PONE-D-23-22881

RE:Synthesis of cationic liposome nanoparticles using a thin film dispersed hydration and extrusion method

PLOS ONE

Dear Dr. Curtin,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Kind regards,

Pradeep Kumar, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: No

Reviewer #2: Yes

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2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: Partly

Reviewer #2: Partly

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3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the article presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript describes the very standard procedure of liposome formation using hydration method by using AVANTI extrusion kit. Generally, the paper describes standardly used procedures but there are already at least 2 protocols at the protocols.io already existing: https://www.protocols.io/view/liposome-encapsulation-of-hydrophilic-and-hydropho-rm7vzymorlx1/v1; https://www.protocols.io/view/two-step-protocol-preparation-and-extrusion-of-pho-n2bvj95xlk5w/v3

These two protocols describe the same procedures as this lab protocol and it is hard for me to see any improvements. On the other hand, in the end of this paper, work with 2D cell lines is showed. Liposomes are used in the cell line tests but on the protocol.io I did not find any lab protocol of this usage. Then the additional value of this protocol may be sufficient.

Specific comments:

26-27 An optimised method was developed ...

What is the optimised of your method? Can you add parameters you have optimised? You are using combination of 100 nm a nd 50 nm membrane, specific concentration of liposomes, etc.

37-38 Lipid nanoparticles, particularly liposomes, have been identified as potentially viable and flexible drug vesicles ...

This sounds to me like refering to a new potential material. Liposomes are 60 years old and nearly 30 years already on the market. I think this should be reflected in the introduction.

72-74 According to available evidence, a liposome diameter of less than 200 nm is the optimal size for effective drug administration, particularly when targeting the brain and crossing the blood brain barrier(13)

Here, you are citing your older version of this protocol?

Generally I have problem with the introduction that it states general knowledge about liposomes that is known for decades and in the same time citing very recent articles that are not primary sources of these informations. From your intraduction, liposomes seem to me like a super new potential material that yet needs to be studied.

99-100 dissolved in a fixed amount of chloroform and stirred for 15 minutes above the lipid’s transition temperature, Tc.

What amount of chloroform and at which temperature? In the protocol, the chloroform amount is stated, temperature not. Were these quantities optimised as it is stated in abstract?

119 ... and surface charge.

I would be very careful with connections of Zeta potential and surface charge. This may vary with ionic strength, size (as you even show)

126 - please check italic/normal characters in the equation

Nowhere in the methods I see the procedure for the doxorubicin encapsulation and purification. If there was purification used, the calculation of EE does not make sence as there was doxorubicin removed from the solution

190 - liposomi

193-194 Here and on many other places you overusing the digits in calculation. There is no point is showing that your measured value is 64.087 ± 33.98. Reasonable amount of digits need to be shown in the whole publication.

3.2 Extrusion study passes: The study you are here performing was surely done many times and event the manual from the manufacturer contains this information, for example here: https://www.ncnr.nist.gov/userlab/pdf/E134extruder.pdf

212-214 ... on the other hand, values less than ±30 can result in particle aggregation, flocculation, and precipitation due to the van der Waal forces of attraction which result in physical instability of the colloidal suspension(15).

Here, you are citing the paper in which this information is stated also with citation. It is very popular to give some border for the Zeta potential but from my point of view this is not correct. The colloidal stability is given by DLVO theory by the combination of electrostatic repulsion and van der Walls attraction. If this value exists it is material specific (in this case for liposomes).

218 - 219 ... confirming the effectiveness of the proposed extrusion method ...

Here it seems like a new method.

223 ... surface charge, 26.7 ± 15.6 mV

Please note, Zeta potential is not a surface charge

3.5 Encapsulation % If I understand correctly, you are using Ultracentrifugation and then without the knowlenge about volumes, you are substracting the concentrations. I think this is not correct approach.

The final part about cytotoxicity is not in the protocol. If something has added value considering the previously published protocols it is probably this part.

In the results of cytotoxicity test, the ones with longer time from synthesis is more toxic for the cells. This probably means that the doxorubicin leaked from the liposomes and killing the cells, but still you are stating that "Storage at 4 ºC appears to be the best, with stability maintained over 16 weeks." (line 312).

Figure 1 is 1:1 copy from Functionalized liposomes for targeted breast cancer drug delivery, Nel et al. Bioactive Materials 2023 and it is not adding any information. I would suggest either cite the source or to add some relevant info.

Reviewer #2: Dear Authors,

The review paper entitled “Synthesis of cationic liposome nanoparticles using a thin film dispersed hydration and extrusion method” by Cazzolla et al. is a nice manuscript describing the synthesis of cationic liposomes. Although this reviewer believes the manuscript contains important data, suitable to be published, unfortunately, in terms of writing and structure, the manuscript is not professional and challenges the reader in many points. Due to some of the major concerns of this reviewer that are listed below, the manuscript in its current form does not meet all of the requirements for publishing.

Some of the concerns listed below need to be answered by the authors to better justify the data and thus, make it clearer for the readers before the publication:

1. The language throughout the manuscript causes significant misunderstandings and more importantly misstatements. It needs more care than this journal requires.

2. There are several senences in the manuscript where the punctation marks are missing thus making it really hard to follow.

a. Line 190, the word liposomes was misspelled

b. Lines 233-234, what does the authors mean by intensity zeta size and number zeta size?

c. The sentence of lines 241-242 needs to be modified.

d. Lines 270-272, “Results in terms of size will be discussed considering the number and not the intensity, to show better the actual increase, if present, of the liposomes size.” needs some simplification.

e. Line 274, what does no encapsulated case mean and in line 277 the author described it as unencapsulated. Please maintain consistency across the manuscript.

f. Line 276, the text in the manuscript needs to be written in past tense.

3. In Figure 4B, the term Zeta Size in the graph needs to change to Size.

4. The text in the manuscript says Figures 5A and 5B, whereas in the actual figures, ther is no A and B in Figure 5.

5. The text says Figures 6A, 6B and 6C, whereas the actual figures are 7A, 7B and 7C.

6. There are several manuscipts describing the synthesis of cationic liposomes, what makes this one unique. The novelty needs to be discussed further in detail.

7. Use of cationic lipids comes with its own challenges and toxicities. The potential toxicities involved need to be mentioned in the introduction.

8. Furhtermore, the stability of the synsthesized liposomes are attributed to the highly positive zeta potential. Longer shelf-life does not make the liposome safe. A safety study such as a hemolysis study needs to be performed to evaluate the toxicities on cell membranes.

9. Authors also need to perform drug release study as a part of stability study as well.

10. The authors need to discuss more about the drop in zeta potential from time zero to 1 week old samples. The zeta potential at time zero was 47.90 mV whereas in 1 week old samples stored in cold were 7.90 mV and 18.30 mv for DOTAP and DOTAP-PI respectively.

11. The justification of such high standard errors in the zeta potential needs to be discussed as well.

12. A justification of why Doxorubicin was used to study the encapsulation % needs to be described in the manuscript and not Propidium Iodide.

13. Cytotoxicity need to be performed with freshly prepared liposomes as well.

14. The authors need to clearly discuss why the bigger size impacted the cytotoxicity.

15. Furthermore, the authors need to study the safety of these cationic liposomes in healthy cells in addition to the cancer cells.

Overall, the listed concerns do not decrease the scientific merit of the submitted manuscript but make it hard to follow. Thus, this reviewer believes the manuscript will be suitable to publish only after major changes.

Sincerely yours.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-23-22881R1RE:Synthesis of cationic liposome nanoparticles using a thin film dispersed hydration and extrusion methodPLOS ONE

Dear Dr. Curtin,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Please take note of the comments and concerns raised by Reviewer 1 in response to your revised manuscript.. 

==============================

Please submit your revised manuscript by Dec 23 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Pradeep Kumar, B.Pharm., M.Pharm., Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: No

Reviewer #2: Yes

**********

2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. If the manuscript contains new data, have the authors made this data fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the article presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Authors,

I appreciate the minor changes you have performed in order to improve the manuscript, such as better phrasing, clarification about Zeta potential, number of digits, reference in figure 1.

Still I have following major issues that were already stated in first response:

1) Introduction still looks like the liposomes are really new and potential material even after you added 2 lines to the text. Prime example 90-91: "These negative consequences have propelled researchers to create advanced, alternative drug delivery systems that use nanotechnology to transport DOXO (15)"

Here, it again is cited very recent article on DOXO-iron particles which is really something new in development. But the liposomes with DOXO are totally not new. There are dozens of already marketed DOXO - liposome products in market. https://go.drugbank.com/drugs/DB00997. First of them nearly 30 years FDA approved.

I would really suggest to perform deep literature review to have a more relevant introduction.

2) There are at least 2 protocols at protocol.io that are dealing with the same problem. Both of them are better than the one here reported.

https://www.protocols.io/view/two-step-protocol-preparation-and-extrusion-of-pho-n2bvj95xlk5w/v3

https://www.protocols.io/view/liposome-encapsulation-of-hydrophilic-and-hydropho-rm7vzymorlx1/v1

I would suggest to add the part with 2D cell culture and cell viability assay in order to form complete protocol which can help students and researchers to do basic tests in liposomes.

3) There is still not stated how the Doxorubicin was encapsulated. There are numerous methods for this, the most common ones use salt gradients: for example review here: 10.3390/pharmaceutics14020254.

A protocol for this method would be beneficial. Furthermore, the equation for EE calculation is adding concentrations as an extensive value. This is not correct. Using ultracentrifugation, the volumes of all fractions need to be known. There is a plenty of research done on the EE experimental evaluation: https://doi.org/10.1016/j.ijpharm.2017.11.035. Your reported EE around 85 % corresponds to the EE with using salt gradients but it is nowhere stated.

4) 96-98 There is now statement about the novelty of the work. I seriously doubt this statement as the liposomes were intensively tested through last 50 years (for example here: Kitamura et al., 1996, https://aacrjournals.org/cancerres/article/56/17/3986/502500/Intrathecal-Chemotherapy-with-1-3-Bis-2) but if this publication should serve as a protocol it is not necessary to have original research.

Reviewer #2: Dear Authors,

The revised paper entitled “Synthesis of cationic liposome nanoparticles using a thin film dispersed hydration and extrusion method” by Cazzolla et al. is a nice manuscript describing the synthesis of cationic liposomes. The reviewer believes the comments and concers were answered appropriately.

Overall, this reviewer believes the manuscript is suitable to publish the revised manuscript.

Sincerely yours.

**********

7. 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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

RE:Synthesis of cationic liposome nanoparticles using a thin film dispersed hydration and extrusion method

PONE-D-23-22881R2

Dear Dr. Curtin,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Pradeep Kumar, Ph.D.

Academic Editor

PLOS ONE