Controlling the Skin Barrier Quality through the Application of Polymeric Films Containing Microspheres with Encapsulated Plant Extract

Round 1

Reviewer 1 Report

Thank you for submitting your manuscript for publication in Process.

In this manuscript, the authors prepared microencapsulate of Calendula officinalis flower extract in gelatin and then incorporation microspheres into thin polymeric films made from sodium alginate or mixture of sodium alginate and starch. It is an interesting research topic and the information could be useful in the related dermocosmeceutical research. The experiments were well designed and the conclusions are convinced.

Comments:

It is necessary the mucoadhessivity of films with the skin. Did you perform mucoadhesiveness assays?

What skin (artificial or natural) type did you use for experiments of release and to determine the barrier quality of the stratum corneum, transepidermal water loss and skin colour measurements?

Author Response

We thank the experts and editor who have helped develop this paper. Thank you for your very careful review of our paper and for the comments, corrections and suggestions that ensued. A major revision of the paper has been carried out to take all of them into account. And in the process, we believe the paper has been significantly improved.

Below we provide detailed answers to the reviewer’ comments. In our manuscript, words or sentences in green are changes in accordance with the Reviewer’ comments.

Best regards,

Justyna Kozlowska

Reviewer #1:

In this manuscript, the authors prepared microencapsulate of Calendula officinalis flower extract in gelatin and then incorporation microspheres into thin polymeric films made from sodium alginate or mixture of sodium alginate and starch. It is an interesting research topic and the information could be useful in the related dermocosmeceutical research. The experiments were well designed and the conclusions are convinced.

Comments:

It is necessary the mucoadhessivity of films with the skin. Did you perform mucoadhesiveness assays?

• We did not perform mucoadhesiveness assays, We used the probes offered by a leading representative of the apparatus for testing skin parameters - Courage + Khazaka electronic GmbH. The main goal of our study is the design, preparation and characterization of a new class of materials having the ability to improve the effectiveness of penetration of active ingredients into the skin. These materials may become the basis for a new cosmetics or dermatological formulation. The polymer film was modified by addition of glycerin which is chemical substance temporarily diminishing the barrier of the skin (accelerant, sorption promoter). Because the obtained materials are not intended for contact with mucosal, but only with the surface layer of the skin (stratum corneum), we have not decided on mucoadhesiveness assays.

What skin (artificial or natural) type did you use for experiments of release and to determine the barrier quality of the stratum corneum, transepidermal water loss and skin colour measurements?

• Section: 2.4.6. Skin Examination: The skin parameters measurements were conducted on the volar forearm skin with participation of five probants with normal skin (women, aged 23-25) in the laboratory in controlled temperature (20–22°C) and humidity (relative humidity 40–60%).

Reviewer 2 Report

The article entitled “Controlling the skin barrier quality though the application of polymeric films containing microspheres with encapsulated plant extract” describes encapsulated Calendula officinalis flower extract has contributed to obtaining materials that have a positive effect on the quality of the skin barrier. Interestingly, the formulated materials allows increased protection against transepidermal water loss.

The paper has to go through minor revision before consideration for publication in Processes.

• In Materials and Methods

1. How the author confirms the formation of water in oil emulsion?
2. The author should explain clearly the loading mechanism of Calendula officinalis flower extract into microspheres.
3. What solvents were used to wash Calendula officinalis flower extract with microspheres after 24 h incubation?
4. How the author confirmed the loading efficacy of the extract into the microsphere after 24 h incubation.
5. How much loading efficacy is changed before and after the washing?

• Page no 3, line no-101

The ‘etanol’ spelling should be changed to ethanol.

• The author should give clear explanation on the expansion of legends mentioned in the figure for the reader.

Author Response

We thank the experts and editor who have helped develop this paper. Thank you for your very careful review of our paper and for the comments, corrections and suggestions that ensued. A major revision of the paper has been carried out to take all of them into account. And in the process, we believe the paper has been significantly improved.

Below we provide detailed answers to the reviewer’ comments. In our manuscript, words or sentences in green are changes in accordance with the Reviewer’ comments.

Best regards,

Justyna Kozlowska

Reviewer #2:

The article entitled “Controlling the skin barrier quality though the application of polymeric films containing microspheres with encapsulated plant extract” describes encapsulated Calendula officinalis flower extract has contributed to obtaining materials that have a positive effect on the quality of the skin barrier. Interestingly, the formulated materials allows increased protection against transepidermal water loss.

The paper has to go through minor revision before consideration for publication in Processes.

• In Materials and Methods

1. How the author confirms the formation of water in oil emulsion?

Two different identification tests for emulsion were conducted:

- Dilution test - if water was added to the emulsion, it was not be mixed. On the other hand, oil mixed well.

- Dye solubility test - if an oil soluble dye (Sudan III) was added to an emulsion, the continuous phase appeared red.

The results indicate  that it is w/o emulsion.

 2. The author should explain clearly the loading mechanism of Calendula officinalis flower extract into microspheres.

Thank you for the right suggestion. The loading mechanism of Calendula officinalis flower extract into microspheres was described in section 4. Loading Capacity and In Vitro Release:

“After immersion, the Calendula officinalis flower extract was absorbed on the surface and inside the pores of the freeze-dried gelatin microspheres. This can be explained by the features of the gelatin, which has a high swelling ability.” 

3. What solvents were used to wash Calendula officinalis flower extract with microspheres after 24 h incubation?

The secton: 2.2. Preparation of Gelatin Microspheres has been compleeted: “Then, any remaining extract was removed and the microspheres were washed with deionized water, frozen and lyophilized.”

4. How the author confirmed the loading efficacy of the extract into the microsphere after 24 h incubation.

Thank you for your comment. The manuscript has been supplemented with a new section with these results:“2.4.4. Loading Capacity of Matrices with Microspheres

The films with extract-loaded gelatin microspheres (1 x 1 cm) were weighed and put into 2mL of 1M NaOH for 1h. After that, the samples was centrifuged (10 000 rpm, 5 min) and the supernatant solution was collected. The polyphenolic compounds were examined by using the Folin-Ciocalteu method [28]. Therefore, 20 µl of sample with extract was mixed with 100 µl of Folin-Ciocalteu’s reagent and 1.58 µl of H₂O. After 4 min, 300 µl of saturated Na₂CO₃ solution was added to the mixture. The samples were kept in the incubator at 40°C for 30 min. Then, the absorbance was measured at 725 nm using a UV-VIS spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). The results are the average of three samples. The presented data of polyphenol content in the matrices with microspheres were calculated based on gallic acid solution using standard curve equation in the concentration rage 0–0.50 mg/mL. The coefficient of linear correlation was R = 0.9992.”

The results are in the section: 3.4. Loading Capacity and In Vitro Release

5. How much loading efficacy is changed before and after the washing?

We did not check it. Incorporation of the extract into the microspheres consists in immersing the microspheres in the extract for 24h. Then the remains of the extract are removed and the microspheres are washed with deionized water. The loading efficacy after the washing was measured. 

• Page no 3, line no-101

The ‘etanol’ spelling should be changed to ethanol.

Thank you. It was corrected.

• The author should give clear explanation on the expansion of legends mentioned in the figure for the reader.

The explanation of the abbreviations is  given in the descriptions of the drawings and tables:

“ALG+1.5G, ALG+2.0G, ALG+2.5G – samples based on sodium alginate containing 1.5, 2 and 2.5% of glycerin, respectively. ALG:S+1.5G, ALG:S+2.0G, ALG:S+2.5G samples based on sodium alginate and starch containing 1.5, 2 and 2.5% of glycerin, respectively.”

Reviewer 3 Report

This paper titled "Controlling the skin barrier quality though the application of polymeric films containing microspheres with encapsulated plant extract" discusses the application of polymeric films blended with starch for medicinal purposes. This paper is well-written and conclusions are supported by the experimental observations. I have a couple of comments that should be addressed before this paper is accepted for publication.

1. In the title, replace "though" with "through".
2. Section 2.4.1.: Please mention the ASTM standard used for testing. 

Author Response

We thank the experts and editor who have helped develop this paper. Thank you for your very careful review of our paper and for the comments, corrections and suggestions that ensued. A major revision of the paper has been carried out to take all of them into account. And in the process, we believe the paper has been significantly improved.

Below we provide detailed answers to the reviewer’ comments. In our manuscript, words or sentences in green are changes in accordance with the Reviewer’ comments.

Best regards,

Justyna Kozlowska

Reviewer #3:

This paper titled "Controlling the skin barrier quality though the application of polymeric films containing microspheres with encapsulated plant extract" discusses the application of polymeric films blended with starch for medicinal purposes. This paper is well-written and conclusions are supported by the experimental observations. I have a couple of comments that should be addressed before this paper is accepted for publication.

1. In the title, replace "though" with "through".

It was corrected.

2. Section 2.4.: Please mention the ASTM standard used for testing. 

Thank you for the suggestion. It has been completed: "The tests were carried out in accordance with ASTM D638 standard."

Reviewer 4 Report

Dear Authors, even if the paper could be of interest for the topic, there are some important missing points that cannot be overlooked, first of all, the absence of a statistical analysis, which makes the discussion of any observed result vain.
I have noted other missing points that you can improve, concerning:

Authors could provide the total polyphenols content of the extract alone, and furthermore the results of the total polyphenols determination in in vitro systems

The methods of calculation of polyphenol release in vitro is not reported and it is not clear to follow the results for this aspect.

Authors are kindly requested to provide more details about the technical aspects of the determination of the skin color: which parameters of the color were considered (L*, a*, b*, Delta E?, etc..), in which area of the harms (a photo could be useful if they have)? Which system of calibration they used?

Furthermore, as authors refer the results of color on arbitrary units (a.u.), they are kindly requested to specify the correspondence between the color parameters (L, a, b, DE, etc) determined by the instruments and the au

The discussion of the results is not satisfactory: the authors could explain the biological-biochemical -chemical basis of the observed results on skin parameters. Which mechanisms or which compounds they thinks are responsible of the effects of the extracts or the formulation, on the skin?

Author Response

We thank the experts and editor who have helped develop this paper. Thank you for your very careful review of our paper and for the comments, corrections and suggestions that ensued. A major revision of the paper has been carried out to take all of them into account. And in the process, we believe the paper has been significantly improved.

Below we provide detailed answers to the reviewer’ comments. In our manuscript, words or sentences in green are changes in accordance with the Reviewer’ comments.

Best regards,

Justyna Kozlowska

Reviewer #4:

Dear Authors, even if the paper could be of interest for the topic, there are some important missing points that cannot be overlooked, first of all, the absence of a statistical analysis, which makes the discussion of any observed result vain. I have noted other missing points that you can improve, concerning:

Authors could provide the total polyphenols content of the extract alone, and furthermore the results of the total polyphenols determination in in vitro systems

Thank you for the right suggestion. Information about the total polyphenols  in the purchased plant extract is given in the section: Materials.

“The hydroglycolic Calendula officinalis flower extract (propylene glycol/water (80:20)) was acquired from Provital S.A. (Barcelona, Spain).  The total polyphenol content of Calendula officinalis flower extract was determined using the Folin–Ciocalteu method [28] with gallic acid as a standard. The absorbance measurements were performed at 725 nm using a UV–VIS spectrophotometer. The results were calculated as gallic acid equivalents (mgGAE/ml). The total polyphenolic content of the extract was 206.6 ± 3.6 mgGAE/ml.”

The results of the total polyphenols determination in in vitro system (loading capacity of matrices with microspheres) are in the section: 3.4. Loading Capacity and In Vitro Release.

The methods of calculation of polyphenol release in vitro is not reported and it is not clear to follow the results for this aspect.

Thank you for your comment. The manuscript has been supplemented with a new section with these results:2.4.4. Loading Capacity of Matrices with Microspheres

"The films with extract-loaded gelatin microspheres (1 x 1 cm) were weighed and put into 2mL of 1M NaOH for 1h. After that, the samples was centrifuged (10 000 rpm, 5 min) and the supernatant solution was collected. The polyphenolic compounds were examined by using the Folin-Ciocalteu method [28]. Therefore, 20 µl of sample with extract was mixed with 100 µl of Folin-Ciocalteu’s reagent and 1.58 µl of H₂O. After 4 min, 300 µl of saturated Na₂CO₃ solution was added to the mixture. The samples were kept in the incubator at 40°C for 30 min. Then, the absorbance was measured at 725 nm using a UV-VIS spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). The results are the average of three samples. The presented data of polyphenol content in the matrices with microspheres were calculated based on gallic acid solution using standard curve equation in the concentration rage 0–0.50 mg/mL. The coefficient of linear correlation was R = 0.9992."

The results are in the section: 3.4. Loading Capacity and In Vitro Release.

Authors are kindly requested to provide more details about the technical aspects of the determination of the skin color: which parameters of the color were considered (L*, a*, b*, Delta E?, etc..), in which area of the harms (a photo could be useful if they have)? Which system of calibration they used? Furthermore, as authors refer the results of color on arbitrary units (a.u.), they are kindly requested to specify the correspondence between the color parameters (L, a, b, DE, etc) determined by the instruments and the au

Thank you for your suggestion.

The section 4.6. Skin Examination has been completed:

"The skin colour and TEWL after application of the sodium alginate and sodium alginate/starch films were examined using the colorimeter (Skin-Colorimeter CL 400, Courage + Khazaka, Germany) and tewameter (Tewameter TM 300, Courage + Khazaka, Germany) with the use of MPA-software."

The section 5. Skin Examination was supplemented:

"The measured skin colour is expressed as an XYZ-value and are calculated into L*a*b related value. L* gives information about the black-white axis and skin brightness, while a* and b* are the coordinates in the colour space – a* locates the values on the red-green axis, whereas b* shows the colour position on the blue-yellow axis. Therefore, the a* values were considered in this research due to their correlation to skin redness, erythema and microcirculation."

The discussion of the results is not satisfactory: the authors could explain the biological-biochemical - chemical basis of the observed results on skin parameters. Which mechanisms or which compounds they thinks are responsible of the effects of the extracts or the formulation, on the skin?

The section 3.5. Skin Examination has been completed:

"Beneficial effect of materials based on sodium alginate or sodium alginate and starch on Beneficial effect of prepared materials based on sodium alginate or sodium alginate and starch on biophisical skin parameters is caused by the whole structure of these materials. A moist polymer matrices loosens the skin structure and increases its permeability owing to occlusion and hydrophilicity of polymers. Materials are also modified by the addition of glycerin which acts as an penetration promoter – it  supports transepidermal penetration, and thus reduce the skin barrier functions. It contributes to the more effective penetration to the deeper skin layers of Calendula officinalis flower extract released from the microparticles. Calendula officinalis flower extract is well known for a long time for its strong healing effects on the skin. Therefore, treatment efficiency of many skin conditions is increased."

Round 2

Reviewer 4 Report

Dear authors, although the additions have contributed significantly to the improvement of some aspects, there still remains the large and serious gap in the statistical validity of the results that you have not filled in the review with the addition of tests that indicate the significance of the differences. Without this evaluation, which is the basis of any scientific work, the validity of any experimental treatment cannot be affirmed with respect to control. This is of vital importance especially in the in vivo experiments, that were carried out on a very limited number of volunteers.

Author Response

Thank you very much for the right comment and we apologize for the lack of response. This is the result of our oversight.We decided to invite for research on biophisical skin parameters a very limited group of probants due to the strict selection for compliance of skin type and phenotype, as well as probants age, gender, race, region of the body under investigation. The experimental conditions were correctly settled, controlled, and monitored. The ambient conditions (room temperature and humidity) were kept constant range. We focused only on probants with a normal skin. The obtained results did not indicate a huge discrepancy. Each measurement was repeated 5 times. The graphs show the average of measurements with standard deviation. Therefore, we agreed that this is a sufficient number of people surveyed as part of the preliminary study. We have added this information in the manuscript: "For this purpose, besides mechanical properties, moisture content, contact angles and in vitro release study, the preliminary tests with the usage of a tewameter and a colorimeter were carried out." – line 82.

Of course, we plan to increase the number of probants with different types of skin in order to further investigate the impact of the obtained materials on skin parameters. However, it is impossible at this time due to the current pandemic of SARS-Cov-2 coronavirus. There are restrictions in Poland that prevent us from leaving home outside the living, work and health goals, and prohibit the organization of any human assembly. Admission to the Nicolaus Copernicus University area is also prohibited. For these reasons, it is not possible today to extend our research.