Experiment on inducing apoptosis of melanoma cells by micro-plasma jet

In recent years, a lot of experiments have shown that atmospheric pressure low-temperature plasma jet (CAP) can induce apoptosis of various cancer cells.^1–3 It is currently believed that the reactive oxygen species (ROS) and reactive nitrogen (RNS) produced by CAP play a leading role in inducing apoptosis of cancer cells.^4–7 At present, the research on plasma jet inducing apoptosis of cancer cells has been fully done. In order to cope with the precision medicine advocated in clinical practice, the research has also gone from macroscopic and large-scale plasma jet to micro-plasma jet. However, although the fine plasma jet has good therapeutic prospects, and there are some experimental studies on the treatment of cancer cells in vitro by plasma jet, there are no studies on the treatment of cancer cells and normal cells in the same environment.^8–10 This shortcoming prompted us to conduct research in this area.

In this article, we designed and manufactured a micro-plasma jet device that can produce a plasma jet of 50 µm size, using this device to treat cancer cells and normal cells under the same conditions.^11–13 In addition, we constructed 3D cell clusters of cancer cells and normal cells, and used the device for processing to study the anti-cancer effect of the device and its impact on normal cells. Different from the other micro-plasma jet that treat cancer cells, we have added the treatment of normal cells, and the treatment of 3D cell clusters of cancer cells and normal cells in the same environment. The micro-plasma jet can accurately target 3D cancer cell clusters for treatment.¹⁴ Due to the lack of plasma jet targeted killing research on melanoma cells, the cancer cells used in this experiment are melanoma cells (CCTCC cell bank).^15–21 

The experimental device used in this research is independently developed by our research group.²² The micro-plasma jet device is shown in Fig. 1(A). The micro-plasma jet device can be divided into four modules: plasma jet, gas flow rate control, data acquisition and storage, and high voltage DC power supply.^23–25 

The HVDC power supply is a custom 12 V DC drive boost module with adjustable output voltage of 0–10 kV and output current of 0.2 mA. Digital oscilloscope and high voltage probe monitor discharge voltage and waveform. Copper was deposited on two perforated printed circuit boards to form two copper rings 1.5 mm wide, 1.6 mm thick, 2.5 mm apart, 10 and 7 mm in diameter, respectively. The former was placed in a coaxial position over the latter, 2.5 mm apart. The 10 mm copper ring serves as a floating electrode to help control the discharge intensity and avoid transition to arc discharge. The 1.1 mm diameter tungsten needle is placed 6 mm above the coaxial position of the two copper rings as the needle electrode, forming a needle-ring-ring electrode structure, which is conducive to synchronous and stable glow discharge, and can inhibit the generation of uneven filamentous discharge. In addition, in order to confirm the reaction species produced by the plasma jet, the optical emission spectrum was measured at the nozzle of the quartz tube.

In this study, the concentration of hydrogen peroxide was determined by ammonia molybdenum acid method: Hydrogen peroxide reacts with ammonium molybdenum acid under acidic conditions to produce a stable peroxymolybdic acid compound.²⁶ Prepare a standard solution of hydrogen peroxide of 10, 20, 40, 50, and 80 µmol/l, take 1 ml of plasma activated water (PAW) and 1 ml of detection solution and mix in a quartz cuvette, add pure water to a fixed capacity of 3 ml, and measure after 10 min of reaction. Determination of nitric acid concentration using sodium hydroxide: Prepare a standard solution of 10, 20, 40, 50, and 80 µmol/l nitric acid. Take 500 µl PAW and 500 µl detection solution and mix in a quartz cuvette, add pure water to a fixed capacity of 3.5 ml, and measure the absorbance at 200 nm. Determination of nitrous acid concentration by naphthalene hexanediamine hydrochloride method: Under weak acid conditions, nitrite and p-aminobenzenesulfonamide are diazotized and coupled with N-1-naphthalene hexanediamine to form a fuchsia dye. After dissolving 9 ml of acetic acid in 21 ml of pure water, dissolve 0.5 g of P-aminobenzenesulfonic acid in it. After heating and dissolving, store in a brown bottle as solution A, and dissolve 0.2 g of N-1-naphthalene diamine hydrochloride in 100 ml of pure water to form solution B. Take 500 µl of PAW and 400 µl of solution A, react for 3 min, then add 200 µl of solution B, react for 15 min, take 500 µl of sample and set the volume to 3 ml in a quartz cuvette to measure the absorbance.

In this part of the study, the cancer cells that were centrifuged (1000 rpm, 10 min) were inoculated in a 6-well plate, the medium (10% four seasons green, 90% DMEM) was added, and then plasma jet treatment was performed at different times. During treatment, the quartz tube of the device is located 2 mm below the liquid level of the medium. The treated cancer cells were characterized to obtain apoptosis. On this basis, select the appropriate time gradient to treat cancer cells and characterize them. Characterization methods include scratch experiment, MTT cell activity detection, and intracellular reactive oxygen species detection. Normal cells were treated and characterized using the same time gradient, and then the experimental results of cancer cells and normal cells were compared.^27–29 

In order to make the experimental results more illustrative of the killing effect of plasma jet on cancer cells, a 50% density cancer cell experiment was conducted. Add two times the medium to the resuspended and centrifuged cells to dilute the cancer cells. After the cancer cells are adherent to the wall, the plasma jet treatment under the same experimental conditions is carried out and characterized.^30,31

The 3D cell clusters are obtained by cell reselection and centrifugation in the T25 culture flask. In order to keep the cell clusters from cracking when treated with a plasma jet, 1000 rpm was used to centrifuge for 15 min. Transfer the obtained cancer cell clusters to one side of the Petri dish, and the normal cell clusters to the other side of the Petri dish. Add the configured medium and place a glass sheet slightly below the liquid level in the middle of the two cell clusters to prevent scattered cells from entering the opposite cell clusters area during the treatment process to the maximum extent possible. After the treatment was completed, the two cell clusters were inoculated into a 6-well plate, labeled with DCFH-DA probe and observed.^32–35 

In order to verify whether the ROS scavenger has an impact on the killing effect of plasma jet, the following experiments are designed:

The cultured cancer cells were resuspended and centrifuged, blown with 5 ml of culture medium, inoculated into a 6-well plate, and cultured overnight until the cells covered the bottom of the 6-well plate. ROS scavenger was added to the orifice plate, and then treated with a plasma jet for 10 min. After treatment, continue to cultivate for 24 h and characterize with scratch experiment. The control group used the same treatment method, but did not add ROS scavenger.

As shown in Fig. 1(B), the driving gas used in the experiment is helium. Since the inner diameter of the elastic quartz tube is 50 µm, the flow rate of the control gas is 0.2 slm. And the effect of low gas flow rate on 3D cell clusters will be reduced. Since a DC power supply is used, when the voltage is applied to 3 kV, a synchronous and stable plasma jet can be seen at the nozzle of the tungsten needle electrode. During the experimental treatment, the plasma jet does not directly contact the liquid level, but pushes the helium-driven active particles to the 50 µm elastic quartz capillary, and the active particles finally flow out of the nozzle. After removing the 50 µm quartz tube, the spectrum of the fine plasma jet device is detected, and the results are shown in Fig. 1(C). It can be clearly seen that the fine plasma jet produces a large number of gas reaction species, the highest peak of which is the peak of helium (587.5 nm); at the same time, it can also be seen that the peak of nitrogen is located at 300–470 nm. As we know, the gaseous reactive species produced by the plasma jet can react with an aqueous solution or cell culture medium to produce a wealth of aqueous reactive species. Therefore, before conducting cell experiments, we tested the concentration of active substances induced by the fine plasma jet device in the medium. As shown in Fig. 1(D). It can be seen that after 10 min of fine plasma treatment, the concentrations of HNO₂, H₂O₂ and HNO₃ were 1.03, 2.66, and 3.43 µmol/l. The result shows that after the micro-plasma jet treatment, the content of reactive nitrogen and reactive oxygen species in the cell culture medium increases, which means that the micro-plasma jet can cause oxidative stress in the cell culture medium.

In order to find a suitable cell processing time, the cultured cells are inoculated into a 6-well plate. After the cells are adherent to the wall, the cells are treated with a plasma jet device with a time gradient of 0, 5, 10, 15, and 20 min, and the treated cells are resuspended and transferred to a new 6-well plate and continued to be cultured for 24 h to obtain cell adherent results, as shown in Fig. 2(A). It can be seen that at the time of processing for 10 min, there are almost no cells that have been re-adherent. Figure 2(B) is a line chart of the percentage of adherent cells. After the cancer cells are treated, MTT detects cell viability, and the results are shown in Fig. 2(C). It can be seen from 0–1 min that the cell vitality is significantly reduced. Starting from 1–7 min, the rate of decline in cell vitality slows down. From 7 to 10 min, the cell vitality is greatly reduced. Therefore, it is concluded that the vitality of cells decreases rapidly as the plasma jet processing time increases. By about 10 min, the cell vitality has basically dropped to zero. Compared with the control group, the proliferation ability and activity of cells showed a tendency to be suppressed in the data processed by each experimental group. Combining the two experiments, we determined that the maximum time for plasma jet to treat cells is 10 min.

After determining the processing time, we conducted a specific plasma jet treatment cell characterization experiment. Figure 3(A) is a comparison chart of the scratch experimental results of A-375(a) and HA1800(b). The cells continued to be cultured for 24 h after plasma jet treatment. Figure 3(B) is a histogram of the average width of the scratch area. The experiment was carried out in a 6-well plate. As can be seen from the figure, for cancer cells, the width of the scratch area increases with the increase of processing time. The cells in the pores treated for 0 min migrated significantly, indicating that cancer cells have a strong ability to divide and migrate without being treated by plasma jet. In the 5 min treatment results, the number of migrated cells was greatly reduced, indicating that the plasma jet had induced most of the apoptosis, but there were still some scratch areas covered by the migrated cells. This may be because some cells were not induced to apoptosis during the 5 min treatment period, so the non-apoptotic cells continued to divide and migrate to repair the scratched area. There was almost no migration of cells in the pores treated for 10 min, indicating that the cells in the pores had basically apoptosis. The experimental results show that the plasma jet can inhibit the migration and repair of melanoma cells, and the inhibitory effect is already quite obvious at 10 min. The scratch area of normal cells for 5 and 10 min healed obviously, and the cells still had a strong ability to divide and migrate, indicating that the plasma jet had a weak effect on the apoptosis of normal cells.

The experimental conditions for reactive oxygen species detection are the same as the previous experimental conditions. The DCFH-DA reagent is used in this experiment. The principle is that DCFH-DA itself has no fluorescence and can freely penetrate the cell membrane. After entering the cell, DCFH can be hydrolyzed by lipases in the cell to produce DCFH, but DCFH cannot pass through the cell membrane. Reactive oxygen species in the cell can oxidize non-fluorescent DCFH to generate fluorescent DCF. Using green fluorescence with an excitation wavelength of 488 nm and an emission wavelength of 525 nm, apoptotic cells labeled by the DCFH-DA probe can be observed under a fluorescence microscope. Figure 4(A) is a comparison chart of the reactive oxygen species experimental results of A-375(a), 50% density A-375(b) and HA1800(c). The proportion of fluorescent cell area can be obtained using Image J. In Fig. 4(A), it can be seen from (a) that there is no reactive oxygen species in cancer cells that have not been treated with plasma jet, so the cells have not apoptosis. The 5 min treatment results showed that the number of cells labeled by the DCFH-DA probe increased significantly, and the area of labeled cancer cells accounted for 18.21%. It can be analyzed that after 5 min of plasma jet treatment, the content of reactive oxygen species in most cells increased and the number of apoptosis increased. After 10 min of treatment, almost all the cells observed under the microscope were covered with green fluorescence, the content of reactive oxygen species in the cells was high, and a large number of cells apoptosis. The area of apoptotic cells is 54.2%. The experimental results show that the active substances induced by medium ion jet in the medium can induce apoptosis of cells. After apoptosis, the cell membrane loses its blocking effect, causing the cells to be labeled by DCFH-DA probes. In Fig. 4(A-b), the 50% density A-375 treatment experiment, we obtained the proportion of fluorescent cell area of 0, 5, and 10 min, which were 0%, 9.76%, and 22.46%, respectively. It can be seen that when the cell density is reduced by half, the plasma jet still has a strong effect on apoptosis. Especially for cells that have been processed for 10 min. When the cell density is reduced by half, the number of cells labeled by the probe is also reduced by about half accordingly. For the treatment results of normal cells, in Fig. 4(A-c), we obtained fluorescent cells accounting for 0%, 0.27%, and 2.77%. Compared with cancer cells, the amount of apoptosis in the treatment results of normal cells in each time period is much lower than cancer cells, which means that the plasma jet generated by the device has a much stronger apoptosis effect on cancer cells than normal cells. This experiment also echoes the previous experiment, reflecting that the micro-plasma jet can induce apoptosis of most cancer cells in about 10 min, and its apoptosis results are significant. Figure 4(B) is a compromise diagram of the area of the fluorescent cells of three experiments.

The experiment of 3D cell clusters is characterized using reactive oxygen species fluorescent labeling, and the characterization results are shown in Fig. 5(A). It can be seen that the cells processed in the same environment were not fluorescently labeled at 0 min. At 5 min, more than half of the A-375 cells had been labeled, while the number of HA1800 cells was relatively small. During 10 min of plasma jet treatment, A-375 cells were basically labeled by probes, and the content of reactive oxygen species in the cells was higher, while the number of HA1800 cells was obviously not as large as that of A-375 cells. Judging from the experimental results, the more appropriate treatment time for plasma jet to induce apoptosis of cancer cells is 10 min. Because the reactive oxygen species test results of cancer cells at this time show that cancer cells have basically been induced to apoptosis, while the number of normal cells is small. It can be directly seen from Fig. 5(B) that the ability of plasma jet to induce apoptosis of cancer cells is much higher than that of normal cells. Combined with the design of this experiment, the two types of cells treated by the plasma jet are located in the same hole and are isolated. At this time, the plasma jet directly treats the cells, which can simulate the location of the junction between cancer cells and normal cells in the human body. Judging from the experimental results, the lethality of plasma jet on cancer cells is greater than normal cells, so the author believes that plasma may have unexpected effects in the treatment of human cancer.

It can be seen from Fig. 6(A) that ROS scavenger has a great impact on the healing area of A-375. It can be seen from the control group that the healing range of cells without ROS scavenger is very small. The experimental group added a ROS scavenger. From the figure, it can be seen that the plasma jet has no significant effect on the cell healing ability, and the cell healing area exceeds half of the scratch area. Using Image J to process the scratch map to obtain the average width of the scratch area [Fig. 6(B)], it can be clearly concluded that ROS scavenger can inhibit the apoptosis-inducing effect of the active substance produced by the plasma jet on cancer cells.

In this paper, plasma jet-induced apoptosis experiments were mainly carried out, and plasma jet treatment and subsequent characterization of A-375 cells and HA1800 cells were carried out respectively. In the scratch experiment, the treatment of A-375 cells showed that within the treatment time of 0–10 min, the cell activity was inversely proportional to the plasma jet treatment time. The longer the plasma jet processing time, the lower the cell activity. Plasma jet treated A-375 cells for 5 min, and the cell activity was only about 30% of that in 0 min. Compared with the scratch experiment of HA1800, the activity of plasma jet treatment A-375 for 5 min is much lower than that of HA1800. Treatment of HA1800 cells for 0–10 min, the longer the treatment time, the slightly reduced cell activity, and the cell activity at 10 min is much higher than that of A-375 cells at 10 min. The MTT experiment and reactive oxygen species detection experiment on A-375 cells showed that the plasma jet has a prominent killing effect on cancer cells, and most cancer cells can be killed in a very short period of time. Reactive oxygen species detection experiments on HA1800 have shown that plasma jet can also inhibit the growth of normal cells. But the inhibitory effect is much lower than that of cancer cells. The results of plasma jet treatment of cancer cells of different densities showed that even if the cell density decreased, the ability of plasma jet to induce apoptosis of cancer cells did not change. At the same time, experiments on ROS scavenger have shown that ROS scavenger can reduce the killing effect of plasma jet on cancer cells. The reason is that ROS scavenger neutralize the ROS induced by plasma jet, resulting in a decrease in the number of cancer cells. After plasma jet treatment of 3D cell clusters of two cells in the same well, it can be seen from the reactive oxygen species detection experiment that under simultaneous treatment, the ability of plasma jet to induce apoptosis of cancer cells is much higher than that of normal cells. The experiment simulates to a certain extent the area where cancer cells bind to normal cells in the human body, and the experimental results are also complementary to the experimental results obtained by processing and characterizing the two types of cells separately.

Based on these experiments, it can be concluded that plasma jet has a significant role in inducing apoptosis. Combining the experimental characterization results of cancer cells and normal cells, it can be seen that plasma jet can induce apoptosis at the interface where normal cells and cancer cells are mixed. Since the basis of normal cells in the human body is relatively large, it is possible to target and treat cancerous sites in the human body at a smaller cost.

In this paper, detailed experiments were conducted on plasma jet-induced apoptosis, and we detailed experimental results were obtained. The author hopes that through this article, it will provide a new direction for cancer treatment and provide sufficient data support.

This work was partially supported by the National Natural Science Foundation of China under Grant Number 62163009 and 61864001; the Natural Science Foundation of GuangXi under Grant Number 2021JJD170019; the Foundation of Guangxi Key Laboratory of Automatic Detecting Technology and Instruments (Guilin University of Electronic Technology) under Grant Number YQ23103;Innovation Project of GuangXi Graduate Education under Grant Nos. YCSW2022277 and 2023YCXS184; Guangxi Major Scientific and Technological Innovation Base (Guilin University of Electronic Technology) under Grant 231002-k.

The authors have no conflicts to disclose.

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Hua Li is currently a professor and doctoral supervisor at Guilin University of Electronic Technology. He received a bachelor’s degree and a master’s degree from North University of China in 2001 and 2004, respectively. He received a Ph.D. from Beijing Institute of Technology in 2007 and a postdoctoral fellowship in Instrument Science and Technology from Tsinghua University in 2010. He is mainly engaged in plasma medicine, MEMS and other research.

Qihao Shi is currently a master’s student in the School of Life and Environmental Sciences at Guilin University of Electronic Technology. He received a B.S. degree in bioinformatics from Southeast University, and his research direction is plasma medicine.

Wenxiang Xiao is currently an associate professor at Guilin University of Electronical Technology. She received a Ph.D. degree from Sichuan University in 2007. Her research interests focus on the luminescent nanomaterials and their application in biomedical sensing, and the development in portable optical sensing systems.