Dear Dr Kazemirad,

Thank you very much for submitting your manuscript "Chitosan nanoparticles improve the effectivity of miltefosine against Acanthamoeba" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Reviewer 2:

I've completely gone through the manuscript titled "Chitosan nanoparticles improve the effectivity of miltefosine against Acanthamoeba" ". The authors report the effects of miltefosine loaded chitosan nanoparticles against the two forms of Acanthamoaba namely, cultured trophozoites and cyst form. The cytotoxicity and hemolytic effects of the MF-CS-NPs were also determined on Vero cell line and human RBCs, respectively. This is a well-conceived and well-carried-out study that have yielded valuable data. However, before this paper can be published, a number of flaws must properly be taken care of. The authors should have a native English speaker revise the manuscript before resubmitting it in order to correct the annoying spelling, grammatical, syntactic, and stylistic errors.

General and specific comments for the authors

Introduction

• The introduction is not for the authors perform a review about the subject but to clearly show why the study is relevant and how the data obtained will contribute to the management of AK. Most of the references cited in this part are outdated.

• The authors are advised to pay special emphasis on recent challenges in treatment options of AK.

Methods

• How the authors confirm the synthesis of NPs

• How the authors standardized the dosing in the experiments

• The authors are advised to perform TEM microscopy of the MF-CS-NPs

• No information is given about how long the isolate of Acanthamoaba used was obtained.

• The authors should clearly mention the positive and negative control in their experiments

• How the authors measured the encapsulation efficacy and drug loading content of the MF-CS-NPs

• Penicillin and streptomycin from which source? company, country and city name

• It would be interesting if the authors could perform the experiments in three incubation periods i-e 24h, 48,72h respectively.

• In In vitro assay the authors used various concentrations of 9.8, 19.6, 39.2, 78.125, 156.25, 312.5, and 199 625 µg/ml against trophozoites while 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml for cyst. What was the reason for choosing these concentrations?

Results

The results are well presented. The readers can easily be able to understand what the figures and tables represents.

Fig 8 must be removed as it not giving any scientific information

Discussion

The discussion must be written more straightforward. Rather than repeating background information in the first alinea that has already been given in the Introduction. The authors should have briefly mentioned the rationale for carrying out the study and give a short version of their findings, and then continue in the subsequent alineas to discuss each finding.

Conclusion

Conclusion must be informative. The authors should mentioned their results in this section.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Aiman Abu Ammar

Guest Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Section Editor

PLOS Neglected Tropical Diseases

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Reviewer 2:

I've completely gone through the manuscript titled "Chitosan nanoparticles improve the effectivity of miltefosine against Acanthamoeba" ". The authors report the effects of miltefosine loaded chitosan nanoparticles against the two forms of Acanthamoaba namely, cultured trophozoites and cyst form. The cytotoxicity and hemolytic effects of the MF-CS-NPs were also determined on Vero cell line and human RBCs, respectively. This is a well-conceived and well-carried-out study that have yielded valuable data. However, before this paper can be published, a number of flaws must properly be taken care of. The authors should have a native English speaker revise the manuscript before resubmitting it in order to correct the annoying spelling, grammatical, syntactic, and stylistic errors.

General and specific comments for the authors

Introduction

• The introduction is not for the authors perform a review about the subject but to clearly show why the study is relevant and how the data obtained will contribute to the management of AK. Most of the references cited in this part are outdated.

• The authors are advised to pay special emphasis on recent challenges in treatment options of AK.

Methods

• How the authors confirm the synthesis of NPs

• How the authors standardized the dosing in the experiments

• The authors are advised to perform TEM microscopy of the MF-CS-NPs

• No information is given about how long the isolate of Acanthamoaba used was obtained.

• The authors should clearly mention the positive and negative control in their experiments

• How the authors measured the encapsulation efficacy and drug loading content of the MF-CS-NPs

• Penicillin and streptomycin from which source? company, country and city name

• It would be interesting if the authors could perform the experiments in three incubation periods i-e 24h, 48,72h respectively.

• In In vitro assay the authors used various concentrations of 9.8, 19.6, 39.2, 78.125, 156.25, 312.5, and 199 625 µg/ml against trophozoites while 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml for cyst. What was the reason for choosing these concentrations?

Results

The results are well presented. The readers can easily be able to understand what the figures and tables represents.

Fig 8 must be removed as it not giving any scientific information

Discussion

The discussion must be written more straightforward. Rather than repeating background information in the first alinea that has already been given in the Introduction. The authors should have briefly mentioned the rationale for carrying out the study and give a short version of their findings, and then continue in the subsequent alineas to discuss each finding.

Conclusion

Conclusion must be informative. The authors should mentioned their results in this section.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Yes, the objectives of the study is clearly articulated with a clear testable hypothesis stated and the study design is appropriate to address the stated objectives.

Reviewer #2: (No Response)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Yes, the results are clearly and completely presented and the figures (Tables, Images) are of sufficient quality for clarity

Reviewer #2: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Yes, the conclusions are supported by the data presented

Reviewer #2: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Nagwa Mostafa El-Sayed

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

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Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr Kazemirad,

We are pleased to inform you that your manuscript 'Chitosan nanoparticles improve the effectivity of miltefosine against Acanthamoeba' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Aiman Abu Ammar

Guest Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Section Editor

PLOS Neglected Tropical Diseases

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