Synergistic Effect of Nanopulse and Cold Plasma in Pancreatic Cancer

Pancreatic adenocarcinoma is a highly aggressive malignancy with disease-related mortality almost equaling its incidence. The notorious resistance of pancreatic cancer not only to conventional cytotoxic therapies but also to almost all targeted agents developed to date continues to puzzle the oncological community and represents one of the biggest hurdles to reduce the death toll from this ominous disease. Currently, we have been focusing on developing nanosecond electric pulse or nano-pulse (NP) technology for cancer therapy. However, due to the demand for high electric fields, all currently available designs of NP delivery systems are only able to treat tumors with small sizes (4-7 mm). To solve this problem, we propose a combination of NP with non-thermal plasma or cold plasma (CP) to treat large tumors. Our hypothesis is that the synergistic effect between NP and CP-produced reactive plasma species can be utilized to treat large tumors with sizes relevant to human cancer. First, the dose-dependent cytotoxicity of NP or CP to Pan02 pancreatic cancer cells was examined by cells treated with different doses of NP or CP. Second, we selected one specific dose of NP for further combination evaluation. The combination treatment was carried out either by treating cells with NP first then CP, or CP first then NP. The synergistic effect was assessed with cell viability and impedance measurement. Our results showed that NP at a very low dose can sensitize cancer cells that will then be killed by CP. Our future goals are to develop a prototype engineering system for both NP and CP delivery to tumor tissue, to evaluate the synergistic effect of NP and CP in vitro and in vivo, and to explore the potential molecular mechanisms of the synergistic effect of NP and CP

Evaluation of Electric Property Changes in Cancer Cells in Vitro Induced by Cold Plasma and Electric Field Treatment

Nonthermal atmospheric pressure plasma jet (APPJ) is a promising method for medical applications, especially in the treatment of tumors in vitro. Its main mechanism of action is it induces temporary or permanent damage to tissue known as reversible electroporation (RE) and irreversible electroporation (IRE), respectively. It has been previously shown that APPJ treatment reduces the proliferation of cancer cells, reduces the size of the tumor, and induces oxidative stress in cancer cells that can lead to the death of the cells via apoptosis [1]. Furthermore, nanosecond pulsed electric field (nsPEF) has equally been reported to cause increase in permeability of cell membrane and/or oxidative stress on cells, which is accompanied with a decrease in the impedance of the cells. It is found that the impedance of compromised or dying cells are different, typically lower than healthy cells [2]. It is therefore possible to analyze the electrical properties of the cells before, during and after treatment to better monitor the treatment outcome on the cells in real time. Here, we report the conductivity change of pancreatic cancer cells (Pan02) in response to the treatment of 200-ns, 9 kV pulsed plasma jet, 60-ns, 50 kV/cm nsPEF and the combination in vitro. The dosage dependence is evaluated by varying the treatment time of the plasma, and pulse numbers of the nsPEF, followed by the comparison of the impedance change of the monolayer Pan02 cells. [1] C. Jiang et al., Synergistic Effects of an Atmospheric-Pressure Plasma Jet and Pulsed Electric Field on Cells and Skin, in IEEE Transactions on Plasma Science. 49(11) (2021). [2] E. Oshin, S. Guo, and C. Jiang “Determining Tissue Conductivity in Tissue Ablation by Nanosecond Pulsed Electric Fields,” Bioelectrochemistry. 143 (2022)

Synergistic Effects of Nanosecond Pulsed Plasma and Electric Field on Inactivation of Pancreatic Cancer Cells In Vitro

Nanosecond pulsed atmospheric pressure plasma jets (ns-APPJs) produce reactive plasma species, including charged particles and reactive oxygen and nitrogen species (RONS), which can induce oxidative stress in biological cells. Nanosecond pulsed electric field (nsPEF) has also been found to cause permeabilization of cell membranes and induce apoptosis or cell death. Combining the treatment of ns-APPJ and nsPEF may enhance the effectiveness of cancer cell inactivation with only moderate doses of both treatments. Employing ns-APPJ powered by 9 kV, 200 ns pulses at 2 kHz and 60-nsPEF of 50 kV/cm at 1 Hz, the synergistic effects on pancreatic cancer cells (Pan02) in vitro were evaluated on the metabolic activities of cells and transcellular electrical resistance (TER). It was observed that treatment with ns-APPJ for \u3e 2 min disrupts Pan02 cell stability and resulted in over 30% cell death. Similarly, applying nsPEF alone, \u3e 20 pulses resulted in over 15% cell death. While the inactivation activity from the individual treatment is moderate, combined treatments resulted in 80% cell death, approximately 3-to-fivefold increase compared to the individual treatment. In addition, reactive oxygen species such as OH and O were identified at the plasma-liquid interface. The gas temperature of the plasma and the temperature of the cell solution during treatments were determined to be near room temperature

Mechanisms of Synergistic Effect Between Nanosecond Electric Pulse and Nonthermal Plasma to Treat Pancreatic Cancer

Pancreatic cancer is a significant cause of cancer-associated mortality. Currently, prevention or early diagnosis at a curable stage is exceedingly difficult. The standard of care is surgery, chemotherapy, and radiation therapy, which are largely ineffective against late-stage pancreatic cancer. New effective therapeutics or drugs are desperately needed for this deadly disease. We previously reported that nanosecond electric pulses (nsEP) could be an effective therapy to treat pancreatic cancer in animal models. However, owing to the demand for high-intensive electric fields, all currently available designs of nsEP delivery systems can only treat small tumors (4-7 mm). To overcome this hurdle, we propose a combination of nsEP with non-thermal plasma (NTP) to treat large tumors with clinically relevant sizes. Previously, we reported that a synergistic effect resulted from moderate nsEP and NTP dosages to treat Pan02 pancreatic cancer cells. In this study we designed experiments to further determine the underlying mechanism. The role of reactive oxygen species (ROS) in cell death and its relation to mitochondrial membrane potential drop were analyzed by flow cytometry and fluorescence microscopy. We demonstrated that ROS generation was nsEP-dose dependent. ROS from both mitochondria and cytoplasm were upregulated with individual treatments compared to the control. Importantly, the combination treatment induced the highest level of ROS increase, indicating that ROS may participate in the cell death mechanism. Ongoing studies include the analysis of cell death pathways by Western Blot and proteomics to further identify specific death pathway(s) involved and its correlation with ROS. Our next step is to develop a prototype nsEP and NTP dual delivery system to effectively treat large pancreatic cancer in animal models and investigate if immune outcomes can be resulted from this combination therapy