Comparison of the Biological Properties of Plasma-Treated Solution and Solution of Chemical Reagents

Round 1

Reviewer 1 Report

The manuscript " Comparison of the Biological Properties of Plasma-Treated Solution and Solution of Chemical Reagents" by Pavlik et al deals with a comparison between the plasma-activated solution and chemically prepared similar solution for cancer therapy purposes.

In this work, the authors prepared solutions with a mixture of nitrites, nitrates, and hydrogen peroxide chemically and compared the effect on the healthy and myeloblastosis cells with that of the plasma-treated solution. Such work could elucidate the inherent phenomenon in the plasma-activated solutions for cancer therapy. However, there is the main drawback to this work as follows:

• The authors restricted the long-lived reactive species to nitrite, nitrate, and hydrogen peroxide in the plasma-treated solution. The characteristic time scale for their experiment to treat solution and the cells are about 5 minutes. During this time scale, many important chemical reactive agents play a crucial role in cell treatment. For example, in the time scale of their experiments,  ozone, hydroxyl, peroxynitrite, and singlet oxygen are reactive species that have a relatively long lived-time and make strong reactions with the cells. Therefore, their prepared solution could not show the exact plasma-activated solution, and the corresponding effects on the cells are obviously different. The authors are requested to discuss this issue and how we can exclude long-lived reactive species such as ozone, hydroxyl, peroxynitrite, hypochlorous acid in the time scale of the experiment. Do the authors suggest a way to prepare a similar chemical solution to the plasma-activated one by taking into account the aforementioned reactive species?
• What was the pH of both solutions( plasma treated and chemically prepared). Did the authors control the same pH for their experiments? 

 

Other comments

 

• The frequency of the piezodischarge was written as 21 Hz in section 2, line 122. Please verify the correctness of the deriving frequency and give the waveform in this line.
• Please indicate the voltage as RMS or peak-to-peak value in line 123, section 2.
• Please mention the discharge power of the cold plasma in section 2.
• The authors mentioned the measurement methods for reactive species in section 2.3 for the plasma-activated solution. Did they measure the prepared chemical solution by this method too? Is there any error with their measurement method when they compare with that of the prepared solutions?
• It is suggested to cover a complete and up-to-date reference about the subject in the introduction. As I see, the work with DOI: https://doi.org/10.3390/app12010128 deals with "the evaluation of the cytotoxic effect of cold plasma or plasma-treated solutions (PTS) on hematologic parameters in the whole blood of CLL patients" could be referenced in the introduction.
• In the abstract, the term "nitrides" should be replaced by "Nitrates".
• In line 70, please correct the "perxinitrite" spelling as "peroxynitrite".

Author Response

Dear reviewer!

Thank you for bringing your attention to some shortcomings in our article.

Mass spectrograph measurements showed that the concentrations of O₃, HOCl and short-lived RONS such as OH, O₂^-, ONOO^- and other, in the tested solution are insignificant and are less than 5 μM for O₃ and HOCl, and less than 1 μМ for the short-lived RONS. These data were also confirmed using absorption spectra - the main components generated by cold plasma are nitrites, nitrates and hydrogen peroxide. Therefore, we created a chemical solution identical to one generated by plasma using the same components). The difference in biological effects can be explained by the higher chemical activity and nonequilibrium in a solution treated with cold plasma compared to a chemically prepared solution.

The pH of the chemically prepared solution was about 4.5, which was the same as the pH of the solution treated with cold plasma. We have added a pH chart to the body of the article.

We apologize for some typos that occurred during the translation. Some of them have already been corrected by the editors. We indicated the discharge power in section 2 and gave a reference where you can find the wave graph.

The prepared chemical solution was made based on the obtained measurements of RONS concentrations in the plasma-treated solution. Verification of the correctness of the preparation of solutions was carried out by the methods already indicated. Thus, we achieved the same concentrations.

We've corrected the introduction having in mind all the remarks made.

Author Response File: Author Response.docx

Reviewer 2 Report

The technical part of this paper is outside my field of expertise.

Minor points: The introduction is rather lengthy and not up to the point. Some shortening might help.

Check/correct Ref. 6

Lines 82-84: nitrites”m”

Fig. 1: Figure should be enlarged, instead of 1 x 4 it should be 2 x 2. Quality (resolution) is too poor.

Line 168-170: it is surely nice to have a software but what is the instrument? Can you show a “comet tail” ?

Line 172: “confines” ?

Figures 7-9: Effect is not really that large. Asterisk: what kind of statistical significance and how does it correlate with the displayed error bars? What are the “white bars”, or whatever is shown there?

Author Response

Dear reviewer!

Thank you for bringing your attention to some shortcomings in our article.

We apologize for some typos that occurred during the translation. They have already been corrected.

We've corrected the introduction focusing on leukemia and RONS. We have also made some changes to English terms and expressions.

We have modified the cell photo drawing and added a photo to illustrate the DNA damage analysis. But, unfortunately, we don’t have the ability to improve the quality of the photos. In the above photographs, one can clearly see the differences at different stages of death.

We have added a more detailed explanation of the * sign and "white bars" to the figures (this is a comparison of two specific points). An asterisk * indicates a statistically significant difference (p < 0.05) from the control, an asterisk above the frame indicates a statistically significant difference (p < 0.05) between different experimental samples.

We tried to take into account all your comments when correcting the article.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors revised and corrected their manuscript based on the comments. Now, it is suitable for publication in the Applied Sciences journal.