Therapeutic Effects of 30 nm Cyclosporin A-Loaded Nanoparticles Using PLGA-PEG-PLGA Triblock Copolymer for Transdermal Delivery in Mouse Models of Psoriasis

Round 1

Reviewer 1 Report

A fascinating article evaluating the effectiveness of poly(DL-lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(DL-lactide-co-glycolide) triblock copolymers (PLGA-PEG-PLGA) as a carrier in the treatment of psoriasis. I found it very interesting also as a clinical dermatologist; I have some queries:

In the conclusion, you state: "is also known that applying  CsA to mouse skin has a hair growth effect [38,39]. Further investigation is required to  determine whether this is due to therapeutic effects or side effects.  ".

Cyclosporine is used in various hair conditions as a treatment in clinical dermatology, so it may be considered a therapeutic effect.

In the introduction, I would expand the paragraph to also discuss psoriasis treatments. You should mention currently available therapies; here are some articles you could add: (doi: 10.1111/dth.14504. doi: 10.1111/dth.13185.doi: 10.1080/09546634.2023.2200868.)

Good Luck!

English was fine. The article was understandable

Author Response

Comment 1:
In the conclusion, you state: "is also known that applying CsA to mouse skin has a hair growth effect [38,39]. Further investigation is required to determine whether this is due to therapeutic effects or side effects.".

Cyclosporine is used in various hair conditions as a treatment in clinical dermatology, so it may be considered a therapeutic effect.

Reply:

Thank you very much for your expert opinion. We have corrected the part you pointed out to a sentence that claims the usefulness of NPs for psoriasis treatment.

Comment 2:
In the introduction, I would expand the paragraph to also discuss psoriasis treatments. You should mention currently available therapies; here are some articles you could add: (doi: 10.1111/dth.14504. doi: 10.1111/dth.13185.doi: 10.1080/09546634.2023.2200868.)

Reply:

Thank you for your advice. At the end of the first paragraph of the "introduction", we have added the suggested references and text about psoriasis treatments.

Reviewer 2 Report

Akira Kagawa et al reported the development of cyclosporin A-loaded nanoparticles using PLGA-PEG-PLGA triblock copolymer, and the therapeutic effect of CsA-loaded PLGA-PEG-PLGA NPs on psoriasis was evaluated in mouse models of psoriasis. I think this manuscript needs major revision and is not suitable for publishing in Applied Sciences in its current form. Here are some comments of this manuscript for authors.
1. Why did the authors use CsA in this study? Is it a commonly used anti- psoriasis agents?

2. How did the authors construct the three-dimensional cultured skin? The authors should investigate the permeability of the nanoparticles into the three-dimensional cultured skin.

3. How did the authors measure the epidermal thickness in Figure 6?

4. The same carrier PLGA-PEG-PLGA triblock copolymer was used in the author’s previous paper (Colloids and Surfaces A, 2020, 600, 12486), and the same agent (CsA) was encapsulated. So the authors should describe the novelty and new findings of this manuscript.

5. PLGA NPs displayed enhanced cellular uptake than PLGA-PEG-PLGA NPs as shown in Figure 2, but PLGA-PEG-PLGA NPs exerted stronger therapeutic effect than PLGA NPs, please explain this inconsistency.

6. There are some typo errors in the manuscript, for example CO2 in line 132, please check and revise.

7. The authors should carefully improve the English language throughout the manuscript.

The authors should carefully improve the English language throughout the manuscript.

Author Response

Comment 1:
Why did the authors use CsA in this study? Is it a commonly used anti- psoriasis agents?

Reply:

Thank you for your comment. CsA is commonly used to treat psoriasis. Oral CsA drugs have a high risk of side effects, so an administration method that allows easy control of blood drug levels is desirable. Therefore, transdermal administration may be suitable, but this is difficult to achieve due to the physical properties of CsA as described in the manuscript. We used CsA in this study because we believed that this problem could be solved by using nanoparticles as drug carriers.

Comment 2:
How did the authors construct the three-dimensional cultured skin? The authors should investigate the permeability of the nanoparticles into the three-dimensional cultured skin.

Reply:

The 3D cultured skin was purchased and used according to the attached manual. This three-dimensional cultured skin model is an alternative to the Draize test and is used to evaluate sample safety but is not used to verify permeability.

Comment 3:
How did the authors measure the epidermal thickness in Figure 6?

Reply:

As described in Section 2.5.2 of the manuscript, the epidermal thickness of each group was randomly selected 15–20 times and size measurements were performed using ImageJ Fiji software.

Comment 4:
The same carrier PLGA-PEG-PLGA triblock copolymer was used in the author’s previous paper (Colloids and Surfaces A, 2020, 600, 12486), and the same agent (CsA) was encapsulated. So the authors should describe the novelty and new findings of this manuscript.

Reply:

The previous paper was a study on the development of nanoparticles and their permeability into normal skin. This study aims to verify whether the previously developed nanoparticles can be used to treat psoriasis. As explained in the second paragraph of the introduction and the conclusion section, the novelty and new findings of this study are that we demonstrated the utility of PLGA-PEG-PLGA as a drug carrier for CsA and presented a new option for psoriasis therapeutics applied to the skin.

Comment 5:
PLGA NPs displayed enhanced cellular uptake than PLGA-PEG-PLGA NPs as shown in Figure 2, but PLGA-PEG-PLGA NPs exerted stronger therapeutic effect than PLGA NPs, please explain this inconsistency.

Reply:

We added an explanation to the first paragraph of the discussion section.

Comment 6:
There are some typo errors in the manuscript, for example CO2 in line 132, please check and revise.

Reply:

Thank you for your advice. We have corrected the typos.

Comment 7:
The authors should carefully improve the English language throughout the manuscript.

Reply:

Thank you for your advice. We have reviewed the English language.

Reviewer 3 Report

In this manuscript, the authors aim to evaluate the therapeutic effects of 30-nm cyclosporin A-loaded nanoparticles using PLGA-PEG-PLGA triblock copolymer as a drug carrier for transdermal delivery in the treatment of psoriasis.

The manuscript is logically organized. However, I believe several key points require major revisions to improve the readability of the work.

Below are my thoughts and some detailed comments that might help you revise the manuscript.

1.      The title does not correspond to the article content. However, cyclosporin A is already used as a drug for psoriasis with administration by the parenteral route, and the manuscript does not deeply study its therapeutic effect.

2.      Some type errors are in the text (for ex. Phrase in line 30) and lack of spaces between number and measure units.

3.      In section 3, Figure 1, why do the 2^nd and 3^rd images, labeled “free coumaririn-6”, show green fluorescence if it is due to the presence of coumarin-6? Furthermore, there is an error in the label and in the caption, the extended names have to be replaced with related acronyms already reported in the main text.

4.      Also in Figure 2B there is an unexplained fluorescence for the free coumarin-6 sample.

5.      What are the blanks in Figure 3? There is a lack in the comment of the Figure.

6.      Figure 4 does not have high relevance. This could be moved to supporting information.

7.       Although the article is well written, innovation, also compared to a previous article of the authors is a little scarse.

Author Response

Comment 1:
The title does not correspond to the article content. However, cyclosporin A is already used as a drug for psoriasis with administration by the parenteral route, and the manuscript does not deeply study its therapeutic effect.

Reply:

This study did not investigate the therapeutic effects of cyclosporin A, but rather verified the therapeutic effects of cyclosporin A-loaded PLGA-PEG-PLGA nanoparticles. Since this is a study on the use of PLGA-PEG-PLGA as a drug carrier, we believe that the current title is acceptable.

Comment 2:
Some type errors are in the text (for ex. Phrase in line 30) and lack of spaces between number and measure units.

Reply:

Thank you for your advice. We have corrected the typos.

Comment 3:
In section 3, Figure 1, why do the 2nd and 3rd images, labeled “free coumaririn-6”, show green fluorescence if it is due to the presence of coumarin-6? Furthermore, there is an error in the label and in the caption, the extended names have to be replaced with related acronyms already reported in the main text.

Reply:

Thank you for pointing this out. The name of nanoparticles containing coumarin 6 was the same as that of nanoparticles that do not contain coumarin 6, so this has been corrected. Even when coumarin 6 is in its free form and not contained in nanoparticles, it cannot be said that there is absolutely no possibility that it will be taken up into cells. In this case, as you pointed out, green fluorescence was observed in Fig. 1, indicating that a small amount of free coumarin 6 was taken up by the cells. This has been added to section 3.1.

Comment 4:
The small amount of fluorescence measured in the free coumarin 6 samples in Figure 2B is consistent with the results in Figure 1.

Reply:

The previous paper was a study on the development of nanoparticles and their permeability into normal skin. This study aims to verify whether the previously developed nanoparticles can be used to treat psoriasis. As explained in the second paragraph of the introduction and the conclusion section, the novelty and new findings of this study are that we demonstrated the utility of PLGA-PEG-PLGA as a drug carrier for CsA and presented a new option for psoriasis therapeutics applied to the skin.

Comment 5:
Thank you for your advice. We have added an explanation to the caption of Figure 3.

Reply:

We added an explanation to the first paragraph of the discussion section.

Comment 6:
Figure 4 does not have high relevance. This could be moved to supporting information.

Reply:

In the previous study (references 11 in the manuscript), we received advice on conducting safety tests for nanoparticles, so we conducted the experiment shown in Fig. 4.

Comment 7:
Although the article is well written, innovation, also compared to a previous article of the authors is a little scarse.

Reviewer 4 Report

The work carried out is very interesting and easy to read. Furthermore, the methodology and equipment used support very well the results found, which are very promising. However, there are some aspects that I consider would help improve the manuscript:

1. Is it possible to have an even more detailed description of the development of Nanoiparticles? Is this so that other researchers can reproduce these procedures?

2. Is it possible to make a complementary scheme for the preparation of nanoparticles?

3. By chance, did you carry out in vitro release kinetic studies to understand the API release mechanism?

4. By chance, did you do physicochemical stability studies of the nanoparticles?

Author Response

Comment 1:
Is it possible to have an even more detailed description of the development of Nanoiparticles? Is this so that other researchers can reproduce these procedures?

Reply:

Thank you for your advice. We have added an explanation to the section 2.2.

Comment 2:
Is it possible to make a complementary scheme for the preparation of nanoparticles?

Reply:

As mentioned in the manuscript, the nanoparticle preparation method itself is not new and has been published, so we think its creation is unnecessary.

Comment 3:
By chance, did you carry out in vitro release kinetic studies to understand the API release mechanism?

Reply:

In our previous study (references 11 in the manuscript), we conducted experiments on drug release behavior from nanoparticles. This study mainly involved therapeutic experiments, and the physicochemical experiments were reported in the previous paper.

Comment 4:
By chance, did you do physicochemical stability studies of the nanoparticles?

Reply:

Although we confirmed that the nanoparticles did not aggregate during the experimental operations, we did not examine their long-term stability.

Round 2

Reviewer 2 Report

The authors addressed most of my concerns.

Reviewer 3 Report

The authors' responses to previous comments are adequate.