Development of a Chitosan-Silver Nanocomposite/β-1,3-Glucan/Hyaluronic Acid Composite as an Antimicrobial System for Wound Healing

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1.  The affliation should be end with Taiwan, China.

2. The role of respective component in the composite gel should be clarified clearly based on the experimental results.

3. The mechanical properties of the gel should be tested.

4. The antibacterial performance of AgNP incorporated gel and chitosan incoporated gel should also compared to illustrated the nessicity of combining AgNP with chitosan.

5. Chitosan is also a natural antibacterial agent. Why the authors do not mention this?

6. Since both AgNP and chitosan are antibacterial agents, the antibacterial mechanism of the gel should be clearly stated.

7. Why the authors do not introduce in-vivo or in-vitro tests to verify the feasibility of the gel?

 

Comments on the Quality of English Language

The English of this manuscript is understandable. 

Author Response

Author's Reply to the Review Report (Reviewer 1)

Comments and Suggestions for Authors

1. The affliation should be end with Taiwan, China.

 Author response:

Thanks for the reviewer’s suggestion. The end of affiliation has been modified as Taiwan, Republic of China.

 

2. The role of respective component in the composite gel should be clarified clearly based on the experimental results.

Author response:

Thanks for the reviewer’s suggestion. The role of respective component in the composite hydrogel has been clarified and shown in the lines 103-108 and lines 443-453. It has indicated that chitosan was used as a reducing and capping agent in the synthesis of chitosan-silver nanocomposites (Ag NP@CHI) to reduce the direct toxicity of Ag NPs on normal cells. To provide a moist environment at the wound site, enhancing granulation and epithelialization, Ag NP@CHI was incorporated into β-1,3-glucan/HA-based hydrogel to form Ag NP@CHI@β-1,3-glucan/HA. Incorporating Ag NP@CHI with β-1,3-glucan/HA significantly improved biocompatibility and hemocompatibility of Ag NP@CHI and promoted blood coagulation and wound healing significantly.

 

3. The mechanical properties of the gel should be tested.

Author response:

Thanks for the reviewer’s suggestion. The prepared composite hydrogel in this study primarily aimed to maintain the antibacterial properties of silver nanoparticle while reducing its direct toxicity to wounds. At the same time, this composite hydrogel enhanced blood clotting and promoted wound healing. Future applications are expected to involve the integration of coating materials; therefore, the mechanical properties will be investigated in the future and are not a primary focus at this stage.

 

4. The antibacterial performance of AgNP incorporated gel and chitosan incoporated gel should also compared to illustrated the nessicity of combining AgNP with chitosan.

Author response:

Thanks for the reviewer’s suggestion. The antibacterial efficiency of Ag NP@CHI@β-1,3-glucan/HA and CHI@β-1,3-glucan/HA had been shown in Figure 7, indicating that although CHI@β-1,3-glucan/HA had antibacterial effect due to the participation of chitosan, its efficiency is still significantly lower than Ag NP@CHI@β-1,3-glucan/HA. In addition to being as a template and stabilizer to synthesize silver nanoparticles, chitosan also significantly reduced the toxicity of silver nanoparticles onto cells (Figure 6a). Therefore, the combining Ag NPs with chitosan was still necessary in this study.

 

5. Chitosan is also a natural antibacterial agent. Why the authors do not mention this?

Author response:

The antibacterial potency of chitosan had been mentioned in the revised article in lines 313-315, and lines 400-402, showing as that "Chitosan generally exhibits stronger effects on gram-positive bacteria (S. aureus) than on gram-negative bacteria (E. coli) [55-57]" and " Since chitosan and β-glucans possess potential antibacterial activity against various gram-positive and gram-negative bacteria [66,67], the CHI@β-1,3-glucan/HA exhibited 42% suppression of E. coli growth", respectively. Although chitosan is a natural antibacterial agent with low cytotoxicity, its antibacterial effect is still far lower than that of silver nanoparticles. Therefore, this study takes advantage of each and reduces its shortcomings to develop a composite material for future wound repair.

 

6. Since both AgNP and chitosan are antibacterial agents, the antibacterial mechanism of the gel should be clearly stated.

Author response:

Thanks for the reviewer’s suggestion. The antibacterial mechanism of the composite gel has been added in the revised manuscript, lines 426-434, and shown as "Chitosan and β-1,3-glucan in the composite hydrogel could initially interact with the negatively charged outer or inner membrane of bacteria through electrostatic interactions, or damage membrane integrity through interactions with phospholipids and proteins, altering the permeability of the bacterial cell membrane. Subsequently, the released silver ions further could damage the cell wall and cytoplasmic membrane, denature bacterial ribosomes, interrupt adenosine triphosphate (ATP) production, interfere with DNA replication, and even cause membrane perforation, leading to the release of organelles from the cell, directly killing the bacteria.".

 

7. Why the authors do not introduce in-vivo or in-vitro tests to verify the feasibility of the gel?

Author response:

Thanks for the reviewer’s suggestion. Through in vitro antibacterial tests, biocompatibility assessment, hemocompatibility evaluation, blood coagulation test, and cell migration experiment, it was demonstrated that the developed Ag NP@CHI@β-1,3-glucan/HA did possess antibacterial properties and the potential to promote wound healing. Future applications will be expected to involve integration with coating materials; therefore, related mechanical properties and in vivo experiments will be conducted after the complete development of the dressing material.

 

8. Comments on the Quality of English Language. The English of this manuscript is understandable. 

Author response:

Thanks for the reviewer’s suggestion. The full article has been carefully proofread, and the grammar in submitted manuscript has been improved by the scientific translation service to prevent misunderstanding.

In conclusion, by this revision, we believe that our research has been presented more clearly and convincingly to make a conclusion that Ag NP@CHI@β-1,3-glucan/HA demonstrates potential as a wound healing hydrogel in the future.

Reviewer 2 Report

Comments and Suggestions for Authors

The article by Cheng-Jung Yao et al. is devoted to one of the current topics, such as the development of a wound healing gel with antimicrobial properties. The gel that the researchers create is a multicomponent system consisting of silver nanoparticles obtained using chitosan, as well as β-1,3-glucan and hyaluronic acid.

 

Overall, the main drawback of this article is that the title talks about gel, but no properties of the gel are studied at all. In my opinion, it is necessary to study the properties of gels, such as porosity, density, swelling ratio and mechanical parameters of the gel, in addition, it is necessary to study the morphology of the gel using SEM. Otherwise, the article is written quite logically.

 

The following are some notes on writing the article:

·       Line 56 Ag NP is better to write together.

·       Line 119 The molecular weight of chitosan must be indicated.

·       Line 123 The molecular weight of β-1,3-glucan must be indicated.

·       Lines 164-171 A reference to the method for determining cytotoxicity must be provided. Since the method using MTT, dissolving the resulting crystals in DMSO, is most often used.

·       Equation 1. In order to determine the percentage of hemolysis, it is necessary to provide an explanation of the terms "absorbance of PC" and "absorbance of NC."

·       Line 207 Gram-negative must be written with a lowercase letter.

·       Figure 2b – what is the reason for the high PdI value at the lowest chitosan concentration?

·       Figure 2d – it is necessary to add the spectrum of the original chitosan to the existing spectra.

·       Figure 2f – the microscopic images are presented very small, it is difficult to examine them, perhaps it is necessary to separate them into a separate figure.

·       Line 322, it is written that the HA spectrum is presented, but in Figure 4a the spectrum is missing, perhaps the β-1,3-glucan/HA spectrum was meant?

·       Figure 5. In the figure captions, it is necessary to add information about which NC and PC were used for each part in Figure 5.

·       Line 378 in vitro italicize.

·       Line 395 E. coli italicize.

·       Line 424 extra period at the end of the sentence.

·       Figure 7c, regarding the data presented in Figure 7c, the activity exhibited by silver nitrate is predictable, since it is a low-molecular compound capable of penetrating through a paper filter. All other samples could show low activity, due to the fact that they could not penetrate through the paper due to the size of their molecules. This is indirectly confirmed by the data presented in Figure 7d, where at least AgNP@CHI and Ag NP@CHI@β-1,3-glucan/HA exhibit activity against bacteria. Thus, I believe that the Agar well diffusion method is not suitable for these samples and can be excluded from the article.

 

·       Line 435 80oС correct.

Author Response

Author's Reply to the Review Report (Reviewer 2)

The article by Cheng-Jung Yao et al. is devoted to one of the current topics, such as the development of a wound healing gel with antimicrobial properties. The gel that the researchers create is a multicomponent system consisting of silver nanoparticles obtained using chitosan, as well as β-1,3-glucan and hyaluronic acid.

Overall, the main drawback of this article is that the title talks about gel, but no properties of the gel are studied at all. In my opinion, it is necessary to study the properties of gels, such as porosity, density, swelling ratio and mechanical parameters of the gel, in addition, it is necessary to study the morphology of the gel using SEM. Otherwise, the article is written quite logically.

Author response:

Thanks for the reviewer’s suggestion. Because the prepared Ag NP@CHI@β-1,3-glucan/HA is in the form of a three-dimensional polymeric network with high water content, the “gel” in the title and article has been revised as “hydrogel” to avoid misunderstanding. This study purpose was to develop an ideal wound dressing to possess biocompatibility, exhibit high antibacterial activity, and promote blood coagulation and would healing. Therefore, the prepared composite hydrogel primarily aimed to maintain the antibacterial properties of silver nanoparticle while reducing its direct toxicity to wounds. At the same time, this composite hydrogel could enhance blood clotting and promoted wound healing. Future applications will be expected to involve integration with coating materials; therefore, related mechanical properties and in vivo experiments will be conducted after the complete development of the dressing material.

 

 The following are some notes on writing the article:

• Line 56 Ag NP is better to write together.

Author response:

Thanks for the reviewer’s suggestion. The literature search shows that both Ag NPs and AgNPs are used as abbreviations for silver nanoparticles, and no correction might be needed here.

 

• Line 119 The molecular weight of chitosan must be indicated.

Author response:

As suggestion, the molecular weight of chitosan has been added in line 118.

 

• Line 123 The molecular weight of β-1,3-glucan must be indicated.

Author response:

As suggestion, the molecular weight of β-1,3-glucan has been added in line 123.

 

• Lines 164-171 A reference to the method for determining cytotoxicity must be provided. Since the method using MTT, dissolving the resulting crystals in DMSO, is most often used.

Author response:

As suggestion, the reference has been added in line 171.

 

• Equation 1. In order to determine the percentage of hemolysis, it is necessary to provide an explanation of the terms "absorbance of PC" and "absorbance of NC."

Author response:

As suggestion, the equation 1 has been modified as “Hemolysis percentage (%) = (OD_{test samples} - OD_{negative control})/( OD_{positive control} - OD_{negative control}) × 100” in the revised manuscript.

 

• Line 207 Gram-negative must be written with a lowercase letter.

 Author response:

As suggestion, the “Gram-negative” has been modified as “gram-negative” in line 207.

 

• Figure 2b – what is the reason for the high PdI value at the lowest chitosan concentration?

Author response:

Thanks for the reviewer’s suggestion. As shown in TEM images (Figure 2f), the chitosan concentration of 0.5 mg/mL was effective as a template for synthesizing chitosan-silver nanocomposites, but it was insufficient to stabilize the chitosan-silver nanocomposites in solution, resulting in a high PdI value.

 

• Figure 2d – it is necessary to add the spectrum of the original chitosan to the existing spectra.

Author response:

As suggestion, FTIR spectra of chitosan has been added in Figure 2d.

 

• Figure 2f – the microscopic images are presented very small, it is difficult to examine them, perhaps it is necessary to separate them into a separate figure.

Author response:

Thanks for the reviewer’s suggestion. The size of TEM images in Figure 2f has been adjusted to be enough for observation of differences.

 

• Line 322, it is written that the HA spectrum is presented, but in Figure 4a the spectrum is missing, perhaps the β-1,3-glucan/HA spectrum was meant?

Author response:

Thanks for the reviewer’s suggestion. The error has been corrected and shown in line 323 as “β-1,3-glucan/HA”.

 

• Figure 5. In the figure captions, it is necessary to add information about which NC and PC were used for each part in Figure 5.

Author response:

As suggestion, the information about NC and PC has been added in the legend of Figure 5, and shown as "NC: CaCl₂ only; PC: HealiAid® Collagen Wound Dressing."

 

• Line 378 in vitro italicize.

Author response:

As suggestion, the error has been corrected in line 380.

 

• Line 395 E. coli italicize.

Author response:

As suggestion, the error has been corrected in line 397.

 

• Line 424 extra period at the end of the sentence.

Author response:

As suggestion, the error has been corrected in line 426.

 

• Figure 7c, regarding the data presented in Figure 7c, the activity exhibited by silver nitrate is predictable, since it is a low-molecular compound capable of penetrating through a paper filter. All other samples could show low activity, due to the fact that they could not penetrate through the paper due to the size of their molecules. This is indirectly confirmed by the data presented in Figure 7d, where at least AgNP@CHI and Ag NP@CHI@β-1,3-glucan/HA exhibit activity against bacteria. Thus, I believe that the Agar well diffusion method is not suitable for these samples and can be excluded from the article.

Author response:

Thanks for the reviewer’s suggestion. The agar well diffusion method may not be the most suitable test for evaluating the antibacterial effects of the prepared Ag NP@CHI and Ag NP@CHI@β-1,3-glucan/HA, but it is a commonly used method to determine antibacterial activity. So it should not be excluded from the article. Furthermore, this testing method also provided us with an idea for future product development. Perhaps Ag NP@CHI@β-1,3-glucan/HA in the form of a hydrogel as a dressing would perform better than in its dry form.

 

• Line 435 80oС correct.

Author response:

As suggestion, the error has been corrected in line 445.

In conclusion, by this revision, we believe that our research has been presented more clearly and convincingly to make a conclusion that Ag NP@CHI@β-1,3-glucan/HA demonstrates potential as a wound healing hydrogel in the future.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have reported the use of silver nanoparticle composite for wound healing, the authors have conducted systematic research and presented the results well. 

Minor comments

I still question the reliability of using Silver nanoparticles at a open wound site . Silver can harm bacteria, ofcourse its toxic to mamalian cells too. Is it wise to use silver? 

Life cycle assessment needs to be done. 

What are the practical implications of the study, can it be used as bandages and wound dressings

Figures are not clear, the SEM images need to be replaced. Figure legends improved. 

Need to work on all these areas. 

LAnguage needs fine tuning

Author Response

Author's Reply to the Review Report (Reviewer 3)

The authors have reported the use of silver nanoparticle composite for wound healing, the authors have conducted systematic research and presented the results well. 

Minor comments

I still question the reliability of using Silver nanoparticles at a open wound site. Silver can harm bacteria, of course its toxic to mamalian cells too. Is it wise to use silver? 

Life cycle assessment needs to be done. 

What are the practical implications of the study, can it be used as bandages and wound dressings

Figures are not clear, the SEM images need to be replaced. Figure legends improved. 

Need to work on all these areas. 

LAnguage needs fine tuning

Author response:

Thanks for the reviewer’s suggestion. Although silver nanoparticles can render a cytotoxic effect on eukaryotic cells and correlate with the concentration and particle size, they are still widely used in different fields of human activity due to the high reactivity and antibacterial activity against microorganisms. It has been indicated that the usage of different polymeric compounds as stabilizers can allow reducing of its cytotoxic effect and saving antibacterial activity [Heliyon. 2019 Mar 7;5(3):e01305.]. Therefore, the prepared Ag NP@CHI in the present study could indeed reduce the cytotoxicity of silver nanoparticles, and then the incorporation of β-1,3-glucan/HA with Ag NP@CHI could effectively improve biocompatibility and promote wound healing while maintaining its antibacterial properties.

Through in vitro antibacterial tests, biocompatibility assessment, hemocompatibility evaluation, blood coagulation test, and cell migration experiment, it was demonstrated that the developed Ag NP@CHI@β-1,3-glucan/HA did possess antibacterial properties and the potential to promote wound healing. Future applications will be expected to involve integration with coating materials; therefore, related mechanical properties, in vivo experiments and life cycle assessment will be conducted after the complete development of the dressing material.

The size of TEM images in Figure 2f has been adjusted to be enough for observation of differences.

Finally, the full article has been carefully proofread, and the grammar in submitted manuscript has been improved by the scientific translation service to prevent misunderstanding.

In conclusion, by this revision, we believe that our research has been presented more clearly and convincingly to make a conclusion that Ag NP@CHI@β-1,3-glucan/HA demonstrates potential as a wound healing hydrogel in the future.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript can be accepted at the current version.

Comments on the Quality of English Language

The English is ok.

Author Response

Comments and Suggestions for Authors

The manuscript can be accepted at the current version.

Comments on the Quality of English Language. The English is ok.

 

Author response:

Thanks for the reviewer’s response.

In conclusion, by this revision, we believe that our research has been presented more clearly and convincingly to make a conclusion that Ag NP@CHI@β-1,3-glucan/HA demonstrates potential as a wound healing composite in the future.

Reviewer 2 Report

Comments and Suggestions for Authors

Round 2

The authors tried and corrected some comments, however, I am sure that there is a need to present the characteristics or properties of such systems as gel or hydrogel in this work.

If the authors do not want to prove and provide the properties of the gel/hydrogel, it is worth using not the terms gel/hydrogel both in the title and in the text of the article itself - but, for example, such a term as "composite".

Line 119. Although the authors indicated the molecular weight, they did not indicate the units of measurement, is it kDa or Da. It is probably Da, then it is more logical to give the molecular weight in kDa for the uniformity of presentation of information, since the molecular weight for β-1,3-glucan is presented in kDa.

Author Response

Comments and Suggestions for Authors

Round 2

The authors tried and corrected some comments, however, I am sure that there is a need to present the characteristics or properties of such systems as gel or hydrogel in this work.

If the authors do not want to prove and provide the properties of the gel/hydrogel, it is worth using not the terms gel/hydrogel both in the title and in the text of the article itself - but, for example, such a term as "composite".

Author response:

Thanks for the reviewer’s suggestion. Because this study purpose was to develop an ideal wound dressing to possess biocompatibility, exhibit high antibacterial activity, and promote blood coagulation and would healing, the terms gel/hydrogel both in the title and in the text of the article have been replaced as “composite”.

Line 119. Although the authors indicated the molecular weight, they did not indicate the units of measurement, is it kDa or Da. It is probably Da, then it is more logical to give the molecular weight in kDa for the uniformity of presentation of information, since the molecular weight for β-1,3-glucan is presented in kDa.

Author response:

As suggestion, the unit of chitosan molecular weight has been added in line 118 as “kDa”.

In conclusion, by this revision, we believe that our research has been presented more clearly and convincingly to make a conclusion that Ag NP@CHI@β-1,3-glucan/HA demonstrates potential as a wound healing composite in the future.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made the necessary corrections and the article can now be published.