Cytokine-Related Effect of Buccal-Delivered Collagen Peptide Incorporated in Mucoadhesive Films to Improve Female Skin Conditions

Round 1

Reviewer 1 Report

- the test on volunteers was carried out only on the female gender, for what reason?
- the authors declared the use of collagen 1 and 3 KDa, on which observations were these dimensions chosen?
- The authors are sure that the application of the double-layer buccal film has always been performed correctly, moreover the observed effect of hydration remains even after the observation period at four weeks.
-Is Raw264.7 cell culture the most suitable for the study?
The text needs to be carefully reviewed due to the presence of numerous typos and grammatical errors.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The Authors presented a study entitled: “Cytokine-related effect of buccal delivered collagen peptide incorporated in mucoadhesive films to improve female skin conditions”.

The manuscript is interesting, however, further improvements of the English form are needed.

Moreover, the Authors should consider the following advices:

Please, check and correct some errors typo as “absortion”, “bucaal”, “Custometer”,  “Al though”, along the manuscript.

In my opinion, for quantifying the concentration of collagen peptide in artificial saliva after permeation through buccal swine mucosa by using Franz diffusion cells, it should be better to perform HPLC experiments.

In the “Materials and Methods” section, please, check and correct the numbering from paragraph “2.4. Test product and placebo”, to “2.7. Statistical analysis”.

In the same paragraph “2.4. Test product and placebo”, please, check the sentence “The test sample collagen peptide (1 KDa) delivery buccal film from formulation of buccal permeation test” at lines 165-166 for clarifying it.

In the “Materials and Methods” section, at paragraph “2.6. Cytokine test”, subparagraph “2.6.1. Cell line”, the full name of the used raw 264.7 cells should be reported.

About the cytokine test, Why the Authors did not performed an experiment onto another group in which DMEM media, collagen peptide, and LPS for 24 h were in use?

Had the Authors performed cytotoxicity experiments on raw 264.7 cells before testing the collagen film in-vivo? And on buccal mucosa cell lines? If not, in my opinion, Cytotoxicity tests on buccal mucosa cell lines, as well as on raw 264.7 cells should be performed.

In the “Results” section, at paragraph “3.2. Characteristics of volunteers”, please check the sentence “All participants were no the reaction of skin irritation to environment such as prickle, itch, and side effects” reported at lines 264-266.

Moreover, at the beginning of this same paragraph, please, check and correct the error typo “Final”, at line 260.

In the “Results” section, at paragraph “3.6. Assessment of safety and Questionnaire”, in my opinion, a robust and rigorous assessment of the side effects following the use of the studied product must be performed for claiming it as safe.

In the “Results” section, at sub-paragraph “3.7.2. Peroxynitrite (ONOO^-) and Intereukin-1 α”, please, clarify the sentence “Totally, relative levels of peroxynitrite were increased treated with all groups” reported at lines 402-403, as well as the sentence “Overall, the trends of IL-1 α levels were increased treated with collagen and LPS” reported at lines 417-418.

Moreover, please check the collagen peptide dosage reported in brackets at line 420, “(50 and 100 μg/mL)”.

Please check and correct the error typo “wight” in the caption of Figure 2.

Please check and correct Figure 8; it should be “Figure 9”.

In the “Discussion” section, from line 443 to line 445, please clarify the sentence “Koo et al. investigated that hydrophilic polymer-based buccal film containing the 2 KDa-3 KDa of collagen hydrolysate can be permeated by performing in vitro human buccal tissues permeation test.”

In the last part of “Discussion” section, from line 497 to line 525, the Authors hypothesized a kind of “Homeopathic effect” attributed to the collagen peptide after its absorption through the buccal mucosa; however, in my opinion, further experiments should be performed for supporting this hypothesis.

Moreover, what the Authors think about the expression levels of matrix metalloproteinase after the collagen peptide intake as supplement? Again, what about the Fibroblast Production, Extracellular Matrix organization, and production of endogenous collagen? And what about the antioxidant properties of the studied collagen peptide film?

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have complied with the requested changes into the manuscript. I just only suggest to check the correct name of the instrument used for performing the skin elasticity measurements; it should be "Cutometer MP580".

Author Response

We corrected the name of instrument from "Cutometer MPA 580" to "Cutometer MP 580".