is limited to gene therapy.