Investigation of Sonosensitizers Based on Phenothiazinium Photosensitizers
, Chia-Feng Hsieh 1, Hsing-Ju Wu 2,3,†, Mohamed Ameen 1 and Tun-Pin Hung 4,*
Round 1
Reviewer 1 Report
This manuscript has already been reviewed by me. Some of my comments and comments of other reviewers were taken into account by the authors. So my opinion remains the same. There were no responses to some comments and no changes in the text. Therefore, I suggest that the authors respond to the comments of the previous review and highlight the changes in the paper.
Due to the fact that the authors synthesized the photosensitizer not for the first time, I propose to replace the word Development in the title with the word Investigation.
Line 21. In the abstract, it is necessary to replace abbreviated names with full ones. (MB6C.)
Line 27. The sentence rephrased. But it's still not true. The main cause of death according to WHO statistics is heart disease, cancer is only in 6th place.
Line 32. Introduction section could be extended. More information needs to be added on the PDT and PDI. The following papers could be cited.
Lebedeva, N. S., Gubarev, Y. A., Koifman, M. O., & Koifman, O. I. (2020). The application of porphyrins and their analogues for inactivation of viruses. Molecules, 25(19), 4368.
Tsolekile, N., Nelana, S., & Oluwafemi, O. S. (2019). Porphyrin as diagnostic and therapeutic agent. Molecules, 24(14), 2669.
Malatesti, N., Munitic, I., & Jurak, I. (2017). Porphyrin-based cationic amphiphilic photosensitisers as potential anticancer, antimicrobial and immunosuppressive agents. Biophysical reviews, 9(2), 149-168.
Line 66. Abbreviations could be deciphered at the first mention (MB6C.)
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript is suitable for publication in Applied Sciences as it is technically good work. However, the authors need to:
1. Correct the entire manuscript using an English language expert.
2. Expand results and discussions as well as the conclusions part.
3. Add the spectras of the compound as a supplementary file to look into the spectral scans.
4. Why MB6C is a potential compound? What structural features of this compound is responsible for its activity. Need to add statements in abstract, results and discussions and conclusions.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The paper is devoted to the study of the activity of methylene blue derivatives in PDT and SDT. The paper is well designed and well done. I recommend publish paper with minor revisions.
Line 22. In the abstract, it is necessary to replace abbreviated names with full ones.
Line 28. The sentence could be rephrased.
Line 32. Introduction section could be extended. More information needs to be added on the PDT and PDI. The following papers could be cited.
Lebedeva, N. S., Gubarev, Y. A., Koifman, M. O., & Koifman, O. I. (2020). The application of porphyrins and their analogues for inactivation of viruses. Molecules, 25(19), 4368.
Tsolekile, N., Nelana, S., & Oluwafemi, O. S. (2019). Porphyrin as diagnostic and therapeutic agent. Molecules, 24(14), 2669.
Malatesti, N., Munitic, I., & Jurak, I. (2017). Porphyrin-based cationic amphiphilic photosensitisers as potential anticancer, antimicrobial and immunosuppressive agents. Biophysical reviews, 9(2), 149-168.
Line 66. Abbreviations could be deciphered at the first mention.
Line 240. “Moieties” is superfluous word
I recommend citing this references with synthesis of 3,7-bis(dihexylamino)-phenothiazin-5-ium iodide and 3,7-bis(di(2-hydroxyethyl)amino)-phenothiazin-5-ium iodide.
Brown, S. B., O'grady, C. C., Griffiths, J., Mellish, K. J., & Vernon, D. I. (2008). U.S. Patent No. 7,371,744. Washington, DC: U.S. Patent and Trademark Office.
De Crozals, G., Farre, C., Sigaud, M., Fortgang, P., Sanglar, C., & Chaix, C. (2015). Methylene blue phosphoramidite for DNA labelling. Chemical Communications, 51(21), 4458-4461.
Reviewer 2 Report
Title: “Development of a novel sonosensitizer for sonodynamic therapy.”
Authors: Cheng-Chung Chang, Chia-Feng Hsieh, Hsing-Ju Wu, Tun-Pin Hung
Affiliation: National Chung Hsing University, Taiwan World Wide Ranking #986 in Clinical Medicine
English is a second language.
Minor concerns: There are many typos and syntax errors which make this article difficult to understand (see line by line review).
Major concerns: The paper is not that novel. The authors developed derivatives of methylene blue (MB) with ranging lipophilicity and compare dark toxicity to light and sonication toxicity and yield of singlet O2. The lipophilic drug has expected better uptake and is more toxic both in the dark and when excited to produce singlet oxygen. There is limited discussion and conclusion to the paper with most of the paper being technical and not clearly presented. I would point out to the investigators that lipophilic photosensitizers localize to biomembranes where singlet oxygen and other ROS have a longer half life and interact with lipids causing lipid peroxidation and therefore enhance toxicity when excited. There is no discussion of how their derivatives compare to others.
Line by line review:
Line 28; “Cancer, the leading cause of death worldwide, has caused the number of cancer patients to increase rapidly.” Syntax suggest ‘Cancer, the leading cause of death worldwide, has seen a rapid increase in the number of patients diagnosed.’ Is there a reference for the incidence worldwide?
Line 21; “Many efforts for cancer treatments involve invasive procedures, including chemotherapy, radiation, and surgery to remove the tumor.” Comment Is chemotherapy and radiotherapy considered invasive? I guess you may be able to make a case for selective arterial delivery of chemotherapy by interventional radiology or brachyseed or needle implant. However, generally would not consider chemo- and radiotherapy invasive.
Line 55; “…and 5-aminolevulinic acid,[27,28] have also been reported as effective sonosensitizers…” Please note that 5-ALA is a prodrug and is not directly a sonosensitizer.
Line 98; “…was added to the 1-containing solution…” Seems to be a typo error. 1- what? Iodide?
Line 121; “…cancer cells were cultured in MEM and cultured in a 37 °C…” What percentage of FBS was used? Were the cells starved of FBS and if so why?
Line 149; “The determinations were repeated three times.2.6. Design of PDT and SDT Apparatus Setting” Appears to be typo error with ‘2.6. Design of PDT and SDT Apparatus Setting’ to be a new heading.
Line 198; “Moreover, we observe that MB and MBOH do not dissolve in EA and 198 toluene as well as MB6C.” Syntax is very awkward. What are you trying to say? What is the EA abbreviation Etoh? Should spell out abbreviations in first instance.
Line 207; “…3,6-dusubstituted…” Typo? I believe you mean ‘…3,6-disubstituted…’
Line 211; “…by the 3.6 substituent…” Typo/Syntax? Are you trying to say 3,6 substituents?
Line 260; “Cellular Toxicity” In this section are you describing the MTT assay for survival or appearance of the cells? It is not clear from the figure legends of Figure 6 or 7. In materials and methods you describe MTT assay. Here you appear to describe cell morphology only?
Reviewer 3 Report
The manuscript Development of a novel sonosensitizer for sonodynamic therapy submitted by Cheng-Chung Chang and Hsing-Ju Wu et al. describes the synthesis and biological evaluation of two phenothiazine derivatives. The phenothiazine derivatives were assessed toward their cytotoxic action in PDT and SDT against lung cancer cells (A549). The research aiming to develop new cancer treatments methods is important. The paper is rather well written and fits the scope of Applied Sciences but not the Biomedical Engineering section.
However, I have some major concerns about this study:
1. The novelty of the study is arguable. All the synthesized compounds are known in the literature and some of them patented – MBOH (https://doi.org/10.1039/C4CC10164B); MB6C (https://doi.org/10.1562/0031-8655(2002)075<0392:IVPAOA>2.0.CO;2 and patent WO2005054217A1); phenothiazinium tetraiodide hydrate (https://doi.org/10.1021/acschemneuro.8b00042; https://doi.org/10.1002/ejoc.200900118; https://doi.org/10.1016/j.jphotobiol.2014.12.017; https://doi.org/10.1039/C5TB02569A; patent US2015011712A1). The evaluation only shows that these compounds are capable of generating ROS and MB6C is active in both PDT and SDT.
2. The characterization of the prepared compounds is insufficient. The 13C NMR data is missing. The MS data should be given to the fourth decimal and the values obtained should not differ by more than 10 ppm from the calculated ones. UV-Vis data is missing. For biological activity experiments the compounds should be confirmed to be at least 95% pure. In the 1H NMR the triplets should also have the coupling constants given. There seems to be some inconsistency in MB6C 1H NMR as the values you present for Hc suggest the presence of 4 protons, not 2; the signal at 0.8-1.0 should be a triplet and correspond to CH3 but is denoted as 20 protons. Please state the temperature the compound was dried at (line 92). The NMR spectra (1-10 ppm for 1H and 0-200 ppm for 13C NMR) should be presented in the supplementary data.
3. How did you confirm that the prepared tetraiodide is a hydrate? No experimental data indicates it.
4. The biological evaluation should also include experiments on healthy cells to indicate the safety of using the new compounds. Also, the experimental section should contain all information necessary for repeating of the experiment (i.e. use of antibiotics, what solutions were used – DMSO or water, what was the DMSO concentration in the culture, etc).
5. The used method for singlet oxygen generation mediation could be quantitative if the used light was monochromatic. Currently it shows only that 1O2 is generated. This is in contrast to the statement in lines 268-269 “singlet oxygen quantum yield of this compound is not as high as that of MB”. Did you consider other ROS to play a role in the biological activity of the tested compounds?
6. In the logP calculation, did you take into consideration the differences in logε in different solvents? If not, the data should be recalculated.
7. “All these MB analogs are localized in the cytoplasm”(324) Did you prove this anyhow? There is no data confirming this statement. Based on the lipophilic character of MB6C it seems that it would be rather present in the biological membranes rather than in cytoplasm.
8. The obtained results should be discussed in terms of SDT of other phenothiazine derivatives.
Other, less significant remarks:
9. It strikes me that the first and third author declare equal contribution. Shouldn’t than the third author be listed as the second one?
10. What was the effect of light alone in the photocytotoxicity study?
11. It would be better to show logε than absorbance in the UV-Vis study.
12. “ Cancer (…) has caused the number of cancer patients to increase rapidly”(lines 28-29). Please rewrite.
13. Stock solutions (line 75) – in what solvent?
14. When the chemical name is complex, two types of brackets () and [] should be used (line 96).
15. The little photographs in Figure 3abc give little information to the reader, all solvents should be stated. Also, please pay attention to the subscript in the solvents listed in the emission part of the Figure 3abc.
16. Please provide citation to the fragment in lines 193-196.
17. Most significant English/style/typos: “values of”(line 23); multiplication signs should be ×, not x (82, 126); deuterated DMSO should be denoted as DMSO-d6 (lines 83, 92, 101, 113); N in substituted amines chemical names should be italicized (88, 97); “methanol was removed under vacuum and purified”(lines 99-100); Me4Si (114); “2.6. design…” (149-150); CL (214); “3.6 substituent” (211); “cells are already slightly dark toxic”(271); cm2 (286).
