PONE-D-20-29628

How alginate properties influence in situ internal gelation in Crosslinked Alginate Microcapsules (CLAMs) formed by spray drying

PLOS ONE

Dear Dr. Jeoh,

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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C. Anandharamakrishnan

Academic Editor

PLOS ONE

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SAS was supported the Agriculture and Food Research Initiative grant 2018-67012-28029 from the USDA National Institute of Food and Agriculture

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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3. Have the authors made all data underlying the findings in their manuscript 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: No

Reviewer #3: Yes

Reviewer #4: No

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4. Is the manuscript 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 note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. 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 Editor,

Thank you for giving me opportunity for review this manuscript.

This manuscript focused on crosslinking of alginates. It is well written and results are explained well and supported by suitable literature. The research question is clearly mentioned. I found few points need to be add which improve the article. For instance

Line 91: What are others methods of crosslinking other than spray drying, kindly mention that

Line 150: Kindly mention reference for this method if authors have developed the method then kindly mention that

Line 187: What is basis for selection of inlet temperature? Does inlet temperature affect the alginate crosslinking?

Line 188: What is outlet temperature? Does outlet temperature affect the alginate crosslinking?

Line 197: Kindly give reference for the method

Line 225: What is yield during spray if any losses are there kindly mention in manuscript

Line 236: Authors have taken calcium phosphate for this study, why calcium phosphate has been considered? Does salt type influence the crosslinking?

Line 249: There is any method to get 100 % crosslinking

Line 256: What are other properties of alginate which may influence the crosslinking

Line 367: What is moisture content of CLAMs does moisture content of CLAMs affect the crosslinking

Line 378: kindly mention the limitations of this study if any

Reviewer #2: The authors studied about “How alginate properties influence in situ internal gelation in Crosslinked Alginate Microcapsules (CLAMs) formed by spray drying”. This study is performed alternative to external gelation to prove the advantage of this study. This represented study has to clarify the following points before acceptance in this journal:

1. The microcapsule preparation method used calcium as crosslinker, the authors should aware the used calcium concentration should not exceed physiological intake limit. Excessive calcium consumption will cause severe health complication specially cardiac complications. Hence, explain in the manuscript how much quantity of calcium used and that quantity are in acceptable limit or not.

2. Ammonium hydroxide is used as a base in this study to maintain higher pH. It is boiling point almost near to room temperature, so it can evaporate in room atmosphere before spray or it can completely evaporate in nozzle head. In this concern how ammonium hydroxide based pH theory works in microcapsule preparation. Please explain.

3. In page no:11, line no: 226 mentioned that crosslinked alginate microencapsulation. What type of molecule is encapsulated into the microcapsule?, like that in graphical abstract mentioned dry cargo-loaded CLAM. What type of cargo is loaded into the CLAM?

4. Extend of crosslinking data of external crosslinked microparticles is not mentioned in the manuscript and not compared with CLAM. Please include that in the manuscript.

Reviewer #3: The paper entitled “Alginate properties impacting gelation in spray-dried crosslinked alginate microcapsules” is related to influence of alginate molecular properties and their effect on extend of cross linking in in-situ gelation in CLAMs produced by spray drying technique.

The approach of the manuscript is interesting and could provide information. The paper is well written and methodology is understandable.

I would like to suggest the Editor to accept the manuscript with minor corrections.

1. Materials and methods require details in explaining the statistical analysis.

2. In table 2, authors explain significant difference only for G/M ratio. But they didn’t say anything about significant difference for other parameters in the table. Are there? Also please use same number of decimals for all values.

3. Figure 5 caption explains the same as in discussion part. Please change it.

Reviewer #4: Review Comments

Manuscript Title: How alginate properties influence in situ internal gelation in Crosslinked Alginate Microcapsules (CLAMs) formed by spray drying

The following aspects may be considered to improve the work.

1. Improve quality of all figures. Some values are just not readable!

2. Elaborate the underlying chemical changes as equations.

3. Improve discussion with recent (2019-20) articles on spray drying + crosslinking (perhaps even other molecules).

4. Add details on the purity and composition of all raw materials. How would they impact the findings of this study?

5. Add details on the drying mechanism in the spray dryer (relevant to the microcapsule formation).

6. What about the post-encapsulation quality?

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6. 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: Yes: PINTU CHOUDHARY

Reviewer #2: No

Reviewer #3: No

Reviewer #4: 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.]

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Attachments

Attachment

Submitted filename: PONE-D-29628.docx

Attachment

Submitted filename: PLOS ONE-review.docx

PONE-D-20-29628R1

How alginate properties influence in situ internal gelation in Crosslinked Alginate Microcapsules (CLAMs) formed by spray drying

PLOS ONE

Dear Dr. Jeoh,

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 submit your revised manuscript by Feb 27 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

C. Anandharamakrishnan

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #5: All comments have been addressed

Reviewer #6: All comments have been addressed

Reviewer #7: All comments have been addressed

Reviewer #8: All comments have been addressed

Reviewer #9: All comments have been addressed

Reviewer #10: (No Response)

Reviewer #11: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

Reviewer #9: Yes

Reviewer #10: Partly

Reviewer #11: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

Reviewer #9: Yes

Reviewer #10: N/A

Reviewer #11: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript 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 #2: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

Reviewer #9: Yes

Reviewer #10: Yes

Reviewer #11: Yes

**********

5. Is the manuscript 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 note any specific errors here.

Reviewer #2: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

Reviewer #9: Yes

Reviewer #10: Yes

Reviewer #11: 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 #2: (No Response)

Reviewer #5: (No Response)

Reviewer #6: The authors did address all the comments by the reviewers sufficiently. I agree with the author that the comment by reviewer 3 regarding the caption of Figure 5 is not applicable since the Figure is very complex and an extensive caption to explain is necessary.

Reviewer #7: This manuscript reported that “How alginate properties influence in situ internal gelation in crosslinked alginate microcapsules(CLAMs) formed by spray drying”. The authors compared the effects of alginic acid molecular weight, size distribution and molecular composition on CLAMs gelation. Overall, the manuscript was well written, experiments were well designed, the data interpretation is sound and the conclusion drawn by authors is firmly supported by the data provided. Therefore, it would be suitable for publication in the journal "PLOS ONE" without further revision.

Reviewer #8: This work studies the impact of alginate properties in crosslinked alginate microcapsules formed by CLAMs process.

The authors described various factors that affect the extent of crosslinking, with a particular focus on the influence of commercially available alginates. Due to lack of information of commercial alginates, the authors performed a characterisation of these to study the impact of their properties on the extent of crosslinking in CLAMs.

Interestingly, the impact of alginate properties is dependent on the method utilised for crosslinking, where spray-dried CLAMs are mainly influenced by the molecular weight or density of alginates at saturating salt concentrations. The paper is well written and the materials and methods are clear and provide sufficient information.

This manuscript has been already reviewed and most of my doubts have been solved during this round of revision. I have only one question regarding the G/M ratio. The authors described that by external gelation, 5% and 93% of crosslinking is achieved with PolyMs and PolyGs, respectively. On the other hand, 50% crosslinking was observed with PolyMs and PolyGs by CLAMs process, which contradicts the expectations of the authors. I believe these results need to be further discussed by the authors. Is this equal crosslinking of polyMs and polyGs influenced by alginic acid? are there any other parameters that may influence the outcomes observed?.

Reviewer #9: The authors have adequately responded to all reviewer comments. The paper is well-written, and will be an important contribution to this area of pharmaceutical development, particle engineering and spray drying.

Reviewer #10: The authors present preparation of crosslinked alginate microcapsules (CLAMs) with alginates of different properties using spray drying technique. The study seems to have been designed well and the experiments are done rigorously. The authors seem to have addressed most of earlier concerns raised be reviewers. However, there are few concerns that need to be fixed before it is finalized.

Specific comments:

In the process of preparation of the CLAMs the feed suspension contains calcium hydrogen phosphate, succinic acid, and ammonium hydroxide along with Alginates. When the crosslinking takes place by calcium ions due to pH change, the crosslinking possibilities are; it can crosslink the alginates directly (alginate-Ca-alginate), or it can crosslink alginate through a succinate-bridge (alginate-Ca-succinate-Ca-alginate), or it can conjugate an alginate to one succinate carboxyl group and the other remains as carboxylic acid, or it can be a mixture of these. The COO- groups in succinates can exist in ionic form of COO- NH4+ (in presence of NH3 and H2O or NH4OH), and COO- H3O+ (on evaporation of NH3), and in equilibrium with COOH.

The mono carboxylic acid of the succinate can also be detectable at ~1710 cm-1 in the FTIR along with alginic acid if present. To confirm if the acid carbonyl observed is due to alginic or succinic or both, the process should be tested with and an alkyl acid containing only one carboxylic acid instead of a dicarboxylic acid in the process. Otherwise, the formation of alginic acid should be confirmed with another method of analysis to eliminate other possibilities.

Reviewer #11: The authors present preparation of crosslinked alginate microcapsules (CLAMs) with alginates of different properties using spray drying technique. The study seems to have been designed well and the experiments are done rigorously. The authors seem to have addressed most of earlier concerns raised be reviewers. However, there are few concerns that need to be fixed before it is finalized.

Specific comments:

In the process of preparation of the CLAMs the feed suspension contains calcium hydrogen phosphate, succinic acid, and ammonium hydroxide along with Alginates. When the crosslinking takes place by calcium ions due to pH change, the crosslinking possibilities are; it can crosslink the alginates directly (alginate-Ca-alginate), or it can crosslink alginate through a succinate-bridge (alginate-Ca-succinate-Ca-alginate), or it can conjugate an alginate to one succinate carboxyl group and the other remains as carboxylic acid, or it can be a mixture of these. The COO- groups in succinates can exist in ionic form of COO- NH4+ (in presence of NH3 and H2O or NH4OH), and COO- H3O+ (on evaporation of NH3), and in equilibrium with COOH.

The mono carboxylic acid of the succinate can also be detectable at ~1710 cm-1 in the FTIR along with alginic acid if present. To confirm if the acid carbonyl observed is due to alginic or succinic or both, the process should be tested with and an alkyl acid containing only one carboxylic acid instead of a dicarboxylic acid in the process. Otherwise, the formation of alginic acid should be confirmed with another method of analysis to eliminate other possibilities.

**********

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 #2: No

Reviewer #5: Yes: elham khodaverdi

Reviewer #6: Yes: Janke Kleynhans

Reviewer #7: No

Reviewer #8: Yes: Paulina Ramírez-García

Reviewer #9: No

Reviewer #10: No

Reviewer #11: 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.

How alginate properties influence in situ internal gelation in Crosslinked Alginate Microcapsules (CLAMs) formed by spray drying

PONE-D-20-29628R2

Dear Dr. Jeoh,

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.

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Kind regards,

C. Anandharamakrishnan

Academic Editor

PLOS ONE