Development of Topical Formulations Containing 20% of Coated and Uncoated Zinc Oxide Nanoparticles: Stability Assessment and Penetration Evaluation by Reflectance Confocal Laser Microscopy
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsI believe this topic is of interest to a broad audience (dermatologists, patients, pharmacists, cosmetologists...). therefore, as this manuscript is well written, tables figures and literature is great, and this study is well conducted, I believe this manuscript on nano particles Zn will add to the body of literature in the cosmetology and dermatology field and that it is acceptable for publication in submitted form.
Author Response
Reviewer #1
Thank you for revising the manuscript and for your considerations.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe Authors presented an article entitled: “Development of topical formulations containing 20% of coated and uncoated zinc oxide nanoparticles: stability assessment and penetration evaluation by Reflectance confocal laser microscopy”.
The manuscript is well written and organized.
The topic is very interesting and useful for the Scientific community in the field.
In the “Materials and methods section”, sub-section “2.2.2. Determination of the hydrodynamic size by Dynamic light scattering (DLS)”, please revise the English form of the following sentence reported at line 129: “were diluted ethyl acetate”; it should be reported as “were diluted in ethyl acetate”.
Moreover, at lines 131 and 132, please, correct the error typo: “ultrassonic bath”; also at line 286 In the “Results and discussions” section.
In the “Results and discussions” section, sub-section “3.1. Characterization of ZnOn”, at lines 259, 260 and 262, please correct the error typo “ration”.
In Figure 1, the panel’s letters a, b, c, and d should be reported on the referred micrograph.
In the “Results and discussions” section, sub-section “3.2. Development and characterization of the topical formulations”, please, improve the English form of the following sentence reported at lines 347-349: “The amount of 20% of ZnOn solubilized in Caprylic Capric Triglycerides was firstly incorporation in the formulation in the end of the process of preparation, which resulted in immediate phase separation”.
In Figure 3 and 4, please, check the correspondence between the “NC” and “UC” abbreviations; in my opinion, it should be better to report the same abbreviation for avoiding misleading in the readers.
In the “Results and discussions” section, sub-section “3.2.2. Stability Evaluation”, sub-sub-section “3.2.2.1 Functional stability”, please, explain the abbreviation “FPS” reported at line 436.
In figure 6 and 7 the scale bars should be reported.
In my opinion, the Supplementary figure 1 is misleading for the readers; it should be better to report as figure 8. Thus, figure 8, scheme 2, and figure 9 should be reported as figure 9, 10, and 11, respectively. Furthermore, in the caption referred to supplementary fig.1, please, specify that the panel c is referred to the uncoated ZnOn samples.
Please, check that the following sentence reported at lines 496-497 is incomplete: “and uncoated (C) Zinc Oxide nanoparticles incubated at 45 °C for 0, 7, 30, 60 and 90 days.”
Please, check the correspondence between the Supplementary fig. 2 and the scheme 2. In this case, I suggest reporting it as figure 10 for avoiding misleading in the readers.
Please, explain the abbreviations “Z cote” and “HP1” reported in Scheme 2. Furthermore, please, check that in panels A and C the x- and y-axis are superimposed on the referred parameter denominations.
In the “Results and discussions” section, sub-section “3.4. Penetration assessment”, please, explain the abbreviation referred to the “RCM” imaging technique.
In the “Results and discussions” section, sub-section “3.4. Penetration assessment”, please, improve the English form of the following sentence reported at lines 588-590: “For many years, the penetration of topically applied substances through the stratum corneum was assumed to be diffusion inside the lipid layers surrounding the corneocytes”.
The “Conclusions” should be further improved.
Comments on the Quality of English LanguageIn the “Materials and methods section”, sub-section “2.2.2. Determination of the hydrodynamic size by Dynamic light scattering (DLS)”, please revise the English form of the following sentence reported at line 129: “were diluted ethyl acetate”; it should be reported as “were diluted in ethyl acetate”.
Moreover, at lines 131 and 132, please, correct the error typo: “ultrassonic bath”; also at line 286 In the “Results and discussions” section.
In the “Results and discussions” section, sub-section “3.1. Characterization of ZnOn”, at lines 259, 260 and 262, please correct the error typo “ration”.
In the “Results and discussions” section, sub-section “3.2. Development and characterization of the topical formulations”, please, improve the English form of the following sentence reported at lines 347-349: “The amount of 20% of ZnOn solubilized in Caprylic Capric Triglycerides was firstly incorporation in the formulation in the end of the process of preparation, which resulted in immediate phase separation”.
In the “Results and discussions” section, sub-section “3.4. Penetration assessment”, please, improve the English form of the following sentence reported at lines 588-590: “For many years, the penetration of topically applied substances through the stratum corneum was assumed to be diffusion inside the lipid layers surrounding the corneocytes”.
Author Response
Reviewer #2
Thank you for your suggestions and your interest in our study. We accepted all the suggestions, as follows:
In the “Materials and methods section”, sub-section “2.2.2. Determination of the hydrodynamic size by Dynamic light scattering (DLS)”, please revise the English form of the following sentence reported at line 129: “were diluted ethyl acetate”; it should be reported as “were diluted in ethyl acetate”.
Answer 1 129 line. Changed "were diluted ethyl acetate" to "were diluted in ethyl acetate";
Moreover, at lines 131 and 132, please, correct the error typo: “ultrassonic bath”; also at line 286 In the “Results and discussions” section.
Answer 2. 131 and 132 lines. Changed “ultrassonic bath” to “ultrasonic bath”;
In the “Results and discussions” section, sub-section “3.1. Characterization of ZnOn”, at lines 259, 260 and 262, please correct the error typo “ration”.
Answer 3When we made the suggested correction, we re-read the paragraph. We found that we could improve it. Thus, the paragraphs between 259-261 lines: "For the coated ZnOn, the size of all the nanoparticles with ration lower than 2 was equal or lower than 200 nm. For the uncoated ZnOn, 94,7% of the nanoparticles with ration lower than 2 exibited size lower than 200nm” have been changed to“ For the coated ZnOn and the uncoated ZnOn diluted less than twice, the size of all the coated nanoparticles was equal to or lower than 200 nm. While 94.7% of the uncovered nanoparticles exhibited a size smaller than 200nm"
The paragraph between 261 “For the uncoated and coated ZnOn, 81,8% and 75,7% of the nanoparticles with ration higher than 2 exhibited size higher than 100 nm” has been changed to “However, when the coated and uncoated nanoparticles were diluted more than twice, 81.8% and 75.7% of the uncoated and coated nanoparticles, respectively, were greater than 100 nm in size”.
In Figure 1, the panel’s letters a, b, c, and d should be reported on the referred micrograph.
Answer 4 Figure 1 Changed “SEM and TEM images of the uncoated (a,c) and coated (b,d) ZnOn” (268 line) to “SEM and TEM images of the uncoated (a,c) and coated (b,d) ZnOn”. The letters a, b, c and d have been referred to in the respective micrographs.
In the “Results and discussions” section, sub-section “3.2. Development and characterization of the topical formulations”, please, improve the English form of the following sentence reported at lines 347-349: “The amount of 20% of ZnOn solubilized in Caprylic Capric Triglycerides was firstly incorporation in the formulation in the end of the process of preparation, which resulted in immediate phase separation”
Answer 5 lines 347-349: “The amount of 20% of ZnOn solubilized in Caprylic Capric Triglycerides was firstly incorporation in the formulation in the end of the process of preparation, which resulted in immediate phase separation” has been changed to"Initially, in the development of the formulation, the dispersed nanoparticles in Caprylic Capric Triglycerides were incorporated at 20% into the formulation, after the emulsification of the oil phase in the aqueous phase. The addition of the ZnOn nanoparticle dispersion after the emulsification of the two phases led to the immediate separation of these phases.
In Figure 3 and 4, please, check the correspondence between the “NC” and “UC” abbreviations; in my opinion, it should be better to report the same abbreviation for avoiding misleading in the readers
Answer6 Thank you, your suggestion will make the figure captions clearer. We have changed everything to C, coated ZnOn nanoparticles and UC, uncoated ZnOn nanoparticles.
In the “Results and discussions” section, sub-section “3.2.2. Stability Evaluation”, sub-sub-section “3.2.2.1 Functional stability”, please, explain the abbreviation “FPS” reported at line 436.
Answer 7 Line 436. Thank you for your suggestion. It really isn't correct. We carried out an in vitro test to assess the ability of the ZnOn formulation to absorb UVA and UVB radiation. Therefore, we cannot refer to the Sun Protection Factor (SPF), which is an in vivo test. We have modified the text as follows: “The absorption capacity of UVA/B radiation by the vehicle of the formulations was very similar to the control, indicating that the components of the vehicle do not absorb this radiation. The coated and uncoated ZnOn nanoparticles are responsible for the absorption”.
In figure 6 and 7 the scale bars should be reported.
Answer 8 We agree with the suggestion. Unfortunately, we carried out the tests in another laboratory on the Ribeirão Preto Campus and this informationwas not given, nor did we request it.
In my opinion, the Supplementary figure 1 is misleading for the readers; it should be better to report as figure 8. Thus, figure 8, scheme 2, and figure 9 should be reported as figure 9, 10, and 11, respectively. Furthermore, in the caption referred to supplementary fig.1, please, specify that the panel c is referred to the uncoated ZnOn samples.
Answer 9 We agree with the suggestion. The figures have been renumbered and we have specified that panel C in figure 8 relates to the data from the uncoated ZnOn samples.
Please, check that the following sentence reported at lines 496-497 is incomplete: “and uncoated (C) Zinc Oxide nanoparticles incubated at 45 °C for 0, 7, 30, 60 and 90 days.”
Answer 10:It was really a typo. Line 496 is part of the title of the Supplementary figure 1 (Figure 8). Therefore, the phrase “and uncoated Zinc Oxide nanoparticles (C) incubated ate 45ºC for 0, 7, 30, 60 and 90 days” will be included in the title of Supplementary figure 1 (Figure 8).
Please, check the correspondence between the Supplementary fig. 2 and the scheme 2. In this case, I suggest reporting it as figure 10 for avoiding misleading in the readers.
Answer 11.We agree with the suggestion. There is a correspondence between Supplementary fig.2 and the scheme 2. It would therefore be more appropriate to list the figure as figure 10.
Please, explain the abbreviations “Z cote” and “HP1” reported in Scheme 2. Furthermore, please, check that in panels A and C the x- and y-axis are superimposed on the referred parameter denominations
Answer 12.Thank you for your comment. Z-cote corresponds to the raw material, coated ZnOn nanoparticle, and HP1, uncoated ZnOn nanoparticle. In order to standardize the abbreviations, formulations with Z cote will be called C formulation, and those with HP1 will be designated UC. Overlaps on the x and y axes will be corrected.
In the “Results and discussions” section, sub-section “3.4. Penetration assessment”, please, explain the abbreviation referred to the “RCM” imaging technique.
Answer 13.The Reviewer is right, once again. The abbreviation RCM, Reflectance Confocal Microscopy, was not cited in either Methods or Results. We've added it in both parts.
In the “Results and discussions” section, sub-section “3.4. Penetration assessment”, please, improve the English form of the following sentence reported at lines 588-590: “For many years, the penetration of topically applied substances through the stratum corneum was assumed to be diffusion inside the lipid layers surrounding the corneocytes”.
Answer 14. Thank you. We have tried to improve the text, but it has been taken from published works. We have tried to interpret the text as faithfully as possible. However, we have made small changes to improve understanding.
“The penetration of substances applied topically to the skin occurs by diffusion through the lipid layer around the corneocytes. However, in recent decades some researchers have shown that hair follicles play an important role in the penetration of substances through the stratum corneum, in addition to the lipid layer”.
The “Conclusions” should be further improved.
Answer 15. We agree, the conclusion could be improved. We rewrite the conclusion as follows:
“The results of the evaluation of formulations C and UC and the vehicle by zeta potential measurement showed lower potential values than the vehicle. Texturometer analysis showed that the formulations with ZnOn nanoparticles coated and uncoated had higher firmness, cohesiveness, consistency and viscosity index values than the vehicle, but the spreadability values were similar to the vehicle. These results were confirmed by the rheological analysis data, which showed pseudoplastic behavior of both formulations and the vehicle. The formulations added with coated and uncoated ZnOn nanoparticles showed similar physicochemical properties.
Evaluation of the physicochemical stability of the formulations showed that both formulations have a decrease in their rheological properties, consistency index, apparent viscosity and hysteresis area, when stored at 45ºC during 7, 15, 30, 60 and 90 days. The formulation added with coated nanoparticle ZnOn showed a greater ability to absorb UVA/UVB radiation when compared to the formulation with uncoated nanoparticles. Therefore, the absorbances observed for the formulations were due to the ZnOn nanoparticles, indicating that the raw materials have the functional capacity to absorb UVA/UVB radiation. The coated ZnOn nanoparticles incorporated into the formulation accumulated in the furrows and hair follicles, but were not found inside the keratinocytes, which suggests that they do not penetrate in the viable epidermis”.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe letters a and b of the related panels in Figure 1 panels are not visible, please check them and improve the visibility; please, also check the panel c of Figure 2.
Usually, the scale bars must be reported in the micrographs prepared for a manuscript to be published; so, you should ask the information about the related scale bars to your colleagues and must report it in figure 6 and 7. Also in Figure 11 the scale bars and the magnification must be reported.
In Figure 9, please check and correct the English form of the reported names, particularly when referring to the vehicle, which has been reported as “veiculo”, and the related caption must be revised, accordingly; furthermore, please, also improve the quality of the figure which it is not so visible, particularly the y-axis.
The quality of Figure 10 must be improved before the publication because is poorly visible.
Comments on the Quality of English LanguageIn Figure 9, please check and correct the English form of the reported names, particularly when referring to the vehicle, which has been reported as “veiculo”, and the related caption must be revised, accordingly; furthermore, please, also improve the quality of the figure which it is not so visible, particularly the y-axis.
Author Response
We thank the reviewer for his careful considerations
- The visual quality of figure 1 A and B has been improved, and figure 2 C has been checked, but we found nothing wrong.
- Figures 6, 7 and 11 have been assessed and justified. The bars were added in Figure 1 because it is a standard representation of SEM and TEM images since not all the microscopical pictures indicate the size of what is being analyzed in the picture. We do not have the original files to add the bars in Figures 6 and 7. The files were sent to our lab as images because we didn't have the program coupled with the microscope. The partner lab doesn't have the original file anymore because they delete the files annually. We don't believe the lack of the bar would result in a compromised understanding of the images or the results because it was not a quantitative analysis. We didn't intend to assess the nanoparticle size, since it would be impossible in an inverted microscope. We merely intended a qualitative assessment of the formulations' stability. The legends of the figures indicate the magnification that was used and the methodology section describes all the details needed to reproduce the experiment.
Figure 11 wasn't a qualitative analysis either. We merely intended to show where the formulation accumulated. Traditionally, RCM of skin doesn't need bars because the depth of the image is indicated by what structures are visible. Again, we can't add the bar because it needs to be done in the equipment during the analysis.
- Figure 9. The abbreviations in figure 9 have been checked and the English corrected.
- Figure 10 . The visibility of figure 10 has been improved
