Photochemical Preparation of Silver Colloids in Hydroxypropyl Methylcellulose for Antibacterial Materials with Controlled Release of Silver

Round 1

Reviewer 1 Report

1. The manuscript coatings-956805 deals with the preparation and characterization of a silver-cellulose derivative composite. The composite is prepared from a mixture of silver nitrate and hydroxypropyl methylcellulose (HPMC), exposed to a LED lamp light source. the influences of HPMC content and time exposure to the light source have been studied. Even if a lot of work and characterizations have been performed, the manuscript suffers from the absence of a clear message. For instance, the title did not allow the reader to know the aim of this article. Thus, the authors should consider giving the title more specific, and rewrite the article following this main idea. 

2. After reading the manuscript coatings-956805, for several times, a question still remains: what is the aim of this manuscript? to discuss the preparation and specificity of the composite or to evaluate the antibacterial activity of it? Without this crucial information, I can not evaluate properly this manuscript. That is for the reason that I reject this manuscript for publication. 

Author Response

Authors: Kvitek et al.

We want to thank the reviewers for thoroughly reviewing our manuscript and giving suggestions for improvements. We have taken all comments into account carefully and made the following changes and corrections in the reworked manuscript.

Thank you very much for your kind assistance in the matter.

With compliments,

Ondřej Kvítek

Ref. 1:

1. The manuscript coatings-956805 deals with the preparation and characterization of a silver-cellulose derivative composite. The composite is prepared from a mixture of silver nitrate and hydroxypropyl methylcellulose (HPMC), exposed to a LED lamp light source. the influences of HPMC content and time exposure to the light source have been studied. Even if a lot of work and characterizations have been performed, the manuscript suffers from the absence of a clear message. For instance, the title did not allow the reader to know the aim of this article. Thus, the authors should consider giving the title more specific, and rewrite the article following this main idea. 

The title of the manuscript was changed to: “Photochemical preparation of silver nanoparticles in hydroxypropyl methylcellulose for antibacterial materials with controlled release of silver”

2. After reading the manuscript coatings-956805, for several times, a question still remains: what is the aim of this manuscript? to discuss the preparation and specificity of the composite or to evaluate the antibacterial activity of it? Without this crucial information, I can not evaluate properly this manuscript. That is for the reason that I reject this manuscript for publication.

The abstract and introduction sections were modified to convey the aims of the work more clearly. The text: “Hydroxypropyl methylcellulose (HPMC) is routinely used for controlled release of medicine thanks to its slow dissolution in water and could be used as a matrix for controlled release of AgNPs, if a method to produce such a material without the need of other reactants was developed.” was added to the abstract of the revised manuscript. The text: “HPMC was chosen as a stabilizing agent due to its slow solubility in water and its capability to form matrix for the AgNPs to be supported in. Thanks to those properties HPMC is often used for controlled release of drugs. The solid samples prepared as a combination of the AgNPs and HPMC could therefore be used for better control of AgNPs release from the functional materials, so the concentration of silver does not reach toxic levels.” was added to the last paragraph of the introduction section.

Reviewer 2 Report

1. The publication presented by Kvitek et al. is devoted to the synthesis of Silver nanoparticles and incorporation of this to materials. AgNPs were obtained photochemical reduction of AgNO3 in hydroxypropyl methylcellulose (HPMC) solutions by eliminating the mixture with an LED bulb. The investigation is quite diverse; the results are presented clearly. However, I was surprised to notice that the references are completely messed up. I believe the paper could be accepted after this technical revision.

Author Response

Authors: Kvitek et al.

We want to thank the reviewers for thoroughly reviewing our manuscript and giving suggestions for improvements. We have taken all comments into account carefully and made the following changes and corrections in the reworked manuscript.

Thank you very much for your kind assistance in the matter.

With compliments,

Ondřej Kvítek

Ref. 2:

1. The publication presented by Kvitek et al. is devoted to the synthesis of Silver nanoparticles and incorporation of this to materials. AgNPs were obtained photochemical reduction of AgNO3 in hydroxypropyl methylcellulose (HPMC) solutions by eliminating the mixture with an LED bulb. The investigation is quite diverse; the results are presented clearly. However, I was surprised to notice that the references are completely messed up. I believe the paper could be accepted after this technical revision.

The formatting of the references got unfortunately disorganized during the reformatting of the manuscript, where the DOIs of the cited articles were assigned separate numbers, which disrupted the numbering of the references. This problem was corrected in the revised manuscript..

Reviewer 3 Report

This paper concerns the incorporation of silver nanoparticles into a HPMC cellulose derivative, using photochemical routes, for potential food protective applications.

The paper is relatively clearly written, well-organised and could potentially be accepted for publication. However, there are a few issues that need to be resolved, requiring major changes to the manuscript. They are:

1. If the AgNP are meant to be used in potential food protective applications (as is mentioned in the introduction and alluded to throughout), there needs to be some degree of leach-testing to be done to ensure that the amount of AgNP removed from the system provides a low toxicity risk

2. NP release studies are increasingly critical to NP-incorporation reports

3. SEM images of the NP-incorporated composite are not particularly definitive (I cannot discern any AgNP presence). Is it possible to use an alternative/newly acquired image with a more definitive presence?

4. There does not seem to have been any statistical significance testing done on the bacterial studies. There does need to be a presentation of the relative significance of the experimental values being presented and so, the value of the material for its proposed function.

5. The referencing is very sloppy. There is no proper format or structure and many references simply have a DOI listed. Please adopt a recognised style and use it consistently throughout. The entire section needs to be re-done. Also, whilst it is difficult to say if the area has been properly covered with the referencing in its current state, the authors rightfully stated the area as being a large one. Thus, the manuscript may be improved through reference to one or more of the following overlapping relevant works:

• • DOI: 10.1021/la950435d
  • DOI: 10.1016/j.matlet.2019.127077
  • DOI: 10.1021/la5006743
  • DOI: 10.1186/s12989-014-0049-1
  • DOI: 10.3390/polym11122000
  • DOI: 10.3390/ijms20040865, etc.

Author Response

Authors: Kvitek et al.

We want to thank the reviewers for thoroughly reviewing our manuscript and giving suggestions for improvements. We have taken all comments into account carefully and made the following changes and corrections in the reworked manuscript.

Thank you very much for your kind assistance in the matter.

With compliments,

Ondřej Kvítek

Ref. 3:

1. This paper concerns the incorporation of silver nanoparticles into a HPMC cellulose derivative, using photochemical routes, for potential food protective applications.

The paper is relatively clearly written, well-organised and could potentially be accepted for publication. However, there are a few issues that need to be resolved, requiring major changes to the manuscript. They are:

• If the AgNP are meant to be used in potential food protective applications (as is mentioned in the introduction and alluded to throughout), there needs to be some degree of leach-testing to be done to ensure that the amount of AgNP removed from the system provides a low toxicity risk

The proposed materials were mainly prepared with the potential application of controlled release of AgNPs for medicinal purpose in e.g. tissue engineering. The aims of the work were explained more clearly in the revised manuscript in the last paragraph of the introduction section the text: : “HPMC was chosen as a stabilizing agent due to its slow solubility in water and its capability to form matrix for the AgNPs to be supported in. Thanks to those properties HPMC is often used for controlled release of drugs. The solid samples prepared as a combination of the AgNPs and HPMC could therefore be used for better control of AgNPs release from the functional materials, so the concentration of silver does not reach toxic levels.” was added. The results of AgNP release tests from the prepared material are supported in Figure 7 and discussion thereof.

2.NP release studies are increasingly critical to NP-incorporation reports

The results of AgNP release tests from the prepared material are supported in Figure 7 and discussion thereof.

3. SEM images of the NP-incorporated composite are not particularly definitive (I cannot discern any AgNP presence). Is it possible to use an alternative/newly acquired image with a more definitive presence?

The AgNPs are incorporated in the volume of the material and can therefore not be seen on the SEM images which show only the surface structure of the prepared material. The AgNPs are however present in the materials in appropriate amount, as is documented by the other performed measurements (TEM, UV-Vis spectroscopy). AgNPs were also observed on the surface of the spin-coated thin films in the newly added AFM images in Figure 11 of the revised manuscript.

4. There does not seem to have been any statistical significance testing done on the bacterial studies. There does need to be a presentation of the relative significance of the experimental values being presented and so, the value of the material for its proposed function.

The statistical significance testing was not necessary to perform, since all the prepared materials that contained the AgNPs caused all the bacterial colony forming units to diminish completely. The complete bacterial elimination can be also seen in the images of the test dishes that were added in Figure 13 in the revised manuscript.

5. The referencing is very sloppy. There is no proper format or structure and many references simply have a DOI listed. Please adopt a recognised style and use it consistently throughout. The entire section needs to be re-done. Also, whilst it is difficult to say if the area has been properly covered with the referencing in its current state, the authors rightfully stated the area as being a large one. Thus, the manuscript may be improved through reference to one or more of the following overlapping relevant works:

• • DOI: 10.1021/la950435d
  • DOI: 10.1016/j.matlet.2019.127077
  • DOI: 10.1021/la5006743
  • DOI: 10.1186/s12989-014-0049-1
  • DOI: 10.3390/polym11122000
  • DOI: 10.3390/ijms20040865, etc.

The formatting of the references got unfortunately disorganized during the reformatting of the manuscript, where the DOIs of the cited articles were assigned separate numbers, which disrupted the numbering of the references. This problem was corrected in the revised manuscript. Also, several of the references from the suggested list were added as reference numbers 8, 9, 12, 31 and 32 in the revised manuscript.

Reviewer 4 Report

I read carefully the manuscript entitled ‘Composites of silver nanoparticles with hydroxypropyl methylcellulose prepared by photochemical reduction for reputed Coatings Journal. In this research work authors proposed the photochemical reduction of AgNO3 using hydroxypropyl methylcellulose (HPMC) as a reducing agent by taking three different concentrations. Further they have prepared foils, sponges and thin films, characterized and finally studied antibacterial effect. This article is comprehensive, fits and suitable to publish in Coatings Journal. This manuscript is generally well written and clearly presented however still need to be addressed many comments and thus require substantial major revision before its acceptance.

1. Title need to modify which can describe whole research work mainly application part

2. In abstract authors should mention the values of results and importance of research work in one or two sentences.

3. In the introduction section, write the novelty of the work and the problem statement clearly. The manuscript is lacking to cite essential review of literature author should refer an cite some recent review articles related to synthesis and application of silver nanoparticles

4. The Introduction section is not described well the advantages of the developed methods and their application in medical sector. Give details of the research objectives at the end of Introduction clearly.

5.It is very confusing why author choose three different concentrations since all concentration showing somehow similar results give proper explanation for the same.

6. Give reaction mechanism of the developed process by adding a schematic representation.

7. In the revised version perform XRD, FTIR and AFM analysis which is quiet expected.

8. In TEM analysis for 0.5% concentration the synthesized AgNPs are not in good form give detailed explanation. Author mentioned that 1% concentration is good however 0.25% gave monodispersed and similar size of synthesized NPs. It is difficult to understand about the research objectives elaborate this point extensively during revision.

9. Actual images of antibacterial activities as a supplementary material is recommended.

10. Correct the references according to Journal style and also refer some recent research articles those are in line with the research work.

11. More discussion of results under discussion section is essential.

12. Comparative table of results with the literature is recommended.

13. Write the practical applications and future research perspectives of this work by adding new section before conclusions

14. The conclusion of the study is not discussed with the specific output obtained from the study, it could be modified with precise outcomes with a take home message.

Author Response

Authors: Kvitek et al.

We want to thank the reviewers for thoroughly reviewing our manuscript and giving suggestions for improvements. We have taken all comments into account carefully and made the following changes and corrections in the reworked manuscript.

Thank you very much for your kind assistance in the matter.

With compliments,

Ondřej Kvítek

Ref. 4:

I read carefully the manuscript entitled ‘Composites of silver nanoparticles with hydroxypropyl methylcellulose prepared by photochemical reduction for reputed Coatings Journal. In this research work authors proposed the photochemical reduction of AgNO3 using hydroxypropyl methylcellulose (HPMC) as a reducing agent by taking three different concentrations. Further they have prepared foils, sponges and thin films, characterized and finally studied antibacterial effect. This article is comprehensive, fits and suitable to publish in Coatings Journal. This manuscript is generally well written and clearly presented however still need to be addressed many comments and thus require substantial major revision before its acceptance.

1. Title need to modify which can describe whole research work mainly application part

The title of the manuscript was changed to: “Photochemical preparation of silver nanoparticles in hydroxypropyl methylcellulose for antibacterial materials with controlled release of silver”

2. In abstract authors should mention the values of results and importance of research work in one or two sentences.

The abstract was modified to convey the aims of the work more clearly. The text: “Hydroxypropyl methylcellulose (HPMC) is routinely used for controlled release of medicine thanks to its slow dissolution in water and could be used as a matrix for controlled release of AgNPs, if a method to produce such a material without the need of other reactants was developed.” was added to the abstract of the revised manuscript.

3. In the introduction section, write the novelty of the work and the problem statement clearly. The manuscript is lacking to cite essential review of literature author should refer an cite some recent review articles related to synthesis and application of silver nanoparticles

The introduction section of the manuscript was improved; reference list was considerably extended to cite recent reviews regarding AgNPs (references number 9, 10, 11, 21, 23, 24, 25, 28 and 29). The text: “AgNPs supported on graphene oxide were infused into polyviscose pads to form antibacterial functional textile [8]. In medicine AgNPs can be used in diagnosis (e.g. as plasmonic nanoantennas, for tunable wavelength imaging or surface enhanced fluorescence [9]) or directly for treatment (e.g. as topical antimicrobial agents, wound dressing, implants or for cancer treatment) [10,11]. On the other hand, high concentrations of silver induce cytotoxicity and lead to argyria due to inappropriate silver accumulation in blood, lungs, liver and kidneys. Size of the AgNPs plays a great role in that smaller nanoparticles show both stronger antibacterial effects and are more toxic [10,12]. It is therefore necessary to control the size of the AgNPs during the preparation process (small enough particles to be effective in antimicrobial action, but big enough to be non-toxic) and to ensure controlled release of the silver from the functional material to maintain safe but effective concentrations of the silver in the area of the medicament action [13].” was added to the first paragraph of introduction section. The text: “Another way to eliminate the need for additional reaction agents is employing physicochemical methods of silver nitrate reduction. Because silver nitrate is very sensitive to visible light, it is possible to induce its reduction by illumination of its solution in a photochemical reduction process [31,32]. It is still necessary for the reaction mixture to contain stabilizing agents to prevent the AgNPs from aggregating when the nitrate gets reduced.” was added to the third paragraph of the introduction section.

4. The Introduction section is not described well the advantages of the developed methods and their application in medical sector. Give details of the research objectives at the end of Introduction clearly.

The introduction was modified to convey the aims of the work more clearly. The text: “HPMC was chosen as a stabilizing agent due to its slow solubility in water and its capability to form matrix for the AgNPs to be supported in. Thanks to those properties HPMC is often used for controlled release of drugs. The solid samples prepared as a combination of the AgNPs and HPMC could therefore be used for better control of AgNPs release from the functional materials, so the concentration of silver does not reach toxic levels.” was added to the last paragraph of the introduction section.

5. It is very confusing why author choose three different concentrations since all concentration showing somehow similar results give proper explanation for the same.

The different concentrations of HPMC solutions lead to very different thicknesses of the spin-coated films, since the polymer concentration is a very important factor in controlling the spin-coating process. The UV-Vis absorption spectra of the samples with different HPMC concentration show considerable differences as well (especially the overall absorption is influenced significantly, but differences can be spotted in the character of the spectra as well). Lastly, different properties of thin film samples prepared from different HPMC concentrations were also observed in the AFM images in the newly added Figure 11 and discussion thereof in the revised manuscript: “The surface morphology of the spin-coated thin films on glass slides was studied by AFM (Figure 11). The films prepared from HPMC solutions without the AgNPs show a very homogeneous and smooth surface. This shows the capability of the HPMC to form delicate thin films from solutions at the chosen preparation conditions. In the case of the films with AgNPs, the top parts of the particles can be seen sticking from the film’s smooth surface in the micrographs. The thicker films prepared from higher HPMC (particularly the 1 % HPMC AgNP sample) concentrations are able to support larger particles that rise up to about 150 nm above the film surface. Many smaller particles can be seen on the images as well, scattered on the film’s surface. With the lower thickness of the films the larger particles could not be captured in the volume of the film, therefore a higher number of smaller particles are sticking from the surface, while the larger particles were washed away from the surface during the spin-coating.”

6. Give reaction mechanism of the developed process by adding a schematic representation.

The reaction to produce AgNPs runs as a simple decomposition of AgNO₃ by the electromagnetic radiation. The reaction schematic can be written as: 2AgNO₃ -> 2Ag + O₂ + 2NO₂. The reaction schematic was added to the experimental section: “The chemical reaction taking place can be written as: 2AgNO₃ -> 2Ag + O₂ + 2NO₂.”

7. In the revised version perform XRD, FTIR and AFM analysis which is quite expected.

The FTIR analysis of the foils and thin films was added in Figures 7 and 12. AFM analysis of the thin films was added in Figure 11 in the revised manuscript. We were not able to perform the XRD analysis on the prepared samples, however, we believe not much new information of the samples could be obtained compared to the already present TEM analysis of the colloids.

8. In TEM analysis for 0.5% concentration the synthesized AgNPs are not in good form give detailed explanation. Author mentioned that 1% concentration is good however 0.25% gave monodispersed and similar size of synthesized NPs. It is difficult to understand about the research objectives elaborate this point extensively during revision.

The text: “ The sample with 0.5 % HPMC solution showed the widest size distribution of the AgNPs and shows the characters of size distributions from the other two studied HPMC concentrations with highest number of particles being 10-40 nm and 80-110 nm in diameter. The wide size distribution is undesirable for the controlled antibacterial effect of the colloid.” was added to the manuscript to comment on the AgNPs prepared in 0.5 % HPMC solution. The text: “The narrowest size distribution was found to be in the case of the 0.25 % HPMC solution, where majority of the particles was found to be 60-120 nm in diameter.” was added to the manuscript to compare the results obtained for AgNPs prepared at 0.25 a 1 % HPMC concentration.

9. Actual images of antibacterial activities as a supplementary material is recommended.

We support the images of the test dishes in the updated Figure 13 in the revised manuscript.

10. Correct the references according to Journal style and also refer some recent research articles those are in line with the research work.

The format of the references was corrected, additional appropriate recent studies are referenced in the revised manuscript. The reference list was extended considerably.

11. More discussion of results under discussion section is essential.

The discussion section of the manuscript was improved. The texts: “The proposed preparation procedure and conditions should be therefore further explored to improve on the size distribution and control of the AgNP size, since in comparison to photochemically reduced AgNPs in polyvinyl pyrrolidone [31,32] the size distribution of the AgNPs was rather wide.”, “The dissolution of HPMC was gradual over the course of several hours to about 1 day, depending on the preparation method of the solid sample. This result is very promising in the sense of controlled release of silver from the functional material.” and “The prepared materials show a promise for new branch of antibacterial materials based on AgNPs. The controlled release of AgNPs from the material would take course over several hours to days and the concentration of the silver in the vicinity of the material could be controlled by the dissolution rate of the HPMC composite. Further development of the proposed material is however needed. The employed concentrations of AgNPs were able to eliminate bacteria in the tests easily, the concentration could therefore be tuned to lower levels of silver content. The size dispersion of the prepared nanoparticles turned out to be rather wide and further improvements to the preparation method could be explored. Lastly, cytotoxicity tests would need to be performed to determine the high end of the desired concentration of these particular AgNPs in the antibacterial materials.”  were added to the discussion section of the revised manuscript.

12. Comparative table of results with the literature is recommended.

We believe that a table of referenced results falls somewhat out of a scope of original research article and would be more suited for a different publication format such as a review.

13. Write the practical applications and future research perspectives of this work by adding new section before conclusions

A new paragraph was added to the discussion section to evaluate possible developments of the proposed preparation methods: “The prepared materials show a promise for new branch of antibacterial materials based on AgNPs. The controlled release of AgNPs from the material would take course over several hours to days and the concentration of the silver in the vicinity of the material could be controlled by the dissolution rate of the HPMC composite. Further development of the proposed material is however needed. The employed concentrations of AgNPs were able to eliminate bacteria in the tests easily, the concentration could therefore be tuned to lower levels of silver content. The size dispersion of the prepared nanoparticles turned out to be rather wide and further improvements to the preparation method could be explored. Lastly, cytotoxicity tests would need to be performed to determine the high end of the desired concentration of these particular AgNPs in the antibacterial materials.”

14. The conclusion of the study is not discussed with the specific output obtained from the study, it could be modified with precise outcomes with a take home message.

The conclusion of the presented research was better explained in the discussion section of the revised manuscript, the text: “The prepared materials show a promise for new branch of antibacterial materials based on AgNPs. The controlled release of AgNPs from the material would take course over several hours to days and the concentration of the silver in the vicinity of the material could be controlled by the dissolution rate of the HPMC composite. Further development of the proposed material is however needed. The employed concentrations of AgNPs were able to eliminate bacteria in the tests easily, the concentration could therefore be tuned to lower levels of silver content. The size dispersion of the prepared nanoparticles turned out to be rather wide and further improvements to the preparation method could be explored. Lastly, cytotoxicity tests would need to be performed to determine the high end of the desired concentration of these particular AgNPs in the antibacterial materials.” was added in the revised manuscript.

Round 2

Reviewer 1 Report

Since the authors modified the manuscript coatings-956805 according to my comments, I could review it more appropriately. The title indicates that the manuscript deals with the photochemical preparation of silver/HPMC composite and the controlled release of silver for antibacterial purposes. Even if the authors modified many points in it, some others still need to be improved. That is for this reason that I recommend major revision for this manuscript. My comments and recommendations are listed below. I hope that they will help the authors.

1. In the title, the authors mentioned the release of silver. However, Lines 51-53, they indicate that silver NPs release Ag+ ions. Is “release of silver” should be replaced by the “release of silver ions”?

2. Line 93: “HPMC) serving both as a reducing agent and capping agent.” The authors mentioned that HPMC is used to stabilize silver particles in the introduction (l85) and that “the reducing power of HPMC” is insufficient to produce silver particles. 

3. Line 104: “2AgNO3 -> 2 Ag + O2 + 2NO2” is there any formation of silver oxide phase?  

4. Lines 196-208: Why is there a UV-vis signal at 0 min of irradiation? As the signals from 0 min to 120 min look like more or less the same, is it an SPR signal or HPMC signature? 

5. Lines 224-225: “the wide size distribution … of the colloid.” So? Can the authors give any ways to improve the size distribution?

6. Fig. 4: What’s about NPs distribution within the HPMC matrix? What’s about sample homogeneity? Moreover, due to the really wide size distribution, I am not sure that nanoparticles adequately describe the samples. I think that Ag particles will be more appropriated. 

7. Line 259: “release of AgNPs” Is it a release of silver, release of silver ions, or release of AgNPs?

8. Fig. 8: where is the end of the caption? 

9. Lines 304-309: “150 nm” is not any more nanoparticles. “Many smaller particles can be seen…” And so what? 

10. Lines 313-317: what kind of useful pieces of information for the discussion give this paragraph at this point? Could it be discussed with Fig 7 (lines 249-256)?

11. Fig. 13: did the authors have tested Ag(+)@HPMC? Is it more or less active than Ag particles@HPMC?

12. Line 350: “mostly lower than 100nm” I disagree with that point. The authors should describe their composites more precisely.

13. Line 362: “release of silver from the functional material.” And Line 367: “controlled release of AgNPs from the materials” see the comments n°1 and n°7.

14. Can the authors add the evaluation of silver ions release in Luria-Bertani growth medium? 

Author Response

Authors: Kvitek et al.

We want to thank the reviewers for thoroughly reviewing our manuscript and giving suggestions for improvements. We have taken all comments into account carefully and made the following changes and corrections in the reworked manuscript.

Thank you very much for your kind assistance in the matter.

With compliments,

Ondřej Kvítek

Ref. 1:

Since the authors modified the manuscript coatings-956805 according to my comments, I could review it more appropriately. The title indicates that the manuscript deals with the photochemical preparation of silver/HPMC composite and the controlled release of silver for antibacterial purposes. Even if the authors modified many points in it, some others still need to be improved. That is for this reason that I recommend major revision for this manuscript. My comments and recommendations are listed below. I hope that they will help the authors. 

1. In the title, the authors mentioned the release of silver. However, Lines 51-53, they indicate that silver NPs release Ag+ ions. Is “release of silver” should be replaced by the “release of silver ions”?

Silver ions get released from silver particles and silver particles are released from the HPMC due to its dissolution. Therefore, it is appropriate the title states the general term – release of silver.

2. Line 93: “HPMC) serving both as a reducing agent and capping agent.” The authors mentioned that HPMC is used to stabilize silver particles in the introduction (l85) and that “the reducing power of HPMC” is insufficient to produce silver particles. 

The sentence on lines 92-93 was modified to: "Silver colloids were prepared by photochemical method with hydroxypropyl methylcellulose (HPMC) serving as a capping agent." The HPMC was tested as a reducing agent but showed to be insufficient. This is described in the first paragraph of the Results section.

3. Line 104: “2AgNO3 -> 2 Ag + O2 + 2NO2” is there any formation of silver oxide phase? 

The silver oxide is less stable than metallic silver under the preparation conditions. Moreover, no significant features of Ag oxides were found in the UV-Vis absorption spectra. Oxidation of the Ag particles after the preparation is prevented by the HPMC stabilization.

4. Lines 196-208: Why is there a UV-vis signal at 0 min of irradiation? As the signals from 0 min to 120 min look like more or less the same, is it an SPR signal or HPMC signature? 

The spectrum of HPMC showed no specific absorption in the measured wavelength range. The absorption at 0 min of irradiation is caused by Ag seeds for the particle growth. The seeds are created in the HPMC solutions during the AgNO3 mixing with HPMC and its heating for 30 minutes. The particles then grow on these seeds, because the photochemical reduction is catalyzed by the presence of these Ag seeds. A sentence was added in the experimental section in chapter 2.1: "During this process, Ag seeds are created that catalyze the photochemical reduction of the AgNO₃ in the next preparation step."

5. Lines 224-225: “the wide size distribution … of the colloid.” So? Can the authors give any ways to improve the size distribution?

The size distribution can be influenced in many ways – the modification of the reactant concentration (e.g. lower AgNO₃ concentration), modification of the seeding process (e.g. longer seeding time, higher temperature) or modification of the photoreduction process (e.g. using more specific wavelengths, stirring or heating during the process). The modification of the photoreduction of AgNO₃ in HPMC to achieve better control of the prepared Ag particle size will be studied further in future, which is described in the Discussion section of the manuscript. The sentence: " The size dispersion of the prepared nanoparticles turned out to be rather wide and further improvements to the preparation method could be explored (e.g. better control of the seeding process, modification of the reactant concentrations or tuning the wavelength, heating and stirring during the photoreduction process). " was added in last paragraph of the Discussion section of the manuscript.

6. What’s about NPs distribution within the HPMC matrix? What’s about sample homogeneity? Moreover, due to the really wide size distribution, I am not sure that nanoparticles adequately describe the samples. I think that Ag particles will be more appropriated. 

The homogeneity of the AgNPs distribution in the HPMC matrix can be evaluated from the photographs and micrographs of the solid samples. The even coloration of the samples suggests no further aggregation of the Ag particles, the UV-Vis spectra of the thin-film samples and the solutes show no signs of aggregation of the Ag particles in the solid samples. Also, a new SEM image was added to the revised manuscript, which proves the homogeneity of the AgNP distribution over the HPMC matrix. The results of TEM show an overwhelming majority of the prepared Ag particles fall in the nanometer-scale range.

7. Line 259: “release of AgNPs” Is it a release of silver, release of silver ions, or release of AgNPs?

The correct term is release of AgNPs, since the release was observed by the UV-Vis absorption spectrum, where the SPR absorption of metallic Ag can be seen.

8. where is the end of the caption? 

The formatting of the Figure 8 caption and the following paragraph was corrected.

9. Lines 304-309: “150 nm” is not any more nanoparticles. “Many smaller particles can be seen…” And so what? 

The results of TEM show an overwhelming majority of the prepared Ag particles fall in the nanometer-scale range. The title of the manuscript was changed to: " Photochemical preparation of silver colloids in hydroxypropyl methylcellulose for antibacterial materials with controlled release of silver".

10. Lines 313-317: what kind of useful pieces of information for the discussion give this paragraph at this point? Could it be discussed with Fig 7 (lines 249-256)?

Figure 12 and the paragraph describing it was merged with Figure 7 and its description.

11. did the authors have tested Ag(+)@HPMC? Is it more or less active than Ag particles@HPMC?

The Ag ions generally possess strong antibacterial effect; however, they are also cytotoxic at higher concentrations. It is therefore advantageous to use Ag particles instead that are able to slowly release Ag ions, which then do not exceed toxic concentrations. Ag particles also show ability to bound selectively to bacterial membranes and disrupting their function. This topic is well described in literature referenced in the manuscript (namely ref 14 and 15) Ag(I) salts are also generally quite unstable – photosensitive and decompose at elevated temperature. This would be disadvantageous during usage of the materials.

12.  “mostly lower than 100nm” I disagree with that point. The authors should describe their composites more precisely.

The sentence in question was changed to: "Most of the particles were found to be smaller than 100 nm in diameter." The particle size distribution analysis shows that overwhelming majority of the prepared Ag particles was smaller than 100 nm in diameter (see Figure 4d). A more detailed description of the particle size results is in the results section. In the discussion section, there is only summary of these results.

13. Line 362: “release of silver from the functional material.” And Line 367: “controlled release of AgNPs from the materials” see the comments n°1 and n°7.

The sentence on line 362 was changed to: "release of AgNPs from the functional material".

14. Can the authors add the evaluation of silver ions release in Luria-Bertani growth medium?

The Ag ions generated from the released AgNPs in the bacterial suspensions were studied only in the sense of the antibacterial effect of the samples, which showed to be very strong. The antibacterial effect of the Ag ions has been evaluated in the past by other research groups and the results were summarized in review articles such as those listed in the references of this manuscript, namely ref. 14 and 15.

Reviewer 3 Report

1. The re-submission addresses most of the issues raised in the previous round. My concern still remains the SEM images however. I believe much better images can and should be obtained. Certainly the capability is there; the researchers have a FEG system at their disposal. So the lack of a quality, zoomed in image is inexplicable. The current SEM images offer little value.

Author Response

Authors: Kvitek et al.

We want to thank the reviewers for thoroughly reviewing our manuscript and giving suggestions for improvements. We have taken all comments into account carefully and made the following changes and corrections in the reworked manuscript.

Thank you very much for your kind assistance in the matter.

With compliments,

Ondřej Kvítek

Ref. 3:

1. The re-submission addresses most of the issues raised in the previous round. My concern still remains the SEM images however. I believe much better images can and should be obtained. Certainly the capability is there; the researchers have a FEG system at their disposal. So the lack of a quality, zoomed in image is inexplicable. The current SEM images offer little value.

New SEM images were added in Figures 5d and 6d. We believe these images better illustrate the homogeneous distribution of the Ag particles in the HPMC matrices.

Reviewer 4 Report

1. Authors have substantially revised the manuscript according to comments. The present form of the manuscript can be accepted and to publish in Coatings.

Author Response

Authors: Kvitek et al.

We want to thank the reviewers for thoroughly reviewing our manuscript and giving suggestions for improvements. We have taken all comments into account carefully and made the following changes and corrections in the reworked manuscript.

Thank you very much for your kind assistance in the matter.

With compliments,

Ondřej Kvítek

Ref. 4:

1. Authors have substantially revised the manuscript according to comments. The present form of the manuscript can be accepted and to publish in Coatings.

We would like to thank the reviewer for the very helpful comments and suggestions during the revision of the manuscript once again.

Round 3

Reviewer 1 Report

The authors have modified their manuscript following my recommendations and have given satisfactory answers to my questions and comments. Therefore, even if a manuscript can always be improved, I believe their manuscript has met the standards for publication in Coatings.