Enhancing pancreatic cancer ablation efficiency: bipolar IRE with conductive MOF

Abstract

Irreversible electroporation (IRE) has emerged as a promising therapeutic modality for pancreatic cancer. However, traditional IRE techniques rely on high-voltage electric fields and require precise alignment of multiple electrodes, which complicates the procedure and increases associated risks. To address these challenges, we developed a novel “bipolar” IRE electrode that combines both the cathode and anode into a single device, simplifying the procedure and potentially reducing operational risks. Additionally, we incorporated a conductive metal–organic framework (MOF) to enhance the electric field distribution of the electric field, thereby improving the efficacy of tumor ablation. Mechanistic studies revealed that this combined approach induces tumor cell apoptosis and improves the consistency of ablation outcomes. Both in vitro and in vivo experiments demonstrated that the bipolar electrode, in combination with the conductive MOF, achieved a significant apoptosis rate of 59.95% ± 2.41 in vitro and resulted in an 85.77% ± 0.21 reduction in tumor volume in in vivo models, without any adverse effects. This approach provides a more optimized and potentially more effective solution for treating pancreatic cancer.

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1 Introduction

Pancreatic cancer is one of the most aggressive malignancies, with a dismal 5-year survival rate of less than 8%.^1–3 Surgical resection remains the gold standard for treatment; however, approximately 80% of patients are ineligible for surgery at the time of initial diagnosis.^4,5 Conventional therapies, such as radiotherapy^6,7 and chemotherapy,^8–11 often yield limited efficacy and cause significant side effects, highlighting the pressing need for alternative treatments options. Minimally invasive interventional approaches have thus emerged as vital strategies for managing pancreatic cancer.^12,13 Among these, irreversible electroporation (IRE) is a promising non-thermal ablation technique. IRE utilizes high-frequency electric fields to induce nanoscale pores in the cell membrane, resulting in the disruption of cellular homeostasis and apoptosis.^14–16 This non-thermal therapy mechanism avoids the “heat sink” effect inherent to thermal ablation where blood flow dissipates heat and compromises treatment efficacy. Unlike traditional thermal ablation methods, such as radiofrequency, microwave, and laser ablation, IRE selectively targets tumor cells while sparing adjacent critical structures, including blood vessels and nerves. This unique advantage significantly reduces the risk of complications, including pancreatic fistula formation and hemorrhage.^17–19 Additionally, IRE promotes the release of tumor antigens, which can activate antitumor immune responses, further enhancing its therapeutic potential.^20,21 IRE's ability to preserve extracellular structures and induce immunogenic cell death makes it particularly effective for tumors near vital organs or resistant to conventional therapies. These properties position IRE as a valuable therapeutic option in the treatment of pancreatic cancer.

Despite its potential, current IRE technology requires precise alignment of two or more electrodes around the tumor, typically guided by imaging techniques such as computed tomography (CT)²² or ultrasound (US),²³ to generate an effective electric field between cathode and anode electrodes.^24–26 This electrode placement is critical, as it directly affects the electric field strength and distribution, thereby influencing the efficacy of tumor ablation.²⁷ In contrast to thermal ablation techniques, which generally require the placement of a single electrode at the tumor core, the complexity, cost, and potential complications associated with IRE are substantially higher.^28,29 These challenges significantly hinder the broader clinical application of IRE in oncology.

In this study, we present a novel bipolar electrode design that integrates the cathode and anode into a single device, allowing for direct insertion into the tumor core. This design achieves the same therapeutic effect as two parallel monopolar electrodes, while reducing both the time and complexity associated with electrode placement. By simplifying the ablation procedure, our bipolar electrode has the potential to enhance the clinical applicability of IRE. To further maximize the ablation efficacy, we also incorporated the local injection of conductive nanomaterials based on metal–organic frameworks (MOFs). MOFs were selected for their tunable electrical conductivity, high biocompatibility, and ability to passively accumulate in tumors via the enhanced permeability and retention (EPR) effect.³⁰ Their porous structure also allows potential co-delivery of therapeutic agents, enabling synergistic tumor-killing mechanisms such as cuproptosis.³¹ These nanomaterials modify the electrical conductivity within and around tumor cells, intensifying and broadening the apoptotic effect, thereby expanding the ablation range with a single electrode insertion.³² The integration of MOFs with IRE leverages their capacity to enhance electric field distribution, addressing historical limitations of multi-electrode alignment.

2 Experimental section

2.1 Materials and chemicals

Ni₃(HITP)₂ powder was purchased from Nanjing XFNANO Materials Tech Co., Ltd. Dopamine hydrochloride was purchased from Aladdin Biochemical Technology Co., Ltd. Electroporation system was jointly developed by the research team and the Zhejiang CuraWay Medical Technology Co., Ltd. A 25% ammonia solution and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. Mito-tracker, CCK-8 cell viability and ROS assay kit were purchased from Beyotime Biotech Inc.

2.2 Design and simulation of bipolar electrodes

The electric field distribution, temperature distribution, and ablation range of bipolar electrodes were simulated using Comsol Multiple Physical Quantities modeling software. The simulated tissue was represented as a cubic model, into which the device was immersed. The applied electric field intensity was set to a static value, and the grid was unstructured, utilizing tetrahedral elements. The computational model was constructed using an electrode that integrates both the cathode and anode into a single device.

2.3 Cell lines and cell culture

KPC cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Gibco DMEM (Thermo Fisher), supplemented with 10% fetal bovine serum, 100 units mL⁻¹ of penicillin, and 100 μg mL⁻¹ of streptomycin. All cells were incubated in a cell culture chamber (Thermo Fisher) at 37 °C and 5% CO₂.

2.4 In vitro therapeutic efficacy of bipolar electrode IRE ablation on potato model

IRE ablation was performed on a potato model using both bipolar and monopolar electrodes under varying parameters. In each experiment, bipolar and monopolar electrodes were inserted along the length of the potato, ensuring a minimum insertion depth of 10 mm for the conductive portion. The electroporation zones were visualized by tissue sections and the extent of ablation was measured 24 hours post-experiment.

2.5 In vitro therapeutic efficacy of bipolar electrode IRE ablation on cells model

IRE ablation was performed on KPC cells model (1 × 10⁵ cells per well) cultured in 24-well plates for 24 hours using bipolar and monopolar electrodes with varying parameters. After the ablation, 1 mL 0.4% Trypan Blue reagent (biosharp Co., Ltd) was added to each well, and the cells were washed three times with PBS. The areas of blue and non-blue cells were observed and measured to evaluate the ablation effect.

2.6 IRE ablation parameters optimization

For the monopolar electrodes, 90 pulse sequences were applied with a pulse duration of 90 μs, an exposed segment length of 5 mm, a needle spacing of 4 mm, and electric field strengths ranging from 750 to 3000 V cm⁻¹. For bipolar electrodes, similar settings were used, with 90 pulse sequences, a pulse duration of 90 μs, an exposed segment length of 6 mm, a needle spacing of 4 mm, and electric field strengths ranging from 750 to 3000 V cm⁻¹.

2.7 Preparation and characterization of Ni₃(HITP)₂@PDA

To prepare Ni₃(HITP)₂@PDA, 5 mg Ni₃(HITP)₂ was dissolved in 5 mL ethanol and subjected to ultrasonic dispersion for 10 minutes. The suspension was then transferred to a 100 mL beaker, and 18 mL of double-distilled water and 3 mL of ethanol were added. The mixture was stirred continuously at room temperature for 10 minutes. Subsequently, 600 μL of 25% ammonia solution was added dropwise, and stirring continued for an additional 20 minutes. Then, 2 mL of a 50 mg mL⁻¹ PDA solution was introduced, and the mixture was stirred for 3 hours until a color change from black to brown was observed, indicating the successful synthesis of Ni₃(HITP)₂@PDA. The resulting product was washed by centrifugation with double-distilled water and ethanol (10 000 × rps, 10 minutes) three times, then dried for further use.

2.8 In vitro evaluation of bipolar electrode IRE ablation with Ni₃(HITP)₂@PDA

KPC cells (1 × 10⁵ cells per dish per well) were cultured in confocal dishes or 6-well plates for 24 hours. The IRE ablation procedures were conducted with both monopolar and bipolar electrodes. Specifically, for the monopolar electrode, the electric field intensity was set at 1500 V cm⁻¹, the electrode spacing was 0.7 cm, the pulse duration was 90 μs, and the number of repetition pulses was 90. In the case of the bipolar electrodes, the electric field intensity was chosen to be 2500 V cm⁻¹, the electrode spacing was 0.4 cm, the pulse duration remained at 90 μs, and similarly, the number of repetition pulses was 90. Ni₃(HITP)₂@PDA was added 2 hours prior to bipolar electrodes IRE ablation. Live/dead cell staining, ROS staining and Mito-tracker staining were conducted on the treated KPC cells in various groups and observed using a confocal laser scanning microscope (CLSM, Leica STELLARIS 5). Transmission electron microscope (JEM-1400, JEOL) was used to examine alterations in the cell membrane and organelles post-treatment. Flow cytometry (FACSCanto II, BD) was employed to assess apoptotic cells according to standard procedure.

2.9 In vivo evaluation of bipolar electrode IRE ablation with Ni₃(HITP)₂@PDA

BALB/c nude mice (male, 4–6 weeks, 15–20 g) was injected 5 × 10⁶ KPC cells (10 μL) into the right thigh to establish a tumor-bearing mouse model. The mice were randomly assigned to control, Ni₃(HITP)₂@PDA, monopolar electrode, bipolar electrodes and bipolar electrodes + Ni₃(HITP)₂@PDA groups. The IRE ablation procedures were conducted with both monopolar and bipolar electrodes. Specifically, for the monopolar electrode, the electric field intensity was set at 1500 V cm⁻¹, the pulse duration was 90 μs, and the number of repetition pulses was 90. In the case of the bipolar electrodes, the electric field intensity was chosen to be 2500 V cm⁻¹, the pulse duration remained at 90 μs, and similarly, the number of repetition pulses was 90. A 100 μL injection of Ni₃(HITP)₂@PDA was added 2 hours prior to bipolar electrodes IRE ablation. Tumor volumes and body weights of the mice were recorded at predetermined time points. After 14 days, the animals were euthanized, and the tumors were resected, photographed, and used for further analyses.

2.10 Biosafety assessments

2.10.1 In vitro cytotoxicity assay. KPC and L929 cells (1 × 10⁴ cells per well) were cultured in 96-well plates and incubated with various concentration of Ni₃(HITP)₂@PDA for 12, 24 hours. Cell viability was assessed using a CCK-8 cell viability assay kit. The cell viability was calculated using the following eqn (1):

2.10.2 In vitro hemolysis assay. Whole blood was collected from healthy BALB/c nude via eyeball blood collection and stored in anticoagulant tubes containing EDTA. After gentle mixing, the blood samples were centrifuged at 3000 rpm for 15 minutes to assess the hemolysis rate of the nanoparticles. Different concentrations of nanomaterials (12.5–200 μg mL⁻¹) were prepared, and 1 mL of each nanomaterial solution, 1 mL of double-distilled water (ddH₂O), and 1 mL of PBS were each mixed with 20 μL of blood. The mixtures were incubated at 37 °C for 4 hours, followed by centrifugation at 3000 rpm for 15 minutes. The samples were leveled, and the images of the hemolysis phenomenon were captured using a mobile phone. The supernatants were carefully extracted, and the absorbance at 542 nm was measured using an enzyme spectrophotometer. The hemolysis ratio was calculated using the following eqn (2):

2.10.3 In vivo organ safety experiment. At day 14, histological analysis of critical organs (heart, liver, spleen, lung, and kidney) was conducted following HE staining to assess the biological safety of different treatments (bipolar electrodes and bipolar electrodes + Ni₃(HITP)₂@PDA), with a PBS group serving as the control.

2.11 Histological analysis and immunostaining

For histological analysis, excised tumors and organs were fixed in 4% paraformaldehyde in phosphate-buffered saline, embedded in paraffin, and sectioned into 5 μm thick slices. Tumors were stained with hematoxylin and eosin (H&E). Subsequently, immunofluorescent staining was performed for Ki-67, P53, TUNEL, BAX, BCL-2 and TNF-α to further evaluate tumor cell proliferation and cell apoptosis. The intensity of positively stained areas for Ki-67, p53, TUNEL, BAX, BCL-2, and TNF-α was quantified using ImageJ software.

2.12 Statistical analysis

Statistical analyses were performed using Prism software version 10.0 (GraphPad). Data are presented as the mean ± standard error of the mean. The statistical significance between groups was evaluated using the independent samples t-test or one-way ANOVA, while the statistical significance within groups was determined using Tukey's test.

2.13 Ethical statement

This research complies with all relevant ethical regulations. Animal studies were conducted in accordance with the National Institute Guide for the Care and Use of Laboratory Animals. The experimental protocols were approved by the Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine (Reference Number: 2022-506). BALB/c nude mice (male, 4–6 weeks, 15–20 g), were purchased from Ziyuan experimental animal Technology Co., Ltd (Hangzhou, China). The animals were raised in specific pathogen-free animal facility and provided with ad libitum access to food and water. All experimental/control animals were co-housed in a habitant under standard conditions (23–26 °C, 40–60% humidity, 12 hours light–dark cycle, and 4–5 mice per cage). At the end point of the study, animal euthanasia was performed via CO₂ inhalation followed by cervical dislocation.

3 Results and discussion

3.1 Design and simulation experiments of bipolar electrode

Building upon the design principles and manufacturing experience developed in our group's previous work with monopolar electrodes (Fig. S1a and c, ESI†), we have created a novel IRE ablation electrode that integrates both the cathode and anode into a single unit. This innovative design features two exposed ends of 3 mm in length, separated by a 4 mm insulating interval (Fig. S1b and d, ESI†). This integrated configuration simplifies the electrode placement process and improves targeting precision by generating a more controlled electric field.

Using COMSOL Multiphysics simulations, we observed that when the bipolar electrode is aligned parallel to the electric field, it produces an elliptical ablation zone, whereas a perpendicular orientation results in a circular ablation zone. This behavior is pivotal for customizing the ablation area according to the shape of the tumor. Further analysis revealed that as the electric field intensity decreases, the ablation zone shrinks in both the horizontal (Fig. 1(a)–(c) and Fig. S2, ESI†) and vertical planes (Fig. 1(d)–(f) and Fig. S3, ESI†), accompanied by a reduction in temperature near the electrode (Fig. 1(d)–(i) and Fig. S4, ESI†).

Fig. 1 COMSOL multiphysics software simulation of the electric field, temperature, and ablation range distribution for a bipolar electrode under varying electric field parameters. Electric field distribution parallel to the electrodes (a)–(c) and perpendicular to the electrodes (d)–(f); ablation temperature distribution (g)–(i); and ablation volume (j)–(l) at electric field strengths of 3000 V cm⁻¹, 2000 V cm⁻¹, and 1000 V cm⁻¹, respectively.

Consequently, the ablation zone transitions from an ellipsoidal to a gourd-like shape (Fig. 1(j)–(l) and Fig. S5, ESI†). These simulation results demonstrate that our newly designed bipolar electrode can be tailored to effectively target tumors of varying shapes and sizes by adjusting the electric field parameters. It is worth noting that the bipolar electrode developed by Zhao et al.³³ generates temperatures more than 50 °C at the electrode tip during high electric field intensity ablation. In contrast, our biopolar electrode maintains the temperature around the electrode below 45 °C, even at a field strength of 3000 V cm⁻¹. This ensures that the electrode does not cause thermal damage to the normal tissues surrounding the tumor during ablation. This feature makes the electrode particularly suitable for treating pancreatic tumors located near critical structures such as blood vessels, nerves, bile ducts, and pancreatic ducts.³⁴

3.2 In vitro therapeutic efficacy of bipolar electrode ablation

To validate the simulation data on the bipolar electrode's ablation capabilities, we conducted in vitro experiments using both potato-models and pancreatic cancer cell lines. In the potato model, we observed that, at a field intensity of 2500 V cm⁻¹, the bipolar electrode produced ablation zones of approximately 2.88 ± 0.08 cm², consistent with the elliptical ablation patterns predicted by the simulation software. Additionally, the ablation area decreased proportionally as the applied electric field intensity was reduced (Fig. S6, ESI†). In subsequent cell experiments, we demonstrated that the bipolar electrode effectively ablated pancreatic cancer cells, with the damage zone closely aligned with the results from both the simulation and potato model. Similarly, the efficacy of ablation diminished as the electric field intensity decreased, in accordance with the pattern observed in the potato model (Fig. S7, ESI†). However, when the electric field intensity fell below 1000 V cm⁻¹, residual survival of KPC cells was observed around the insulating layer of the bipolar electrode. This finding suggests that further intervention or optimization is required to fully inactivate tumors in these regions and prevent incomplete ablation.

To better understand the differences between monopolar and bipolar electrodes, we compared their ablation effects using both potato and cell models. The results indicated that, at a field intensity of 2500 V cm⁻¹, the bipolar electrode achieved a similar ablation area to that of a monopolar electrode at a field intensity of 1500 V cm⁻¹ (Fig. S8–S10, ESI†). These findings suggest that while the bipolar electrode may require a higher field intensity to match the ablation area of a monopolar electrode, it offers the advantage of simplifying the ablation procedure by integrating both the cathode and anode into a single unit. However, applying more than 1500 V cm⁻¹ to the bipolar electrode may pose a risk of electrical hazards.³⁵ Additionally, the low conductivity and heterogeneity of tumor cells can result in an uneven electric field distribution, undermining the efficacy of IRE ablation.³⁶ Recent advances in nanotechnology have shown that nanomaterials can greatly boost the precision and effectiveness of tumor diagnosis and treatment.³⁷ However, the differences in cellular electrical conductivity have become a major obstacle. To solve this, we’ve applied a conductive nanomaterial around the tumor. This improves both internal and peripheral conductivity, making the electric field more uniform. As a result, the therapeutic efficacy of bipolar IRE ablation is significantly enhanced without needing to increase the electric field intensity. Nanomaterial around the tumor to enhance both internal and peripheral conductivity, thereby improving the uniformity of the electric field and enhancing the therapeutic effectiveness of bipolar IRE ablation without necessitating an increase in electric field intensity.

3.3 Characterization of conductive MOF

Metal–organic frameworks (MOFs) represent a highly versatile class of nanocarriers, gaining significant attention in oncology for their potential in tumor diagnosis and treatment.³⁸ MOF are distinguished by their exceptional drug-loading capabilities, which allow for efficient delivery of therapeutic agents.³⁸ Moreover, their intrinsic electrical and thermal properties further enhance their functionality,³⁹ presenting promising avenues for improving the precision and efficacy of cancer therapies. Ni₃(HITP)₂ is a sheet-like MOF (Fig. 2(a)–(c)), demonstrates excellent electrical conductivity, making it particularly suitable for tumor treatment when combined with electroablation technology.^40–42 Furthermore, Guo et al.⁴³ reported that Ni²⁺ can stimulate the production of reactive oxygen species (ROS) and induce apoptosis. Therefore, under electrical stimulation, Ni₃(HITP)₂@PDA releases Ni²⁺, amplifying the apoptotic response. However, Ni₃(HITP)₂ suffers from limited water dispersibility, poor biocompatibility, and inherent cytotoxicity, which restrict its direct application in biological systems.^44,45 To overcome these limitations, we modified the surface of Ni₃(HITP)₂ with a polydopamine (PDA) coating to improve its dispersibility and biosafety. Dynamic light scattering (DLS) analysis of Ni₃(HITP)₂ and its modified counterpart Ni₃(HITP)₂@PDA revealed distinct differences in polydispersity index (PDI): the pristine Ni₃(HITP)₂ exhibited a PDI of 0.4878 ± 0.04, whereas the PDA-modified sample showed a significantly lower PDI of 0.4318 ± 0.02 (p < 0.05, Fig. S11, ESI†). This statistically significant reduction in PDI demonstrates that surface functionalization with PDA effectively improves the dispersibility of Ni₃(HITP)₂. The successful deposition of the polydopamine (PDA) layer on Ni₃(HITP)₂ was systematically validated through multi-modal characterization: morphological analysis via scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed a conformal PDA coating on the MOF surface with preserved structural integrity (Fig. 2(d) and (e)); elemental verification by energy-dispersive X-ray spectroscopy (EDS) mapping demonstrated homogeneous nitrogen distribution characteristic of PDA incorporation (Fig. 2(f)); and X-ray diffraction (XRD) patterns confirmed retained crystallinity of the MOF framework post-modification. Critically, Fourier-transform infrared (FTIR) spectroscopy provided complementary chemical evidence through the emergence of N–H stretching vibrations at 3310 cm⁻¹ and C=O/C–N bending modes near 1578 cm⁻¹. These spectral fingerprints, distinctly absent in pristine Ni₃(HITP)₂, conclusively confirm successful surface functionalization (Fig. S12, ESI†). The resulting Ni₃(HITP)₂@PDA nanoparticles exhibited a diameter of 426.90 ± 7.18 nm (Fig. 2(h)) and a zeta potential of −31.71 ± 2.75 mV (Fig. S13, ESI†), supporting their improved stability in solution. XPS analysis further elucidated the elemental composition: the atomic ratios of C, N, and Ni in pristine Ni₃(HITP)₂ were determined to be 81.40%, 15.18%, and 3.42%, respectively. After PDA encapsulation, these ratios shifted to 86.64% (C), 10.60% (N), and 2.76% (Ni). Moreover, conductivity measurements demonstrated that Ni₃(HITP)₂ retained its conductive properties following PDA modification (Fig. S14, ESI†), with conductivity increasing as the concentration rose (Fig. 2(i)). This retention of conductivity, combined with improved dispersibility and biocompatibility, makes Ni₃(HITP)₂@PDA a promising candidate for enhancing the effectiveness of IRE-based tumor therapies.

Fig. 2 Characterization of conductive MOFs. Scanning electron microscopy (a), transmission electron microscopy (b), energy dispersive spectroscopy (c) of Ni₃(HITP)₂; scanning electron microscopy (d), transmission electron microscopy (e), and energy dispersive spectroscopy (f), X-ray diffraction (g), dynamic Light scattering (h), and conductivity (i) of Ni₃(HITP)₂@PDA.

3.4 Biosafety of Ni₃(HITP)₂@PDA and in vitro therapeutic efficacy of bipolar electrode IRE ablation combined with Ni₃(HITP)₂@PDA

To assess the biosafety of Ni₃(HITP)₂@PDA, we conducted both in vitro and in vivo evaluations. In vitro, the 12 and 24-hours Cell Counting Kit-8 (CCK-8) assay revealed no significant cytotoxicity at concentrations below 100 μg mL⁻¹ (P > 0.05) (Fig. 3(a) and Fig. S16a, ESI†). In vivo hemolysis assays in mice demonstrated a hemolysis rate below 3% at concentrations under 200 μg mL⁻¹ (Fig. 3(b) and Fig. S16b, ESI†). Furthermore, one week after local injection of Ni₃(HITP)₂@PDA into a mouse subcutaneous tumor model, histological analysis of major organs (heart, liver, spleen, lungs, kidneys) showed no signs of damage (Fig. 3(c)). Peripheral blood analysis of tumor-bearing mice indicated that Ni₃(HITP)₂@PDA had no adverse effects on liver, kidney, or heart function (Fig. S17, ESI†). These results confirm that Ni₃(HITP)₂@PDA exhibits excellent biosafety in both in vitro and in vivo settings. After confirmation its biosafety, we evaluated the antitumor effects of bipolar electrode IRE ablation The results of live–dead cell staining experiments demonstrated that the bipolar electrode could obtain the same tumor cell killing effect as the monopolar electrode. The tumor killing effect of the bipolar electrode can be further enhanced by the combination of Ni₃(HITP)₂@PDA (Fig. 3(d)).

Fig. 3 Biosafety evaluation of Ni₃(HITP)₂@PDA and antitumor effect of bipolar electrodes + Ni₃(HITP)₂@PDA ablation in a cell model. (a) Results of the 24-hour CCK-8 assay, with “ns” indicating no significant difference. (b) Hemolysis assay results. (c) HE-stained histological sections of internal organs (Heart, liver, spleen, lungs, kidneys) from mice with subcutaneous tumors. Groups I, II and III represent the control, bipolar electrodes and bipolar electrodes + Ni₃(HITP)₂@PDA, respectively. (d) Live/dead staining of control, Ni₃(HITP)₂@PDA, monopolar electrode, bipolar electrodes and bipolar electrodes + Ni₃(HITP)₂@PDA groups.

3.5 In vitro therapeutic mechanism of bipolar electrode IRE ablation combined with Ni₃(HITP)₂@PDA

To elucidate the mechanism underlying the combination of bipolar electrodes and Ni₃(HITP)₂@PDA for the treatment of pancreatic tumors, we systematically established a KPC cell model. The results of mitochondrial membrane potential experiments clearly demonstrated that bipolar electrode ablation could effectively damage the mitochondria of tumor cells, thereby triggering apoptotic and necrotic processes. This effect was found to be comparable to that of monopolar electrode ablation. Moreover, the addition of Ni₃(HITP)₂@PDA was shown to significantly augment both the number and the severity of mitochondrial damage (Fig. 4(a)). In addition, as demonstrated in Fig. S18 (ESI†), both bipolar and monopolar electrode ablation triggered substantial reactive oxygen species (ROS) generation in KPC cells. This ROS surge induced mitochondrial homeostatic imbalance, ultimately driving apoptotic progression. Notably, co-administration of Ni₃(HITP)₂@PDA markedly amplified intracellular ROS accumulation, resulting in more extensive mitochondrial structural compromise and dysfunction. Flow cytometry analysis revealed that the apoptosis-inducing effect on tumor cells was comparable between bipolar and monopolar ablation modes (45.55% vs. 48.57%). This finding is similar to the tumor cell apoptosis-inducing effect of the balloon electrode developed by Mohammad et al.⁴⁶ When Ni₃(HITP)₂@PDA was added, the apoptosis rate induced by bipolar electrode ablation increased from 45.52% ± 1.83 to 59.95% ± 2.41 (Fig. 4(b) and Fig. S19, ESI†). Transmission electron microscopy (TEM) findings revealed that both monopolar and bipolar electrode ablation inflicted irreversible damage on cell membranes and mitochondria. This structural disruption led to an imbalance in intracellular homeostasis, thereby inducing tumor cell apoptosis and necrosis. These findings are consistent with previous studies by Jeon⁴⁷ and McNicoll.⁴⁸ The addition of Ni₃(HITP)₂@PDA significantly increased the extent of both membrane and mitochondrial damage, thereby further enhancing the antitumor efficacy of the ablation (Fig. 4(c)). This heightened cytotoxicity may be attributed to the accumulation of Ni₃(HITP)₂@PDA around the tumor cells, which alters the local conductive environment and increases cellular conductivity. The resulting conductivity enhancement intensifies membrane and mitochondrial damage, thereby augmenting the cytotoxic effects on pancreatic tumor cells. In summary, the therapeutic efficacy of bipolar electrode ablation on pancreatic tumor cells operates through a dual mechanism: disruption of cell membrane integrity and mitochondrial damage. The incorporation of Ni₃(HITP)₂@PDA amplifies membrane injury and stimulates an excessive generation of intracellular ROS, which exacerbates mitochondrial dysfunction and ultimately induces apoptosis in pancreatic cancer cells.

Fig. 4 Antitumor mechanism in KPC cell model. (a) Mito-tracker staining of Control, Ni₃(HITP)₂@PDA, monopolar electrode, bipolar electrodes and bipolar electrodes + Ni₃(HITP)₂@PDA groups. (b) Flow cytometry was used to quantitatively evaluate the proportion of apoptotic in control, Ni₃(HITP)₂@PDA, monopolar electrode, bipolar electrodes and bipolar electrodes + Ni₃(HITP)₂@PDA groups. (c) TEM image of control, monopolar electrode, bipolar electrodes and bipolar electrodes + Ni₃(HITP)₂@PDA groups. Monopolar electrode parameters: pulse intensity = 1500 V cm⁻¹, pulse duration = 90 μs, number of pulses = 90; bipolar electrodes parameters: pulse intensity = 2500 V cm⁻¹, pulse duration = 90 μs, number of pulses = 90. The red arrow indicates the cell membrane, and the orange arrow indicates the mitochondria.

3.6 In vivo therapeutic efficacy of bipolar electrode IRE ablation combined with Ni₃(HITP)₂@PDA

Fig. 5(a) illustrates the KPC tumor-bearing mouse model and the associated antitumor effects achieved through localized injection of Ni₃(HITP)₂@PDA in combination with bipolar electrode IRE ablation. Tumor volume curves showed a steady increase in the control group, whereas both the bipolar electrodes and bipolar electrodes + Ni₃(HITP)₂@PDA groups exhibited consistent reductions in tumor volume over the 14-day observation period (Fig. 5(b) and Fig. S20, ESI†). Notably, the combined treatment group (bipolar electrodes + Ni₃(HITP)₂@PDA) demonstrated an 85.77% ± 0.21 reduction in tumor volume in the in vivo models, with no observed adverse effects, indicating a significantly enhanced therapeutic effect. Body weight remained stable across all groups, with no significant differences observed during the 14-day period (Fig. 5(c)), indicating minimal systemic toxicity. Photographs taken on day 14 of the post-treatment significantly smaller tumors in the bipolar electrodes + Ni₃(HITP)₂@PDA group compared to the control, monopolar electrode and bipolar electrodes groups (Fig. 5(d)). These observations were corroborated by gross examination and weight measurements of the excised tumors, which confirmed clear size differences among the groups. Tumors in the control group was the largest, whereas tumors in the bipolar electrodes + Ni₃(HITP)₂@PDA group were distinctly smaller (Fig. S21, ESI†). Although the monopolar electrode and bipolar electrodes groups led to tumor size and weight reductions, these changes were less pronounced compared to the combined treatment group (Fig. 5(e) and (f)). As shown in Fig. 5(g), hematoxylin and eosin (HE) staining revealed dense clusters of tumor cells in the control group. In contrast, monopolar electrode and bipolar electrodes, and bipolar electrodes + Ni₃(HITP)₂@PDA groups exhibited visibly reduced tumor cell density. Immunohistochemical staining for Ki-67, a marker of cellular proliferation, indicated the highest positive staining in the control group, followed by the monopolar electrode and bipolar electrodes groups, with minimal staining observed in the bipolar electrodes + Ni₃(HITP)₂@PDA group, suggesting a substantial reduction in tumor cell proliferation. In addition, the immunohistochemical analysis of mouse tumor pathology further confirmed that both bipolar and monopolar electrode ablation therapies achieve the goal of tumor cell killing by inducing significant expression of apoptosis- and necrosis-related proteins, including TUNEL, BAX, BCL-2, P53, and TNF-α. Moreover, the local injection of Ni₃(HITP)₂@PDA could further enhance the degree and extent of apoptosis and necrosis, thereby amplifying the therapeutic effect. Quantitative analysis of staining intensities for Ki-67, TUNEL, P53, BAX, BCL-2, and TNF-α (Fig. S22, ESI†) further substantiated these findings, establishing that the combined treatment achieves optimal antitumor effects.

Fig. 5 Anti-tumor evaluation in a murine subcutaneous tumor mode. (a) Schematic illustration for subcutaneous establishment of a KPC tumor-bearing murine model and the anti-tumor effect in treating tumor-bearing murine model. (b) Tumor volume in groups I, II, III and IV over 14 days post-treatment. (c) Body weight in groups I, II, III and IV over 14 days post-treatment. (d) General observation of tumor volume changes in subcutaneous tumor model of mice in groups I, II, III and IV pre and post treatment. (e) Tumor weight in groups I, II, III and IV, ***p < 0.001, *p < 0.05, ns means ns means no significant difference. (f) Tumor volume in groups I, II, III and IV at day 14th post-treatment, ***p < 0.001, *p < 0.05, ns means ns means no significant difference. (g) HE stains, immunohistochemical Ki-67, TUNEL, P53, BAX, BCL2 and TNF-α staining in groups I, II, III and IV at day 14th post-treatment. Groups I, II, III and IV represent the control, monopolar electrode, bipolar electrodes and bipolar electrodes + Ni₃(HITP)₂@PDA, respectively.

Overall, both vivo and in vitro experimental results show that bipolar electrodes are able to safely and effectively ablate pancreatic tumors with similar therapeutic efficacy as monopolar electrodes, but with much reduced handling difficulty. When combined with conductive Ni₃(HITP)₂@PDA, the ablation effect of the bipolar electrode can be further enhanced, providing a new method and idea to improve the efficacy of pancreatic cancer treatment. However, it should also be noted that this study still has certain limitations, and there is a long way to go before it can be translated into clinical application. Firstly, in this study, we used local injection of Ni₃(HITP)₂@PDA combined with IRE ablation for pancreatic cancer treatment research. This is a local treatment method and cannot treat tumors outside the ablation area, which is different from the conventional tumor treatment methods in clinical practice. Nevertheless, as an adjunctive treatment method, it has certain clinical significance for the treatment of postoperative tumor recurrence and advanced tumor relief. Secondly, the research on the treatment of pancreatic cancer with Ni₃(HITP)₂@PDA combined with IRE ablation is still in its initial stage and requires a large amount of preclinical experimental data to verify its effectiveness and safety. It should be compared with the current standard treatment methods for pancreatic cancer, such as surgery, chemotherapy, and radiotherapy, as well as emerging treatment methods like immunotherapy and targeted drug therapy. These research results will provide more robust evidence for the clinical application of this technology.

4 Conclusions

The IRE ablation technique has garnered significant attention from researchers as a novel strategy for treating pancreatic cancer. However, existing IRE methods require the placement of two or more electrodes in parallel on the tumor, which poses technical challenges, even when guided by ultrasound or CT imaging. Moreover, residual tumor tissue following ablation presents a substantial issue, both of which impede the clinical application of IRE ablation. In this study, we have designed an innovative “bipolar electrode” that integrates both the cathode and anode into a single electrode, thereby considerably simplifying the procedural complexity. Furthermore, results from ex vivo and in vivo experiments have demonstrated that the local injection of conductive MOF (Ni₃(HITP)₂@PDA) can notably enhance the efficacy of IRE ablation. Nonetheless, the combination of these therapeutic modalities still necessitates extensive preclinical research to verify its safety and effectiveness before it can be effectively translated into clinical practice. In summary, although there is still some distance to use this combination of bipolar IRE and Ni₃(HITP)₂@PDA for the treatment of pancreatic cancer in clinical therapy, it is undeniable that the results of this study provide a new therapy and strategy for the treatment of pancreatic cancer.

Author contributions

Lei Xu, Liting Xie, QiYu Zhao, Jie Lin, Aiguo Wu, and Tianan Jiang, conceived and designed research; Lei Xu, Wenjing Lou, and Fan Xu. performed most experiments; Chengyue Zhang, Haoyu Liu, Yue Hu, Xinyu Miao, Zhiwei Hou, and Wenyuan Ma, helped to perform research; Lei Xu, Wenjing Lou. Chengyue Zhang, Aochi Liu, and Fan Xu. analyzed data; Lei Xu, Wenjing Lou, Fan Xu, Qiyu Zhao, Yujiao Xie, Jie Lin, Aiguo Wu, and Tianan Jiang wrote and revised the manuscript.

Data availability

The datasets supporting this article have been uploaded as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by National Science and Technology Major Project (2023ZD0500902), the Development Project of National Major Scientific Research Instrument of China, Grant/Award Number: 82027803, National Natural Science Foundation of China (no. 32025021, 12374390, 81971623, 82202151), Youth National Natural Science Foundation of China (no. 82171937), Zhejiang Provincial Natural Science Foundation of China (no. Y24H180007), Medical Science and Technology Project of Zhejiang Province (no. 2023574233), the member of Youth Innovation Promotion Association Foundation of CAS, China (2023310), and the Key Scientific and Technological Special Project of Ningbo City (2023Z209), Ningbo Youth Science and Technology Innovation Leading Talents Project (2024QL029).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4qm01041h

‡ These authors contributed equally to this work.

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