Design of Prototype Formulations for In Vitro Dermal Delivery of the Natural Antioxidant Ferulic Acid Based on Ethosomal Colloidal Systems
Round 1
Reviewer 1 Report
General comment
This manuscript deals with the preparation and characterization of ethosomes loaded with ferulic acid (FA) for topical administration. The authors selected optimized ethosomes formulations on which they performed in vitro studies to evaluate FA release/skin permeation.
The experimental protocol is not properly designed making the scientific soundness of the results questionable. In addition, this manuscript is too wordy and there many repetitions that should be avoided. The authors should shorten the manuscript, removing all repetitions.
Specific comments
In the title and in the keywords, the term “transdermal” is inappropriate. Transdermal formulations are designed to achieve a systemic effect while cosmetic formulations are intended only for topical effect. Therefore, the term “transdermal” should be changed to dermal.
Line 17. The term “aspect ratio” is unclear. Please, clarify the meaning of this terminology
Line 78. The term transdermal should be changed to dermal.
References 15 and 16 should be checked.
Line 181. The authors should specify the solvents used to obtain free FA solutions.
Line 190. The description of stability tests performed on ethosomal dispersions and semisolid products is unclear. What was the amount of ethosomal dispersions and free FA used to obtain semisolid products? How did the authors extract FA from these fromulations? Did the formulation ingredients interfere with the UHPLC analyses?
Line 195. Generally, triethanolamine is used to neutralize Carbopol. Why did the authors use trimethylamine?
Line 262. The meaning of the sentence “Moreover, they are more readily absorbed as compared to neutral one”. The authors designed these ethosomal formulations for topical application. Therefore, the skin is supposed to be the target organ through which diffusion takes place. As the skin surface is negatively charged, why should negative ethosomes be absorbed more readily?
Fig.2 is unreadable. Please, provide a figure with a better resolution.
In Fig. 3B, the authors reported the thermogram of an ethosomal suspension containing FA but they did not report the thermogram of empty ethosomes. The comparison between empty and FA loaded ethosomes would have provided useful information about the interactions between FA and the ethosomal bilayers. The authors should add the thermogram of empty ethosomes and comment the differences in the thermal behavior of empty and FA loaded ethosomes.
Line 107 (page 13) In vitro permeability tests. The authors reported many experimental details that they had already reported in the materials and methods section. All these repeated details should be removed. The authors reported the use of a cellulose membrane coated with a thin layer of soy lecithin but they did not explain how they prepared this membrane. Such information should be provided. All data depicted in Fig. 5 and reported in Table 2 were provided without any standard deviation. How many replicates did the authors perform for each experiment? Are the differences (among the data reported in Table 2 and in the text) statistically significant? The incorporation of drug loaded colloidal suspension into semisolid vehicle is known to affect drug release/skin permeation but the authors did not perform in vitro release/skin permeation experiments on the gels containing FA loaded ethosomes. Why?
Author Response
Article: Design of prototype formulations for in-vitro dermal delivery of the natural antioxidant ferulic acid based on ethosomal colloidal systems
Response to Reviewer 1
The authors thank the reviewer for the comments and suggestions made. Likewise, we indicate that the suggested changes will be reported in red to facilitate the review process. At last, the manuscript was submitted to a language editing service (the certificate is attached).
Specific comments
1. Reviewer Comments: In the title and in the keywords, the term “transdermal” is inappropriate. Transdermal formulations are designed to achieve a systemic effect while cosmetic formulations are intended only for topical effect. Therefore, the term “transdermal” should be changed to dermal.
Author response: we agree, and the change was made (lines 2-27-78)
2. Reviewer Comments: Line 17. The term “aspect ratio” is unclear. Please, clarify the meaning of this terminology
Author response: we agree, and the change was made (line 17)
3. Reviewer Comments: Line 78. The term transdermal should be changed to dermal.
Author response: we agree, and the change was made (line 78)
4. Reviewer Comments: References 15 and 16 should be checked.
Author response: it was decided to delete this part because the references are not available.
5. Reviewer Comments: Line 181. The authors should specify the solvents used to obtain free FA solutions.
Author response: we agree, and the change was made (line 203)
6. Reviewer Comments: Line 190. The description of stability tests performed on ethosomal dispersions and semisolid products is unclear. What was the amount of ethosomal dispersions and free FA used to obtain semisolid products? How did the authors extract FA from these fromulations? Did the formulation ingredients interfere with the UHPLC analyses?
Author response: we agree, and the change was made (line 218-221-225)
7. Reviewer Comments: Line 195. Generally, triethanolamine is used to neutralize Carbopol. Why did the authors use trimethylamine?
Author response: In deed, it was a spelling mistake. So, the change was made (line 221).
8. Reviewer Comments: Line 262. The meaning of the sentence “Moreover, they are more readily absorbed as compared to neutral one”. The authors designed these ethosomal formulations for topical application. Therefore, the skin is supposed to be the target organ through which diffusion takes place. As the skin surface is negatively charged, why should negative ethosomes be absorbed more readily?
Author response: Regarding this comment, we decided to delete the sentence to avoid confusion.
9. Reviewer Comments: Fig.2 is unreadable. Please, provide a figure with a better resolution.
Author response: we agree, and the change was made
10. Reviewer Comments: In Fig. 3B, the authors reported the thermogram of an ethosomal suspension containing FA but they did not report the thermogram of empty ethosomes. The comparison between empty and FA loaded ethosomes would have provided useful information about the interactions between FA and the ethosomal bilayers. The authors should add the thermogram of empty ethosomes and comment the differences in the thermal behavior of empty and FA loaded ethosomes.
Author response: we agree, and the change was made
11. Reviewer Comments: Line 107 (page 13) In vitro permeability tests. The authors reported many experimental details that they had already reported in the materials and methods section. All these repeated details should be removed.
Author response: we agree, and the change was made
12. Reviewer Comments: The authors reported the use of a cellulose membrane coated with a thin layer of soy lecithin, but they did not explain how they prepared this membrane. Such information should be provided.
Author response: we agree, and the change was made (line 193)
13. Reviewer Comments: All data depicted in Fig. 5 and reported in Table 2 were provided without any standard deviation. How many replicates did the authors perform for each experiment? Are the differences (among the data reported in Table 2 and in the text) statistically significant?
Author response: In this case, we made one replica for each test because we only wanted to be stablished a comparison between the permeability of free and encapsulated AF used two kinds of membranes.
14. Reviewer Comments: The incorporation of drug loaded colloidal suspension into semisolid vehicle is known to affect drug release/skin permeation, but the authors did not perform in vitro release/skin permeation experiments on the gels containing FA loaded ethosomes. Why?
Author response: first, the permeability of free AF and the ethosomal dispersion were evaluated using different membranes. Subsequently, it was evaluated the behavior in different formulations.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In this research, the advantages of the encapsulation of ferulic acid in ethossomes has been established.
There are no objective errors in the methods or results, and the conclusions are supported by the data presented.
In the introduction, authors should explain the advantages of ethossomes and explain why they didn’t choose another encapsulation system such as SLN's or NLC's, for instance.
In the 2.2.8 section of materials and methods, flow should be replaced with Flux. The last sentence of the stability tests section seems to be out of context.
In the results 3.6.1 section, line 122, the expression “free encapsulated” is confusing. In table 2, AUC is flux? If yes, it should be corrected. Authors claim in the discussion of results in table 2 that the release of FA from the cellulose membranes is not biphasic, but this behaviour does not mimic the in vitro and in vivo skin. What is the advantage?
Author Response
Article: Design of prototype formulations for in-vitro dermal delivery of the natural antioxidant ferulic acid based on ethosomal colloidal systems
Response to Reviewer 2
The authors thank the reviewer for the comments and suggestions made. Likewise, we indicate that the suggested changes will be reported in red to facilitate the review process. At last, the manuscript was submitted to a language editing service (the certificate is attached).
Specific comments
1. Reviewer Comments: In this research, the advantage of the encapsulation of ferulic acid in ethosomes has been established. There are no objective errors in the methods or results, and the conclusions are supported by the data presented.
Author response: we agree
2. Reviewer Comments: In the introduction, authors should explain the advantages of ethossomes and explain why they didn’t choose another encapsulation system such as SLN's or NLC's, for instance.
Author response: we agree and the change was made (line 59)
3. Reviewer Comments: In the 2.2.8 section of materials and methods, flow should be replaced with Flux.
Author response: we agree and the change was made (lines 208-225-403)
4. Reviewer Comments: The last sentence of the stability tests section seems to be out of context.
Author response: we agree and the change was made (line 225)
5. Reviewer Comments: In the results 3.6.1 section, line 122, the expression “free encapsulated” is confusing.
Author response: we agree and the change was made (line 453)
6. Reviewer Comments: In table 2, AUC is flux? If yes, it should be corrected.
Author response: we agree and the change was made (table 2)
7. Reviewer Comments: Authors claim in the discussion of results in table 2 that the release of FA from the cellulose membranes is not biphasic, but this behavior does not mimic the in vitro and in vivo skin. What is the advantage?
Author response: In this work, a comparison was made between the Strat-M® membrane that simulates human skin and a cellulose membrane to know the permeability of free and encapsulated FA.
Author Response File: Author Response.docx
Reviewer 3 Report
The article presents some considerations, especially in support analytical techniques that are not scientifically correct.
For example the PDI setting is completely wrong. I advise you to study the DLS measurement technique of particle size.Please see the International Standard ISO22412 Particle Size Analysis – Dynamic Light Scattering, International Organisation for Standardisation (ISO) 2008. Or in alternative please read the manual of the equipment.
Author Response
Article: Design of prototype formulations for in-vitro dermal delivery of the natural antioxidant ferulic acid based on ethosomal colloidal systems
Response to Reviewer 3
The authors thank the reviewer for the comments and suggestions made. Likewise, we indicate that the suggested changes will be reported in red to facilitate the review process. At last, the manuscript was submitted to a language editing service (the certificate is attached).
Specific comments
1. Reviewer Comments: The article presents some considerations, especially in support analytical techniques that are not scientifically correct. For example the PDI setting is completely wrong. I advise you to study the DLS measurement technique of particle size. Please see the International Standard ISO22412 Particle Size Analysis – Dynamic Light Scattering, International Organisation for Standardisation (ISO) 2008. Or in alternative please read the manual of the equipment.
Author response: we agree with the comment and we have reviewed the ISO standard document 13321:1996 E and ISO 22412:2008 for clarifying concepts about PDI and cumulant analysis. (lines 149-321)
Author Response File: Author Response.docx
Reviewer 4 Report
The manuscript "Design of Prototype Formulations for In-Vitro Transdermal Delivery of The Natural Antioxidant Ferulic Acid Based on Ethosomal Colloidal Systems" is an interesting work that investigates the protection effect of ferulic acid loaded ethosomes against different environmental factors. Formulation was optimized by a two-level fractional factorial design considering the particle size, zeta potential, encapsulation efficiency, polydispersity index and aspect ratio as response variables. The optimal formulation was fully characterized. The topic of the research is interesting, but the manuscript is poorly written, the results are not exhaustively presented and some information and data need to be added. I recommend minor revision before publication in Cosmetics.
In the abstract, line 20, in the Experimental section, line 139, in the results section, line22, the word “encapsulation” should be replaced with “incorporation” because the authors report that ferulic acid is a weak acid with hydrophobic characteristic.
In the Introduction, lines 57-59 and 61, the words “moreover and additionally” are repeated and should be eliminated.
In the Introduction, lines 61-64, the important results reported by Manca et al on the effects provide by ethanol in phospholipid assembling and vesicle ability to drug delivery should be reported (see: ML Manca, I Castangia, P Matricardi, S Lampis, X Fernàndez-Busquets, AM Fadda, Maria Manconi. Molecular arrangements and interconnected bilayer formation induced by alcohol or polyalcohol in phospholipid vesicles. Colloids and Surfaces B: Biointerfaces 117 (2014) 360–367).
In the Experimental section, lines 107-107, please report the final FA concentration in the vesicle dispersion
In the Experimental section, lines 115-118, please report references for phospholipid vesicles prepared for direct addition of water co-solvents to phospholipid such as I Castangia, ML Manca, P Matricardi, C Sinico, S Lampis, X Fernàndez-Busquets, AM Fadda, Maria Manconi. Effect of diclofenac and glycol intercalation on structural assembly of phospholipid lamellar vesicles. International Journal of Pharmaceutics 456 (2013) 1-9.
In the Experimental section, lines 122-127, the authors report that liposomes were dried at room temperature. As well known, the dry process involves the break and fusion of vesicles. I can not understand how the authors can observe the vesicles surface. Is possible that propylene glycol acts a dehydration-protect. This Issus should be reported in the discussion.
Author Response
Article: Design of prototype formulations for in-vitro dermal delivery of the natural antioxidant ferulic acid based on ethosomal colloidal systems
Response to Reviewer 4
The authors thank the reviewer for the comments and suggestions made. Likewise, we indicate that the suggested changes will be reported in red to facilitate the review process. At last, the manuscript was submitted to a language editing service (the certificate is attached).
Specific comments
Comments and Suggestions for Authors
1. Reviewer Comments: The manuscript "Design of Prototype Formulations for In-Vitro Transdermal Delivery of The Natural Antioxidant Ferulic Acid Based on Ethosomal Colloidal Systems" is an interesting work that investigates the protection effect of ferulic acid loaded ethosomes against different environmental factors. Formulation was optimized by a two-level fractional factorial design considering the particle size, zeta potential, encapsulation efficiency, polydispersity index and aspect ratio as response variables. The optimal formulation was fully characterized. The topic of the research is interesting, but the manuscript is poorly written, the results are not exhaustively presented and some information and data need to be added. I recommend minor revision before publication in Cosmetics.
Author response: we agree and the manuscript was sent to an editing service to improve writing and translation
2. Reviewer Comments: In the abstract, line 20, in the Experimental section, line 139, in the results section, line22, the word “encapsulation” should be replaced with “incorporation” because the authors report that ferulic acid is a weak acid with hydrophobic characteristic.
Author response: we agree and the change was made (lines 16-52-79)
3. Reviewer Comments: In the Introduction, lines 57-59 and 61, the words “moreover and additionally” are repeated and should be eliminated.
Author response: we agree and the change was made
4. Reviewer Comments: In the Introduction, lines 61-64, the important results reported by Manca et al on the effects provide by ethanol in phospholipid assembling and vesicle ability to drug delivery should be reported (see: ML Manca, I Castangia, P Matricardi, S Lampis, X Fernàndez-Busquets, AM Fadda, Maria Manconi. Molecular arrangements and interconnected bilayer formation induced by alcohol or polyalcohol in phospholipid vesicles. Colloids and Surfaces B: Biointerfaces 117 (2014) 360–367).
Author response: we agree and the change was made
5. Reviewer Comments: In the Experimental section, lines 107-107, please report the final FA concentration in the vesicle dispersion
Author response: we agree and the change was made (line 96)
6. Reviewer Comments: In the Experimental section, lines 115-118, please report references for phospholipid vesicles prepared for direct addition of water co-solvents to phospholipid such as I Castangia, ML Manca, P Matricardi, C Sinico, S Lampis, X Fernàndez-Busquets, AM Fadda, Maria Manconi. Effect of diclofenac and glycol intercalation on structural assembly of phospholipid lamellar vesicles. International Journal of Pharmaceutics 456 (2013) 1-9.
Author response: we agree and the change was made
7. Reviewer Comments: In the Experimental section, lines 122-127, the authors report that liposomes were dried at room temperature. As well known, the dry process involves the break and fusion of vesicles. I can not understand how the authors can observe the vesicles surface. Is possible that propylene glycol acts a dehydration-protect. This Issus should be reported in the discussion.
Author response: we agree and the change was made (line 269)
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors addressed properly most comments. However, the authors did not answer adequately the comments listed below.
The authors were asked to address the following comment:
In Fig. 3B, the authors reported the thermogram of an ethosomal suspension containing FA but they did not report the thermogram of empty ethosomes. The comparison between empty and FA loaded ethosomes would have provided useful information about the interactions between FA and the ethosomal bilayers. The authors should add the thermogram of empty ethosomes and comment the differences in the thermal behavior of empty and FA loaded ethosomes.
The authors’ answer was: “we agree, and the change was made”.
In the revised version, Fig. 3B does not show the thermograms of empty and FA loaded ethosomes. In addition, the authors did not add any comment about the differences in the thermal behavior of empty and FA loaded ethosomes. Therefore, the authors’ answer is not satisfactory. The authors has still to address the previous comment.
The authors were asked to report the standard deviations of the data illustrated in Table 2 and the number of replicates they performed for each experiments. The authors’ answer was: “In this case, we made one replica for each test because we only wanted to be stablished a comparison between the permeability of free and encapsulated AF used two kinds of membranes.”
I wonder what scientific and statistical meaning has single data (without no replicates and no standard deviation). In addition, how did the authors obtain the standard deviations of some data reported in Fig. 5 if they did not perform any replicate? Experiments should be performed at least in triplicate to obtain statistically significant results. Therefore, the authors’ response is not satisfactory and the authors has to provide data with proper statistical significance.
The authors were asked to address the following comment:
The incorporation of drug loaded colloidal suspension into semisolid vehicle is known to affect drug release/skin permeation, but the authors did not perform in vitro release/skin permeation experiments on the gels containing FA loaded ethosomes. Why?
The authors’ answer was: “first, the permeability of free AF and the ethosomal dispersion were evaluated using different membranes. Subsequently, it was evaluated the behavior in different formulations.”
This answer is unclear. The authors did not perform in vitro release/skin permeation experiments on the gels containing FA loaded ethosomes. They performed only stability tests on these gel formulations. As semisolid vehicles are known to affect drug release/skin permeation, what is the point of performing only stability tests without carrying out in vitro release studies to demonstrate that these formulations allow a suitable FA release? If the authors are not going to provide information about FA release from gel formulations, these vehicles should be removed from the text.
Author Response
The response information is attached in the word document
Author Response File: Author Response.docx
Reviewer 3 Report
The authors answered the questions and modified the text accordingly.
Author Response
I am not clear about the suggestions and recommendations for round 2.
