Multilayered Polyelectrolyte Structures Deposited on Corona-Charged Substrate Blends as Potential Drug Delivery Systems

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is well written and the experimental part is sufficiently developed both in the preparation part and in the characterization.  

-Introduction and abstract sufficiently good

-could be helpful drawings showing the layer by layer deposition mechanism and  formulas of the involved polymers

-146:better explain more extensively  the layer by layer deposition eventually with the help of a scheme.

-157: what do you mean with ‘order’? you mean ‘sequence’?

-in discussing fig.5-7 (time decay curves) and fig.8-9 (steady state values), you anticipate crystallinity and morphological infos that will be presented later (DSC and SEM). Add a phrase saying that this is an anticipation of what will presented later or move these interpretations after DSC and SEM chapters.

-fig10 scale missing. are the scale the same for all the SEM images? Necessary to label the images in fig10 for a better understanding of the following discussion. 

-fig.10: images too dark. Improve luminosity and contrast.

-345-370: it’s difficult to distinguish the methods used in this article to create pores and alternative method used in the literature. is your method lyophilization for PDLA lyophilized/ PEC lyophilized and a posteriori solvent treatement. Better separate, in the discussion, what you have done and better refer to fig10 (with labels!). only after illustrating your methods, extend discussion to literature.

-are you sure that a distribution of pores with bigger diameter and a lower density  gives rise to a higher surface area available compared to pores smaller and more densely distributed?

-490: BH release explain. in conclusions explain the acronyms

- XRD could be helpful for crystallinity in the future as well as use of kelvin probe for surface potential.

-SEM images of the substrate after layer by layer deposition could be helpful in this manuscript for better understanding the final morphology

-conclusions too synthetic and not well developed and cared for. Should be written more extensively as in the abstract.

Author Response

Reviewer 1

The manuscript is well written and the experimental part is sufficiently developed both in the preparation part and in the characterization.  

-Introduction and abstract sufficiently good – We thank the reviewer for the comment

-could be helpful drawings showing the layer by layer deposition mechanism and  formulas of the involved polymers – Schematic of the layer deposition process has been added. The deposition was carried out in an automated slide stainer mentioned in the text.

-146:better explain more extensively  the layer by layer deposition eventually with the help of a scheme – Further explanation has been added and a schematic of the process has been added.

-157: what do you mean with ‘order’? you mean ‘sequence’? – Yes, the proper wording should have been ‘sequence’. The text has been edited accordingly.

-in discussing fig.5-7 (time decay curves) and fig.8-9 (steady state values), you anticipate crystallinity and morphological infos that will be presented later (DSC and SEM). Add a phrase saying that this is an anticipation of what will presented later or move these interpretations after DSC and SEM chapters. – The part has been edited following the recommendation.

-fig10 scale missing. are the scale the same for all the SEM images? Necessary to label the images in fig10 for a better understanding of the following discussion.  – The scale of the figure is added. Due to time constrains and scheduling we were not able to send all of our samples for SEM imaging, and because of this the two places we used provided us with pictures, taken at different scales. All calculations for the pore sizes were done by taking into account the scale of each image so that the results can be compared numerically, regardless of the difference in scale

-fig.10: images too dark. Improve luminosity and contrast. – We improved the images

-345-370: it’s difficult to distinguish the methods used in this article to create pores and alternative method used in the literature. is your method lyophilization for PDLA lyophilized/ PEC lyophilized and a posteriori solvent treatement. Better separate, in the discussion, what you have done and better refer to fig10 (with labels!). only after illustrating your methods, extend discussion to literature.

- In our review we investigate two different types of porous composite structures. The first type is created with the use of lyophilization, where the pores form naturally during the drying process, while the second type relies on the interactions between the water soluble PEG and the dichloromethane, in which the polymers are dissolved, for its pore creation. The samples containing PEG were dried under normal atmospheric pressure and room temperature. The lyophilized samples were not treated with any other solvent after drying. In the section of the text you are referring to we are using the cited literature to back our observation, that the existence of pores in the structure affects the crystallinity of the samples. We have added further explanation on each type of composite film in both the Materials and Methods, as well as in the beginning of the Results section.

-are you sure that a distribution of pores with bigger diameter and a lower density  gives rise to a higher surface area available compared to pores smaller and more densely distributed? – In our research we used our two methods for creation of porous films without investigating the effects of the pore sizes. We used the substrates without further modifications and assumed that the surface areas would be comparable for both porous samples. The sizes of the pores were not a major focus of our research, and such investigation on the effect of pore size on the available surface area can be done in future research projects.

-490: BH release explain. in conclusions explain the acronyms – Acronyms have been replaced with full names where needed and the rest have been explained.

- XRD could be helpful for crystallinity in the future as well as use of kelvin probe for surface potential. – We are thankful for the suggestion and will keep them in mind for any future investigations

-SEM images of the substrate after layer by layer deposition could be helpful in this manuscript for better understanding the final morphology – SEM images of the final morphology of the substrates after layer deposition were taken. As they show very similar morphology to the initial substrates we decided to provide only the initial SEM images to avoid repetition. As the focus was on the differences in the types of substrates we chose those images instead of the ones after the layer deposition, as the deposited layers are the same for all types of substrates.

-conclusions too synthetic and not well developed and cared for. Should be written more extensively as in the abstract. – The conclusion and abstract were extended and improved as recommended.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This research explores an innovative use of biodegradable polymers (PLA/PEC) in multilayer drug delivery systems, which is a timely and relevant topic. The integration of corona charging adds a novel aspect to substrate modification for biomedical applications.

1. Abstract should clarify the novelty of the study, particularly how the use of corona charging uniquely contributes to the field, and include the quantitative results to strengthen the impact of the abstract.

2. Introduction should clearly define the research gap this study addresses and the novelty of this study. PLA and PDLA are the same, so you should only use one or the other. Reduce redundancy when discussing the properties of PLA, PEC, and PEG.

3. Methods (or discussion) should include justifications for the choice of materials (e.g., why chitosan and casein were selected over other polyelectrolytes), expand on the rationale for using specific corona charging conditions, and add information on statistical analyses used to validate experimental results.

Technique for loading BH into polymer substrate was not presented in the method part. Amount of BH loading was not noted.

Surface free energy calculation was not described in the method part. Only water contact angle was noted.

Time of drug release was noted as 8 hr, but all results showed only 6 hr. The release kinetics were not mentioned in the method part. Model of spectrophotometer should be noted.

4. Results and Discussion

Firstly, non-porous and porous composite substrates from different preparation methods should be described in this part.

The multilayered films were not confirmed by any technique.

The effect of charges on film properties, especially in drug release pattern, should be discuss deeply.

The SEM images of 1:1 composite substrate should present in Figure 10.

Page 14: “The values of the surface free energy significantly increased for the porous films 389 compared to non-porous ones…” however, the red bar charts were lower than the black bar charts in all condition. Please confirm.

Encapsulated BH data were different from each formulation. Please discuss.

Enhance clarity by consolidating figures with overlapping content (e.g., combine release profiles for different substrates into one comparative graph). The patterns of each line in the figures should be similar.

Discuss the biological implications of the results, particularly for drug delivery systems.

Highlight how the findings compare to or advance existing literature.

Missing a compelling argument for why this method/system is superior to existing drug delivery systems.

5. Ensure consistency in citation format (e.g., uniform use of DOIs or full journal names).

6. Additional Suggestions:

Review for grammatical errors, such as inconsistent tense usage and punctuation.

Be careful with abbreviations. When used, it should be used throughout the entire article.

Comments on the Quality of English Language

Review for grammatical errors, such as inconsistent tense usage and punctuation.

Be careful with abbreviations. When used, it should be used throughout the entire article.

Author Response

Reviewer 2

This research explores an innovative use of biodegradable polymers (PLA/PEC) in multilayer drug delivery systems, which is a timely and relevant topic. The integration of corona charging adds a novel aspect to substrate modification for biomedical applications.

1. Abstract should clarify the novelty of the study, particularly how the use of corona charging uniquely contributes to the field, and include the quantitative results to strengthen the impact of the abstract. –Abstract has been edited per recommendation.

2. Introduction should clearly define the research gap this study addresses and the novelty of this study. PLA and PDLA are the same, so you should only use one or the other. Reduce redundancy when discussing the properties of PLA, PEC, and PEG. – This study represents a part of a larger research that our team has done on the impact of different modification techniques and layer deposition conditions on the controlled release of bioactive substances from multilayer films, based on biodegradable polymers. Our main focus is Poly-lactic acid as a base material for the multilayers, and we have chosen the specific type of corona treatment as a standard modification, that allows for the creation of the investigated multilayers. We have investigated other methods of surface modification, however, the corona treatment is always utilized as a base that can be used in the comparison of the results. In terms of research gap we have focused our efforts in the research of completely biodegradable systems, that have not been researched extensively as far as we have been able to find.

We have changed the text to only use PDLA and have reduced any redundancies as required.

3. Methods(or discussion) should include justifications for the choice of materials (e.g., why chitosan and casein were selected over other polyelectrolytes), expand on the rationale for using specific corona charging conditions, and add information on statistical analyses used to validate experimental results.

The text in the manuscript is extended as follows:

Polyelectrolyte multilayer films are constructed from two biopolyelectrolytes, namely chitosan and casein. Chitosan is the only polycation in nature that is known as a biocompatible biodegradable polysaccharide with proven antimicrobial properties [Aranaz, I., Alcántara, A. R., Civera, M. C., Arias, C., Elorza, B., Heras Caballero, A., & Acosta, N. (2021). Chitosan: An overview of its properties and applications. Polymers, 13(19), 3256.]. in summary, casein enhances the functionality and versatility of PEMs due to its biocompatibility, anionic nature, and ability to enable controlled release and adhesion. This makes it a valuable component in drug delivery systems and biomedical coatings. In our previous research we have already developed polyelectrolyte multilayers from chitosan and casein [1.         Pilicheva, B., Uzunova, Y., & Marudova, M. (2022). Polyelectrolyte Multilayer Films as a Potential Buccal Platform for Drug Delivery. Polymers, 14(4), 734.

2. Marudova, M., Exner, G., Pilicheva, B., Marinova, A., Viraneva, A., Bodurov, I., ... & Yovcheva, T. (2019). Effect of assembly pH and ionic strength of chitosan/casein multilayers on benzydamine hydrochloride release. International Journal of Polymeric Materials and Polymeric Biomaterials, 68(1-3), 90-98.]. They offer a versatile and promising approach for developing drug delivery systems, particularly for buccal administration. Their properties can be finely tuned by adjusting assembly conditions, enabling the design of tailored delivery platforms for a range of therapeutic agents. (lines 239 to 249)

In order to determine the optimal conditions for charging, preliminary tests were carried out. The influence of charging time on the surface potential of the electrets was investigated. For this, samples of PDLA were studied at different charging times  – 1, 5, 10, 20 and 30 minutes.

Table Surface potential for samples of PDLA at different charging times

The obtained results show, that the charging time does not impact the surface potential, and because of this the charging time used in our study was set to 1 minute.

By calculating the electrical fields, that are created by the electret in the gaps between the electret and corona electrode, it was determined, that the maximal voltage, that can be applied to the corona electrode (without causing a break in the dielectric) at the distances, used in the apparatus (distance between the grid and ground electrode was 10 mm, and the distance between grid and corona electrode was 7 mm) can be approximately 7 kV. To ensure that no break in the dielectric occurred the voltage was set at 5 kV.

A number of preliminary tests were carried out at different voltages of the grid (350V, 500V, 650V, 800V and 1000V) and the voltage, that had to be applied to the grid for our study, was set at the highest value, due to the fact, that biodegradable materials have weaker electret properties and are charged to a value, lower than that of the grid, which is not the case for materials such as polypropylene, Teflon, polyethylene terephthalate etc.

Technique for loading BH into polymer substrate was not presented in the method part. Amount of BH loading was not noted. - The loading technique is described in the Materials and methods section (line 132 to 134). The loading amount is shown in Table 2.

Surface free energy calculation was not described in the method part. Only water contact angle was noted. – The method of calculation of the surface free energy was added to the methods.

Time of drug release was noted as 8 hr, but all results showed only 6 hr. The release kinetics were not mentioned in the method part. Model of spectrophotometer should be noted – The time of drug release was indeed 6 not 8 hours and the text has been fixed (the number had been a technical error not noticed by the authors). The section in Materials and methods has been extended to cover the used kinetic models. The model of the spectrophotometer has been added.

4. Results and Discussion

Firstly, non-porous and porous composite substrates from different preparation methods should be described in this part. – The required description has been added.

The multilayered films were not confirmed by any technique.

Our previous studies have already confirmed the polyelectrolyte multilayer built-up on similar substrates [Yovcheva, T., Viraneva, A., Marinova, A., Sotirov, S., Exner, G., Bodurov, I., ... & Vlaeva, I. (2018). Insulating chitosan/casein multilayers on corona charged polylactic acid substrates. IEEE Transactions on Dielectrics and Electrical Insulation, 25(3), 766-771.]. The present study builds on our previous work by recapitulating the influence of support on drug substance incorporation.

The effect of charges on film properties, especially in drug release pattern, should be discuss deeply. The text is extended per recommendation.

The SEM images of 1:1 composite substrate should present in Figure 10. – The SEM images of 1:1 composite substrate were added in the paper

Page 14: “The values of the surface free energy significantly increased for the porous films 389 compared to non-porous ones…” however, the red bar charts were lower than the black bar charts in all condition. Please confirm. – We thank the reviewer for the comment. The values of the free surface energy are higher only for porous films, created by addition of PEG, when compared to non-porous ones. For the lyophilized films the values of the surface energy are lower than those for non-porous samples, which can also be observed from the numerical values given in the text before the figure. We have added clarifications in the text to better address the results.

Encapsulated BH data were different from each formulation. Please discuss.

Additional discussion has been added in the text after Table 2.

Enhance clarity by consolidating figures with overlapping content (e.g., combine release profiles for different substrates into one comparative graph). The patterns of each line in the figures should be similar. – Edits have been made per recommendation.

Discuss the biological implications of the results, particularly for drug delivery systems.

Highlight how the findings compare to or advance existing literature.

Missing a compelling argument for why this method/system is superior to existing drug delivery systems. - Additional discussion has been added in the text

5. 5. Ensure consistency in citation format (e.g., uniform use of DOIs or full journal names).- All citations have been edited to fit the paper’s requirements.

6. Additional Suggestions:

Review for grammatical errors, such as inconsistent tense usage and punctuation.

Be careful with abbreviations. When used, it should be used throughout the entire article.

– Paper has been revised to remove any grammatical errors and missing punctuation. Abbreviations have been made consistent throughout the article.

Comments on the Quality of English Language

Review for grammatical errors, such as inconsistent tense usage and punctuation.

Be careful with abbreviations. When used, it should be used throughout the entire article.

– Paper has been revised to remove any grammatical errors and missing punctuation. Abbreviations have been made consistent throughout the article.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Viraneva et al reports on the surface functionalization of polyester films by surface activation in a corona discharge setup, followed by layer-by-layer coating with polyelectrolytes.

The reviewer finds it confusing that the authors seem to mix up a research paper and a review paper. While being submitted as a review paper (also stated e.g. in lines 82, 109 etc), the paper contains a materials and methods section and appears to primarily present new data. At least, there is no indication that this would be a reprint of previous publications as there are no citations along with the presented data.

Specific remarks:

1.       Check wording. For instance, “doesn’t” is used in line 34, which is colloquial speech and also wrong grammar in this specific sentence (correct: “…drawbacks that do not ….”).

2.       The more common abbreviation for poly(epsilon-caprolactone) is PCL.

3.       Not clear why PEG is introduced in lines 51-57. Suggest to remove. PEG seems to be used as a porogen, at least it can be expected that hydrophilic PEG will leach out from the polyesters upon water contact.

4.       Not clear what the statements on poor mechanical properties in lines 58-68 have to do with the scope of this study. Suggest to remove.

5.       Despite freeze drying being used as a method, the paragraph on freeze drying (lines 69-73) in the introduction seems out of scope when it comes to describing the state of the art associated with this study. Suggest to remove. Instead, it is suggested to better describe different surface modification approaches, their shortcomings, and the basis of decisions that made the authors select their approach.

6.       The introduction lacks a description of surface activation techniques like their corona discharging, as well as of alternative methods.

7.       The authors use the term composite (e.g. line 127). Please check the definition of this term. The term blend may be more common for this polymer mixture unless there is a macro-phase separation. Finding separate thermal transitions in the sample does not mean that there is a macrophase separation.

8.       Fig. 1, Section 2.2.2: If this is a self-build instrument, this should be stated giving more details about the instrument parts. Otherwise, the commercial source should be named. A citation and/or instrument name for the vibrating electrode method should be added. At current, it will not be possible to reproduce this by others based on the provided description.

9.       Section 2.2.3.: The authors described a washing step only after each double layer coating, but not in between. With this process, it is likely to contaminate a polyelectrolyte stock solution with the other polyelectrolyte.

10.   Section 2.2.6: Is this a self-build instrument?

11.   Section 2.2.7: Closed vessels are preferred over release tests in open beakers at 37°C.

12.   Dots must be used in the English language to separate digits, e.g. pH 7.4, labeling of figure axis, etc.

13.   Figures should be organized in multi-panel assemblies, e.g. Fig 2-7 can become one figure.

14.   For those figures, it is not clear what was the initial surface potential. The data also lack a control for untreated samples.

15.   Fig 8-9: These figures seem to represent the same data as the previous figures. There is a lack of information about the number of repetitions and a lack of error bars in Fig8-9.

16.   The discussion of thermal properties on page 11 is confusing. The authors did not provide information on the thermal events detected for pure PDLA (“were close to those reported by other authors”). The authors also did not find a glass transition of PCL, which is not surprising giving the range of their heating program, but concluded that the absence of a detectable glass transition indicates very high crystallinity.

17.   Lines 323-334: The authors discuss increasing crystallinity in the context of increasing sample porosity. The reviewer has doubts that macroporosity created by freeze drying or PEG leaching may be directly linked to increased free volumes on the molecular level in the polymer phase.

18.   The authors lack to provide sufficient information on their materials in the materials section, e.g. the molecular weights of PEG, PCL and PDLA are not provided. Without this information, it is impossible to conclude if PEG may predominately act as a plasticizer (very low molecular weight) or a porogen (higher molecular weight). At line 351, the term PEG 400 is stated, suggesting low molecular weights.

19.   SEM Images: The authors should explain the principle of pore formation in the PEG-containing samples. As per the method description, the films are casted from DCM solutions with slow solvent evaporation. This should create compact films. It is also unclear what has been the source of moisture, as the authors had to remove moisture (i.e. condensed water) from the sample surface (see line 125-126). In line 359, the authors provide a citation that pore formation of PDLA operates through plastic deformation in aqueous condition. So has there been an exposure to water for the samples of the manuscript? The reviewer is also not convinced about the statements in line 362 that PEG 400 and chloroform are immiscible, thus causing pore formation during evaporation.

20.   As porosity seem to have a substantial effect, it may be relevant to provide a quantitative measure of porosity be determining the surface areas of the sample e.g. by mercury porosimetry. These values may help to discuss all subsequent data.

21.   Fig 11: Information on the number of repetitions and error bars missing.

22.   Lines 372-375: Measuring water contact angle by droplet analysis is most common for flat surfaces. How did the authors included the porous architecture into their model to calculate surface free energies?

23.   Lines 378-388: Reporting values without units is inappropriate. Please also use proper rounding to relevant digits and dots (not commas) to separate digits. There are also dots missing at the end of some of the sentences in this paragraph.

24.   Table 2: The stated value of payload must be set in the context of the surface area. The information on the dimension if samples used for LbL coating is missing. Where the film free standing or on a substrate?

25.   The discussion on the differences in drug loading levels in lines 407-423 is hard to follow without a detailed method description, how the drug was loaded. There is a statement of “the chosen bioactive substance” in line 152. If this is the drug, the authors may need to justify to which of the polyelectrolyte solution the drug was added and why. Higher drug loading – which here is a surface deposition process based on charge interaction -is hard to connect to crystallinity, metastability, and other terms suggested by the authors in line 420-424.

26.   How do you see the relevance of the loaded drug in relation to therapeutic doses?

27.   Also, more information on the data analysis should be provided in the methods section based on which the release mechanism as presented in Table 2 has been concluded.

28.   The authors conclude that the polarity of corona discharge, i.e. the type of charges introduced to the surface in the pretreatment phase, affect properties like drug release behavior. In the reviewers understanding, this surface activation is only a first step to allow the binding of the polyelectrolytes, which (by chance) also incorporate the drug as a cargo. Without a more detailed understanding of the LbL deposition process, namely the order by which the different layers have been added, how contaminations of stock solutions have been excluded, which layer thicknesses etc. have been obtained, it appears very speculative to discuss the observed results mechanistically.

Author Response

Reviewer 3

The manuscript by Viraneva et al reports on the surface functionalization of polyester films by surface activation in a corona discharge setup, followed by layer-by-layer coating with polyelectrolytes.

The reviewer finds it confusing that the authors seem to mix up a research paper and a review paper. While being submitted as a review paper (also stated e.g. in lines 82, 109 etc), the paper contains a materials and methods section and appears to primarily present new data. At least, there is no indication that this would be a reprint of previous publications as there are no citations along with the presented data.

Specific remarks:

1. Check wording. For instance, “doesn’t” is used in line 34, which is colloquial speech and also wrong grammar in this specific sentence (correct: “…drawbacks that do not ….”). – The language of the paper has been checked and the necessary edits have been made.

2. The more common abbreviation for poly(epsilon-caprolactone) is PCL. – All abbreviations have been changed accordingly

3. Not clear why PEG is introduced in lines 51-57. Suggest to remove. PEG seems to be used as a porogen, at least it can be expected that hydrophilic PEG will leach out from the polyesters upon water contact. – Listed part has been removed.

4. Not clear what the statements on poor mechanical properties in lines 58-68 have to do with the scope of this study. Suggest to remove. – Listed part has been removed.

5. Despite freeze drying being used as a method, the paragraph on freeze drying (lines 69-73) in the introduction seems out of scope when it comes to describing the state of the art associated with this study. Suggest to remove. Instead, it is suggested to better describe different surface modification approaches, their shortcomings, and the basis of decisions that made the authors select their approach. – Listed part has been removed.

6. The introduction lacks a description of surface activation techniques like their corona discharging, as well as of alternative methods. – The surface activation technique that we are using has been developed from previous research results. We have chosen this specific method of surface modification as a base method in all of our research, so that we are able to compare the results from a variety of papers with this parameter as a basis. This review is a part of a larger research effort focused on Poly-lactic acid as a base for the creation of multilayers and we have standardized this specific methodology of corona charging for all of our research. In other papers we have investigated other surface activation techniques, such as chemical modification, however as in this paper we were focusing on the variation in the composition of the base substrate, we did not look into other alternative methods for surface modification.

7. 7.       The authors use the term composite (e.g. line 127). Please check the definition of this term. The term blend may be more common for this polymer mixture unless there is a macro-phase separation. Finding separate thermal transitions in the sample does not mean that there is a macrophase separation. – The term has been changed following the comment of the reviewer.

8. Fig. 1, Section 2.2.2: If this is a self-build instrument, this should be stated giving more details about the instrument parts. Otherwise, the commercial source should be named. A citation and/or instrument name for the vibrating electrode method should be added. At current, it will not be possible to reproduce this by others based on the provided description - The instrument, used for the charging of the samples under corona discharge is made of a corona electrode (needle), grounded plate electrode and a grid (controlling electrode) between them. The distance between the grid and ground electrode was 10 mm and the distance between grid and corona electrode was 7 mm. The sample is placed on the ground electrode and set voltage is applied to the corona electrode from a high voltage source NB-850. The controlling electrode (grid), determines the surface potential, to which the sample can be charged. The grid is powered with the use of a high voltage source NV 825 or alternatively with the use of a voltage splitter.

Vibrating electrode with compensation method:

The surface potential of the created electrets was measures with the use of the vibrating electrode with compensation method with an error, no higher than 5%.

The setup of the instrument is show in the figure below.

Fig. Instrument for measurement of the surface potential of electrets

The instrument is made of:

Circuit for supplying compensating voltage, which is made of a high voltage source 1, resistor R1 and condenser C. The high voltage source used is NB-825;

The measurement circuit includes two electrodes 4 and 6, measured sample 5, zero indicator 7 and digital voltmeter (V), that measures the compensating voltage. The condenser C1 protects the zero indicator from the high voltage. A voltmeter ВН-3 is used as a zero indicator and the digital voltmeter used is 1АМ202;

The vibrating electrode circuit includes a sound generator type ГЗ109 – 2 and a dynamic high speaker – 3.

9. Section 2.2.3.: The authors described a washing step only after each double layer coating, but not in between. With this process, it is likely to contaminate a polyelectrolyte stock solution with the other polyelectrolyte. – The text has been expanded and edited to further clarify and explain the layer deposition method.

10. Section 2.2.6: Is this a self-build instrument? – For the measurements of the water contact angle, all of the small droplets were placed on the surface of the substrates by hand using a microsyringe, and then a profile/side image of them was taken with the use of a high resolution camera, that has the capability of capturing macro images. Those images were later processed with the program described in the section. This setup is self-build.

11. Section 2.2.7: Closed vessels are preferred over release tests in open beakers at 37°C. The beakers were covered during the release test.

12. Dots must be used in the English language to separate digits, e.g. pH 7.4, labeling of figure axis, etc. – All numbers have been changed, following the reviewers comment.

13. Figures should be organized in multi-panel assemblies, e.g. Fig 2-7 can become one figure. – All figures were combined in a multi-panel assembly as requested.

14. For those figures, it is not clear what was the initial surface potential. The data also lack a control for untreated samples - We have provided the initial values of the surface potential in a separate table. Measurements of non-charged samples showed, that all types possess practically zero potential on their surface before charging, and that is why we decided not to include them in the paper.

15. Fig 8-9: These figures seem to represent the same data as the previous figures. There is a lack of information about the number of repetitions and a lack of error bars in Fig8-9. –The data in these figures represent the values of the normalized potential at the 6^th hour, after which they become stable. We have presented them in a separate figure to better show the difference in values for all substrates. All values were an average value from 5 samples. Error bars were added to the figures as requested.

16. The discussion of thermal properties on page 11 is confusing. The authors did not provide information on the thermal events detected for pure PDLA (“were close to those reported by other authors”). The authors also did not find a glass transition of PCL, which is not surprising giving the range of their heating program, but concluded that the absence of a detectable glass transition indicates very high crystallinity.

The authors did not provide information on the thermal events detected for pure PDLA (“were close to those reported by other authors”). The authors also did not find a glass transition of PCL, which is not surprising giving the range of their heating program, but concluded that the absence of a detectable glass transition indicates very high crystallinity. Actually this experiment was done from -80 degree Celsius and perfect base line was achieved. But even in this case the glass transition was not detected.

17. Lines 323-334: The authors discuss increasing crystallinity in the context of increasing sample porosity. The reviewer has doubts that macroporosity created by freeze drying or PEG leaching may be directly linked to increased free volumes on the molecular level in the polymer phase - The porous structure significantly increases the free surface of the polymer, creating heterogeneities that favor the emergence of crystal nuclei.

18. The authors lack to provide sufficient information on their materials in the materials section, e.g. the molecular weights of PEG, PCL and PDLA are not provided. Without this information, it is impossible to conclude if PEG may predominately act as a plasticizer (very low molecular weight) or a porogen (higher molecular weight). At line 351, the term PEG 400 is stated, suggesting low molecular weights - The values for the molecular weights have been added in the materials section.

19. SEM Images: The authors should explain the principle of pore formation in the PEG-containing samples. As per the method description, the films are casted from DCM solutions with slow solvent evaporation. This should create compact films. It is also unclear what has been the source of moisture, as the authors had to remove moisture (i.e. condensed water) from the sample surface (see line 125-126). In line 359, the authors provide a citation that pore formation of PDLA operates through plastic deformation in aqueous condition. So has there been an exposure to water for the samples of the manuscript? The reviewer is also not convinced about the statements in line 362 that PEG 400 and chloroform are immiscible, thus causing pore formation during evaporation - In row 363 DCM should have been written instead of chloroform. The text has been edited to address this. The mechanism proposed for the pore formation and the immiscibility of DCM and PEG 400 is also commented and researched by other authors, namely: Phaechamud, T., & Chitrattha, S. (2016). Pore formation mechanism of porous poly (dl-lactic acid) matrix membrane. Materials Science and Engineering: C, 61, 744-752., namely: “This result indicated that the pore formation mechanism of PLA porous membrane related with non-solvent inducing a phase separation. The different pore size of those films related with the viscosity of non-solvents (Table 1) which the higher viscous liquid promoted the higher pore size.” As a result of the immiscibility of PEG 400 and PDLA solution in DCM, during the drying process pores of pockets of PEG 400 are formed in the dried out PDLA film.

20. As porosity seem to have a substantial effect, it may be relevant to provide a quantitative measure of porosity be determining the surface areas of the sample e.g. by mercury porosimetry. These values may help to discuss all subsequent data. - We are thankful for the suggestion and will keep them in mind for any future investigations

21. Fig 11: Information on the number of repetitions and error bars missing. - All values were an average value from 6 samples. Error bars were added to the figures as requested.

22. Lines 372-375: Measuring water contact angle by droplet analysis is most common for flat surfaces. How did the authors included the porous architecture into their model to calculate surface free energies? –As the pore sizes of the created porous structures are very small, the model for the calculation of the surface free energy doesn’t require any modification. The utilized model has also been used by other authors for the calculation of the pore sizes of porous structures.

23. Lines 378-388: Reporting values without units is inappropriate. Please also use proper rounding to relevant digits and dots (not commas) to separate digits. There are also dots missing at the end of some of the sentences in this paragraph – Units have been added to all of the given values. The numbers have been edited to fit the requirements and all missing dots have been added.

24. Table 2: The stated value of payload must be set in the context of the surface area. The information on the dimension if samples used for LbL coating is missing. Where the film free standing or on a substrate?

- Clarification has been added in the methods section that describes the deposition process.

25. The discussion on the differences in drug loading levels in lines 407-423 is hard to follow without a detailed method description, how the drug was loaded. There is a statement of “the chosen bioactive substance” in line 152. If this is the drug, the authors may need to justify to which of the polyelectrolyte solution the drug was added and why. Higher drug loading – which here is a surface deposition process based on charge interaction -is hard to connect to crystallinity, metastability, and other terms suggested by the authors in line 420-424. - Additional discussion has been added in the text

26. How do you see the relevance of the loaded drug in relation to therapeutic doses? – Answer has been provided in the text.

27. Also, more information on the data analysis should be provided in the methods section based on which the release mechanism as presented in Table 2 has been concluded. – The required models have been provided in the text.

28. The authors conclude that the polarity of corona discharge, i.e. the type of charges introduced to the surface in the pretreatment phase, affect properties like drug release behavior. In the reviewers understanding, this surface activation is only a first step to allow the binding of the polyelectrolytes, which (by chance) also incorporate the drug as a cargo. Without a more detailed understanding of the LbL deposition process, namely the order by which the different layers have been added, how contaminations of stock solutions have been excluded, which layer thicknesses etc. have been obtained, it appears very speculative to discuss the observed results mechanistically. – Due to the small number of layers, created on the surface of the substrates, their morphology and structure is dependent on that of the substrates themselves.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I recommend to design the picture layout of Figure 8 as 3x3 position. The first is non-porous, the second is lyophilized, and the last line is composite.

Author Response

Comment 1: I recommend to design the picture layout of Figure 8 as 3x3 position. The first is non-porous, the second is lyophilized, and the last line is composite.

Response 1: Thank you for the recommendation. We have formatted figure 8 as you have described.

Reviewer 3 Report

Comments and Suggestions for Authors

The reviewer notices some significant attempts of the authors to improve the manuscript and lift the data presentation and methodological description to a common minimum standard.

However, the reviewer also notices:

- some edits are incomplete, e.g. the consistent use of the abbreviation PCL or the correction of the word "composite"

- some old figures are not removed when new figures were added

- The authors' explanation of the absence of an effect of porosity of contact angle measurements is not convincing, as the corresponding figure shows such effects of porosity of porous vs. non-porous films

- The discussion of the pore formation mechanisms is still not fully convincing for the reviewer. As PEG400 is not removed, it is not clear where the voids in the material originate from.

- The author's claim this to be a review paper, which is not clear. It seems the be a research paper with original data.

- The authors did not add more information on different surface modification techniques in the introduction and did not motivate their research appropriately.

- It is not the corona discharging that makes certain release pattern, but the architecture of the multilayers encapsulating the drug and their stability. Not studying the layer formation and characteristics remains a shortcoming. The discussion of the corona discharging treatment to justify certain release pattern can be considered as misleading.

Author Response