Nanosecond pulsed electric fields (nsPEFs) are a pulsed power technology known for ablating tumors, but they also modulate diverse biological mechanisms. Here we show that nsPEFs regulate trans-plasma membrane electron transport (tPMET) rates in the plasma membrane redox system (PMRS) shown as a reduction of the cell-impermeable, WST-8 tetrazolium dye. At lower charging conditions, nsPEFs enhance, and at higher charging conditions inhibit tPMET in H9c2 non-cancerous cardiac myoblasts and 4T1-luc breast cancer cells. This biphasic nsPEF-induced modulation of tPMET is typical of a hormetic stimulus that is beneficial and stress-adaptive at lower levels and damaging at higher levels. NsPEFs also attenuated mitochondrial electron transport system (ETS) activity (O2 consumption) at Complex I when coupled and uncoupled to oxidative phosphorylation. NsPEFs generated more reactive oxygen species (ROS) in mitochondria (mROS) than in the cytosol (cROS) in non-cancer H9c2 heart cells but more cROS than mROS in 4T1-luc cancer cells. Under lower charging conditions, nsPEFs support glycolysis while under higher charging conditions, nsPEFs inhibit electron transport in the PMRS and the mitochondrial ETS producing ROS, ultimately causing cell death. The impact of nsPEF on ETS presents a new paradigm for considering nsPEF modulation of redox functions, including redox homeostasis and metabolism.

We previously reported that nano-pulse treatment (NPT), a pulsed power technology, resulted in 4T1-luc mammary tumor elimination and a strong in situ vaccination, thereby completely protecting tumor-free animals against a second live tumor challenge. The mechanism whereby NPT mounts effective antitumor immune responses in the 4T1 breast cancer predominantly immunosuppressive tumor microenvironment (TME) remains unanswered. In this study, orthotopic 4T1 mouse breast tumors were treated with NPT (100 ns, 50 kV/cm, 1000 pulses, 3 Hz). Blood, spleen, draining lymph nodes, and tumors were harvested at 4-h, 8-h, 1-day, 3-day, 7-day, and 3-month post-treatment intervals for the analysis of frequencies, death, and functional markers of various immune cells in addition to the suppressor function of regulatory T cells (Tregs). NPT was verified to elicit strong in situ vaccination (ISV) against breast cancer and promote both acute and long-term T cell memory. NPT abolished immunosuppressive dominance systemically and in the TME by substantially reducing Tregs, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). NPT induced apoptosis in Tregs and TAMs. It also functionally diminished the Treg suppression capacity, explained by the downregulation of activation markers, particularly 4-1BB and TGFβ, and a phenotypic shift from predominantly activated (CD44+CD62L−) to naïve (CD44−CD62L+) Tregs. Importantly, NPT selectively induced apoptosis in activated Tregs and spared effector CD4+ and CD8+ T cells. These changes were followed by a concomitant rise in CD8+CD103+ tissue-resident memory T cells and TAM M1 polarization. These findings indicate that NPT effectively switches the TME and secondary lymphatic systems from an immunosuppressive to an immunostimulatory state, allowing cytotoxic T cell function and immune memory formation to eliminate cancer cells and account for the NPT in situ vaccination.

High-intensity nanosecond pulse electric fields (nsPEF) can preferentially induce various effects, most notably regulated cell death and tumor elimination. These effects have almost exclusively been shown to be associated with nsPEF waveforms defined by pulse duration, rise time, amplitude (electric field), and pulse number. Other factors, such as low-intensity post-pulse waveform, have been completely overlooked. In this study, we show that post-pulse waveforms can alter the cell responses produced by the primary pulse waveform and can even elicit unique cellular responses, despite the primary pulse waveform being nearly identical. We employed two commonly used pulse generator designs, namely the Blumlein line (BL) and the pulse forming line (PFL), both featuring nearly identical 100 ns pulse durations, to investigate various cellular effects. Although the primary pulse waveforms were nearly identical in electric field and frequency distribution, the post-pulses differed between the two designs. The BL's post-pulse was relatively long-lasting (~50 µs) and had an opposite polarity to the main pulse, whereas the PFL's post-pulse was much shorter (~2 µs) and had the same polarity as the main pulse. Both post-pulse amplitudes were less than 5% of the main pulse, but the different post-pulses caused distinctly different cellular responses. The thresholds for dissipation of the mitochondrial membrane potential, loss of viability, and increase in plasma membrane PI permeability all occurred at lower pulsing numbers for the PFL than the BL, while mitochondrial reactive oxygen species generation occurred at similar pulsing numbers for both pulser designs. The PFL decreased spare respiratory capacity (SRC), whereas the BL increased SRC. Only the PFL caused a biphasic effect on trans-plasma membrane electron transport (tPMET). These studies demonstrate, for the first time, that conditions resulting from low post-pulse intensity charging have a significant impact on cell responses and should be considered when comparing the results from similar pulse waveforms.

Nonthermal atmospheric pressure plasma jets (APPJ) produce reactive agents including charged particles, and reactive species, which induce oxidative stress, suggesting their application in cancer therapy. Similarly, nanosecond pulsed electric field (nsPEF) have been studied largely for tumor ablation because of its effect to permeabilize the plasma membrane allowing Ca 2+ influx, and dissipate the mitochondrial membrane potential (ΔΨm) leading to apoptosis or regulated cell death. We have previously shown that synergistic treatments of a nanosecond pulsed plasma jet (ns-APPJ) and nsPEF enhance the inactivation effect on pancreatic cancer cells (Pan02) [1] . This study further examines the effects of the ns-APPJ and nsPEF on Pan02 cells in vitro. Cells were exposed to a single needle-electrode helium plasma jet driven by 200 ns, 9 kV pulses at 2 kHz for ≤ 6 min and 60 ns, 50 kV/cm nsPEF treatment at 1 Hz for ≤ 2 min consecutively. The intracellular ROS and loss of ΔΨm induced by the ns-APPJ and nsPEF are evaluated with respect to cell inactivation using flow cytometry. In addition, the roles of gas-phase ROS produced by the ns-APPJ are assessed using optical emission spectroscopy (OES).

One of the earliest possible medical applications for usEPs was to ablate tumors. Many laboratories carried out many studies to investigate the potential for usEPs to serve as a cancer therapy. Like simulations with cells in suspension, usEPs also passed through cells in tumor tissues forming high-density nanopores in all cell membranes as supraelectroporation, distinct from conventional electroporation. The first studies were smaller in scope, but showed proof of principle that usEPs could reduce fibrosarcoma tumor volume in mice. More extensive studies followed, showing that usEPs could treat B16f10 melanoma tumors in mice, although some tumors required more than one and as many as six 300 ns, 40 kV/cm treatment (6 × 1.2 Vs/cm). Later studies showed that as many as 5–6 Vs/cm was required to eliminate tumors completely. The blood supply to these melanoma tumors was also reduced, as was revascularization, as shown using endothelial markers. Other studies showed that usEPs ablated mouse liver tumors. While all of these studies investigated ectopic tumors within mice's flanks, studies also showed that usEPs could also completely ablate orthotopic rat liver tumors, using a 5-needle array with heterogeneous electric fields. Other studies demonstrated that usEPs could eliminate many different tumor types in mice, including some human tumors in immunodeficient mice. One study showed decreased tumor sizes in dog osteosarcoma.

As the largest intracellular structure in mammalian cells, the nucleus, its double phospholipid nuclear envelope, and its chromatin/DNA content were suspected targets for usEPs. Many different methods were used to determine DNA/ nuclear damage including analyses with the comet assay, DNA migration on agarose gels, mitotic indices, and chromatid structures, fluorescent in situ hybridization (FISH). In Jurkat cells exposed to (3.6 × 10−3 Vs/cm), the telomers were displaced from the nucleus, and nuclear membranes were sheared from the nucleus. SV40 fibroblasts did not show this apparent telomer and nuclear membrane damage, indicating cell-type differences., It was also shown as adherent cells were less susceptible to usEP-induced damage. Other studies showed that usEP induced significant physical damage to the nuclear membrane, cytoskeleton, and telomers, which form protein–protein or protein-DNA interactions with the nuclear envelope. Some usEP-induced nuclear/DNA damages were suspected due to effects similar to ionizing radiation caused by reactive oxygen species (ROS). Other studies using stably transfected cells with fluorescently labeled Histone-2b (H2B), which is tightly wound with DNA, and PCNA (proliferating cell nuclear antigen), which is loosely associated with DNA, indicated that H2B remained in the nucleus. In contrast, translocation of PCNA from the nucleus to the cytoplasm showed permeabilization of the nuclear membrane. So, usEPs had relatively severe and seemly rapid effects on nuclear structures. Yet, when phosphorylated Histone 2AX (γH2AX) was used as an early indicator of DNA damage in Jurkat cells, the damage appeared to be related to apoptosis's end stages since it was caspase-dependent. While all these methods are valid indicators of effects on DNA and/or the nucleus, the results between the comet assay and γH2AX under similar conditions with the same cell type are not readily reconcilable. One other study demonstrated that usEP also had effects on nuclear substructures called nuclear speckles, which are part of splicing factors and small nuclear ribonucleoproteins (snRNPs) that exhibit roles to provide splicing factors at transcription sites. So, there are apparent effects of usEPs on DNA, the nucleus, and subnuclear factors; the full extent of these effects requires additional experimentation.

Cells employs many different mechanisms for their expiration. The best known and studied regulated cell death mechanism is apoptosis. Cell death subtypes, including regulated cell death (RCD), programmed cell death (PCD), and accidental cell death, are discussed. A discussion on and caution for the use of the term “necrosis” is included. Apoptosis was the earliest RCD mechanism shown in Jurkat cell responses to usEPs as determined by cytochrome c release and caspase activation. This apoptotic cell death was enhanced by usEP-induced supraelectroporation, as electric fields with nanosecond durations and short(fast) rise-fall times passed through the cell, while pulses with microsecond duration go around cells. However, using Jurkat clones that did and did not express APAF-1, which is an essential protein for apoptosome formation as a platform for caspase-9 and caspase-3 activation, it was also shown that usEPs induced caspase-dependent and caspase-independent cell death. A role for caspases depended on the usEP charging intensity with lower usEP impact causing caspase-dependent cell death while higher charging caused caspase-independent cell death. Although a full discussion of all RCD mechanisms is not included, evidence is presented that not all cell types responded to usEP by apoptotic cell death. The presence or absence of Ca2+ has an impact on the RCD mechanisms. Human triple-negative breast cancer cells expressed either or both necroptosis and parthanatos. Necroptosis is sometimes considered regulated necrosis because plasma membrane pores form from intracellular proteins. Finally, usEPs are also shown to induce cell responses downstream of toll-like receptors (TLRs). Considerations for immunogenic cell death (ICD) are also considered.

The endoplasmic reticulum (ER) is a tubular membranous labyrinth throughout the cytoplasm that is continuous with the nuclear membrane, exhibits contact sites with mitochondria and the plasma membrane, and displays domains for specific functions. It is smooth or rough with ribosomes for protein synthesis and is the home for folding proteins in their proper tertiary structures. The ER includes cellular stress response sensors that can lead to unfolded protein response (UPR), leading to regulated cell death (RCD). The ER is also a primary storage site for Ca2+ that can be used for scores of Ca2+ mediated signal transduction responses such as neurotransmitter release, muscle contraction, or contributors to RCD, among others. Since usEPs were unique for intracellular electric field effects, usEP-induced Ca2+ release was an excellent way to define these intracellular effects, albeit not without caveats. Because usEPs also induced plasma membrane permeabilization for Ca2+ influx, which occurred at lower charging conditions than ER-induced nanopore formation and Ca2+ release; because some cells expressed voltage-gated Ca2+ channels (VGCC), which could be directly activated or activated due to usEP-induced plasma membrane depolarization; because capacitative Ca2+ increases could increase intracellular Ca2+; and Ca2+ increases from Ca2+-induced Ca2+ release, significant care, and experimental manipulations were required to be sure that increases in intracellular Ca2+ were due to release from internal stores. Also, because there were other intracellular stores for Ca2+, other approaches were needed to ensure that the source of intracellular Ca2+ release was from the ER. This chapter provides details from several studies using many different experimental techniques that lead to the conclusion that usEPs could induce Ca2+ release from the ER by forming ER nanopores. Notably, another series of experimental studies supported theoretical evidence that shorter pulse durations lead to more significant increases in intracellular Ca2+ than longer pulse durations.

Receptors, channels, glycoprotein, and transport proteins, among other integral proteins, load the plasma membrane lipid bilayer, which receives reinforcement from cytoskeletal filaments as the boundaries of cellular life. Early studies suggested that usEPs did not induce plasma membrane permeabilization until relatively high amplitude conditions. As will be discussed here and elsewhere, this was because the usEP-induced plasma membrane pores were so small that they did not allow entry of propidium iodide, a commonly used marker for permeability, or calcein exit from the cell. The plasma membrane contained nanoelectropores or nanopores, so-called because they were ~1 nm in diameter. These lipid nanopore structures exhibited complex conductances similar to classic pores, which are common in protein structures. Another finding presented some confounding information regarding plasma membrane phosphatidylserine (PS) externalization, commonly used as a marker for apoptosis. usEPs “pulled” PS through these nanopores. Now PS externalization was an ambiguous marker for apoptosis. Although plasma membrane lipids were clear usEP targets, plasma membrane channels were also targets for usEPs. As will be discussed, these effects could be due indirectly to nanopore formation or possibly directly on the channels themselves, although more evidence for direct effects is necessary. In any event, patch-clamp techniques provided new, exciting, and valuable information about usEP effects on plasma membranes. As might be expected, cells have mechanisms that confront nanopore formation and other possible usEP-induced injuries, which will be presented. Finally, in relatively uncharted territory, usEPs were also shown to affect redox systems in plasma membranes as electron carriers. Although usEPs are unique for their effects on intracellular structures and functions, their impact on the plasma membrane has provided a wealth of information about how they can be used as tools in biology and medicine.

The endocytotic and symbiotic inclusion of a prokaryote by an early eukaryote, its subsequent evolution as mitochondria, and its collaboration with the nucleus provided these new symbiotes with enough ATP to evolve a new world of extraordinarily diverse organisms. Mitochondria assumed roles for lives replete with energy from ATP and control over the death of cells when their usefulness was finished or when they malfunctioned or were injured beyond repair. The outer mitochondrial membrane (OMM) protects the electron transport chain (ETC) in the inner mitochondrial membrane and the mitochondria’s DNA, which is used for some of the proteins in the ETC. The ETC is supplied with electrons from NADH, FADH2 produced by oxidative phosphorylation (OXPHOS) as Complexes I, III, and IV pump proton (H +) out of the matrix to generate a proton motive force and a mitochondrial membrane potential (ΔΨm). H + reenter the matrix through ATP synthase for the production of ATP. All this complexity provides usEPs with multiple targets for effects on cell life and death. UsEP’s role in cytochrome c release in apoptosis and other regulated cell death (RCD) mechanisms in cancer ablation has been a significant application with clinical medicine, which is still in developmental stages in clinical trials. UsEPs increase reactive oxygen species (ROS) and dissipate the ΔΨm, which can occur without permeabilization of the IMM, especially in the presence of Ca2+ that enters cells through nanopores in the plasma membrane. This loss of ΔΨm is facilitated by usEP effects on the Ca2+-dependent and redox-sensitive protein cyclophilin D (CypD). CypD regulates the mitochondrial permeability transition pore (mPTP) that dissipates the ΔΨm, leading to regulated cell death and apoptosis if mitochondria release cytochrome c into the cytoplasm to activate caspases. We also discuss the possible identity of the mPTP as ATP synthase. Experiments continue to test this hypothesis. Experiments here also show that usEPs with a shorter (faster) rise-fall time are more effective to dissipate ΔΨm than usEPs with a longer (slower) rise-fall time. It also appears that over-expression of BCL-xl and BCL2 cannot protect the mitochondria from the effects of usEPs. Experiments measuring oxygen consumption in cells treated or not with usEPs indicate that the usEPs attenuate oxygen consumption in Complexes I and IV of the ETC. These results suggest that usEPs inhibit electron transport in the ETC. We also show that usEPs that ultimately lead to cell death in 4T1-luc mammary cancer cells up-regulates essential subunits in the ETC. Thus, usEPs target several mitochondrial components, including those that regulate ΔΨm and electron transport in the ETC.

A dielectric biconical antenna (DiBiCA) for radiating subnanosecond pulses to treat subcutaneous tissue was designed, constructed, and tested. It is composed of a conical wave launcher and truncated conical emitter. In between, there is a short cylinder that provides a space for a ring terminating resistor. The material of the antenna has a dielectric constant of 28, so its size is small (length: 7 cm and aperture diameter: 2.2 cm). It was housed in an oil container to withstand high voltages and avoid surface flashover. The radiated electric field, measured in water, increased as the input voltage increased up to 30 kV but leveled off for higher voltages up to 50 kV, presumably because of losses in the antenna dielectric. The maximum field was 1.5 kV/cm for a depth of 5 mm and 1.0 kV/cm for a depth of 20 mm. Although the dielectric loss mechanism remains to be investigated, the antenna can be useful for noninvasive delivery of subnanosecond pulses to induce biological responses on subcutaneous targets. The DiBiCA radiated pulses were shown to change the viabilities of dendritic cells and macrophages for 10‐min exposure. Bioelectromagnetics. 2020;41:413–424. © 2020 Bioelectromagnetics Society.

In this study, 9-anthraldehyde-N(4)-methylthiosemicarbazone (MeATSC) 1 and [Co(phen)(2)(O2CO)]Cl center dot 6H(2)O 2 (where phen = 1,10-phenanthroline) were synthesized. [Co(phen)(2)(O2CO)]Cl6H2O 2 was used to produce anhydrous [Co(phen)(2)(H2O)(2)](NO3)(3) 3. Subsequently, anhydrous [Co(phen)(2)(H2O)(2)](NO3)(3) 3 was reacted with MeATSC 1 to produce [Co(phen)(2)(MeATSC)](NO3)(3)center dot 1.5H(2)O center dot C2H5OH 4. The ligand, MeATSC 1 and all complexes were characterized by elemental analysis, FT IR, UV visible, and multinuclear NMR (H-1, C-13, and Co-59) spectroscopy, along with HRMS, and conductivity measurements, where appropriate. Interactions of MeATSC 1 and complex 4 with calf thymus DNA (ctDNA) were investigated by carrying out UV visible spectrophotometric studies. UV visible spectrophotometric studies revealed weak interactions between ctDNA and the analytes, MeATSC 1 and complex 4 (K-b = 8.1 x 10(5) and 1.6 x 10(4)M(-1), respectively). Topoisomerase inhibition assays and cleavage studies proved that complex 4 was an efficient catalytic inhibitor of human topoisomerases I and II alpha. Based upon the results obtained from the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTS) assay on 4T1-luc metastatic mammary breast cancer cells (IC50 = 34.4 +/- 5.2 mu M when compared to IC50 = 13.75 +/- 1.08 mu M for the control, cisplatin), further investigations into the molecular events initiated by exposure to complex 4 were investigated. Studies have shown that complex 4 activated both the apoptotic and autophagic signaling pathways in addition to causing dissipation of the mitochondrial membrane potential (Delta psi(m)). Furthermore, activation of cysteine-aspartic proteases3 (caspase 3) in a time- and concentration-dependent manner coupled with the Delta psi(m), studies implicated the intrinsic apoptotic pathway as the major regulator of cell death mechanism.

An electrical engineering technology, nanosecond electric pulse (nsEP), has been studied to ablate local tumor in a non-thermal and minimally invasive manner. Recently, several groups including our lab discovered that animals with complete tumor ablation following nsEP treatment were protected from a second tumor challenge. Therefore, nsEP tumor ablation is also a novel in situ vaccination approach. Breast cancer is considered a low immunogenic tumor with only a small subset of patients responsive to immune checkpoint inhibitors. We found nsEP treatment resulted in not only a potent in situ vaccine effect in a poorly immunogenic 4T1 mouse breast cancer model but also an abscopal effect, a rejection of distant untreated tumor lesion. We further examined the impact of nsEP on cancer cell death, tumor microenvironment (TME) modification, the dynamic changes of local and systemic immune response. 4T1 cells treated with nsEP did not show apoptotic death markers but released damage-associated molecular patterns, including calreticulin, high mobility group protein B1, and ATP. In contrast to abundant immune suppressor cells in untreated breast TME, nsEP treatment led to a significant reduction of both myeloid-derived suppressor cells and regulatory T cells. Conversely, the ratio of tumor associated macrophages M1-like verse M2-like was reversed. An increase of tissue-resident memory T cells was observed in draining lymph nodes. In summary, our results suggest that nsEP is a novel non-drug immunogenic cell death inducer and in situ vaccination approach. Further elucidation of its mechanisms and the development of an in vivo nsEP delivery system suitable for patients is critical to warrant this novel technology toward a clinical trial.

Nano-pulse stimulation (NPS), previously called nsPEFs, induced a vaccine-like effect after ablation of orthotopic N1-S1 hepatocellular carcinoma (HCC), protecting rats from subsequent challenges with N1-S1 cells. To determine immunity, immune cell phenotypes were analyzed in naïve, treated and protected rats. NPS provides a positive, post-ablation immuno-therapeutic outcome by alleviating immunosuppressive T regulatory cells (Treg) in the tumor microenvironment (TME), allowing dendritic cell influx and inducing dynamic changes in natural killer cells (NKs), NKT-cells and T-lymphocytes in blood, spleen and liver. NPS induced specific increases in NKs and NKT-cells expressing CD8 and activation receptors CD314-NKG2D and CD161 (NK1.1) in the TME after treatment, as well as some variable changes in CD4+ and CD8+ effector (Tem) and central memory (Tem) lymphocytes in blood and spleen. After orthotopic challenge, CD8+ T-cells were cytotoxic, inducing apoptosis in N1-S1 cells; additionally, in contrast to post-treatment immune responses, CD4+ and CD8+ memory precursor effector cells (MPECs) and short-lived effector cells (SLECs) were present, while still including CD8+ CD161 NK cells, but not involving CD8+ CD314-NKG2D+ NKs. This immunity was N1-S1-specific and was sustained for at least 8 months. NPS vaccinates rats in vivo against HCC by activating innate and adaptive immune memory mechanisms that prevent HCC recurrence.

Electric pulse based technology has been developed and studied as a non-thermal ablation method for local control of pancreatic cancer. Irreversible electroporation (IRE) has shown a significant survival benefit for local advanced pancreatic cancer in clinical trials. However, incomplete ablation with local recurrence and major complications limit the potential of this new technology. We have developed an integrated moderate heating electric pulse delivery system which consists of controllable tumor heating, multi-parameter monitoring and electric pulse delivery. The impedance of tumor is greatly decreased after moderate heating at 42°C for 1–2 min, which does not cause any cell death. Moderate heating significantly enlarges the ablation zone of tumor treated with IRE. In contrast to IRE alone, moderate heating assisted IRE results in a high rate of complete tumor regression and a significant longer median survival. Another electric pulse technology, nanosecond electric pulses, has been assessed for the treatment of pancreatic cancer as well. Nanosecond electric pulse treatment achieves more survival benefit in animals with partial tumor ablation than those treated with IRE and leads to a vaccine-like protective effect in animals with complete local ablation. More studies are needed to demonstrate the advantages and translational feasibility of the enhanced electric pulse technologies.

A Pancreatic cancer is a notorious malignant neoplasm with an extremely poor prognosis. Current standard of care is rarely effective against late-stage pancreatic cancer. In this study, we assessed nanopulse stimulation (NPS) as a local treatment for pancreatic cancer in a syngeneic mouse Pan02 pancreatic cancer model and characterized corresponding changes in the immune profile. A single NPS treatment either achieved complete tumor regression or prolonged overall survival in animals with partial tumor regression. While this is very encouraging, we also explored if this local ablation effect could also result in immune stimulation, as was observed when NPS led to the induction of immune-mediated protection from a second tumor challenge in orthotopic mouse breast and rat liver cancer models. In the Pan02 model, there were insufficient abscopal effects (1/10) and vaccine-like protective effects (1/15) suggesting that NPS-induced immune mechanisms in this model were limited. To evaluate this further, the immune landscape was analyzed. The numbers of both T regulatory cells (Tregs) and myeloid derived suppressor cells (MDSCs) in blood were significantly reduced, but memory (CD44+) T-cells were absent. Furthermore, the numbers of Tregs and MDSCs did not reduce in spleens compared to tumor-bearing mice. Very few T-cells, but large numbers of MDSCs were present in the NPS treated tumor microenvironment (TME). The number of dendritic cells in the TME was increased and multiple activation markers were upregulated following NPS treatment. Overall, NPS treatments used here are effective for pancreatic tumor ablation, but require further optimization for induction of immunity or the need to include effective combinational NPS therapeutic strategy for pancreatic cancer.

It is now well established that ruthenium complexes are attractive alternatives to platinum-based anticancer agents. Most of the ruthenium compounds currently under investigation contain a single metal center. The synthesis of multinuclear analogues may provide access to novel complexes with enhanced biological activity. In this work, we have synthesized a set of three trinuclear complexes containing organometallic ruthenium fragments—(arene)RuCl—coordinated to a 2,4,6-tris(di-2-pyridylamino)-1,3,5-triazine core [(Arene = benzene (2), p-cymene (1), or hexamethylbenzene (3)]. The interaction of the complexes with DNA was extensively studied using a variety of biophysical probes as well as by molecular docking. The complexes bind strongly to DNA with apparent binding constants ranging from 2.20 to 4.79 × 104 M−1. The binding constants from electronic absorption titrations were an order of magnitude greater. The mode of binding to the nucleic acid was not definitively determined, but the evidence pointed to some kind of non-specific electrostatic interaction. None of the complexes displayed any significant antimicrobial activity against the organisms that were studied and exhibited anticancer activity only at high (> 100 μM) concentration.

Pancreatic cancer is a notorious malignancy with a very poor prognosis. Without significant advance in the treatment and early diagnosis, pancreatic cancer will continue to be a serious global health issue. Nanosecond electric pulses (nsEPs) or nanosecond pulsed electric fields (nsPEFs) have been demonstrated to result in completely local ablation and protection from the second tumor challenge in several cancer models. Here, we assessed the potential advantages of nsEPs for pancreatic cancer in a syngeneic mouse Pan02 pancreatic cancer model and characterized its immune profile. We found that a single nsEP treatment could achieve complete tumor regression without any mortality. Animals with partial regression prolonged survival in those treated with nsEPs more than those treated with irreversible electroporation (IRE). Low rate of abscopal effect (10%) and vaccine-like protective effect (1/15) was observed in animals treated with nsEPs but not with surgery or IRE. Significant reduction of both T regulatory cells and myeloid derived suppressor cells (MDSCs) was seen in the blood of tumor animals after nsEP treatment. Heterogeneous T cell responses following nsEP treatment were found. Very few T cells were infiltrated in the tumor but large amount of MDSCs existed in tumor. The number of dendritic cells in the tumor was increased and activated with upregulation of multiple activation markers following nsEP treatment. Our findings could help design a more effective combinational nsEP therapeutic strategy for pancreatic cancer.