Review Reports - Bemotrizinol-Loaded Lipid Nanoparticles for the Development of Sunscreen Emulsions

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

Manuscript accepted in this version.

Author Response

Manuscript accepted in this version.

We would like to thank the reviewer for taking the time to review our manuscript.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Research in the field of sun protection and formulation based on nanotechnology is highly studied these days. This manuscript proposes the inclusion of bemotrizinol, a photoprotective active ingredient, in a nanostructured lipid system, and the DSC evaluation of the influence of the composition on the possibility of a higher percentage of active ingredient loading in the system. Additionally, the nanoparticles were studied for size, polydispersity index, and morphology. These nanoparticles were subsequently incorporated into an emulsion. The SPF, release, and permeation of the emulsified systems with and without nanoparticles were evaluated. Additionally, the stability of these systems and of the dispersions containing the nanoparticles were also assessed.

Similar studies have been conducted with other photoprotective agents, including those developed by the same research group, which are referenced throughout this study. Next, I have some comments about the weaknesses of the manuscript.

Major comments

Objective

The objective of this study is unclear, as it is presented in a disorganized manner in the introduction, referring more to a methodological summary than to an objective. It is unclear what knowledge gap this research seeks to fill.

Methodology

The methodology is not described in detail. In many sections, it is unclear what the methodological conditions were based on; often, they only mention is a study the research group conducted. For example, what was the basis for defining the composition of the emulsion and its processing conditions? What was the basis for defining the conditions for the DSC tests? Stability studies mention room temperature, but they do not define what that temperature was and whether it was controlled or not. Additionally, these studies do not establish the number of replicates or at least repetitions in the tests performed. The focus of the stability study was on pH, viscosity, and appearance; it was important to monitor chemical stability or its SPF, considering that BMZ is unstable at high temperatures, such as those it was subjected to in the nanoparticle production process. Ideally, a statistical experimental design would have been implemented, or at least response variables would have been available to support this type of questioning.

Why do pseudo-sink conditions mean? Sink conditions were not discussed or demonstrated in the release and permeation tests. How long was the skin stored at 4°C? The loading efficiency assessment is justified by the translucency of the system, ensuring that no precipitation occurs in the external phase. I believe that at least an assessment of the active ingredient concentration in the dispersion should have been considered, and the difference in concentrations between the external and the trapped phase should have corroborated what is being stated.

The methodology doesn't explain in detail how the photoprotection assessment was performed using the Mansur equation, especially when using the emulsion system that included the nanoparticles. Was there an extraction process for the active ingredient? Was the dispersion translucent at the time of reading? As I understand it, only the effect of the presence of suspended nanoparticles is being evaluated.

Results

Regarding the evaluation of the photoprotective effect of the formulation, it is not clear to me what improvement is proposed by including the nanoparticulate system containing the active ingredient, since no statistical evaluation is carried out to determine whether they are the same or different.

Regarding occlusivity, they say there was no influence of the composition on the occlusivity attributed to the added oils. There was no statistical analysis to confirm this, but I don't see that the test performed was sensitive enough to confirm this either.

The results of the stability study at the different sampling times proposed in the methodology were not presented and therefore no discussion was held on the matter.

In the release studies, it is mentioned that the sensitivity of the analytical method did not allow quantification before three hours. Is the analytical method adequate? On the other hand, would the lack of sink conditions be a consequence of this?

Regarding the results of the permeation studies, it is mentioned that the active ingredient did not pass into the receptor compartment, which would support the product's safety. This statement should be evaluated, considering that the sink conditions may not have existed, especially considering the active ingredient's low solubility in water. Of course, this is a hydroalcoholic medium and not only water, but it would be necessary to demonstrate that these conditions were met.

Conclusions

Since the research problem addressed by this study is not entirely clear, the conclusions focus on the specific aspects achieved in each of the trials conducted.

Author Response

REVIEWER 2

Answer

We would like to thank the reviewer for taking the time to review our manuscript.

Major comments

Objective

Answer

Thank you for your comment. The objective of the study has been revised to be more concise in order to improve clarity and accessibility.

Methodology

The methodology is not described in detail. In many sections, it is unclear what the methodological conditions were based on; often, they only mention is a study the research group conducted.

Answer

As reported in the Instructions for authors of the Journal Colloids and Interfaces, new methods and protocols should be described in detail while well-established methods can be briefly described and appropriately cited. Therefore, to comply with the Instructions for authors, we reported only a brief description of the experiments that we have already described in details in previous publications.

For example, what was the basis for defining the composition of the emulsion and its processing conditions?

Answer

The ingredients used to prepare the emulsions were chosen because they are:

Commonly used to prepare cosmetic emulsions
Easily available in the market
Low-cost

Emulsions were prepared using the method that is normally used to obtain this type of formulations.

What was the basis for defining the conditions for the DSC tests?

Answer

DSC tests were performed in the same experimental conditions we used in previous works to characterize lipid nanoparticles.

Stability studies mention room temperature, but they do not define what that temperature was and whether it was controlled or not. Additionally, these studies do not establish the number of replicates or at least repetitions in the tests performed. The focus of the stability study was on pH, viscosity, and appearance; it was important to monitor chemical stability or its SPF, considering that BMZ is unstable at high temperatures, such as those it was subjected to in the nanoparticle production process. Ideally, a statistical experimental design would have been implemented, or at least response variables would have been available to support this type of questioning.

According to the literature [Doiron T. 20 °C-A Short History of the Standard Reference Temperature for Industrial Dimensional Measurements. J Res Natl Inst Stand Technol. 2007 Feb 1;112(1):1-23. doi: 10.6028/jres.112.001], the room temperature is defined at 20 °C. Therefore, the stability studies were conducted at 20 ± 1 °C, and each measurement was performed in triplicate. Of course, the temperature was controlled. This information has now been included in Section 2.5. A recent study (Determination of Bemotrizinol degradation in different stress condition by high performance liquid chromatography in sunscreen preparations, https://doi.org/10.21203/rs.3.rs-2629843/v1) demonstrated BMTZ stability at temperatures as high as 105°C for six hours. Medeiros et al., 2020 (reference 23 in the manuscript) reported that bemotrizinol remained stable during typical cosmetic manufacturing processes, maintaining its efficacy when exposed to moderate temperatures for short durations. Therefore, in the experimental conditions we used no BMTZ thermal degradation could be supposed to occur. Furthermore, its encapsulation within delivery systems, such as nanostructured lipid carriers (NLCs), can enhance the overall stability of the active compound by protecting it from thermal degradation and photo-oxidative stress (Santonocito et al., reference 52 in the manuscript).

Why do pseudo-sink conditions mean? Sink conditions were not discussed or demonstrated in the release and permeation tests.

Answer

The term "sink conditions" refers to a situation where the concentration of a drug in a solution is kept below a certain threshold, typically less than 10% of its maximum solubility. Sink conditions are ensured when the drug is continuously absorbed or removed from the system without reaching saturation. In in vitro skin permeation experiments, as BMTZ did not permeate, sink conditions were undoubtedly met, as no accumulation of the UV-filterg occurred in the receiving solution. In in vitro release experiments, the use of a receiving consisting of water/ethanol 50/50 ensured skin conditions as BMTZ is soluble in ethanol. In the manuscript, the term "pseudo-sink conditions" is used to describe a system that mimics in vivo conditions, but being static it lacks the typical biological dynamics of the human body.

How long was the skin stored at 4°C?

Answer

The skin (SCE) was stored at 4 ± 1 °C for a period not exceeding three months. This information has been inserted in the text.

The loading efficiency assessment is justified by the translucency of the system, ensuring that no precipitation occurs in the external phase. I believe that at least an assessment of the active ingredient concentration in the dispersion should have been considered, and the difference in concentrations between the external and the trapped phase should have corroborated what is being stated.

Answer

As reported in the manuscript, the loading capacity was assessed as the maximum amount of BMTZ that could be encapsulated into NLC providing a colloidal suspension that did not show signs of precipitation, as already reported for other active ingredients. Bemotrizinol is a water-insoluble compound being its water solubility less than 5 ng/L at 25°C according to the Scientific Committee on Consumer Safety. When a water-insoluble compound is incorporated into lipid nanoparticles, if the resulting colloidal dispersion is clear, the maximum amount of compound that could be present in the water phase of the colloidal suspension is the amount corresponding to its water solubility while all compound is virtually in the lipid core of the nanocarrier. Therefore, in clear colloidal dispersion, the incorporation rate (entrapment efficiency) of a water-insoluble substance is, for all practical purposes, 100%. In such conditions, the experimental determination of the entrapment efficiency by separating the nanocarrier from the aqueous phase is a mere academic exercise that would not provide any practical benefit. We explain this concept by taking into account BMTZ incorporation into the investigated NLC. When we added 7% w/w of BMTZ into the colloidal suspension and we obtained a clear dispersion, we had maximum 0.005 ng/ml of BMTZ in the aqueous phase and about 7x10⁷ ng/ml in the lipid core of the NLC. Therefore, when we obtained clear lipid nanoparticles dispersions, we considered that all added drug was entrapped into the nanoparticle lipid core and the entrapment efficiency was practically 100%.

The methodology doesn't explain in detail how the photoprotection assessment was performed using the Mansur equation, especially when using the emulsion system that included the nanoparticles. Was there an extraction process for the active ingredient? Was the dispersion translucent at the time of reading? As I understand it, only the effect of the presence of suspended nanoparticles is being evaluated.

Answer

The assessment of photoprotection was performed using the Mansur equation, which is a well-established in vitro method to estimate the SPF (Sun Protection Factor) of sunscreen formulations. This method relies on measuring the UV absorbance of the product in a specific wavelength range (290–320 nm) and applying a mathematical equation (equation 3) that takes into account the erythemal effectiveness (EE) of the UV radiation, the solar irradiance (I), and the absorbance of the formulation at each wavelength. To perform the measurement, we properly diluted the emulsions containing free BMTZ and BMTZ-NLC in deionized water. We did not extract the active ingredient or nanoparticles from the formulations. The absorbance was then measured in the UV-Vis range (290–320 nm) using a spectrophotometer. The resulting absorbance data was input into the Mansur equation to estimate the SPF of the formulation. Thus, by following this standard procedure, we were able to estimate the SPF of the nanoparticle-enriched emulsion, and the obtained values reflect the photoprotection properties of the formulation. This method is widely used for quick and effective SPF estimation and is particularly useful for comparative analysis between different sunscreen formulations, making it ideal for this study. The samples we analyzed by spectrophotometry were perfectly clear. Translucent samples could not be analyzed spectrophotometrically.

Results

Regarding the evaluation of the photoprotective effect of the formulation, it is not clear to me what improvement is proposed by including the nanoparticulate system containing the active ingredient, since no statistical evaluation is carried out to determine whether they are the same or different.

Answer

As reported in literature [Wissing, S.A.; Lippacher, A.; Muller, R.H. Investigations on the occlusive properties of solid lipid nanoparticles (SLN). Journal of Cosmetic Science 2001, 52 (5), 313–324; Pissavini, M.; Tricaud, C.; Wiener, G.; Lauer, A.; Contier, M.; Kolbe, L.; Trullás Cabanas, C.; Boyer, F.; Meredith, E.; de Lapuente, J.; Dietrich, E.; Matts, P.J. Validation of a new in vitro Sun Protection Factor method to include a wide range of sunscreen product emulsion types. International Journal of Cosmetic Science 2020, 42 (5), 421-428], formulations containing UV-filters loaded into lipid nanoparticles showed an improvement (about 20%) of SPF value in comparison with the corresponding emulsions containing the same amount of free UV-filters. These results support the ability of lipid nanoparticles to behave as UV-blockers. Therefore, incorporating BMTZ into suitable NLC could be a promising strategy to develop sunscreen formulations with reduced content of synthetic UV-filter while achieving higher SPF values. The results of our study are in good agreement with the data reported in the literature as about a 20% increase of SPF values was observed. To address the reviewer’s concern about the lack of statistical analysis, we inserted in the text the following sentences: “In particular, all formulations containing free BMTZ or BMTZ loaded into NLC showed SPF values that were significantly different (p<0.05) compared with the blank emulsion. Statistically significant differences (p<0.05) were observed for the following comparisons: emulsion B vs emulsion B_NLC, emulsion C vs emulsion C_NLC, emulsion D vs emulsion D_NLC.

Regarding occlusivity, they say there was no influence of the composition on the occlusivity attributed to the added oils. There was no statistical analysis to confirm this, but I don't see that the test performed was sensitive enough to confirm this either.

Answer

The in vitro occlusion test is a well-recognized method for assessing the occlusive properties of a formulation by predicting its ability to reduce transepidermal water loss (TEWL). This test provides reliable, reproducible, and objective data on how effectively a product forms a barrier on the skin, helping to improve hydration. Using this test allows for consistent comparisons between different formulations, and it offers an efficient way to evaluate the occlusive effect early in the development process. We did not specify the results of the statistical analyses because we thought that it was clear that there were not statistically significant differences among the obtained data. To address the criticism raised by the reviewer, we inserted in the text the following statement: “No statistically significant difference among the occlusion factor values was observed (p>0.05)”.

The results of the stability study at the different sampling times proposed in the methodology were not presented and therefore no discussion was held on the matter.

Answer

To address the reviewer’s comment, we inserted in the text the following sentence: “All formulations showed good stability at room temperature as no significant changes of appearance, pH and viscosity was observed at all sampling times (data not shown).

In the release studies, it is mentioned that the sensitivity of the analytical method did not allow quantification before three hours. Is the analytical method adequate? On the other hand, would the lack of sink conditions be a consequence of this?

Answer

As reported in the manuscript, the analytical method we used as a limit of detection of 0.01 μg/mL that is similar to that of an HPLC method that performs analyte detection spectrophotometrically. Pseudo-sink conditions were ensured by the use of a receiving phase consisting of water/ethanol 50/50 as BMTZ is soluble in ethanol.

Regarding the results of the permeation studies, it is mentioned that the active ingredient did not pass into the receptor compartment, which would support the product's safety. This statement should be evaluated, considering that the sink conditions may not have existed, especially considering the active ingredient's low solubility in water. Of course, this is a hydroalcoholic medium and not only water, but it would be necessary to demonstrate that these conditions were met.

Answer

Other authors, performing an in vitro skin permeation study on BMTZ and other UV-filters, showed that no BMTZ permeation was observed after 24 h (Souza C, Maia Campos PMBG. Development of a HPLC method for determination of four UV filters in sunscreen and its application to skin penetration studies. Biomed Chromatogr. 2017 Dec;31(12). doi: 10.1002/bmc.4029). The results of our experiments are in perfect agreement with the data reported by Souza and Campos. Therefore, the lack of BMTZ skin permeation could not be attributed to the absence of sink conditions but it depended on BMTZ poor ability to permeate through the skin.

Conclusions

Since the research problem addressed by this study is not entirely clear, the conclusions focus on the specific aspects achieved in each of the trials conducted.

Answer

The aim of the work has been better explained, therefore in the revised version of the manuscript the conclusions should be clearer.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Query:

Have you tested the PIT method on larger batches to confirm its scalability?
Since the zeta potential is around -10 mV, how do you ensure long-term stability of the NLCs?
Is a 4-hour duration sufficient for the skin permeation study considering sunscreen application times?
Did you compare the in vitro SPF values with any in vivo data or validated methods?
Did you evaluate the effect of varying Brij 72 and Brij 721 concentrations on NLC properties?
Are the DSC thermal transitions consistent across multiple batches?
Did you calculate the encapsulation efficiency of BMTZ in the NLCs?
Why was a cellulose membrane used for release studies instead of a more skin-like membrane?
Was data normality tested before applying the Student’s t-test?
Have you tested the photostability of BMTZ in NLC compared to free BMTZ?

Suggestions

Include scalability data or discuss potential scale-up challenges of the PIT method for industrial application.
Provide additional stability studies (e.g., centrifugation, freeze-thaw cycles) to support the use of NLCs with low zeta potential.
Extend the skin permeation study duration or include a justification for the 4-hour time frame.
Validate the in vitro SPF results by comparing them with in vivo SPF values or regulatory standards.
Investigate the effect of different surfactant-to-lipid ratios on particle size, stability, and skin feel.
Report batch-to-batch consistency for DSC curves to support reproducibility of thermal behavior.
Clearly differentiate between drug loading and encapsulation efficiency and provide data for both.
Consider using synthetic membranes like Strat-M® or porcine skin for more biologically relevant release studies.
Perform ANOVA or other suitable statistical tests for multi-sample comparisons and confirm assumptions of normality.
Add a photostability study to evaluate whether BMTZ remains stable under UV exposure when encapsulated in NLC.

This is a well-executed and relevant study that combines innovative nanocarrier formulation with practical sunscreen application. The experimental design is detailed and methodical, covering multiple formulation parameters and characterization techniques.

However, a few improvements could strengthen the manuscript:

Photostability and long-term NLC stability need further attention.
Encapsulation efficiency and in vivo SPF correlation would add depth.
Justification of certain choices, like membrane type or statistical methods, would improve scientific rigor.

The manuscript contributes meaningfully to the field of cosmetic nanotechnology and shows promise for developing safer, more effective sunscreens. With some additional data and clarifications, it could be a strong candidate for publication.

Author Response

REVIEWER 3

We would like to thank the reviewer for taking the time to review our manuscript.

Have you tested the PIT method on larger batches to confirm its scalability?

Answer

We did not test the PIT method on larger batches. However, the scalability of the PIT method has already been reported in the literature (Anton N, Benoit JP, Saulnier P. Design and production of nanoparticles formulated from nano-emulsion templates-a review. J Control Release. 2008 Jun 24;128(3):185-99. doi: 10.1016/j.jconrel.2008.02.007).

Since the zeta potential is around -10 mV, how do you ensure long-term stability of the NLCs?

Answer

a variation of temperature. The so-called transitional phase inversion occurs, when, at a fixed composition, the relative affinity of the surfactant for the different phases is changed and controlled by the temperature”. The resulting nanoemulsion shows a great stability.” This great stability is due to the fact that the steric stabilization prevents the droplets flocculation and therefore coalescence”. As reported in the manuscript, in a previous work [29], the good stability of NLC with ζ-potential values lower than 30 mV was attributed to a steric stabilization provided by the long polyoxyethylene chains of oleth-20 located on the surface of the nanoparticles. Steric stabilization of nanoparticle suspensions has already been well described in the literature (Tadros, T. (2013). Steric Stabilization. In: Tadros, T. (eds) Encyclopedia of Colloid and Interface Science. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-20665-8_146).

Is a 4-hour duration sufficient for the skin permeation study considering sunscreen application times?

Answer

Under normal conditions of use, sunscreen formulations are not supposed to remain on the skin surface for a period exceeding four hours. In fact, consumers are recommended to apply frequently the product to ensure a proper photoprotective activity. In addition, other authors, performing a skin permeation study on BMTZ and other UV-filters, showed that no BMTZ permeation was observed after 24 h (Souza C, Maia Campos PMBG. Development of a HPLC method for determination of four UV filters in sunscreen and its application to skin penetration studies. Biomed Chromatogr. 2017 Dec;31(12). doi: 10.1002/bmc.4029).

Did you compare the in vitro SPF values with any in vivo data or validated methods?

Answer

As reported in the manuscript, we compared the in vitro SPF values of the emulsions containing free BMTZ with the in-silico SPF values calculated using the software BASF sunscreen simulator. As shown in the manuscript, we observed a very good agreement between the in vitro SPF values and the in-silico SPF, thus supporting the reliability of the in vitro method we used.

Did you evaluate the effect of varying Brij 72 and Brij 721 concentrations on NLC properties?

Answer

To prepare O/W emulsions, we used the percentages of Brij72 and Brij 721 that are commonly used to obtain stable formulations. We did not change the percentages of these emulsifiers as such changes could result in lower stability of the emulsion. However, NLC were incorporated into the formulations after obtaining the emulsion. Therefore, no effect of the emulsifier systems could be expected.

Are the DSC thermal transitions consistent across multiple batches?

Answer

We tested different batches of the same NLC suspensions and we obtained reproducible results.

Did you calculate the encapsulation efficiency of BMTZ in the NLCs?

Answer

As reported in the manuscript, loading capacity was assessed as the maximum amount of BMTZ that could be encapsulated into NLC providing a colloidal suspension that did not show signs of precipitation, as already reported for other active ingredients. Bemotrizinol is a water-insoluble compound being its water solubility less than 5 ng/L at 25°C according to the Scientific Committee on Consumer Safety. When a water-insoluble compound is incorporated into lipid nanoparticles, if the resulting colloidal dispersion is clear, the maximum amount of compound that could be present in the water phase of the colloidal suspension is the amount corresponding to its water solubility while all compound is virtually in the lipid core of the nanocarrier. Therefore, in clear colloidal dispersion, the incorporation rate (entrapment efficiency) of a water-insoluble substance is, for all practical purposes, 100%. In such conditions, the experimental determination of the entrapment efficiency by separating the nanocarrier from the aqueous phase is a mere academic exercise that would not provide any practical benefit. We explain this concept by taking into account BMTZ incorporation into the investigated NLC. When we added 7% w/w of BMTZ into the colloidal suspension and we obtained a clear dispersion, we had maximum 0.005 ng/ml of BMTZ in the aqueous phase and about 7x10⁷ ng/ml in the lipid core of the NLC. Therefore, when we obtained clear lipid nanoparticles dispersions, we considered that all added drug was entrapped into the nanoparticle lipid core and the entrapment was practically 100%.

Why was a cellulose membrane used for release studies instead of a more skin-like membrane?

Answer

As pointed out in the very initial studies on dermal and transdermal drug delivery, in vitro release and in vitro skin permeation are two different steps in the percutaneous absorption process (Wester RC, Maibach HI. Cutaneous pharmacokinetics: 10 steps to percutaneous absorption. Drug Metab Rev. 1983;14(2):169-205. doi: 10.3109/03602538308991388). In vitro release refers to a method used to study the release of a drug from the vehicle in which the drug is incorporated (e.g. cream, gel, lotion etc.). After topical application, a drug must first be released from the vehicle to penetrate through the skin. If no drug release occurs, no drug skin penetration/permeation could be expected. Generally, in vitro release studies are performed using dialysis membranes with an adequately large molecular weight cut off (MWCO) to allow drug transport. For instance, Gupta and Trivedi (Vandana Gupta, Piyush Trivedi, Chapter 15 - In vitro and in vivo characterization of pharmaceutical topical nanocarriers containing anticancer drugs for skin cancer treatment,Editor(s): Alexandru Mihai Grumezescu, Lipid Nanocarriers for Drug Targeting, William Andrew Publishing, 2018, Pages 563-627, ISBN 9780128136874, https://doi.org/10.1016/B978-0-12-813687-4.00015-3) evaluated the in vitro drug release for topical nanocarriers by Franz diffusion cell using Spectra/Por regenerated cellulose membrane of MWCO 10,000 g/mol and discussed the different methods applied to measure drug release from nano-sized drug delivery systems. All methods involved the use of dialysis membranes, like Spectra/Por.

Therefore, we performed in vitro release studies using the type of membrane that is normally used in this type of experiments.

We performed in vitro skin permeation experiments using human skin samples as such samples were available in our labs. Therefore, we had no need to use synthetic membranes like Strat-M® that are used when human skin samples are not available. In addition, we point out that our research group works in the European Union and that the European legislation does not allow to test cosmetic ingredients or cosmetic formulations on animals.

Was data normality tested before applying the Student’s t-test?

Answer

Data normality was tested before applying the Student’s t-test, although we did not specify this evaluation in the text. As we had small sample sizes, we thought it was understood that data normal distribution had been checked prior to performing the Student’s t-test. To comply with the reviewer’s request, we inserted this information in the text.

Have you tested the photostability of BMTZ in NLC compared to free BMTZ?

Answer

A recent study demonstrated the high photostability and thermal stability of BMTZ (Determination of Bemotrizinol degradation in different stress condition by high performance liquid chromatography in sunscreen preparations, https://doi.org/10.21203/rs.3.rs-2629843/v1). Therefore, in the experimental conditions we used, no BMTZ photodegradation could be supposed to occur.

Suggestions

Include scalability data or discuss potential scale-up challenges of the PIT method for industrial application.

We have already answered this comment at point 1.

Provide additional stability studies (e.g., centrifugation, freeze-thaw cycles) to support the use of NLCs with low zeta potential.

We have already addressed this comment at point 2.

Extend the skin permeation study duration or include a justification for the 4-hour time frame.

We have already addressed this comment at point 3.

Validate the in vitro SPF results by comparing them with in vivo SPF values or regulatory standards.

We have already addressed this comment at point 4.

Investigate the effect of different surfactant-to-lipid ratios on particle size, stability, and skin feel.

As reported in the literature (Anton N, Gayet P, Benoit JP, Saulnier P. Nano-emulsions and nanocapsules by the PIT method: an investigation on the role of the temperature cycling on the emulsion phase inversion. Int J Pharm. 2007 Nov 1;344(1-2):44-52. doi: 10.1016/j.ijpharm.2007.04.027), using the PIT method, stable nanoemulsions are obtained only when a proper balance between surfactants and lipids is achieved. When we started working on lipid nanoparticles, we investigated different types of surfactants and lipids to obtain lipid nanoparticles with good technological properties (small sizes, low polydispersity index, good stability under storage). These investigations allowed us to set the optimal type of surfactant and lipid and their concentrations that we used to perform the work under review. Therefore, using different surfactant-to-lipid ratios would not provide lipid nanoparticles with suitable technological properties.

Report batch-to-batch consistency for DSC curves to support reproducibility of thermal behavior.

Batch-to batch consistency has been inserted in the manuscript.

Clearly differentiate between drug loading and encapsulation efficiency and provide data for both.

We have already addressed this comment at point 7.

Consider using synthetic membranes like Strat-M® or porcine skin for more biologically relevant release studies.

We have already addressed this comment at point 8

Perform ANOVA or other suitable statistical tests for multi-sample comparisons and confirm assumptions of normality.

We have already addressed this comment at point 9.

Add a photostability study to evaluate whether BMTZ remains stable under UV exposure when encapsulated in NLC.

We already addressed this comment at point 10.

However, a few improvements could strengthen the manuscript:

Photostability and long-term NLC stability need further attention.

Encapsulation efficiency and in vivo SPF correlation would add depth.

Justification of certain choices, like membrane type or statistical methods, would improve scientific rigor.

Reviewer 4 Report (New Reviewer)

Comments and Suggestions for Authors

Comments are attached on file

Comments for author File: Comments.pdf

Author Response

REVIEWER 4

The manuscript (submitted to Colloids and Interfaces) Bemotrizinol-loaded lipid nanoparticles for the development of sunscreen emulsions Sarpietro M.G. et al., describes the use of nanostructured lipid carriers as bemotrizinol delivery in skin applications. In manuscript, process of preparation and evaluation of bemotrizinol-loaded NLC and sunscreen emulsions was described. In my opinion, the work presents an interesting approach to a new form of bemotrizinol formulation for skin applications, but it contains some major shortcomings that should be correct

Answer

We would like to thank the reviewer for taking the time to review our manuscript.

1) Please explain why such concentrations of bemotrizinol were chosen? Are any products with this substances available on the pharmaceutical or cosmetic market? If so, in what concentrations?

Answer

Bemotrizinol is highly photostable and thermally stable UV-filter that is widely used in commercial sunscreen products. Our research group works in the European Union. The maximum bemotrizinol concentration in cosmetic products that is allowed by the European legislation is 10% w/w. Therefore, we performed our study using bemotrizinol concentrations lower than 10% w/w.

2) Why did the authors use emulsifier (Oleth-20) at a concentration of 8.7% ? Was any previous research done to assess this amount?

Answer

Why DSC study was performed from 10 to 60 C, while bemotrizinol melting point according to the literature is around 80 . DSC analysis at a higher temperature could allow detailed determination of the thermal behavior of drug and NLC.

Answer

When the first experiments were carried out, the scans were performed from 10 to 85°C; however, a flat line was obtained from about 50 °C to 85 °C, apart from the scan performed on pure bemotrizinol. Considering that the crucible may open at elevated temperatures during the scan—requiring the measurement to be repeated—and that temperatures above 60 °C would not provide additional information, the scans were performed up to 60 °C.

Interactions between NLC components should be confirmed by other methods, e.g. FTIR analysis.

Answer

The use of other techniques to investigate the interactions among NLC components was beyond the scope of this work. In addition, due to the nature of the ingredients used to obtain NLC, FTIR analyses would not provide useful information. In fact, oleth-20 (the surfactant used to prepare NLC) contains a long polyoxyethylene chain (20 units) and a long unsaturated alkyl chain (18 C). The FTIR spectrum of this molecule would show a large and broad absorption band in the range 3200-3600 cm⁻¹, many bands in the range 2850-3000 cm⁻¹ due to the stretching vibrations of the aliphatic C-H bonds in the oleyl chain and the polyethylene glycol chain and absorption bands in the range 1000-1500 cm⁻¹ due to the ether bond in the polyethylene glycol chain (C-O-C), the C-O bond in the oleyl alcohol and bending vibrations of the methylene (CH₂). These absorption bands would cover those of bemotrizinol making the interpretation of NLC component interactions a hard task.

The authors should conduct and present DSC curves for pure ingredients and drug (attached in the Supplementary Files). Please improve the quality of the figures presenting DSC curves.

Answer

As requested by the reviewer, we inserted the DSC curves of cetyl palmitate and bemotrizinol in the Supplementary files. DSC curves of other components have not been reported as they produced a flat line.

In addition, we improved the quality of the figures showing DSC curves.

4) Please prepare a graphic of the preparation method of o/w emulsion, in Section 2.6. This

will allow to clearly present the described method.

Answer

Unfortunately, it is unclear what type of graph we are supposed to prepare. We prepared O/W emulsions using the commonly used method that is reported in any textbook dealing with pharmaceutical technology.

Therefore, we believe that a graph depicting such a trivial method is not suitable for a research article.

5) Section 2.10 why was the viscosity measurement performed 1h after placing the sample in the instrument? Why was this assay not conducted immediately after placing the sample?

Using a mixture of water and ethanol (50% ethanol) is not a suitable acceptor fluid to ensure sink conditions. Ethanol is a known permeation enhancer and can affect the obtained results. This acceptor fluid does not reflect the physiological environment. Surfactants are a better choice as component of the acceptor fluid.

Answer

We performed viscosity measurements 1h after placing the sample in the instrument to allow the sample to settle down as emulsions are known to be thixotropic.

In previous in vitro skin permeation studies on poor water-soluble UV-filters (Montenegro, L.; Carbone, C.; Paolino, D.; Drago, R.; Stancampiano, A.H.; Puglisi, G. In vitro skin permeation of sunscreen agents from O/W emulsions. Int. J. Cosmet. Sci. 2008, 30, 57–65; Montenegro, L.; Turnaturi, R.; Parenti, C.; Pasquinucci, L. In Vitro Evaluation of Sunscreen Safety: Effects of the Vehicle and Repeated Applications on Skin Permeation from Topical Formulations. Pharmaceutics 2018, 10, 27), we used a receiving solution consisting of water/ethanol 50/50 to ensure sink conditions. We demonstrated that this receiving solution did not alter the skin barrier properties while ensuring sink conditions. Therefore, a mixture consisting of water/ethanol 50/50 could be regarded as a suitable receiving solution to assess in vitro skin permeation of poor-water soluble compounds.

7) Include the other TEM images in the Supplementary Files.

Answer

Unfortunately, we cannot comply with the reviewer’s request. We performed TEM analyses several years ago and the computer where these images were stored no longer works. Therefore, we have only the images we selected for the manuscript.

8) Lines 342-346: HLB values of IPM and DO, MYR are similar (around 11) and this cannot be used as an explanation for the smaller size of NLC obtained using IPM. Please correct this lines.

Answer

We deleted the sentence “to the higher HLB values of this liquid oil (HLB IPM = 11.5; HLB DO and MYR = 11) that could lead”.

Lines 562-564: pH values of the obtained emulsions ranged from 6.5 to 7.5, and this is too high according to physiological conditions. The pH of prepared emulsion should be modified, to lower values (around 5.0).

Answer

A recent study BMTZ (Determination of Bemotrizinol degradation in different stress condition by high performance liquid chromatography in sunscreen preparations, https://doi.org/10.21203/rs.3.rs-2629843/v1) pointed out that bemotrizinol is instable in an acidic environment. Therefore, we preferred to prepare emulsions with a pH close to physiological values.

10) The in vitro release test and permeation experiments (2.11 and 2.12) should be analyzed by a high-sensitivity method, e.g. HPLC. No recorded concentration of drug during the first 3 h of drug release and during the permeation study was probably a consequence of an inappropriate method with low sensitivity. This fact suggests the low quality of obtained results and the determinations should be repeated.

Answer

Other authors, performing an in vitro skin permeation study on BMTZ and other UV-filters using a validated HPL C method to determine the permeated amount, showed that no BMTZ permeation was observed after 24 h (Souza C, Maia Campos PMBG. Development of a HPLC method for determination of four UV filters in sunscreen and its application to skin penetration studies. Biomed Chromatogr. 2017 Dec;31(12). doi: 10.1002/bmc.4029). The results of our experiments are in perfect agreement with the data reported by Souza and Campos. Therefore, the lack of BMTZ skin permeation could not be attributed to the lack of sensitivity of the analytical methods but it depended on BMTZ poor ability to permeate through the skin.

Round 2

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Comments are addressed point wise. Good to go!

Author Response

We would like to thank the reviewer for taking the time to review our manuscript.

Reviewer 4 Report (New Reviewer)

Comments and Suggestions for Authors

Thank you for preparing the revised manuscript. The manuscript can be submitted for publication.

Author Response

We would like to thank the reviewer for taking the time to review our manuscript.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript covers an interesting application of bemotrizinol-loaded nanostructured lipid carriers in sunscreens. Indeed, it systematically describes the analysis of the composition of lipids, the preparation and characterization of NLCs, and their incorporation into emulsions. Though this research is promising for the development of sunscreen technology, several key scientific points in the manuscript need further clarification and development.

The hypothesis that NLCs can synergistically act together with UV filters to give improved sun protection is well-posed. In the introductory section, however, no in-depth discussion about the shortcomings of existing formulation methodologies and how the approach presented in this study innovatively addresses such deficiencies is presented. New related publications are advised to cite in the manuscript, such as: Pharmaceutics, 2024, 16(3): 427; Current Bioactive Compounds, 2024, 20(5): 31-42; Food Hydrocolloids, 2024, 150: 109668; Pharmaceutics, 2024, 16(10): 1270; Industrial Crops and Products, 2024, 214: 118494.
The reported values of ζ-potential of about -10 mV are suspicious regarding colloidal stability, since according to the theory, such low values would predict instability. Considering the trends in stability that were observed during storage, one may consider whether steric stabilization or specific molecular interactions could be at play and how this quantitatively could be duly validated by means of advanced techniques, including atomic force microscopy.
The melting enthalpy (ΔH) and transition temperature (Tm) are reduced with the increase in liquid lipid percentage in the DSC profiles, which evidences lower crystallinity quantified by a linearly decreasing recrystallization index (RI). Based on their findings, can the authors comment on whether the observed enthalpy reductions are only due to the lipid composition or are influenced by experimental inconsistencies such as heating/cooling rate variations?
At higher BMTZ concentrations, such as 7% w/w, double peaks signify phase separation. How would the distribution of BMTZ within the lipid matrix be quantified? Could additional techniques such as SAXS confirm the presence of different phases?
Fig. 7 Viscosity vs. Spreadability: The linear relationship between viscosity and spreadability is intriguing; however, this might be an over-simplification of the relationship. The spreadability relates to other rheological properties such as elasticity or yield stress. Were those measured or considered? Does flow curve analysis or oscillatory rheology support the finding?
Table 8: While the incorporated amount of BMTZ-loaded NLC increased, the SPF values reached 6.34 in the case of DNLC formulations. It can be seen that the occlusion factor of all samples remains around a constant value. This could be due to the nature of the emulsifier used masking the variation generally brought about by the lipids. To what extent does the improvement in SPF compare with the theoretical improvement in UV absorption efficiency calculated from optical properties?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is the transferred version of the rejected version of the submitted version "Bemotrizinol-loaded lipid nanoparticles for the development of sunscreen emulsions” presented by Grazia Sarpietro and coworkers.

The authors did not followed my comments and the submitted version has not changed significantly.

When it was requested that the sections were merged, the authors did not follow the instructions. Comparably, they did not separate the sections accordingly.

It was requested to provide TEM images with better resolution but they have not changed at all.

Tables remain long and various data can be illustrated into graphs, specially DLS results.

The references remain outdated.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript focuses on the formulation and characterization of different types of nanostructured lipid carriers (NLC) containing bemotrizinol (BMTZ), as an UV-filter. It is difficult to understand the novelty of the present work as the same authors have submitted a very similar paper intitled “Bemotrizinol-Loaded Nanostructured Lipid Carriers for the Development of Sunscreen Emulsions” mentioned in “Santonocito, D.; Puglia, C.; Montenegro, L. Effects of Lipid Phase Content on the Technological and Sensory Properties of O/W Emulsions Containing Bemotrizinol-Loaded Nanostructured Lipid Carriers. Cosmetics 2024, 11, 123.” Suggestion to be considered before publication: In the section "Materials and Methods":
Materials. Page 2, line 98: check spelling.
Add section for Encapsulation Efficiency and Loading Capacity of the BMTZ in the NLC
In vitro skin permeation experiments. Avoid rewriting conditions already explained in the release test. Explain how integrity of the membrane was checked. Add information about analytical interferences due to skin composition. In the section “Results and discussion”:
Move details regarding formulation in the table or figure legend (ex. Page 9, line 305 – 311; page 14, line 415 - 419)
Line 324. Check PDI value. Conclusion section, too long.

Author Response

Please see the attachment

Author Response File: Author Response.pdf