Formulation of Topical Antioxidant Creams with Hydroxycitrate or Aglianico Del Vulture Red Wine Extract for the In Vitro Prevention of Blue Light-Induced Oxidative Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents research on two natural antioxidant cream formulations containing either hydroxycitrate (HCA) or Aglianico del Vulture red wine powder (RWP) for protection against blue light-induced oxidative stress. While the topic addresses an emerging concern in cosmetic science regarding blue light exposure, several issues require attention to strengthen the scientific rigor and clarity of the work. Detailed comments are given below.

1. The manuscript frequently confuses "blue light" (visible light, 380-500 nm) with "UV radiation" throughout the text. Section headings (3.5), figure captions (Figure 7), and methodology (section 2.4) refer to "UV-induced ROS" despite the study's focus on blue light. This creates significant confusion about what type of light exposure was actually tested. It is suggested to make the terminology consistent throughout the manuscript.
2. The study uses Winstor and DUNE90 lamps but fails to provide spectral emission profiles of these light sources. Without this critical information, it's impossible to determine which specific wavelengths were used in the study. The claim that "blue light under 453 nm" was tested lacks verification.
3. The paper does not explain how the concentrations in the cream (8.5 mg/5g HCA and 20 mg/5g RWP) were translated to the cell culture concentrations (500 µM cHCA and 400 µg/mL cRWP). The dilution factor and extraction method for testing the creams in vitro need clarification.
4. The study is limited to in vitro HaCaT cell testing. For a cosmetic product claim, some form of ex vivo skin model or preliminary in vivo testing would significantly strengthen the relevance of the findings. Why authors didn't consider skin models or animal models?
5. The paper does not compare the formulated creams with existing commercial blue light protection products or standard antioxidants (like vitamin C or E), making it difficult to assess the relative efficacy and novelty of the proposed formulations. It is suggested to add a comparison table.
6. No evaluation of skin penetration for HCA or RWP is provided, which is critical for determining whether these compounds can reach target cells in viable concentrations when applied topically.
7. A topical formulation significantly affects the biophysical parameters of kin. Considering assessment of biophysical parameters will strengthen the claims about topical formulations. Authors may overview the literature for the said experiments, e.g. “DOI: 1016/j.ejpb.2022.10.016” and “DOI: 10.3390/pharmaceutics15010164”.
8. Stability claims can be further reinforced by supplementing the experiments concerning measurements of active pharmaceutical ingredient and activity of formulation during accelerated storage stability evaluation. Authors may overview the literature for the said experiments, e.g. “DOI10.1016/j.jsps.2018.07.005” and “DOI: 10.3390/pharmaceutics15010164”.
9. The paper does not sufficiently differentiate this work from existing literature on red wine polyphenols in cosmetics. A clearer statement of what makes Aglianico del Vulture wine uniquely suitable for this application would strengthen the novelty claim.
10. The text describes hydrating "A4 in A5" (lines 100-101), but Table 1 does not include an "A5" component in Phase A, creating confusion in the formulation methodology.
11. Unclear Viscosity Reporting: Tables 2-5 report viscosity as "14,910- 74.5%" which is ambiguous. Is this 14,910 mPa·s or 74.5%? The formatting and meaning need clarification.
12. Figure 1 claims to show "Evaluation of smell, texture and skin feel" but appears to be a generic cream image without methodological details on how these sensory properties were quantitatively assessed. All figures would benefit from higher resolution and clearer labeling.
13. While the statistical methods mention Dunnett's test for multiple comparisons, the figure captions (lines 211, 226, 251, 269, 284) do not consistently specify which group served as the control for comparisons.
14. Lines 147-148 state, "exposed to blue light with the two kinds of lamp", which should be "lamps" (plural). Multiple instances show inconsistent spacing around asterisks for significance markers (e.g., "* p< 0.05" should be "*p < 0.05").
15. The rationale for selecting 8.5 mg/5 g HCA and 20 mg/5 g RWP for the cream formulations is not explained, nor is there dose-response data specifically for the formulated creams.
16. The paper reports a "slight reduction of approximately 20% in proliferation" after blue light exposure but doesn't discuss whether this magnitude of effect has meaningful clinical implications for skin health.

Author Response

This manuscript presents research on two natural antioxidant cream formulations containing either hydroxycitrate (HCA) or Aglianico del Vulture red wine powder (RWP) for protection against blue light-induced oxidative stress. While the topic addresses an emerging concern in cosmetic science regarding blue light exposure, several issues require attention to strengthen the scientific rigor and clarity of the work. Detailed comments are given below.

1. The manuscript frequently confuses "blue light" (visible light, 380-500 nm) with "UV radiation" throughout the text. Section headings (3.5), figure captions (Figure 7), and methodology (section 2.4) refer to "UV-induced ROS" despite the study's focus on blue light. This creates significant confusion about what type of light exposure was actually tested. It is suggested to make the terminology consistent throughout the manuscript.

Response 1: We thank the reviewer for pointing out this inconsistency. Our study specifically investigated the effects of blue light exposure, rather than UV radiation. We have carefully revised the manuscript to ensure consistent use of the correct terminology. Specifically:

• Section headings (e.g., Section 3.5) have been updated to refer to "blue light-induced ROS" instead of "UV-induced ROS."
• Figures 5 and 7 and their captions have been corrected to reflect blue light exposure.
• The methodology section (2.4) has been clarified to specify that blue light, not UV radiation, was used in all experimental setups.

2. The study uses Winstor and DUNE90 lamps but fails to provide spectral emission profiles of these light sources. Without this critical information, it's impossible to determine which specific wavelengths were used in the study. The claim that "blue light under 453 nm" was tested lacks verification.

Response 2: We sincerely apologize for the oversight in the manuscript. The previous mention of Winstor and DUNE90 lamps was incorrect. The experiments were really conducted using light-emitting diodes (Honglitronic, Guangzhou, PRC), wavelength 450 nm, irradiance 53 mW/cm² at 9 cm distance, and a blue light-emitting LED (BLE LED, Philips GmbH Innovative Technologies), wavelength 453 nm, irradiance 50 mW/cm². We have corrected the manuscript accordingly and greatly appreciate the reviewer for bringing this error to our attention.

3. The paper does not explain how the concentrations in the cream (8.5 mg/5g HCA and 20 mg/5g RWP) were translated to the cell culture concentrations (500 µM cHCA and 400 µg/mL cRWP). The dilution factor and extraction method for testing the creams in vitro need clarification.

Response3: We sincerely thank the reviewer for raising this point. To clarify, in order to test the creams at concentrations equivalent to those used in vitro, 10× stock solutions were prepared: 4 mg/mL for RWP and 5 mM for HCA. Assuming a density of 1 g/mL and a total cream weight of 5 g, the formulations were prepared as follows:

RWP cream: 4 mg/g (10×), totaling 20 mg/5 g;

HCA cream: 5 mM (10×), 1.7 mg/g, totaling 8.5 mg/5 g.

Given that RWP is water-soluble and HCA is soluble in cell culture medium, these solvents were employed as vehicles for the respective emulsions. This strategy ensured that the active concentrations in the creams corresponded directly to the effective concentrations tested in vitro (500 µM cHCA and 400 µg/mL cRWP), thereby maintaining consistency between the in vitro and cream-based experiments.

4. The study is limited to in vitro HaCaT cell testing. For a cosmetic product claim, some form of ex vivo skin model or preliminary in vivo testing would significantly strengthen the relevance of the findings. Why authors didn't consider skin models or animal models?

Response 4: We thank the reviewer for this valuable comment. This study represents a preliminary investigation conducted on HaCaT cells to evaluate the cellular effects of the tested formulations. In future work, we plan to proceed with the next step by employing various ex vivo skin models and/or animal models to further validate the findings and strengthen their relevance for cosmetic applications. To clearly indicate the scope of the current study, we have also added “in vitro” to the manuscript title.

5. The paper does not compare the formulated creams with existing commercial blue light protection products or standard antioxidants (like vitamin C or E), making it difficult to assess the relative efficacy and novelty of the proposed formulations. It is suggested to add a comparison table.

Response 5: Riteniamo che questo confronto mirato affronti efficacemente la questione scientifica fondamentale dello studio, mentre lavori futuri potrebbero ampliarsi con analisi comparative più ampie, se necessario.

6. No evaluation of skin penetration for HCA or RWP is provided, which is critical for determining whether these compounds can reach target cells in viable concentrations when applied topically.

Response 6: We thank the reviewer for this important comment. In the present preliminary study, we did not assess skin penetration of HCA or RWP. In our future work, we plan to include a comparison with liposomal formulations and evaluate skin penetration, in order to provide a more detailed and comprehensive understanding of the efficacy and bioavailability of these compounds when applied topically.

7. A topical formulation significantly affects the biophysical parameters of skin. Considering assessment of biophysical parameters will strengthen the claims about topical formulations. Authors may overview the literature for the said experiments, e.g. “DOI: 1016/j.ejpb.2022.10.016” and “DOI: 10.3390/pharmaceutics15010164”.

Response 7: We sincerely thank the reviewer for providing these valuable references, which offer important insights and guidance for designing future studies. Both were added to the manuscript as references to paragraph 2.3. While we are currently unable to incorporate such biophysical assessments in the present work, we have clarified the scope of the study by adding “in vitro” to the manuscript title. We believe this appropriately frames the study and sets the stage for subsequent investigations that may include these additional evaluations.

8. Stability claims can be further reinforced by supplementing the experiments concerning measurements of active pharmaceutical ingredient and activity of formulation during accelerated storage stability evaluation. Authors may overview the literature for the said experiments, e.g. “DOI10.1016/j.jsps.2018.07.005” and “DOI: 10.3390/pharmaceutics15010164”.

Response 8: We sincerely thank the reviewer for this valuable suggestion and for providing the relevant references, which have been promptly incorporated into the manuscript. While these analyses were beyond the scope of the present study, we plan to perform further stability assessments in our upcoming formulation studies with liposomal systems, which will allow a more comprehensive evaluation of both the active ingredients and the activity of the formulations over time.

9. The paper does not sufficiently differentiate this work from existing literature on red wine polyphenols in cosmetics. A clearer statement of what makes Aglianico del Vulture wine uniquely suitable for this application would strengthen the novelty claim.

Response 9: We thank the reviewer for this valuable suggestion. We have addressed the comment by adding relevant information and literature on Aglianico del Vulture wine in the Introduction (references https://doi.org/10.1016/j.foodchem.2024.142573 and https://doi.org/10.3390/antiox9080708 and https://doi.org/10.1155/2021/5533793), which highlights the potential of Aglianico extracts, including its pomace, for applications in the nutraceutical and pharmaceutical fields. These extracts are particularly rich in polyphenolic compounds and exhibit strong antioxidant activity, supporting their unique suitability for cosmetic formulations and reinforcing the novelty of our study.

10. The text describes hydrating "A4 in A5" (lines 100-101), but Table 1 does not include an "A5" component in Phase A, creating confusion in the formulation methodology.

Response 10: We thank the reviewer for pointing out this error. The A5 has now been corrected to A1 in the manuscript.

11. Unclear Viscosity Reporting: Tables 2-5 report viscosity as "14,910- 74.5%" which is ambiguous. Is this 14,910 mPa·s or 74.5%? The formatting and meaning need clarification.

Response 11: We thank the reviewer for pointing out this issue. The reported viscosity values correspond to 14,910 mPa·s, which is equivalent to 74.5%. The tables have now been corrected to clearly reflect this information.

12. Figure 1 claims to show "Evaluation of smell, texture and skin feel" but appears to be a generic cream image without methodological details on how these sensory properties were quantitatively assessed. All figures would benefit from higher resolution and clearer labeling.

Response 12: We thank the reviewer for this comment. The term “smell” has been removed from the caption of Figure 1 to better reflect the content. According to the reviewer's suggestion, all figures have been revised to have clearer labeling and higher resolution (600 dpi or higher).

13. While the statistical methods mention Dunnett's test for multiple comparisons, the figure captions (lines 211, 226, 251, 269, 284) do not consistently specify which group served as the control for comparisons.

Response 13: We appreciate the reviewer’s insightful comment, which has helped us improve the presentation of statistical significance. We have added further details about the statistical analyses in Section 2.7. The captions of Figures 3-7 have been carefully revised to specify the statistical method employed. Where Dunnett’s post-hoc test was performed, we have clearly indicated which group served as the control in each comparison.

14. Lines 147-148 state, "exposed to blue light with the two kinds of lamp", which should be "lamps" (plural). Multiple instances show inconsistent spacing around asterisks for significance markers (e.g., "* p< 0.05" should be "*p < 0.05").

Response14: Thank you for pointing this out. We have corrected the grammatical error on lines 147–148 by changing "lamp" to "lamps" (plural). Additionally, we have reviewed and standardized the formatting of all significance markers throughout the manuscript to ensure consistent spacing (e.g., "*p < 0.05").

15. The rationale for selecting 8.5 mg/5 g HCA and 20 mg/5 g RWP for the cream formulations is not explained, nor is there dose-response data specifically for the formulated creams.

Response 15: We chose to evaluate, in topical formulations, only the concentrations that had shown the greatest efficacy in in vitro tests. To clarify, in order to test the creams at concentrations equivalent to those used in vitro, 10× stock solutions were prepared: 4 mg/mL for RWP and 5 mM for HCA. Assuming a density of 1 g/mL and a total cream weight of 5 g, the formulations were prepared as follows:

RWP cream: 4 mg/g (10×), totaling 20 mg/5 g;

HCA cream: 5 mM (10×), 1.7 mg/g, totaling 8.5 mg/5 g.

Given that RWP is water-soluble and HCA is soluble in cell culture medium, these solvents were employed as vehicles for the respective emulsions. This strategy ensured that the active concentrations in the creams corresponded directly to the effective concentrations tested in vitro (500 µM cHCA and 400 µg/mL cRWP), thereby maintaining consistency between the in vitro and cream-based experiments.

16. The paper reports a "slight reduction of approximately 20% in proliferation" after blue light exposure but doesn't discuss whether this magnitude of effect has meaningful clinical implications for skin health.

Response 16: Thank you for your insightful comment. We agree that discussing the clinical relevance of the observed ~20% reduction in cell proliferation is important. In the revised manuscript, we have added a brief discussion highlighting that while a 20% decrease may appear modest, it could be significant in the context of cumulative or repeated blue light exposure, particularly in individuals with compromised skin barrier function or pre-existing conditions. We have also emphasized the need for further in vivo studies to better understand the clinical implications of this finding.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study by Pappalardo et al. is of considerable interest and, in my view, may merit publication after some important revisions aimed at clarifying methodological aspects.

Overall, the experiments are logically conceived and competently executed. The manuscript is well written, clearly structured, and supported by an appropriate number and quality of references.

Major points:

-Please clarify the rationale for selecting the concentrations of HCA and RWP incorporated into the final cream formulations.

-Provide a more detailed description of the microbiological assay performed on the creams, including the original references for the method employed.

-The presentation of statistical significance in the figures should be improved, as it is not immediately clear which comparisons are statistically significant.

-Two technical issues arise in the section “Effect of topical creams enriched with RWP or HCA on HaCaT cell proliferation”: 1) The authors state that “RWP (20 mg/5 g) and HCA (8.5 mg/5 g) were added to the basic formulation, thus obtaining a cream enriched with HCA (cHCA) or RWP (cRWP).” However, on page 9, line 254, they report: “… we evaluated the effects of 400 μg/mL cRWP and 500 μM cHCA…” Please explain how the concentrations expressed as weight of RWP and HCA in the creams correspond to the concentrations applied to the cells (400 μg/mL cRWP and 500 μM cHCA). 2) In the same section (page 9, lines 255–256), the authors report that cells were pretreated with topical formulations “dissolved in culture medium for cHCA or sterile water for RWP.” Given the hydrophilic nature of the cream, please clarify how these experiments were carried out. Was an emulsion generated?

-Importantly, regarding the role of tocopherol in the formulation: the inclusion of a negative control consisting of the cream without HCA and RWP in the experiments presented in Figures 6 and 7 seems essential to rule out that the observed effects are attributable to tocopherol or other cream components rather than to HCA or RWP.

Author Response

The study by Pappalardo et al. is of considerable interest and, in my view, may merit publication after some important revisions aimed at clarifying methodological aspects.

Overall, the experiments are logically conceived and competently executed. The manuscript is well written, clearly structured, and supported by an appropriate number and quality of references.

Major points:

- Please clarify the rationale for selecting the concentrations of HCA and RWP incorporated into the final cream formulations.

Response: We chose to evaluate, in topical formulations, only the concentrations that had shown the greatest efficacy in in vitro tests. For details on the calculations, see below. To clarify, in order to test the creams at concentrations equivalent to those used in vitro, 10× stock solutions were prepared: 4 mg/mL for RWP and 5 mM for HCA. Assuming a density of 1 g/mL and a total cream weight of 5 g, the formulations were prepared as follows:

RWP cream: 4 mg/g (10×), totaling 20 mg/5 g;

HCA cream: 5 mM (10×), 1.7 mg/g, totaling 8.5 mg/5 g.

Given that RWP is water-soluble and HCA is soluble in cell culture medium, these solvents were employed as vehicles for the respective emulsions. This strategy ensured that the active concentrations in the creams corresponded directly to the effective concentrations tested in vitro (500 µM cHCA and 400 µg/mL cRWP), thereby maintaining consistency between the in vitro and cream-based experiments.

 

- Provide a more detailed description of the microbiological assay performed on the creams, including the original references for the method employed.

Response: We appreciate the reviewer's suggestion. The requested information regarding Contact Slide 1 and Contact Slide 2 has now been added to the corresponding paragraph 2.3 in the manuscript.

 

- The presentation of statistical significance in the figures should be improved, as it is not immediately clear which comparisons are statistically significant.

Response: We thank the reviewer for this valuable remark. In the previous version, we performed one-way ANOVA for all experiments, and when significance was detected (p < 0.05), we applied Dunnett’s post-hoc test to compare all treatment groups against the untreated controls. As also noted by another reviewer, the figure legends did not clearly specify which group was used as the control. To improve clarity, we have now: (i) added further details on statistical methods in Section 2.7, (ii) revised figure captions to explicitly indicate the statistical test used in each experiment, and (iii) clarified, where appropriate, which group served as the control.

In line with this reviewer’s suggestion, for the experiments on ROS production (Figures 5 and 7), we have adopted Tukey’s post-hoc test to compare all treatment groups with each other, rather than only against the untreated cells. Statistical significance is now represented using letter-based labeling, where groups sharing the same letter are not significantly different, and groups with different letters are significantly different (p < 0.05).

- Two technical issues arise in the section “Effect of topical creams enriched with RWP or HCA on HaCaT cell proliferation”: 1) The authors state that “RWP (20 mg/5 g) and HCA (8.5 mg/5 g) were added to the basic formulation, thus obtaining a cream enriched with HCA (cHCA) or RWP (cRWP).” However, on page 9, line 254, they report: “… we evaluated the effects of 400 μg/mL cRWP and 500 μM cHCA…” Please explain how the concentrations expressed as weight of RWP and HCA in the creams correspond to the concentrations applied to the cells (400 μg/mL cRWP and 500 μM cHCA). 2) In the same section (page 9, lines 255–256), the authors report that cells were pretreated with topical formulations “dissolved in culture medium for cHCA or sterile water for RWP.” Given the hydrophilic nature of the cream, please clarify how these experiments were carried out. Was an emulsion generated?

Response: In order to test the same concentrations used in in vitro tests in topical formulations, 10x stock solutions were prepared: 4 mg/ml for rwp and 5 mm for hca. based on dr. miraglia's instructions, according to which 1 ml of cream equals 1 g and each sample would have a total weight of 5 g, the following formulations were created:

• RWP: stock solution at 4 mg/g (10x), equal to 20 mg in 5 g of cream;
• HCA: stock solution at 5 mm (10x), corresponding to 1.7 mg/g, or 8.5 mg in 5 g of cream.

Considering the solubility of rwp in water and that of hca in the culture medium, these solvents were used respectively for the emulsion of the two creams.

 

- Importantly, regarding the role of tocopherol in the formulation: the inclusion of a negative control consisting of the cream without HCA and RWP in the experiments presented in Figures 6 and 7 seems essential to rule out that the observed effects are attributable to tocopherol or other cream components rather than to HCA or RWP.

Response: We thank the reviewer for this important comment. Indeed, the suggestion to include a negative control consisting of the cream without HCA and RWP is valid. However, due to the tight timeline for this revision, it was not feasible to perform this experiment.

A brief review of the literature indicates that tocopherol has photoprotective properties, suggesting that it or other cream components could contribute to the observed effects. Nonetheless, the main objective of our study was to evaluate which of RWP or HCA, when added to the cream, could most effectively reduce blue light-induced oxidative stress, independent of the base cream itself.

In this context, it is noteworthy that RWP demonstrated a higher capacity to reduce ROS compared to HCA, likely due to the presence of multiple antioxidant compounds acting synergistically (characterization of RWP is detailed in https://doi.org/10.1155/2021/5533793). In various biological contexts — including innate immunity, inflammation, and several conditions with a strong inflammatory component (e.g., Down syndrome, MASH, sepsis) — both HCA and RWP, individually or in combination, have been shown to reduce oxidative stress.

Therefore, we expect that the base cream may contribute to some reduction of oxidative stress upon blue light exposure, but the observed effects are primarily attributable to RWP and HCA.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have revised the manuscript according to recommendations. Hence, the manuscript can be accepted for publication.

Author Response

Authors have revised the manuscript according to recommendations. Hence, the manuscript can be accepted for publication.

Response 1:      Thank you very much for your positive feedback and for recommending our manuscript for publication. We sincerely appreciate the time and effort you dedicated to reviewing our work and for your valuable suggestions, which have significantly improved the quality of the paper.

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the Authors for their kind replies to my queries, which are largely satisfactory. However, a few methodological points remain to be clarified before I can recommend final acceptance:

-In the rebuttal, the term “mm” appears in several instances, which I believe is intended to mean “mM” (millimolar). Please confirm and correct accordingly.

-I still do not fully understand how the experiments in Figures 6 and 7 were technically performed. While it is clear that RWP and HCA are soluble in cell culture medium, in these experiments, the Authors employed creams enriched with HCA (cHCA) or RWP (cRWP). I doubt that such formulations can be directly solubilized in cell culture medium. Please clarify this point and ensure that it is explicitly explained in the manuscript.

-Regarding the absence of appropriate controls for the presence of tocopherol: I remain unconvinced that the justification “due to the tight timeline for this revision, it was not feasible to perform this experiment” is fully valid. The Authors could have requested an extension in order to perform the requested experiments. Nevertheless, I will not insist further and can accept the rebuttal provided.

-Finally, while the Authors declare no conflicts of interest, it should be noted that several affiliations are linked to private sector entities. This point may warrant further consideration by the Editors.

Author Response

I thank the Authors for their kind replies to my queries, which are largely satisfactory. However, a few methodological points remain to be clarified before I can recommend final acceptance:

 

- In the rebuttal, the term “mm” appears in several instances, which I believe is intended to mean “mM” (millimolar). Please confirm and correct accordingly.

Response: We thank the reviewer for carefully pointing out this oversight. Indeed, the intended unit was “mM” (millimolar), and we have corrected all instances accordingly in the revised manuscript.

- I still do not fully understand how the experiments in Figures 6 and 7 were technically performed. While it is clear that RWP and HCA are soluble in cell culture medium, in these experiments, the Authors employed creams enriched with HCA (cHCA) or RWP (cRWP). I doubt that such formulations can be directly solubilized in cell culture medium. Please clarify this point and ensure that it is explicitly explained in the manuscript.

Response: We thank the reviewer for raising this important point. We confirm that both RWP and HCA were soluble under the experimental conditions used. Specifically, the creams were formulated using water (for RWP) and culture medium (for HCA) as vehicles, which ensured that the active compounds could be readily dispersed and solubilized when added to the cell culture medium. We have now revised the manuscript to explicitly clarify this aspect, in order to avoid any ambiguity regarding the experimental procedures. We have added this clarification in the relevant paragraph 2.5 of the revised manuscript.

 

- Regarding the absence of appropriate controls for the presence of tocopherol: I remain unconvinced that the justification “due to the tight timeline for this revision, it was not feasible to perform this experiment” is fully valid. The Authors could have requested an extension in order to perform the requested experiments. Nevertheless, I will not insist further and can accept the rebuttal provided.

Response: We sincerely thank the reviewer for their understanding and for accepting our rebuttal, and we are grateful for his flexibility and constructive feedback.

 

- Finally, while the Authors declare no conflicts of interest, it should be noted that several affiliations are linked to private sector entities. This point may warrant further consideration by the Editors.

Response: We thank the reviewer for highlighting this point. We confirm that, despite some affiliations being linked to private sector entities, there are no conflicts of interest affecting the present work, as already declared in the form submitted to the editor.

Moreover, we send the conflict of interest form signed by all authors after the submission of the manuscript, as requested by the Editor, in order to declare the no conflicts.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

No further comments