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Article

Optimization of Indirect CAP Exposure as an Effective Osteosarcoma Cells Treatment with Cytotoxic Effects

by
Alexandru Bogdan Stache
1,2,
Ilarion Mihăilă
3,
Ioana Cristina Gerber
3,
Loredana Mihaiela Dragoș
4,
Cosmin Teodor Mihai
5,
Iuliu Cristian Ivanov
4,
Ionuț Topală
6,* and
Dragoș-Lucian Gorgan
1,*
1
Faculty of Biology, Alexandru Ioan Cuza University of Iasi, 700505 Iasi, Romania
2
Department of Molecular Genetics, Center of Fundamental Research and Experimental Development in Translational Medicine—TRANSCEND, Regional Institute of Oncology, 700483 Iasi, Romania
3
Integrated Center of Environmental Science Studies in the North-Eastern Development Region (CERNESIM), Alexandru Ioan Cuza University of Iasi, 700506 Iasi, Romania
4
Molecular Diagnosis Department, Regional Institute of Oncology, 700483 Iasi, Romania
5
Praxis Medical Investigations, 700376 Iasi, Romania
6
Faculty of Physics, Alexandru Ioan Cuza University of Iasi, 700506 Iasi, Romania
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2023, 13(13), 7803; https://doi.org/10.3390/app13137803
Submission received: 12 May 2023 / Revised: 22 June 2023 / Accepted: 28 June 2023 / Published: 2 July 2023
(This article belongs to the Special Issue Recent Advances in Atmospheric-Pressure Plasma Technology, 2nd Volume)
Browse Figures
Versions Notes

Abstract

:
Over the past decade, cold atmospheric plasma (CAP) has undergone extensive research as a promising therapeutic approach in oncology, with different treatment methods and exposure configurations being investigated and resulting in various biological effects, most of them after long exposure or treatment durations. This study aimed to evaluate the potential of a custom-made CAP generation source to produce plasma-activated medium (PAM) with cytotoxic effects and subsequently to establish the optimal exposure and treatment parameters. The exposure’s electrical parameters, as well as pH and NO 2 − content of PAM were analyzed. The cytotoxic potential and optimal parameters of the treatment were established by evaluating the viability of human osteosarcoma cells (HOS cell line) and human osteoblasts (HOB cell line) treated with PAM under different conditions. Our results showed that indirect treatment with CAP presents selective dose-dependent cytotoxic effects, while the cell viability decrease was not found to be correlated with the PAM acidification due to CAP exposure. The Griess assay revealed very high and long-term stable concentrations of NO 2 − in PAM. Overall, this study presents a simple and faster method of producing PAM treatment with cytotoxic effects on HOS cells, by using a custom-built CAP source.

1. Introduction

Cancer, a large collection of related diseases that develop over time and involve abnormal cell growth with an invasive or spreading potential [1], is the second cause of death worldwide behind cardiovascular disease [2,3]. As World Health Organization reports, one of six deaths is cancer-related; this disease is responsible for approximately 10 million deaths per year [4]. Osteosarcoma (OS), a malignant neoplasm that occurs as a solid osteoid-producing tumor, is the most frequent primary malignant bone tumor in children and young adults [5,6]. OS usually develops at the level of long bone metaphysis, but its appearance in other areas of the body that are more difficult to access or that have sensitive structures in the vicinity constitutes a challenge for current therapy methods. In addition, the acquisition of resistance to classical chemotherapy and the cancer recurrence due to postoperative micro residual tumors are adding even more obstacles to OS treatment [7,8,9]. Therefore, the development of new therapeutic methods for OS treatment is necessary and represents a research domain of interest [10].
Cold atmospheric plasma (CAP) is a partially ionized gas close to room temperature, consisting of photons, ions, electrons, free radicals and excited or neutral molecules and is usually generated by applying an electrical discharge to a gas [11,12,13].
According to Chen et al. [14] the interest in cold plasmas started in the late 1970s with atmospheric pressure plasma. The reduced production costs of atmospheric pressure plasma have led to its rapid development and relevance in various research areas [14,15]. Researchers demonstrated in the mid-1990s that CAP can be used to inactivate bacteria, thus making the technology relevant to life sciences and medical fields [16]. The reports of in vitro experiments on mammalian cells from the middle of the 2000s, in addition to several experimental studies on the antimicrobial activity of CAP, marked the start of an intense research period on plasma-biological target interactions using different types of microorganisms, plants, mammalian cells and tissue models [17,18]. Today, several CAP applications in biology and medicine are included in the field of plasma medicine, as follows: decontamination and sterilization [19,20,21,22,23], wound healing [24,25,26,27], angiogenesis [28,29,30,31], dentistry [32,33,34,35], pharmacology [36,37,38,39,40,41], biocompatibility of implants [42,43,44] and oncology [45,46,47,48].
One of the most frequently used techniques for producing CAP is dielectric barrier discharge (DBD) [49,50,51]. Under conventional laboratory circumstances, CAP can easily be generated by providing high voltage electrical power (in the form of alternating or pulsed electric field) to a device with one or two electrodes, at least one of which must be insulated with a dielectric barrier [49,51,52,53,54]. The electrodes are known as power and counter electrodes, the first one being attached to the high-voltage power source and the second one, which can be optional, is grounded. A strong electric field is thus produced, resulting in electric discharges that partly ionize the gas in which they occur, generating CAP [52]. An essential feature of all CAP production methods is the prevention of arcing [55]. With each pulse in a DBD discharge, the electrical voltage drops due to the charges gathering on the dielectric layer covering the electrode/electrodes, stopping the discharge. As a result, DBD is an externally pulsed or self-pulsed discharge that prevents the discharge current to increase to a point where arcing could occur [56,57].
Regardless of the configuration, with one or two electrodes, there are two types of DBD that can be distinguished: DBD without flow, in which the plasma is produced and remains to the space between the electrodes, and DBD with flow, also known as a plasma jet or atmospheric pressure plasma jet (APPJ), in which the plasma is located both in the space between the electrodes and in the post-electrode region [52,58]. Yan et al. [59] asserted that these two forms of plasma discharge may also be classified as direct discharge sources (DBD without flow) and indirect discharge sources (DBD with flow), based on the discharge mode presented above.
Over the past decade, both DBD techniques have been explored for their potential use in the field of oncology, and they have been shown to be a promising alternative to traditional cancer therapies [48]. Regarding the treatment of tumor cells, two methods of CAP treatments are widely used, namely direct treatment and indirect treatment. In direct treatment, cells are directly subjected to CAP discharges, whereas in indirect treatment, plasma discharges are applied to a liquid which is then administered to the cells [12].
Turrini et al. assumed that the biological effects of CAP result in an interaction between cells and the plasma’s physical (UV, electromagnetic field, heat) and chemical (short- and longed-lived reactive oxygen and nitrogen species (RONS)) elements, with RONS being responsible for oxidative damage, membrane alterations, apoptosis and double-strand breaks in DNA [60]. When a plasma discharge occurs over a liquid, the RONS formed at interfaces are partially transferred into the liquid, where, due to the interaction between them and the molecules in the liquid, secondary reactive species are formed [3,9].
Khlyustova et al. state that CAP direct treatment only allows for a maximum plasma penetration depth of 60 µm for anti-tumor effects, making it challenging to treat oncology patients using this technique. On the other hand, indirect treatment with plasma-activated medium (PAM) offers a significant advantage as it allows for targeting various regions of the body or tumor by injecting PAM at the therapeutic location. Moreover, from a technical standpoint, producing and storing PAM as an anti-tumor treatment is easier and more convenient. [61]. Therefore, from a translational perspective, the indirect method of treatment with CAP appears to be more promising. Viewed from the same perspective, it is important for a potential cancer-fighting therapy like CAP to possess certain essential qualities in addition to its targeted antitumor effects. These characteristics may include a reduced production time, the ability to be stored for subsequent use, an optimal balance between treatment duration and its efficacy and cost-effective production.
Considering the previously mentioned aspects, the purpose of this study is to evaluate the potential of our custom-made CAP source to produce PAM, to establish the optimal exposure and treatment parameters in order to create a fast-obtaining treatment with rapid and efficient cytotoxic effect on HOS cells, as well as to characterize the produced PAM as a possible treatment in OS therapy.

2. Materials and Methods

2.1. Experimental Setup and PAM Production

The DBD CAP generation system is made up of a custom-made high voltage AC power supply with a sinusoidal waveform (maximum voltage 15 kV), a high voltage electrode (HVE), a dielectric layer and a grounding electrode (GE). The power supply is composed of a Variac transformer (0 to 240 V) and a 20 kV peak-to-peak neon transformer with a maximum average current of 30 mA. The HVE consists of four stainless steel cylindrical subunits, each with a 14 mm diameter interconnected by a copper plate. The GE electrode is represented by a copper plate with a thickness of 1.5 mm, covered with a 0.1 mm thick dielectric layer [62,63]. In order to expose the medium to CAP discharges and to create the PAM, 24 well flat-bottomed cell culture plates (cat. 92024, TPP Techno Plastic Products AG, Trasadingen, Switzerland) containing 150 μL exposure medium/well were used as a support/discharge vessel. The HVE was introduced into the cell culture plate without allowing the electrode subunits to come into contact with the internal walls of the wells. The height of the exposure medium column was 0.8 mm, the gap between the electrode subunits and the base of each well, as well as the distance between the electrode and the exposure medium surface was consistently maintained at 3 mm and 2.2 mm, respectively. Completing the exposure assembly was conducted by attaching the dielectric layer and the GE under the cell culture plate (Figure 1A). The number of cylindrical subunits in the HVE composition is equal to the number of wells in the column, thus exposing the medium from all four wells from a column to the CAP discharge at the same time. In order to avoid the appearance of possible side effects of the discharge that may have as a consequence of the modification of the exposure parameters, columns 1 and 6 from the exposure plate were not used (Figure 1B). In all experiments, the medium was exposed to CAP for different periods of time (30 s, 60 s and 90 s), known as exposure time (Texp), this being the period in which the source was turned on, generating DBD plasma in the air trapped between the lower end of each cylindrical subunit of the HVE and the medium in the well. In this study we will refer to the solutions exposed to plasma for the previously presented time periods as PAM-medium* 30 s, PAM-medium* 60 s and PAM-medium* 90 s (* where medium represent the exposed solution, i.e., PBS or RPMI 1640). Following the exposure, the PAM from each well was collected and mixed with the PAM from the other wells within the same exposed column to create a uniform treatment.

2.2. Cell Culture

In order to evaluate the biological effects of CAP treatment, the human osteosarcoma (HOS) cell line (CRL-1543, American Type Culture Collection, Manassas, VA, USA) and Human Osteoblasts (HOB) cell line (406-05a, Cell Applications, Inc, San Diego, CA, USA) were used in this study. The HOS cells were grown in RPMI 1640 medium (Capricorn Scientific GmbH, Ebsdorfergrund, Germany), supplemented with 10% (v/v) fetal bovine serum (Sigma Aldrich, St. Louis, MO, USA) and 1% (v/v) penicillin/streptomycin (Biological Industries, Kibbutz Beit-Haemek, Israel), and the HOB cells were grown in HOB Growth Medium (Cell Applications, Inc, San Diego, CA, USA). Both HOS and HOB cells were cultured in a climate-controlled incubator CERTOMAT®CO2 Incubator (Sartorius Stedim Biotech GmbH, Göttingen, Germany) at 37 °C, in an atmosphere with 5% CO2 and 95% humidity.

2.3. CAP Indirect Treatment

This study evaluated the effects of indirect CAP treatment on HOS cells, comparing two different types of exposure medium, three Texp and four treatment times (Ttr).
Twenty-four hours before treatment, 1 × 105 cells were transferred to a 96-well cell culture plate (TPP Techno Plastic Products AG, Trasadingen, Switzerland). To perform the indirect CAP treatment, the growth medium was discarded from each cell culture plate well and the cells were washed with PBS (Corning, Corning, NY, USA) to completely remove the medium and cell debris. The treatment consisted of adding 100 μL of PAM to the each well of the cell culture plate, except for the control cells where was added the same amount of untreated medium. Cell culture plates were incubated at 37 °C, in a 5% CO2 atmosphere and 95% humidity, for 5 min to 30 min, a period of time also known as treatment time (Ttr). Subsequently, the treatment was removed and, after a washing step of the cells with PBS, 100 μL of complete medium was added to each well. The plates were incubated for 2 h (or 24 h for the treatment selectivity evaluation) under the same conditions previously described.

2.4. CAP Characterization

The voltage and current characterizations of the CAP discharges were analyzed by using Tektronix P6015A (Tektronix, Inc., Beaverton, OR, USA) and Pearson 6585 (Pearson Electronics, Palo Alto, CA, USA) probes connected to TDS5034 digital scope (Tektronix, Inc., Beaverton, OR, USA). Electrical measures for each Texp were carried out during RPMI 1640 exposures to CAP.
Optical emission spectra (OES) of the constituent elements of CAP were detected using a monochromator equipped with a CCD detector (Horiba Triax 550 with Symphony CCD Detector) within a wavelength range of 300–430 nm. The optical fiber was oriented perpendicularly to the CAP discharges, at a distance of 1 cm from the exposure plate wall, with the acquisition time being set at 5 s.
Temperature variation inside the wells was measured using a multimeter and a type K thermometer. The wells were filled with 150 μL of RPMI 1640 and temperature was measured immediately after each treatment time, both in the treated and neighboring columns.

2.5. Cell Viability Assay

In order to evaluate the viability of the cells following the plasma treatment, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was performed. The mitochondria of live cells metabolize this tetrazolium salt to formazan, an insoluble blue chemical that may be detected spectrophotometrically after being solubilized in an SDS or DMSO solution [64].
After the CAP post-treatment incubation period, the medium was removed from the culture plates and the cells were then washed with PBS. Subsequently, the cells were covered with 110 µL of MTT working solution composed of 10 μL of MTT solution (5 mg/mL) (Merck, Darmstadt, Germany) and 100 μL fresh RPMI 1640 medium, and were incubated for 2 h at 37 °C, in a 5% CO2 atmosphere and 95% humidity. After incubation, a volume of 90 μL MTT medium was carefully removed from each well and 70 μL DMSO (Sigma Aldrich, St. Louis, MO, USA) was added, to dissolve the formazan crystals. The samples were incubated for 10 min in previously described conditions, then spectrophotometrically analyzed at 570 nm wavelength, using the multimodal plate reader FilterMax F5 (Molecular Devices LLC, San Jose, CA, USA). All assays were performed in triplicate.

2.6. pH Analysis of PAM

In order to evaluate the change in PAM pH, 3 mL of medium/Texp were exposed to CAP and transferred into a 15 mL tube. The pH measurements were made using a PH 210 Microprocessor pH Meter (Hanna Instruments, Woonsocket, RI, USA).

2.7. Quantification of Nitrite and Nitrate Concentrations in PAM

The levels of nitrites ( NO 2 − ) and nitrates ( NO 3 − ) in PAM were assessed using the Griess Reagent System (Promega, Madison, WI, USA). The nitrites detection using the Griess reagent system is based on a chemical reaction of sulfanilamide solution (SS) and N-1-naphthylethylenediamine dihydrochloride (NED) under acidic conditions (phosphoric acid). For each sample, 50 µL of PAM was mixed with 50 µL of SS and incubated for 10 min in the dark at room temperature. Afterward, 50 µL NED was added to each sample, followed by a new incubation stage of 10 min in the dark at room temperature. The samples were read at 540 nm with the multimodal plate reader FilterMax F5 (Molecular Devices LLC, San Jose, CA, USA). In order to detect nitrates, the NO 3 − was reduced to NO 2 − with vanadium chloride [65], then, the reduced nitrates cumulated with the nitrites initially present in the sample were spectrophotometrically evaluated as previously described. Afterward, NO 3 − levels have been identified by subtraction of NO 2 − levels from the total amount of reduced NO 3 − plus NO 2 − levels.

3. Results

3.1. Electrical Characterisation of CAP

As shown in Figure 2, after the ignition of CAP producing device, a typical current–voltage waveform is observed. From an electrical point of view, the discharges in the dielectric barrier are presented in the form of packages with a frequency of 100 Hz. Each package can be described as having an initial zone (I) in which the voltage begins to rise, a median zone (II) in which the voltage reaches its maximum value and remains constant, a zone of progressive decrease in voltage (III) and a zone final where the voltage drops to the minimum value (IV) (Figure 3). The frequency of the electrical voltage identified in the packages was 23 kHz.
By examining the shape of the voltage waveform in each package, it was determined that regions II and III are the determining elements for the plasma discharge. To gain a clearer understanding of the discharge properties, we analyzed the number and amplitude of the currents in these two areas across 10 packages. The currents were found to have intensities ranging from 0.06 A to 0.72 A (Table S1), with the most common intensity being 0.06 A, averaging around 977 currents on the positive half-cycle and 632 currents on the negative half-cycle (Figure 4).

3.2. Optical Emission Spectroscopy

Optical emission spectra obtained for CAP discharges show several characteristic lines of N2 s positive system (Figure 5), identified at the wavelengths 313.51, 315.78, 328.55, 337.28, 349.86, 353.73, 357.70, 366.89, 371.21, 375.62, 380.48, 389.43, 391.46, 394.15, 399.56, 405.92, 413.91 and 420.22 nm.

3.3. CAP-Discharges Temperatures

In order to determine the rotational temperature of nitrogen during the moment of CAP discharge, high-resolution spectra in the range of 331 nm to 338 nm (0.017 nm resolution) were acquired. The OES data obtained were subjected to analysis using the molecular nitrogen second positive C-B system (band head at 337.28 nm) as a simulation model for data fitting with the MassiveOES software [66]. As a result, using 5 s integration time we were able to monitor the rotational temperature values during the 30 s, 60 s, and 90 s exposures. No significant differences were observed for the different tree exposure times; no temperature increase during exposure was registered. The average value of rotational temperature is 1163 ± 24 K, higher than the values observed for diffuse planar dielectric barrier discharges, but comparable with values observed for filamentary discharges or sparks.
Monitoring the temperature of the solution subjected to CAP discharges, we noted that the exposure only caused minor temperature increases. Starting from the temperature of 16.5 °C of the control sample, the temperature of the medium exposed for 30 s was 19 °C, that of the medium exposed for 60 s was 23 °C, and the highest temperature value of 27 °C was identified for the medium exposed for 90 s. Additionally, we noted that the exposure had a negligible effect on the temperatures of the other columns except for the first adjacent column, which had a slight temperature increase, as illustrated in Figure 6.

3.4. The Impact of PAM-PBS and PAM-RPMI Treatments on HOS Cells Viability

To compare the response of cells to the treatment, this study assessed the viability of HOS cells indirectly treated with CAP via two distinct plasma-exposed solutions: PBS and RPMI 1640.
For this, three variants of treatment were performed, for each type of solution, depending on the CAP exposure time: 30, 60 and 90 s. Control groups were used for both PAM-PBS and PAM-RPMI treatment. The cells belonging to this group were identically handled as the cells from the PAM-treated groups. Two hours after the treatment cell viability was assessed.
PAM-PBS treatment resulted in a decrease in cell viability by 4.5%, 15.9%, and 17.2%, respectively, for the three experimental variants. However, the results of Dunnett’s multiple comparisons test indicate that the observed differences are not statistically significant (Figure S1). By comparison, the use of PAM-RPMI caused a decrease in cell viability by 12.1% for 30 s, 21.5% for 60 s, and 78% for 90 s exposure time. These reductions were statistically significant, as determined by Dunnett’s multiple comparisons test (Figure S2). On the other hand, if we compare the results obtained by both treatments for each experimental variant separately, Šídák’s post hoc multiple comparison test indicates a significant difference between the effects of the treatments only for the exposure time of 90 s (Figure 7).

3.5. Identifying the Optimal Treatment Time

In order to identify the optimal treatment time of indirect HOS exposure to CAP, in this study we performed a cell viability analysis, using three PAM-RPMI doses (with Texp of 30, 60 and 90 s). The treatment times analyzed were 5, 10, 20 and 30 min, for all experimental variants also control groups were used. Cell viability was assessed at 2 h post-treatment.
PAM-RPMI treatment exhibited cytotoxic effects for all experimental variants, these ranged from mild to moderate and strong depending on the combination of exposure time and treatment time (Figure 8).
After treating the cells with PAM-RPMI 30 s, the viability of HOS cells experienced a slight decrease, as follows: 92.22% for 5 min Ttr, 88.19% for 10 min Ttr, 87.09% for 20 min Ttr and 78.35% for 30 min Ttr. For the PAM-RPMI 60 s treatment, the trend of decreasing viability was the same, reaching values of 89.83% for 5 min Ttr, 84.22% for 10 min Ttr, 87.29% for 20 min Ttr and 75.78% for 30 min Ttr. The last experimental variant evaluated in this study, PAM-RPMI 90 s treatment, was most influenced by the treatment time, the HOS cell viability values being 87.55% for 5 min Ttr, 77.85% for 10 min Ttr, 49.32% for 20 min Ttr and 27.70% for 30 min Ttr.
When evaluating the impact of the different treatment times on HOS cell viability it can be observed that an increase in the duration in which the cells are in contact with PAM-RPMI has as a consequence a stronger impact on cell viability, resulting in a direct correlation between the treatment time and its cytotoxicity. According to the Tukey multiple comparison tests, it can be observed that for the PAM-RPMI 30 s and PAM-RPMI 60 s the only statistically significant difference regarding the decrease in cell viability is between the samples treated with PAM-RPMI for 30 min and those treated for 5 min, while, in the case of PAM-RPMI 90 s, there is a statistically significant decrease in cell viability for all experimental variants, with the exception of the Ttr’s of 5 and 10 min, between which the differences are statistically not significant. According to the results previously presented, the treatment time of 30 min had the strongest cytotoxic effect, regardless of the exposure time used.

3.6. pH Evaluation of PAM-RPMI

Compared with the control group represented by RPMI 1640, which presented a pH of 7.42, the PAM-RPMI samples suffered gradual dose-dependent acidification after exposure to CAP (Figure 9), as follows: pH 7.13 for PAM-RPMI 30 s, pH 6.78 for PAM-RPMI 60 s and pH 6.26 for PAM-RPMI. All the differences observed between the CAP-treated samples and the control samples were statistically significant.

3.7. Evaluation of PAM pH Influence on Cell Viability

To investigate the impact of acidification of CAP-exposed medium on cells, two batches of HOS cells were used. One batch was treated with PAM-RPMI in three experimental variants (with Texp of 30, 60, and 90 s), while the other batch was treated with simple RPMI 1640 medium that had been adjusted with hydrochloric acid to match the pH of the PAM-RPMI solutions. Thus, three equivalent treatment options with pH values matching those of the PAM-RPMI solutions used in the first batch (pH RPMI 30 s, pH RPMI 60 s, and pH RPMI 90 s) were created. The Ttr used in this experiment was 30 min. Control groups consisting of untreated cells were used for both experimental batches.
After treatment with PAM-RPMI cell viability decreased to 80.59% for PAM-RPMI 30 s, 80.59% for PAM-RPMI 60 s, and 25.05% for PAM-RPMI 90 s. However, for the cells treated with pH-RPMI, changes in cell viability were limited to 98.41% for pH-RPMI 30 s, 97.92% for pH-RPMI 60 s and 100.89% for pH-RPMI 90 s. Furthermore, only the changes in cell viability caused by the PAM-RPMI treatment were found to be statistically significant (Figure 10).

3.8. Analysis of PAM-RPMI Treatment Selectivity

The selectivity of indirect CAP treatment was assessed by treating both HOS and HOB cell lines with PAM-RPMI and evaluating their viability at two different time points. While both at 2- and 24-h post-treatment the viability of HOB cells presented minor modifications compared with the control samples’ viability, HOS cells exhibited a continuous viability decrease (Figure 11 and Figure 12), as follows: from 80.59% at 2 h to 62.88% at 24 h for the 30 s exposure time, from 80.59% at 2 h to 56.07% at 24 h for the 60 s exposure time and from 25.05% at 2 h to 5.57% at 24 h post-treatment for the 90 s exposure time.

3.9. Assessing the Stability of PAM pH at Various Storage Temperatures

PAM-RPMI solutions had relatively stable pH values for 3 months at storage temperatures of −20 °C and −80 °C. On the other hand, after remaining relatively constant for two months at 4 °C, the pH level of PAM-RPMI underwent a sudden decrease during the third month of storage (Figure 13).
Figure 14 displays the pH fluctuations of PAM-RPMI stored solutions, these pH variations being determined by calculating the difference between the highest and lowest pH values observed (Table S2). The pH fluctuations ranged from 0.22 to 0.46 at 4 °C, from 0.03 to 0.10 at −20 °C, and from 0.04 to 0.06 at −80 °C. Control samples also showed pH fluctuations of 0.19 at 4 °C, 0.04 at −20 °C, and 0.03 at −80 °C.

3.10. Nitrite and Nitrate Concentration in PAM-RPMI

The gradual increases in NO 2 − and NO 3 − in the PAM-RPMI solutions, which are CAP dose-dependent, is presented in Figure 15. The results suggest that CAP generates millimolar concentrations of NO 2 − and NO 3 − in all the experimental variants of PAM-RPMI. Thus, the 30 s exposure to CAP yielded 1.6 mM of NO 2 − and 1.57 mM of NO 3 − and the 60 s exposure to CAP generated 2.76 mM of NO 2 − and 2.03 mM of NO 3 − . The maximum values of both NO 2 − and NO 3 − (3.59 mM and 2.36 mM, respectively) were identified in the PAM-RPMI exposed to CAP for 90 s.

3.11. Nitrite Stability in PAM-RPMI Stored at −80 °C

Considering the key role of NO 2 − played in PAM’s anti-tumor efficacy, it is crucial to preserve its stability during storage. Therefore, we aimed to investigate if there were any changes in the levels of NO 2 − in our self-produced PAM-RPMI treatment after being stored at low temperatures for a prolonged period.
As shown in Figure 16, despite the duration of exposure to CAP, the levels of NO 2 − in the PAM-RPMI stored at a temperature of −80 °C remained constant during the monitoring period. The slight variations observed were not statistically significant.

4. Discussion

While the treatment of osteosarcoma (OS) has undergone significant improvements over the course of recent years, the 5-year overall survival rate remains notably low at approximately 60% [7,8,9]. The present therapy for OS involves surgical intervention, chemotherapy and radiotherapy [8,67]. While OS typically develops in the metaphysis of long bones, detecting tumors in other regions, such as the skull or spine, can raise difficulties due to the proximity of neurovascular structures [7]. According to Xu et al., the existence of micro residual tumors, intraoperative contamination during radical surgery and residual microlesions in peritumoral tissue that cannot be entirely excised are the primary factors responsible for local recurrence in patients with OS [8]. Moreover, chemotherapy can lead to drug resistance and unwanted side effects caused by its non-specific toxicity [9]. Therefore, it is imperative to explore novel treatment methods that can supplement or partially replace the existing therapies, thereby reducing recurrence rates and improving the survival rates of patients [10].
Mateu-Sanz et al. presented CAP as a promising anti-tumor treatment due to its demonstrated efficacy and selectivity in treating more than 20 different cancer types [9]. Over the past few years, different plasma devices have exhibited the capability to induce cytotoxic effects in vitro on various OS cell lines, either through direct or indirect treatment, offering a new potential direction in treating OS. For OS treatment, plasma-activated solutions are a particularly promising option as they not only have similar antitumor effects as a direct treatment but also offer the potential to bypass the need for open surgical interventions to expose tumors to CAP [9]. Moreover, PAM provides a feasible approach for aiming tumors deeply embedded in the tissue or for treating cancer cells where the physical effects of CAP need to be avoided [68].
The objective of this study was to provide a brief characterization of the CAP generated by a custom-made plasma source, as well as to analyze the resulting PAM and its potential anti-tumoral effects. Furthermore, the study aimed to identify the optimal exposure parameters for inducing a cytotoxic effect on HOS cells.
The use of indirect CAP therapy at atmospheric pressure is described in the specialized literature as having potential in the development of new antitumor future strategies. The most common way to provide this medication is with physiologically adapted plasma-activated solutions, including growth medium, phosphate-buffered saline, or Ringer’s solution [69]. Although Yan et al. observed that CAP-exposed PBS consistently had lower cytotoxic effects compared to CAP-exposed culture medium [70], Zhang et al. study comparing the effects of two solutions exposed to CAP, namely PBS and RPMI 1640, on A549 cells, demonstrated a more robust cytotoxic effect of CAP-exposed PBS [71]. Hence, in this study, we evaluated the antitumoral effects of two distinct solutions exposed to CAP generated by our custom-built source, Phosphate-Buffered Saline (PBS) and Roswell Park Memorial Institute 1640 medium (RPMI 1640), the aim was to determine which of these two CAP-exposed solutions had a superior cytotoxic effect on HOS cells. Our findings indicated that for exposure times of 30 and 60 s, the cytotoxic effects were relatively comparable between the two solutions, with a slightly stronger cytotoxic effect observed for PAM-RPMI. However, for an exposure time of 90 s, a significant difference was observed between the two solutions, with PAM-RPMI 90 s resulting in a reduction in HOS cell viability of approximately 78%. Previous studies have demonstrated that the exposure of different culture media to CAP can induce modifications of their components [72,73,74]. Moreover, while Tornin et al. showed that the presence of pyruvate in PAM inhibits its cytotoxic activity, Tanaka et al. demonstrated that the only constituent element in Ringer’s lactate solution whose exposure to plasma gives the entire solution anti-tumor characteristics is L-sodium lactate [75,76]. The possible reactivity between CAP and the multitude of constituent elements of the RPMI medium, in contrast to the simple PBS solution, may be the cause of the higher cytotoxicity observed in this study when using PAM-RPMI. This assumption should be verified in the future by analyzing which element or combination of elements following CAP exposure, in the same concentration or concentrations as in the evaluated RPMI 1640, gain cytotoxic properties similar to PAM-RPMI. Therefore, according to our results, RPMI 1640 was selected as the solution of choice for producing PAM in the subsequent stages of this study.
An important parameter in indirect CAP treatment is the interaction between the PAM and the target material (i.e., HOS cells), also called treatment time. According to the specialized literature, in previous studies different treatment times were used, starting from 10 min, up to 72 h [3,9,60,75,77,78,79,80,81,82,83,84].
According to Mateu-Sanz et al., the indirect method typically requires significantly longer treatment time than direct treatments with CAP in order to achieve the same level of efficiency [85]. As a result, most studies using the indirect method for OS employ treatment durations of 2 h or more, resulting in bone tumor cell viability decreasing between 80% and 0% at 24, 48, or 72 h post-treatment [9,86,87,88]. In these cases, the effectiveness of the treatments depended on various factors, including exposure and treatment times, PAM solution type, cell type, and also the time between treatment and evaluation. Our study found that even with a short 30-min PAM treatment, we were able to achieve a viability decrease in HOS cells down to 28% in just 2 h after the treatment. These results are comparable to the results of other studies that used the direct method [12,89,90,91,92], which is generally accepted as the most effective method for reducing cell viability with CAP treatment. However, our study achieved these results with a shorter exposure and treatment times than those typically used in the indirect method.
Observing that the color of the RPMI 1640 medium (containing phenol red) changed gradually during the exposure to CAP discharges, depending on the Texp used, the subsequent stage of our investigation was to assess the pH of PAM-RPMI. The results show that the exposure of the RPMI 1640 to CAP determines its acidification, in a dose-dependent manner. Previous studies have observed similar [78,93] or even stronger acidification [3,94] of solutions exposed to CAP. However, Mateu-Sanz et al. and Liu et al. were able to produce PAM without any alterations to the pH levels. The main difference in their approach was the addition of FBS to the solution that was exposed to CAP, either before or after the exposure [9,81]. According to Subramanian et al., the pH reduction observed following CAP exposure is a result of increased hydrogen ion concentration [3].
After observing the acidification of PAM-RPMI, our objective was to determine the impact of pH variations caused by treatment on the viability of HOS cells indirectly exposed to CAP. Specifically, we aimed to differentiate whether the decrease in cell viability was due to the effects of CAP treatment or to the acidification of the treatment solution. Thus, we concluded that only the low pH RPMI 1640 does not influence the viability of HOS cells, the cytotoxic effects being, therefore, determined by the exposure of RPMI 1640 to CAP. The results support Subramanian et al. affirmation that acidification of the treatment solution alone is insufficient to induce cancer cell death [3].
Analyzing the viability of HOB cells treated with PAM-RPMI it can be observed that the treatments had minor to no effects on it both at 2- and 24-h post-treatment. Interestingly, the viability of HOS-treated cells continued to drop after the first evaluation at 2 h after the treatment even though PAM-RPMI was not in contact with the cells, suggesting that the treatment induce in HOS cells triggers continuous long-term cytotoxic effects. Therefore, the indirect PAM-RPMI treatment produced by our custom-built CAP system is able to selectively kill HOS cells. According to Schneider et al., the relevant reactive species in the composition of PAM, as well as their cytotoxic effects, are present only in acidic conditions [95]. Therefore, we evaluated the dynamics of the pH levels of the PAM-RPMI solutions over a period of 3 months. Additionally, we examined the impact of storage temperature on the stability of the pH of the PAM-RPMI solutions. Figure 11 and Figure 12 demonstrate that the acidification of RPMI 1640 due to CAP exposure remains stable for 3 months, indicating that −80 °C is the optimal storage method of PAM-RPMI treatment due to its under 0.1 pH variation. However, given that the pH of PAM stored at −20 °C does not undergo significant variations, this storage temperature remains a viable option, especially when considering factors such as cost or the availability of equipment in some laboratories. Analyzing the pH stability of PAM stored at different temperatures (25 °C, 4 °C, −20 °C, and −80 °C) for 7 days, Fan et al. did not identify significant changes [96]. By comparison, in the present study, no significant variations were recorded during the first 2 months of storage, with changes being identified in the third month.
CAP is known to generate a significant amount of reactive nitrogen species [11,78], and the levels of these species in PAM solutions used for cancer cell treatment can impact the treatment’s antitumor effectiveness [11,46]. Moreover, the main contributor to the acidification of the PAM solution is attributed to the generation of nitrites ( NO 2 − ) and nitrates ( NO 3 − ) resulting from the interaction between the short-lived reactive nitrogen species generated by CAP and the exposed solution [3,9]. Thus, after establishing the optimal parameters for the production of PAM with antitumor effect by using our custom-made plasma generation device, the analysis of NO 2 − and NO 3 − from PAM-RPMI was carried out in order to identify both NO 2 − and NO 3 − levels and the influence that the exposure time to CAP can have on them. The results indicated that exposure of RPMI 1640 medium to CAP resulted in a dose-dependent increase in PAM-RPMI NO 2 − and NO 3 − concentrations, ranging from 1.6 mM to approximately 3.6 mM for NO 2 − and from 1.5 mM to approximately 2.3 mM for NO 3 − . The levels of NO 2 − and NO 3 − identified are considerably higher compared to those reported in previous studies where analyzing different solutions exposed to CAP for different periods of time and using different plasma production methods generally detected concentrations of both NO 2 − and NO 3 − between several µM and 1 mM [3,61,71,73,75,77,80,97,98]. Moreover, apart from the observed high concentrations of NO 2 − in PAM-RPMI, it was observed that these concentrations remained stable for up to 3 months when PAM-RPMI was stored at −80 degrees Celsius.
Compared to other studies, our optimized treatment is able to selectively kill approximative 75% of osteosarcoma cells in just two hours by 30 min cell treatment time with PAM-RPMI exposed to CAP for 90 s.
Further studies regarding the effects of the treatment on other tumor cell lines are necessary in order to assess PAM-RPMI produced by the presented CAP system as a potential new treatment in cancer therapy. Additionally, the molecular mechanism of the cell death activated by the treatment, along with the implication of such high levels of NO 2 − and NO 3 − in the observed cytotoxicity of the treatment should be evaluated. According to Kim et al., in order to make advancements regarding the introduction and standardization of CAP indirect treatment in the medical field it is necessary to clarify and establish certain aspects which, among others, include the discharge method, exposure time, the exposed liquid type and dosage [99]. Therefore, we assume that the results of this study can bring PAM treatment a step closer to its usage in cancer therapy.

5. Conclusions

This study presents a rapid, selective and effective cytotoxic treatment of HOS cells with PAM, by exposing RPMI 1640 medium to our custom-built CAP source. In the presented configuration of generating PAM, the optimal HOS cells treatment parameters identified are the 90 s exposure of RPMI 1640 to CAP with a treatment time of 30 min.
The recorded pH variations of the treatment do not impair cell viability, hence, the treatment’s observed effects can be exclusively attributed to the CAP indirect exposure.
RPMI 1640 exposures to CAP generates very high and stable levels of NO 2 − , which, to the best of the authors’ knowledge, are the highest concentrations reported so far among studies targeting the effects of indirect CAP treatments in oncology.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app13137803/s1, Table S1. Average number of currents per discharge package; Figure S1: Viability of HOS cells treated with PAM-PBS. Values are means ± SEM (n = 3). Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (ns—nonsignificant); Figure S2. Viability of HOS cells treated with PAM-RPMI. Values are means ± SEM (n = 3). Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (** p = 0.0055, *** p = 0.0001, **** p < 0.0001); Table S2. The lowest and highest pH values identified over 30 days of PAM-RPMI storage at different temperatures.

Author Contributions

Conceptualization and methodology, D.-L.G. and A.B.S.; software, A.B.S. and I.M.; validation, I.T. and D.-L.G.; formal analysis, C.T.M.; investigation, A.B.S.; resources, L.M.D.; data curation, A.B.S. and I.M.; writing—original draft preparation, A.B.S.; writing—review and editing, I.T. and D.-L.G.; visualization, I.C.I. and I.C.G.; supervision, D.-L.G.; project administration, A.B.S.; funding acquisition, D.-L.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work is related to COST Action CA20114—Therapeutical applications of Cold Plasmas (PlasTHER), supported by COST (European Cooperation in Science and Technology)—www.cost.eu (accessed on 30 June 2023).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Assembly diagram of the plasma generation device. (A) Longitudinal section through the plasma-activated medium (PAM) generation assembly. (B) High voltage electrode (HVE) positioning on the exposure plate and wells used to produce PAM.
Figure 1. Assembly diagram of the plasma generation device. (A) Longitudinal section through the plasma-activated medium (PAM) generation assembly. (B) High voltage electrode (HVE) positioning on the exposure plate and wells used to produce PAM.
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Figure 2. The pattern of dielectric barrier discharge (DBD) pulses.
Figure 2. The pattern of dielectric barrier discharge (DBD) pulses.
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Figure 3. Current−voltage waveforms in (A) a discharge package and (B) each of the four defined regions within the package.
Figure 3. Current−voltage waveforms in (A) a discharge package and (B) each of the four defined regions within the package.
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Figure 4. Average number of different amplitude currents per discharge package.
Figure 4. Average number of different amplitude currents per discharge package.
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Figure 5. The optical emission spectrum of cold atmospheric plasma (CAP) at 300–427 nm.
Figure 5. The optical emission spectrum of cold atmospheric plasma (CAP) at 300–427 nm.
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Figure 6. Temperature of the PAM-RPMI and the medium in the adjacent columns on CAP exposure plate.
Figure 6. Temperature of the PAM-RPMI and the medium in the adjacent columns on CAP exposure plate.
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Figure 7. Viability of human osteosarcoma cells (HOS) treated with PAM-PBS and PAM-RPMI. Values are means ± SEM (n = 3). Statistical significance was determined by two-way ANOVA followed by Šídák’s post hoc multiple comparison test (ns—nonsignificant, **** p < 0.0001).
Figure 7. Viability of human osteosarcoma cells (HOS) treated with PAM-PBS and PAM-RPMI. Values are means ± SEM (n = 3). Statistical significance was determined by two-way ANOVA followed by Šídák’s post hoc multiple comparison test (ns—nonsignificant, **** p < 0.0001).
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Figure 8. Comparative analysis of the PAM-RPMI cytotoxicity influenced by the Ttr. ((A) PAM exposure time of 30 s; (B) PAM exposure time of 60 s; (C) PAM exposure time of 90 s) Values are means ± SEM (n = 4) and represent the viability of CAP-treated samples compared to control samples. Statistical significance was determined by two-way ANOVA followed by Tuckey post hoc multiple comparison test (ns—nonsignificant, * p < 0.05, *** p = 0.0003, **** p < 0.0001).
Figure 8. Comparative analysis of the PAM-RPMI cytotoxicity influenced by the Ttr. ((A) PAM exposure time of 30 s; (B) PAM exposure time of 60 s; (C) PAM exposure time of 90 s) Values are means ± SEM (n = 4) and represent the viability of CAP-treated samples compared to control samples. Statistical significance was determined by two-way ANOVA followed by Tuckey post hoc multiple comparison test (ns—nonsignificant, * p < 0.05, *** p = 0.0003, **** p < 0.0001).
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Figure 9. pH analysis of the PAM-RPMI solution after exposure. Values are means ± SEM (n = 3). Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc multiple comparison tests (**** p < 0.0001).
Figure 9. pH analysis of the PAM-RPMI solution after exposure. Values are means ± SEM (n = 3). Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc multiple comparison tests (**** p < 0.0001).
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Figure 10. Viability of HOS cells treated with PAM-RPMI and pH modified RPMI 1640 solution. Values are means ± SEM (n = 4). Statistical significance was determined by two-way ANOVA followed by Šídák’s post hoc multiple comparison test (ns—nonsignificant, **** p < 0.0001).
Figure 10. Viability of HOS cells treated with PAM-RPMI and pH modified RPMI 1640 solution. Values are means ± SEM (n = 4). Statistical significance was determined by two-way ANOVA followed by Šídák’s post hoc multiple comparison test (ns—nonsignificant, **** p < 0.0001).
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Figure 11. Viability of HOS cells and HOB treated with PAM-RPMI at 2-h post-treatment. Values are means ±SEM (n = 4). Statistical significance was determined by two-way ANOVA followed by Šídák’s post hoc multiple comparison test (ns—nonsignificant, **** p < 0.0001).
Figure 11. Viability of HOS cells and HOB treated with PAM-RPMI at 2-h post-treatment. Values are means ±SEM (n = 4). Statistical significance was determined by two-way ANOVA followed by Šídák’s post hoc multiple comparison test (ns—nonsignificant, **** p < 0.0001).
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Figure 12. Viability of HOS cells and HOB treated with PAM-RPMI at 24-h post-treatment. Values are means ± SEM (n = 4). Statistical significance was determined by two-way ANOVA followed by Šídák’s post hoc multiple comparison test (ns—nonsignificant, **** p < 0.0001).
Figure 12. Viability of HOS cells and HOB treated with PAM-RPMI at 24-h post-treatment. Values are means ± SEM (n = 4). Statistical significance was determined by two-way ANOVA followed by Šídák’s post hoc multiple comparison test (ns—nonsignificant, **** p < 0.0001).
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Figure 13. pH analysis of PAM-RPMI preserved for three months at different storage temperatures (4 °C, −20 °C and −80 °C).
Figure 13. pH analysis of PAM-RPMI preserved for three months at different storage temperatures (4 °C, −20 °C and −80 °C).
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Figure 14. The differences between the lowest and the highest pH values identified over 3 months of PAM-RPMI storage at different temperatures.
Figure 14. The differences between the lowest and the highest pH values identified over 3 months of PAM-RPMI storage at different temperatures.
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Figure 15. The analysis of NO 2 − (A) and NO 3 − (B) concentrations in PAM-RPMI. Values are means ± SEM (n = 3). Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc multiple comparison test (**** p < 0.0001).
Figure 15. The analysis of NO 2 − (A) and NO 3 − (B) concentrations in PAM-RPMI. Values are means ± SEM (n = 3). Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc multiple comparison test (**** p < 0.0001).
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Figure 16. Stability of NO 2 − from PAM-RPMI preserved for up to 3 months at −80 °C. Values are means ± SEM (n = 3). Statistical significance was determined by two-way ANOVA followed by Dunnett’s post hoc multiple comparison test.
Figure 16. Stability of NO 2 − from PAM-RPMI preserved for up to 3 months at −80 °C. Values are means ± SEM (n = 3). Statistical significance was determined by two-way ANOVA followed by Dunnett’s post hoc multiple comparison test.
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Stache, A.B.; Mihăilă, I.; Gerber, I.C.; Dragoș, L.M.; Mihai, C.T.; Ivanov, I.C.; Topală, I.; Gorgan, D.-L. Optimization of Indirect CAP Exposure as an Effective Osteosarcoma Cells Treatment with Cytotoxic Effects. Appl. Sci. 2023, 13, 7803. https://doi.org/10.3390/app13137803

AMA Style

Stache AB, Mihăilă I, Gerber IC, Dragoș LM, Mihai CT, Ivanov IC, Topală I, Gorgan D-L. Optimization of Indirect CAP Exposure as an Effective Osteosarcoma Cells Treatment with Cytotoxic Effects. Applied Sciences. 2023; 13(13):7803. https://doi.org/10.3390/app13137803

Chicago/Turabian Style

Stache, Alexandru Bogdan, Ilarion Mihăilă, Ioana Cristina Gerber, Loredana Mihaiela Dragoș, Cosmin Teodor Mihai, Iuliu Cristian Ivanov, Ionuț Topală, and Dragoș-Lucian Gorgan. 2023. "Optimization of Indirect CAP Exposure as an Effective Osteosarcoma Cells Treatment with Cytotoxic Effects" Applied Sciences 13, no. 13: 7803. https://doi.org/10.3390/app13137803

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