PONE-D-21-05079

Formulation of tunable size PLGA-PEG nanoparticles for drug delivery using microfluidic technology

PLOS ONE

Dear Dr. Albertazzi,

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Academic Editor

PLOS ONE

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: I Don't Know

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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: Yes

Reviewer #3: 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

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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: In this work, the authors described the preparation of PLGA-PEG nanoparticles by microfluidic technique. I appreciate the manuscript that focuses on the research close to a real application, not only for the very sophisticated things, however, cannot be repeated or large scale synthesis is rather doubtful. This is an example of the paper in which the guidelines of well-known PLGA-PEG NPs preparation is described, however, in the microfluidic channels. Nevertheless, I have found some drawbacks that still required corrections. Therefore, after a major correction and required corrections, the manuscript could be published in PLOS ONE.

The author should discuss and compare their founding’s with these two papers already existing in the literature:

Journal of Colloid and Interface Science 475 (2016) 136–148

International Journal of Pharmaceutics 548 (2018) 530–539

Applied Nanoscience 8 (2018) 905–914

Why authors choose the acetonitrile (ACN) for the preparation of NPs? I recommend comparing the mixing parameters of ACN with water and other solvents as it was shown in the paper: Colloids and Surf. B 2021, 201, 111598.

Moreover, in future work, it is good to consider the more volatile solvent which is easier to remove from NPs, for instance, THF.

DiI, this abbreviation is not described in the section about the encapsulation of fluorescent compounds.

You should describe why there is so low DOX encapsulation?

I would recommend reading these two papers: Colloids Surfaces B Biointerfaces. 141 (2016) 187–195; Sci. Rep. 7 (2017) 1–12. I believe you can find the answer there.

I won’t start the sentence with: Thanks to…

I am confused with the results of cellular uptake. Why authors didn’t show the uptake for NPs loaded with DOX? Maybe, the reason for their low efficiency is correlated with the fact that these NPs cannot reach the nucleus of the cell?

Therefore, I will recommend repeating the preparation of NPs loaded with DOX to obtain the higher EE and, subsequently, their cellular uptake should be also conducted.

The relevant reviews in the field should be cited:

Adv Drug Deliv Rev. 2018 Mar 15;128:101-114, doi: 10.1016/j.addr.2017.12.015.

RSC Adv., 2019, 9, 2055-2072, DOI: 10.1039/C8RA08972H

Polym. Int. 68 (2019) 997-1014, https://doi.org/10.1002/pi.5753

Lab Chip, 2017, 17, 209-226, DOI: 10.1039/C6LC01049K

Nanoscale, 2016,8, 12430-12443, https://doi.org/10.1039/C5NR07964K

Reviewer #2: The synthesis of PLGA nanoparticles using microfluidics was compared to a bulk method of nanoparticle production. This has been done before for PLGA nanoparticles and so the authors should note the point of difference with their study and what has been done previously. A reference that is missing from this article that must be included is;

Streck S et al. (2019). Comparison of bulk and microfluidics for the formulation of functionalized poly-lactic-co-glycolic acid PLGA nanoparticles modified with cell-penetrating peptides of different architectures. International Journal of Pharmaceutics X https://doi.org/10.1016/j.ijpx.2019.100030

The authors encapsulated three different dye molecules and progressed to evaluate the toxicity of their formulation nanoparticles on a human breast epithelial cell line. This data was not mentioned in the abstract or conclusion however, and should be included.

How many independent batches were measured to obtain values for size and PDI of the nanoformulations?

Why was zeta-potential not measured?

The first paragraph of the introduction should be reduced because this information is VERY well established in the literature.

Specific comments

Abstract

Line 4. Review the use of English here “…allows to obtain…”.

Fig. 1. S and AS should be defined in the figure caption.

Fig. 2. HFF should be defined in the figure caption.

Paragraph “Particle diameter tunability using microfluidic chip” second sentence (“Faster diffusion yields smaller NPs…”). Please provide a reference citation or a citation to data in the manuscript for this statement.

“It is especially important to control the particle size, as their performance and cellular uptake depends on this feature.” Please provide a reference citation for this statement.

“This is a common strategy to encapsulate drugs into PLGA formulations.” Please provide reference citations for this statement.

Materials and methods, page 1, line 7 and elsewhere. Please correct ‘miliQ water’ to ‘purified water’.

Reviewer #3: The authors described the comparison of the PLGA-based nanoparticles prepared using the typical manual bulk mixing and a microfluidic chip-assisted nanoprecipitation. Peglated PLGA nanoparticles prepared by both methods have been widely documented in the literature. The authors claimed the tuneability of the particle size by microfluidic method. Again this is already very well-documented in the literature, particularly for PLGA nanoparticles. Therefore, it is difficult to extract the novelty of this study. It maybe beneficial for the authors to re-consider the focus of the study. Specific comments are shown below:

The first section of the Results and discussion, namely “Microfluidic hydrodynamic flow focusing and manual bulk formulation”, does not seem to be based on any data. It is not clear what the purpose of this section.

In this section, the authors claimed that CFD simulation was used to generate the Figure 1c. However, there was no CFD described in the method section. No real data to confirm the CFD prediction/simulation.

In section named “Particle diameter tunability using microfluidic chip” the authors only used one S/AS ratio for the conventional bulk method, which achieved similar particle size as the microfluidic method, but no other S/AS was used in the bulk method. This limited the quality of the comparison considering the significant differences in throughput rate of the two method. Such limited comparison seem inadequate for drawing any conclusion.

The cellular studies were only performed on microfluidic particles, but not particles prepared by bulk method. The EE and single particle analysis also indicated that there is no significant difference in bulk and microfluidic method when the same A/SA ratios were used. These findings that are directly relevant to the comparison are not mentioned in the conclusion.

No statistical analysis was described in the method section.

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Reviewer #1: Yes: Marek Brzeziński

Reviewer #2: No

Reviewer #3: No

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Formulation of tunable size PLGA-PEG nanoparticles for drug delivery using microfluidic technology

PONE-D-21-05079R1

Dear Dr. Albertazzi,

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,

José das Neves

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #1: All comments have been addressed

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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 #1: Yes

**********

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

Reviewer #1: Yes

**********

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 #1: 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 #1: 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: The authors made all required corrections. Therefore, I recommend the publication of this manusctipt.

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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: Yes: Marek Brzeziński